Methods for diagnosing and treating multiple sclerosis

Abstract

Provided herein are methods and immune biomarkers for identifying multiple sclerosis (MS) progression and treatment options. Materials and methods for prognosing, staging, and monitoring MS in a sample are also provided, including methods for determining a subject as at risk for developing MS. The methods include detecting at least one biomarker, such as CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3, in the cerebrospinal fluid (CSF) of a subject, and administering a pharmaceutically effective amount of a therapy (e.g., trebrutinib and potentially one or more additional therapies, e.g., an anti-CD20 therapy) to the subject.

Description

[Technical Field]

[0001] Government Interests Parties to the Joint Research Agreement This invention is the subject matter made jointly under the Public Health Service Cooperative Research and Development Agreement (PHS-CRADA Ref. No. 2020-0226) between the National Institute of Neurological Disorders and Stroke (NINDS) at the National Institutes of Health and Sanofi-Genzyme. The U.S. Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 482,714, filed February 1, 2023, and U.S. Provisional Patent Application No. 63 / 623,681, filed January 22, 2024, which are incorporated by reference in their entireties for any purpose.

[0003] Electronic Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on January 29, 2024, is named "01183-0270-00PCT-PRN.xml" and is 19,427 bytes in size. The Sequence Listing is included therein. The XML file is a part of the present specification and is incorporated herein by reference in its entirety.

[0004] The present disclosure generally relates to screening, detection, prognosis, and treatment of subjects with multiple sclerosis by detecting one or more biomarkers, including CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3, in the cerebrospinal fluid (CSF) of the patient. [Background technology]

[0005] Multiple sclerosis (MS) is a neurological disease affecting over one million people worldwide. It is the most common cause of neurological disability in young and middle-aged adults and has a significant physical, psychological, social, and economic impact on patients and their families. MS involves an immune-mediated process in which an abnormal response of the body's immune system is directed against the central nervous system (CNS). During the progression of the disease, sclerosis, or lesions or scars, appear in the myelin sheath of nerve cells, interfering with the transmission of electrical signals. The sclerosis accumulates over time, resulting in the debilitating symptoms experienced by MS patients. MS patients generally experience one of four clinical courses: clinically isolated syndrome, relapsing-remitting, secondary progressive, and primary progressive, with mild, moderate, or severe disease. Approximately 85% of MS patients have the relapsing-remitting form of the disease, experiencing clearly defined relapses (also called exacerbations or exacerbations), which are episodes of acute deterioration of neurological function, followed by periods of partial or complete recovery (remission) without disease progression.

[0006] Treatments such as trebrutinib have been developed. See, for example, U.S. Patent No. 9,688,676, U.S. Patent Application Publication No. 2021 / 0244720, and U.S. Patent Application Publication No. 2022 / 140511, each of which is incorporated by reference in its entirety. With the inclusion of various treatments, it is necessary to understand the effectiveness of such treatments. Therefore, molecular biomarkers are necessary to measure MS disease activity and evaluate treatment efficacy. Therefore, it is necessary to develop highly sensitive molecular tests to identify and measure molecular biomarkers associated with MS in order to diagnose and monitor the progression and severity of MS. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure has identified that proteins measured in cerebrospinal fluid (CSF) can serve as a window into neuroinflammation and can be used to assess the prognosis of disease progression and to help provide evidence of therapeutic response after treatment. In the aforementioned examples, the present disclosure demonstrates that Olink Proteomics can be used to assess proteome expression using a high-throughput multiplex immunoassay technology that allows for the measurement of over 1,000 proteins from a small sample volume. This type of assay can be performed on a variety of subjects, including those undergoing treatment, those for whom treatment is being determined, and those being monitored for the development of multiple sclerosis. Physiological improvements and impairments, such as changes in lesions (e.g., active lesions measured by magnetic resonance imaging (MRI)), can be simultaneously monitored to assess correlations between proteome expression in CSF and clinical outcomes. Furthermore, therapeutic treatments can be modified and / or tested for determined efficacy. For example, in some examples disclosed herein, changes to the MS CSF proteome upon therapeutic intervention with either the B cell-depleting agent ocrelizumab or the brain-penetrant Bruton's tyrosine kinase (BTK) inhibitor trebrutinib can be assessed, providing insight into both the pathophysiology of the disease and the efficacy of therapeutic intervention.

[0008] The present disclosure relates to the detection of at least one biomarker to predict or confirm treatment response for MS, wherein the at least one biomarker is selected from a protein listed in Tables 1-3, e.g., CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, the at least one biomarker is selected from a protein listed in Table 3. In some examples, the present disclosure relates to the detection of at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more to predict or confirm treatment response for MS. The present disclosure has also identified that certain treatments can result in changes in expression of at least one, at least two, at least three, at least four, at least five, at least six, or more biomarkers. In particular, one example disclosed herein includes a treatment change (e.g., from an anti-CD20 antibody, such as ocrelizumab (Ocrevus®), to trebrutinib). The present disclosure identifies that the biomarkers disclosed herein can be used for prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of biomarker protein expression (e.g., reduction of biomarkers) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting at least one, at least two, at least three, at least four, at least five, at least six, or more biomarkers (both RNA and protein), which can indicate the severity of MS, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of a subject with MS.

[0009] Thus, in one embodiment, provided herein is a method of treating a subject with MS, comprising: (a) detecting at least one biomarker in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting at least one biomarker in a biological sample from the subject, including CSF; (b) identifying the subject as having MS if the subject expresses at least one biomarker in the biological sample; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method comprises detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a 120 mg dose. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody comprises ocrelizumab or rituximab. In some examples, the initial anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject is administered IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered prior to treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered following treatment with trebrutinib.

[0010] In another embodiment, disclosed herein is a method of identifying a patient as suitable for participation in a clinical trial for MS, the method comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses the at least one biomarker in the biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS. In some examples, the method comprises detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0011] In yet another embodiment, disclosed herein are methods of diagnosing a subject as having MS, the methods comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from the subject; and (b) identifying the subject as having MS if the subject expresses the at least one biomarker in the biological sample. In some examples, the methods comprise detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0012] In yet another embodiment, disclosed herein are methods of identifying a subject with MS who expresses at least one biomarker in a biological sample comprising CSF, the methods comprising: (a) detecting at least one biomarker in the biological sample; and (b) identifying a subject with MS who expresses at least one biomarker in the biological sample. In some examples, the methods comprise identifying a subject with MS as expressing at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0013] Also disclosed herein are methods for identifying a subject as likely to develop MS, the methods comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing at least one biomarker in the biological sample as likely to develop MS. In some examples, the methods comprise detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0014] In another embodiment, disclosed herein is a method of identifying a subject who is likely to develop MS, the method comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses the at least one biomarker in the biological sample as likely to develop MS. In some examples, the method comprises detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0015] In some examples, the biomarker is increased compared to the biomarker in the reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further comprises obtaining a biological sample from the subject.

[0016] In some examples, the disclosure also provides methods of monitoring the progression of MS in a subject over time, comprising: (a) detecting at least one biomarker in a first biological sample obtained from the subject at a first time point; (b) detecting at least one biomarker in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying the subject as having progressive MS if at least one biomarker is increased at the second time point compared to the first time point, or (ii) identifying the subject as having static or regressing MS if at least one biomarker is about the same or decreased at the second time point compared to the first time point. In some examples, the method comprises detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more, and comparing the biomarkers.

[0017] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the initial anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0018] In some examples, methods disclosed herein include methods of evaluating the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) at least one biomarker in a first biological sample comprising CSF obtained from the subject at a first time point and (ii) at least one biomarker in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of the treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and the at least one biomarker in the second biological sample compared to at least one biomarker in a sample obtained from an untreated patient, wherein the abundance of the at least one biomarker in the second biological sample is approximately the same or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS. In some examples, the method comprises detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more, and determining a correlation between the effectiveness of the treatment and the biomarkers. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject, and assessing the effectiveness of the treatment with the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0019] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, at least one biomarker comprises RNA. In some examples, the at least one biomarker RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, the at least one biomarker comprises a protein. In some examples, the at least one biomarker is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of MS onset or a treatment to reduce the likelihood or susceptibility to developing MS.

[0020] In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3.

[0021] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib and an anti-CD20 antibody.

[0022] In some instances, the subject has been treated for MS prior to detecting the at least one biomarker, hi some instances, the subject has been previously treated with an anti-CD20 therapy.

[0023] In some examples, the anti-CD20 therapy is ocrelizumab or rituximab.

[0024] In some instances, the subject has not been treated for MS prior to detecting the at least one biomarker and / or has not been previously treated with an anti-CD20 therapy.

[0025] In some examples, the subject comprises one or more brain lesions.

[0026] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib plus one or more doses of anti-CD20 therapy and / or a booster of anti-CD20 therapy.

[0027] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0028] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0029] The present disclosure relates to the detection of CXCL13 as a biomarker for predicting or confirming treatment response in MS. The present disclosure also identifies that certain treatments can result in changes in CXCL13 biomarker expression. In particular, one embodiment disclosed herein includes a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure identifies that CXCL13 can be used as a biomarker for prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of CXCL13 protein expression (e.g., reduction of CXCL13) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting CXCL13 (both RNA and protein) as a biomarker indicative of MS severity, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of a subject with MS.

[0030] Thus, in one aspect, provided herein is a method of treating a subject with MS, comprising: (a) detecting CXCL13 in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting CXCL13 in a biological sample from the subject, including CSF; (b) identifying the subject as having MS if the subject expresses CXCL13 in the biological sample; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody comprises ocrelizumab or rituximab. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject is administered the IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0031] In another embodiment, disclosed herein is a method for identifying a patient as suitable for participating in a clinical trial for MS, the method comprising: (a) detecting CXCL13 in a biological sample comprising CSF from the subject; and (b) identifying a subject that expresses CXCL13 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS.

[0032] In yet another embodiment, disclosed herein is a method of diagnosing a subject as having MS, the method comprising: (a) detecting CXCL13 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CXCL13 in the biological sample as having MS.

[0033] In yet another embodiment, disclosed herein is a method for identifying a subject with MS who expresses CXCL13 in a biological sample comprising CSF, the method comprising: (a) detecting CXCL13 in the biological sample; and (b) identifying a subject with MS who expresses CXCL13 in the biological sample.

[0034] Also disclosed herein is a method for identifying a subject as having a high likelihood of developing MS, the method comprising: (a) detecting CXCL13 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CXCL13 in the biological sample as having a high likelihood of developing MS.

[0035] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting CXCL13 in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses CXCL13 in the biological sample as having a high likelihood of developing MS.

[0036] In some examples, CXCL13 is increased compared to CXCL13 in a reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further comprises obtaining a biological sample from the subject.

[0037] In some examples, the present disclosure also provides a method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL13 in a first biological sample obtained from the subject at a first time point; (b) detecting CXCL13 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CXCL13 at the second time point compared to CXCL13 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CXCL13 at the second time point compared to CXCL13 at the first time point as having static or regressing MS.

[0038] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0039] In some examples, the methods disclosed herein include a method of evaluating the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting CXCL13 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of the therapy between the first and second time points; and (b) determining a correlation between the efficacy of the therapy and CXCL13 in the second biological sample compared to CXCL13 in a sample obtained from an untreated patient, wherein CXCL13 in the second biological sample is approximately the same as or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the therapy is effective for the subject's MS. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and evaluating the therapeutic efficacy of the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0040] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the CXCL13 comprises CXCL13 RNA. In some examples, CXCL13 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, CXCL13 comprises CXCL13 protein. In some examples, CXCL13 is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of MS onset or a treatment to reduce the likelihood or susceptibility of developing MS.

[0041] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods further include administering to the subject a further or increased dose of an anti-CD20 antibody.

[0042] In some examples, the subject has been treated for MS prior to detecting CXCL13. In some examples, the subject has been previously treated with an anti-CD20 therapy.

[0043] In some instances, the anti-CD20 therapy is ocrelizumab. In some instances, the anti-CD20 therapy is rituximab.

[0044] In some instances, the subject has not been treated for MS prior to detecting CXCL13 and / or has not been previously treated with an anti-CD20 therapy.

[0045] In some examples, the subject comprises one or more brain lesions.

[0046] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib plus one or more doses of anti-CD20 therapy and / or a booster of anti-CD20 therapy.

[0047] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0048] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0049] The present disclosure relates to the detection of CXCL10 as a biomarker for predicting or confirming treatment response in MS. C-X-C motif chemokine ligand 10 (CXCL10), also known as interferon-γ-inducible protein 10 (IP-10) or hypo-inducible cytokine B10, is an 8.7 kDa protein encoded by the CXCL10 gene in humans. Luster et al., Nature. 315(6021):672-6 (1985); Luster et al., Proceedings of the National Academy of Sciences of the United States of America. 84(9):2868-71 (May 1987). The present disclosure also identifies that certain treatments can result in changes in CXCL10 biomarker expression. In particular, one embodiment disclosed herein includes a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure has identified that CXCL10 can be used as a biomarker for prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of CXCL10 protein expression (e.g., reduction of CXCL10) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting CXCL10 (both RNA and protein) as a biomarker for MS severity, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of subjects with MS.

[0050] Thus, in one aspect, provided herein is a method of treating a subject with MS, comprising: (a) detecting CXCL10 in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from the subject; (b) identifying the subject as having MS if the subject expresses CXCL10 in the biological sample; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody comprises ocrelizumab or rituximab. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject is administered the IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0051] In another embodiment, disclosed herein is a method for identifying a patient as suitable for participating in a clinical trial for MS, the method comprising: (a) detecting CXCL10 in a biological sample comprising CSF from the subject; and (b) identifying a subject that expresses CXCL10 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS.

[0052] In yet another embodiment, disclosed herein is a method for diagnosing a subject as having MS, the method comprising: (a) detecting CXCL10 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CXCL10 in the biological sample as having MS.

[0053] In yet another embodiment, disclosed herein is a method for identifying a subject with MS who expresses CXCL10 in a biological sample comprising CSF, the method comprising: (a) detecting CXCL10 in the biological sample; and (b) identifying a subject with MS who expresses CXCL10 in the biological sample.

[0054] Also disclosed herein is a method for identifying a subject as having a high likelihood of developing MS, the method comprising: (a) detecting CXCL10 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CXCL10 in the biological sample as having a high likelihood of developing MS.

[0055] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting CXCL10 in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses CXCL10 in the biological sample as having a high likelihood of developing MS.

[0056] In some examples, CXCL10 is increased compared to CXCL10 in a reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further comprises obtaining a biological sample from the subject.

[0057] In some examples, the present disclosure also provides a method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL10 in a first biological sample obtained from the subject at a first time point; (b) detecting CXCL10 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CXCL10 at the second time point compared to CXCL10 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CXCL10 at the second time point compared to CXCL10 at the first time point as having static or regressing MS.

[0058] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered simultaneously during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0059] In some examples, the methods disclosed herein include a method of evaluating the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting CXCL10 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of the therapy between the first and second time points; and (b) determining a correlation between the efficacy of the therapy and CXCL10 in the second biological sample compared to CXCL10 in a sample obtained from an untreated patient, wherein CXCL10 in the second biological sample is approximately the same as or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the therapy is effective for the subject's MS. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and evaluating the therapeutic efficacy of the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0060] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the CXCL10 comprises CXCL10 RNA. In some examples, CXCL10 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, CXCL10 comprises CXCL10 protein. In some examples, CXCL10 is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment for reducing the rate of progression of the onset of MS or a treatment for reducing the likelihood or susceptibility of developing MS.

[0061] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib and an anti-CD20 antibody.

[0062] In some instances, the subject has been treated for MS prior to detecting CXCL10. In some instances, the subject has been previously treated with an anti-CD20 therapy.

[0063] In some instances, the anti-CD20 therapy is ocrelizumab. In some instances, the anti-CD20 therapy is rituximab.

[0064] In some examples, the subject comprises one or more brain lesions.

[0065] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib plus one or more doses of anti-CD20 therapy and / or a booster of anti-CD20 therapy.

[0066] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0067] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0068] The present disclosure relates to the detection of CD27 as a biomarker for predicting or confirming treatment response in MS. The present disclosure also identifies that certain treatments can result in changes in CD27 biomarker expression. In particular, one embodiment disclosed herein includes a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure identifies that CD27 can be used as a biomarker for prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of CD27 protein expression (e.g., reduction of CD27) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting CD27 (both RNA and protein) as a biomarker indicative of MS severity, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of a subject with MS.

[0069] Thus, in one aspect, provided herein is a method of treating a subject with MS, comprising: (a) detecting CD27 in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting CD27 in a biological sample containing CSF from the subject; (b) identifying the subject expressing CD27 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject receives the IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0070] In another embodiment, disclosed herein is a method of identifying a patient as suitable for participation in a clinical trial for MS, the method comprising: (a) detecting CD27 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CD27 in the biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS.

[0071] In yet another embodiment, disclosed herein is a method of diagnosing a subject as having MS, the method comprising: (a) detecting CD27 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CD27 in the biological sample as having MS.

[0072] In yet another embodiment, disclosed herein is a method for identifying a subject with MS who expresses CD27 in a biological sample comprising CSF, the method comprising: (a) detecting CD27 in the biological sample; and (b) identifying a subject with MS who expresses CD27 in the biological sample.

[0073] Also disclosed herein is a method for identifying a subject as having a high likelihood of developing MS, the method comprising: (a) detecting CD27 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CD27 in the biological sample as having a high likelihood of developing MS.

[0074] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting CD27 in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses CD27 in the biological sample as having a high likelihood of developing MS.

[0075] In some examples, CD27 is increased compared to CD27 in the reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further comprises obtaining a biological sample from the subject.

[0076] In some examples, the present disclosure also provides methods of monitoring the progression of MS in a subject over time, the methods including: (a) detecting CD27 in a first biological sample obtained from the subject at a first time point; (b) detecting CD27 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CD27 at the second time point compared to CD27 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CD27 at the second time point compared to CD27 at the first time point as having static or regressing MS.

[0077] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0078] In some examples, methods disclosed herein include a method of evaluating the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting CD27 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of the therapy between the first and second time points; and (b) determining a correlation between the efficacy of the therapy and CD27 in the second biological sample compared to CD27 in a sample obtained from an untreated patient, wherein CD27 in the second biological sample is approximately the same as or decreased compared to the abundance in the sample from the untreated patient, thereby indicating that the therapy is effective for the subject's MS. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and evaluating the therapeutic efficacy of the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0079] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the CD27 comprises CD27 RNA. In some examples, CD27 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, CD27 comprises CD27 protein. In some examples, CD27 is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of MS onset or a treatment to reduce the likelihood or susceptibility to onset of MS.

[0080] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib.

[0081] In some instances, the subject has been treated for MS prior to detecting CD27. In some instances, the subject has been previously treated with an anti-CD20 therapy.

[0082] In some examples, the anti-CD20 therapy is ocrelizumab or rituximab.

[0083] In some examples, the subject comprises one or more brain lesions.

[0084] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib plus one or more doses of anti-CD20 therapy and / or a booster of anti-CD20 therapy.

[0085] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0086] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0087] The present disclosure relates to the detection of NEFL as a biomarker for predicting or confirming treatment response in MS. The present disclosure also identifies that certain treatments can result in changes in NEFL biomarker expression. In particular, one embodiment disclosed herein includes a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure identifies that NEFL can be used as a biomarker of prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of NEFL protein expression (e.g., reduction of NEFL) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting NEFL (both RNA and protein) as a biomarker indicative of MS severity, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of a subject with MS.

[0088] Thus, in one aspect, provided herein is a method of treating a subject with MS, comprising: (a) detecting NEFL in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting NEFL in a biological sample from the subject, including CSF; (b) identifying the subject expressing NEFL in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject receives the IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0089] In another embodiment, disclosed herein is a method for identifying a patient as suitable for participation in a clinical trial for MS, the method comprising: (a) detecting NEFL in a biological sample comprising CSF from the subject; and (b) identifying a subject that expresses NEFL in the biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS.

[0090] In yet another embodiment, disclosed herein is a method for diagnosing a subject as having MS, the method comprising: (a) detecting NEFL in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing NEFL in the biological sample as having MS.

[0091] In yet another embodiment, disclosed herein is a method for identifying a subject with MS who expresses NEFL in a biological sample including CSF, the method comprising: (a) detecting NEFL in the biological sample; and (b) identifying a subject with MS who expresses NEFL in the biological sample.

[0092] Also disclosed herein is a method for identifying a subject as having a high likelihood of developing MS, the method comprising: (a) detecting NEFL in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing NEFL in the biological sample as having a high likelihood of developing MS.

[0093] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting NEFL in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses NEFL in the biological sample as having a high likelihood of developing MS.

[0094] In some instances, NEFL is increased compared to NEFL in a reference sample. In some instances, the reference sample is derived from a second subject. In some instances, the second subject does not have MS. In some instances, the reference sample comprises CSF. In some instances, the method further comprises obtaining a biological sample from the subject.

[0095] In some examples, the present disclosure also provides a method for monitoring the progression of MS in a subject over time, comprising: (a) detecting NEFL in a first biological sample obtained from the subject at a first time point; (b) detecting NEFL in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject whose NEFL has increased at the second time point compared to the NEFL at the first time point as having progressive MS, or (ii) identifying a subject whose NEFL has approximately the same or decreased at the second time point compared to the NEFL at the first time point as having static or regressive MS.

[0096] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered simultaneously with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from trebrutinib. In some examples, the anti-CD20 antibody is administered before trebrutinib. In some examples, the anti-CD20 antibody is administered after trebrutinib.

[0097] In some examples, the methods disclosed herein include a method of evaluating the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting NEFL in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of the treatment between the first and second time points; and (b) determining a correlation between the efficacy of the treatment and NEFL in the second biological sample compared to NEFL in a sample obtained from an untreated patient, wherein NEFL in the second biological sample is approximately the same as or reduced compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for the subject's MS. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and evaluating the therapeutic efficacy of the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0098] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the NEFL comprises NEFL RNA. In some examples, NEFL RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, NEFL comprises NEFL protein. In some examples, NEFL is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of MS onset or a treatment to reduce the likelihood or susceptibility of developing MS.

[0099] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib and an anti-CD20 antibody.

[0100] In some instances, the subject has been treated for MS prior to detecting NEFL. In some instances, the subject has been previously treated with an anti-CD20 therapy.

[0101] In some examples, the anti-CD20 therapy is ocrelizumab or rituximab.

[0102] In some examples, the subject comprises one or more brain lesions.

[0103] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib in addition to one or more doses of an anti-CD20 therapy and / or one or more booster doses of an anti-CD20 therapy.

[0104] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0105] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0106] The present disclosure relates to the detection of CCL4 as a biomarker for predicting or confirming treatment response in MS. The present disclosure also identifies that certain treatments can result in changes in CCL4 biomarker expression. In particular, one embodiment disclosed herein includes a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure identifies that CCL4 can be used as a biomarker of prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of CCL4 protein expression (e.g., reduction of CCL4) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting CCL4 (both RNA and protein) as a biomarker indicative of MS severity, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of a subject with MS.

[0107] Thus, in one aspect, provided herein is a method of treating a subject with MS, comprising: (a) detecting CCL4 in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting CCL4 in a biological sample from the subject, including CSF; (b) identifying the subject expressing CCL4 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody comprises ocrelizumab or rituximab. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject is administered the IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0108] In another embodiment, disclosed herein is a method of identifying a patient as suitable for participating in a clinical trial for MS, the method comprising: (a) detecting CCL4 in a biological sample comprising CSF from the subject; and (b) identifying a subject that expresses CCL4 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS.

[0109] In yet another embodiment, disclosed herein is a method of diagnosing a subject as having MS, the method comprising: (a) detecting CCL4 in a biological sample comprising CSF from the subject; and (b) identifying the subject as having MS if the subject expresses CCL4 in the biological sample.

[0110] In yet another embodiment, disclosed herein is a method for identifying a subject with MS as expressing CCL4 in a biological sample comprising CSF, the method comprising: (a) detecting CCL4 in the biological sample; and (b) identifying a subject with MS who expresses CCL4 in the biological sample.

[0111] Disclosed herein is a method for identifying a subject as having a high likelihood of developing MS, the method comprising: (a) detecting CCL4 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CCL4 in the biological sample as having a high likelihood of developing MS.

[0112] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting CCL4 in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses CCL4 in the biological sample as having a high likelihood of developing MS.

[0113] In some examples, CCL4 is increased compared to CCL4 in the reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further comprises obtaining a biological sample from the subject.

[0114] In some examples, the disclosure also provides methods of monitoring the progression of MS in a subject over time, comprising: (a) detecting CCL4 in a first biological sample obtained from the subject at a first time point; (b) detecting CCL4 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CCL4 at the second time point compared to CCL4 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CCL4 at the second time point compared to CCL4 at the first time point as having static or regressing MS.

[0115] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0116] In some examples, the methods disclosed herein include a method of evaluating the effectiveness of a treatment with a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting CCL4 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CCL4 in the second biological sample compared to CCL4 in a sample obtained from an untreated patient, wherein the CCL4 abundance in the second biological sample is approximately the same as or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and evaluating the effectiveness of the treatment with the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0117] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the CCL4 comprises CCL4 RNA. In some examples, CCL4 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, CCL4 comprises CCL4 protein. In some examples, CCL4 is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of MS onset or a treatment to reduce the likelihood or susceptibility of developing MS.

[0118] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib and an anti-CD20 antibody.

[0119] In some instances, the subject has been treated for MS prior to detecting CCL4, hi some instances, the subject has been previously treated with an anti-CD20 therapy.

[0120] In some examples, the anti-CD20 therapy is ocrelizumab or rituximab.

[0121] In some examples, the subject comprises one or more brain lesions.

[0122] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib in addition to one or more doses of an anti-CD20 therapy and / or one or more booster doses of an anti-CD20 therapy.

[0123] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0124] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0125] The present disclosure relates to the detection of CCL3 as a biomarker for predicting or confirming treatment response in MS. The present disclosure also identifies that certain treatments can result in changes in CCL3 biomarker expression. In particular, one embodiment disclosed herein includes a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure identifies that CCL3 can be used as a biomarker for prognosis, onset, and treatment efficacy in the cerebrospinal fluid (CSF) of subjects with MS. Modulation of CCL3 protein expression (e.g., reduction of CCL3) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting CCL3 (both RNA and protein) as a biomarker indicative of MS severity, including progression, regression, or static presence of lesions (e.g., active lesions) in the brain of a subject with MS.

[0126] Thus, in one aspect, provided herein is a method of treating a subject with MS, comprising: (a) detecting CCL3 in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, comprising: (a) detecting CCL3 in a biological sample from the subject, including CSF; (b) identifying the subject expressing CCL3 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, a pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody comprises ocrelizumab or rituximab. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject is administered IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered prior to treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered following treatment with trebrutinib.

[0127] In another embodiment, disclosed herein is a method of identifying a patient as suitable for participating in a clinical trial for MS, the method comprising: (a) detecting CCL3 in a biological sample comprising CSF from the subject; and (b) identifying a subject that expresses CCL3 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS.

[0128] In yet another embodiment, disclosed herein is a method of diagnosing a subject as having MS, the method comprising: (a) detecting CCL3 in a biological sample comprising CSF from the subject; and (b) identifying the subject as having MS if the subject expresses CCL3 in the biological sample.

[0129] In yet another embodiment, disclosed herein is a method of identifying a subject with MS as expressing CCL3 in a biological sample comprising CSF, the method comprising: (a) detecting CCL3 in the biological sample; and (b) identifying a subject with MS who expresses CCL3 in the biological sample.

[0130] Disclosed herein is a method for identifying a subject as having a high likelihood of developing MS, the method comprising: (a) detecting CCL3 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CCL3 in the biological sample as having a high likelihood of developing MS.

[0131] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting CCL3 in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses CCL3 in the biological sample as having a high likelihood of developing MS.

[0132] In some examples, CCL3 is increased compared to CCL3 in the reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further comprises obtaining a biological sample from the subject.

[0133] In some examples, the disclosure also provides methods of monitoring the progression of MS in a subject over time, the methods including: (a) detecting CCL3 in a first biological sample obtained from the subject at a first time point; (b) detecting CCL3 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CCL3 at the second time point compared to CCL3 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CCL3 at the second time point compared to CCL3 at the first time point as having static or regressing MS.

[0134] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0135] In some examples, methods disclosed herein include a method of evaluating the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting CCL3 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CCL3 in the second biological sample compared to CCL3 in a sample obtained from an untreated patient, wherein the CCL3 in the second biological sample is approximately the same as or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and evaluating the effectiveness of the treatment of the pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0136] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the CCL3 comprises CCL3 RNA. In some examples, CCL3 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, CCL3 comprises CCL3 protein. In some examples, CCL3 is determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of MS onset or a treatment to reduce the likelihood or susceptibility of developing MS.

[0137] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib and an anti-CD20 antibody.

[0138] In some instances, the subject has been treated for MS prior to detecting CCL3. In some instances, the subject has been previously treated with an anti-CD20 therapy.

[0139] In some examples, the anti-CD20 therapy is ocrelizumab or rituximab.

[0140] In some examples, the subject comprises one or more brain lesions.

[0141] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib plus one or more doses of anti-CD20 therapy and / or a booster of anti-CD20 therapy.

[0142] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0143] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0144] The present disclosure also relates to the detection of CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 as biomarkers for predicting or confirming treatment response in MS. The present disclosure also identifies that certain treatments can result in changes in CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 biomarker expression. In particular, one embodiment disclosed herein involves a treatment change (e.g., from ocrelizumab (Ocrevus®) to trebrutinib). The present disclosure identifies that CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 can be used as biomarkers of prognosis, development, and treatment efficacy in cerebrospinal fluid (CSF) of subjects with MS. Modulation of CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 protein expression (e.g., decreased CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3) is associated with a more favorable outcome of MS. Accordingly, provided herein are methods for detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 (RNA and protein) as biomarkers indicative of MS severity, including the presence of progressive, regressive, or static lesions (e.g., active lesions) in the brain of a subject with MS.

[0145] Thus, in one aspect, provided herein is a method of treating a subject with MS, the method comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the subject's cerebrospinal fluid (CSF); and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In a second aspect, disclosed herein is a method of treating a subject with MS, the method comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a biological sample from the subject, including CSF; (b) identifying the subject as having MS if the subject expresses CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the biological sample; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a 60 mg dose. In some examples, the pharmaceutically effective amount of trebrutinib is a 120 mg dose. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some embodiments, the method of treating a subject with MS further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the anti-CD20 antibody comprises ocrelizumab or rituximab. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some examples, the subject is administered an IV infusion at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks). In some examples, the anti-CD20 antibody is administered during the course of treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0146] In another embodiment, disclosed herein is a method of identifying a patient as suitable for participating in a clinical trial for MS, the method comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS.

[0147] In yet another embodiment, disclosed herein is a method of diagnosing a subject as having MS, the method comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a biological sample comprising CSF from the subject; and (b) identifying the subject as having MS if the subject expresses CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the biological sample.

[0148] In yet another embodiment, disclosed herein is a method for identifying a subject with MS who expresses CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a biological sample comprising CSF, the method comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the biological sample; and (b) identifying a subject with MS who expresses CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the biological sample.

[0149] Disclosed herein are methods for identifying a subject as having a high likelihood of developing MS, the methods comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a biological sample comprising CSF from the subject; and (b) identifying a subject expressing CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the biological sample as having a high likelihood of developing MS.

[0150] In another embodiment, disclosed herein is a method for identifying a subject who is likely to develop MS, the method comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in a biological sample comprising CSF from the subject; and (b) identifying a subject who expresses CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in the biological sample as having a high likelihood of developing MS.

[0151] In some examples, CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 are increased compared to CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a reference sample. In some examples, the reference sample is from a second subject. In some examples, the second subject does not have MS. In some examples, the reference sample comprises CSF. In some examples, the method further includes obtaining a biological sample from the subject.

[0152] In some examples, the disclosure also provides a method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a first biological sample obtained from the subject at a first time point; (b) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a second biological sample obtained from the subject at a second time point; and (c) (i) detecting CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in the first biological sample obtained from the subject at a second time point. and / or CCL3 at the first time point, or (ii) identifying as having progressive MS those subjects with increased CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 at the second time point compared to CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 at the second time point, or (iii) identifying as having static or regressing MS those subjects with approximately the same or decreased CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 at the second time point compared to CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 at the first time point.

[0153] In some examples, the method includes administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In one embodiment, the method further includes administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject. In some examples, the anti-CD20 antibody dose includes one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, the anti-CD20 antibody is administered during treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered separately from treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered before treatment with trebrutinib. In some examples, the anti-CD20 antibody is administered after treatment with trebrutinib.

[0154] In some examples, the methods disclosed herein include methods for assessing the efficacy of a pharmaceutically effective amount of trebrutinib treatment in a subject with MS, comprising: (a) detecting (i) CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a first biological sample comprising CSF obtained from the subject at a first time point; and (ii) CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points. and (b) determining a correlation between the effectiveness of the treatment and CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in the second biological sample compared to CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in a sample obtained from an untreated patient, wherein CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3 in the second biological sample are approximately the same or decreased compared to their abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for treating MS in the subject. In one embodiment, the method further comprises administering a pharmaceutically effective amount of an anti-CD20 antibody to the subject and assessing the effectiveness of treatment with pharmaceutically effective amounts of trebrutinib and the anti-CD20 antibody.

[0155] In some examples, the difference between the first time point and the second time point is about 1 month to about 2 years. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 60 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered at a dose of 120 mg. In some examples, the pharmaceutically effective amount of trebrutinib is administered orally. In some examples, the pharmaceutically effective amount of trebrutinib is administered daily. In some examples, the anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least 6 months). In some examples, the subject is administered IV infusions at intervals of about 1 week to about 3 weeks (e.g., about 1 week, about 2 weeks, or about 3 weeks). In some examples, CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 comprise CXCL10, CXCL13 RNA, CD27, NEFL, CCL4, and / or CCL3. In some examples, CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 RNA are determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. In some examples, CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 comprise CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 protein. In some examples, CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 are determined by flow cytometry or Western blot. In some examples, the method further includes monitoring the subject for the onset of MS symptoms. In some examples, the method further includes administering to the subject a treatment to reduce the rate of progression of the onset of MS or to reduce the likelihood or susceptibility of developing MS.

[0156] In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. In some examples, the methods include administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib and an anti-CD20 therapy.

[0157] In some examples, the subject has been treated for MS prior to detecting CXCL10, CXCL13, CD27, NEFL, CCL4 and / or CCL3. In some examples, the subject has been previously treated with an anti-CD20 therapy.

[0158] In some instances, the anti-CD20 therapy is ocrelizumab. In some instances, the anti-CD20 therapy is rituximab.

[0159] In some examples, the subject comprises one or more brain lesions.

[0160] In some examples, the subject is administered a pharmaceutically effective amount of trebrutinib in addition to an anti-CD20 therapy and / or one or more doses of a booster of the anti-CD20 therapy.

[0161] In some instances, administration of a pharmaceutically effective amount of trebrutinib (and potentially with one or more additional therapies, such as anti-CD20 therapy) results in one or more of the following: a reduction in brain pathology; a reduction in the amount of iron in the subject's brain tissue; and / or a decrease in synaptic density. In some instances, the reduction or decrease (d) is compared to a previous amount from the same subject.

[0162] In some instances, the MS is relapsing-remitting multiple sclerosis. In some instances, the MS is secondary progressive multiple sclerosis.

[0163] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information were specifically and individually indicated to be incorporated by reference. To the extent that the publications, patents, patent applications, and items of information incorporated by reference conflict with the disclosure contained herein, the present specification supersedes and / or takes precedence over any such conflicting material.

[0164] Where values ​​are described in terms of ranges, the description should be understood to include disclosure of all possible subranges within such ranges, as well as specific numerical values ​​falling within such ranges, whether or not a specific numerical value or specific subrange is explicitly recited.

[0165] The term "each," when used in reference to a collection of items, is intended to identify each individual item in the set, but does not necessarily refer to every item in the set unless specifically stated otherwise or the context of the usage clearly indicates otherwise.

[0166] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "conjugate" includes a plurality of conjugates, reference to a "cell" includes a plurality of cells, etc.

[0167] Numerical ranges are inclusive of the numbers defining the range. Measurements and measurable values ​​are understood to be approximations, taking into account significant digits and error associated with measurement. Also, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," "including" is not intended to be limiting. It is to be understood that both the foregoing general and detailed descriptions are exemplary and explanatory only and are not restrictive of the teachings.

[0168] Unless otherwise stated in the specification above, embodiments herein that recite various components as "comprising" are also contemplated as "consisting of" or "consisting essentially of" the recited components; embodiments herein that recite various components as "consisting of" are also contemplated as "comprising" or "consisting essentially of" the recited components; and embodiments herein that recite various components as "consisting essentially of" are also contemplated as "consisting of" or "comprising" the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0169] As used herein, the terms "or a combination thereof" and "or combinations thereof" refer to any and all permutations and combinations of the listed terms preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular situation, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations including repeats of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those of skill in the art will understand that typically there is no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0170] "Or" is used in its inclusive sense, ie, equivalent to "and / or," unless the context requires otherwise.

[0171] Various embodiments of the features of the present disclosure are described herein. However, it should be understood that such embodiments are provided by way of example only, and that many variations, changes, and substitutions may occur to those skilled in the art without departing from the scope of the present disclosure. It should also be understood that various alternatives to the specific embodiments described herein are within the scope of the present disclosure.

[0172] Embodiment 1. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting CXCL13 in cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with multiple sclerosis (MS), comprising: Embodiment 2. A method of treating a subject with MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL13 in a biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising: Embodiment 3. The method of embodiment 1 or 2, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 4. The method of embodiment 3, wherein the pharmaceutically effective amount of trebrutinib is a 60 mg dose. Embodiment 5. The method of any one of embodiments 1-4, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 6. The method of any one of embodiments 1-5, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 7. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from a subject; (b) identifying subjects that express CXCL13 in a biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; 1. A method for identifying a patient as suitable for participation in a clinical trial for MS, comprising: Embodiment 8. A method of diagnosing a subject as having MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL13 in a biological sample as having MS; and A method of diagnosing a subject as having MS, comprising: Embodiment 9. A method of identifying a subject with MS as expressing CXCL13 in a biological sample comprising CSF, comprising: (a) detecting CXCL13 in a biological sample; (b) identifying a subject with MS who expresses CXCL13 in a biological sample; and 1. A method for identifying a subject with MS as expressing CXCL13 in a biological sample comprising CSF, the method comprising: Embodiment 10. A method of identifying a subject as likely to develop MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL13 in a biological sample as being likely to develop MS; and 20. A method of identifying a subject as likely to develop MS, comprising: Embodiment 11. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL13 in a biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 12. The method of any one of embodiments 1 to 11, wherein CXCL13 is increased compared to CXCL13 in a reference sample. Embodiment 13 The method of embodiment 12, wherein the reference sample is from a second subject. Embodiment 14 The method of embodiment 13, wherein the second subject does not have MS. Embodiment 15. The method of any one of embodiments 12 to 14, wherein the reference sample comprises CSF. Embodiment 16. The method of any one of embodiments 1 to 15, further comprising obtaining a biological sample from the subject. Embodiment 17. A method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL13 in a first biological sample obtained from the subject at a first time point; (b) detecting CXCL13 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CXCL13 at a second time point compared to CXCL13 at a first time point as a subject with progressive MS; or (ii) identifying subjects with approximately the same or decreased CXCL13 at the second time point compared to CXCL13 at the first time point as having static or regressing MS; 20. A method for monitoring the progression of MS in a subject over time, comprising: Embodiment 18. The method of embodiments 7-17, further comprising administering a pharmaceutically effective amount of trebrutinib to the subject. Embodiment 19. The method of embodiment 18, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 20. The method of embodiment 18 or 19, wherein the pharmaceutically effective amount of trebrutinib is a 60 mg dose. Embodiment 21. The method of any one of embodiments 7 to 20, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 22. The method of any one of embodiments 7 to 21, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 23. A method for assessing the efficacy of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CXCL13 in a first biological sample comprising CSF obtained from a subject at a first time point, and (ii) CXCL13 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CXCL13 in the second biological sample relative to CXCL13 in a sample obtained from an untreated patient, wherein the CXCL13 in the second biological sample is approximately the same as or reduced relative to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective against MS in the subject; A method for assessing the efficacy of a therapeutically effective amount of trebrutinib in a subject with MS, comprising: Embodiment 24. The method of embodiment 23, wherein the difference between the first time point and the second time point is about 1 month to about 2 years. Embodiment 25. The method of embodiment 23 or 24, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 26. The method of embodiment 25, wherein the pharmaceutically effective amount of trebrutinib is a 60 mg dose. Embodiment 27. The method of any one of embodiments 23-26, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 28. The method of any one of embodiments 23-27, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 29. The method of any one of embodiments 1 to 28, wherein the CXCL13 comprises CXCL13 RNA. Embodiment 30. The method of embodiment 29, wherein CXCL13 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 31 The method of any one of embodiments 1 to 30, wherein the CXCL13 comprises a CXCL13 protein. Embodiment 32 The method of embodiment 31, wherein CXCL13 is determined by flow cytometry or Western blot. Embodiment 33 The method of any one of embodiments 1 to 32, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 34. The method of any one of embodiments 1 to 33, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS, or reducing the likelihood or susceptibility to developing MS. Embodiment 35. The method of any one of embodiments 1 to 34, further comprising administering to the subject an additional or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 36 The method of any one of embodiments 1 to 35, wherein the subject is undergoing treatment for MS prior to detecting CXCL13. Embodiment 37. The method of embodiment 36, wherein the subject has been previously treated with an anti-CD20 therapy. Embodiment 38 The method of embodiment 37, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 39. The method of any one of embodiments 1 to 38, wherein the subject comprises one or more brain lesions. Embodiment 40. The method of any one of embodiments 1-6 or 18-39, wherein the subject is administered one or more booster doses of anti-CD20 therapy administering a pharmaceutically effective amount of trebrutinib. Embodiment 41. Administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density 41. The method of any one of embodiments 1 to 6 or 18 to 40, resulting in one or more of: Embodiment 42. The method of any one of embodiments 1 to 41, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 43. The method of any one of embodiments 1 to 41, wherein the MS is secondary progressive multiple sclerosis. Embodiment 44. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting CXCL10 in cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with multiple sclerosis (MS), comprising: Embodiment 45. A method of treating a subject with MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL10 in a biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising: Embodiment 46. The method of embodiment 44 or 45, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 47. The method of embodiment 46, wherein the pharmaceutically effective amount of trebrutinib is a 60 mg dose. Embodiment 48. The method of any one of claims 44-47, wherein a pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 49. The method of any one of claims 44-48, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 50. A method of identifying a patient as suitable for participation in a clinical trial of MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from a subject; (b) identifying subjects that express CXCL10 in a biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; 1. A method for identifying a patient as suitable for participation in a clinical trial for MS, comprising: Embodiment 51. A method of diagnosing a subject as having MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL10 in a biological sample as having MS; and A method of diagnosing a subject as having MS, comprising: Embodiment 52. A method of identifying a subject with MS as expressing CXCL10 in a biological sample comprising CSF, comprising: (a) detecting CXCL10 in a biological sample; (b) identifying a subject with MS who expresses CXCL10 in a biological sample; and 1. A method for identifying a subject with MS as expressing CXCL10 in a biological sample comprising CSF, the method comprising: Embodiment 53. A method of identifying a subject as likely to develop MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL10 in a biological sample as being likely to develop MS; and 20. A method of identifying a subject as likely to develop MS, comprising: Embodiment 54. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CXCL10 in a biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 55. The method of any one of embodiments 44 to 55, wherein CXCL10 is increased compared to CXCL10 in a reference sample. Embodiment 56 The method of embodiment 55, wherein the reference sample is from a second subject. Embodiment 57. The method of embodiment 56, wherein the second subject does not have MS. Embodiment 58. The method of any one of embodiments 55 to 57, wherein the reference sample comprises CSF. Embodiment 59. The method of any one of embodiments 44 to 58, further comprising obtaining a biological sample from the subject. Embodiment 60. A method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL10 in a first biological sample obtained from the subject at a first time point; (b) detecting CXCL10 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CXCL10 at a second time point compared to CXCL10 at a first time point as a subject with progressive MS; or (ii) identifying subjects with approximately the same or decreased CXCL10 at the second time point compared to CXCL10 at the first time point as having static or regressing MS; 20. A method for monitoring the progression of MS in a subject over time, comprising: Embodiment 61 The method of embodiments 50-60, further comprising administering a pharmaceutically effective amount of trebrutinib to the subject. Embodiment 62. The method of embodiment 60, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 63. The method of embodiment 61 or 62, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 64. The method of any one of embodiments 50-63, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 65. The method of any one of embodiments 50-64, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 66. A method for assessing the efficacy of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CXCL10 in a first biological sample comprising CSF obtained from a subject at a first time point, and (ii) CXCL10 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CXCL10 in the second biological sample relative to CXCL10 in a sample obtained from an untreated patient, wherein the CXCL10 in the second biological sample is approximately the same as or reduced relative to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective against MS in the subject; A method for assessing the efficacy of a therapeutically effective amount of trebrutinib in a subject with MS, comprising: Embodiment 67. The method of embodiment 66, wherein the difference between the first time point and the second time point is about 1 month to about 2 years. Embodiment 68. The method of embodiment 66 or 67, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 69. The method of embodiment 68, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 70. The method of any one of embodiments 66-69, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 71. The method of any one of embodiments 66-70, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 72. The method of any one of embodiments 44 to 71, wherein the CXCL10 comprises CXCL10 RNA. Embodiment 73 The method of embodiment 72, wherein CXCL10 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 74. The method of any one of embodiments 44 to 73, wherein the CXCL10 comprises a CXCL10 protein. Embodiment 75. The method of embodiment 74, wherein CXCL10 is determined by flow cytometry or Western blot. Embodiment 76. The method of any one of embodiments 44 to 75, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 77. The method of any one of embodiments 44 to 76, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS, or for reducing the likelihood or susceptibility to developing MS. Embodiment 78. The method of any one of embodiments 44 to 77, further comprising administering to the subject a further or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 79. The method of any one of embodiments 44 to 78, wherein the subject is undergoing treatment for MS prior to detecting CXCL10. Embodiment 80. The method of embodiment 79, wherein the subject has been previously treated with an anti-CD20 therapy. Embodiment 81 The method of embodiment 80, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 82. The method of any one of embodiments 44 to 81, wherein the subject comprises one or more brain lesions. Embodiment 83. The method of any one of embodiments 44-49 or 61-83, wherein the subject is administered one or more booster doses of anti-CD20 therapy administering a pharmaceutically effective amount of trebrutinib. Embodiment 84. Administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density The method of any one of embodiments 44 to 49 or 61 to 83, resulting in one or more of: Embodiment 85. The method of any one of embodiments 44-84, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 86. The method of any one of embodiments 44-85, wherein the MS is secondary progressive multiple sclerosis. Embodiment 87. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting at least one biomarker in cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method of treating a subject with multiple sclerosis (MS). Embodiment 88. A method of treating a subject with MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying a subject as having MS who expresses at least one biomarker in the biological sample; (c) administering a pharmaceutically effective amount of trebrutinib to the subject; Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method of treating a subject with MS. Embodiment 89. The method of embodiment 87 or 88, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 90. The method of embodiment 89, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 91. The method of any one of embodiments 87-90, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 92. The method of any one of embodiments 87-91, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 93. A method of identifying a patient as suitable for participation in a clinical trial of MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying subjects who express at least one biomarker in the biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method for identifying patients as suitable for participation in a clinical trial for MS. Embodiment 94. A method of diagnosing a subject as having MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying a subject as having MS who expresses at least one biomarker in the biological sample; At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method of diagnosing a subject as having MS. Embodiment 95. A method of identifying a subject with MS as expressing at least one biomarker in a biological sample comprising CSF, comprising: (a) detecting at least one biomarker in a biological sample; (b) identifying a subject with MS who expresses at least one biomarker in a biological sample; and Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method for identifying a subject with MS as expressing at least one biomarker in a biological sample, including CSF. Embodiment 96. A method of identifying a subject as likely to develop MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying a subject who expresses at least one biomarker in the biological sample as likely to develop MS; and Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method for identifying a subject as likely to develop MS. Embodiment 97. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying a subject who expresses at least one biomarker in the biological sample as likely to develop MS; and Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method for identifying a subject who is likely to develop MS. Embodiment 98. The method of any one of embodiments 87 to 97, wherein at least one biomarker is increased compared to at least one biomarker in a reference sample. Embodiment 99. The method of embodiment 98, wherein the reference sample is from a second subject. Embodiment 100. The method of embodiment 99, wherein the second subject does not have MS. Embodiment 101. The method of any one of embodiments 98 to 100, wherein the reference sample comprises CSF. Embodiment 102. The method of any one of embodiments 87 to 101, further comprising obtaining a biological sample from the subject. Embodiment 103. A method of monitoring the progression of MS in a subject over time, comprising: (a) detecting at least one biomarker in a first biological sample obtained from a subject at a first time point; (b) detecting at least one biomarker in a second biological sample obtained from the subject at a second time point; (c) (i) identifying a subject as having progressive MS who has an increase in at least one biomarker at a second time point compared to at least one biomarker at a first time point; or (ii) identifying subjects as having static or degenerative MS if the subjects have at least one biomarker that is about the same or decreased at the second time point compared to the at least one biomarker at the first time point; Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method for monitoring the progression of MS in a subject over time. Embodiment 104. The method of embodiments 93-105, further comprising administering to the subject a pharmaceutically effective amount of trebrutinib. Embodiment 105. The method of embodiment 104, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 106. The method of embodiment 104 or 105, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 107. The method of any one of embodiments 93-106, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 108. The method of any one of embodiments 93 to 107, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 109. A method for assessing the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) at least one biomarker in a first biological sample comprising CSF obtained from a subject at a first time point and (ii) at least one biomarker in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and at least one biomarker in the second biological sample compared to at least one biomarker in a sample obtained from an untreated patient, wherein the abundance of the at least one biomarker in the second biological sample is approximately the same or decreased compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for treating MS in the subject; Including, At least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4 and CCL3; A method for assessing the efficacy of treatment with a pharmaceutically effective amount of trebrutinib in a subject with MS. Embodiment 110. The method of embodiment 109, wherein the difference between the first time point and the second time point is from about 1 month to about 2 years. Embodiment 111. The method of embodiment 109 or 110, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 112. The method of embodiment 111, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 113. The method of any one of embodiments 109-112, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 114. The method of any one of embodiments 109-113, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 115. The method of any one of embodiments 87 to 114, wherein at least one biomarker comprises RNA. Embodiment 116. The method of embodiment 115, wherein at least one biomarker RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 117. The method of any one of embodiments 87 to 114, wherein at least one biomarker comprises a protein. Embodiment 118. The method of embodiment 117, wherein at least one biomarker is determined by flow cytometry or Western blot. Embodiment 119. The method of any one of embodiments 87 to 118, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 120. The method of any one of embodiments 87 to 119, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS or reducing the likelihood or susceptibility to developing MS. Embodiment 121. The method of any one of embodiments 87 to 120, further comprising administering to the subject an additional or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 122. The method of any one of embodiments 87 to 121, wherein the subject has received treatment for MS prior to detecting at least one biomarker. Embodiment 123. The method of embodiment 122, wherein the subject has been previously treated with an anti-CD20 therapy. Embodiment 124. The method of embodiment 123, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 125. The method of any one of embodiments 87 to 124, wherein the subject comprises one or more brain lesions. Embodiment 126. The method of any one of embodiments 87-92 or 94-125, wherein the subject is administered one or more doses of an anti-CD20 therapy in addition to the pharmaceutically effective amount of trebrutinib. Embodiment 127. Administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density The method of any one of embodiments 87 to 92 or 94 to 126, resulting in one or more of: Embodiment 128. The method of any one of embodiments 87-127, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 129. The method of any one of embodiments 87-128, wherein the MS is secondary progressive multiple sclerosis. Embodiment 130. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting CD27 in the cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with multiple sclerosis (MS), comprising: Embodiment 131. A method of treating a subject with MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CD27 in a biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising: Embodiment 132. The method of embodiment 130 or 131, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 133. The method of embodiment 132, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 134. The method of any one of embodiments 130-133, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 135. The method of any one of embodiments 130-134, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 136. A method of identifying a patient as suitable for participation in a clinical trial of MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from a subject; (b) identifying subjects that express CD27 in a biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; 1. A method for identifying a patient as suitable for participation in a clinical trial for MS, comprising: Embodiment 137. A method of diagnosing a subject as having MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CD27 in a biological sample as having MS; and A method of diagnosing a subject as having MS, comprising: Embodiment 138. A method for identifying a subject with MS as expressing CD27 in a biological sample comprising CSF, comprising: (a) detecting CD27 in a biological sample; (b) identifying a subject with MS who expresses CD27 in a biological sample; and 1. A method for identifying a subject with MS as expressing CD27 in a biological sample comprising CSF, the method comprising: Embodiment 139. A method for identifying a subject as likely to develop MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CD27 in a biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 140. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CD27 in a biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 141. The method of any one of embodiments 130 to 140, wherein CD27 is increased compared to CD27 in a reference sample. Embodiment 142. The method of embodiment 141, wherein the reference sample is derived from a second subject. Embodiment 143. The method of embodiment 142, wherein the second subject does not have MS. Embodiment 144. The method of any one of embodiments 141 to 143, wherein the reference sample comprises CSF. Embodiment 145. The method of any one of embodiments 130 to 144, further comprising obtaining a biological sample from the subject. Embodiment 146. A method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CD27 in a first biological sample obtained from the subject at a first time point; (b) detecting CD27 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CD27 at a second time point compared to CD27 at a first time point as a subject with progressive MS; or (ii) identifying subjects with about the same or decreased CD27 at the second time point compared to CD27 at the first time point as having static or regressing MS; 20. A method for monitoring the progression of MS in a subject over time, comprising: Embodiment 147. The method of embodiments 136-146, further comprising administering to the subject a pharmaceutically effective amount of trebrutinib. Embodiment 148. The method of embodiment 147, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 149. The method of embodiment 147 or 148, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 150. The method of any one of embodiments 136-149, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 151. The method of any one of embodiments 136-150, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 152. A method for assessing the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CD27 in a first biological sample comprising CSF obtained from a subject at a first time point, and (ii) CD27 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CD27 in the second biological sample relative to CD27 in a sample obtained from an untreated patient, wherein the CD27 in the second biological sample is approximately the same or is decreased relative to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective against MS in the subject; A method for assessing the efficacy of a therapeutically effective amount of trebrutinib in a subject with MS, comprising: Embodiment 153. The method of embodiment 152, wherein the difference between the first time point and the second time point is from about 1 month to about 2 years. Embodiment 154. The method of embodiment 152 or 153, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 155. The method of embodiment 154, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 156. The method of any one of embodiments 152-156, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 157. The method of any one of embodiments 152 to 156, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 158. The method of any one of embodiments 130 to 157, wherein the CD27 comprises CD27 RNA. Embodiment 159. The method of embodiment 158, wherein CD27 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 160. The method of any one of embodiments 130 to 157, wherein the CD27 comprises a CD27 protein. Embodiment 161. The method of embodiment 160, wherein CD27 is determined by flow cytometry or Western blot. Embodiment 162. The method of any one of embodiments 130 to 161, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 163. The method of any one of embodiments 130-162, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS or reducing the likelihood or susceptibility to developing MS. Embodiment 164. The method of any one of embodiments 130 to 163, further comprising administering to the subject an additional or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 165. The method of any one of embodiments 130 to 164, wherein the subject is undergoing treatment for MS before detecting CD27. Embodiment 166. The method of embodiment 165, wherein the subject has been previously treated with anti-CD20 therapy. Embodiment 167. The method of embodiment 166, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 168. The method of any one of embodiments 130 to 167, wherein the subject comprises one or more brain lesions. Embodiment 169. The method of any one of embodiments 130-135 or 137-168, wherein the subject is administered one or more doses of an anti-CD20 therapy in addition to a pharmaceutically effective amount of trebrutinib. Embodiment 170. Administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density The method of any one of embodiments 130 to 135 or 137 to 168, resulting in one or more of: Embodiment 171. The method of any one of embodiments 130-170, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 172. The method of any one of embodiments 130-171, wherein the MS is secondary progressive multiple sclerosis. Embodiment 173. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting NEFL in cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with multiple sclerosis (MS), comprising: Embodiment 174. A method of treating a subject with MS, comprising: (a) detecting NEFL in a biological sample containing CSF from a subject; (b) identifying a subject expressing NEFL in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising: Embodiment 175. The method of embodiment 173 or 174, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 176. The method of embodiment 175, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 177. The method of any one of embodiments 173 to 176, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 178. The method of any one of embodiments 173 to 177, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 179. A method for identifying a patient as suitable for participation in a clinical trial of MS, comprising: (a) detecting NEFL in a biological sample containing CSF from a subject; (b) identifying subjects that express NEFL in a biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; 1. A method for identifying a patient as suitable for participation in a clinical trial for MS, comprising: Embodiment 180. A method of diagnosing a subject as having MS, comprising: (a) detecting NEFL in a biological sample containing CSF from a subject; (b) identifying a subject expressing NEFL in the biological sample as having MS; and A method of diagnosing a subject as having MS, comprising: Embodiment 181. A method for identifying a subject with MS as expressing NEFL in a biological sample comprising CSF, comprising: (a) detecting NEFL in a biological sample; (b) identifying a subject with MS who expresses NEFL in a biological sample; and 1. A method for identifying a subject with MS as expressing NEFL in a biological sample comprising CSF, the method comprising: Embodiment 182. A method for identifying a subject as likely to develop MS, comprising: (a) detecting NEFL in a biological sample containing CSF from a subject; (b) identifying a subject expressing NEFL in the biological sample as likely to develop MS; and 20. A method of identifying a subject as likely to develop MS, comprising: Embodiment 183. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting NEFL in a biological sample containing CSF from a subject; (b) identifying a subject expressing NEFL in the biological sample as likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 184. The method of any one of embodiments 173 to 183, wherein the NEFL is increased compared to the NEFL in a reference sample. Embodiment 185. The method of embodiment 184, wherein the reference sample is derived from a second subject. Embodiment 186. The method of embodiment 185, wherein the second subject does not have MS. Embodiment 187. The method of any one of embodiments 184 to 186, wherein the reference sample comprises CSF. Embodiment 188. The method of any one of embodiments 173 to 187, further comprising obtaining a biological sample from the subject. Embodiment 189. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting NEFL in a first biological sample obtained from a subject at a first time point; (b) detecting NEFL in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying as a subject with progressive MS a subject whose NEFL is increased at a second time point compared to a first time point; or (ii) identifying subjects with a NEFL that is about the same or decreased at the second time point compared to the NEFL at the first time point as having static or degenerative MS; 20. A method for monitoring the progression of MS in a subject over time, comprising: Embodiment 190. The method of embodiments 173-189, further comprising administering to the subject a pharmaceutically effective amount of trebrutinib. Embodiment 191. The method of embodiment 190, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 192. The method of embodiment 190 or 191, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 193. The method of any one of embodiments 179-192, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 194. The method of any one of embodiments 179 to 193, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 195. A method for assessing the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting NEFL in (i) a first biological sample comprising CSF obtained from a subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and NEFL in the second biological sample compared to NEFL in a sample obtained from an untreated patient, wherein the NEFL in the second biological sample is approximately the same as or reduced compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective against MS in the subject; A method for assessing the efficacy of a therapeutically effective amount of trebrutinib in a subject with MS, comprising: Embodiment 196. The method of embodiment 195, wherein the difference between the first time point and the second time point is from about 1 month to about 2 years. Embodiment 197. The method of embodiment 195 or 196, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 198. The method of embodiment 197, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 199. The method of any one of embodiments 195-198, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 200. The method of any one of embodiments 195-199, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 201. The method of any one of embodiments 173 to 200, wherein the NEFL comprises NEFL RNA. Embodiment 202. The method of embodiment 201, wherein NEFL RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 203. The method of any one of embodiments 173 to 200, wherein the NEFL comprises a NEFL protein. Embodiment 204 The method of embodiment 203, wherein NEFL is determined by flow cytometry or Western blot. Embodiment 205. The method of any one of embodiments 173-204, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 206. The method of any one of embodiments 173 to 205, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS or reducing the likelihood or susceptibility to developing MS. Embodiment 207. The method of any one of embodiments 173 to 206, further comprising administering to the subject an additional or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 208. The method of any one of embodiments 173 to 207, wherein the subject is undergoing treatment for MS prior to detecting NEFL. Embodiment 209. The method of embodiment 208, wherein the subject has been previously treated with an anti-CD20 therapy. Embodiment 210. The method of embodiment 209, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 211. The method of any one of embodiments 173 to 210, wherein the subject comprises one or more brain lesions. Embodiment 212. The method of any one of embodiments 173-178 or 180-211, wherein the subject is administered one or more doses of an anti-CD20 therapy in addition to a pharmaceutically effective amount of trebrutinib. Embodiment 213. The administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density The method according to any one of embodiments 173 to 178 or 180 to 212, resulting in one or more of: Embodiment 214. The method of any one of embodiments 173 to 213, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 215. The method of any one of embodiments 173-214, wherein the MS is secondary progressive multiple sclerosis. Embodiment 216. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting CCL4 in the cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with multiple sclerosis (MS), comprising: Embodiment 217. A method of treating a subject with MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL4 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising: Embodiment 218. The method of embodiment 216 or 217, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 219. The method of embodiment 218, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 220. The method of any one of embodiments 216-219, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 221. The method of any one of embodiments 216-220, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 222. A method of identifying a patient as suitable for participation in a clinical trial of MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from a subject; (b) identifying subjects that express CCL4 in the biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; 1. A method for identifying a patient as suitable for participation in a clinical trial for MS, comprising: Embodiment 223. A method of diagnosing a subject as having MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL4 in the biological sample as having MS; and A method of diagnosing a subject as having MS, comprising: Embodiment 224. A method for identifying a subject with MS as expressing CCL4 in a biological sample comprising CSF, comprising: (a) detecting CCL4 in a biological sample; (b) identifying a subject with MS who expresses CCL4 in a biological sample; and 1. A method for identifying a subject with MS as expressing CCL4 in a biological sample comprising CSF, the method comprising: Embodiment 225. A method for identifying a subject as likely to develop MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL4 in the biological sample as being likely to develop MS; and 20. A method of identifying a subject as likely to develop MS, comprising: Embodiment 226. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL4 in the biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 227. The method of any one of embodiments 216 to 226, wherein CCL4 is increased compared to CCL4 in a reference sample. Embodiment 228. The method of embodiment 227, wherein the reference sample is derived from a second subject. Embodiment 229. The method of embodiment 228, wherein the second subject does not have MS. Embodiment 230. The method of any one of embodiments 216 to 229, wherein the reference sample comprises CSF. Embodiment 231. The method of any one of embodiments 216 to 230, further comprising obtaining a biological sample from the subject. Embodiment 232. A method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CCL4 in a first biological sample obtained from the subject at a first time point; (b) detecting CCL4 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying as a subject with progressive MS a subject having increased CCL4 at a second time point compared to CCL4 at a first time point; or (ii) identifying subjects with about the same or decreased CCL4 at the second time point compared to the CCL4 at the first time point as having static or regressing MS; 20. A method for monitoring the progression of MS in a subject over time, comprising: Embodiment 233. The method of embodiments 222-232, further comprising administering to the subject a pharmaceutically effective amount of trebrutinib. Embodiment 234. The method of embodiment 233, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 235. The method of embodiment 233 or 234, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 236. The method of any one of embodiments 222 to 235, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 237. The method of any one of embodiments 222 to 236, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 238. A method for assessing the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CCL4 in a first biological sample comprising CSF obtained from a subject at a first time point, and (ii) CCL4 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CCL4 in the second biological sample relative to CCL4 in a sample obtained from an untreated patient, wherein the CCL4 in the second biological sample is approximately the same as or reduced relative to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective against MS in the subject; A method for assessing the efficacy of a therapeutically effective amount of trebrutinib in a subject with MS, comprising: Embodiment 239. The method of embodiment 238, wherein the difference between the first time point and the second time point is about 1 month to about 2 years. Embodiment 240. The method of embodiment 238 or 239, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 241. The method of embodiment 240, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 242. The method of any one of embodiments 238-241, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 243. The method of any one of embodiments 238-242, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 244. The method of any one of embodiments 216 to 243, wherein the CCL4 comprises CCL4 RNA. Embodiment 245. The method of embodiment 244, wherein CCL4 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 246. The method of any one of embodiments 216 to 243, wherein the CCL4 comprises a CCL4 protein. Embodiment 247. The method of embodiment 246, wherein CCL4 is determined by flow cytometry or Western blot. Embodiment 248. The method of any one of embodiments 216 to 247, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 249. The method of any one of embodiments 216 to 248, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS or reducing the likelihood or susceptibility to developing MS. Embodiment 250. The method of any one of embodiments 216 to 249, further comprising administering to the subject an additional or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 251. The method of any one of embodiments 216 to 250, wherein the subject is undergoing treatment for MS prior to detecting CCL4. Embodiment 252. The method of embodiment 251, wherein the subject has previously been treated with anti-CD20 therapy. Embodiment 253. The method of embodiment 252, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 254. The method of any one of embodiments 216 to 253, wherein the subject comprises one or more brain lesions. Embodiment 255. The method of any one of embodiments 216-221 or 223-254, wherein the subject is administered one or more doses of an anti-CD20 therapy in addition to the pharmaceutically effective amount of trebrutinib. Embodiment 256. Administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density The method of any one of embodiments 216 to 221 or 223 to 255, resulting in one or more of: Embodiment 257. The method of any one of embodiments 216-256, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 258. The method of any one of embodiments 216-257, wherein the MS is secondary progressive multiple sclerosis. Embodiment 259. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting CCL3 in cerebrospinal fluid (CSF) of a subject; (b) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with multiple sclerosis (MS), comprising: Embodiment 260. A method of treating a subject having MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL3 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising: Embodiment 261. The method of embodiment 259 or 260, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 262. The method of embodiment 261, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 263. The method of any one of embodiments 259-262, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 264. The method of any one of embodiments 259 to 263, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 265. A method for identifying a patient as suitable for participation in a clinical trial of MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from a subject; (b) identifying subjects that express CCL3 in the biological sample, thereby identifying the patient as suitable for participation in a clinical trial for MS; 1. A method for identifying a patient as suitable for participation in a clinical trial for MS, comprising: Embodiment 266. A method of diagnosing a subject as having MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL3 in the biological sample as having MS; and A method of diagnosing a subject as having MS, comprising: Embodiment 267. A method for identifying a subject with MS as expressing CCL3 in a biological sample comprising CSF, comprising: (a) detecting CCL3 in a biological sample; (b) identifying a subject with MS who expresses CCL3 in a biological sample; and 1. A method for identifying a subject with MS as expressing CCL3 in a biological sample comprising CSF, the method comprising: Embodiment 268. A method for identifying a subject as likely to develop MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL3 in the biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 269. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL3 in the biological sample as being likely to develop MS; and A method for identifying a subject who is likely to develop MS, comprising: Embodiment 270. The method of any one of embodiments 259 to 269, wherein CCL4 is increased compared to CCL4 in a reference sample. Embodiment 271. The method of embodiment 270, wherein the reference sample is derived from a second subject. Embodiment 272. The method of embodiment 271, wherein the second subject does not have MS. Embodiment 273. The method of any one of embodiments 270 to 272, wherein the reference sample comprises CSF. Embodiment 274. The method of any one of embodiments 259 to 273, further comprising obtaining a biological sample from the subject. Embodiment 275. A method of monitoring the progression of MS in a subject over time, comprising: (a) detecting CCL3 in a first biological sample obtained from the subject at a first time point; (b) detecting CCL3 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying as a subject with progressive MS a subject having increased CCL3 at a second time point compared to CCL3 at a first time point; or (ii) identifying subjects with about the same or decreased CCL3 at the second time point compared to the CCL3 at the first time point as having static or regressing MS; 20. A method for monitoring the progression of MS in a subject over time, comprising: Embodiment 276. The method of embodiments 265-275, further comprising administering to the subject a pharmaceutically effective amount of trebrutinib. Embodiment 277. The method of embodiment 276, wherein the pharmaceutically effective amount of trebrutinib is a dose of about 50 mg to about 130 mg. Embodiment 278. The method of embodiment 276 or 277, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 279. The method of any one of embodiments 265 to 278, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 280. The method of any one of embodiments 265 to 279, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 281. A method for assessing the effectiveness of a treatment of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CCL3 in a first biological sample comprising CSF obtained from a subject at a first time point, and (ii) CCL3 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of a treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CCL3 in the second biological sample relative to CCL3 in a sample obtained from an untreated patient, wherein the CCL3 in the second biological sample is approximately the same as or reduced relative to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective against MS in the subject; A method for assessing the efficacy of a therapeutically effective amount of trebrutinib in a subject with MS, comprising: Embodiment 282. The method of embodiment 281, wherein the difference between the first time point and the second time point is about 1 month to about 2 years. Embodiment 283. The method of embodiment 281 or 282, wherein the pharmaceutically effective amount of trebrutinib is administered at a dose of about 50 mg to about 130 mg. Embodiment 284. The method of embodiment 283, wherein the pharmaceutically effective amount of trebrutinib is a dose of 60 mg. Embodiment 285. The method of any one of embodiments 281 to 284, wherein the pharmaceutically effective amount of trebrutinib is administered orally. Embodiment 286. The method of any one of embodiments 281 to 285, wherein a pharmaceutically effective amount of trebrutinib is administered daily. Embodiment 287. The method of any one of embodiments 259 to 286, wherein the CCL3 comprises CCL3 RNA. Embodiment 288. The method of embodiment 287, wherein CCL3 RNA is determined by polymerase chain reaction, quantitative polymerase chain reaction, or Northern blot. Embodiment 289. The method of any one of embodiments 259 to 286, wherein the CCL3 comprises a CCL3 protein. Embodiment 290. The method of embodiment 289, wherein CCL3 is determined by flow cytometry or Western blot. Embodiment 291. The method of any one of embodiments 259-290, wherein the method further comprises monitoring the subject for the onset of symptoms of MS. Embodiment 292. The method of any one of embodiments 259 to 291, further comprising administering to the subject a treatment for reducing the rate of progression of the onset of MS or reducing the likelihood or susceptibility to developing MS. Embodiment 293. The method of any one of embodiments 259 to 292, further comprising administering to the subject an additional or increased dose of a pharmaceutically effective amount of trebrutinib. Embodiment 294. The method of any one of embodiments 259 to 293, wherein the subject is undergoing treatment for MS prior to detecting CCL3. Embodiment 295. The method of embodiment 294, wherein the subject has been previously treated with anti-CD20 therapy. Embodiment 296. The method of embodiment 295, wherein the anti-CD20 therapy is ocrelizumab. Embodiment 297. The method of any one of embodiments 259 to 296, wherein the subject comprises one or more brain lesions. Embodiment 298. The method of any one of embodiments 259-264 or 266-297, wherein the subject is administered one or more doses of an anti-CD20 therapy in addition to a pharmaceutically effective amount of trebrutinib. Embodiment 299. Administration of a pharmaceutically effective amount of trebrutinib is Decreased brain lesions; a reduction in the amount of iron in the subject's brain tissue; and / or Decreased synaptic density The method of any one of embodiments 259 to 264 or 266 to 298, resulting in one or more of: Embodiment 300. The method of any one of embodiments 259-299, wherein the MS is relapsing-remitting multiple sclerosis. Embodiment 301. The method of any one of embodiments 259-300, wherein the MS is secondary progressive multiple sclerosis.

[0173] The following drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any way. Like reference symbols indicate similar elements in the drawings. [Brief explanation of the drawings]

[0174] [Figure 1] FIG. 1 shows a heat map of the Olink Proteomics dataset for each sample. [Figure 2A] Figure 1 shows differential abundance analysis of untreated multiple sclerosis (MS) subjects (n=71) (left) compared to healthy volunteers (n=31) (right). Ring dots are proteins with decreased abundance in untreated MS subjects compared to healthy volunteers. Solid dots are proteins with increased abundance in untreated MS subjects compared to healthy volunteers. [Figure 2B] Pathway analysis of differentially abundant proteins in untreated MS subjects. Circle size indicates the ratio of genes in the dataset to pathway size. [Figure 2C-2D] 2A-2E show examples of proteins elevated in the CSF of subjects with untreated MS: MZB1 (FIG. 2C), CD79B (FIG. 2D), and TNFRSF13B (FIG. 2E). [Figure 2E] 2A-2E show examples of proteins elevated in the CSF of subjects with untreated MS: MZB1 (FIG. 2C), CD79B (FIG. 2D), and TNFRSF13B (FIG. 2E). [Figure 3] 1 shows differential abundance analysis of MS subjects treated with a B cell depleting agent compared to untreated MS subjects. Ring dots are proteins with decreased abundance in subjects treated with a B cell depleting agent compared to untreated subjects. Solid dots are proteins with increased abundance in subjects treated with a B cell depleting agent compared to untreated subjects. [Figure 4A] Differential abundance analysis of MS subjects treated with a BTK inhibitor (n=6) (left) for 48 weeks after transitioning from a B cell-depleting agent (anti-CD20 antibody) compared to MS subjects treated with a B cell-depleting agent (anti-CD20 antibody) (n=7) (right). Ring dots represent proteins with decreased abundance in subjects treated with a BTK inhibitor after transitioning from a B cell-depleting agent compared to subjects treated with a B cell-depleting agent. [Figure 4B]Principal component analysis of the Olink Proteomics dataset showing treatment: HV: healthy volunteers; Untreated: untreated MS subjects; Anti-CD20: MS subjects treated with anti-CD20 antibodies; 12wk BTKi: MS subjects treated with a BTK inhibitor for 12 weeks after transition from an anti-CD20 antibody; 48wk BTKi: MS subjects treated with a BTK inhibitor for 48 weeks after transition from an anti-CD20 antibody. [Figure 4C-4D] Figure 4 shows examples of disease-reversing proteins 12 and 48 weeks after transitioning from anti-CD20 therapy to trebrutinib: NEFL (Figure 4C), CXCL13 (Figure 4D), CXCL10 (Figure 4E), CD27 (Figure 4F), CCL4 (Figure 4G), CCL3 (Figure 4H). [Figures 4E-4F] Figure 4 shows examples of disease-reversing proteins 12 and 48 weeks after transitioning from anti-CD20 therapy to trebrutinib: NEFL (Figure 4C), CXCL13 (Figure 4D), CXCL10 (Figure 4E), CD27 (Figure 4F), CCL4 (Figure 4G), CCL3 (Figure 4H). [Figure 4G-4H] Figure 4 shows examples of disease-reversing proteins 12 and 48 weeks after transitioning from anti-CD20 therapy to trebrutinib: NEFL (Figure 4C), CXCL13 (Figure 4D), CXCL10 (Figure 4E), CD27 (Figure 4F), CCL4 (Figure 4G), CCL3 (Figure 4H). [Figure 5] Figure 1 shows CXCL13 protein expression in various subjects. HV: healthy volunteers; HAM / TSP: HTLV-1 associated myelopathy / tropical spastic paraparesis without treatment; MS_untreated: subjects with MS but not treated; MS_ocrelizumab: subjects treated with anti-CD20 antibody only; MS_trebrutinib: subjects treated with a daily dose of trebrutinib; NPX: protein expression. [Figure 6]Figure 1 shows CXCL13 protein expression in various subjects. HV: healthy volunteers; HAM / TSP: HTLV-1 associated myelopathy / tropical spastic paraparesis without treatment; MS_untreated: subjects with MS but not treated; MS_ocrelizumab: subjects treated with anti-CD20 antibody only; MS_trebrutinib: subjects treated with a daily dose of trebrutinib. This data is measured via MSD. [Figure 7] These data show that CXCL10 was downregulated in subjects who switched from ocrelizumab to trebrutinib. These data suggest that treatment with trebrutinib, especially after a course of ocrelizumab, may result in decreased CXCL10 protein levels, which is associated with more favorable clinical outcomes, including active disease. MS_ocrelizumab_baseline: subjects treated with ocrelizumab alone for at least 6 months; MS_trebrutinib_t1: subjects treated with trebrutinib for 12 weeks; MS_trebrutinib_t2: subjects treated with trebrutinib for 48 weeks. DETAILED DESCRIPTION OF THE INVENTION

[0175] The present disclosure is based on the discovery that at least one biomarker is associated with poor prognosis and onset of MS. In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more are associated with poor prognosis and onset of MS. Methods provided herein include identifying at least one biomarker. In some examples, the method includes treating a subject with MS by (a) detecting at least one biomarker in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more. In some examples, the method further includes administering one or more additional therapies, such as an anti-CD20 antibody.

[0176] In some embodiments, CXCL13 is associated with poor prognosis and onset of MS. Chemokine (C-X-C motif) ligand 13 (CXCL13), also known as B-lymphocyte chemoattractant (BLC) or B-cell attractant chemokine 1 (BCA-1), is a protein ligand encoded by the CXCL13 gene in humans. Legler DF et al., J. Exp. Med. 187(4):655-60 (February 1998); Gunn et al., Nature. 391(6669):799-803 (February 1998). Provided herein are methods that involve identifying CXCL13. In some examples, the method includes treating a subject with MS by (a) detecting CXCL13 in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes administering one or more additional therapies, such as an anti-CD20 antibody.

[0177] In some embodiments, CXCL10 is associated with poor prognosis and onset of MS. C-X-C motif chemokine ligand 10 (CXCL10), also known as interferon-gamma-inducible protein 10 (IP-10) or poorly inducible cytokine B10, is an 8.7 kDa protein encoded by the CXCL10 gene in humans. Luster et al., Nature. 315(6021):672-6 (1985); Luster et al., Proceedings of the National Academy of Sciences of the United States of America. 84(9):2868-71 (May 1987). Provided herein are methods that involve identifying CXCL10. In some examples, the method includes treating a subject with MS by (a) detecting CXCL10 in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes administering one or more additional therapies, such as an anti-CD20 antibody.

[0178] In some examples, CD27 is associated with poor prognosis and onset of MS. Provided herein are methods that involve identifying CD27. In some examples, the method involves treating a subject with MS by (a) detecting CD27 in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes administering one or more additional therapies, such as an anti-CD20 antibody.

[0179] In some examples, NEFL is associated with poor prognosis and onset of MS. Provided herein are methods that involve identifying NEFL. In some examples, the methods involve treating a subject with MS by (a) detecting NEFL in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the methods further include administering one or more additional therapies, such as an anti-CD20 antibody.

[0180] In some examples, CCL4 is associated with poor prognosis and onset of MS. Provided herein are methods that involve identifying CCL4. In some examples, the methods involve treating a subject with MS by (a) detecting CCL4 in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the methods further include administering one or more additional therapies, such as an anti-CD20 antibody.

[0181] In some examples, CCL3 is associated with poor prognosis and onset of MS. Provided herein are methods that involve identifying CCL3. In some examples, the methods involve treating a subject with MS by (a) detecting CCL3 in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the methods further include administering one or more additional therapies, such as an anti-CD20 antibody.

[0182] In some examples, CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 are associated with poor prognosis and onset of disease. In some examples, CXCL13, CXCL10, CD27, NEFL, CCL4, or CCL3 are associated with poor prognosis and onset of disease. In some examples, CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3 are associated with poor prognosis and onset of disease. Provided herein are methods that involve identifying CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 in the subject's CSF, and (b) treating a subject with MS by administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes administering one or more additional therapies, such as an anti-CD20 antibody.

[0183] The methods provided herein also include identifying at least one biomarker. In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, the methods provided also include identifying at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, or at least six biomarkers, or more. The methods provided herein also include identifying CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3.

[0184] In some examples, the method comprises treating a subject with MS by (a) detecting at least one biomarker in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the biomarker is selected from Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some embodiments, the at least one biomarker is selected from CXCL1, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, the method comprises detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more in the subject's CSF. In some examples, the method comprises treating a subject with MS by (a) detecting CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 in the subject's CSF and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further comprises administering one or more additional therapies, such as an anti-CD20 antibody.

[0185] MS is classified into three clinical phenotypes: relapsing-remitting (RRMS), secondary-progressive (SPMS), and primary-progressive (PPMS) (Lublin et al. (2014) Neurology. 83:278-86). These three phenotypes are further subdivided into active and inactive forms based on the presence or absence of disease activity, defined by clinical relapses and / or the presence of so-called active lesions on MRI scans. Active MRI lesions are gadolinium-enhancing lesions on T1-weighted scans (T1Gd+) or new / enlarged T2-weighted lesions. Relapsing MS (RMS) forms encompass RRMS and active SPMS, while progressive MS (PMS) forms comprise inactive SPMS and PPMS. The methods disclosed herein evaluate at least one biomarker in each MS subgroup. In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, the methods disclosed herein evaluate at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more in the CSF of a subject. The methods disclosed herein evaluate CXCL13 for each MS subgroup. The methods disclosed herein evaluate CXCL10 for each MS subgroup. The methods disclosed herein evaluate CD27 for each MS subgroup. The methods disclosed herein evaluate NEFL for each MS subgroup. The methods disclosed herein evaluate CCL4 for each MS subgroup. The methods disclosed herein evaluate CCL3 for each MS subgroup. The methods disclosed herein assess CXCL13, CXCL10, CD27, NEFL, CCL4 and / or CCL3 in each MS subgroup.

[0186] I. Definition As used herein, "BTK inhibitor," "BTK inhibitor compound," and "compound" can refer to trebrutinib, which can also be considered to be (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-3-(4-phenoxyphenyl)-1H-imidazo[4,5-c]pyridin-2(3H)-one, having the following structure: [ka] It has the following structure: 4-amino-3-(4-phenoxyphenyl)-1-[(3R)-1-(prop-2-enoyl)piperidin-3-yl]-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] and / or a pharmaceutically acceptable salt thereof.

[0187] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a carrier or excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, and includes carriers or excipients that are acceptable for veterinary use as well as for human pharmaceutical use. A pharmaceutically acceptable carrier / excipient includes both one and more than one such excipient. A pharmaceutically acceptable carrier or adjuvant does not destroy its pharmacological activity and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the therapy / drug.

[0188] In some examples, the pharmaceutical compositions of the present disclosure include one or more acetate, citrate, and / or maleate salts. In some examples, the pharmaceutical composition can include water or phosphate buffered saline (PBS). In some examples, the pharmaceutical composition can include chitosan. The pharmaceutical compositions disclosed herein can include one or more pharmaceutically acceptable salts. In some examples, pharmaceutically acceptable salts include salts containing hydrochloric acid, sodium, sulfate, acetate, phosphate or diphosphate, chloride, potassium, maleic acid, calcium, citrate, mesylate, nitrate, tartaric acid, aluminum, gluconic acid, and any combination thereof.

[0189] The pharmaceutical compositions of the present disclosure may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle.In some cases, the pH of the formulation can be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.In some examples, the pharmaceutical composition is administered orally.

[0190] "Treating" a disease or "treatment" of a disease includes: (1) preventing the disease, e.g., preventing clinical symptoms of the disease from developing in a mammal that has been exposed to or may be susceptible to the disease but has not yet experienced or manifested symptoms of the disease; (2) inhibiting the disease, e.g., arresting or reducing the development of the disease or its clinical symptoms; and / or (3) alleviating the disease, e.g., causing regression of the disease or its clinical symptoms.

[0191] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0192] A "therapeutically effective amount" means the amount of a BTK inhibitor compound that, when administered to a mammal for treating a disease, is sufficient to affect such treatment for the disease. A "therapeutically effective amount" will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the mammal being treated.

[0193] A "subject" or "patient" herein refers to a human subject or patient. Generally, the subject or patient is eligible for treatment for multiple sclerosis. For purposes herein, such eligible subjects or patients are those who are experiencing, have experienced, or may experience one or more signs, symptoms, or other indicators of multiple sclerosis, e.g., those who have been diagnosed with and / or are at risk for developing multiple sclerosis, whether newly diagnosed (having "new-onset" MS), previously diagnosed with a new relapse or exacerbation, previously diagnosed and in remission, etc. Persons suffering from or at risk for developing multiple sclerosis can optionally be identified as those who have been screened for high levels of CD20-positive B cells in serum, cerebrospinal fluid (CSF), and / or MS lesions, and / or screened for the use of assays to detect autoantibodies, assessed qualitatively and preferably quantitatively. Exemplary such autoantibodies associated with multiple sclerosis include anti-myelin basic protein (MBP), anti-myelin oligodendrocyte glycoprotein (MOG), anti-ganglioside, and / or anti-neurofibrillar antibodies. Such autoantibodies may be detected in a subject's serum, cerebrospinal fluid (CSF), and / or MS lesions. "Elevated" autoantibody or B-cell levels herein refer to levels of such autoantibodies or B-cells that significantly exceed levels in individuals without MS.

[0194] In some embodiments, subjects with MS have at least one documented relapse within the past year and / or more than two documented relapses within the past two years, and / or more than one active brain lesion in the past six months and on an MRI scan prior to screening.

[0195] In some examples, the methods disclosed herein delay or slow the progression of MS. As used herein, "delaying" or "slowing" the progression of multiple sclerosis means preventing, prolonging, impeding, delaying, delaying, stabilizing, and / or postponing the onset of the disease. This delay can be of varying lengths of time depending on the history of the disease and / or the individual being treated.

[0196] In some instances, the subject has one or more symptoms of MS. A "symptom" of MS is a morbid phenomenon or deviation from normal in structure, function, or sensation experienced by a subject that is indicative of MS.

[0197] A subject described herein may have one of the various forms of MS. "Multiple sclerosis" (MS) refers to a chronic inflammatory, often disabling disease of the central nervous system characterized by demyelination and neurodegeneration. There are three internationally recognized forms of MS: primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), and secondary progressive multiple sclerosis (SPMS).

[0198] Within the scope of this disclosure, "relapsing multiple sclerosis," "relapsing MS," or "RMS" can include clinically isolated syndrome (CIS), relapsing remitting multiple sclerosis (RRMS), and relapsing secondary progressive multiple sclerosis (R-SPMS). See, e.g., Lublin et al., Defining the clinical course of multiple sclerosis; the 2013 revisions, Neurology 2014;83:278-286.

[0199] As used herein, "progressive multiple sclerosis" refers to primary progressive multiple sclerosis (PPMS) and secondary progressive multiple sclerosis (SPMS). In some embodiments, progressive multiple sclerosis is characterized by a documented, irreversible loss of neurological function that persists for six months or more and is not attributable to a clinical relapse.

[0200] "Primary progressive multiple sclerosis" or "PPMS" is characterized by gradual progression of the disease from its onset, with rare overlapping relapses and remissions. There may be periods of plateauing disease activity, and there may be good days and bad days or weeks. PPMS differs from RRMS and SPMS in that onset typically occurs in the late 30s or early 40s, men are just as likely as women to develop it, and initial disease activity is often in the spinal cord rather than the brain. PPMS disease activity can also be observed (or found) in the brain. PPMS is the subtype of MS least likely to show inflammatory (gadolinium-enhancing) lesions on MRI scans. The primary progressive form of the disease affects approximately 15% of all people with multiple sclerosis. PPMS can be defined according to the criteria of Thompson et al. (2018) Lancet 7(2):162-173. Subjects with PPMS treated herein are typically those with a suspected or confirmed diagnosis of PPMS. In some embodiments, the multiple sclerosis is primary progressive multiple sclerosis (PPMS). In some embodiments, the patient has been diagnosed with PPMS according to the criteria set forth in Thompson et al. (2018) Lancet Neurol. 17:162-73. In some embodiments, the patient has PPMS, and the treatment results in a reduced risk of 12-week complex disability progression (cCDP).

[0201] "Relapsing-remitting multiple sclerosis" or "RRMS" is characterized by relapses (also known as exacerbations), during which new symptoms may appear and old symptoms may return or worsen. Relapses are followed by periods of remission, during which the person fully or partially recovers from the deficits acquired during the relapse. Relapses can last for days, weeks, or months, and recovery can be slow and gradual or nearly instantaneous. The majority of people with MS (approximately 85%) are first diagnosed with RRMS. This is typically when they are in their 20s or 30s, although much earlier and later diagnoses have been noted. Women are twice as likely as men to have this subtype of MS. During relapses, myelin, the protective insulating sheath around nerve fibers (neurons) in the white matter regions of the central nervous system (CNS), can be damaged in an inflammatory response by the body's own immune system. This causes a wide variety of neurological symptoms, which vary considerably depending on which areas of the CNS are damaged. Shortly after a relapse, the inflammatory response subsides, and a special type of glial cell in the CNS (called oligodendrocytes) can support remyelination, thereby repairing the myelin sheath around axons. This remyelination may be involved in remission. Approximately 50% of patients with RRMS convert to SPMS within 10 years of disease onset. After 30 years, this number rises to 90%. At any given time, the relapsing-remitting form of the disease accounts for approximately 55% of all people with MS.

[0202] In some embodiments, the MS is relapsing multiple sclerosis (RMS). In some embodiments, the patient has been diagnosed with RMS according to the criteria set forth in Thompson et al. (2018) Lancet Neurol. 17:162-73. In some embodiments, the patient has RMS, and the treatment results in a 12-week risk reduction of combined disability progression (cCDP). In some embodiments, the 12-week risk reduction of cCDP is measured as an increase in the time to onset of cCDP that is sustained for at least 12 weeks. In some embodiments, the time to onset of cCDP refers to the first occurrence of a confirmed progression event according to one of the following three criteria: (i) Confirmed Disability Progression (CDP); (ii) A sustained increase of 20% in the Timed 25-Foot Walk Test (T25FWT) score compared to the T25FWT score at or immediately prior to treatment initiation (e.g., within any one of 6, 5, 4, 3, 2, or 1 month, or within any one of 4, 3, 2, or 1 week, or within 7, 6, 5, 4, 3, 2, or 1 day prior to treatment initiation); or (iii) A sustained increase of 20% in the 9-Hole Peg Test (9-HPT) score compared to the 9-HPT score at or immediately prior to treatment initiation (e.g., within any one of 6, 5, 4, 3, 2, or 1 month, or within any one of 4, 3, 2, or 1 week, or within 7, 6, 5, 4, 3, 2, or 1 day prior to treatment initiation). In some embodiments, CDP refers to a sustained increase in EDSS score of 1.0 point in patients with an EDSS score of 5.5 at or shortly before the start of treatment, or a sustained increase of 0.5 point in patients with an EDSS score >5.5 at or shortly before the start of treatment.

[0203] Also disclosed herein are methods of detecting one or more proteins in a sample (e.g., in a sample comprising CSF). In some examples, the protein is selected from the proteins listed in Tables 1-3. In some examples, at least one biomarker is selected from the proteins listed in Table 3. In some examples, the protein is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, disclosed herein are methods of detecting two or more, three or more, four or more, five or more, or six or more proteins in a sample (e.g., in a sample comprising CSF).

[0204] In some examples, the method includes detecting CXCL13. Homeostatic B cell-attracting chemokine 1 (BCA-1), also known as CXCL13 (or ANGIE, BLC, BLR1L, ANGIE2, or Scyb13), is constitutively expressed by follicular dendritic cells (FDCs) and macrophages in secondary lymphoid organs (e.g., spleen, lymph nodes, and Peyer's patches). See Gunn et al., Nature 391:799-803 (1998) and Carlsen et al., Blood 104(10):3021-3027 (2004). CXCL13 acts primarily through the G protein-coupled CXCR5 receptor (Burkitt's lymphoma receptor 1). CXCR5 is expressed, for example, on mature B lymphocytes, CD4+ follicular helper T cells (Thf cells), a minor subset of CD8+ T cells, and activated tonsillar Treg cells. See Legler et al., J. Exp. Med. 187:655-660 (1998); Forster et al., Blood 84:830-840 (1994); Fazilleau et al., Immunity 30:324-335 (2009); Ansel et al., J. Exp. Med. 190:1123-1134 (1999); Lim et al., J. Clin. Invest. 114(11):1640-1649 (2004); and R. Forster, Chapter in Academic Press Cytokine Reference, August 2000.

[0205] CXCL13 is a potent B cell chemoattractant that guides naive B cells to the follicles of secondary lymphoid organs and is constitutively expressed by follicular dendritic cells (FDCs) and stromal cells in B cell-rich regions of secondary lymphoid organs. CXCL13 is also known as B cell-attracting chemokine 1 (BCA-1). CXCL13 signals through its receptor, CXCR5. CXCR5 is a seven-transmembrane G protein-coupled receptor and a member of the CXC chemokine receptor subfamily of the class 1 GPCR family. CXCR5 is expressed at high levels on naive and activated B cells, including peripheral blood and tonsillar B cells. It is also expressed on a subset of activated peripheral blood CD4+ T cells and the majority of CD4+ cells in secondary lymphoid tissues. CXCL13 is the only known ligand for CXCR5.

[0206] CXCL13 plays a role in the development of peripheral lymphoid organs; for example, Ansel et al. showed that CXCL13-deficient mice have severe defects in peripheral lymph node development. CXCL13 induces membrane lymphotoxin α1β2 expression on naive B cells recruited to follicles, which promotes FDC maturation and further enhances CXCL13 production. CXCL13-deficient mice immunized with T cell-dependent antigens develop germinal centers in lymph nodes and spleens, but these are small and irregular in structure, suggesting that CXCL13 is required for the recruitment and correct positioning of B cells within follicles.

[0207] CXCL13 also plays a role in innate immunity, and CXCL13-deficient mice lack both peritoneal and pleural cavity B1 cells and are defective in the production of natural antibodies against body cavity bacterial antigens (Ansel, K M. et al. Immunity, 16:67-76, 2002).

[0208] In some examples, the method includes detecting CXCL10. C-X-C motif chemokine ligand 10 (CXCL10), also known as interferon-gamma-inducible protein 10 (IP-10) or hypo-inducible cytokine B10, is an 8.7 kDa protein encoded by the CXCL10 gene in humans. Luster et al., Nature. 315(6021):672-6 (1985); Luster et al., Proceedings of the National Academy of Sciences of the United States of America. 84(9):2868-71 (May 1987).

[0209] In some examples, the method includes detecting CD27. In some examples, the method includes detecting NEFL. In some examples, the method includes detecting CCL4. In some examples, the method includes detecting CCL3.

[0210] In some examples, the method includes detecting CXCL13, CXCL10, CD27, NEFL, CCL4 and / or CCL3.

[0211] II. Detection Method The methods provided herein include detecting the level of at least one biomarker (e.g., RNA or protein) in the CSF of a subject. In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, the methods include detecting the levels of at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more in the CSF of the subject. The methods provided herein include detecting the level of CXCL13 (e.g., RNA or protein) in the CSF of the subject. The methods provided herein include detecting the level of CXCL10 (e.g., RNA or protein) in the CSF of the subject. The methods provided herein include detecting the level of CD27 (e.g., RNA or protein) in the CSF of the subject. The methods provided herein include detecting the level of NEFL (e.g., RNA or protein) in the CSF of a subject. The methods provided herein include detecting the level of CCL4 (e.g., RNA or protein) in the CSF of a subject. The methods provided herein include detecting the level of CCL33 (e.g., RNA or protein) in the CSF of a subject. The methods provided herein include detecting the level of CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 (e.g., RNA or protein) in the CSF of a subject.

[0212] In some examples, the method includes identifying a patient as suitable for participation in a clinical trial for MS. In some examples, the method includes (a) detecting at least one biomarker in a biological sample from the subject, including CSF, and (b) identifying the subject in the biological sample expressing the at least one biomarker, thereby identifying the patient as suitable for participation in a clinical trial for MS. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more biomarkers.

[0213] In some examples, the method includes diagnosing a subject as having MS. In some examples, the method includes (a) detecting the level of at least one biomarker in a biological sample comprising CSF from the subject, and (b) identifying the subject as having MS who expresses the at least one biomarker in the biological sample. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0214] In some examples, the method includes identifying a subject with MS as expressing at least one biomarker in a biological sample comprising CSF. In some examples, the method includes (a) detecting at least one biomarker in the biological sample and (b) identifying a subject with MS who expresses at least one biomarker in the biological sample. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0215] In some examples, the method includes identifying a subject as likely to develop MS. In some examples, the method includes (a) detecting at least one biomarker in a biological sample comprising CSF from the subject, and (b) identifying the subject expressing at least one biomarker in the biological sample as likely to develop MS. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more biomarkers.

[0216] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting at least one biomarker in a biological sample comprising CSF from the subject, and (b) identifying the subject expressing at least one biomarker in the biological sample as being likely to develop MS. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0217] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting at least one biomarker in a first biological sample obtained from the subject at a first time point, (b) detecting at least one biomarker in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying the subject as having progressive MS if at least one biomarker at the second time point is increased compared to the at least one biomarker at the first time point, or (ii) identifying the subject as having static or regressing MS if at least one biomarker at the second time point is approximately the same or decreased compared to the at least one biomarker at the first time point. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more.

[0218] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting (i) at least one biomarker in a first biological sample comprising CSF obtained from the subject at a first time point and (ii) at least one biomarker in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and the at least one biomarker in the second biological sample compared to at least one biomarker in a sample obtained from an untreated patient, wherein the abundance of the at least one biomarker in the second biological sample is approximately the same as or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more, and determining a correlation between the effectiveness of the treatment and the biomarkers.

[0219] In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3.

[0220] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting CXCL13 in a biological sample including CSF from the subject, and (b) identifying a subject who expresses CXCL13 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting CXCL13 in a biological sample including CSF from the subject, and (b) identifying a subject who expresses CXCL13 in the biological sample as having MS.

[0221] In some examples, the method includes identifying a subject with MS as expressing CXCL13 in a biological sample including CSF. In some examples, the method includes (a) detecting CXCL13 in the biological sample and (b) identifying a subject with MS who expresses CXCL13 in the biological sample. In some examples, the method includes identifying a subject as having a high likelihood of developing MS. In some examples, the method includes (a) detecting CXCL13 in a biological sample including CSF from the subject and (b) identifying a subject who expresses CXCL13 in the biological sample as having a high likelihood of developing MS.

[0222] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting CXCL13 in a biological sample including CSF from the subject, and (b) identifying the subject who expresses CXCL13 in the biological sample as being likely to develop MS.

[0223] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting CXCL13 in a first biological sample obtained from the subject at a first time point, (b) detecting CXCL13 in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying a subject with increased CXCL13 at the second time point compared to CXCL13 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CXCL13 at the second time point compared to CXCL13 at the first time point as having static or regressing MS.

[0224] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting CXCL13 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of a treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CXCL13 in the second biological sample compared to CXCL13 in a sample obtained from an untreated patient, wherein CXCL13 in the second biological sample is approximately the same as or reduced compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS.

[0225] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting CXCL10 in a biological sample including CSF from the subject, and (b) identifying a subject that expresses CXCL10 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting CXCL10 in a biological sample including CSF from the subject, and (b) identifying a subject that expresses CXCL10 in the biological sample as having MS.

[0226] In some examples, the method includes identifying a subject with MS as expressing CXCL10 in a biological sample including CSF. In some examples, the method includes (a) detecting CXCL10 in the biological sample and (b) identifying a subject with MS who expresses CXCL10 in the biological sample. In some examples, the method includes identifying a subject as having a high likelihood of developing MS. In some examples, the method includes (a) detecting CXCL10 in a biological sample including CSF from the subject and (b) identifying a subject who expresses CXCL10 in the biological sample as having a high likelihood of developing MS.

[0227] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting CXCL10 in a biological sample including CSF from the subject, and (b) identifying the subject who expresses CXCL10 in the biological sample as being likely to develop MS.

[0228] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting CXCL10 in a first biological sample obtained from the subject at a first time point, (b) detecting CXCL10 in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying a subject with increased CXCL13 at the second time point compared to CXCL10 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CXCL13 at the second time point compared to CXCL10 at the first time point as having static or regressing MS.

[0229] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting CXCL10 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of a treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CXCL10 in the second biological sample compared to CXCL10 in a sample obtained from an untreated patient, wherein CXCL10 in the second biological sample is approximately the same as or decreased compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS.

[0230] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting CD27 in a biological sample including CSF from the subject, and (b) identifying a subject expressing CD27 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting CD27 in a biological sample including CSF from the subject, and (b) identifying a subject expressing CD27 in the biological sample as having MS.

[0231] In some examples, the method includes identifying a subject with MS as expressing CD27 in a biological sample including CSF. In some examples, the method includes (a) detecting CD27 in the biological sample and (b) identifying a subject with MS who expresses CD27 in the biological sample. In some examples, the method includes identifying a subject as having a high likelihood of developing MS. In some examples, the method includes (a) detecting CD27 in a biological sample including CSF from the subject and (b) identifying a subject who expresses CD27 in the biological sample as having a high likelihood of developing MS.

[0232] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting CD27 in a biological sample comprising CSF from the subject, and (b) identifying the subject who expresses CD27 in the biological sample as being likely to develop MS.

[0233] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting CD27 in a first biological sample obtained from the subject at a first time point, (b) detecting CD27 in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying a subject with increased CD27 at the second time point compared to CD27 at the first time point as having progressive MS, or (ii) identifying a subject with about the same or decreased CD27 at the second time point compared to CD27 at the first time point as having static or regressing MS.

[0234] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting CD27 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CD27 in the second biological sample compared to CD27 in a sample obtained from an untreated patient, wherein CD27 in the second biological sample is approximately the same as or decreased compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for the subject's MS.

[0235] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting NEFL in a biological sample including CSF from the subject, and (b) identifying a subject expressing NEFL in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting NEFL in a biological sample including CSF from the subject, and (b) identifying a subject expressing NEFL in the biological sample as having MS.

[0236] In some examples, the method includes identifying a subject with MS as expressing NEFL in a biological sample including CSF. In some examples, the method includes (a) detecting NEFL in the biological sample and (b) identifying a subject with MS who expresses NEFL in the biological sample. In some examples, the method includes identifying a subject as having a high likelihood of developing MS. In some examples, the method includes (a) detecting NEFL in a biological sample including CSF from the subject and (b) identifying a subject who expresses NEFL in the biological sample as having a high likelihood of developing MS.

[0237] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting NEFL in a biological sample from the subject, the biological sample including CSF, and (b) identifying the subject who expresses NEFL in the biological sample as likely to develop MS.

[0238] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting NEFL in a first biological sample obtained from the subject at a first time point, (b) detecting NEFL in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying a subject with an increased NEFL at the second time point compared to the NEFL at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased NEFL at the second time point compared to the NEFL at the first time point as having static or regressing MS.

[0239] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting NEFL in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and NEFL in the second biological sample compared to NEFL in a sample obtained from an untreated patient, wherein NEFL in the second biological sample is approximately the same as or reduced compared to the amount present in the sample from the untreated patient, thereby indicating that the treatment is effective for the subject's MS.

[0240] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting CCL4 in a biological sample including CSF from the subject, and (b) identifying the subject expressing CCL4 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting CCL4 in a biological sample including CSF from the subject, and (b) identifying the subject expressing CCL4 in the biological sample as having MS.

[0241] In some examples, the method includes identifying a subject with MS as expressing CCL4 in a biological sample including CSF. In some examples, the method includes (a) detecting CCL4 in the biological sample and (b) identifying a subject with MS who expresses CCL4 in the biological sample. In some examples, the method includes identifying a subject as likely to develop MS. In some examples, the method includes (a) detecting CCL4 in a biological sample including CSF from the subject and (b) identifying a subject who expresses CCL4 in the biological sample as likely to develop MS.

[0242] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting CCL4 in a biological sample comprising CSF from the subject, and (b) identifying the subject who expresses CCL4 in the biological sample as being likely to develop MS.

[0243] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting CCL4 in a first biological sample obtained from the subject at a first time point, (b) detecting CCL4 in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying a subject with increased CCL4 at the second time point compared to CCL4 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CCL4 at the second time point compared to CCL4 at the first time point as having static or regressing MS.

[0244] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting CCL4 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of a treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CCL4 in the second biological sample compared to CCL4 in a sample obtained from an untreated patient, wherein the CCL4 in the second biological sample is approximately the same as or reduced compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS.

[0245] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting CCL3 in a biological sample including CSF from the subject, and (b) identifying a subject expressing CCL3 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting CCL3 in a biological sample including CSF from the subject, and (b) identifying a subject expressing CCL3 in the biological sample as having MS.

[0246] In some examples, the method includes identifying a subject with MS as expressing CCL3 in a biological sample including CSF. In some examples, the method includes (a) detecting CCL3 in the biological sample and (b) identifying a subject with MS who expresses CCL3 in the biological sample. In some examples, the method includes identifying a subject as likely to develop MS. In some examples, the method includes (a) detecting CCL3 in a biological sample including CSF from the subject and (b) identifying a subject who expresses CCL3 in the biological sample as likely to develop MS.

[0247] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting CCL3 in a biological sample comprising CSF from the subject, and (b) identifying the subject who expresses CCL3 in the biological sample as likely to develop MS.

[0248] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting CCL3 in a first biological sample obtained from the subject at a first time point, (b) detecting CCL3 in a second biological sample obtained from the subject at a second time point, and (c) (i) identifying a subject with increased CCL3 at the second time point compared to CCL3 at the first time point as having progressive MS, or (ii) identifying a subject with approximately the same or decreased CCL3 at the second time point compared to CCL3 at the first time point as having static or regressing MS.

[0249] In some examples, the method includes evaluating the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting CCL3 in (i) a first biological sample comprising CSF obtained from the subject at a first time point and (ii) a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject is administered one or more doses of a treatment between the first and second time points; and (b) determining a correlation between the effectiveness of the treatment and CCL3 in the second biological sample compared to CCL3 in a sample obtained from an untreated patient, wherein the CCL3 in the second biological sample is approximately the same as or reduced compared to the abundance in a sample from an untreated patient, thereby indicating that the treatment is effective for the subject's MS.

[0250] In some examples, the method includes identifying a patient as suitable for participating in a clinical trial for MS. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 in a biological sample containing CSF from the subject, and (b) identifying a subject expressing CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in the biological sample, thereby identifying the patient as suitable for participating in a clinical trial for MS. In some examples, the method includes diagnosing the subject as having MS. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a biological sample containing CSF from the subject, and (b) identifying a subject expressing CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in the biological sample as having MS.

[0251] In some examples, the method includes identifying a subject with MS as expressing CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a biological sample comprising CSF. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in the biological sample, and (b) identifying a subject with MS who expresses CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in the biological sample. In some examples, the method includes identifying a subject as likely to develop MS. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a biological sample comprising CSF from the subject, and (b) identifying a subject who expresses CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in the biological sample as likely to develop MS.

[0252] In some examples, the method includes identifying a subject who is likely to develop MS. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a biological sample comprising CSF from the subject, and (b) identifying the subject as likely to develop MS if the subject expresses CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in the biological sample.

[0253] In some examples, the method includes monitoring the progression of MS in a subject over time. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a first biological sample obtained from the subject at a first time point, (b) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a second biological sample obtained from the subject at a second time point, and (c) (i) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a second biological sample obtained from the subject at a second time point compared to CXCL13, CXCL10, CD27, CCL4, and / or CCL3 at the first time point. and (ii) identifying a subject as having progressive MS if the subject has increased CXCL13, CXCL10, CD27, CCL4 and / or CCL3 at the second time point, or (ii) identifying a subject as having static or regressing MS if the subject has similar or decreased CXCL13, CXCL10, CD27, CCL4 and / or CCL3 at the second time point compared to the first time point.

[0254] In some examples, the method includes assessing the effectiveness of a treatment in a subject with MS. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib. In some examples, the treatment includes a pharmaceutically effective amount of trebrutinib and a pharmaceutically effective amount of an anti-CD20 antibody. In some examples, the method includes (a) detecting (i) CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a first biological sample comprising CSF obtained from a subject at a first time point, and (ii) CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a second biological sample comprising CSF obtained from a subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; and (b) detecting CXCL13, CXCL10, CD27, CCL4, and / or CCL3 in a first biological sample comprising CSF obtained from a subject at a first time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points. and determining a correlation between the effectiveness of the treatment and CXCL13, CXCL10, CD27, CCL4 and / or CCL3 in the second biological sample compared to CXCL13, CXCL10, CD27, CCL4 and / or CCL3 in the sample from the untreated patient, wherein the CXCL13, CXCL10, CD27, CCL4 and / or CCL3 in the second biological sample are approximately the same as or decreased compared to their abundance in a sample from an untreated patient, thereby indicating that the treatment is effective against MS in the subject.

[0255] The methods provided herein include methods of extracting samples from a subject. In some examples, the methods include extracting CSF from a subject. The method of extracting CSF from a subject includes a lumbar puncture (i.e., spinal tap) procedure, in which a needle is inserted into the spinal canal to collect cerebrospinal fluid for testing.

[0256] In some examples, the method detects protein (e.g., CXCL13 protein, CXCL10 protein, CD27 protein, NEFL protein, CCL4 protein, and / or CCL3 protein) abundance. In some examples, the method detects RNA (e.g., CXCL13 RNA, CXCL10 RNA, CD27 RNA, NEFL RNA, CCL4 RNA, and / or CCL3 RNA) abundance. Collectively, proteins and nucleic acids (e.g., RNA) can be referred to as analytes. The disclosure and methods described herein can be used to detect and analyze a wide variety of different analytes. For purposes of this disclosure, "analyte" can include any biological substance, structure, moiety, or component to be analyzed. The term "target" can similarly refer to the analyte of interest.

[0257] Analytes can be broadly classified into two groups: nucleic acid analytes and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidated variants of proteins, hydroxylated variants of proteins, methylated variants of proteins, ubiquitinated variants of proteins, sulfated variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte may be a cellular organelle (e.g., a nucleus or mitochondria). In some examples, the non-nucleic acid analyte is a CXCL13 protein. In some examples, the non-nucleic acid analyte is a CXCL10 protein. In some examples, the non-nucleic acid analyte is a CD27 protein. In some examples, the non-nucleic acid analyte is a NEFL protein. In some examples, the non-nucleic acid analyte is a CCL4 protein. In some examples, the non-nucleic acid analyte is a CCL3 protein. In some examples, the non-nucleic acid analyte is a CXCL10 protein, a CXCL13 protein, a CD27 protein, a NEFL protein, a CCL4 protein, and / or a CCL4 protein.

[0258] Cell surface features corresponding to the analyte may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, cluster of differentiation proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation) status of cell surface proteins, gap junctions, and adherens junctions.

[0259] The analyte may originate from a particular type of cell and / or a particular intracellular region. For example, the analyte may originate from the cytosol, the nucleus, mitochondria, microsomes, or more generally any other compartment, organelle, or part of a cell. Permeabilizing agents that specifically target particular cellular compartments and organelles can be used to selectively release the analyte from the cell for analysis.

[0260] Proteomics has recently emerged and has been developed for the large-scale study of protein patterns in living organisms. The typical goal of proteome analysis is to identify and quantify proteins present in specific tissues under specific conditions. Proteomics techniques, combined with bioinformatics, are powerful tools for protein identification and characterization. Generally, two-dimensional (2D) electrophoresis is used for protein separation, and mass spectrometry followed by databank searching is used for protein identification. Up to 10,000 proteins can be studied simultaneously. In some instances, proteomes are evaluated using Olink Proteomics (www.olink.com). See Deutsch et al., J Proteome Res. 2021 Dec 3;20(12):5241-5263; and Cui et al., Lab Invest, 2022 Nov;102(11):1170-1181, each of which is incorporated by reference in its entirety.

[0261] The detection and quantification of individual proteins is one of the fundamental aspects of proteomics. Immunologically based methods, such as quantitative enzyme-linked immunosorbent assay (ELISA), Western blotting, and dot blotting, are very common and highly sensitive assays for protein detection, and use antibodies that specifically react with whole proteins or specific epitopes (e.g., fusion tags) after cell lysis. Detection techniques are typically based on chemiluminescence or fluorescence.

[0262] Examples of measurement methods for assessing protein expression (e.g., CXCL13 protein expression) include LC-MS, immunoassays, enzyme activity assays, and capillary electrophoresis. In some examples, qualitative or quantitative approaches can be used, including LC-MS; and enzyme immunoassays, two-antibody sandwich ELISAs, colloidal gold assays, radioimmunoassays, latex agglutination immunoassays, fluorescent immunoassays, Western blots, immunohistochemistry, surface plasmon resonance spectroscopy (SPR), and quartz crystal microbalance (QCM) using monoclonal or polyclonal antibodies specific for CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3.

[0263] As another example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide bonds with reducing agents and detergent washes, chaotropic salt treatment, treatment with antigen retrieval solutions, and treatment with acidic glycine buffers. Methods for staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017;65(8):431-444, Lin et al., Nat Commun. 2015;6:8390, Pirici et al., J. Histochem. Cytochem. 2009;57:567-75, and Glassetal., J. Histochem. Cytochem. 2009;57:899-905, the entire contents of each of which are incorporated herein by reference.

[0264] In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) can be performed as part of or in addition to the exemplary workflows presented herein. For example, samples can be fixed and immunofluorescence protocols can be used to probe for analytes (e.g., proteins). For example, samples can be rehydrated, blocked, and permeabilized (3xSSC, 2% BSA, 0.1% Triton X, 1U / µl RNAse inhibitor, 10 min at 4°C) before staining with a fluorescent primary antibody (1:100 in 3xSSC, 2% BSA, 0.1% Triton X, 1U / µl RNAse inhibitor, 30 min at 4°C). Biological samples can be washed, coverslipped (in glycerol + 1U / µl RNAse inhibitor), imaged (e.g., using a confocal microscope or other device capable of fluorescence detection), washed, and processed.

[0265] Any of a variety of staining and imaging techniques described herein or known in the art can be used in accordance with the methods described herein. In some embodiments, the stain comprises an optical label described herein, including, but not limited to, a fluorescent, radioactive, chemiluminescent, calorimetric, or colorimetric detectable label. In some embodiments, the stain comprises a fluorescent antibody against a target analyte (e.g., a cell surface or intracellular protein) in the biological sample. In some embodiments, the stain comprises an immunohistochemical stain against a target analyte (e.g., a cell surface or intracellular protein) in the biological sample. In some embodiments, the stain comprises a chemical stain such as hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS).

[0266] Examples of nucleic acid analytes include DNA analytes such as genomic DNA, methylated DNA, differentially methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. In some examples, the nucleic acid analyte is a CXCL13 DNA molecule. In some examples, the nucleic acid analyte is a CXCL13 RNA molecule. In some examples, the nucleic acid analyte is a CXCL10 DNA molecule. In some examples, the nucleic acid analyte is a CXCL10 RNA molecule. In some examples, the nucleic acid analyte is a CD27 DNA molecule. In some examples, the nucleic acid analyte is a CD27 RNA molecule. In some examples, the nucleic acid analyte is a NEFL DNA molecule. In some examples, the nucleic acid analyte is a NEFL RNA molecule. In some examples, the nucleic acid analyte is a CCL4 DNA molecule. In some examples, the nucleic acid analyte is a CCL4 RNA molecule. In some examples, the nucleic acid analyte is a CCL3 DNA molecule. In some examples, the nucleic acid analyte is a CCL3 RNA molecule.

[0267] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. RNA can be a transcript (e.g., present in a tissue section). RNA can be small (e.g., less than 200 nucleobases in length) or large (e.g., RNA more than 200 nucleobases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), small tRNA-derived RNA (tsRNA), and small rRNA-derived RNA (srRNA). RNA can be double-stranded or single-stranded RNA. RNA can be circular RNA. The RNA can be bacterial rRNA (eg, 16s rRNA or 23s rRNA).

[0268] Detection of at least one biomarker, such as CXCL13 RNA, CXCL10 RNA, CD27 RNA, NEFL RNA, CCL4 RNA, and / or CCL3 RNA, can be performed by methods known in the art. For example, the abundance of CXCL13 RNA, CXCL10 RNA, CD27 RNA, NEFL RNA, CCL4 RNA, and / or CCL3 RNA can be determined using qPCR. In some embodiments, quantification of RNA and / or DNA is performed by real-time PCR (also known as quantitative PCR or qPCR), using techniques well known in the art, such as, but not limited to, TAQMAN™, or dyes such as SYBR®, or in capillaries (LightCycler® Capillaries). In some embodiments, quantification of genetic material is determined by optical absorbance and real-time PCR. In some embodiments, quantification of genetic material is determined by digital PCR. In some embodiments, to compare the expression levels of target nucleic acids, the analyzed genes can be compared to reference nucleic acid extracts (DNA and RNA) corresponding in expression (mRNA) and abundance (DNA).

[0269] "PCR amplification" refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for performing PCR are described, for example, in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of each of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture contains the genetic material to be amplified, an enzyme, one or more primers used in a primer extension reaction, and reagents for the reaction. The oligonucleotide primers are of sufficient length to provide hybridization to complementary genetic material under annealing conditions. The length of the primer generally depends on the length of the amplification domain, but is typically at least 4 base pairs (bp), at least 5 base pairs (bp), at least 6 base pairs (bp), at least 8 base pairs (bp), at least 9 base pairs (bp), at least 10 base pairs (bp), at least 11 base pairs (bp), at least 12 base pairs (bp), at least 13 base pairs (bp), at least 14 base pairs (bp), at least 15 base pairs (bp), at least 16 base pairs (bp), at least 17 base pairs (bp), at least 18 base pairs (bp), at least 19 base pairs (bp), at least 20 base pairs (bp), at least 25 base pairs (bp), at least 30 base pairs (bp), at least 35 base pairs (bp), and may be 40 bp or longer, with primer lengths generally ranging from 18 to 50 bp. The genetic material can be contacted with a single primer or a set of two primers (a forward primer and a reverse primer), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.

[0270] In some embodiments, PCR amplification can include reactions such as, but not limited to, strand displacement amplification reactions, rolling circle amplification reactions, ligase chain reactions, transcription-mediated amplification reactions, isothermal amplification reactions, and / or loop-mediated amplification reactions.

[0271] In some examples, sequencing can be performed to determine the abundance of at least one biomarker RNA, such as CXCL13 RNA, CXCL10 RNA, CD27 RNA, NEFL RNA, CCL4 RNA, and / or CCL3 RNA. Polynucleotide sequencing can be performed by a variety of systems. More commonly, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real-time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR), and / or isothermal amplification. Non-limiting examples of methods for sequencing genetic material include, but are not limited to, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion, Sanger sequencing, next-generation sequencing methods (e.g., single-molecule real-time sequencing, nanopore sequencing, and polony sequencing), ligation methods, and microarray methods.

[0272] In some embodiments, the genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The desired reverse transcriptase activity can be provided by one or more different reverse transcriptases (i.e., RNA-dependent DNA polymerases), suitable examples of which include, but are not limited to, M-MLV, MuLV, AMV, HIV, ArrayScript™, MultiScribe™, ThermoScript™, and SuperScript® I, II, III, and IV enzymes. "Reverse transcriptase" includes naturally occurring enzymes as well as all such modified derivatives thereof, including derivatives of naturally occurring reverse transcriptases.

[0273] In some examples, at least one biomarker expression, such as CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 expression (RNA or protein), is measured and compared to a reference sample or reference amount. In some examples, CXCL10, CXCL13, CD27, NEFL, CCL4, and / or CCL3 expression (RNA or protein) is measured and compared to a reference sample or reference amount. The reference amount of a nucleic acid / protein can be any suitable reference amount. In some embodiments, the reference amount of a nucleic acid / protein can be determined based on the amount of the nucleic acid / protein in a corresponding sample at a corresponding location (e.g., a reference sample such as a control subject who has not been diagnosed with MS, does not exhibit any symptoms of MS, has no family history of MS, and does not have any known risk factors for MS). In some embodiments, the reference amount of a nucleic acid / protein can be a composite amount or an average amount (e.g., an average amount in a population of people with and without MS).

[0274] In some embodiments, the reference amount can be based on a reference amount published by an appropriate agency (e.g., a government agency (e.g., the United States Food and Drug Administration) or a professional organization (e.g., the American Medical Association or the American Psychiatric Association)), e.g., a reference amount that is a threshold amount of a nucleic acid / protein at a location in a tissue of interest.

[0275] In some embodiments, the reference amount of a nucleic acid / protein can be determined based on any appropriate criteria. For example, in some embodiments, the reference amount of a nucleic acid / protein can be derived from an age-matched healthy subject. In some embodiments, the reference amount of a nucleic acid / protein can be derived from a gender-matched healthy subject or a gender-matched population of healthy subjects. In some embodiments, the reference amount of a nucleic acid / protein can be derived from an age-matched, gender-matched healthy subject or a population of age-matched, gender-matched healthy subjects. In some embodiments, the reference amount of a nucleic acid / protein can be derived from an aggregate sample (e.g., an average of 2 or more individuals) of healthy subjects (e.g., age-matched and / or gender-matched).

[0276] A healthy subject can be any suitable healthy subject. In some embodiments, a healthy subject does not have MS, does not have symptoms of MS, does not have a genetic mutation associated with MS, does not have a family history of MS, does not have behavioral risk factors for MS, or a combination thereof. For example, in some embodiments, a healthy subject has one or more of: no known brain disorder; no symptoms; or no more than three (e.g., no more than two, or no more than one) of: no brain disorder, no known genetic mutation associated with the risk of brain disorder, no family history of brain disorder, and no behavioral risk factors for brain disorder. Other non-limiting examples of healthy subjects are those who do not have a disorder of the subject's biological system (e.g., circulatory system, digestive and excretory system, endocrine system, integumentary or exocrine system, immune and lymphatic system, muscular system, nervous system, see the example of the brain above, renal and urinary system, reproductive system, respiratory system, skeletal system, or a combination thereof), do not have symptoms of a disorder, do not have a genetic mutation associated with the subject's disorder, do not have a family history of the subject's disorder, do not have behavioral risk factors for the subject's disorder, or a combination thereof.

[0277] In some cases, the amount of a nucleic acid / protein can be elevated relative to a reference amount. For example, the amount of a nucleic acid / protein can be at least 0.2-fold (e.g., at least 0.4-fold, at least 0.6-fold, at least 0.8-fold, at least 1-fold, at least 1.3-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more) greater than a reference amount (e.g., any of the exemplary reference amounts described herein or known in the art).

[0278] In some cases, the amount of nucleic acid / protein can be decreased relative to a reference amount. For example, the amount of nucleic acid / protein can be at least 0.2-fold (e.g., at least 0.4-fold, at least 0.6-fold, at least 0.8-fold, at least 1-fold, at least 1.3-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 18-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold or more) less than a reference amount (e.g., any of the exemplary reference amounts described herein or known in the art).

[0279] In some cases, the amount of nucleic acid / protein may be elevated relative to a reference amount. For example, the amount of nucleic acid may be at least 5% more, at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40% more, at least 45% more, at least 50% more, at least 55%, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, at least 10 ...00% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, at least 100% more, % increase (e.g., about 5% to about 99% increase, about 5% to about 80% increase, about 5% to about 60% increase, about 5% to about 40% increase, about 5% to about 20% increase, about 20% to about 95% increase, about 20% to about 80% increase, about 20% to about 60% increase, about 20% to about 40% increase, about 40% to about 99% increase, about 40% to about 80% increase, about 40% to about 60% increase, about 60% to about 99% increase, about 60% to about 80% increase, about 80% to about 99% increase).

[0280] In some cases, the amount of nucleic acid can be reduced relative to a reference amount. For example, the amount of nucleic acid / protein can be at least 5% less, at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55%, at least 60% less, at least 65% less, at least 70% less, at least 75% less, or at least 80% less compared to a reference amount (e.g., any of the exemplary reference amounts described herein). , at least 85% less, at least 90% less, or at least 95% less (e.g., about 5% to about 99% less, about 5% to about 80% less, about 5% to about 60% less, about 5% to about 40% less, about 5% to about 20% less, about 20% to about 95% less, about 20% to about 80% less, about 20% to about 60% less, about 20% to about 40% less, about 40% to about 99% less, about 40% to about 80% less, about 40% to about 60% less, about 60% to about 99% less, about 60% to about 80% less, or about 80% to about 99% less). Other suitable reference amounts and methods for determining them will be apparent to those skilled in the art.

[0281] III. Treatment method Disclosed herein are methods of treating a subject with multiple sclerosis (MS). In some examples, the method includes detecting at least one biomarker in the subject's cerebrospinal fluid (CSF). In some examples, the biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. In some examples, the method includes (a) detecting at least one biomarker, at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more in the subject's cerebrospinal fluid (CSF), and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting at least one biomarker in a biological sample comprising CSF from the subject, (b) identifying the subject as having MS, wherein the subject expresses at least one biomarker in the biological sample, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes detecting at least two biomarkers, at least three biomarkers, at least four biomarkers, at least five biomarkers, at least six biomarkers, or more. In some examples, the method further includes at least one or more additional treatments, such as a pharmaceutically effective amount of an anti-CD20 antibody.

[0282] Disclosed herein are methods for treating a subject with multiple sclerosis (MS). In some examples, the method includes (a) detecting CXCL13 in the subject's cerebrospinal fluid (CSF), and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting CXCL13 in a biological sample containing CSF from the subject, (b) identifying the subject expressing CXCL13 in the biological sample as having MS, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0283] In some examples, the method includes (a) detecting CXCL10 in cerebrospinal fluid (CSF) of the subject, and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting CXCL10 in a biological sample including CSF from the subject, (b) identifying the subject as having MS if the subject expresses CXCL13 in the biological sample, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0284] In some examples, the method includes (a) detecting CD27 in cerebrospinal fluid (CSF) of a subject, and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting CD27 in a biological sample containing CSF from the subject, (b) identifying the subject expressing CD27 in the biological sample as having MS, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0285] In some examples, the method includes (a) detecting NEFL in cerebrospinal fluid (CSF) of a subject, and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting NEFL in a biological sample from the subject, including CSF, (b) identifying the subject expressing NEFL in the biological sample as having MS, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0286] In some examples, the method includes (a) detecting CCL4 in cerebrospinal fluid (CSF) of the subject, and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting CCL4 in a biological sample from the subject, including CSF, (b) identifying the subject expressing CCL4 in the biological sample as having MS, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0287] In some examples, the method includes (a) detecting CCL3 in cerebrospinal fluid (CSF) of the subject, and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting CCL3 in a biological sample from the subject, including CSF, (b) identifying the subject expressing CCL3 in the biological sample as having MS, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0288] In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 in the cerebrospinal fluid (CSF) of the subject, and (b) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method includes (a) detecting CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 in a biological sample from the subject, including CSF, (b) identifying the subject as having MS who expresses CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3 in the biological sample, and (c) administering a pharmaceutically effective amount of trebrutinib to the subject. In some examples, the method further includes one or more additional therapies, such as an anti-CD20 antibody.

[0289] In some cases, the subject has been treated with one or more additional therapies. For example, the subject may be treated with an anti-CD20 antibody for some time (e.g., several weeks or months) before treatment with trebrutinib. In some cases, it may be beneficial to treat the subject with one or more doses of an anti-CD20 antibody during treatment with trebrutinib.

[0290] In some embodiments, administration of trebrutinib reduces the total number of lesions after 12 or 48 weeks of trebrutinib treatment. In some embodiments, administration of trebrutinib maintains, but does not increase, the total number of lesions after 12 or 48 weeks of trebrutinib treatment. In some examples, administration of trebrutinib and one or more additional therapies (such as anti-CD20 antibodies) reduces the total number of lesions after 12 or 48 weeks of treatment. In some examples, administration of trebrutinib and one or more additional therapies (such as anti-CD20 antibodies) maintains, but does not increase, the total number of lesions after 12 or 48 weeks of treatment.

[0291] In some embodiments, the BTK inhibitor compound (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-3-(4-phenoxyphenyl)-1H-imidazo[4,5-c]pyridin-2(3H)-one is administered to treat relapsing multiple sclerosis (RMS) in a subject in need thereof. In some embodiments, the BTK inhibitor compound is a pharmaceutically acceptable salt of (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-3-(4-phenoxyphenyl)-1H-imidazo[4,5-c]pyridin-2(3H)-one. In some embodiments, a therapeutically effective amount of a BTK inhibitor (e.g., trebrutinib) is administered. In some embodiments, a dose of 5 to 60 mg of the BTK inhibitor (e.g., trebrutinib) is administered.

[0292] BTK inhibitors (e.g., trebrutinib) can be prepared according to the methods and schemes described, for example, in U.S. Pat. No. 9,688,676 and U.S. Patent Application Publication No. 20210244720, each of which is incorporated by reference in its entirety.

[0293] After detecting at least one biomarker in the cerebrospinal fluid (CSF) of a subject (e.g., using any one of the methods described herein), the present disclosure provides methods of treating MS in a subject by administering a therapeutically effective amount of a BTK inhibitor (e.g., trebrutinib). In some examples, the at least one biomarker is selected from the proteins listed in Tables 1-3. In some examples, the at least one biomarker is selected from the proteins listed in Table 3. In some examples, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3. After detecting at least one biomarker, such as CXCL13, CXCL10, CD27, NEFL, CCL4, and / or CCL3, in the cerebrospinal fluid (CSF) of a subject (e.g., using any one of the methods described herein), the present disclosure provides methods of treating MS in a subject by administering a therapeutically effective amount of a BTK inhibitor (e.g., trebrutinib). Accordingly, provided herein, following detection, is a method of treating multiple sclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of a BTK inhibitor comprising (R)-1-(1-acryloylpiperidin-3-yl)-4-amino-3-(4-phenoxyphenyl)-1H-imidazo[4,5-c]pyridin-2(3H)-one (e.g., trebrutinib) and / or a pharmaceutically acceptable salt thereof.

[0294] In some embodiments, the therapeutically effective amount is about 5 to about 60 mg. In some embodiments, a dose of about 5 to 10 mg, 10 to 15 mg, 15 to 20 mg, 20 to 25 mg, 25 to 30 mg, 30 to 35 mg, 35 to 40 mg, 40 to 45 mg, 45 to 50 mg, 50 to 55 mg, or 55 to 60 mg is administered. In some embodiments, the dose is 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, or 60 mg. In some embodiments, the dose is 5 mg. In some embodiments, the dose is 15 mg. In some embodiments, the dose is 30 mg. In some embodiments, the dose is 60 mg.

[0295] In some embodiments, the dose is administered daily. The daily dose can be delivered as a single dose or can be divided into multiple portions. For example, in some embodiments, the dose is administered once daily (e.g., about every 24 hours). In some embodiments, the dose is administered twice daily. In some embodiments, the dose is subdivided into two portions that are administered twice daily (e.g., about every 12 hours). In some embodiments, the dose is subdivided into three portions that are administered three times daily (e.g., about every 8 hours). In some embodiments, the dose is subdivided into four portions that are administered four times daily (e.g., about every 6 hours).

[0296] In some embodiments, the dose is administered orally. In some embodiments, the dose is administered in the form of a tablet. In some embodiments, the dose is administered in the form of a pill, capsule, semisolid, powder, sustained release formulation, solution, suspension, elixir, aerosol, or any other suitable composition.

[0297] In some embodiments, the subject is administered the BTK inhibitor (e.g., trebrutinib) for a period of about 4, 8, 12, 16, 20, or 48 weeks. In some embodiments, the subject is administered the BTK inhibitor (e.g., trebrutinib) for a period of about 12 weeks. In some embodiments, the subject is administered the BTK inhibitor (e.g., trebrutinib) for a period of about 48 weeks. In some embodiments, the dose is once daily.

[0298] In some embodiments, the dose is administered with a meal. In some embodiments, the dose is administered once daily with a meal. In some embodiments, a 5 mg, 15 mg, 30 mg, or 60 mg dose is administered with a meal. In some embodiments, a 5 mg, 15 mg, 30 mg, or 60 mg dose is administered once daily with a meal. In some embodiments, a 60 mg dose is administered once daily with a meal. In some embodiments, the dose is administered via oral solution or tablet. In some embodiments, the dose is administered via oral solution or tablet with a meal. In some embodiments, the dose is administered once daily via oral solution or tablet. In some embodiments, the dose is administered once daily via oral solution or tablet with a meal. In some embodiments, a 60 mg dose is administered via oral solution or tablet. In some embodiments, a 60 mg dose is administered via oral solution or tablet with a meal. In some embodiments, a 60 mg dose is administered via oral solution or tablet with a meal. In some embodiments, a 60 mg dose is administered via oral solution or tablet once daily. In some embodiments, the 60 mg dose is administered once daily with a meal in an oral solution or tablet.

[0299] Subjects with multiple sclerosis may, in some instances, have conventional T1 and / or T2 weighted (T2w) lesions (eg, as detected by magnetic resonance (MR) imaging).

[0300] In some embodiments, administration of a BTK inhibitor (e.g., trebrutinib) reduces new active brain lesions. In some embodiments, administration of a BTK inhibitor reduces new active lesions. In some embodiments, administration of a BTK inhibitor (e.g., trebrutinib) reduces new or enlarging lesions. In some instances, administration of a BTK inhibitor inhibits the formation of new active brain lesions as measured by MRI.

[0301] In some embodiments, administration of a BTK inhibitor (e.g., trebrutinib) reduces the number of new T1 lesions as measured by MRI. In some embodiments, the number of new T1 lesions is less than 1. In some embodiments, the number of new T1 lesions is equal to or less than 0.77, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. In some embodiments, no new T1 lesions form after 12 weeks of BTK inhibitor (e.g., trebrutinib) treatment.

[0302] In some embodiments, administration of a BTK inhibitor (e.g., trebrutinib) reduces the number of new or hypertrophic T2 lesions as measured by MRI. In some embodiments, the number of new or hypertrophic T2 lesions is 2 or less. In some embodiments, the number of new or hypertrophic T2 lesions is 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 or less. In some embodiments, after 12 weeks of BTK inhibitor (e.g., trebrutinib) treatment, no new or hypertrophic T2 lesions form.

[0303] In some instances, the BTK inhibitor compound is administered as a monotherapy.

[0304] In some instances, the subject is treated with one or more additional therapies. For example, the additional therapy can be an anti-CD20 antibody. The "CD20" antigen, or "CD20," is a non-glycosylated phosphoprotein of approximately 35 kDa found on the surface of more than 90% of B cells derived from peripheral blood or lymphoid organs. CD20 is present on both normal and malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include "B lymphocyte-restricted antigen" and "Bp35." The CD20 antigen is described, for example, in Clark et al. Proc. Natl. Acad. Sci. (USA) 82:1766 (1985).

[0305] In some embodiments, the anti-CD20 antibody is ocrelizumab. CDR L1 sequence: (SEQ ID NO: 1): RASSSVSYMH CDR L2 sequence (SEQ ID NO: 2): APSNLAS CDR L3 sequence (SEQ ID NO: 3): QQWSFNPPT CDR H1 sequence (SEQ ID NO: 4): GYTFTSYNMH CDR H2 sequence (SEQ ID NO: 5): AIYPGNGDTSYNQKFKG CDR H3 sequence (SEQ ID NO: 6): VVYYSNSYWYFDV

[0306] Ocrelizumab has a variable light chain sequence: [ka] and the variable heavy chain sequence: [ka] Includes.

[0307] Ocrelizumab has a light chain amino acid sequence: [ka] and the heavy chain amino acid sequence: [ka] or the heavy chain amino acid sequence: [ka] Includes.

[0308] In some examples, the anti-CD20 antibody is humanized. In some examples, the anti-CD20 is included in a pharmaceutical formulation or composition. Anti-CD20 antibodies, compositions and methods related thereto are described in U.S. Patent Application Publication No. 2022 / 0064320, which is incorporated by reference in its entirety.

[0309] In some embodiments, the amino acid K at the C-terminus of the heavy chain is removed.

[0310] The term ocrelizumab (CAS Registry Number 637334-45-3), as used herein, refers to a genetically engineered humanized monoclonal antibody against the CD20 antigen, including fragments thereof that retain the ability to bind to CD20, comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO: 9, and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 11. Ocrelizumab is available from Genentech.

[0311] In some embodiments, the anti-CD20 antibody is rituximab (CAS Registry Number 174722-31-7). Rituximab refers to a genetically engineered humanized monoclonal antibody against the CD20 antigen, including fragments thereof that retain the ability to bind to CD20, comprising (a) a light chain comprising the amino acid sequence of SEQ ID NO: 12, and (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 13.

[0312] Rituximab has a light chain amino acid sequence: [ka] and the heavy chain amino acid sequence: [ka] Includes.

[0313] In certain embodiments, methods are provided for treating multiple sclerosis in a patient, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 8 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 7. In some examples, the anti-CD20 antibody comprises a human IgG1 constant region. In some embodiments, the CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD20 antibody is ocrelizumab (CAS Registry Number 637334-45-3).

[0314] In certain embodiments, methods are provided for treating multiple sclerosis in a patient, comprising administering to the patient an effective amount of an anti-CD20 antibody, wherein the anti-CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-CD20 antibody is rituximab (CAS Registry Number 174722-31-7).

[0315] In some embodiments, the initial anti-CD20 antibody dose comprises one or more intravenous infusions (e.g., intravenous (IV) infusions) of the anti-CD20 antibody. The one or more IV infusions can be administered over a period of time (e.g., at least six months). In some embodiments, the subject receives the IV infusions at intervals of about one week to about three weeks (e.g., about one week, about two weeks, or about three weeks).

[0316] In one embodiment, the patient has not been previously treated with drugs, such as immunosuppressants, to treat multiple sclerosis, and / or has not been previously treated with antibodies against B-cell surface markers (e.g., has not been previously treated with a CD20 antibody).

[0317] In certain embodiments, the patient is premedicated prior to infusion of the anti-CD20 antibody. In certain embodiments, the patient is premedicated with methylprednisolone (or equivalent) about 30 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is premedicated with 100 mg IV methylprednisolone (or equivalent) about 30 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is additionally (or alternatively) premedicated with an antihistamine (e.g., diphenhydramine) about 30-60 minutes prior to each infusion of the anti-CD20 antibody. In certain embodiments, the patient is additionally (or alternatively) premedicated with an antipyretic (e.g., acetaminophen / paracetamol).

[0318] The CD20 antibody may be the only drug administered to a patient to treat multiple sclerosis, although optionally, a second pharmaceutical agent, e.g., a second multiple sclerosis disease-modifying drug (DMT), such as a cytotoxic agent, chemotherapeutic agent, immunosuppressant, cytokine, cytokine antagonist or antibody, growth factor, hormone, integrin, integrin antagonist or antibody (e.g., an LFA-1 antibody, or an alpha-4 integrin antibody, e.g., natalizumab (TYSABRI®) available from Biogen Idec / Elan Pharmaceuticals, Inc.), may be administered along with the antibody that binds to a B-cell surface marker (e.g., a CD20 antibody). [Example]

[0319] Example 1. Evaluation of large-scale proteome changes in cerebrospinal fluid from multiple sclerosis patients Example 1A. Background Molecular biomarkers are necessary to measure multiple sclerosis (MS) disease activity and evaluate treatment efficacy. In many cases, samples (e.g., blood samples) collected from a subject do not correlate with the biological activity occurring in the subject's brain. Therefore, proteins from cerebrospinal fluid (CSF) were examined herein to examine biomarkers for specific pathophysiology, such as neuroinflammation. It was hypothesized that such biomarkers could serve as prognostic indicators of disease progression and provide evidence of therapeutic response after treatment.

[0320] This study used platform technology (Olink® Proteomics). This study utilizes a high-throughput multiplexed immunoassay technology that allows for the measurement of over 1,000 proteins from small sample volumes. See, e.g., Assarsson E, et al. PLoS One 2014;9(4):e95192; and Cui M, et al. Lab Invest 2022, each incorporated by reference in its entirety. Two sets of subjects were analyzed. First, Olink Proteomics technology was used to profile the baseline CSF proteome of treatment-naive MS patients. Second, changes to the MS CSF proteome were assessed upon therapeutic intervention, either with a B-cell depleting agent alone or after transition to trebrutinib, a brain-penetrant Bruton's tyrosine kinase (BTK) inhibitor.

[0321] Example 1B. Baseline CSF proteome of untreated MS patients To establish a baseline of proteome expression, we performed Olink proteomics assays on CSF from multiple subject groups, including healthy volunteers (HVs), patients with HTLV-1-associated myelopathy / tropical spastic paraparesis (HAM / TSP), patients with radiologically isolated syndrome (RIS), patients with clinically isolated syndrome (CIS), and patients with multiple sclerosis (MS), including those with relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), and primary progressive MS (PPMS). CSF samples were collected from 31 healthy volunteers and 71 treatment-naive subjects with MS. The CSF proteome was examined using the Cardiometabolic, Inflammation, Neurology, and Oncology Olink Explore 384 panels (1463 analytes total). A heatmap is shown in Figure 1.

[0322] Pathway analysis was performed using Ingenuity Pathway Analysis. Data were categorized into untreated MS subjects compared to healthy volunteers (Figure 2A). Untreated MS patients showed differential abundance compared to healthy volunteers. As seen in Table 1, certain proteins were either under- or overexpressed in untreated MS subjects compared to healthy volunteers.

[0323] [Table 1]

[0324] [Table 2]

[0325] [Table 3]

[0326] As can be seen in Table 1, Olink analysis detected 64 proteins whose levels in the CSF were altered in untreated MS subjects (4 proteins showed decreased abundance in untreated MS subjects and 60 proteins showed increased abundance in untreated MS subjects).

[0327] Olink data showed that MS patients displayed unique proteins and pathways compared to healthy volunteers (Figure 2B). Pathway analysis in Figure 2B showed increases in markers of macrophage, B, and T cell activation in MS CSF. As specific examples, MZB1, CD79B, and TNFRSF13B, which showed increases from the Olink Proteomic data, are shown as individual biomarkers, demonstrating the ability to examine single proteins using these methods (see Figures 2C-E). Collectively, these data demonstrate the ability to detect proteins in CSF and show that MS subjects and healthy individuals have distinct global proteome expression in CSF.

[0328] Example 1C. Treatments involving intervention with either a B cell depleting agent alone or following transition to trebrutinib. Next, we investigated whether treatment of patients with MS with different therapies results in differential proteome expression. Groups of subjects with MS were treated with either an anti-CD20 antibody alone or an anti-CD20 antibody, then transitioned to trebrutinib, and their proteomes were examined using similar techniques as described in Example 1B. More specifically, the cohort of MS patients in this example included treatment-naïve patients, patients treated with a B-cell depleting agent for at least 6 months, and patients 12 and 48 weeks after transitioning from B-cell depleting therapy to trebrutinib in a clinical trial (NCT04742400).

[0329] Several participants (age [mean ± standard deviation]: 48.2 ± 7.9 years; gender: 2 women) who had been treated with ocrelizumab for more than 6 months (median: 3.2 years; range: 1.8-4.0 years) were included. Participants had no signs of acute focal inflammation based on magnetic resonance imaging. CSF samples were collected before baseline (within 6 months of the last ocrelizumab infusion) and at weeks 12 and 48 after transition to 60 mg trebrutinib.

[0330] The data were categorized into two sets of comparisons. The comparisons included: (1) subjects treated with anti-CD20 antibodies compared to untreated MS subjects (Figure 3), and (2) subjects treated with trebrutinib after transition from anti-CD20 antibodies compared to untreated MS subjects (Figure 4A). Compared to untreated MS patients, each group showed differential abundance. As seen in Table 2, certain proteins were either underexpressed or overexpressed in subjects treated with anti-CD20 antibodies compared to untreated MS subjects.

[0331] [Table 4]

[0332] As a specific example, FCRL1 showed an almost 3-fold decrease from the Olink Proteomic data. See Table 2.

[0333] Similar to the anti-CD20 results, data were generated for subjects treated with trebrutinib after transition from an anti-CD20 antibody compared to subjects treated with an anti-CD20 antibody. As shown in Figure 4A, MS subjects treated with trebrutinib after transition from an anti-CD20 antibody showed differential protein abundance compared to MS subjects treated with an anti-CD20 antibody.

[0334] [Table 5]

[0335] [Table 6]

[0336] [Table 7]

[0337] As seen in Table 3, 60 proteins showed a decrease in abundance 48 weeks after transition to trebrutinib, and 0 proteins showed an increase in abundance 48 weeks after transition to trebrutinib. Thirty proteins whose abundance increased in subjects with MS (compared to HV) were reversed in samples taken 48 weeks after transition to trebrutinib compared to baseline anti-CD20 levels. The CSF proteome of people with MS changed 48 weeks after transition from B cell-depleting therapy to trebrutinib, with 30 disease-associated proteins (47%) returning toward levels observed in healthy volunteers.

[0338] A principal component analysis (PCA) of the Olink Proteomics dataset, colored by treatment, is shown in Figure 4B. Six exemplary biomarkers, NEFL (Figure 4C), CXCL13 (Figure 4D), CXCL10 (Figure 4E), CD27 (Figure 4F), CCL4 (Figure 4G), and CCL3 (Figure 4H), were differentially expressed, demonstrating the ability to test single proteins using these methods. The biomarkers are examples of disease-reversing proteins 48 weeks after transitioning from anti-CD20 therapy to trebrutinib.

[0339] Example 2. Identification of CXCL13 in CSF as a biomarker for multiple sclerosis. Using the same data set as that generated in Example 1C, a cohort of subjects was examined for proteome changes.In this cohort, subjects were administered ocrelizumab for up to 6 months.Subsequently, a subset of patients were switched from ocrelizumab treatment to trebrutinib treatment for up to 48 weeks.After that, CSF was extracted and proteome analysis was performed using Olink Proteome Assay.

[0340] This assay identified CXCL13 as downregulated in subjects who switched from ocrelizumab to trebrutinib. Compared with subjects with HTLV-1-associated myelopathy / tropical spastic paraparesis or untreated subjects, subjects treated with a 12-week and 48-week course of trebrutinib showed reduced CSF CXCL13. Similarly, this reduction was observed when subjects who continued treatment with an anti-CD20 antibody (ocrelizumab) were compared with subjects treated with a 12-week and 48-week course of trebrutinib. See Figures 4D, 5, and 6. These data suggest that treatment with trebrutinib, particularly after a course of ocrelizumab, may result in reduced CXCL13 protein levels, which is associated with more favorable clinical outcomes, including active disease. Figure 6 is a separate method for measuring protein levels (MSD - Mesoscale Discovery), so we see these shifts in CXCL13 in multiple modalities.

[0341] Example 3. Identification of CXCL10 in CSF as a biomarker for multiple sclerosis. Using the same data set as that generated in Example 1C, a cohort of subjects was examined for proteome changes.In this cohort, subjects were administered ocrelizumab for up to 6 months.Subsequently, a subset of patients were switched from ocrelizumab treatment to trebrutinib treatment for up to 48 weeks.After that, CSF was extracted and proteome analysis was performed using Olink Proteome Assay.

[0342] Figures 4E and 7 herein show that CXCL10 was measured in the cerebrospinal fluid (CSF) of multiple sclerosis patients via Olink proteomics technology. In Figure 4E, the cohort of MS patients included patients treated with ocrelizumab (anti-CD20) for at least 6 months, patients transitioned from B cell-depleting therapy to trebrutinib 12 weeks later (12wk BTKi), and patients transitioned from B cell-depleting therapy to trebrutinib 48 weeks later (48wk BTKi). In Figure 7, the cohort of MS patients included patients treated with ocrelizumab for at least 6 months in a phase 2 clinical trial (NCT04742400) (MS_ocrelizumab_baseline), patients 12 weeks after transitioning from B-cell depletion therapy to trebrutinib (MS_trebrutinib_t1), and patients 48 weeks after transitioning from B-cell depletion therapy to trebrutinib (MS_trebrutinib_t2).

[0343] This assay identified CXCL10 as downregulated in subjects who switched from ocrelizumab to trebrutinib. These data suggest that treatment with trebrutinib, especially after a course of ocrelizumab, may result in decreased CXCL10 protein levels, which is associated with more favorable clinical outcomes, including active disease.

[0344] Other embodiments While the present disclosure has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, not limiting, of the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method of treating a subject with MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from said subject; (b) identifying the subject expressing CXCL13 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising:

2. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting CXCL13 in a biological sample comprising CSF from said subject; (b) identifying said subject expressing CXCL13 in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising:

3. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL13 in a first biological sample obtained from a subject at a first time point; (b) detecting CXCL13 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CXCL13 at the second time point compared to CXCL13 at the first time point as a subject with progressive MS; or (ii) identifying subjects with approximately the same or decreased CXCL13 at the second time point compared to CXCL13 at the first time point as having static or regressing MS; 1. A method for monitoring the progression of MS in a subject over time, comprising:

4. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CXCL13 in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) CXCL13 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CXCL13 in the second biological sample compared to CXCL13 in a sample obtained from an untreated patient, wherein the CXCL13 in the second biological sample is approximately the same as or reduced compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for MS in the subject; 1. A method for assessing the efficacy of a treatment of a subject with MS with a pharmaceutically effective amount of trebrutinib, comprising:

5. 1. A method of treating a subject with MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from said subject; (b) identifying the subject expressing CXCL10 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising:

6. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting CXCL10 in a biological sample comprising CSF from said subject; (b) identifying said subject expressing CXCL10 in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising:

7. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting CXCL10 in a first biological sample obtained from a subject at a first time point; (b) detecting CXCL10 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CXCL10 at the second time point compared to CXCL10 at the first time point as a subject with progressive MS; or (ii) identifying subjects with approximately the same or decreased CXCL10 at the second time point compared to CXCL10 at the first time point as having static or regressing MS; 1. A method for monitoring the progression of MS in a subject over time, comprising:

8. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CXCL10 in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) CXCL10 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CXCL10 in the second biological sample compared to CXCL10 in a sample obtained from an untreated patient, wherein the CXCL10 in the second biological sample is approximately the same as or reduced in abundance compared to the amount in the sample from the untreated patient, thereby indicating that the treatment is effective for MS in the subject; 1. A method for assessing the efficacy of a treatment of a subject with MS with a pharmaceutically effective amount of trebrutinib, comprising:

9. 1. A method of treating a subject with multiple sclerosis (MS), comprising: (a) detecting at least one biomarker in cerebrospinal fluid (CSF) of said subject; (b) administering to the subject a pharmaceutically effective amount of trebrutinib; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method of treating a subject with multiple sclerosis (MS).

10. 1. A method of treating a subject with MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from said subject; (b) identifying the subject expressing the at least one biomarker in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method of treating a subject with MS.

11. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from said subject; (b) identifying said subject expressing said at least one biomarker in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method for identifying patients as suitable for participation in a clinical trial for MS.

12. 1. A method of diagnosing a subject as having MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from said subject; (b) identifying the subject expressing the at least one biomarker in the biological sample as having MS; and Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method of diagnosing a subject as having MS.

13. 1. A method for identifying a subject with MS as expressing at least one biomarker in a biological sample comprising CSF, the method comprising: (a) detecting said at least one biomarker in said biological sample; (b) identifying the subject with MS who expresses the at least one biomarker in the biological sample; and Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method for identifying a subject with MS as expressing at least one biomarker in a biological sample, including CSF.

14. 1. A method of identifying a subject as having a high likelihood of developing MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying a subject expressing said at least one biomarker in said biological sample as having a high likelihood of developing MS; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method for identifying a subject as likely to develop MS.

15. 1. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting at least one biomarker in a biological sample comprising CSF from a subject; (b) identifying a subject expressing said at least one biomarker in said biological sample as having a high likelihood of developing MS; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method for identifying a subject who is likely to develop MS.

16. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting at least one biomarker in a first biological sample obtained from a subject at a first time point; (b) detecting the at least one biomarker in a second biological sample obtained from the subject at a second time point; (c) (i) identifying a subject as having progressive MS in which at least one biomarker is increased at the second time point compared to the at least one biomarker at the first time point; or (ii) identifying subjects as having static or regressing MS who have at least one biomarker that is about the same or decreased at the second time point compared to the at least one biomarker at the first time point; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method for monitoring the progression of MS in a subject over time.

17. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) at least one biomarker in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) the at least one biomarker in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and the at least one biomarker in the second biological sample compared to the at least one biomarker in a sample obtained from an untreated patient, wherein the at least one biomarker in the second biological sample is approximately the same or is decreased in abundance compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for treating MS in the subject; Including, the at least one biomarker is selected from CXCL13, CXCL10, CD27, NEFL, CCL4, and CCL3; A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS.

18. 1. A method of treating a subject with MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from said subject; (b) identifying the subject expressing CD27 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising:

19. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting CD27 in a biological sample comprising CSF from said subject; (b) identifying said subject expressing CD27 in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising:

20. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting CD27 in a first biological sample obtained from the subject at a first time point; (b) detecting CD27 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CD27 at the second time point compared to CD27 at the first time point as a subject with progressive MS; or (ii) identifying subjects with about the same or decreased CD27 at the second time point compared to CD27 at the first time point as having static or regressing MS; 1. A method for monitoring the progression of MS in a subject over time, comprising:

21. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CD27 in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) CD27 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CD27 in the second biological sample relative to CD27 in a sample obtained from an untreated patient, wherein the CD27 in the second biological sample is approximately the same as or reduced in abundance relative to the sample from the untreated patient, thereby indicating that the treatment is effective in treating MS in the subject; 1. A method for assessing the efficacy of a treatment of a subject with MS with a pharmaceutically effective amount of trebrutinib, comprising:

22. 1. A method of treating a subject with MS, comprising: (a) detecting NEFL in a biological sample comprising CSF from said subject; (b) identifying the subject expressing NEFL in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising:

23. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting NEFL in a biological sample comprising CSF from said subject; (b) identifying said subject as expressing NEFL in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising:

24. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting NEFL in a first biological sample obtained from a subject at a first time point; (b) detecting NEFL in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying as a subject with progressing MS a subject whose NEFL is increased at the second time point compared to the NEFL at the first time point; or (ii) identifying subjects with a NEFL that is about the same or decreased at the second time point compared to the NEFL at the first time point as having static or regressing MS; 1. A method for monitoring the progression of MS in a subject over time, comprising:

25. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) NEFL in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) NEFL in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and NEFL in the second biological sample compared to NEFL in a sample obtained from an untreated patient, wherein the NEFL in the second biological sample is approximately the same as or reduced compared to the abundance in the sample from the untreated patient, thereby indicating that the treatment is effective for MS in the subject; 1. A method for assessing the efficacy of a treatment of a subject with MS with a pharmaceutically effective amount of trebrutinib, comprising:

26. 1. A method of treating a subject with MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from said subject; (b) identifying said subject expressing CCL4 in said biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising:

27. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from said subject; (b) identifying said subject expressing CCL4 in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising:

28. 1. A method for identifying a subject who is likely to develop MS, comprising: (a) detecting CCL4 in a biological sample comprising CSF from a subject; (b) identifying a subject expressing CCL4 in said biological sample as having a high likelihood of developing MS; 1. A method for identifying a subject who is likely to develop MS, comprising:

29. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting CCL4 in a first biological sample obtained from a subject at a first time point; (b) detecting CCL4 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CCL4 at the second time point compared to CCL4 at the first time point as a subject with progressive MS; or (ii) identifying subjects with about the same or decreased CCL4 at the second time point compared to CCL4 at the first time point as having static or regressing MS; 1. A method for monitoring the progression of MS in a subject over time, comprising:

30. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CCL4 in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) CCL4 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CCL4 in the second biological sample compared to CCL4 in a sample obtained from an untreated patient, wherein the CCL4 in the second biological sample is approximately the same as or reduced in abundance compared to the sample from the untreated patient, thereby indicating that the treatment is effective for MS in the subject; 1. A method for assessing the efficacy of a treatment of a subject with MS with a pharmaceutically effective amount of trebrutinib, comprising:

31. 1. A method of treating a subject with MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from said subject; (b) identifying the subject expressing CCL3 in the biological sample as having MS; and (c) administering a pharmaceutically effective amount of trebrutinib to the subject; 1. A method of treating a subject with MS, comprising:

32. 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising: (a) detecting CCL3 in a biological sample comprising CSF from said subject; (b) identifying said subject expressing CCL3 in said biological sample, thereby identifying said patient as suitable for participation in a clinical trial for MS; 1. A method of identifying a patient as suitable for participation in a clinical trial for MS, comprising:

33. 1. A method for monitoring the progression of MS in a subject over time, comprising: (a) detecting CCL3 in a first biological sample obtained from a subject at a first time point; (b) detecting CCL3 in a second biological sample obtained from the subject at a second time point; and (c) (i) identifying a subject with increased CCL3 at the second time point compared to CCL3 at the first time point as a subject with progressive MS; or (ii) identifying subjects with about the same or decreased CCL3 at the second time point compared to CCL3 at the first time point as having static or regressing MS; 1. A method for monitoring the progression of MS in a subject over time, comprising:

34. 1. A method for assessing the therapeutic efficacy of a pharmaceutically effective amount of trebrutinib in a subject with MS, comprising: (a) detecting (i) CCL3 in a first biological sample comprising CSF obtained from the subject at a first time point, and (ii) CCL3 in a second biological sample comprising CSF obtained from the subject at a second time point, wherein the subject has been administered one or more doses of the treatment between the first and second time points; (b) determining a correlation between the effectiveness of the treatment and CCL3 in the second biological sample relative to CCL3 in a sample obtained from an untreated patient, wherein the CCL3 in the second biological sample is approximately the same as or reduced in abundance relative to the sample from the untreated patient, thereby indicating that the treatment is effective for MS in the subject; 1. A method for assessing the efficacy of a treatment of a subject with MS with a pharmaceutically effective amount of trebrutinib, comprising:

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