WO2015063604A2

WO2015063604A2 - BIOMARKERS PREDICTIVE OF THERAPEUTIC RESPONSIVENESS TO IFNβ AND USES THEREOF

Info

Publication number: WO2015063604A2
Application number: PCT/IB2014/002979
Authority: WO
Inventors: Luisa IMBERTI; Federico SERANA; Ruggero CAPRA
Original assignee: Imberti Luisa; Serana Federico; Capra Ruggero
Priority date: 2013-11-01
Filing date: 2014-10-31
Publication date: 2015-05-07
Also published as: WO2015063604A3

Abstract

Methods, assays and kits for the identification, assessment and/or treatment of a subject having multiple sclerosis (MS) are disclosed.

Description

BIOMARKERS PREDICTIVE OF THERAPEUTIC

RESPONSIVENESS TO ΙΕΝβ AND USES THEREOF

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/899,126, filed

November 1, 2013, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is an inflammatory disease of the brain and spinal cord characterized by recurrent foci of inflammation that lead to destruction of the myelin sheath. In many areas, nerve fibers are also damaged. Inflammatory activity in MS patients tends to be highest in the initial phase of disease.

Emerging data demonstrate that irreversible axonal loss occurs early in the course of MS. Transected axons fail to regenerate in the central nervous system (CNS). Therefore, early treatment aimed at suppressing MS lesion formation is of significant importance. As early as disease onset, axons are transected in lesions with active inflammation (Trapp et al., (1998) N Engl J Med 338: 278-285; Bjartmar et al., (2001) Curr Opin Neurol 14: 271-278; Ferguson et al., (1997) Brain 120: 393-399). The degree of demyelination is related to the degree of

inflammation and the exposure of demyelinated axons to the inflammatory environment, as well as non-inflammatory mediators (Trapp et al., (1998) N Engl J Med 338: 278-285; Kornek et al., (2000) Am J Pathol 157: 267-276; Bitsch et al., (2000) Brain 123: 1174-1183). There is also destruction of oligodendrocytes with impaired remyelination in demyelinating lesions (Peterson et al., (2002) J Neuropathol Exp Neurol 61: 539-546; Chang et al., (2002) N Engl J Med 346: 165-173). The loss of oligodendrocytes leads to a reduction in the capacity to re-myelinate and may result in the loss of trophic factors that support neurons and axons (Bjartmar et al., (1999) J Neurocytol 28: 383-395).

Given the destructive effects of inflammatory MS lesions, the need exists for identifying and/or assessing a patient or patient population having multiple sclerosis that would benefit from treatment with an interferon-β (IFN-β) agent in the course of disease, or identifying a patient or patient population as responding or not responding to an IFN-β agent. SUMMARY OF THE INVENTION

The present invention provides, at least in part, methods, assays and kits for the identification, assessment and/or treatment of a subject having multiple sclerosis (MS). In one embodiment, responsiveness of a subject to an interferon beta agent (referred to interchangeably herein as an "IFN-β," "IFN-b," "ΙΡΝβ," or "IFNb," agent), e.g., an IFN-β la molecule or an IFN-β lb molecule, is predicted by evaluating an alteration (e.g., an increased or decreased level) of an MS biomarker in a sample, e.g., a serum sample obtained from an MS patient. In certain embodiments, the MS biomarker evaluated is Myxovirus protein-A (MxA), e.g., a change in the level of expression of MxA. In one embodiment, an increase in the level of MxA mRNA (e.g., an increase in MxA mRNA induction after administration of an interferon-b agent of > 16 relative units, e.g., relative to a healthy sample) is indicative of an improved outcome, e.g., reduced probability of disability progression. In one embodiment, the subject is an MS patient who has experienced a relapse (e.g., a severe or mild relapse), also referred to herein as "a subject with relapsing MS." Thus, the invention can, therefore, be used, for example: to evaluate responsiveness to, or monitor, a therapy or treatment that includes an IFN-b agent; identify a patient as likely to benefit from a therapy or treatment that includes an IFN-b agent; stratify patient populations (e.g., stratify patients as being likely or unlikely to respond (e.g., responders or disease non-progressors vs. non-responders or disease progressors) to a therapy or treatment that includes an IFN-b agent; and/or more effectively monitor, treat multiple sclerosis, or prevent progression of disease and/or relapse. In one embodiment, the invention can be used to evaluate the risk of disease progression in a subject with MS who has experienced a relapse (e.g., a severe or mild relapse). Accordingly, in one aspect, the invention features a method of, or assay for, evaluating a sample, e.g., a sample from an MS subject (e.g., a subject with relapsing MS). The method includes detecting an alteration (e.g., an increased or decreased level) of an MS biomarker in the sample. In one embodiment, the MS biomarker evaluated includes MxA. In other embodiments, the MS biomarker includes an antibody to the IFN-b agent, e.g., a binding antibody (BAb) and/or a neutralizing antibody (NAb).

The method, or assay, can further include one or more of the following: (i) identifying a subject (e.g., a patient, a patient group or population), having MS (e.g., a subject with relapsing MS), or at risk of developing MS, as having an increased or a decreased likelihood to respond to an MS treatment (or an MS therapy, as used interchangeably herein), e.g. , identifying a subject as a responder or a non-responder to the MS treatment, or as a disease non-progressors vs. disease progressors;

(ii) determining a treatment regimen upon evaluation of the sample (e.g., selecting, or altering the course of, a therapy or treatment, a dose, a treatment schedule or time course, and/or the use of an alternative MS therapy, e.g., altering a therapy from a first-line of MS therapy to a second- line of MS therapy);

(iii) analyzing MS disease progression in the subject; and/or

(iv) treating the subject (e.g., administering an MS therapy (e.g., a first-line or a second- line MS therapy) to the subject).

In one embodiment, the MS therapy (e.g., the first-line MS therapy) includes a treatment with an IFN-b agent. In one embodiment, the second-line MS therapy is chosen from one or more of: an antibody or fragment thereof against alpha-4 integrin (e.g., natalizumab

(TYSABRI®)); a fingolimod (e.g., FTY720; GILENYA®); a dimethyl fumarate (e.g., an oral dimethyl fumarate (TECFIDERA®)); or an antibody against CD52 (e.g., alemtuzumab

(LEMTRADA®)).

In one embodiment, one or more of (i)-(iv) are determined in response to the detection of the alteration. An alteration (e.g., a change, e.g., an increased or a decreased level) in the sample in one or more of the aforesaid MS biomarkers relative to a specified parameter (e.g., a reference value or sample; a sample obtained from a healthy subject; or a sample obtained from the subject at a different time interval, e.g., prior to, during, or after treatment), indicates one or more of: an increased or decreased responsiveness of the subject to the IFN-b agent; identifies the subject as having an increased or decreased likelihood to respond to the treatment with the IFN-b agent; determines the treatment to be used; and/or analyzes or predicts the progression of the MS disease.

In one embodiment, the alteration or change is detected at a single-time point, e.g., prior to an MS therapy; at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve months after initiation of an MS therapy. In other embodiments, the alteration or change is detected at predetermined time intervals, e.g., every 6, 12, 24, 36 or more months after initiation of an MS therapy. In yet other embodiments, an average value of the change or alteration is acquired, e.g., an average value acquired over 6 to 24- month period.

In one embodiment, an increase in the level in MxA, e.g., an increase in the induction of MxA mRNA, in response to an IFN-b agent, in a subject with MS who has experienced a relapse (e.g., a severe or mild relapse) is indicative of an improved outcome, e.g., reduced probability of disability progression. In one embodiment, the increase in MxA (induced MxA mRNA in response to the IFN-b agent) is from about 10 to 20- fold (e.g., at least 10, 12, 14, 16, 18 or 20) relative to reference value (e.g., a healthy control). In one embodiment, the increase in MxA is indicative of a lower risk of an increase in EDSS score (e.g., a lower risk of a 1 -point EDSS increase). In another embodiment, the increase in MxA (induced MxA mRNA in response to the IFN-b agent) is indicative of a reduced probability of disease progression (e.g., a progression similar to a relapse-free patient). Alternatively, either no response or a lower increase in the level of MxA in response to the IFN-b agent (e.g., an increase of less than 10-fold) relative to a reference value (e.g., a healthy control) is indicative of an increased probability of disability progression in the subject, e.g., a subject with MS who has experienced a relapse (e.g., a severe or mild relapse).

In another aspect, the invention features a method of, or assay for, identifying a subject

(e.g., a patient, a patient group or population), having MS (e.g., a subject having relapsing MS), or at risk for developing MS, as having an increased or decreased likelihood to respond to an MS treatment, e.g. , an MS treatment with an IFN-b agent; or as being a disease progressor or non- progressor. The method includes:

acquiring a value (e.g., obtaining possession of, determining, detecting, or evaluating, the level) of an MS biomarker in a subject (e.g., a sample from the subject), and

responsive to said value, identifying the subject having MS, or at risk for developing MS, as being likely or less likely to respond to an IFN-b agent; or as a disease progressor or non- progressor.

In one embodiment, the MS biomarker evaluated includes MxA, and optionally, one or more of: a binding antibody (BAb) or a neutralizing antibody (NAb).

An alteration (e.g., a change, e.g., an increased or a decreased level) in one or more of the aforesaid MS biomarkers relative to a specified parameter (e.g., a reference value or sample; a sample obtained from a healthy subject; or a sample obtained from the subject at a different time interval, e.g., prior to, during, or after treatment), indicates one or more of: an increased or decreased responsiveness of the subject to the IFN-b agent; identifies the subject as having an increased or decreased likelihood to respond to the treatment with the IFN-b agent; or as being a disease progressor or non-progressor.

In one embodiment, an increase in the level in MxA, e.g., an increase in the induction of MxA mRNA, in response to an IFN-b agent, in a subject with MS who has experienced a relapse (e.g., a severe or mild relapse) is indicative of an improved outcome, e.g., reduced probability of disability progression. In one embodiment, the increase in MxA (induced MxA mRNA in response to the IFN-b agent) is from about 10 to 20- fold (e.g., at least 10, 12, 14, 16, 18 or 20) relative to reference value (e.g., a healthy control). In one embodiment, the increase in MxA is indicative of a lower risk of an increase in EDSS score (e.g., a lower risk of a 1 -point EDSS increase). In another embodiment, the increase in MxA (induced MxA mRNA in response to the IFN-b agent) is indicative of a reduced probability of disease progression (e.g., a progression similar to a relapse-free patient). Alternatively, either no response or a lower increase in the level of MxA in response to the IFN-b agent (e.g., an increase of less than 10-fold) relative to a reference value (e.g., a healthy control) is indicative of an increased probability of disability progression in the subject, e.g., a subject with MS who has experienced a relapse (e.g., a severe or mild relap se) .

In another aspect, the invention features a method of, or assay for, evaluating or monitoring a treatment (e.g., an MS treatment, e.g. , an MS treatment with an IFN-b agent) in a subject (e.g., a patient, a patient group or population), having MS (e.g., a subject with relapsing MS), or at risk for developing MS. The method includes: acquiring a value (e.g., obtaining possession of, determining, detecting, or evaluating, the level) of an MS biomarker in a subject (e.g., a sample from the subject); and

(optionally) responsive to said value, treating, selecting and/or altering one or more of the course of the MS treatment, the dosing of the MS treatment, the schedule or time course of the MS treatment, or administration of a second, alternative MS therapy.

In one embodiment, the method includes comparing the value of the MS biomarker to a specified parameter (e.g., a reference value or sample; a sample obtained from a healthy subject; or a sample obtained from the subject at a different time interval, e.g., prior to, during, or after treatment). The method can be used, e.g., to evaluate the suitability of, or to choose between alternative treatments, e.g., a particular dosage, mode of delivery, time of delivery, or generally to determine the subject's probable drug response.

In yet another aspect, the invention features a method of, or assay for, evaluating a subject' s prognosis or MS disease progression, in a subject (e.g., a patient, a patient group or population), having MS (e.g., a subject with relapsing MS), or at risk for developing MS. The method includes:

acquiring a value (e.g., obtaining possession of, determining, detecting, or evaluating, the level) of an MS biomarker in a subject (e.g., a sample from the subject); and

(optionally) comparing the value of the MS biomarker to a specified parameter (e.g., a reference value or sample; a sample obtained from a healthy subject; or a sample obtained from the subject at different time intervals, e.g., prior to, during, or after treatment, e.g. , an MS treatment, e.g. , an MS treatment with an IFN-b agent).

An alteration (e.g., a change, e.g., an increased or a decreased level) in one or more of the aforesaid MS biomarkers relative to a specified parameter indicates one or more of: an increased or decreased responsiveness of the subject to the IFN-b agent; identifies the subject as having an increased or decreased likelihood to respond to the treatment with the IFN-b agent; or as being a disease progressor or non-progressor. In one embodiment, the alteration or change is detected at a single-time point, e.g., prior to an MS therapy; at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve months after initiation of an MS therapy. In other embodiments, the alteration or change is detected at predetermined time intervals, e.g., every 6, 12, 24, 36 or more months after initiation of an MS therapy. In yet other embodiments, an average value of the change or alteration is acquired, e.g., an average value acquired over 6 to 24- month period.

Treatment

In other embodiments, any of the aforesaid methods further include treating, or preventing in, a subject having MS one or more symptoms associated with MS. In certain embodiments, the treatment includes reducing, retarding or preventing, a relapse, or the progression of a disability, in the MS subject. In one embodiment, the method includes, responsive to an MS biomarker value (e.g., an MS biomarker value obtained as described herein), administering to the subject (e.g. , a patient with relap sing-remitting multiple sclerosis (RRMS)) a therapy for MS (also referred to herein as an "MS therapy"), e.g. , an MS therapy with an IFN-b agent, in an amount sufficient to reduce one or more symptoms associated with MS. Alternatively, or in combination with the methods described herein, the invention features a method of treating, or preventing in, MS (e.g., one or more symptoms associated with MS), in a subject having MS (e.g., a subject with MS who has experienced a relapse (e.g., a severe or mild relapse)), or at risk for developing MS. In one embodiment, the subject is identified as having an increased or decreased probability of showing disability progression, using the methods, or assays, described herein.

In certain embodiments, the method includes:

acquiring a value (e.g., obtaining possession of, determining, detecting, or evaluating the level) of an MS biomarker chosen from MxA, and optionally, one or more of: a binding antibody (BAb) or a neutralizing antibody (NAb), in a subject;

responsive to said value, administering to a subject (e.g. , a patient with relapsing MS, e.g., relapsing remitting multiple sclerosis (RRMS)) a therapy for MS (also referred to herein as an "MS therapy"), e.g. , an MS therapy with an IFN-b agent, in an amount sufficient to reduce one or more symptoms associated with MS.

In certain embodiments, the method of treatment includes an MS therapy (e.g., a first-line MS therapy), e.g. , an MS therapy that includes an ΙΡΝβ agent (e.g., an IFN-β l a molecule or an IFN-β lb molecule, including analogues and derivatives thereof (e.g., pegylated variants thereof)). In one embodiment, the MS therapy includes an IFN-β la agent (e.g., AVONEX®, REBIF®). In another embodiment, the MS therapy includes an INF-β lb agent (e.g.,

BETASERON®, BETAFERON®). In another embodiment, the MS therapy is an alternative therapy (e.g., a therapy selected when a patient is non-responsive to an INF-β therapy).

In one embodiment, the MS therapy is an alternative therapy to the IFN-β agent, e.g., a second-line MS therapy. In one embodiment, the second-line MS therapy is chosen from one or more of: an antibody or fragment thereof against alpha-4 integrin (e.g., natalizumab

(TYSABRI®)); a fingolimod (e.g., FTY720; GILENYA®) or a S IP-agonist; a dimethyl fumarate (e.g., an oral dimethyl fumarate (TECFIDERA®)); or an antibody against CD52 (e.g., alemtuzumab (LEMTRADA®)).

In certain embodiments, the alternative therapy to the IFN-β agent includes a polymer of four amino acids found in myelin basic protein, e.g., a polymer of glutamic acid, lysine, alanine and tyrosine (e.g., glatiramer (COPAXONE®)). In other embodiments, the alternative therapy includes an antibody or fragment thereof against alpha-4 integrin (e.g., natalizumab (TYSABRI®). In yet other embodiments, the alternative therapy includes an anthracenedione molecule (e.g., mitoxantrone (NOVANTRONE®)). In yet another embodiment, the alternative therapy includes a fingolimod (e.g., FTY720; GILENYA®). In one embodiment, the alternative therapy is a dimethyl fumarate (e.g., an oral dimethyl fumarate (TECFIDERA®)). In other embodiments, the alternative therapy is an antibody to the alpha subunit of the IL-2 receptor of T cells (e.g., Daclizumab). In yet other embodiments, the alternative therapy is an antibody against CD52 (e.g., alemtuzumab (LEMTRADA®)).

In certain embodiments, the method further includes the use of one or more symptom management therapies, such as antidepressants, analgesics, anti-tremor agents, among others.

Additional embodiments or features are as follows:

Subjects

For any of the methods or assays disclosed herein, the subject treated, or the subject from which the sample is obtained, is a subject having, or at risk of having MS at any stage of treatment. In certain embodiment, the subject has experienced one or more relapses (e.g., mild or severe relapses). In one embodiment, the subject has a negative status for an interferon antibody or a neutralizing antibody. In one embodiment, the subject is NAb-BAb-. In other embodiments, the subject has a positive status for an interferon antibody or a neutralizing antibody, or both (e.g., NAb+BAb-; NAb-BAb+, or NAb+BAb+).

In other embodiments, the MS patient is chosen from a patient having one or more of: Benign MS, RRMS (e.g., quiescent RRMS, active RRMS), primary progressive MS, or secondary progressive MS. In other embodiments, the subject has MS-like symptoms, such as those having clinically isolated syndrome (CIS) or clinically defined MS (CDMS). In one embodiment, the subject is an MS patient (e.g. , a patient with RRMS) after administration of an MS therapy described herein (e.g., after administration of an IFN-b agent).

In one embodiment, the subject has a relapsing form of MS (e.g., RRMS or relapsing SPMS). In one embodiment, the subject has RRMS and has one or more ongoing clinical exacerbations and/or subclinical activity, e.g., as shown by gadolinium (Gd) enhancement or development of new and/or enlarged T2/FLAIR lesions on magnetic resonance imaging (e.g., brain or spinal cord MRI). In another embodiment, the subject has SPMS and has one or more ongoing clinical exacerbations and/or subclinical activity, e.g., as shown by gadolinium (Gd) enhancement or development of new and/or enlarged T2/FLAIR lesions on magnetic resonance imaging (e.g., brain or spinal cord MRI). In one embodiment, the subject has an active form of MS, e.g., an active RRMS. In other embodiments, the MS subject has at least one newly developed lesion. In other embodiment, the MS subject has at least one pre-existing lesion. In one embodiment, the subject has RRMS, and has one or more newly developed or pre-existing lesions, or a combination thereof. In other embodiments, the subject has a baseline EDSS score of 1.5 to 7.

Evaluation Intervals

In other embodiments, the subject is an MS patient after administration of the MS therapy for one, two weeks, one month, two months, three months, four months, six months, one year or more.

In one embodiment, the alteration or change is detected at a single-time point, e.g., prior to an MS therapy; at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve months after initiation of an MS therapy. In other embodiments, the alteration or change is detected at predetermined time intervals, e.g, every 6, 12, 24, 36 or more months after initiation of an MS therapy. In yet other embodiments, an average value of the change or alteration is acquired, e.g., an average value acquired over 6 to 24- month period.

The MxA level can be evaluated at one or more intervals post-injection of the IFN-b agent. In one embodiment, the MxA level is evaluated 9 to 15 hours, or 10- 12 hours, after injection of the IFN-b agent, e.g., post-injection of IFN-β 1 agent chosen from AVONEX®, REBIF®, BETASERON®, or BETAFERON®. In other embodiments, the MxA level is evaluated 10 to 60 hours, or 12 to 48 hours post-injection of a pegylated form of the IFN-β 1 agent.

In one embodiment, the method or assay includes comparing the value (e.g., level) of one or more MS biomarkers to a specified parameter (e.g., a reference value or sample; a sample obtained from a healthy subject; a sample obtained from a patient at different treatment intervals). For example, a sample can be analyzed at any stage of treatment, but preferably, prior to, during, or after terminating, administration of the MS therapy, to thereby determine appropriate dosage(s) and treatment regimen(s) of the MS therapy (e.g., amount per treatment or frequency of treatments) for prophylactic or therapeutic treatment of the subject. In certain embodiments, the methods, or assays, of the invention include the step of detecting the level of one or more MS biomarkers in the subject, prior to, or after, administering the MS therapy, to the subject.

Evaluation Markers

In certain embodiments, the method, or assay, further includes the step of obtaining the sample, e.g., a biological sample, from the subject. In one embodiment, the method, or assay, includes the step of obtaining a predominantly non-cellular fraction of a body fluid from the subject. The non-cellular fraction can be plasma, serum, or other non-cellular body fluid. In one embodiment, the sample is a serum sample.

In certain embodiments, the detection or determining steps of the methods or assays described herein include determining quantitatively the value (e.g., level) (e.g., amount or concentration) of an MS biomarker (e.g., one or more of the MS biomarkers described herein) from a sample, e.g., a sample of plasma, serum, or other non-cellular body fluid, wherein the amount or concentration of the MS biomarker, thereby provides a value (also referred to herein as a "determined," or "detected," "value"). In certain embodiments, the determined or detected value is compared to a specified parameter (e.g., a reference value; a control sample; a sample obtained from a healthy subject; a sample obtained from the subject at different time intervals, e.g., prior to, during, or after treatment, or a pre-determined value). In alternative embodiments, the sample is assayed for qualitative, or both quantitative and qualitative determination of the MS biomarker level. In certain embodiments, methods or assays of the invention relate to determining quantitatively the amount or concentration of the MS biomarker from plasma or serum of the subject, wherein the plasma or serum is obtained from the blood of the subject, for example.

In certain embodiments, the MS biomarker evaluated is a gene or gene product, e.g., cDNA, RNA (e.g., mRNA), or a polypeptide. In other embodiments where the MS biomarker is a nucleic acid, the nucleic acid can be detected, or the level determined, by any means of nucleic acid detection, or detection of the expression level of the nucleic acids, including but not limited to, nucleic acid hybridization assay, amplification-based assays (e.g., polymerase chain reaction), or sequencing. In one embodiment, the MS biomarker is evaluated using a real-time PCR assay.

In yet another embodiment, the one or more MS biomarkers are assessed at predetermined intervals, e.g., a first point in time and at least at a subsequent point in time. In one embodiment, a time course is measured by determining the time between significant events in the course of a patient's disease, wherein the measurement is predictive of whether a patient has a long time course. In another embodiment, the significant event is the progression from primary diagnosis to death. In another embodiment, the significant event is the progression from primary diagnosis to disease progression. In another embodiment, the significant event is the progression from primary diagnosis to relapse. In another embodiment, the significant event is the progression from secondary MS to death. In another embodiment, the significant event is the progression from remission to relapse. In another embodiment, the significant event is the progression from relapse to death. In certain embodiments, the time course is measured with respect to one or more overall survival rate, time to progression and/or using the EDSS or other assessment criteria. The method or assays disclosed herein can further include one or more steps of:

performing a neurological examination, evaluating the subject' s status on the Expanded Disability Status Scale (EDSS), or detecting the subject's lesion status (e.g., as assessed using an MRI).

In other embodiments, the methods, assays, and/or kits described herein further include providing or generating, and/or transmitting information, e.g. , a report, containing data of the evaluation or treatment determined by the methods, assays, and/or kits as described herein. The information can be transmitted to a report-receiving party or entity (e.g., a patient, a health care provider, a diagnostic provider, and/or a regulatory agency, e.g., the FDA), or otherwise submitting information about the methods, assays and kits disclosed herein to another party. The method can relate to compliance with a regulatory requirement, e.g., a pre- or post approval requirement of a regulatory agency, e.g., the FDA. In one embodiment, the report-receiving party or entity can determine if a predetermined requirement or reference value is met by the data, and, optionally, a response from the report-receiving entity or party is received, e.g., by a physician, patient, diagnostic provider.

In another aspect, the invention features kits for evaluating a sample, e.g., a sample from an MS patient, to detect or determine the level of one or more MS biomarkers.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A is a schematic depicting exemplary longitudinal analysis of participation of patients treated with IFNP, including exemplary times and/or reasons for exclusion from the study. Patients' disposition and dropouts during the 3-year study period are shown. Reported as adverse events are the following (comprising both drug-related and drug-independent clinical events): injection site reaction (1), thyroiditis (1), liver enzyme elevation (4), leucopenia (1), kidney stones (1), migraine (1), pregnancy (1), breast nodule (1), and breast cancer (1).

Figure IB is a table showing dropout distribution, a: see Figure 1A for more details, b: early dropouts were excluded from the analysis because only one post-therapy MxA

determination was available. T12, T24, T36: 12, 24, 36 months post-therapy initiation.

Figure 1C is a table showing baseline characteristics of the 100 patients analyzed.

*p<0.01 at the Kruskall-Wallis test. **p<0.05 at the Kruskall-Wallis test.

Figures 2A-2C depict exemplary levels of MxA (relative units, Figure 2A), BAb (% activity, Figure 2B) and NAb (titer, Figure 2C) in patients treated with IFNP over a 36 month time period. MxA values (Figure 2A) in patients treated with IFNP-la i.m. (clear circles, dashed grey line), IFNP-la s.c. (clear squares, solid grey line), IFNP-lb s.c. (clear diamonds, solid black line). Average kinetics of BAb production in all samples (Figure 2B) and of NAb (Figure 2C) production in selected samples in patients treated with IFNP-la i.m. (dashed grey line), IFNP-la s.c. (solid grey line), IFNP-lb s.c. (solid black line). Lines connect predicted means. Arrows indicate the cut-offs. For BAbs, 95% confidence intervals are shown; for NAbs, standard deviations are shown because confidence intervals were not calculated (no statistical inference was performed due to selection bias). MxA: myxovirus-resistance protein A; NR: normalization ratio; BAbs: binding antibodies; NAbs: neutralizing antibodies; ΙΡΝβ: interferon beta.

Figure 3 depicts exemplary BAb levels (% total activity) at TO in patients that were naive for therapy with IFNP as compared to healthy donors. BAb: binding antibodies.

Figure 4 depicts exemplary levels of log BAb and MxA (relative units) in patients treated with ΙΡΝβ-la i.m., ΙΡΝβ-la s.c. 44μg, or ΙΡΝβ-lb s.c. MxA and anti-ΙΡΝβ antibodies in all samples of all patients. The lines indicate the cut-offs. MxA: myxovirus-resistance protein A; NR: normalization ratio; BAbs: binding antibodies; NAbs: neutralizing antibodies.

Figures 5A-5B depict exemplary quantitation of mRNA expression of IFNAR2.2 over time in patients treated with ΙΡΝβ-la i.m., ΙΡΝβ-la s.c. 44μg, or ΙΡΝβ-lb (Figure 5A); and an exemplary multivariate regression model performed after correcting for the presence of anti- ΙΡΝβ antibodies and type of ΙΡΝβ received, to show average MxA mRNA levels (Figure 5B). Average decrease of IFNAR2.2 mRNA (Figure 5A) in patients treated with ΙΡΝβ-la i.m. (clear circles, dashed grey line), ΙΡΝβ-la s.c. (clear squares, solid grey line), ΙΡΝβ-lb s.c. (clear diamonds, solid black line). Lines connect predicted means; 95% confidence intervals are shown. Average change in MxA mRNA induction over time in dependence of different levels of expression of IFNAR2.3 mRNA (Figure 5B), as determined by multivariable mixed-model regression. IFNAR: interferon receptor; IFNP: interferon beta; MxA: myxovirus-resistance protein A; NR: normalization ratio.

Figure 5C is a table showing exemplary characteristics of the patients and classification according to IFNP-bioactivity profiles. IFNP: interferon beta; MxA: myxovirus-resistence protein A; BAb: binding antibodies; NAb: neutralizing antibodies.

Figure 5D is a table showing exemplary markers of disease activity/progression in the patients with the different IFNP-bioactivity profiles, a: excluding the group of patients with intermediate features; BAb: binding antibodies; Abs: antibodies; ARR: annualized replapse rate; AEDSS: variation in Expanded Disability Status Scale.

Figures 6A-6C depict exemplary effects of MxA on the probability of EDSS increase at various timepoints. Figure 6A depicts an exemplary comparison of MxA values obtained across multiple observations in patients with or without disability progression. Figure 6B depicts exemplary area under the curve (AUC) analysis for MxA in patients at T24 months in patients with a change in EDSS score of less than one or an EDSS score of greater than one. Figure 6C depicts exemplary risk of change in EDSS score at T24 months in patients as a function of MxA (relative units) in patients without relapses in 24 months or in patients with at least one relapse in 24 months. Comparison of the log₂MxA values (Figure 6A) and of the area under the curve for non-transformed MxA mRNA levels calculated over the first two years of treatment (Figure 6B) between patients with or without at least 1 -point EDSS increase in the same period. Predicted probability of 1 -point EDSS increase after 2 years of treatment in patients with at least one relapse (filled circles) vs. relapse-free patients (clear circles) in the same period (Figure 6C). In (Figure 6A) 95% confidence intervals are shown, while the shown p-value refers to the main effect of the AN OVA factor "1 -point EDSS increase" (the interaction with the "time-point" factor was non- significant). In (Figure 6B) the median, interquartile range, and range are shown as box-and-whisker plot. AUC: area under the curve; MxA: myxovirus-resistance protein A; NR: normalization ratio; EDSS: Expanded Disability Status Scale.

Figure 7 is a table showing an exemplary logistic model for EDSS increase.

Figures 8A depicts exemplary analysis of IFNP-bioactive patients who were MxA+,

BAb-, and NAb- at all time-points. Figure 8B depicts exemplary analysis of patients who displayed sporadic (non-consecutive) MxA- samples without antibodies and were considered bona fide bioactive patients. Figure 8C depicts exemplary analysis of IFNP-treated patients who were BAb+ at several timepoints, but had conserved MxA induction. Figure 8D depicts

exemplary analysis of IFNP-non-bioactive patients who were MxA- and BAb+ (and Nab+ if

performed) in at least three consecutive timepoints.

Figure 9 depicts exemplary analysis of the effect of IFNAR2.3 on MxA.

Figure 10 depicts exemplary analysis of the effect of MxA on the probability of 24- month EDSS increase. DETAILED DESCRIPTION OF THE INVENTION

Methods, assays and kits for the identification, assessment and/or treatment of a subject having multiple sclerosis (MS) (e.g. , a patient with relapsing MS)) are disclosed. In one

embodiment, responsiveness of a subject to an interferon beta ("IFN-β" or "IFN-b") agent (e.g., an IFN-β l a molecule or an IFN-β lb molecule) is determined by evaluating an alteration (e.g., an increased or decreased level) of an MS biomarker in a sample, e.g., a serum sample obtained from an MS patient. In certain embodiments, the MS biomarker evaluated is MxA.

Various aspects of the invention are described in further detail in the following

subsections. Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

"Acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity (e.g., a sample, a polypeptide, a nucleic acid, or a sequence), or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following:

separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent.

The term "altered level of expression" of a biomarker as described herein (e.g., MxA) refers to an increase (or decrease) in the expression level of a marker in a test sample, such as a sample derived from a patient suffering from multiple sclerosis or a similar disorder (e.g., clinically isolated syndrome (CIS), benign MS), that is greater or less than the standard error of the assay employed to assess expression. In embodiments, the alteration can be at least twice, at least twice three, at least twice four, at least twice five, or at least twice ten or more times greater than or less than the expression level of the biomarkers in a control sample (e.g., a sample from a healthy subject not having the associated disease), or the average expression level in several control samples. An "altered level of expression" can be determined at the protein or nucleic acid (e.g., mRNA) level.

"Binding compound" shall refer to a binding composition, such as a small molecule, an antibody, a peptide, a peptide or non-peptide ligand, a protein, an oligonucleotide, an oligonucleotide analog, such as a peptide nucleic acid, a lectin, or any other molecular entity that is capable of specifically binding to a target protein or molecule or stable complex formation with an analyte of interest, such as a complex of proteins.

"Binding moiety" means any molecule to which molecular tags can be directly or indirectly attached that is capable of specifically binding to an analyte. Binding moieties include, but are not limited to, antibodies, antibody binding compositions, peptides, proteins, nucleic acids and organic molecules having a molecular weight of up to about 1000 daltons and containing atoms selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur and phosphorus.

A "biomarker" or "marker" is a gene, mRNA, or protein that undergoes alterations in expression that are associated with multiple sclerosis or responsiveness to treatment with IFN-β. The alteration can be in amount and/or activity in a biological sample (e.g., a blood, plasma, or a serum sample) obtained from a subject having multiple sclerosis, as compared to its amount and/or activity, in a biological sample obtained from a healthy subject (e.g., a control); such alterations in expression and/or activity are associated with a disease state, such as multiple sclerosis. For example, a marker of the invention which is associated with multiple sclerosis or predictive of responsiveness to IFN-β therapeutics can have an altered expression level, protein level, or protein activity, in a biological sample obtained from a subject having, or suspected of having, multiple sclerosis as compared to a biological sample obtained from a control subject (e.g., a healthy individual).

A "nucleic acid" "marker" or "biomarker" is a nucleic acid (e.g. , DNA, mRNA, cDNA) encoded by or corresponding to a marker as described herein. For example, such marker nucleic acid molecules include DNA (e.g. , genomic DNA and cDNA) comprising the entire or a partial sequence of any of the nucleic acid sequences set forth herein (e.g., in Table 1), or the complement or hybridizing fragment of such a sequence. The marker nucleic acid molecules also include RNA comprising the entire or a partial sequence of any of the nucleic acid sequences set forth herein, or the complement of such a sequence, wherein all thymidine residues are replaced with uridine residues. A "marker protein" is a protein encoded by or corresponding to a marker of the invention. A marker protein comprises the entire or a partial sequence of a protein encoded by any of the sequences set forth herein, or a fragment thereof. The terms "protein" and "polypeptide" are used interchangeably herein. A marker is "fixed" to a substrate if it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g., standard saline citrate, pH 7.4) without a substantial fraction of the marker dissociating from the substrate.

The terms "homology" or "identity," as used interchangeably herein, refer to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases "percent identity or homology" and "% identity or homology" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value there between. Identity or similarity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences. The term "substantial homology," as used herein, refers to homology of at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more.

Multiple sclerosis is "treated," "inhibited" or "reduced," if at least one symptom of the disease is reduced, alleviated, terminated, slowed, or prevented. As used herein, multiple sclerosis is also "treated," "inhibited," or "reduced," if recurrence or relapse of the disease is reduced, slowed, delayed, or prevented. Exemplary clinical symptoms of multiple sclerosis that can be used to aid in determining the disease status in a subject can include e.g., tingling, numbness, muscle weakness, loss of balance, blurred or double vision, slurred speech, sudden onset paralysis, lack of coordination, cognitive difficulties, fatigue, heat sensitivity, spasticity, dizziness, tremors, gait abnormalities, speech/swallowing difficulties, and extent of lesions assessed by imaging techniques, e.g., MRI. Clinical symptoms of MS are routinely classified and standardized, e.g., using an EDSS rating system. Typically, a decrease of one full step indicates an effective MS treatment (Kurtzke, Ann. Neurol. 36:573-79, 1994), while an increase of one full step will indicate the progression or worsening of the disease (e.g., exacerbation).

The terms "therapy" or "treatment" (e.g., MS therapy or MS treatment) are used interchangeably herein.

As used herein, the "Expanded Disability Status Scale" or "EDSS" is intended to have its customary meaning in the medical practice. EDSS is a rating system that is frequently used for classifying and standardizing MS. The accepted scores range from 0 (normal) to 10 (death due to MS). Typically patients having an EDSS score of about 6 will have moderate disability (e.g., walk with a cane), whereas patients having an EDSS score of about 7 or 8 will have severe disability (e.g., will require a wheelchair). More specifically, EDSS scores in the range of 1-3 refer to an MS patient who is fully ambulatory, but has some signs in one or more functional systems; EDSS scores in the range higher than 3 to 4.5 show moderate to relatively severe disability; an EDSS score of 5 to 5.5 refers to a disability imparing or precluding full daily activities; EDSS scores of 6 to 6.5 refer to an MS patient requiring intermittent to constant, or unilateral to bilateral constant assistance (cane, crutch or brace) to walk; EDSS scores of 7 to 7.5 means that the MS patient is unable to walk beyond five meters even with aid, and is essentially restricted to a wheelchair; EDSS scores of 8 to 8.5 refer to patients that are restricted to bed; and EDSS scores of 9 to 10 mean that the MS patient is confined to bed, and progressively is unable to communicate effectively or eat and swallow, until death due to MS.

An "overexpression" or "significantly higher level of expression" of the gene products refers to an expression level or copy number in a test sample that is greater than the standard error of the assay employed to assess the level of expression. In embodiments, the

overexpression can be at least two, at least three, at least four, at least five, or at least ten or more times the expression level of the gene products in a control sample (e.g., a sample from a healthy subject not afflicted with multiple sclerosis), or the average expression level of gene products in several control samples.

The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example a marker of the invention. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes can be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic monomers.

"Responsiveness," to "respond" to treatment, and other forms of this verb, as used herein, refer to the reaction of a subject to treatment with an MS therapy, e.g., a therapy including an IFN-β agent. As an example, a subject responds to treatment with an IFN-β agent if at least one symptom of multiple sclerosis (e.g., relapse rate) in the subject is reduced or retarded by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In another example, a subject responds to treatment with an IFN-β agent, if at least one symptom of multiple sclerosis in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50% or more as determined by any appropriate measure, e.g., Expanded Disability Status Scale (EDSS) or determining the extent of other symptoms such as relapse rate, muscle weakness, tingling, and numbness. In another example, a subject responds to treatment with an IFN-β agent, if the subject experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted if no treatment is administered. In another example, a subject responds to treatment with an IFN-β agent, if the subject has an increased disease-free survival, overall survival or increased time to progression. Several methods can be used to determine if a patient responds to a treatment including the EDSS criteria, as set forth above.

A "responder" refers to a subject, e.g., an MS patient, if in response to an MS therapy (e.g., IFN beta therapy), at least one symptom of multiple sclerosis in the subject is reduced by about 5%, 10%, 20%, 30%, 40%, 50% or more as determined by any appropriate measure, e.g., EDSS or determining the extent of other symptoms such as relapse rate, muscle weakness, tingling, and numbness. In one embodiment, a responder is defined as a subject with no confirmed relapses and no evidence of sustained disability progression (by EDSS) during the first three years of treatment (e.g., clinical remission).

A "non-responder" refers to a subject, e.g., an MS patient, if in response to an MS therapy (e.g., IFN beta therapy), at least one symptom of multiple sclerosis in the subject is reduced by less than about 5%, as determined by any appropriate measure, e.g., EDSS or determining the extent of other symptoms such as relapse rate, muscle weakness, tingling, and numbness. In one embodiment, a non-responder is defined as those subjects that have active disease on therapy including subjects with at least 3 relapses, development of a 6-month sustained progression in disability defined as a 1.0 point increase in EDSS score from baseline in subjects with a baseline score of < 5.5. Subjects were excluded for having > 10 MRI T2 lesions in the remission or permanently testing positive for NAB starting from year 1 at any titer or NAB titers > 20 in either group.

"Likely to" or "increased likelihood," as used herein, refers to an increased probability that an item, object, thing or person will occur. Thus, in one example, a subject that is likely to respond to treatment with an IFN-β agent to treat multiple sclerosis has an increased probability of responding to treatment with an IFN-β agent to treat multiple sclerosis, relative to a reference subject or group of subjects.

"Unlikely to" refers to a decreased probability that an event, item, object, thing or person will occur with respect to a reference. Thus, a subject that is unlikely to respond to treatment with an IFN-β agent has a decreased probability of responding to treatment with an IFN-β agent relative to a reference subject or group of subjects.

"Sample," "tissue sample," "patient sample," "patient cell or tissue sample" or "specimen" each refers to a biological sample obtained from a tissue or bodily fluid of a subject or patient. The source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents (e.g., serum, plasma); bodily fluids such as cerebral spinal fluid, whole blood, plasma and serum. The sample can include a non-cellular fraction (e.g., plasma, serum, or other non-cellular body fluid). In one embodiment, the sample is a serum sample. In other embodiments, the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood). In certain embodiments, the blood can be further processed to obtain plasma or serum. In another embodiment, the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)). For example, the sample can be a fine needle biopsy sample, an archival sample (e.g., an archived sample with a known diagnosis and/or treatment history), a histological section (e.g., a frozen or formalin-fixed section, e.g., after long term storage), among others. The term sample includes any material obtained and/or derived from a biological sample, including a polypeptide, and nucleic acid (e.g., genomic DNA, cDNA, RNA) purified or processed from the sample. Purification and/or processing of the sample can involve one or more of extraction,

concentration, antibody isolation, sorting, concentration, fixation, addition of reagents and the like. The sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. The amount of a biomarker, e.g., expression of gene products (e.g. , one or more the biomarkers described herein), in a subject is "significantly" higher or lower than the normal amount of a marker, if the amount of the marker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, or at least two, three, four, five, ten or more times that amount. Alternatively, the amount of the marker in the subject can be considered "significantly" higher or lower than the normal amount if the amount is at least about 1.5, two, at least about three, at least about four, or at least about five times, higher or lower, respectively, than the normal amount of the marker.

As used herein, "significant event" shall refer to an event in a patient's disease that is important as determined by one skilled in the art. Examples of significant events include, for example, without limitation, primary diagnosis, death, recurrence, remission, relapse of a patient's disease or the progression of a patient's disease from any one of the above noted stages to another. A significant event can be any important event used determine disease status using e.g., EDSS or other symptom criteria, as determined by one skilled in the art.

As used herein, "time course" shall refer to the amount of time between an initial event and a subsequent event. For example, with respect to a patient's disease, time course can relate to a patient's disease and can be measured by gauging significant events in the course of the disease, wherein the first event can be diagnosis and the subsequent event can be remission or relapse, for example.

A "transcribed polynucleotide" is a polynucleotide (e.g., an RNA, a cDNA, or an analog of one of an RNA or cDNA) which is complementary to or homologous with all or a portion of a mature RNA made by transcription of a marker of the invention and normal post-transcriptional processing (e.g., splicing), if any, of the transcript, and reverse transcription of the transcript.

An "underexpression" or "significantly lower level of expression" of products (e.g. , the markers set forth herein) refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, for example, at least 1.5, twice, at least three, at least four, at least five, or at least ten or more times less than the expression level of the gene products in a control sample (e.g. , a sample from a healthy subject not afflicted with multiple sclerosis), or the average expression level of gene products in several control samples.

Various aspects of the invention are described in further detail below. Additional definitions are set out throughout the specification. Multiple Sclerosis and Methods of Diagnosis

Multiple sclerosis (MS) is a central nervous system disease that is characterized by inflammation and loss of myelin sheaths.

Patients having MS can be identified by clinical criteria establishing a diagnosis of clinically definite MS as defined by Poser et al., Ann. Neurol. 13:227, 1983. Briefly, an individual with clinically definite MS has had two attacks and clinical evidence of either two lesions or clinical evidence of one lesion and paraclinical evidence of another, separate lesion. Definite MS may also be diagnosed by evidence of two attacks and oligoclonal bands of IgG in cerebrospinal fluid or by combination of an attack, clinical evidence of two lesions and oligoclonal band of IgG in cerebrospinal fluid. The McDonald criteria can also be used to diagnose MS. (McDonald et al., 2001, Recomended diagnostic criteria for Multiple sclerosis: guidelines from the International Panel on the Diagnosis of Multiple Sclerosis, Ann Neurol 50: 121-127). The McDonald criteria include the use of MRI evidence of CNS impairment over time to be used in diagnosis of MS, in the absence of multiple clinical attacks. Effective treatment of multiple sclerosis may be evaluated in several different ways. The following parameters can be used to gauge effectiveness of treatment. Two exemplary criteria include: EDSS (extended disability status scale), and appearance of exacerbations on MRI (magnetic resonance imaging).

The EDSS is a means to grade clinical impairment due to MS (Kurtzke, Neurology

33: 1444, 1983). Eight functional systems are evaluated for the type and severity of neurologic impairment. Briefly, prior to treatment, patients are evaluated for impairment in the following systems: pyramidal, cerebella, brainstem, sensory, bowel and bladder, visual, cerebral, and other. Follow-ups are conducted at defined intervals. The scale ranges from 0 (normal) to 10 (death due to MS). A decrease of one full step indicates an effective treatment (Kurtzke, Ann. Neurol.

36:573-79, 1994), while an increase of one full step will indicate the progression or worsening of disease (e.g., exacerbation). Typically patients having an EDSS score of about 6 will have moderate disability (e.g., walk with a cane), whereas patients having an EDSS score of about 7 or 8 will have severe disability (e.g., will require a wheelchair).

Exacerbations are defined as the appearance of a new symptom that is attributable to MS and accompanied by an appropriate new neurologic abnormality (IFNB MS Study Group, supra). In addition, the exacerbation must last at least 24 hours and be preceded by stability or improvement for at least 30 days. Briefly, patients are given a standard neurological examination by clinicians. Exacerbations are mild, moderate, or severe according to changes in a

Neurological Rating Scale (Sipe et al., Neurology 34: 1368, 1984). An annual exacerbation rate and proportion of exacerbation-free patients are determined.

Therapy can be deemed to be effective using a clinical measure if there is a statistically significant difference in the rate or proportion of exacerbation-free or relapse-free patients between the treated group and the placebo group for either of these measurements. In addition, time to first exacerbation and exacerbation duration and severity may also be measured. A measure of effectiveness as therapy in this regard is a statistically significant difference in the time to first exacerbation or duration and severity in the treated group compared to control group. An exacerbation-free or relapse-free period of greater than one year, 18 months, or 20 months is particularly noteworthy. Clinical measurements include the relapse rate in one and two-year intervals, and a change in EDSS, including time to progression from baseline of 1.0 unit on the EDSS that persists for six months. On a Kaplan-Meier curve, a delay in sustained progression of disability shows efficacy. Other criteria include a change in area and volume of T2 images on MRI, and the number and volume of lesions determined by gadolinium enhanced images.

MRI can be used to measure active lesions using gadolinium-DTPA-enhanced imaging (McDonald et al., Ann. Neurol. 36: 14, 1994) or the location and extent of lesions using T2- weighted techniques. Briefly, baseline MRIs are obtained. The same imaging plane and patient position are used for each subsequent study. Positioning and imaging sequences can be chosen to maximize lesion detection and facilitate lesion tracing. The same positioning and imaging sequences can be used on subsequent studies. The presence, location and extent of MS lesions can be determined by radiologists. Areas of lesions can be outlined and summed slice by slice for total lesion area. Three analyses may be done: evidence of new lesions, rate of appearance of active lesions, percentage change in lesion area (Paty et al., Neurology 43:665, 1993).

Improvement due to therapy can be established by a statistically significant improvement in an individual patient compared to baseline or in a treated group versus a placebo group.

Exemplary symptoms associated with multiple sclerosis, which can be treated with the methods described herein or managed using symptom management therapies, include: optic neuritis, diplopia, nystagmus, ocular dysmetria, internuclear opthalmoplegia, movement and sound phosphenes, afferent pupillary defect, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps, hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop, dysfunctional reflexes, paraesthesia, anaesthesia, neuralgia, neuropathic and neurogenic pain, l'hermitte's, proprioceptive dysfunction, trigeminal neuralgia, ataxia, intention tremor, dysmetria, vestibular ataxia, vertigo, speech ataxia, dystonia, dysdiadochokinesia, frequent micturation, bladder spasticity, flaccid bladder, detrusor- sphincter dyssynergia, erectile dysfunction, anorgasmy, frigidity, constipation, fecal urgency, fecal incontinence, depression, cognitive dysfunction, dementia, mood swings, emotional lability, euphoria, bipolar syndrome, anxiety, aphasia, dysphasia, fatigue, uhthoff s symptom, gastroesophageal reflux, and sleeping disorders.

Each case of MS displays one of several patterns of presentation and subsequent course. Most commonly, MS first manifests itself as a series of attacks followed by complete or partial remissions as symptoms mysteriously lessen, only to return later after a period of stability. This is called relap sing-remitting MS (RRMS). Primary-progressive MS (PPMS) is characterized by a gradual clinical decline with no distinct remissions, although there may be temporary plateaus or minor relief from symptoms. Secondary-progressive MS (SPMS) begins with a relapsing- remitting course followed by a later primary-progressive course. Rarely, patients may have a progressive-relapsing (PRMS) course in which the disease takes a progressive path punctuated by acute attacks. PPMS, SPMS, and PRMS are sometimes lumped together and called chronic progressive MS.

A few patients experience malignant MS, defined as a swift and relentless decline resulting in significant disability or even death shortly after disease onset. This decline may be arrested or decelerated by determining the likelihood of the patient to respond to a therapy early in the therapeutic regime and switching the patient to an agent that they have the highest likelihood of responding to.

Analysis of MS Biomarkers

Analysis of levels of expression and/or activity of gene products in the IFN-β signaling pathway has led to the identification of MxA as a biomarker, which correlates with the efficacy of IFN-β agents, alone or in combination, e.g., in combination with another agent for treating multiple sclerosis, in a subject. For example, the present invention provides methods for evaluation of expression level, protein level, protein activity of e.g., MxA.

In some embodiments, methods of the present invention can be used to determine the responsiveness of a subject to treatment with an IFN-β agent (e.g., an ΙΕΝβ-ΙΑ, an ΙΕΝβ-ΙΒ, or a derivative thereof (e.g., a PEGylated derivative)), wherein if a sample in a subject has a significant increase in the amount, e.g., expression, and/or activity of a marker disclosed herein relative to a standard, e.g., the level of expression and/or activity in a healthy subject then the disease is more likely to respond to treatment with an the IFN-β agent, alone or in combination with other therapies for multiple sclerosis, and vice versa.

Myxovirus protein- A (MxA) The nucleotide and protein sequences of human MxA are described, e.g., Aebi et al. (1989) Mol. Cell. Biol. 9:5062-5072; Horisberger et al. (1990) J.

Virol. (1990) 64: 1171-1181; Ku et al. (2011) Immunol. Cell Biol. 89: 173-182; and Weitz et al. (1989) J. Interferon Res. 9:679-689. Commercial antibodies for MxA can be obtained from e.g., SIGMA-ALDRICH®, ORIGENE™, ABNOVA™, and SANTA CRUZ BIOTECHNOLOGY®.

The methods provided herein are particularly useful for identifying subjects that are likely to respond to ΙΕΝβ treatment (e.g. ΙΕΝβ-ΙΑ, ΙΕΝβ-ΙΒ, or a derivative thereof (e.g., a pegylated derivative)) prior to initiation of such treatment (e.g., pre-therapy) or early in the therapeutic regimen. In some embodiments, expression of MxA measured in a subject at least 2 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year after initiation of therapy. In some embodiments, it is preferred that expression of MxA is measured less than 6 months after initiation of therapy to permit the skilled practitioner to switch the subject to a different therapeutic strategy. Thus, in some embodiments it is preferred that expression of MxA is measured within 1-6 months, 1-5 months, 1-4 months, 1-3 months, 1-2 months, 2-6 months, 3- 6 months, 4-6 months, 5-6 months, 2-3 months, 3-4 months, or 4-5 months of initiation of ΙΕΝβ therapy. In some embodiments, the expression of MxA is determined 3-6 months after initiation of therapy (e.g., 3 months, 3.5 months, 4 months, 4.5 months, 5 months, 5.5 months, 6 months).

The methods described herein can also be used to monitor a positive response of a subject to treatment with ΙΕΝβ. Such methods are useful for early detection of tolerance to ΙΕΝβ therapy or to predict whether a subject will shift from a responder to a non-responder phenotype. In such embodiments, the level (e.g., expression) of MxA is determined e.g., at least every week, at least every 2 weeks, at least every month, at least every 2 months, at least every 3 months, at least every 4 months, at least every 5 months, at least every 6 months, at least every 7 months, at least every 8 months, at least every 9 months, at least every 10 months, at least every 11 months, at least every year, at least every 18 months, at least every 2 years, at least every 3 years, at least every 5 years or more. It is also contemplated that expression of the biomarkers is at irregular intervals e.g., biomarkers can be detected in an individual at 3 months of treatment, at 6 months of treatment, and at 7 months of treatment. Thus, in some embodiments, the expression of the biomarkers is determined when deemed necessary by the skilled physician monitoring treatment of the subject.

The methods described herein can be used in any subject having multiple sclerosis including sub-types such as benign MS, quiescent relapsing-remitting MS, active relapsing- remitting MS, primary progressive MS, and secondary progressive MS. It is also contemplated, in other embodiments, that the methods can be used in subjects having MS-like symptoms, such as those having clinically isolated syndrome (CIS) or clinically defined MS (CDMS). Clinically isolated syndrome (CIS) refers to the detection of a single clinical episode of demyelination or other monophasic CNS inflammatory disorder (e.g., Spinal Cord Syndrome,

Brainstem/Cerebellar Syndrome, and others described below). Frohman et al. (2003) Neurology 2003 61(5):602-1 1 report that, in subjects with CIS, three or more white matter lesions on a T2- weighted MRI scan (especially if one of these lesions is located in the periventricular region) is a very sensitive predictor (>80%) of the subsequent development of CDMS within the next 7 to 10 years. In a preferred embodiment, the methods described herein are used to assess expression of one or more biomarkers of Table 1 in a subject having RRMS.

A subject that is identified as a responder using the methods described herein can be treated with any ΙΡΝβ agent known in the art presently or to be developed (e.g. ΙΡΝβ-ΙΑ, ΙΡΝβ- 1B, or a derivative thereof (e.g., a pegylated derivative)). In one embodiment, the ΙΡΝβ agent is an ΙΡΝβ- lA agent (e.g., AVONPX®, RPBIF®). In another embodiment, the ΙΡΝβ agent is an ΙΡΝβ-lB agent (e.g., BPTASPRON®, BPTAFPRON®).

In some embodiments, the amount of the biomarker determined in a serum sample from a subject is quantified as an absolute measurement (e.g., ng/mL). Absolute measurements can easily be compared to a reference value or cut-off value. For example, a cut-off value can be determined that represents a non-responder status; any absolute values falling either above (i.e., for biomarkers that increase expression with MS) or falling below (i.e., for biomarkers with decreased expression in MS) the cut-off value are likely to be non-responders to ΙΡΝβ therapy.

Alternatively, the relative amount of a biomarker is determined. In one embodiment, the relative amount is determined by comparing the expression of one or more serum biomarkers in a subject with MS to the expression of the serum biomarkers in a healthy control subject. In another embodiment, the relative amount is determined by comparing the expression of MxA in a subject with MS at two or more timepoints (e.g., at baseline and monthly beginning 6 months after initiation of therapy).

The present invention also pertains to the field of predictive medicine in which diagnostic assays, pharmacogenomics, and monitoring clinical trials are used for predictive purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to assays for determining the amount, structure, and/or activity of polypeptides or nucleic acids corresponding to one or more markers of the invention, in order to determine whether an individual having multiple sclerosis or at risk of developing multiple sclerosis will be more likely to respond to IFN^-mediated therapy.

Accordingly, in one aspect, the invention is drawn to a method for determining whether a subject with multiple sclerosis is likely to respond to treatment with an IFN-β agent. In another aspect, the invention is drawn to a method for predicting a time course of disease. In still another aspect, the method is drawn to a method for predicting a probability of a significant event in the time course of the disease (e.g., relapse or shift from responder to non-responder status). In certain embodiments, the method comprises detecting a biomarker or combination of biomarkers associated with responsiveness to treatment with an IFN-β agent as described herein and determining whether the subject is likely to respond to treatment with the IFN-β agent

(e.g. ΙΡΝβ- ΙΑ, ΙΡΝβ-ΙΒ, or a derivative thereof (e.g., a pegylated derivative)).

In some embodiments, the methods involve evaluation of a biological sample e.g., a serum sample from a subject, e.g., a patient who has been diagnosed with or is suspected of having multiple sclerosis (e.g. , presents with symptoms of multiple sclerosis) to detect changes in MxA (e.g., gene expression or polypeptide levels).

The results of the screening method and the interpretation thereof are predictive of the patient's response to treatment with IFN-β agents (e.g. , AVONEX®, REBIF®, BETASERON®, BETAFERON®), alone or in combination with symptom management agents. According to the present invention, alterations in expression of MxA is indicative that treatment with IFN-β agents will provide enhanced therapeutic benefit for patients with multiple sclerosis relative to healthy controls.

In yet another embodiment, the one or more alterations, e.g., alterations in biomarker expression are assessed at pre-determined intervals, e.g., a first point in time and at least at a subsequent point in time. In one embodiment, a time course is measured by determining the time between significant events in the course of a patient's disease, wherein the measurement is predictive of whether a patient has a long time course. In another embodiment, the significant event is the progression from primary diagnosis to death. In another embodiment, the significant event is the progression from primary diagnosis to worsening disease. In another embodiment, the significant event is the progression from primary diagnosis to relapse. In another

embodiment, the significant event is the progression from secondary MS to death. In another embodiment, the significant event is the progression from remission to relapse. In another embodiment, the significant event is the progression from relapse to death. In certain

embodiments, the time course is measured with respect to one or more overall survival rate, time to progression and/or using the EDSS or other assessment criteria.

Methods for Detection of Gene Expression

Marker expression level can also be assayed. Expression of a marker of the invention can be assessed by any of a wide variety of well known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

In certain embodiments, activity of a particular gene is characterized by a measure of gene transcript (e.g., mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Marker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.

Methods of detecting and/or quantifying the gene transcript (mRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art (see e.g., Sambrook et al. supra). For example, one method for evaluating the presence, absence, or quantity of cDNA involves a Southern transfer as described above. Briefly, the mRNA is isolated {e.g., using an acid guanidinium-phenol-chloroform extraction method, Sambrook et al. supra.) and reverse transcribed to produce cDNA. The cDNA is then optionally digested and run on a gel in buffer and transferred to membranes. Hybridization is then carried out using the nucleic acid probes specific for the target cDNA.

A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that can contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay.

In order to conduct assays with the above-mentioned approaches, the non-immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components can be removed {e.g. , by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein.

In another embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art. It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule can simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label can be differentiated from that of the 'donor' . Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial

relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art {e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C, 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA" or "surface plasmon resonance" is a technology for studying biospecific interactions in real time, without labeling any of the interactants {e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography,

electrophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes can be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., 1993, Trends Biochem Sci. 18(8):284-7).

Standard chromatographic techniques can also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex can be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components can be exploited to differentiate the complex from uncomplexed components, for example, through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N.H., 1998, J. Mol. Recognit. Winter 11(1-6): 141-8; Hage, D.S., and Tweed, S.A. J Chromatogr B Biomed Sci Appl 1997 Oct 10;699(l-2):499-525). Gel electrophoresis can also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et ah, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typical. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

In a particular embodiment, the level of mRNA corresponding to the marker can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term "biological sample" is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et ah, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Patent No. 4,843,155). The isolated nucleic acid can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full- length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the markers of the present invention.

The probes can be full length or less than the full length of the nucleic acid sequence encoding the protein. Shorter probes are empirically tested for specificity. Exemplary nucleic acid probes are 20 bases or longer in length (See, e.g., Sambrook et al. for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization). Visualization of the hybridized portions allows the qualitative determination of the presence or absence of cDNA.

An alternative method for determining the level of a transcript corresponding to a marker of the present invention in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88: 189-193), self sustained sequence replication (Guatelli et al, 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173- 1177), Q-Beta Replicase (Lizardi et al. , 1988, Bio/Technology 6: 1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Fluorogenic rtPCR can also be used in the methods of the invention. In fluorogenic rtPCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the cells prior to detection.

In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absolute expression level of the marker, determinations can be based on the normalized expression level of the marker.

Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a subject sample, to another sample, e.g., a healthy subject, or between samples from different sources.

Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus MS isolates, or even 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level.

In certain embodiments, the samples used in the baseline determination will be from samples derived from a subject having multiple sclerosis versus samples from a healthy subject of the same tissue type. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is specific to the tissue from which the cell was derived (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data.

Expression data from normal cells provides a means for grading the severity of the multiple sclerosis disease state.

In another embodiment, expression of a marker is assessed by preparing genomic DNA or mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in a subject sample, and by hybridizing the genomic DNA or mRNA/cDNA with a reference polynucleotide which is a complement of a polynucleotide comprising the marker, and fragments thereof. cDNA can, optionally, be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more markers can likewise be detected using quantitative PCR (QPCR) to assess the level of expression of the marker(s). Alternatively, any of the many known methods of detecting mutations or variants (e.g., single nucleotide polymorphisms, deletions, etc.) of a marker of the invention can be used to detect occurrence of a mutated marker in a subject.

In a related embodiment, a mixture of transcribed polynucleotides obtained from the sample is contacted with a substrate having fixed thereto a polynucleotide complementary to or homologous with at least a portion (e.g., at least 7, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 500, or more nucleotide residues) of a marker of the invention. If polynucleotides complementary to or homologous with a marker of the invention are differentially detectable on the substrate (e.g., detectable using different chromophores or fluorophores, or fixed to different selected positions), then the levels of expression of a plurality of markers can be assessed simultaneously using a single substrate (e.g., a "gene chip" microarray of polynucleotides fixed at selected positions). When a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, the hybridization can be performed under stringent hybridization conditions.

In another embodiment, a combination of methods to assess the expression of a marker is utilized. Because the compositions, kits, and methods of the invention rely on detection of a difference in expression levels of one or more markers of the invention, in certain embodiments the level of expression of the marker is significantly greater than the minimum detection limit of the method used to assess expression in at least one of a biological sample from a subject with MS or a healthy control.

Nucleic Acid Molecules and Probes

One aspect of the invention pertains to isolated nucleic acid molecules that correspond to one or markers of the invention, including nucleic acids which encode a polypeptide

corresponding to one or more markers of the invention or a portion of such a polypeptide. The nucleic acid molecules of the invention include those nucleic acid molecules which reside in genomic regions identified herein. Isolated nucleic acid molecules of the invention also include nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules that correspond to a marker of the invention, including nucleic acid molecules which encode a polypeptide corresponding to a marker of the invention, and fragments of such nucleic acid molecules, e.g., those suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules {e.g., cDNA or genomic DNA) and RNA molecules {e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single- stranded or double-stranded; in certain embodiments the nucleic acid molecule is double- stranded DNA.

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. In certain embodiments, an "isolated" nucleic acid molecule is free of sequences (such as protein-encoding sequences) which naturally flank the nucleic acid {i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.

The language "substantially free of other cellular material or culture medium" includes preparations of nucleic acid molecule in which the molecule is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, nucleic acid molecule that is substantially free of cellular material includes preparations of nucleic acid molecule having less than about 30%, less than about 20%, less than about 10%, or less than about 5% (by dry weight) of other cellular material or culture medium.

If so desired, a nucleic acid molecule of the present invention, e.g., the marker gene products identified herein, can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

A nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts (e.g., mRNA) or genomic sequences corresponding to one or more markers of the invention. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted. Polypeptide Detection

Methods to measure biomarkers of this invention, include, but are not limited to: Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, liquid chromatography mass spectrometry (LC-MS), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, laser scanning cytometry, hematology analyzer and assays based on a property of the protein including but not limited to DNA binding, ligand binding, or interaction with other protein partners.

The activity or level of a marker protein can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These can include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high

performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, immunohistochemistry and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining the expression level of one or more biomarkers in a serum sample.

Another agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide corresponding to a marker of the invention, e.g., an antibody with a detectable label. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e. , physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

In another embodiment, the antibody is labeled, e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody. In another embodiment, an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair { e.g., biotin-streptavidin}), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically with a protein corresponding to the marker, such as the protein encoded by the open reading frame corresponding to the marker or such a protein which has undergone all or a portion of its normal post-translational modification, is used.

Proteins from cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

In one format, antibodies, or antibody fragments, can be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, one can immobilize either the antibody or proteins on a solid support. Suitable solid phase supports or carriers include any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.

In another embodiment, the polypeptide is detected using an immunoassay. As used herein, an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte. The immunoassay is thus characterized by detection of specific binding of a polypeptide to an anti-antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.

The polypeptide is detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

In another embodiment, the polypeptide is detected and/or quantified using Luminex™ assay technology. The LUMINEX™ assay separates tiny color-coded beads into e.g., distinct sets that are each coated with a reagent for a particular bioassay, allowing the capture and detection of specific analytes from a sample in a multiplex manner. The LUMINEX™ assay technology can be compared to a multiplex ELISA assay using bead-based fluorescence cytometry to detect analytes such as biomarkers.

The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid corresponding to a marker of the invention in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. Such kits can be used to determine if a subject is suffering from or is at increased risk of developing multiple sclerosis. For example, the kit can comprise a labeled compound or agent capable of detecting a polypeptide or an mRNA encoding a polypeptide corresponding to a marker of the invention in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g. , an antibody which binds the

polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the

polypeptide). Kits can also include instructions for interpreting the results obtained using the kit.

The invention thus includes a kit for assessing the responsiveness of a subject having multiple sclerosis to treatment using an IFN-β agent (e.g., in a sample such as a serum sample). Suitable reagents for binding with a polypeptide corresponding to a marker of the invention include antibodies, antibody derivatives, antibody fragments, and the like. Suitable reagents for binding with a nucleic acid (e.g., a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleic acids. For example, the nucleic acid reagents can include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.

The kit of the invention can optionally comprise additional components useful for performing the methods of the invention. By way of example, the kit can comprise fluids (e.g., SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample compartments, an instructional material which describes performance of a method of the invention, a reference sample for comparison of expression levels of the biomarkers described herein, and the like.

A kit of the invention can comprise a reagent useful for determining protein level or protein activity of a marker.

MS Therapeutic Agents, Compositions and Administration

There are several medications presently used to modify the course of multiple sclerosis in patients. Such agents include, but are not limited to, Beta interferons (e.g., AVONEX®,

REBIF®, BETASERON®, BETAFERON®, among others)), glatiramer (COPAXONE®), natalizumab (TYSABRI®), and mitoxantrone (NOVANTRONE®). IFN-β agents (Beta interferons)

One known therapy for MS includes treatment with interferon beta. Interferons (IFNs) are natural proteins produced by the cells of the immune systems of most animals in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells. Interferons belong to the large class of glycoproteins known as cytokines. Interferon beta has 165 amino acids. Interferons alpha and beta are produced by many cell types, including T-cells and B-cells, macrophages, fibroblasts, endothelial cells, osteoblasts and others, and stimulate both

macrophages and NK cells. Interferon gamma is involved in the regulation of immune and inflammatory responses. It is produced by activated T-cells and Thl cells.

Several different types of interferon are now approved for use in humans. Interferon alpha (including forms interferon alpha-2a, interferon alpha- 2b, and interferon alfacon-1) was approved by the United States Food and Drug Administration (FDA) as a treatment for Hepatitis C. There are two currently FDA-approved types of interferon beta. Interferon beta la

(AVONEX®) is identical to interferon beta found naturally in humans, and interferon beta lb (BETASERON®) differs in certain ways from interferon beta la found naturally in humans, including that it contains a serine residue in place of a cysteine residue at position 17. Other uses of interferon beta have included treatment of AIDS, cutaneous T-cell lymphoma, Acute Hepatitis C (non-A, non-B), Kaposi's sarcoma, malignant melanoma, and metastatic renal cell carcinoma.

IFN-β agents can be administered to the subject by any method known in the art, including systemically {e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or intracavitary installation). Typically, the IFN-β agents are administered subcutaneously, or intramuscularly.

IFN-β agents can be used to treat those subjects determined to be "responders" using the methods described herein. In one embodiment, the IFN-β agents are used as a monotherapy although the treatment regimen can further comprise the use of "symptom management therapies" such as antidepressants, analgesics, anti-tremor agents, etc. In one embodiment, the IFN-β agent is an ΙΡΝβ-lA agent (e.g., AVONEX®, REBIF®). In another embodiment, the INF-β agent is an ΙΝΡβ-lB agent (e.g., BETASERON®, BETAFERON®).

AVONEX®, an Interferon β-la, is indicated for the treatment of patients with relapsing forms of MS that are determined to be responders using the methods described herein to slow the accumulation of physical disability and decrease the frequency of clinical exacerbations. AVONEX® (Interferon beta- la) is a 166 amino acid glycoprotein with a predicted molecular weight of approximately 22,500 daltons. It is produced by recombinant DNA technology using genetically engineered Chinese Hamster Ovary cells into which the human interferon beta gene has been introduced. The amino acid sequence of AVONEX® is identical to that of natural human interferon beta. The recommended dosage of AVONEX® (Interferon beta- la) is 30 meg injected intramuscularly once a week. AVONEX® is commercially available as a 30 meg lyophilized powder vial or as a 30 meg prefilled syringe.

Interferon beta la (AVONEX®) is identical to interferon beta found naturally in humans (AVONEX®, i.e., Interferon beta la (SwissProt Accession No. P01574 and gi:50593016). The sequence of interferon beta is:

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRM NFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQIN HLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNF YFINRLTGYLRN

(SEQ ID NO: l).

In other embodiments, the IFN-b agent includes an amino acid sequence substantially identical to SEQ ID NO: l, e.g., at least 70%, 80%, 90% or 95% identical to SEQ ID NO: l.

Methods for making Avonex® are known in the art.

Treatment of responders identified using the methods described herein further

contemplates that compositions (e.g., IFN beta la molecules) having biological activity that is substantially similar to that of AVONEX® will permit successful treatment similar to treatment with AVONEX® when administered in a similar manner. Such other compositions include, e.g., other interferons and fragments, analogues, homologues, derivatives, and natural variants thereof with substantially similar biological activity. In one embodiment, the INF-β agent is modified to increase one or more pharmacokinetic properties. For example, the INF-β agent can be a modified form of interferon la to include a pegylated moiety. PEGylated forms of interferon beta la are described in, e.g., Baker, D.P. et al. (2006) Bioconjug Chem 17(1): 179-88; Arduini, RM et al. (2004) Protein Expr Purif 34(2) :229-42; Pepinsky, RB et al. (2001) J. Pharmacol. Exp. Ther. 297(3): 1059-66; Baker, D.P. et al. (2010) J Interferon Cytokine Res 30(10):777-85 (all of which are incorporated herein by reference in their entirety, and describe a human interferon beta la modified at its N-terminal alpha amino acid to include a PEG moiety, e.g., a 20 kDa mPEG-O-2-methylpropionaldehyde moiety). Pegylated forms of IFN beta la can be administered by, e.g., injectable routes of administration (e.g., subcutaneously).

REBIF® is also an Interferon β-la agent, while BETASERON® and BETAFERON® are Interferon β lb agents. Both REBIF® and BETASERON® are formulated for administration by subcutaneous injection.

Dosages of IFN-β agents to administer can be determined by one of skill in the art, and include clinically acceptable amounts to administer based on the specific interferon-beta agent used. For example, AVONEX® is typically administered at 30 microgram once a week via intramuscular injection. Other forms of interferon beta la, specifically REBIF®, is administered, for example, at 22 microgram three times a week or 44 micrograms once a week, via

subcutaneous injection. Interferon beta- 1A can be administered, e.g., intramuscularly, in an amount of between 10 and 50 μg. For example, AVONEX® can be administered every five to ten days, e.g., once a week, while REBIF® can be administered three times a week. Non-IFN-β agents

In other embodiments, alternative therapies to the IFN-β agent can be administered. For example, in subjects determined to be non-responders using the methods described herein, a skilled physician can select a therapy that includes a non-IFN-β agent that can include, e.g., glatiramer (COPAXONE®), natalizumab (TYSABRI®, ANTEGREN®), and mitoxantrone (NOVANTRONE®).

Anti-VLA4 antibody (e.g., Natalizumab (TYSABRI®))

Anti-VLA4 antibodies (e.g., Natalizumab) inhibit the migration of leukocytes from the blood to the central nervous system. These antibodies bind to VLA-4 (also called α4β1) on the surface of activated T-cells and other mononuclear leukocytes. They can disrupt adhesion between the T-cell and endothelial cells, and thus prevent migration of mononuclear leukocytes across the endothelium and into the parenchyma. As a result, the levels of pro-inflammatory cytokines can also be reduced. Natalizumab can decrease the number of brain lesions and clinical relapses and accumulation of disability in patients with relapse remitting multiple sclerosis and relapsing secondary-progressive multiple sclerosis. Natalizumab and related VLA-4 binding antibodies are described, e.g., in U.S. Pat. No. 5,840,299. Monoclonal antibodies 21.6 and HP1/2 are exemplary murine monoclonal antibodies that bind VLA-4. Natalizumab is a humanized version of murine monoclonal antibody 21.6 (see, e.g., U.S. Pat. No. 5,840,299). A humanized version of HP 1/2 has also been described (see, e.g., U.S. Pat. No. 6,602,503). Several additional VLA-4 binding monoclonal antibodies, such as

HP2/1, HP2/4, L25 and P4C2, are described, e.g., in U.S. Pat. No. 6,602,503; Sanchez-Madrid et al, (1986) Eur. J. Immunol 16: 1343-1349; Hemler et al, (1987) J Biol. Chem. 2: 11478-11485; Issekutz et al. (1991) J Immunol 147: 109 (TA-2 mab); Pulido et al. (1991) J Biol. Chem. 266: 10241-10245; and U.S. Pat. No. 5,888,507). The contents of the aforesaid publications

(including the antibody compositions, dosages, methods of administration and production) are incorporated herein by reference in their entirety.

Dimethyl Fumarate ( TECFIDERA ® )

Dimethyl fumarate, DMF, (TECFIDERA®) is a fumaric acid ester. DMF is thought to decrease leukocyte passage through the blood brain barrier and exert neuroprotective effects by the activation of antioxidative pathways, specifically through activation of the Nrf-2 pathway (Lee et al. (2008) Int MS Journal 15: 12-18). Research also suggests that BG-12® has the potential to reduce the activity and impact of inflammatory cells on the CNS and induce direct cytoprotective responses in CNS cells. These effects may enhance the CNS cells' ability to mitigate the toxic inflammatory and oxidative stress that plays a role in MS pathophysiology.

Glatiramer acetate (COPAXONE®)

COPAXONE® (glatiramer acetate) consists of the acetate salts of synthetic polypeptides, specifically the four naturally occurring amino acids: L-glutamic acid, L-alanine, L-tyrosine, and L-lysine (Bornstein et al. (1987) N Engl J Med. 317: 408-414). COPAXONE® exhibits structural similarity to myelin basic protein and is thought to function as an immune modulator by shifting the T helper cell type 1 response towards a T helper cell type 2 response (Duda et al. (2000) J Clin Invest 105: 967-976; Nicholas et al. (2011) Drug Design, Development, and Therapy 5: 255-274). Mitoxantrone (NOVANTRONE®, an anthracenedione molecule)

Mitoxantrone is an anthracenedione molecule (l,4-dihydroxy-5,8-bis[2-(2- hydroxyethylamino) ethylamino]-anthracene-9,10-dione) and a type II topoisomerase inhibitor that disrupts DNA synthesis and repair of cells. It is used to treat cancers and MS. Mitoxantrone is used to treat several forms of advancing MS, including secondary progressive MS, progressive relapsing MS, and advanced relapsing-remitting MS. For example, mitoxantrone is effective in slowing the progression of secondary progressive MS and extending the time between relapses in relapsing-remitting MS and progressive relapsing MS (Fox E (2006) Clin Ther 28 (4): 461-74). Fingolimod (GILENYA®; sphingosine 1-phosphate receptor modulator)

Fingolimod is an immunomodulating drug, approved for treating MS. It has reduced the rate of relapses in relapsing-remitting multiple sclerosis by over half, but may have serious adverse effects. Fingolimod is a sphingosine 1-phosphate receptor modulator, which sequesters lymphocytes in lymph nodes, preventing them from moving to the central nervous system for autoimmune responses in MS. Exemplary Sphingosine 1-phosphate (S IP) modulating agents are described, for example, in WO 2012/109108, incorporated herein by reference.

Antibodies to the alpha subunit of the IL-2 receptor of T cells (daclizumab; ZEN AP AX® )

An antibody to the alpha subunit of the IL-2 receptor of T cells, e.g., daclizumab, can be used in the methods and compositions disclosed herein. Daclizumab is a therapeutic humanized monoclonal antibody to the alpha subunit of the IL-2 receptor of T cells. Daclizumab was effective in reducing lesions and improving clinical scores in patients with multiple sclerosis not controlled with interferon (Rose JW et al. (2007). Neurology 69 (8): 785-789). Antibody against CD 52, e.g., alemtuzumab

Antibodies against CD52, e.g., alemtuzumab (currently under further development as LEMTRADA®), bind to CD52, which is a protein present on the surface of mature lymphocytes, but not on stem cells. Phase III studies reported positive results comparing alemtuzumab with REBIF® (high-dose subcutaneous interferon beta- la) in the treatment of patients with relapsing - remitting MS (RRMS). Alemtuzumab has been approved in Europe. Antibody to CD20, e.g., ocrelizumab

Antibodies against CD20, e.g., ocrelizumab, rituximab, ofatumumab, target mature B lymphocytes. Phase 2 clinical studies of rituximab and ocrelizumab in relapse remitting MS have demonstrated a statistically significant reduction in disease activity measured by brain lesions {e.g., measured by MRI scans) and relapse rate compared to placebo.

Inhibitors of dihydroorotate dehydrogenase, e.g., teriflunomide

Inhibitors of dihydroorotate dehydrogenase, e.g., teriflunomide, inhibit pyrimidine synthesis. Teriflunomide (also known as A77 1726 or ) is an active metabolite of leflunomide. Teriflunomide inhibits rapidly dividing cells, including activated T cells, which are thought to drive the disease process in MS. Teriflunomide was investigated in clinical trials as a medication for treating MS. (Vollmer EMS News (May 28, 2009)).

Steroids

Steroids, e.g., corticosteroid, and ACTH agents can be used to treat acute relapses in relapsing -remitting MS or secondary progressive MS. Such agents include, but are not limited to, DEPO-MEDROL®, SOLU-MEDROL®, DELTASONE®, DELTA-CORTEF®,

MEDROL®, DECADRON®, and ACTHAR®.

Doses and modes of administration of the non- ΙΕΝβ agent are known in the art.

Symptom management

In certain embodiments, the method further includes the use of one or more symptom management therapies, such as antidepressants, analgesics, anti-tremor agents, among others. Treatment of a subject with a disease modifying IFN-β agent or non-IFN-β agent can be combined with one or more of the following therapies often used in symptom management of subjects having MS: IMURAN® (azathioprine), CYTOXAN® (cyclophosphamide), NEOSAR® (cyclophosphamide), SANDIMMUNE® (cyclosporine), methotrexate, LEUSTATIN®

(cladribine), TEGRETOL® (carbamazepine), EPITOL® (carbamazepine), ATRETOL®

(carbamazepine), CARBATROL® (carbamazepine), NEURONTIN® (gabapentin),

TOPAMAX® (topiramate), ZONEGRAN® (zonisamide), DILANTIN® (phenytoin),

NORPRAMIN® (desipramine), ELAVIL® (amitriptyline), TOFRANIL® (imipramine), IMAVATE® (imipramine), JANIMINE® (imipramine), SINEQUAN® (doxepine), ADAPIN® (doxepine), TRIADAPIN® (doxepine), ZONALON® (doxepine), VIVACTIL® (protriptyline), MARINOL® (synthetic cannabinoids), TRENTAL® (pentoxifylline), NEUROFEN®

(ibuprofen), aspirin, acetaminophen, ATARAX® (hydroxyzine), PROZAC® (fluoxetine), ZOLOFT® (sertraline), LUSTRAL® (sertraline), EFFEXOR XR® (venlafaxine), CELEXA® (citalopram), PAXIL®, SEROXAT®, DESYREL® (trazodone), TRIALODINE® (trazodone), PAMELOR® (nortriptyline), AVENTYL® (imipramine), PROTHIADEN® (dothiepin), GAMANIL® (lofepramine), PARNATE® (tranylcypromine), MANERIX® (moclobemide), AURORIX® (moclobemide), WELLBUTRIN SR® (bupropion), AMFEBUTAMONE® (bupropion), SERZONE® (nefazodone), REMERON® (mirtazapine), AMBIEN® (Zolpidem), XANAX® (alprazolam), RESTORIL® (temazepam), VALIUM® (diazepam), BUSPAR® (buspirone), SYMMETREL® (amantadine), CYLERT® (pemoline), PROVIGIL® (modafinil), DITROPAN XL® (oxybutynin), DDAVP® (desmopressin, vasopressin), DETROL®

(tolterodine), URECHOLINE® (bethane), DIBENZYLINE® (phenoxybenzamine), HYTRIN® (terazosin), PRO-BANTHINE® (propantheline), URISPAS® (hyoscyamine), CYSTOPAS® (hyoscyamine), LIORESAL® (baclofen), HIPREX® (methenamine), MANDELAMINE® (metheneamine), MACRODANTIN® (nitrofurantoin), PYRIDIUM® (phenazopyridine), CIPRO® (ciprofloxacin), DULCOLAX® (bisacodyl), BISACOLAX® (bisacodyl), SANI- SUPP® (glycerin), METAMUCIL® (psyllium hydrophilic mucilloid), FLEET ENEMA® (sodium phosphate), COLACE® (docusate), THEREVAC PLUS®, KLONOPIN®

(clonazepam), RIVOTRIL® (clonazepam), DANTRIUM® (dantrolen sodium), CATAPRES® (clonidine), BOTOX® (botulinum toxin), NEUROBLOC® (botulinum toxin), ZANAFLEX® (tizanidine), SIRDALUD® (tizanidine), MYSOLINE® (primidone), DIAMOX®

(acetozolamide), SINEMET® (levodopa, carbidopa), LANIAZID® (isoniazid), NYDRAZID® (isoniazid), ANTIVERT® (meclizine), BON AMINE® (meclizine), DRAMAMINE®

(dimenhydrinate), COMPAZINE® (prochlorperazine), TRANSDERM® (scopolamine), BENADRYL® (diphenhydramine), ANTEGREN® (natalizumab), CAMPATH-1H®

(alemtuzumab), FAMPRIDINE® (4-aminopyridine), GAMMAGARD® (IV immunoglobulin), GAMMAR-IV® (IV immunoglobulin), GAMIMUNE N® (IV immunoglobulin), IVEEGAM® (IV immunoglobulin), PANGLOBULIN® (IV immunoglobulin), SANDOGLOBULIN® (IV immunoglobulin), VENOBLOGULIN® (IV immunoglobulin), pregabalin, ziconotide, and ANERGIX-MS®.

It is also contemplated herein that a subject identified as a non-responder will be treated with one or more agents described herein to manage symptoms.

Therapeutic Methods

"Treat," "treatment," and other forms of this word refer to the administration of an IFN-β agent, alone or in combination with one or more symptom management agents, to a subject, e.g., an MS patient, to impede progression of multiple sclerosis, to induce remission, to extend the expected survival time of the subject and or reduce the need for medical interventions (e.g., hospitalizations). In those subjects, treatment can include, but is not limited to, inhibiting or reducing one or more symptoms such as numbness, tingling, muscle weakness; reducing relapse rate, reducing size or number of sclerotic lesions; inhibiting or retarding the development of new lesions; prolonging survival, or prolonging progression-free survival, and/or enhanced quality of life.

As used herein, unless otherwise specified, the terms "prevent," "preventing" and "prevention" contemplate an action that occurs before a subject begins to suffer from the a multiple sclerosis relapse and/or which inhibits or reduces the severity of the disease.

As used herein, and unless otherwise specified, the terms "manage," "managing" and "management" encompass preventing the progression of MS symptoms in a patient who has already suffered from the disease, and/or lengthening the time that a patient who has suffered from MS remains in remission. The terms encompass modulating the threshold, development and/or duration of MS, or changing the way that a patient responds to the disease.

As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or

management of multiple sclerosis, or to delay or minimize one or more symptoms associated with MS. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of MS. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease, or enhances the therapeutic efficacy of another therapeutic agent. As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent relapse of MS, or one or more symptoms associated with the disease, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of the compound, alone or in combination with other therapeutic agents, which provides a prophylactic benefit in the prevention of MS relapse. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

As used herein, the term "patient" or "subject" refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey), that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with

administration of a compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.

The methods described herein permit one of skill in the art to identify a monotherapy that an MS patient is most likely to respond to, thus eliminating the need for administration of multiple therapies to the patient to ensure that a therapeutic effect is observed. However, in one embodiment, combination treatment of an individual with MS is contemplated. It will be appreciated that the IFN-β agent, as described above and herein, can be administered in combination with one or more additional therapies to treat and/or reduce the symptoms of MS described herein, particularly to treat patients with moderate to severe disability (e.g., EDSS score of 5.5 or higher). The pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved. In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some

embodiments, the levels utilized in combination will be lower than those utilized individually.

The invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, figures, sequence listing, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXEMPLIFICATION

Example 1: MxA as a Biomarker: MxA mRNA quantification allows the prediction of disability progression in IFNp-treated multiple sclerosis patients

The present example describes a 3-year prospective longitudinal study that was performed in subjects naive for treatment initiating IFNP therapy at the time of study inclusion. Among other things, the study permitted the analysis of IFNP bioactivity modulation in individual patients in the conditions of a real-life setting. The primary outcome was the analysis of the kinetics of IFNP bioactivity loss, defined according to MxA mRNA induction, and of anti- IFNP antibody production. Secondary objectives included evaluation of whether the expression of the mRNA for the IFNP receptor (IFNAR) subunits and isoforms had an impact on bioactivity loss; and correlating the markers of IFNP bioactivity with the measures of clinical disease activity, to determine whether biomarkers can predict IFNP therapy effectiveness. Summary

118 multiple sclerosis patients naive for treatment were enrolled for a 3-year longitudinal observational study mimicking the conditions of a real-world setting. In order to evaluate the kinetics of bioactivity loss in blood samples obtained every 6 months after therapy initiation, MxA and interferon receptor isoform/subunit mRNA were quantified by real-time PCR, anti- IFNP binding antibodies were detected by radioimmunoprecipitation, and neutralizing antibodies by cytopathic effect inhibition assay. Clinical measures of disease activity and disability progression were also obtained at all time points. At the individual-patient level, the response to IFNP therapy was extremely heterogeneous, including patients with stable or transitory, early or late loss of IFNP bioactivity, and patients with samples lacking MxA mRNA induction in spite of absence of antibodies. No interferon receptor isoform alterations that could explain these findings were found. At the group level, none of these biological features correlated with the measures of clinical disease activity or progression. However, when MxA mRNA was evaluated not at the single time point as a dichotomic marker (induced vs. non-induced), but as the mean of its values measured over the 6-to-24 month period, the increasing average MxA predicted a decreasing risk of short-term disability progression, independently from the presence of relapses. Therefore, a more bioactive treatment, even if unable to suppress relapses, reduces their severity by an amount that is proportional to MxA levels. Together with its feasibility in the routine laboratory setting, these data warrant the quantification of MxA mRNA as a primary tool for a routine monitoring of IFNP therapy.

Methods

Patients

For this prospective longitudinal observational study, 118 patients (36 men and 82 women, between 18 and 64 years of age), with a diagnosis of relap sing-remitting MS according to the McDonald criteria (McDonald 2001) were consecutively enrolled. To be included, patients were required to have an Expanded Disability Status Scale (EDSS) ranging from 0 to 4.5, and to be naive for IFNP therapy. After enrolment they received either intramuscular or subcutaneous (44 μg) IFNP- la (42 and 40 patients, respectively) or IFN - lb (36 patients), according to the principles of good clinical practice. The study lasted 36 months; visits were performed at baseline (TO), and then at 3 (T3), 6 (T6), 12 (T12), 24 (T24), 30 (T30) and 36 (T36) months of therapy. Blood was drawn 12 hours (+1 hour) after the patient-declared timing of the last IFNP injection. Patients were not receiving steroid therapy nor showed signs of viral infection at the moment of blood draw.

At all time points, standard neurological assessments, including reporting of relapses and careful EDSS evaluation, were also required. After the end of the study, EDSS calculation was validated by a neurostatus level-C certified neurologist (http://www.neurostatus.net/ ).

MxA, BAb and NAb quantification

IFN bioactivity analysis was performed by real-time reverse transcriptase PCR assay that measures MxA mRNA expression in patients' whole blood samples, as previously described (Capra 2007). Accordingly, MxA mRNA induction was expressed as relative units, also called "normalization ratio" (NR), which represents the fold-change in respect to MxA mRNA expression in a standard sample of a healthy donor. BAb and NAb quantification were performed as previously described, using a radioimmunoprecipitation assay (RIPA) for BAbs (Capra 2007), and a cytopathic effect (CPE) inhibition assay for NAbs (Antonelli 1998). For BAb analysis, in order to correct for the different amount of total radioactivity obtained after each radiolabeling session, the count per minute read for each serum was normalized as the percentage of the total activity of that session; thus, the reported BAb levels are expressed as "% total activity". BAb and MxA quantification was done in all patients at all time points, exception made for MxA, which was not done at T3; NAb titer was analyzed at several time-points in selected samples (149 determinations from 84 patients). The levels of Bab were expressed as count per minutes (cpm), those of Nab as ten-fold reduction units (TRU), and MxA as normalization rate (NR). Quantification oflFNAR subunits and isoforms

Primers and probes for IFNAR2 and IFNAR2.2 were from Vitale et al. (2006), while those for IFNAR1, IFNAR2.1 and IFNAR2.3 mRNA expression were designed with Primer Express software version 3.0 (Applied Biosystems) and were reported in Serana et al (2008), together with the employed real-time PCR protocol. The NR was used to express the results of IFNAR mRNA expression. Statistical analysis

The longitudinal analysis comparing the means between the groups of patients treated with the three drugs over the follow-up was performed by ANOVA for repeated measures based on linear mixed models, which were fitted with a random intercept. These models also permitted control for covariates, as well as use of all available data despite patients were progressively dropping-out ("unbalanced design"), thus enhancing the power of the analysis and providing a less biased picture of changes over time. For these analyses, the values of MxA, IFNAR and BAb were log-transformed in order to obtain an approximately normal distribution. Median EDSS changes between subgroups were compared by the Kruskal-Wallis and Mann- Whitney tests (used also to compare the area under the curve of non-transformed MxA values); annualized relapse rates were analyzed by negative binomial regression; proportions were compared by the Fisher's exact test. To identify factors associated to the risk of disability progression, logistic multivariable regression was performed. Several covariates including the age, basal EDSS, type of IFN treatment, presence of at least one relapse at T24, arithmetic mean of all log2- transfomred MxA levels measured between T6 and T24, BAb levels, and IFNAR expression levels, were first tested as possible predictors in univariate fashion. When P<0.10, variables were retained and tested (together with their interactions) in multivariable models. The chosen model included as predictors the presence of at least one relapse at T24 and the "average" log₂ MxA level between T6 and T24 (their interaction was not significant and thus excluded). Based on this model, and considering that a back-transformation of the arithmetic mean of log-values corresponds to the geometric mean of the original values, the predicted probability of experiencing a disability increase after 2 years in relation to the geometric mean of non- transformed MxA NR values was calculated. P-value threshold was set at the level of 0.05. Results

Study population

To perform a reliable longitudinal analysis of IFNP bioactivity over the study period, only patients with at least two consecutive laboratory determinations for each marker after therapy beginning were evaluated. Therefore, 18 patients who left the study before T12 and had undergone only one MxA determination after therapy initiation were considered early dropouts and thus excluded from the analysis. Of the remaining 100 patients, 17 terminated the study before reaching T24, and 26 additional patients did not complete the study extension at T36, yielding a total dropout count of 43 subjects, which, added to the early dropouts, makes a total of 61 dropouts (52%), with 57 subjects (48%) completing the 3-year study period. Reasons for dropout are listed in Figures 1A-1B, and baseline characteristics of analyzed patients in Figure 1C.

MxA, BAb, and NAb levels during treatment

The median MxA mRNA level at baseline was similar to that observed in a group of 100 healthy donors (0.78; IQR:0.78-1.34 vs. 0.78; IQR: 0.46-1.44; p=NS) (Zanotti, 2010).

Therefore, these MxA values were pooled together in order to determine the 99th percentile (3.83 NR), which was considered the cut-off used to classify patients as MxA mRNA induced {i.e., above the cut-off, MxA⁺) or MxA mRNA non-induced {i.e., below the cut-off, MxA-). After therapy initiation, the level of MxA mRNA increased similarly in the three treatment groups, and stayed constantly elevated, with minimal fluctuations, over the study period (Figure 2A). The only significant differences were observed at T6, when the MxA levels increased in patients receiving IFNP-la 44μg s.c, and at T18, when MxA decreased in those receiving IFNP- lb in comparison to the other two treatment groups.

Comparison of BAb level between healthy donors (HD) and multiple sclerosis patients before therapy initiation is shown in Figure 3. Even if patients were naive for therapy, the levels of BAbs in the samples of MS patients obtained at TO were significantly higher than that found in 140 healthy donors (median of normalized cpm: 0.98% vs. 0.62%; p<0.0001., Figure 3). Without wishing to be bound by any particular theory, it was thought that this may reflect the polyclonal increase of antibody production in MS, and accordingly, only the distribution of BAb values of MS patients at TO was used to calculate the 99th percentile, which represented the cut- off for BAbs (normalized cpm: 1.73%). The analysis of the kinetics of BAb production showed that the average BAb level during the course of treatment was higher in patients receiving ΙΕΝβ- lb in comparison to that of patients receiving ΙΕΝβ-la (p<0.001 at all time points from T3 to T30), with a sharp and quick increase starting as early as 3 months after therapy initiation, a peak at T6, and then a slow decrease over the rest of the study period until reaching levels similar to those seen in ΙΕΝβ- la- treated patients at T36 (Figure 2B). This average decrease was likely not only due to the increasing number of dropouts during the study period, because only three BAb positive (BAb⁺) patients left the study between T6 and T24, which was the period characterized by the sharpest fall of BAb levels, and because an identical pattern of decrease over time was seen if patients were divided in those who dropped out and those who completed the study (p=NS, not shown). Considering all samples pooled together, the sensitivity of BAb

measurement in respect to detecting MxA samples was 46%, while the specificity was 75%.

When the CPE assay was performed, a sample was considered NAb-positive (Nab⁺) if it had a titer of at least 20 ten-fold reduction units (TRUs), according to Antonelli et al. (1998). Because NAb levels were assayed only at selected time-points, it was not possible to infer statistically tested conclusions. However, because NAb quantification was performed at least once for each patient, and more than once in many of the patients in whom the other two tests suggested a likely loss of bioactivity, a descriptive picture of the results can be drawn, indicating that the kinetics of NAb appearance was similar to that of BAbs, with an early rise, in particular in subjects treated with IFNb-lb (Figure 2C).

The two classes of anti-IFNP antibodies were only partially overlapping because while NAbs were absent in all BAb-negative (BAb-) samples that were tested, they were found in only 32% of BAb⁺ patients, most of whom (90%) were treated with IFNb-lb (Figure 4). mRNA expression oflFNAR subunits/isoforms

In order to test whether IFNAR subunits modulation could have a significant impact on IFNP bioactivity, especially after 3 years of continuous receptor stimulation, as suggested in the case of the subunit IFNAR 1 (Serana 2008, Oliver 2007), and of the isoforms IFNAR2.2 and IFNAR2.3 (Gilli 2007, Gilli 2008), the mRNA expression of all IFNAR subunits and isoforms was quantified at all time points. Figures 5A-5B show the role of IFNAR2.2 and IFNAR2.3 isoforms on IFNP bioactivity. A significant effect was a global decrease in IFNAR2.2 mRNA over time (Figure 5A), independently from the type of IFNP administered. However, a multivariate regression model, performed after correcting for the presence of anti-IFNP antibodies and type of IFNP received, showed that, on average, the MxA mRNA levels were not influenced by modifications in the level of the isoform IFNAR2.2, but, unexpectedly, they increased with the growth of the soluble isoform IFNAR2.3 (Figure 5B). Analysis of the bioactivity profile at the individual-patient level

The kinetics of MxA mRNA induction, of BAb and NAb (when available) production was evaluated at the individual patient-level. A conserved ΙΡΝβ bioactivity would require the simultaneous presence of MxA-induction and, preferably, absence of BAbs and NAbs, according to the aforementioned cut-offs. Thus, the potentially "full biological responders" to IFNP therapy would be those patients with a conserved ΙΡΝβ bioactivity at all time points. However, several intermediate situations (i.e. sporadic MxA non-inductions or isolated antibody positivity) and changes of bioactivity status were observed in the distinct patients during the follow-up period. Categories that could represent the observed spectrum of bioactivity, which allowed for classification of patients into more homogeneous subgroups, were defined (Figure 5C).

A first group of 63 biological responders (63%) was made up by 37 patients whose samples were MxA⁺, BAb-, NAb^~ at all time points, and by 19 individuals that had only one isolated, sporadic non-induced MxA sample, in the context of a profile of high MxA-induction and constant lack of antibodies. Of note, all of these were being treated with IFNP-la (14 with IFNP-la i.m and 5 with sc IFNP-la). The 7 remaining patients of this group (all but one treated with IFNP-la i.m.), who presented with two or three non-consecutive, sporadic MxA non- inductions, were still considered biological responders because of the intermittent nature of their MxA non-induction in the total absence of Nabs or other factors, suggesting a truly significant loss of IFNP bioactivity.

A second group of likely "biological responders" was made by 24 patients that were characterized, for the most part, by a conserved MxA induction, despite the presence of antibodies of the BAb class in at least four of the seven time-points analyzed, with totally absent or under-the-cut off (<20 TRU) NAbs. In particular, MxA mRNA was induced at all time-points regardless of BAbs in 10 patients, while one MxA non-induced sample was found in 14 of them. In most patients BAbs appeared as early as 3 months after therapy beginning; peaked in the subsequent time-point; and then gradually declined up to undetectable values at the end of the follow up, so that only 7 patients had BAbs at T36. Eighteen patients belonging to this group were treated with IFNP-lb.

A third group of 10 patients was considered biologically non-responder due to several consecutive MxA non inductions, and was further divided into two subcategories because of the heterogeneity in the seeming reasons of bioactivity loss (presence vs. absence of anti- IFNP antibodies). Indeed, the first subcategory was made by 8 patients whose samples resulted not only MxA-non-induced in two or more consecutive occasions, but were also simultaneously BAbs⁺ and NAbs⁺; however, NAbs, which were tested at several time-points, resulted high (>200 TRU) only in one patient and very high (>400 TRU) in another one, and in their remaining samples their level of MxA induction was just above the cut-off or stayed fairly low (4<MxA<16 NR). These biologically non-responders had been treated with the highest dose of IFNP (7 with IFNP-lb and 1 with IFNP-la 44 μg s.c). The second subcategory of biologically non-responders was a puzzling subgroup of 2 individuals treated with IFNP-la i.m who were MxA non-induced at nearly all time-points of the follow-up, in the absence of both BAbs and NAbs. Any altered IFNAR subunit/isoform expression that could explain this seeming bioactivity loss was not found (data not shown). In order to exclude the presence of genetic polymorphisms that could render the MxA assay falsely-negative, the area bound by the realtime PCR MxA primer and probes was also sequenced, without finding any differences from the reference sequence (not shown). The kinetics of bioactivity loss was peculiar in at least 4 of the 10 biological non-responders: in one patient the bioactivity loss appeared late in the course of treatment (after 30 months); in two patients treated with IFNP-lb both BAbs and NAbs disappeared and MxA reverted back to induction at T24 or at T30, suggesting that a late recovery of bioactivity is possible; also in one of the patients who had been repeatedly MxA-non-induced (but was BAb7NAb^~), MxA reverted back to induction, albeit at a low level, at T36.

Finally, it was identified a fourth group of 3 patients, 2 of whom treated with IFNP-lb and 1 with IFNP-la i.m., who showed intermediate features and could not be included in any of the previous schemes. Indeed, their MxA, even if sometimes low, was always above the cut-off, despite they had BAbs, in at least two consecutive samples, and they had both BAbs and NAbs at one time-point. BAbs and NAbs, however, declined to levels below the cut-off at the following time points, even if in one of these patients NAbs were high-titred (>200 TRU). This suggests that bioactivity, if not fully, was conserved for the most part of the study period.

MxA as marker of disability progression

The annualized relapse rate and the proportion of relapse-free patients after 24 or 36 months of study period were calculated and used as markers of disease activity, while an EDSS change of at least 1 -point over a period of at least two years was considered a significant marker of disability progression (Noseworthy 1990, Rudick 2010). As expected, the proportion of relapse-free patients after 2 years of treatment was significantly higher among those who did not show disability progression in the same time lapse (82% vs. 20%, p<0.001). However, no significant associations were found between these clinical parameters and the presence of BAbs or the absence of MxA induction, even if patients were grouped according to the different bioactivity profiles obtained by the individual-patient level analysis, or if groups were pooled together to compare all biological responder vs. all biological non-responder patients. The only exception was a significantly different (between the four groups) proportion of subjects with an increased EDSS after 2 years, which was likely due to the two biologically non-responder patients without antibodies, who both showed a clinical worsening (Figure 5D). These results suggest that the risk of disease evolution is not clearly predicted by classifying of patients on the basis of the MxA induction and the presence of antibodies.

However, the analysis was done considering MxA as a binary, dichotomic variable (MxA⁺ vs. MxA-), determined only according to MxA mRNA level variation across a conventional threshold, while the amount of MxA mRNA varies in a continuous fashion (van der Voort 2010, Malucchi 2010). Thereafter, by repeated measure analysis the MxA values that were obtained across multiple observations in the patients with or without disability progression were compared (10 [12%] vs. 73 [88%] patients), finding that patients showing a 1-point EDSS increase after 2 years of treatment had a significantly lower average level of MxA induction (Figure 6A). More importantly, this difference was independent of the type of ΙΕΝβ treatment. A similar result was obtained by calculating the area under the curve for MxA expression from baseline to T24, which resulted lower in patients with an increased 1-point EDSS after 2 years (Figure 6B). These observations led to the hypothesis that a higher level of MxA induction could be associated to a better therapeutic response to IFNP, ultimately affecting the likelihood of EDSS worsening. Therefore, in order to verify to what extent a higher average MxA level could determine a reduced risk of 1-point EDSS increase, the relation between the two variables was studied by multivariable logistic regression. The best fitting model showed that each 1-unit in the "average"log₂MxA levels (or, in other words, each doubling in the geometric mean of non- transformed MxA) predicts a reduction of 47% in the risk of 1-point EDSS increase (OR: 0.53, p=0.02; CI: 0.30-0.92), and that the presence of at least one relapse in the first 2 years is a very strong independent predictor of EDSS worsening risk (OR 22.40, p=0.001, CL3.63-138.i l). No other covariates, including the type of IFNP treatment, basal EDSS, bioactivity profile, and IFNAR expression levels, had any significant impact on this risk. In particular, with this model, it was possible to calculate that relapsed patients with a geometric mean of MxA >16 NR have a probability of EDSS worsening significantly lower than 50%, which is similar to that calculated for patients without relapses (Figure 6C). A logistic model for EDSS increase in shown in

Figure 7. These results show the average MxA as a potential marker of disability progression. No other covariates, including the type of ΙΕΝβ treatment, basal EDSS, bioactivity profile, and IFNAR expression levels, had any significant impact on this risk.

As shown in Figure 2A, even though, on average, MxA mRNA grew similarly in the three groups of treatment with minimal fluctuations, the overall IFNP bioactivity profile of the single individual was rather heterogeneous across the consecutive timepoints and between distinct patients. As shown in Figure 8A, IFNP-bioactive patients were considered being MxA+, BAb-, and NAb- at all time-points. A few exceptions were patients who displayed sporadic (non-consecutive) MxA- samples without antibodies and were considered bona fide always bioactive (Figure 8B). Many IFNP-lb treated patients were BAb-i- at several time-points, but had a conserved MxA induction (Figure 8C). Surely IFNP-non-bioactive were considered patients being MxA- and BAb-i- (and NAb+, if performed) in at least three consecutive time- points (Figure 8D). Two patients were always MxA- without any antibodies. Different combinations of MxA/BAb/NAb status changing over the follow up gave rise to intermediate categories. Even if in some individual patients (as those shown in Figures 8A-8D) there was a correlation between the measures of IFNP bioactivity and the clinical picture, on average, weak correlations with the measures of disease activity or progression were found.

IFNAR subunit/isoform modulation was not associated to decreased IFNP bioactivity, even in patients resulting MxA- without antibodies; indeed, higher amounts of soluble isoform IFNAR2.3 mRNA were associated to increased MxA mRNA levels (Figure 9). In two patients, altered IFNAR subunits/isoforms expression was not found, although INFAR could have a role in determining bioactivity loss. When MxA was evaluated not at the single-time-point level, but as mean of the values measured over the 6-to- 24 month period, increasing MxA predicted a lower risk of 1-point-EDSS increase after 24 months (O.R. for each log2MxA: 0.53; c.L: 0.31- 0.93); the presence of at least one relapse had an independent and strong additive impact on this risk (O.R.: 23.30; c.L: 3.77-144.10). In particular, for an average MxA>16, the probability of disability progression was below 50% (similar to relapse-free patients), even in patients with relapses (Figure 10).

Discussion

This 3-year prospective longitudinal observational study was primarily aimed to study

IFNP bioactivity in a real-life setting. A primary tool to define each patient's IFNP bioactivity status was the quantification of MxA mRNA induction. Its quantification can be performed by a simple and reproducible real-time PCR assay (Pachner 2003), which can be easily transferred to the routine clinical practice (Zanotti 2010).

By applying this technique to samples obtained from 118 naive MS patients beginning

IFNP therapy and followed up clinically for 3 years, it was found that after therapy initiation MxA mRNA was stably induced to a similar extent by IFNP-la i.m., IFNP-la 44μg s.c. and IFNP-lb preparations, with an average value that, at the group-level, does not decrease, but fluctuates only slightly over time. IFNP bioactivity variations were then studied at the individual-patient level, by considering MxA expression as an all-or-nothing phenomenon and defining IFNP bioactivity as lost when MxA mRNA levels resulted below a cut-off determined in healthy subjects and MS patients naive for therapy. Accordingly, it was found that a total of 90/100 patients had an MxA-expression pattern suggestive of a conserved IFNP bioactivity, while 10/100 of patients were biological non-responders. In the majority of cases, the loss of IFNP bioactivity was evident as early as 12 months after therapy start, whereas only in one subject this happened after 30 months of treatment.

To identify the potential causes of IFNP bioactivity loss, a RIPA assay that is capable of detecting all anti-IFNP antibodies was performed. A CPE assay to search for NAbs in selected samples was also performed, in order to resolve uncertain cases in which a partial or fluctuating bioactivity was observed. The pattern and kinetics of antibody production was rather heterogeneous. In 8 of the 10 non bioactive patients, who were all treated with either one of the two IFNP injected subcutaneously, absence of MxA induction was always accompanied by the presence of BAbs, while NAbs were above their cut-off in the majority but not all (14 out of the 18) tested samples. This result confirms that anti-IFNP antibodies are the main determinant of IFNP bioactivity loss, even though Nabs may sometimes be undetected. This can be due to technical problems regarding the detection and quantification of IFNP-induced NAbs that may lead to NAb status misclassification in as much as 30% of samples, even in reference laboratories (Hartung 2012). In the remaining 2 biological (and clinical) non-responder subjects, both treated with IFNP-la i.m., repeated MxA non-inductions were observed in total absence of anti-IFNP antibodies. While impairments of IFNAR receptor system have been hypothesized as a possible determinant of bioactivity loss in patients with similar features (Gilli 2007), no peculiar alterations of IFNAR subunit/isoform were present in the two patients that could explain the loss of bioactivity. Overall, the data indicated that IFNAR modulation does not play a significant role in determining a bioactivity loss and the slight average down-modulation of the active isoform IFNAR2.2 mRNA level did not affect the average MxA induction. Overall, in the study population, IFNAR mRNA modulation was not correlated to IFNP bioactivity loss. These findings partially agree with those reported by Gilli et al (2008), which indicated a conserved IFNP bioactivity during a long-term therapy despite a gradual reduction in the functional IFNAR2.2 (defined as IFNAR-2c in their paper) isoform mRNA expression. The results herein support the agonistic role of the soluble isoform because higher levels of IFNAR2.3 mRNA were associated to higher MxA values in absence, however, of a univocal modulation of IFNAR2.3 over time.

Overall, in the study in which IFNAR subunits/isoforms are studied at the mRNA-level, the role of IFNAR modulation appears negligible, and its quantification does not seem useful to monitor IFNP bioactivity. Among the patients treated with IFNP-la i.m. that were classified as biological responders, there were samples sporadically resulting MxA-non-induced within a background of repeated MxA induction, without any kind of antibodies, nor any peculiar alterations of IFNAR subunit/isoforms expression. Because IFNP-la i.m. is administered once a week, the most likely explanation of this result could be an occasionally wrong sample taking more than a true, sporadic loss of bioactivity. We also evaluated whether the absent MxA mRNA induction could be due to a concurrent statin treatment, following the observations recently published by Feng et al (2012) that statins, in vitro, can reduce the production of MxA. But only one patient assumed a drug of the statin class within a time frame concomitant with a sample resulting ΜχΑ-BAb-NAb-, which was at the last point of his follow up, therefore no other data are available in support of this hypothesis. Another possibility is that a genetic alteration in the IFNP response genes, such as single nucleotide polymorphisms or mutations in the MX1 gene promoter or in other regulatory regions, could have altered the MxA response (Hemmer 2009, Cunningham 2005).

Finally, the repeated time -points' analysis in the individual patients, that allowed us to identify 3 patients in which MxA induction is recovered (although not at high levels) and antibodies disappeared as late as 24-36 months from therapy start, suggested that a very late restoration of IFNP bioactivity is possible.

Despite the heterogeneity of bioactivity profiles, one would expect that disease activity would vary between the groups of patients according to the different profiles of bioactivity, particularly in the light of a previous retrospective study indicating that MxA quantification, when analyzed at the group level, can predict clinical disease activity in patients with MxA levels under the cut-off (Malucchi 2008). This prospective study did not confirm this association as, when analyzed at the group level, the bioactivity profiles were for the most part unlinked from the clinical disease activity. Besides differences in the study designs (prospective vs. retrospective), a possible explanation of this discrepancy is that this strategy of studying IFNP bioactivity as an-all-or nothing phenomenon (MxA induced (MxA⁺) vs. MxA non-induced (MxA-)) may not be sensitive enough to detect small changes in disease activity or disability progression, especially on this temporal scale and relatively smaller sample size. It has to be taken into account that in some of our 10 patients classified as non-bioactive, there were sporadic samples, at certain time-points during the follow-up, in which MxA was slightly over the cut-off in presence of low/intermediate titers of NAbs, indicating that bioactivity can sometimes fluctuate between being completely lost and partially reduced but not totally absent, as if the amount of NAbs were not always sufficient to fully inhibit all IFNP binding. Similarly, it had also been previously observed by Malucchi et al (2010) that samples with levels of MxA falling into an intermediate, "grey-zone" had a reduced, yet not completely lost bioactivity. Therefore, it was assumed that measure of bioactivity on a continuously changing, quantitative scale could be more appropriate to represent the underlying phenomena. Accordingly, it was found that in the first 2 years of study period the average amount of MxA mRNA, considered on its original quantitative scale, was increased in patients with a lower risk of disability progression, expressed in terms of a 1 -point EDSS increase, which is an EDSS variation considered significant in describing a disability progression (Noseworthy 1990, Rudick 2010). A multivariate logistic regression model demonstrated that the proportion of the risk of 2-year progression that could be attributed to MxA was independent from the presence of clinically apparent relapses, which, by themselves, contributed as expected to this risk. More specifically, it could be estimated that patients showing an "average" value of MxA above 16 (or 4-log₂) relative units in the first 2 years of treatement, even in presence of clinically apparent relapses, had an estimated probability of disability progression lower than 50%, which was similar to the low probability observed in relapse-free patients. These data indicated that the levels of MxA, after being shown predictive of the relapse rate, can also be linked to a clinical measure of disability accumulation that, in turn, is predictive of long-term disability (Rio 2006, Rudick 2010). Thus, if similar results can be confirmed by larger studies, an evaluation of MxA on a quantitative scale may prove an efficient tool for identifying patients at high risk of progression, in addition to the commonly employed MRI lesion load and relapse rate.

A number of questions may arise from these findings. First of all, considering the independence of the MxA-effect from the number of relapses, which are the main clinical epiphenomenon of the underlying inflammatory activity, one may wonder which immunological protective mechanism is the increased MxA a marker of. Because it is obvious that not only the number but also the localization and the severity of relapses are crucial determinants of disability accumulation, the most likely explanation of our data could be that a more bioactive treatment, even if unable to suppress their occurrence, can reduce relapse severity (and thus the risk of EDSS worsening) by an extent that appears correlated to increasing average MxA levels.

Another possibility could be that the quantitative monitoring of MxA over time may be a more sensitive and realistic marker of the magnitude of IFNP protective effects against the basal "background" of inflammatory activity, which is not an all-or-nothing phenomenon but a continuous process that can provoke a slow worsening of the disease independent on the number of clinically manifest relapses. In other words, even if therapy can not completely suppress inflammation, a higher MxA over time is the marker of a more sustained IFNP-mediated protection, finally leading to a less severe damage. Another innovation is that MxA assay can help the clinicians to overcome the NAb dilemma. It is now widely recognized that NAbs have an impact on therapy success at the population-level (Polman 2010, Chiu 2009), and, indeed, Nabs appeared as the cause of MxA non-induction in the majority of the patients in this study, even if sometimes they were low tittered and underdetected. Furthermore, the consensus reached between the leading North American and European neurological societies after a decade of debates concluded that "at the individual patient level interpretation of NAb measurement can be complicated and will depend on the specific clinical situation for each patient" and that, in patients with intermediate or low NAb titres, additional information are needed, which can be provided by MxA bioactivity measurements (Polman 2010). Finally, inconsistent results regarding the same patient's NAb status were obtained in four reference laboratories in as much as one-third of the samples (Hartung 2012). On the contrary, the MxA mRNA assay appears as a more practical and convenient assay, easily standardized for a routine practice (Pachner 2003, Zanotti 2013); and the results in this example suggest that it is possible to set reference values indicating a higher probability of therapy success.

In conclusion, the average MxA can be considered as a reliable marker of the IFNP response long-term response, because it encompasses severity of relapses and disability, probably due to being a better marker of the in vivo IFNP effect, which is not an all-or-nothing phenomenon that the mere count of relapses can represent. IFNP bioactivity profile was heterogenous in the different patients. After three years of therapy IFNP bioactivity is conserved in for most patients. BAbs alone cannot define bioactivity loss, e.g., with IFNP-lb s.c. therapy. IFNAR subunit/isoform mRNA modulation is not associated to a loss of IFNP bioactivity over time. Lower average MxA levels during treatment are associated to a higher risk of disability increase after 24 months, which can be independent from the presence of clinically apparent relapses. Therefore, MxA mRNA quantification can stand as a primary tool to routinely monitor IFNP therapy.

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Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby

incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the worldwide web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the worldwide web at ncbi.nlm.nih.gov.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed.

Claims

What is claimed is:

1. A method of treating or preventing one or more symptoms associated with multiple sclerosis (MS), in a subject with MS who has experienced a relapse, comprising: acquiring a value of one or more MS biomarkers, said one or more MS

biomarkers comprising MxA, in the subject,

wherein: (i) an increased value in the level of MxA of at least 10, 12, 14, 16, 18 or 20-fold relative to a reference value, in response to an IFN-b agent, in the subject is indicative of a reduced probability of disability progression; and

(ii) a decreased value in the level of MxA compared to the level in (i), in response to the IFN-b agent, is indicative of an increased probability of disability progression; and

responsive to said increased value, continuing to administer to the subject the IFN-b agent; or

responsive to said decreased value, administration of the IFN-b agent is modified, thereby reducing one or more symptoms associated with MS.

2. A method for evaluating or monitoring an MS treatment that includes an IFN-b agent in a subject with MS who has experienced a relapse, comprising:

acquiring a value of one or more MS biomarkers, said one or more MS

biomarkers comprising MxA, in the subject,

wherein: (i) an increased value in the level of MxA of at least 10, 12, 14, 16, 18 or 20- fold relative to a reference value (e.g., a healthy control), in response to the IFN-b agent, in the subject is indicative of a reduced probability of disability progression; and

(ii) a decreased value in the level of MxA compared to the level in (i), in response to the IFN-b agent, is indicative of an increased probability of disability progression, thereby evaluating or monitoring the MS treatment.

3. The method of claim 1 or 2, wherein the level of MxA is the level of MxA mRNA.

4. The method of any of claims 1-3, wherein the increased value in the level of MxA is indicative of a lower risk of an increase in EDSS score.

5. The method of claim 4, wherein the increase in EDSS score is a 1 -point EDSS increase.

6. The method of any of claims 1-5, wherein the increased value in the level of MxA is indicative of a progression similar to a relapse-free patient.

7. The method of any of claims 1-6, wherein the reference value is obtained from one or more of: a healthy control, a healthy subject population, an MS subject population, an MS population having a relapsing form of MS, or the subject at a different time interval.

8. The method of any of claims 1-7, wherein the reference value is obtained from a healthy control.

9. The method of any of claims 1-8, wherein the IFNb agent is chosen from an IFN-b la molecule, an IFN-blb molecule, or a pegylated variant of an IFN-b la molecule or an IFN-b lb molecule.

10. The method of claim 9, wherein the IFNb- la molecule is Avonex® or Rebif®; and the IFNb- lb molecule is BETASERON® or BETAFERON®.

11. The method of any of claims 1-10, wherein the administration of the IFN-b agent is modified by administering a second alternative MS treatment.

12. The method of claim 11, wherein the second alternative MS therapy is chosen from:

(i) an antibody or fragment thereof against alpha-4 integrin or natalizumab;

(ii) an anthracenedione molecule or mitoxantrone; (iii) a fingolimod or FTY720;

(iv) a dimethyl fumarate or an oral dimethyl fumarate

(v) an antibody to the alpha subunit of the IL-2 receptor of T cells or daclizumab; or

(vi) an antibody against CD52 or alemtuzumab.

13. The method of any of claims 1-12, wherein the subject is a patient having one of: benign MS, relapsing-remitting multiple sclerosis (RRMS), primary progressive MS, or secondary progressive MS; clinically isolated syndrome (CIS) or clinically defined MS (CDMS).

14. The method of any of claims 1-13, wherein the subject is a patient with a relapsing form of MS.

15. The method of claim 14, wherein the relapsing form of MS is RRMS or relapsing secondary-progressive MS (relapsing SPMS).

16. The method of any of claims 1-15, wherein the value is acquired at a single- time point, or as an average value detected at predetermined time intervals.

17. The method claim 16, wherein the value is detected every 6, 12, 24, or 36 or more months after initiation of an MS therapy.

18. The method of any of claims 1-17, wherein said treating or preventing comprises reducing, retarding or preventing, a relapse, or the worsening of a disability, in the MS subject.

19. The method of any of claims 1-18, further comprising one or more of:

performing a neurological examination, evaluating the subject's status on the Expanded Disability Status Scale (EDSS), or detecting the subject's lesion status as assessed using an MRI.

20. The method of any of claims 1-19, further comprising obtaining a sample from the subject, wherein the sample is chosen from a non-cellular body fluid.

21. The method of claim 20, wherein the non-cellular fraction is chosen from plasma or serum.

22. The method of any of claims 1-21, wherein the subject is monitored in one or more of the following periods: prior to beginning of treatment; during the treatment; or after the MS treatment has been administered.

23. A kit for evaluating a sample from an MS patient, to detect or determine the value of one or more MS biomarkers, comprising, detecting the level of MxA with instruction indicating a value of MxA responsive to an IFN-b therapy.

24. The kit of claim 23, wherein said evaluating further comprises providing or transmitting information or a report, containing data of the evaluation or treatment to a report-receiving party or entity chosen from a patient, a health care provider, a diagnostic provider, or a regulatory agency.