WO2001014846A2

WO2001014846A2 - Methods and assay kits for detecting a pro-ms immune response via assay of b cells

Info

Publication number: WO2001014846A2
Application number: PCT/US2000/023130
Authority: WO
Inventors: Emilio Barbera-Guillem; M. Bud Nelson
Original assignee: Biocrystal Ltd.
Priority date: 1999-08-23
Filing date: 2000-08-23
Publication date: 2001-03-01
Also published as: AU6799100A; WO2001014846A3

Abstract

Provided are kits and methods for determining one or more B cell subpopulations as an indicator of the presence in an individual of a disease condition comprising multiple sclerosis (MS) and a pro-MS immune response, or a pro-MS immune response. The method comprises determining an amount of the one or more B cell subpopulations in a body fluid of an individual, comparing the amount determined to a comparative value selected from the group consisting of a reference value or a baseline value, for the one or more B cell subpopulations determined; wherein a difference in the amount of one or more B cell subpopulations determined from the individual when compared to the comparative value comprises the indicator of the disease condition.

Description

METHODS AND ASSAY KITS FOR DETECTING A PRO-MS IMMUNE RESPONSE VIA ASSAY OF B CELLS

FIELD OF THE INVENTION The present invention is related to biological testing, and in particular to methods and compositions for detecting a disease condition comprising multiple sclerosis by determining the presence or quantity of one or more selected B lymphocyte subpopulations. More particularly, the present invention is related to the discovery and detection of one or more selected B cell subpopulations in B cell -containing body fluids of individuals having an immune response which may promote disease progression in multiple sclerosis (a "pro-MS immune response"). The presence of the one or more selected B cell subpopulations may be used singly, or in combination, as a diagnostic marker in screening for the presence of a pro-MS immune response and/or MS in humans, and as a prognostic indicator to monitor disease progression or to monitor efficacy of treatment of an individual's pro-MS immune response and/or multiple sclerosis .

BACKGROUND OF THE INVENTION

Multiple sclerosis ("MS") is a chronic inflamma- tory disease of the central nervous system. The characteristic pathological feature, and still used as the primary basis for diagnosis of MS, is demyelination of the myelin sheath of neurons in the central nervous system. MS affects 250,000 to 350,000 in the United States, and approximately 1 million people worldwide. Typically, MS begins as a relapsing-remitting disease (RRMS) with periodic episodes of associated symptoms (e.g. various forms of neuritis). Often RRMS eventually changes to a progressive course of disease, secondary progressive MS (SPMS) , characterized by more CNS tissue damage which results in more debilitating symptoms. However, in 10 to 20% of individuals, the disease initially develops in a progressive form known as primary progressive MS (PPMS) . Depending on the classification scheme used, there may be additional categories of the MS disease process (e.g., "benign" MS, and "acute progressive" MS).

There is no clear understanding of the immuno- pathogenic processes associated with MS; and, to date, their lacks evidence of a unique immunologic abnormality in individuals with MS. Because of the incomplete understanding of the pathogenesis of MS, therapeutic advances have been slow to emerge. The myelin sheath and oligodendrocytes are believed to be main targets of autoreactive T cells which, when activated and reach the central nervous system, are thought to secrete proinflammatory cytokines . These cyto- kines are believed to induce astrocytes and leukocytes (including by activating microglia and macrophages) to secrete enzymes which damage myelin, and result in inflammation, demyelination, and axonal damage in the central nervous system characteristic in MS. Thus, studies have implicated a cell-mediated immune response, T cells recognizing epitopes of myelin basic protein (MBP) , in the pathogenesis of MS. Accordingly, there are various reports of determinations of T cell subsets in MS. However, it has been reported that individuals having MS displayed normal levels of B cells in peripheral blood as compared with healthy individuals.

Therefore, a need exists for methods which may be used to screen for the presence of markers that may be used as indicators as related to the progression of MS; and for methods to monitor efficacy of treatment for MS, such as inhibiting the progression or preventing further development of MS.

SUMMARY OF THE INVENTION According to a primary object of the present invention, the presence or quantity of a selected B cell subpopulation is determined by a method in which one or more determinants, preferentially expressed by cells of the selected B cell subpopulation, is specifically bound by one or more affinity ligands, thereby facilitating detection and quantification of the selected B cell subpopulation.

It is another object of the present invention to provide a method for screening for the presence of a pro-MS immune response in an individual by determining the relative number (e.g., expressed in cell number, or as a percentage) of one or more B subpopulations that may be circulating in peripheral blood, or B cell containing-body fluids other than peripheral blood (e.g., cerebrospinal fluid, CSF) of the individual. In a preferred embodiment, the one or more B cell subpopulations: (a) are an indicator of a chronic immune, inflammatory process which may contribute to the CNS tissue damage characteristic of MS; and/or (b) are markers for a pro-MS immune response and/or MS, which markers may be used in one or more of diagnostics and prognostics. According to a further object of the present invention, assay kits are provided for performing the above described screening methods . The assay kits may include various components, depending on the complexity of the screening method utilized for assaying for the one or more B cell subpopulations according to the present invention. Assay kits would typically contain a combination of reagents, with each reagent comprising an affinity ligand capable of binding to a determinant that is expressed by the one or more B cell subpopulations, and that facilitates determination of the one or more B cell subpopulations present in the sample analyzed. More specifically, the assay kits may include one or more affinity ligands that detects most B lymphocytes (e.g., specifically binds to a pan B cell determinant) , one or more affinity ligands that detects a B cell subset determinant (e.g., binds specific- ally for a determinant characteristic of mature B cells and/or activation by antigen or in an immune response) , and an affinity ligand that detects sTn (e.g., binds specifically to sTn) for purposes of detection of a B cell sub- population according to the present invention, and may further comprise: one or more reagents comprising a known amount of reference B cells comprising one or more B cell subpopulations according to the present invention for use as a positive control (for testing the ability of the one more detection reagents to specifically bind the determinant (s) for which the detection reagent has binding specificity; i.e., to determine if the detection reagents are still functional for their intended purpose or if they have lost some of their binding ability such as due to improper storage conditions) or as a standard (in representing a known amount that falls within the normal range for the one or more B cell subpopulations) for the method; instructions for use of the assay kit and components; and optionally, other accessories useful in carrying out the methods of the present invention. The foregoing objects are achieved because of the unexpected discovery of and demonstration that there can exist one or more B cell subpopulations in amounts that differ in individuals having a pro-MS immune response, as compared to a comparative amount in healthy controls or in individuals having inflammatory diseases other than MS. For example, the one or more B cell subpopulations can be detected or detected and quantitated (collectively or individually referred to as "determined"), and the resultant value can be used as an indicator for identifying individuals that may have a pro-MS immune response and MS. The above and other objects, features, and advantages of the present invention will be apparent in the following Detailed Description of the Invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term "one or more B cell subpopulations" is used herein, for purposes of the specification and claims, to mean a B lymphocyte subtype selected from the group consisting of an overall population of (all or the majority of subsets of) B cells, sTn+ B cells, sTn+ Bl cells, sTn+ memory B cells, and a combination thereof. "Memory" B cells (e.g., antigen-stimulated B lymphocytes, or progeny thereof, which are not antibody secretors) is a term that is used herein to also encompass "mature" B cells as known to those skilled in the art. Memory B cells, in general, are known to those skilled in the art to be capable of surviving long periods of time (e.g., years), and to circulate freely between the peripheral blood and lymphoid tissues. Memory B cells (e.g., CD5- B cells), as known to those skilled in the art, and for purposes of the present invention, are not inclusive of Bl cells (CD5+ B cells) . Bl cells are known to those skilled in the art as a subset of B cells which produce primarily low affinity IgM and respond primarily to T-independent antigens.

The terms "affinity ligand" and "detection reagent" are used herein, for purposes of the specification and claims, to mean a molecule which has binding specificity and avidity for a determinant associated with, and which can be used for determination of, the one or more B cell subpopulations; and in a preferred embodiment, the molecule may further comprise a detectable moiety which is used to label (or is conjugated to) the molecule to facilitate detection and quantitation for diagnostic and/or prognostic purposes. For example, one type of affinity ligand, specific for a pan B cell marker (e.g., CD19+) , may be used alone to detect an overall B cell population (e.g., CD19+ cells). In continuing with this example, this type of affinity ligand (against a pan B cell marker) may also be used in combination with other affinity ligands (e.g., specific for CD21+ to detect memory B cells; CD19+ CD21+ cells). In general, affinity ligands are known to those skilled in the art to include, but are not limited to, lectins, antibodies, immunoreactive fragments produced or derivatives derived from antibodies, peptides, and aptamers. Immunoreactive fragments produced, or derivatives derived, from an antibody molecule are frag- ments which retain all or a portion of the binding function of the whole antibody molecule, and are known to those skilled in the art to include F(ab')₂, Fab¹, Fab, Fv, scFV, Fd' and Fd fragments. Methods for producing the various fragments from MAbs are well known in the art. For example, F(ab')₂ ^can be produced by pepsin digestion of the monoclonal antibody, Fab' may be produced by reducing the disulfide bridges of F (ab¹ ) ₂ fragments, and Fab fragments can be produced by papain digestion of the monoclonal antibody. In a preferred embodiment, affinity ligands that may be used for assaying for the one or more B cell subpopulations according to the present invention include, but are not limited to: anti-CD19 antibody, including use alone to detect most B cells, or in combination with anti-CD21 antibody and anti-sTn antibody to detect memory B cells that are CD19+ CD21+ sTn+ cells, or anti-CD19 in combination with anti-sTn antibody to detect activated B cells that are CD19+ sTn-t- cells, or in combination with anti-CD5 antibody and anti-sTn antibody to detect Bl cells that are CD19+ CD5+ sTn+ cells; anti-CD21 antibody; anti-CD22 antibody; anti-sTn antibody; Lym-1 antibody (antibody against the B cell deter- minant recognized by Lym-1; see, e.g., U.S. Patent No. 5,789,554), CDIM antibody (antibody against the B cell determinant recognized by CDIM; see, e.g., U.S. Patent No. 5,593,676), anti-CD45R (RAhi or RO) antibody, and a combination thereof. An affinity ligand may further comprise a detectable moiety which has been coupled (using covalent or noncovalent or other means known in the art) to the affinity ligand. The term "detectable moiety" is used herein, for purposes of the specification and claims, to mean a label molecule that is directly or indirectly detectable, and wherein the detectable moiety (when coupled to affinity ligand) may be indicative of the presence of a B cell subpopulation sought to be detected in a sample according to the method of the present invention. Detectable moieties may include, but not limited to, enzymes (e.g., peroxidase, alkaline phosphatase, etc.), radioisotopes, haptens (e.g., biotin, avidin, etc.), chromophores, fluorescent molecules, and functionalized nanocrystals, as known to those skilled in the art of diagnostics. In a preferred embodiment, the detectable moiety comprises a fluorescent molecule comprising water soluble functionalized nanocrystals (e.g., CdSe core, ZnS shell) ; or a fluorophore which may include, but is not limited to, fluorescein (isothiocyanate) , fluorescein derivatives, pthalocyanine dyes, phycoerythrin, up-converting phosphors, peridinin-chlorophyll protein, fluorescamine, dansyl chloride, rhodamine, Texas red tandem, phycocyanin tandem, allophycocyanin tandem, and coumarin derivatives.

The presence of the affinity ligand in the method according to the present invention may be detected directly when the it further comprises a detectable moiety, or indirectly when a secondary affinity ligand (e.g., a secondary antibody, as known to those skilled in the art) which is labeled with the detectable moiety is then used to specifically bind to an unlabelled (primary) affinity ligand (e.g., a combination of primary antibody, and labeled secondary antibody) .

The term "clinical sample" is used herein, for purposes of the specification and claims, to mean a body fluid comprising peripheral blood, or a body fluid other than peripheral blood (particularly cellular effusions associated with multiple sclerosis such as CSF) . A preferred body fluid may be used to the exclusion of a body fluid other than the preferred body fluid. The term "clinical sample" also encompasses a preparation which is derived from the clinical sample, and which is enriched for one or more B cell subpopulations, as will be more apparent from the following descriptions. Likewise, the term "peripheral blood" also encompasses a preparation which is derived from peripheral blood, and which is enriched for one or more B cell subpopulations, as will be more apparent from the following descriptions. As illustrative examples, a mononuclear cell preparation may be derived from peripheral blood using methods known to those skilled in the art (e.g., a com- mercially available medium for gradient centrifugation) ; and individual B cell subpopulations may be enriched for by magnetic separation using commercially available kits for cell separation.

The term "determinant" with reference to one or more B cell subpopulations to be determined, is used herein, for purposes of the specification and claims, to mean one or more cell-associated molecules which may be used to determine an amount of B cells expressing that determinant in a clinical sample (e.g., in distinguishing the one or more B cell subpopulations from mononuclear cell populations (e.g., T cells or monocytes) other than the one or more B cell subpopulations) ; and in a preferred embodiment, one or more molecules expressed by one or more B cell subpopulations which can be used as markers for diagnostic and/or prog- nostic purposes as related to a pro-MS immune response, wherein the one or more molecules is involved in and responsible for binding to one or more affinity ligands having binding specificity and avidity for the determinant. As will be apparent to one skilled in the art, there are various determinants which may be used to detect one or more B cell subpopulations. For example, one type of determinant (a pan B cell determinant) comprises a molecule whose preferential cellular expression is on all or most B cells. In a preferred embodiment, a pan B cell determinant is known by those skilled in the art to include CD19, CD20, CD22,

CD72, Lym-1, CDIM, and the like; and therefore, may be used by itself in a manner to detect an overall B cell population (e.g., a combination of B cell subpopulations) that may be present in a clinical sample. Another type of determinant (a "B cell subset" determinant) is a molecule whose prefer- ential cellular expression is generally limited to a particular subset (subpopulation) of B cells. A B cell subset determinant is not inclusive of CD80 and/or CD86, which are molecules not expressed preferentially by a B cell subpopulation but rather are expressed by dendritic cells, B cells, and monocytes (antigen presenting cells) . In a preferred embodiment, one or more B cell subset determinants is used in combination with a pan B cell determinant to identify a B cell subpopulation, as will be described herein in more detail. In a preferred embodiment, a B cell subset determinant is known by those skilled in the art to include CD5 (which in combination with a pan B cell determinant is known to identify Bl cells) , CD45RA (which in combination with a pan B cell determinant is known to identify a B cell subpopulation which is immunologically naϊve) , CD45R0 (which in combination with a pan B cell determinant is known to identify an activated B cell subpopulation such as activated by an immune response, cytokine, or antigen) , CD21 or CD75 or CD79a or CD79b or RP105 (each known to identify mature B cells), and a combination thereof. Another type of deter- minant is the sTn epitope. It is believed that expression of the sTn epitope by B cells represents a subset of B cells that may be an indicator for the presence of a chronically- activated immune response, such as in a pro-MS immune response in a progressive MS disease process (e.g., in SPMS) . In a preferred embodiment a determinant may comprise a cell-surface molecule such as a receptor, an adhesion molecule, a ligand, an antigen, and a combination thereof. A preferred determinant my be used to the exclusion of a determinant other than the preferred determinant .

The term "individual" is used herein, for purposes of the specification and claims, to mean a mammal, and preferably a human, and more preferably a human who is being screened for, or at risk of developing, or has developed, MS and/or a pro-MS immune response. This may include one or more of an individual having MS (e.g., PPMS, RRMS, or SPMS or other category of MS) ; an individual having indicators of a chronically activated humoral immune response (e.g., as determined by the presence of the one or more B cell subpopulations and the method according to the present invention) that accompanies clinically evident MS; and an individual who has been treated for MS, and therefore, for whom the monitoring of efficacy of treatment is desired. In a preferred embodiment of the present invention, provided is a method for determining the one or more B cell subpopulations in a clinical sample from an individual at risk for developing, or has developed a disease condition selected from the group consisting of MS, a pro-MS immune response, and a combination thereof. The presence of the one or more B cell subpopulations according to the present invention which differs in a body fluid of an individual having MS or suspected of having MS, as compared to a comparative value determined from healthy individuals, may be utilized for a use selected from the group consisting of as an indicator for staging the MS disease process, as an indicator for progressing (e.g., progressive, clinically worsening) of the MS disease, as an indicator of a humoral immune response and

B cell involvement that accompanies the MS disease process (a "pro-MS immune response"), and a combination thereof. Note that it may be possible in treated individuals that clinical manifestations may be significantly improved (e.g., by treatment of T cell involvement and/or immune effector cell involvement) that the individual may be pronounced to be free of an active MS disease process, yet still harbor a pro-MS immune response as indicated by the presence of the one or more B cell subpopulations according to the present invention in an amount that differs from a comparative reference value.

The term "amount" is used herein, for purposes of the specification and claims, a number which is expressive of a quantity of a B cell subpopulation according to the present invention determined from a clinical sample. For example, the amount of a B cell subpopulation from the determination may be expressed as the actual (e.g., absolute) number of B cells of that subpopulation by itself. Alternatively, amount of a B cell subpopulation from the determination may be expressed in relation to a certain parameter (as a relative value); e.g., number of B cells of that subpopulation determined in relation to the quantity of blood or fluid (e.g., number of cells/ml), or in relation to the number of a total cell population (e.g., expressed as a percentage of the number of total white blood cells, or as a percentage of the number of overall B cells, or as a percentage of the number of total lymphocytes) , or in relation to a reference value (e.g., in relation to a pre-determined clinical value) .

The term "differs" or "difference" is used herein relative to a comparison between an amount of a B cell subpopulation determined in a clinical sample and that of a reference value, for purposes of the specification and claims, to mean that the amount of the B cell subpopulation determined in a sample falls outside the range of normal clinical values for that B cell subpopulation that is established by clinical studies (the range of normal clinical values being a "reference value") . Hence, where the indicator (e.g., amount of one or more B cell subpopulations) determined according to the present invention differs with respect to a reference value, such an indicator (e.g., amount of the one or more B cell subpopulations) may be indicative of a disease condition comprising MS, a pro-MS immune response, and a combination thereof. In a preferred use, the term "differs" is used herein, for purposes of the specification and claims, to mean that there is a statis- tically significant difference between an indicator determined according to the present invention ("first value") from a first sample, and an indicator determined (e.g., of the same type or population, or determined by the same combination of affinity ligands) to which it is compared ("second value") obtained from a sample other than the first sample. For example, a first value that is of statistical significant difference as compared to a second value may comprise the first value being a number that is at least about two standard deviations outside the mean of the second value. In an embodiment in which a value from a sample

(first value) is compared to a second value, the first value and the second value may be obtained from the same individual at different points in time, e.g., to monitor the course of the disease condition, or to test the efficacy of treat- ment of the disease condition. Alternatively, the second value may represent a relative number (e.g., mean or median) as established from individuals lacking MS and/or a pro-MS immune response, as will be more apparent from the following embodiments. In general, the methods, assay formats, and compositions, of the present invention are used to generate indicators to identify individuals as having or lacking a disease condition selected from the group consisting of MS, a pro-MS immune response, and a combination thereof; in providing an additional parameter to a competent health professional in making a medical opinion. The term "pro-MS immune response" is used herein, for purposes of the specification and claims, to mean a humoral immune response induced against an epitope comprising a terminal alpha 2,6 linked sialic acid (e.g., comprising sialyl Tn or sTn which comprises a terminal sialic acid alpha 2,6 linked to GalNAc) of a shed antigen (glyco- molecule) , resulting in production of IgG antibody against the epitope ( "anti-α (2, 6) NeuAc Ab" ) , and immune complexes comprised of the shed antigen comprising the epitope com- plexed to anti-α (2 , 6)NeuAc Ab; wherein the shed antigen is released or produced particularly in relation to CNS tissue damage characteristic of MS during the MS disease process. In a preferred embodiment, the resultant immune complexes bind to and induce Fc receptor-expressing cells (e.g., one or more cell types selected from the group consisting of granulocytes, macrophages, microglia, activated mast cells, astrocytes, oligodendrocytes) which results in the release of inflammatory mediators (e.g., cytokines and/or tissue degradative enzymes) which may promote (contribute to) CNS tissue damage characteristic of MS (e.g., demyelination and plaques characteristic of MS) . The present inventors have discovered and described a similar immune response, a pro- tumor immune response. In a preferred embodiment, the anti- α(2,6) NeuAc Ab is induced by a shed antigen comprising glycolipid; and in a more preferred embodiment, glycolipid selected from one or more of the alpha series of ganglio- sides (e.g., GDlα, GTlaα, GQlbα, derivatives thereof which contain one or more additional terminal sialic acids alpha 2,6 linked to GalNAc, and a combination thereof) .

Currently the diagnosis of MS is imprecise. Imaging that detects what appears to be plaques in CNS tissue is typically insufficient, by itself, to give a definitive diagnosis of MS. Often, diagnosis of MS is made only after both detection of plaques and of clinically evident neuropathy. Additionally, staging of MS is typical- ly done by subjective measurements of exacerbation of symptoms, as well of other clinical manifestations. There are difficulties in diagnosis and staging because symptoms vary widely among individuals and change frequently within the individual. Thus, there is the need for tests which can aid in the diagnosis and staging of MS. Further, presently there are no commercially available tests to evaluate for the presence of a pro-MS immune response. There is a need for laboratory tests that distinguish individuals who are more likely to have a favorable prognosis (e.g., one or more of stable remission; limited, localized disease progression; response to anti-MS therapy that either stabilizes or reduces the rate of disease progression) from individuals who are likely to have an unfavorable prognosis (e.g., individuals having undergone anti-MS therapy but who still have indications of a pro-MS immune response, and are thus still at risk for progression of the disease process; individuals having both clinically evident MS and a pro-MS immune response) . Additionally, there is a need for markers that can be used as indicators for predicting whether a particular therapeutic (e.g., drug or immunotherapeutic) can effectively reduce a pro-MS immune response.

In that regard, the present invention relates to a discovery that selected B cell subpopulations may be present in the body fluids of individuals having a disease condition selected from the group consisting of a pro-MS immune response, MS, and a combination thereof; wherein the amount of the one or more B cell subpopulations in these individuals differs when compared to an amount of a comparative reference value determined from healthy individuals (e.g., without evident MS disease) . In a preferred embodiment of the present invention, an indicator for the presence of a pro-MS immune response which can be used for diagnosis, prognosis, and staging in the MS disease process, may comprise detection of one or more B cell subpopulations selected from the group consisting of an overall B cell population, sTn+ B cells, sTn+ memory B cells, sTn+ Bl cells. The one or more B cell subpopulations may be found in circulating in peripheral blood; or alternatively, may be found in any cellular effusions associated with MS (e.g., cerebrospinal fluid) . Thus, a kit to detect one or more B cell subpopulations, in a sample from an individual having or suspected of having MS, comprises a first affinity ligand which specifically binds a pan B cell determinant, and a second affinity ligand which specifically binds sTn. The kit may further comprise one or more third affinity ligands which specifically bind a B cell subset determinant. In a more preferred embodiment, the kit comprises a first affinity ligand which specifically binds a pan B cell determinant, and a second affinity ligand which specifically binds sTn, and one or more third affinity ligands which specifically bind a B cell subset determinant. For purposes of illustration, the first affinity ligand may be selected from the group consisting of an affinity ligand which specifically binds CD19, an affinity ligand which specifically binds CD20, an affinity ligand which specifically binds CD22, an affinity ligand which specifically binds CD72, an affinity ligand which specifically binds Lym-1, an affinity ligand which specifically binds CDIM, and a combination thereof. There are numerous anti-sTn antibodies that may be obtained commercially (e.g., monoclonal antibodies B72.3 and HBStn) . The third affinity ligand may be selected from the group consisting of an affinity ligand which specifically binds CD5, an affinity ligand which specifically binds CD45RA, an affinity ligand which specifically binds CD45RO, an affinity ligand which specifically binds CD21, an affinity ligand which speci- fically binds CD75 , an affinity ligand which specifically binds CD79a, an affinity ligand which specifically binds or CD79b, an affinity ligand which specifically binds RP105, and a combination thereof. Cells detectable by this kit comprise an overall B cell population, sTn+ B cells, sTn+ memory B cells, sTn+ Bl cells.

In one embodiment of the present invention, a diagnostic indicator for a disease condition comprising a pro-MS immune response, which may be used for diagnosis, prognosis, or staging of MS, in an individual may differ (i.e., comprise a significant decrease) in an amount determined of the overall population of B cells in the peripheral blood (e.g., B cells measured by using a pan B cell marker such as CD19+ cells) as compared to the reference value (e.g., the amount of overall population of B cells in peripheral blood of apparently healthy individuals) . In another embodiment of the present invention, a diagnostic indicator for a disease condition comprising a pro-MS immune response, which may be used for diagnosis, prognosis, or staging of MS, in an individual may differ (comprise a significant increase) in an amount determined of one or more B cell subpopulations (selected from the group consisting of sTn+ B cells, sTn+ memory cells, sTn+ Bl cells, and a combination thereof) in peripheral blood as compared to the reference value for the respective B cell subpopulations. In another embodiment, the diagnostic indicator comprises, in the peripheral blood of an individual, a decrease in an overall B cell population, and an increase in one or more B cell subpopulations (selected from the group consisting of sTn-t- B cells, sTn+ memory cells, sTn+ Bl cells, and a combination thereof) as compared to the reference values for the respective B cell subpopulations.

In another embodiment of the present invention, a prognostic indicator for an individual (e.g., as to a criterion for eligibility for treatment of a disease condition comprising a pro-MS immune response and MS) , comprises determining an amount of the one or more B cell subpopulations in a clinical sample from an individual, wherein a difference in that amount as compared to a reference value (range determined from healthy individuals) or a baseline value (a value previously determined from the same individu- al for the one or more B cell subpopulations) for the one or more B cell subpopulations comprises the prognostic indicator.

In another embodiment of the present invention, as an indicator for monitoring efficacy of treatment of an individual who is undergoing or has undergone treatment of a disease condition selected from the group consisting of MS, a pro-MS immune response, and a combination thereof, determined from successive samples (e.g., one or more samples pre-treatment, and one or more samples post-treatment) from the individual is the amount of one or more B cell subpopulations according to the present invention. By comparing the effect of treatment on the amount of one or more of B cell subpopulations determined, information relating to the efficacy of the therapy (including possible need for modification of the treatment regimen) may be obtained.

In accordance with one embodiment of the method according to the present invention, the method comprises determining an amount of the one or more B cell subpopulations in a body fluid of an individual; comparing the amount determined to a comparative value selected from the group consisting of a reference value (comprising a predetermined value or a range of values found in the same type of body fluid of apparently healthy individuals) for the one or more B cell subpopulations determined, a baseline value (compris- ing a predetermined value from one or more previous samples of the same type of body fluid obtained from the same individual being tested) for the one or more B cells sub- populations determined, and a combination thereof; wherein a difference in the amount of one or more B cell subpopula- tions determined from the individual when compared to the comparative value comprises an indicator of a disease condition selected from the group consisting of MS and a pro-MS immune response, and a pro-MS immune response. More specifically, the indicator comprises a difference in the amount of one or more B cell subpopulations selected from the group consisting of an overall B cell population, sTn+ B cells, sTn+ memory cells, sTn-i- Bl cells, and a combination thereof .

In a preferred embodiment, provided is a method for screening for the presence of a disease condition selected from the group consisting of MS and a pro-MS immune response, and a pro-MS immune response, the method comprising: (a) obtaining a clinical sample comprising peripheral blood from an individual; (b) contacting the sample with one or more affinity ligands which specifically binds to one or more B cell subpopulations; (c) determining an amount of the one or more B cell subpopulations (e.g., by detecting and quantitating the one or more affinity ligands which binds to the B cells) present in the sample; (d) comparing the amount of the one or more B cell subpopulations to a reference value for the one or more B cell subpopulations determined; wherein a difference in the amount of one or more B cell subpopulations determined from the individual when compared to the reference value comprises an indicator of the presence of the disease condition. More specifically, the indicator comprises a difference (with respect to the individual as compared to the reference value) in the amount of one or more B cell subpopulations selected from the group consisting of a decrease in amount of an overall B cell population, an increase in amount of sTn+ B cells, an increase in amount of sTn+ memory cells, an increase in amount of sTn+ Bl cells, and a combination thereof. As apparent to one skilled in the art, the comparison should be made with samples of the same type (i.e., the amount in peripheral blood of the individual should be compared to a reference value established for peripheral blood; and the amount in cerebrospinal fluid of the individual should be compared to a reference value established for cerebrospinal fluid) . In this method, and other methods of the present invention, a preferred combination of B cell subpopulations may be used to the exclusion of a combination of B cell subpopulations other than the preferred combination.

In another preferred embodiment, provided is a method of monitoring the course (progression, stabilization, or reduction) of a disease condition selected from group consisting of MS and a pro-MS immune response, and a pro-MS immune response, in a prognostic manner (e.g., at different points of time relative to the individual such as during or after treatment of the individual's disease), the method comprises: (a) obtaining a first clinical sample (preferably comprising peripheral blood) from an individual; (b) con- tacting the first sample with one or more affinity ligands which specifically bind one or more B cell subpopulations, and determining an amount of the one or more B cell sub- populations in the first sample; (c) obtaining a second clinical sample (of the same body fluid type as the first clinical sample) from the individual, wherein the second sample is obtained at a different point in time than the obtaining of the first sample; (d) contacting the second sample with one or more affinity ligands which specifically bind the one or more B cell subpopulations, and determining an amount of the one or more B cell subpopulations in the second sample; (e) comparing the amount of the one or more B cell subpopulations determined in the first sample to the one or more B cell subpopulations determined in the second sample; wherein a difference in the amount determined from the second sample as compared to the amount determined from the first sample may be used as an indicator of a change in the course of the disease condition.

For example, in samples comprising peripheral blood, where the second sample shows a significant decrease in amount of one or more of sTn+ B cells, sTn+ memory B cells, sTn+ Bl cells, as compared to the an amount of respective one or more types of these B cell subpopulations in the first sample, such a decrease may comprise an indicator that the course of the disease condition is being suppressed or reduced, which may also be an indicator of favorable prognosis. In another illustrative example of this embodiment, wherein the amount of overall B cells is increased and the amounts of sTn-i- Bl cells and sTn-i- memory B cells are decreased in the second sample as compared to the amounts for the respective B cell types in the first sample, such differences may comprise an indicator that the MS disease and/or pro-MS immune response is being suppressed or reduced, which may also be an indicator of favorable prognosis. In a further illustrative embodiment, wherein the amounts of sTn+ B cells and sTn+ Bl cells determined from the second sample are significantly increased as compared to the relative amounts of these B cell subpopulations determined from the first sample, such a significant increase may comprise an indicator that the course of the disease condition is progressing (e.g., a worsening of the disease con- dition which may reflect an increase in clinical manifesta- tions associated with MS) , and which may also be an indicator of an unfavorable prognosis.

Thus, an indicator of a change in the course of MS and/or a pro-MS immune response may be used as a prognostic indicator relative to: (a) the effect, if any, of treatment which the individual may be undergoing, when the indicator is generated as a result of comparison between successive samples (e.g., one or more clinical samples obtained from the individual before treatment as compared to one or more clinical samples obtained after treatment; one or more clinical samples obtained from the individual before treatment as compared to one or more clinical samples obtained during or after a phase of treatment; one or more clinical samples obtained from the individual during or after a phase of treatment before treatment as compared to one or more clinical samples obtained after completion of treatment) ; (b) stabilization or improvement or worsening of the pathological condition. As apparent from the disclosure herein, any such treatment may include, but is not limited to, anti- MS therapy, therapy to reduce a pro-MS immune response, therapy to reset an immune system which currently is programmed with a pro-MS immune response, and a combination thereof. As known to those skilled in the art, treatment of MS and/or a pro-MS immune response ("anti-MS therapy") may include, but is not limited to, chemotherapy, vaccine therapy, tolerization therapy, immunotherapy (e.g., β- interferon) , and a combination thereof. This prognostic indicator may be used in the prediction of treatment outcomes of individuals having a disease condition selected from the group consisting of MS, a pro-MS immune response, and a combination thereof, following administration of treatment to the individual. Thus, the amounts of one or more B cell subpopulations according to the present invention may be useful in monitoring the effectiveness of treatment wherein such treatment is intended to impact (indirect- ly or directly) on the disease condition of an individual. Generally, a prognostic indicator provides an additional parameter to a competent health professional in making a medical decision concerning the efficacy of, or need for additional , treatment . For purposes of the description, the methods and compositions of the present invention will be illustrated in the following examples.

EXAMPLE 1 This Example illustrates that a clinical sample, to be assayed for an amount of one or more B cell subpopulations according to the present invention, may be used as obtained or may processed in a manner that includes, but is not limited to, enrichment for mononuclear cells (e.g., containing a plurality of lymphocyte types including T cells and B cells) , and enrichment for an overall lymphocyte type (e.g., overall population of B cells). For example, methods for enriching for mononuclear cells are well known in the art, and include overlaying peripheral blood on a density gradient medium (e.g., Ficoll-Hypaque or Percoll or

Lymphocyte Separation Medium) , and then performing density gradient centrifugation. Depending on the density gradient medium used, typically the mononuclear cells may be harvested from the interface or buffy layer of the gradient. The mononuclear cell population may be further processed to obtain a cell preparation enriched in B cells using one of several methods known to those skilled in the art. For example, neuraminidase-treated sheep red blood cells may be added to the mononuclear cell population, and the mixture may be centrifuged in a density gradient medium. T cells will bind (rosette) with the sheep red blood cells, and therefore are found in the cell pellet. In contrast, a lymphocyte preparation enriched in B cells would remain at the interface and can be harvested. Alternatively, a lymphocyte preparation enriched in B cells may be obtained in a negative selection process. For example, the mononuclear cell population may be mixed with magnetic beads coated with one or more antibodies that bind to T lymphocytes (e.g., anti-CD2 mAb, anti-CD3 mAb, anti-CD4 mAb, anti- CD8 mAb, anti-CD28 mAb, or a combination thereof) . A magnetic field is then applied, thereby immobilizing the T lymphocytes. The rest of the cell suspension (portion of the mononuclear cells which are not immobilized) comprise a lymphocyte preparation enriched in B cells.

In a method of positive selection, a lymphocyte preparation enriched in B cells may be obtained from the clinical sample. For example, the clinical sample is mixed with magnetic beads coated with antibodies that bind to most B lymphocytes (e.g., anti-CD19 mAb, anti-CD20 mAb) . A magnetic field is then applied, thereby immobilizing the B lymphocytes. The rest of the cell suspension (portion of the mononuclear cells which are not immobilized) is discarded. The magnetic beads-B cell complex may be applied directly in an assay for determining an amount of the one or more B cell subpopulations, or the B cells may first be eluted from the magnetic beads using methods known to those skilled in the art (e.g., competition with free ligand) and then assayed for the one or more B cell subpopulations.

EXAMPLE 2 This is an illustrate example for determining an amount of the one or more B cell subpopulations according to the present invention. In order to best perform the methods of the present invention, the clinical sample must be analyzed using a process for determining an amount of at least one of the B cell subpopulations according to the present invention. Such processes include, but are not limited to, immunofluorescence using affinity ligands which binding specifically to the one or more B cell subpopulations to be assayed, and a fluorescent microscope; chemi- luminescence; flow cytometry using affinity ligands with binding specificity for the one or more B cell subpopulations to be determined; and a cell-based assay such as a cell-based enzyme-linked immunosorbent assay ("cELISA"). In a cELISA, cells containing the one or more B cell subpopula- tions to be determined are fixed to the wells of an ELISA plate. For example, each well of a 96 well plate may be incubated with a lOOμl of a solution of poly-L-lysine hydro- bromide (5 mg/ ml) in a buffered saline solution for 30 minutes at room temperature. After removing the solution, the cells (about 100,000 to 200 , 000/well) are plated, the plate is then centrifuged (e.g., at 100 x g for 5 minutes) , and the supernatant is then removed. Glutaraldehyde in buffer (0.25%) is added to each well, and then incubated for 5 minutes at room temperature. The wells containing fixed cells may then be washed with a Tris-buffered saline or other suitable solution, and affinity ligand may then be added in accordance with an ELISA protocol in assaying for the one or more B cell subpopulations to be determined (e.g., incubation with affinity ligand, one or more washes, and subsequent detection of affinity ligand bound to the fixed cells) . As will be apparent to one skilled in the art, using a method according to the present invention, the one or more B cell subpopulations may be determined, and then the amount may be expressed in one of several ways . For example, an amount of each of the one or more B cell subpopulations may be expressed as an absolute number, or a relative number expressed in relation to a certain parameter; e.g., number of cells/quantity of blood (e.g., ml) , or number of cells/number of total cell population (e.g., number of total white blood cells, or mononuclear cells or lymphocytes) , or number of a B cell subpopula- tion/overall B cells where it is a subpopulation of B cells being determined.

In one preferred embodiment, an amount of sTn+ memory B cells may be expressed in relation to the overall number of memory B cells. Thus, for example, sTn+ memory B cells may be expressed as a percentage of memory B cells determined by a pan B cell determinant and a B cell subset determinant which identifies mature B cells. Illustrative examples of a combination of a pan B cell determinant and a B cell subset determinant representative of memory B cells include, but are not limited to, CD19+ CD21+ cells, CD20+ CD21+ cells, CD19+ CD22+ cells, or CD20+ CD22+ cells. As an illustrative example, sTn+ memory B cells detected as CD19+ CD21+ sTn+ cells may be expressed as a percentage of memory B cells detected as CD19+ CD21+ cells. In another preferred embodiment, sTn+ B cells may be expressed in relation to the overall number of B cells determined by a pan B cell determinant. Thus, for example, sTn+ B cells detected as CD19+ sTn+ cells may be expressed as a percentage of overall B cells detected as CD19+ cells. In another preferred embodi- ment, sTn+ memory B cells may be expressed in relation to the overall number of B cells. Thus, for example, sTn-i- memory B cells expressing a pan B cell determinant, a B cell subset determinant , and sTn may be expressed as a percentage of overall B cells expressing a pan B cell determinant. As an illustrative example, sTn+ memory B cells detected as

CD19+ CD21+ sTn+ cells may be expressed as a percentage of B cells detected as CD19+ cells. In another preferred embodiment, sTn+ Bl cells may be expressed in relation to the overall number of Bl cells. Thus, for example, sTn+ Bl cells detected as CD19+ CD5+ sTn+ cells may be expressed as a percentage of Bl cells detected as CD19+ CD5+ cells. Further, it will be apparent to one skilled in the art that the amount determined of a B cell subpopulation will vary depending on factors which include, but are not limited to, the specific processes (methodology) used to determine the B cell subpopulation, the nature of the one or more affinity ligands used in the determination, the origin and processing of the clinical sample tested for the B cell subpopulation, and the laboratory personnel and instruments used to perform the assays used to determine the B cell subpopulation.

In a preferred and illustrative embodiment, one or more B cell subpopulations is assayed for by flow cytometry. The general principles involved in flow cytometry are well known in the art . Parameters that may be used in the process of flow cytometry include (a) light scatter for determining one or more cell properties, including shape, size (forward scatter) , index of refraction, granularity (side scatter) , and roughness; and (b) changes in electrical impedance as each cell passes through the sensing region, which can be used to determine cell volume. Thus, light scatter can be used to gate on (select for) lymphocytes based on the size, granularity and cell volume of cells of the lymphocyte subpopulation.

Where a fluorescent molecule is used as the detectable moiety of an affinity ligand having binding specificity for a B cell subpopulation to be determined, as cells bound to affinity ligand move (typically one by one) through a beam of excitation light from a light source (typically a laser or arc lamp) , a fluorescence emission is detected at a wavelength which is typically different than the wavelength of excitation. The intensity of the fluorescence emission detected is generally proportional to the amount of the affinity ligand bound to the cell; and hence can be used to determine the relative expression by or association with that cell of the determinant bound, and an amount of cells of the one or more B cell subpopulations expressing or having associated therewith that determinant. While the type of light source used will bear on the choice of fluorescent molecules used as a detectable moiety, it should be apparent to those skilled in the art that a wide latitude of choice can be exercised in selecting one or more suitable light sources for use in the methods of the present invention. For example, multiple fluorescent analyses (e.g., detecting and measuring more than one fluorescence emission from a cell having bound thereto 2 types of affinity ligand with each affinity ligand recognizing a different determinant on the B cell subpopulation to be deter- mined than the other affinity ligand) may be performed using either a dual light source or a single light source. In that regard, two different detectable moieties (comprising two different fluorescent molecules) may be used, provided there is sufficient spectral spacing between the emission optima of two different fluorescent molecules to permit individual detection without substantial spectral overlap. This may be achieved by a combination of filters (and mirror) to permit simultaneous detection of the spectrally separated emissions of two different, excited fluorescent molecules to provide information on multiple detector molecule use in a single flow cytometric run. For example, an argon laser can be used to excite a combination of a number of fluorescent molecules, the emissions of which can be spectrally separated with different filters and photo- detectors (e.g., FITC: fluorescein isothiocyanate at 525nm; Pe : phycoerythrin at 575nm, and Pe-Cy5: phyco-erythrin- cyaninδ at 640 nm; PCP : peridinin-chlorophyll protein at 680nm; Texas red tandem at 610 nm, phycocyanin tandem at 640nm, and allo-phycocyanin at 660nm) . Regarding a dual light source, it is known to those skilled in the art that flow cytometers useful for detecting cells can include two channels which can detect cells specifically labeled with two fluorescent molecules; and using a separate light source to excite each of the two different fluorescent molecules. In one embodiment of determining one or more B cell subpopulations according to the present invention in a clinical sample, mononuclear cells were isolated from the clinical sample using a density gradient medium and by density gradient centrifugation. Aliquots, each of approximately 1 million cells, were treated in one of several dif- ferent ways. A first aliquot of cells was left unstained, so as to act as a control for possible auto-fluorescence . A second aliquot of cells was mixed in a staining process with isotype affinity ligand. In that regard, it is widely accepted by those skilled in the art that a desirable control for setting the negative region markers (to account for the fluorescence due to non-specific background observed with the staining process) is to stain with a mAb of the same subclass as the mAb used in the testing, but with an irrelevant specificity (e.g., does not specifically recognize a determinant on the B cell subpopulation to be determined) . Thus, the second aliquot was mixed with an IgGl control antibody labeled with FITC, an IgGl control antibody labeled with Pe, and an IgGl control antibody labeled with Pe-Cy5. This treated second aliquot serves as a negative control relative to any non-specific binding of the isotype (IgGl) antibodies to the cells to be detected. A third aliquot of cells may be stained with one or more affinity ligands having binding specificity for the B cell subpopula- tion to be determined. As an illustrative example, where the B cell subpopulation to be determined is sTn+ memory B cells, the third aliquot of cells is double-stained (stained jointly) with an antibody against a pan B cell determinant (e.g., anti-CD19 antibody (IgGl mAb)) labeled with Pe-Cy5, and an antibody against a B cell subset determinant (e.g., anti-CD21 antibody (IgGl mAb)) labeled with FITC, and an antibody against sTn (e.g., an anti-sTn IgGl mAb labeled with Pe) .

Any one of several available staining protocols may be used for cell staining. For example, for the first aliquot of cells which remain unstained, the cells were mixed with staining buffer alone (e.g., 50 μl of a physiologically acceptable buffer) . The staining buffer utilized was phosphate buffered saline containing 2% fetal calf serum and 0.1% sodium azide. In general, and for mixing an aliquot of cells with a detector molecule, the cells (e.g., sample volume ranging from 20 μl to 100 μl) were incubated with the one or more different, pre-titered detector molecules for 20-40 minutes at 4°C. After this incubation, optional further washes may be made in physiologically acceptable buffer. The sample may be further diluted to a final volume for analysis on the flow cytometer. In this embodiment, the second aliquot of cells (as the negative control for the staining process) was mixed with staining buffer and with 1:10 dilutions of an isotype IgGl labeled with FITC, an isotype IgGl labeled with Pe, and an isotype IgGl labeled with Pe-Cy5; and then incubated for 30 minutes in the dark at 4°C. The mixture was then centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and a wash solution (e.g., 150 μl of a physiologically acceptable solution) was used to suspend the cell pellet, and then the mixture was centrifuged (a wash step) . The wash step may be repeated one or more times. The cell pellet from the final wash is then taken up in a physiologically acceptable solution in a sufficient volume for flow cytometric analysis (e.g., 200- 250 μl) .

Preferred staining combinations

Memory B cells and sTn+ memory B cells

In determining memory B cells, an aliquot of cells was double-stained using essentially the same protocol sum- marized above, except that the antibodies mixed with the cells in this aliquot were anti-CD19 antibody (IgGl mAb) labeled with Pe-Cy5, and an anti-CD21 antibody (IgGl mAb) labeled with FITC (final dilution of each mAb was 1:10) . For determining sTn-i- memory B cells, an aliquot of cells was triple- stained wherein the cells were first mixed and incu- bated with anti-sTn antibody (IgGl mAb) , and then washed; followed by mixing and incubating with a secondary rabbit anti-mouse IgG antibody labeled with Pe, and then washed; followed by a double-staining with anti-CD19 antibody labeled with Pe-Cy5, and an anti-CD21 antibody labeled with FITC, and then washed. Additionally, by gating on the appropriate parameters, overall B cells (CD19+ cells) may be determined. By gating on the appropriate parameters, a B cell subpopulation comprising sTn-i- B cells (CD19+ sTn+ cells) may be determined. B cells and sTn+ B cells

In determining an amount of overall B cells, an aliquot of cells was double-stained using essentially the same protocol as summarized above, except that the antibodies mixed with the cells of the this aliquot were anti-CD19 antibody (IgGl mAb) labeled with Pe-Cy5 (final dilution of each mAb was 1:10) . Alternatively, for sTn+ B cells, the cells were first mixed and incubated with anti-sTn antibody (IgGl mAb) , and then washed; followed by mixing and incubating with a secondary rabbit anti-mouse IgG antibody labeled with Pe, and then washed; followed by staining with anti-CD19 antibody labeled with Pe-Cy5, and then washed. This alternative staining protocol, with the appropriate gating, allows for determination of both overall B cells (CD19+ cells) and sTn+ B cells (CD19+ sTn+ cells) . sTn+ Bl cells

In determining a B cell subpopulation comprising sTn+ Bl cells, an aliquot of cells was triple-stained, wherein the cells were first mixed and incubated with anti-sTn antibody (IgGl mAb) , and then washed; followed by mixing and incubating with a secondary rabbit anti-mouse IgG antibody labeled with Pe, and then washed; followed by a double-staining with anti-CD19 antibody labeled with Pe-Cy5, and an anti-CD5 antibody labeled with FITC, and then washed. Staining and incubation times were similar to those described above. Additionally, by gating on the appropriate parameters, overall B cells (CD19+ cells) may be determined. Also by gating on the appropriate parameters, overall Bl cells (CD19+ CD5+ cells) may be determined. As will be apparent to one skilled in the art, various combinations of stainings may be performed to determine a combination of B cell subpopulations according to the present invention. A number of commercially available flow cytometers can be used as the instrument on which is performed the method of the present invention. Desirably, the flow cytometer has a single laser source; and in a preferred embodiment, the single laser source is an argon laser tuned at 488 nanometers (nm) . Additionally, the flow cytometer is operatively connected to appropriate operating software and data management systems.

According to the method of the present invention, determined were amounts of B cell subpopulations in clinical samples obtained from individuals having a disease condition selected from the group consisting of MS, and MS and a pro- MS immune response; and, as shown in Table 1, the results were compared to comparative values of the same types of B cell subpopulations determined in clinical samples obtained from apparently healthy individuals ("reference value"; expressed as the mean ± std. deviation) and determined in clinical samples obtained from individuals having solid, nonlymphoid tumors and/or a pro-tumor immune response

(expressed as the mean + std. deviation) . In this illustration, the clinical samples comprised peripheral blood obtained by venipuncture into blood collection tubes, wherein peripheral blood mononuclear cells were isolated and then analyzed; and the determinations were performed using flow cytometric methods by the techniques disclosed herein. Light scatter was used as a parameter to gate on primarily lymphocytes based on the size, granularity and cell volume of lymphocytes. In addition to gating for light scatter, each sample undergoing the staining process was gated for respective fluorescence emission(s). For example, when memory B cells were quantitated by double-staining (e.g., for CD19 and CD21) , the analysis was gated on those cells positive for CD19 expression as determined by detection of Pe-Cy5 fluorescent emission. CD19 positive lymphocytes were considered to represent the relative overall population of B cells in the clinical sample analyzed. CD19 positive lymphocytes were then gated for those cells also positive for CD21 expression as determined by detection of FITC fluorescent emission. Lymphocytes double stained for both CD19 and CD21 were considered to represent memory B cells. Such CD19+ CD21+ B cells were then expressed as a percentage of overall B cells by using the formula:

(the relative number of CD19+ CD21+ cells/ relative number of CD19+ cells) X 100. A similar procedure was also used to determine sTn-t- B cell subpopulation (e.g., CD19+ sTn+ B cells) . Such

CD19+ sTn-i- B cells were expressed as a percentage of overall

B cells by using the formula:

(the relative number of CD19+ sTn+ cells/ relative number of CD19+ cells) X 100.

A similar procedure was also used to determine sTn+ memory B cell subpopulation (e.g., CD19+ CD21+ sTn+ B cells) . Such CD19+ CD21+ sTn+ B cells were expressed as a percentage of memory B cells by using the formula:

(the relative number of CD19+ CD21+ sTn+ cells/ relative number of CD19+ CD21+ cells) X 100.

A similar procedure was also used to determine a Bl cell subpopulation (e.g., CD19+ CD5+ B cells). Such CD19+ CD5+ B cells were expressed as a percentage of overall B cells by using the formula:

(the relative number of CD19+ CD5+ cells/ relative number of CD19+ cells) X 100.

A similar procedure was also used to determine an sTn+ Bl cell subpopulation (e.g., CD19+ CD5+ sTn+ cells). Such CD19+ CD5+ sTn+ B cells were expressed as a percentage of overall Bl cells by using the formula:

(the relative number of CD19+ CD5+ sTn+ cells/ relative number of CD19+ CD5+ cells) X 100.

In Table 1, shown are amounts of B cell subpopulations determined from apparently healthy individuals (Table 1, "Reference value"), from individuals having a pro-tumor immune response and solid, nonlymphoid tumor (Table 1, "Tumor/PTIR" ) , from an individual with secondary progressive MS (SPMS) and with evidence of a pro-MS immune response (PMSIR) as determined by immunoassays (Table 1, "SPMS/ PMSIR"), from an individual with relapsing remitting MS (RRMS) and with evidence of a pro-MS immune response (PMSIR) as determined by immunoassays (Table 1, RRMS/PMSIR) . Individuals with primary progressive MS (PPMS) fail to show evidence of a pro-MS immune response, and hence typically demonstrate B cell subpopulations within the range of reference values; further confirming the corollary that PPMS may be a less inflammatory form of MS than forms other than PPMS.

Table 1

Determinations of one or more B cell subpopulations, as illustrated in Table 1, show that a difference in amounts of the one or more B cell subpopulations may be associated with the more inflammatory forms of MS (e.g., secondary progressive MS) and a pro-MS immune response (e.g., with the difference in one or more B cell subpopulations appearing after serological evidence of a pro-MS immune response first appears) . Note that there is a sig- nificant decrease in an of overall B cells (CD19+ cells) in an individual having secondary progressive MS and pro-MS immune response (SPMS/PMSIR) as compared to values in apparently healthy individuals (reference value) . There is a significant increase in amount of sTn+ B cells (CD19+ sTn+ cells) in an individual having SPMS/PMSIR as compared to values in the reference value. While no significant difference was noted in memory B cells (CD19+ CD21+ cells) , there is a significant increase in amount of sTn+ memory B cells (CD19+ CD21+ sTn+ cells) in an individual having SPMS/PMSIR as compared to reference value. While no significant difference was noted in the amount of Bl cells (CD19+ CD5+ cells) , there is a significant increase in the amount of sTn-t- Bl cells (CD19+ CD5+ sTn+ cells) in an individual having SPMS/PMSIR as compared to reference value. Note also from Table 1 that the pattern of B cell subpopulations according to the present invention in individual having SPMS/PMSIR is distinguishable from that of individuals having a pro-tumor immune response (e.g., Tumor/ PTIR) , particularly in the amount of one or more B cell subpopula- tions selected from the group consisting of sTn+ B cells

(e.g., CD19+ sTn+ cells), memory B cells (e.g., CD19+ CD21+ cells) , sTn+ memory B cells (e.g., CD19+ CD21+ sTn+ cells), sTn+ Bl cells (e.g., CD19+ CD5+ sTn+ cells), and a combination thereof. Also illustrated in Table 1 are amounts of B cells from the peripheral blood of an individual newly diagnosed with relapsing-remitting MS (RRMS) , and with few clinically evident symptoms of MS-related neuropathy to date. Thus, the differing (as compared to the reference values) amounts of the B cell subpopulations according to the present invention becomes evident as the course of the stage of MS progresses from the initial stages of RRMS to a more inflammatory stage, as represented by SPMS. Therefore, the B cell subpopulations may be used in monitoring the course of progression of MS (since a significant number of individuals with RRMS progress to SPMS) and for staging of the disease condition comprising MS (e.g., in distinguishing SPMS from PPMS and RRMS, wherein SPMS represents a more inflammatory form than PPMS or RRMS) , or a pro-MS immune response (e.g., wherein RRMS presents with serological markers for a pro-MS immune response, but whereas SPMS can present with both serological markers and cellular markers (see, Table 1) of a pro-MS immune response) .

EXAMPLE 3

This Example illustrates embodiments of assay kits according to the present invention for performing methods for determining one or more B cells subpopulations in a clinical sample from an individual. As apparent to those skilled in the art, the assay kits may include various components, depending on the complexity of assay method utilized. An assay kit contains affinity ligands that facilitate determination of the one or more B cell subpopulations present in the sample analyzed. More specifically, the assay kits may include one or more affinity ligands that detects most B lymphocytes (e.g., specifically binds to a pan B cell determinant) , one or more affinity ligands that detects a B cell subset determinant (e.g., binds specifically to a determinant characteristic of mature B cells and/ or activation by antigen or in an immune response) , and an affinity ligand that detects sTn (e.g., binds specifically to sTn) , for purposes of detection of a B cell subpopulation according to the present invention; and may further comprise: one or more reagents comprising a known amount of reference B cells comprising one or more B cell subpopulations according to the present invention for use as a positive control (for testing the ability of the one more affinity ligands to specifically bind the determinant (s) for which the affinity ligand has binding specificity; i.e., to determine if the affinity ligands are still functional for their intended purpose or if they have lost some of their binding ability such as due to improper storage conditions) or as a standard (in representing a known amount that falls within the reference value (normal range) for the one or more B cell subpopulations) in the method; instructions for use of the assay kit and components; and optionally, other accessories useful in carrying out the methods of the present invention.

In a preferred embodiment, the kit according to the present invention comprises at least one affinity ligand which specifically binds a pan B cell determinant (e.g., wherein the determinant comprises CD19, CD20, CD22, CD72 , Lym-1, CDIM, or the like); at least one affinity ligand which specifically binds a B cell subset determinant (e.g., wherein the determinant comprises CD5 , CD45RA, CD45RO, CD21, CD75W, CD79a, CD79b, RP105, or the like); and at least one affinity ligand which specifically binds to sTn. The affi- nity ligands of the kit may further comprise a detectable moiety coupled thereto. In a preferred embodiment, the kit comprises an anti-CD19 monoclonal antibody as the at least one affinity ligand which specifically binds a pan B cell determinant; an anti-CD 21 monoclonal antibody and an anti- CD5 monoclonal antibody as the least one affinity ligand which specifically binds a B cell subset determinant; and an anti-sTn monoclonal antibody as the at least one affinity ligand which specifically binds to sTn. Thus, in this preferred embodiment , the kit may be used to determine one or more B cell subpopulations selected from overall B cells (CD19+ cells) , sTn+ B cells (CD19+ sTn+ cells) , and sTn+ memory B cells (CD19+ CD21+ sTn+ cells) .

Additionally, wherein the assay kit comprises affinity ligands comprised of monoclonal antibodies, the kit may further comprise a monoclonal antibody comprising an isotype control monoclonal antibody which is the same isotype as the one or more affinity ligands in the kit for binding the one or more B cell subpopulations. For example, and for purposes of illustration but not limitation, wherein the assay kit comprises a labeled anti-CD19 mAb of IgGl sub- class, a labeled anti-CD21 mAb of IgGl subclass) , and an anti-sTn mAb of IgGl subclass; an isotype control antibody may comprise a labeled IgGl mAb that lacks binding specificity for B cells. Thus for analyses of a clinical sample, the isotype control antibody may be used as a control to rule out non-specific staining by affinity ligands of B cells during the assay process. In another illustrative example, wherein an aptamer is used as the affinity ligand, an aptamer of the same general backbone sequence (e.g., differing primarily only in the sequence conferring binding specificity) may be used as an isotype control.

The assay kit according to the present invention may further comprise a known amount of reference B cells selected from the group consisting of a known amount of B cells, a known amount of sTn+ B cells, a known amount of memory B cells, a known amount of sTn+ memory B cells, a known amount of Bl cells, a known amount of sTn+ Bl cells, and a combination thereof. There may be a separate container for each of the reference B cells according to the present invention; or there may be a single container for a combination of a reference comprising more than one B cell subpopulation. The known amount of reference B cells may be stored in a solution, or may be lyophilized for reconstitu- tion, frozen, or a combination thereof. The reference B cells may be fixed by prior treatment with any one of a number of solutions known in the art to include, but are not limited to, 1% paraformaldehyde, methanol, methanol/acetone, acetone, 2% (v/v) paraformaldehyde and acetone, and 70% ethanol. The reference B cells may be used to test the efficacy of the staining process used for determining the amount of the one or more B cell subpopulations in a clini- cal sample, and may also be reacted with isotype control monoclonal antibody, as previously described herein in more detail. The known amount of reference B cells may comprise a standard that is representative of a threshold value characteristic of a B cell subpopulation in an individual having a disease condition selected from the group consisting of MS, a pro-MS immune response, and a combination thereof; or is representative of a threshold value characteristic of a reference value; wherein exemplary respective threshold values are listed in Table 1. It will be apparent to one skilled in the art that cells comprising the known amount of reference B cells for the assay kit according to the present invention are readily available. For example, B-cell lines expressing CD19, CD21, and CD22 have been described previously; an EBV-positive B cell line ("BEVA") expresses CD19, CD20, and CD21; whereas an EBV-positive B cell line ( "Jijoye-P3HR-l" ) strongly expresses CD19 and CD20 with weak expression of CD21; and B cell lines expressing CD19, CD20, and CD21 have been described previously ("YOS-B") .

The foregoing description of the specific embodiments of the present invention have been described in detail for purposes of illustration. In view of the descriptions and illustrations, others skilled in the art can, by applying, current knowledge, readily modify and/or adapt the present invention for various applications without departing from the basic concept, and therefore such modifications and/or adaptations are intended to be within the meaning and scope of the appended claims .

What is claimed is:

Claims

1. An assay kit comprising:

(a) one or more affinity ligands which specifically bind a determinant comprising a pan B cell determinant;

(b) one or more affinity ligands which specifically bind a B cell subset determinant; and

(c) one or more affinity ligands which specifically bind a determinant comprising sialyl Tn (sTn) .

2. The kit according to claim 1, wherein the one or more affinity ligands further comprises a detectable moiety.

3. The kit according to claim 1, wherein the one or more affinity ligands which specifically binds a determinant comprising a pan B cell determinant comprises an affinity ligand which specifically binds CD19.

4. The kit according to claim 1, wherein the one or more affinity ligands which specifically binds a determinant comprising a B cell subset determinant is selected from the group consisting of an affinity ligand which specifically binds CD21, an affinity ligand which specifically binds to CD5, and a combination thereof.

5. The kit according to claim 1, wherein the one or more affinity ligands which specifically binds a determinant comprising a pan B cell determinant comprises an anti-CD19 monoclonal antibody; wherein the one or more affinity ligands which specifically binds a determinant comprising a B cell subset determinant is selected from the group consisting of an anti-CD21 monoclonal antibody, an anti-CD5 monoclonal antibody, and a combination thereof; and wherein the one or more affinity ligands which specifically bind a determinant comprising sTn comprises an anti-sTn monoclonal antibody.

6. The kit according to claim 5, wherein the one or more affinity ligands which specifically binds a determinant comprising a B cell subset determinant comprises an anti- CD21 monoclonal antibody, and an anti-CD5 monoclonal antibody.

7. The kit according to claim 6, wherein one or more B cell subpopulations detectable by the kit comprise one or more B cell subpopulations selected from the group consisting of overall B cells (CD19+ cells) , sTn+ B cells (CD19+ sTn+ cells) , sTn+ Bl cells (CD19+ CD5+ sTn+ cells) , sTn+ memory B cells (CD19+ CD21+ sTn+ cells), and a combination thereof.

8. The kit according to claim 5, further comprising a reagent selected from the group consisting of an isotype control monoclonal antibody for each isotype of affinity ligand included in the kit, reference B cells comprising a known amount of the one or more B cell subpopulations, and a combination thereof.

9. The kit according to claim 1, further comprising a reagent comprising reference B cells comprising a known amount of the one or more B cell subpopulations for a use selected from the group consisting of as an assay staining control, as an assay standard, and a combination thereof.

10. The kit according to claim 7 further comprising a reagent comprising reference B cells comprising a known amount of the one or more B cell subpopulations detectable by the kit for a use selected from the group consisting of as an assay staining control, as an assay standard, and a combination thereof.

11. A method for screening for a disease condition selected from the group consisting of MS and a pro-MS immune response, and a pro-MS immune response, the method comprising:

(a) obtaining a clinical sample from an individual;

(b) contacting the sample with one or more affinity ligands which specifically binds to one or more B cell subpopulations;

(c) determining an amount of the one or more B cell subpopulations present in the sample by measuring the one or more affinity ligands bound;

(d) comparing the amount of the one or more B cell subpopulations to a reference value for the one or more B cell subpopulations determined; wherein a difference in the amount of one or more B cell subpopulations determined from the individual, when compared to the reference value, comprises an indicator of the presence of the disease condition.

12. The method according to claim 11, wherein the one or more affinity ligands comprises one or more affinity ligands which specifically bind a determinant comprising a pan B cell determinant, one or more affinity ligands which specifically bind a B cell subset determinant, and one or more affinity ligands which specifically bind a determinant comprising sTn.

13. The method according to claim 12, wherein the clinical sample comprises peripheral blood, and wherein the indicator is selected from the group consisting of a decrease in an overall B cell population, an increase in sTn+ B cells, an increase in sTn+ memory cells, an increase in sTn+ Bl cells, and a combination thereof.

14. The method according to claim 13, wherein the indicator is selected from the group consisting of a decrease in CD19+ cells, an increase in CD19+ sTn+ cells, an increase in CD19+ CD5+ sTn+ cells, an increase in CD19+ CD21+ sTn+ cells, and a combination thereof.

15. The method according to claim 12, wherein the one or more affinity ligands further comprises a detectable moiety.

16. A method of monitoring the course of a disease condition selected from group consisting of MS and a pro-MS immune response, and a pro-MS immune response, the method comprises : (a) obtaining a first clinical sample from an individual; (b) contacting the first sample with one or more affinity ligands which specifically bind one or more B cell subpopulations present in the first sample, and determining an amount of the one or more B cell subpopulations in the first sample by measuring the one or more affinity ligands bound; (c) obtaining a second clinical sample from the individual, wherein the second sample comprises the same body fluid type as the first sample and is obtained at a different point in time than the obtaining of the first sample; (d) contacting the second sample with one or more affinity ligands which specifically bind the one or more B cell subpopulations in the second sample, and determining an amount of the one or more B cell subpopulations in the second sample by measuring the one or more affinity ligands bound;

(e) comparing the amount of the one or more B cell subpopulations determined in the first sample to the one or more B cell subpopulations determined in the second sample; wherein a difference in the amount determined from the second sample as compared to the amount determined from the first sample may be used as an indicator of a change in the course of the disease condition.

17. The method according to claim 16, wherein the indicator is used in a prognosis for the individual.

18. The method according to claim 16, wherein the indicator is used in monitoring treatment of the disease condition of the individual .

19. The method according to claim 16, wherein the one or more affinity ligands comprises one or more affinity ligands which specifically bind a determinant comprising a pan B cell determinant, one or more affinity ligands which specifically bind a B cell subset determinant, and one or more affinity ligands which specifically bind a determinant comprising sTn.

20. The method according to claim 19, wherein the first sample and second sample each comprise peripheral blood, and wherein the indicator comprises a change in one or more B cell subpopulations selected from the group consisting of an overall B cell population, sTn-t- B cells, sTn+ memory cells, sTn-i- Bl cells, and a combination thereof.

21. The method according to claim 20, wherein the indicator comprises a change in one or more B cell subpopulations selected from the group consisting of CD19+ cells, CD19+ sTn+ cells, CD19+ CD5+ sTn+ cells, CD19+ CD21+ sTn+ cells, and a combination thereof.

22. The method according to claim 16, wherein the one or more affinity ligands further comprises a detectable moiety.

23. A method for detecting a disease condition selected from the group consisting of MS and a pro-MS immune response, and a pro-MS immune response, the method comprises:

(a) determining an amount of one or more B cell subpopulations in a body fluid of an individual by contacting the sample with one or more affinity ligands which binds specifically to the one or more B cell subpopulations, and then measuring the one or more affinity ligands bound;

(b) comparing the amount determined of the one or more B cell subpopulations to a comparative value for the one or more B cell subpopulations determined, wherein the comparative value is selected from the group consisting of a reference value, a baseline value, and a combination thereof; wherein a difference in the amount of one or more B cell subpopulations determined from the sample when compared to the comparative value comprises an indicator of a disease condition selected from the group consisting of MS and a pro-MS immune response, and a pro-MS immune response.

24. The method according to claim 23, wherein the one or more affinity ligands comprises one or more affinity ligands which specifically bind a determinant comprising a pan B cell determinant, one or more affinity ligands which specifically bind a B cell subset determinant, and one or more affinity ligands which specifically bind a determinant comprising sTn.

25. The method according to claim 24, wherein the clinical sample comprises peripheral blood, and wherein the indicator comprises a change in one or more B cell subpopulations selected from the group consisting of an overall B cell population, sTn+ B cells, sTn+ memory cells, sTn+ Bl cells, and a combination thereof.

26. The method according to claim 25, wherein the indicator comprises a change in one or more B cell subpopulations is selected from the group consisting of CD19+ cells, CD19+ sTn+ cells, CD19+ CD5+ sTn+ cells, CD19+ CD21+ sTn+ cells, and a combination thereof.

27. The method according to claim 23, wherein the one or more affinity ligands further comprises a detectable moiety.