How to assess the risks of PML

Abstract

To provide methods of assessing a patient's risk of developing progressive multifocal leukoencephalopathy (PML).SOLUTION: A method of evaluating the level of an anti-JCV antibody in a sample comprises: (a) forming a first reaction mixture comprising a first aliquot of the sample and a substrate on which HPVLP is disposed; and (b) detecting the level of the anti-JCV antibody bound to the substrate on which HPVLP is disposed by detecting a labeled detection reagent bound to the anti-JCV antibody bound to the substrate; where one or both of the following are met: (i) 20 ngs to 60 ngs of HPVP are disposed on the substrate and (ii) the ratio of the sample to the substrate is between 1:50 and 1:30; thereby evaluating the level of the anti-JCV antibody in the sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 61 / 491,810 filed on 31 May 2011, U.S. Provisional Application No. 61 / 508,584 filed on 15 July 2011, U.S. Provisional Application No. 61 / 550,257 filed on 21 October 2011, and U.S. Provisional Application No. 61 / 636,588 filed on 20 April 2012. The prior applications are incorporated herein by reference in their entirety.

[0002] Field of Invention The present invention relates to a method for assessing the risk of developing progressive multifocal leukoencephalopathy (PML) in patients. [Background technology]

[0003] Natalizumab, an anti-VLA-4 (latest antigen 4) antibody therapy, is indicated for the treatment of multiple sclerosis (MS) and relapsing forms of moderate to severe Crohn's disease. Natalizumab treatment, however, carries an increased risk of progressive multifocal leukoencephalopathy (PML), an opportunistic brain infection caused by the JC virus (JCV). PML occurs primarily in immunocompromised individuals and in patients receiving certain immunomodulatory therapies, including natalizumab. PML is hypothesized to be the result of complex interactions between host and viral factors, leading to the reactivation and mutation of latent prototype JCV into a neurotrophic form that can infect oligodendrocytes in the central nervous system. [Overview of the project] [Means for solving the problem]

[0004] The present invention relates, in particular, to an optimized, analytically validated, and highly sensitive assay for detecting the presence of JCV antibodies in biological fluids, such as serum or plasma, as well as to various other methods including methods for evaluating and / or treating patients.

[0005] Therefore, in one embodiment, the present invention features a method for evaluating the level of anti-JCV antibody in a sample, the method comprising one or more or all of the following steps: (a) A step of forming a first reaction mixture comprising a first aliquot of the sample and a substrate on which HPVLP (highly purified virus-like particles, e.g., highly purified VP1 particles) are placed, e.g., a high signal-to-noise HPVLP substrate, (b) A step of detecting the level of anti-JCV antibody bound to a substrate on which HPVLP is placed, for example, a high-signal-to-noise HPVLP substrate, by detecting a labeled detection reagent, for example, an enzyme-labeled anti-IgG antibody bound to the anti-JCV antibody bound to the substrate, thereby evaluating the level of anti-JCV antibody in the sample (as considered herein, the method may include classifying or assigning a value indicating the level of anti-JCV antibody in the sample, the value which is sometimes referred to herein as an index value. The value which can be used to evaluate the sample or patient, and in embodiments, to determine whether to proceed to a further step of the method, for example, step (c) below), and (c) Form a second reaction mixture containing a second aliquot of the sample and solution phase HPVLP, and detect the level of unbound anti-JCV antibody in the second reaction mixture by detecting an anti-JCV antibody that can bind to a substrate on which HPVLP is placed, for example, a high-signal-to-noise HPVLP substrate (as discussed herein, the method may include classifying or assigning a value to the sample that indicates the degree to which incubation with soluble phase HPVLP reduces the level of unbound anti-JCV antibody in the second reaction mixture, the value which is sometimes referred to herein as inhibition, inhibition%, etc. This value can be used to evaluate the sample or patient), This step involves evaluating the level of anti-JCV antibody in the sample.

[0006] In one embodiment, the method further includes: (d) Under conditions that the anti-JCV antibody in the sample is not bound by HPVLP or other antigens, a third reaction mixture containing a third aliquot is formed, and the level of anti-JCV antibody in the third reaction mixture is detected, for example, by detecting an anti-JCV antibody that can bind to a substrate on which HPVLP is placed, such as a high-signal-to-noise HPVLP substrate. Inhibition or inhibition % can be calculated as a function of the degree to which incubation with soluble phase HPVLP (step (c)) reduces the amount of unbound anti-JCV antibody compared to the result in step (d).

[0007] In one embodiment, the method includes steps (a) and (b) and optionally providing the results to another entity, such as a healthcare provider.

[0008] In one embodiment, the method includes steps (a), (b), and (c), and optionally providing the results to another entity, such as a healthcare provider.

[0009] In one embodiment, the method includes steps (a), (b), (c), and (d), and optionally providing the results to another entity, such as a healthcare provider.

[0010] In one embodiment, the method includes step (c) and optionally providing the results to another entity, such as a healthcare provider.

[0011] In one embodiment, the method includes steps (c) and (d) and optionally providing the results to another entity, such as a healthcare provider.

[0012] The methods described herein enable improved performance by using optimized levels and amounts of reagents. Therefore, in some embodiments, 20 ngs–60 ngs, 30 ngs–50 ngs, 20 ngs–40 ngs, or 35 ngs–45 ngs of HPVLP are placed on the substrate of the first reaction mixture. In other embodiments, approximately 20 ngs, 30 ngs, 40 ngs, 50 ngs, or 60 ngs of HPVLP are placed on the substrate. Typically, a multi-substrate device, such as a multi-well plate, such as a polystyrene multi-well plate, would have the amounts of HPVLP specified herein on each of the multiple substrates. A typical substrate is the interior of the wells on a multi-well plate.

[0013] The methods described herein enable improved performance by using optimized ratios of reagent and sample. In some embodiments, the ratio of μL of sample in the first reactant (this refers to the amount of undiluted sample or sample in a dilution, so 1 μL:100 μL of 100 μL would be 1 μL of sample), e.g., serum or plasma, to HPVLP of ngs placed on the substrate is 1:100~1:20, 1:80~1:30, 1:60~1:20, 1:20~1:60, 1:30~1:50. In some embodiments, the ratio of μL of sample, e.g., serum or plasma, to HPVLP of ngs placed on the substrate is approximately 1:30, 1:40, or 1:50. In one embodiment, the ratio of μL of sample, e.g., serum or plasma, to HPVLP of ngs placed on the substrate is approximately (0.08-1.2):30, (0.08-1.2):40, or (0.08-1.2):50.

[0014] In one embodiment, a sample for the first reactant, e.g., serum, is diluted in a buffer, e.g., about 100-fold, before contact with a substrate on which HPVLP is placed, e.g., a high-signal-to-noise HPVLP substrate. In one embodiment, detection is performed using an enzyme-labeled antibody, e.g., enzyme-labeled IgG, such as IgG labeled with HRP (horseradish peroxidase). In another embodiment, the detection reagent, e.g., HRP-labeled IgG, is added at concentrations of at least 0.01 μg / mL, 0.02 μg / mL, 0.03 μg / mL, 0.04 μg / mL, 0.05 μg / mL, 0.06 μg / mL, or 0.08 μg / mL. In one embodiment, the detection reagent is provided in an excess of 10 to 100 times relative to the antibody bound to the substrate. In some embodiments, the detection reagent is provided in an amount that results in an excess of 10, 20, 50, 75, or 100 times or more compared to the antibody bound to the substrate.

[0015] In one embodiment, the solution phase HPVLP in (c) is present in a 2- to 100-fold excess of particles relative to the anti-JCV antibody in the second reaction mixture or sample. In one embodiment, the excess particles relative to the anti-JCV antibody in the second reaction mixture or sample are 2-fold or more, 4-fold or more, 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 40-fold or more, 50-fold or more, 70-fold or more, 80-fold or more, 100-fold or more, or 110-fold or more.

[0016] In one embodiment, 20 ngs–60 ngs, 30 ngs–50 ngs, 20 ngs–40 ngs, or 35 ngs–45 ngs of HPVLP are placed on the substrate of the second reaction mixture. In another embodiment, approximately 20 ngs, 30 ngs, 40 ngs, 50 ngs, or 60 ngs of HPVLP are placed on the substrate. Typically, a multi-substrate device, such as a multi-well plate, such as a polystyrene multi-well plate, would have the amounts of HPVLP specified herein on each of the multiple substrates. A typical substrate is the inside of the wells on a multi-well plate.

[0017] In one embodiment, with respect to the second reaction mixture, the sample is brought into contact with the soluble HPVLP phase, and then the unbound anti-JVC antibody is allowed to bind to the HPVLP positioned on the substrate. In another embodiment, with respect to the second reaction mixture, the sample is in contact with the soluble HPVLP phase positioned on the substrate and the HPVLP simultaneously.

[0018] In some embodiments, the ratio of a μL sample (this refers to the amount of undiluted sample or sample in a dilution, so a 1 μL:100 μL dilution of 100 μL would be 1 μL of sample), e.g., serum or plasma, to HPVLP of ngs placed on the substrate is 1:100~1:20, 1:80~1:30, 1:60~1:20, 1:20~1:60, 1:30~1:50. In some embodiments, the ratio of a μL sample, e.g., serum or plasma, to HPVLP of ngs placed on the substrate is approximately 1:30, 1:40, or 1:50. In some embodiments, the ratio of a μL sample, e.g., serum or plasma, to HPVLP of ngs placed on the substrate is approximately (0.08~1.2):30, (0.08~1.2):40, or (0.08~1.2):50.

[0019] In one embodiment, the sample, e.g., serum, is diluted, e.g., about 100-fold in a buffer before contact with a substrate on which HPVLP is placed, e.g., a high-signal-to-noise HPVLP substrate. In one embodiment, detection is performed using an enzyme-labeled antibody, e.g., HRP-labeled IgG. In another embodiment, the detection reagent, e.g., HRP-labeled IgG, is added at concentrations of at least 0.01 μg / mL, 0.02 μg / mL, 0.03 μg / mL, 0.04 μg / mL, 0.05 μg / mL, 0.06 μg / mL, or 0.08 μg / mL. In one embodiment, the detection reagent is provided in an excess of 10 to 100 times relative to the antibody bound to the substrate. In some embodiments, the detection reagent is provided in an amount that results in an excess of 10-, 20-, 50-, 75-, or 100-fold relative to the antibody bound to the substrate.

[0020] In one embodiment, step (c) and / or (d) is performed in response to the level of anti-JCV antibody detected in step (b).

[0021] In one embodiment, in response to the level of anti-JCV antibody detected in step (b), for example, the index level (nOD) being greater than 0.2 and less than 0.4, then steps (c) and (d) are performed.

[0022] In one embodiment, the sample, for example, serum or plasma, is diluted in, for example, a buffer, in an amount of about 50-fold or more, about 100-fold or more, or about 150-fold or more, etc., before forming the second reaction mixture. In another embodiment, the sample, for example, serum or plasma, is diluted in, for example, a buffer, in an amount of about 50-fold or more, about 100-fold or more, or about 150-fold or more, etc., before forming the third reaction mixture. In another embodiment, the detection of one or both of the second and third reaction mixtures uses an antibody labeled with an enzyme, for example, IgG labeled with an enzyme, for example, IgG labeled with HRP.

[0023] The detection of one or both of the second and third reaction mixtures can use IgG labeled with HRP added at a concentration of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, or 0.08 μg / mL. In one embodiment, the detection reagent is provided in an excess of 10-fold to 100-fold (for example, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold) compared to the antibody bound to the substrate.

[0024] In one embodiment, evaluating the level of anti-JCV antibody in a sample further includes, in step b, evaluating a standard material such as a cutoff calibration sample having an index of about 1 (e.g., an optical density of 1, where a positive control has an optical density of 1.3 and a negative control has an optical density of 0.1) and a signal-to-noise ratio of 15 to 20 times or more (e.g., 16 times or more, 17 times or more, 18 times or more, or 19 times or more). Another embodiment includes, in step b, evaluating a standard material having a score of about 1.3, such as a positive control. In yet another embodiment, the method further includes, in step b, evaluating a standard material such as a negative control having a score of about 0.1.

[0025] In one embodiment, the method includes determining the amount by which binding to the soluble phase HPVLP particles inhibits or reduces binding to the HPVLP particle-configuring substrate compared to binding to the HPVLP particle-configuring substrate in the first aliquot. The results of the first step of the two-step assay (steps (a) and (b) above) are typically expressed as a normalized OD (nOD, or “index”) value. The results of the second step of the two-step assay (steps (c) and optionally (d) above) are typically expressed as “inhibition percentage”. In one embodiment, nOD is OD 450 In one embodiment, the inhibition is at a default value, for example, 45% or less, and the sample is classified as negative.

[0026] In one embodiment, the inhibition is greater than a default value, for example, 45%, and the sample is classified as positive.

[0027] In one embodiment, the cutoff calibration sample (CO) is adjusted to have a reactivity index (nOD) of approximately 1.0, and the positive control (PC) is adjusted to have a reactivity index of approximately 1.3. The CO and PC solutions are prepared by mixing serum positive for JCV antibodies with serum negative for JCV antibodies. For example, for a negative control (NC), which may be a vial of anti-JCV antibody-negative serum, the index (nOD) target is approximately 0.1.

[0028] In one embodiment, the JCV antigen is VLP particles such as HPVLP that are purified by chromatography before use in the assay characterized by the present invention.

[0029] In certain embodiments, the sample is a serum sample, urine sample, plasma sample, blood sample, or cerebrospinal fluid (CSF) sample. In one embodiment, the sample is a serum sample, which is diluted 1:101 before forming a first reaction mixture containing a first aliquot of the sample and a substrate on which HPVLP is placed.

[0030] In another embodiment, the secondary detection reagent (e.g., anti-human IgG) is conjugated with a detectable agent, such as a peroxidase like HRP. In one embodiment, the secondary detection reagent may be anti-human IgG, which is conjugated with HRP. In another embodiment, the detection reagent solution containing IgG-HRP is used at 0.04 μg / mL. For example, a 0.8 mg / mL stock solution of IgG-HRP is diluted 1:15,000, 1:20,000, 1:30,000 or more before being used in an assay to detect the level of anti-JCV antibody conjugated to HPVLP. In another embodiment, the concentration of the secondary detection reagent is adjusted for a new lot to match the signal to a previous lot, and the incubation time with the conjugate is only 30 minutes. In one embodiment, TMB (tetramethylbenzidine) and hydrogen peroxide in buffer are incubated with a reaction mixture containing the HRP-IgG mixture conjugated to anti-JCV antibody for 20 minutes (±2 minutes).

[0031] In one embodiment, a decrease in the detected level in a secondary assay sample compared to a sample that was not pre-incubated indicates that the sample is positive for anti-JCV antibodies, while a change in the detected level below a specified percentage indicates that no JCV-specific antibodies are present in the sample.

[0032] In one embodiment, after a first step of the assay, which involves forming a first reaction mixture comprising a first aliquot of the sample and a substrate on which the HPVLP is placed, e.g., a high-signal-to-noise HPVLP substrate, and detecting the level of anti-JCV antibody bound to the substrate on which the HPVLP is placed, e.g., the high-signal-to-noise HPVLP substrate, the sample is determined to have an index value (i.e., nOD value) greater than 0.2 and less than 0.4 ("uncertain zone"). A second aliquot of the sample can then be tested in a second step of the assay, which involves detecting unbound anti-JCV antibody in the second mixture by contacting the second mixture with the substrate on which the HPVLP is placed, e.g., a high-signal-to-noise HPVLP substrate, after forming a second mixture between the second aliquot and the solution phase HPVLP.

[0033] In another embodiment, if a sample is determined to have an index value of less than 0.2 after the first step of the assay, the sample is determined to be negative for anti-JCV antibodies. In one embodiment, a sample determined to be negative for anti-JCV antibodies is not evaluated using the second step of the assay.

[0034] In another embodiment, if a sample is determined to have an index value greater than 0.4 after the first step of the assay, the sample is determined to be positive for anti-JCV antibodies. In one embodiment, a sample determined to be positive for anti-JCV antibodies is not evaluated using the second step of the assay.

[0035] In one embodiment, the present invention includes obtaining a biological sample from a subject (e.g., plasma, serum, blood, urine, or cerebrospinal fluid (CSF)) and correlating the detected level with the reference substance so that the reference substance is selected to exhibit a false negative rate of 3% or less and minimal cross-reactivity to other polyomaviruses, such as BK virus (BKV). In some embodiments, the reference substance derived from a control sample or a set of control samples is processed with the sample from the subject. In some embodiments, the reference substance is selected so that the false negative rate of the assay is 1% or less. The assay can be performed so that the HPVLP is placed on a solid substrate such as a microtiter plate or slide. In some embodiments, the HPVLP essentially consists of the VP1 viral protein. The HPVLP may further contain at least one of other viral proteins, such as VP2 or VP3. The viral protein(s) in the HPVLP may be recombinant (e.g., MAD1 VP1) or naturally occurring viral protein (e.g., derived from a naturally occurring source). This method can be performed, for example, using biological samples obtained from subjects currently being treated with immunomodulatory drugs, subjects being considered for initiation of immunomodulatory drug treatment, or subjects suspected of having progressive multifocal leukoencephalopathy (PML).

[0036] In another embodiment, the present invention features a kit containing a substrate on which HPVLP is placed, for example, a high signal-to-noise HPVLP substrate. The substrate may include a multiwall plate, such as a 96-well plate. In one embodiment, the kit comprises one or more of the following: a substrate such as a plate, where the wells are coated with a JCV antigen substrate, such as HPVLP; a lyophilized or solution-based JCV antigen, such as HPVLP; a JCV cutoff calibration sample, an anti-JCV antibody-positive control, and a JCV-negative control, which are serum samples such as human serum. In one embodiment, the kit comprises one or more reagents for detecting a complex containing an anti-JCV antibody bound to the antigen, or further comprising one or more reagents, the reagents comprising, for example, a JCV complex, a casein sample, a detectable reagent such as TMB (tetramethylbenzidine), a wash buffer, and a stop reagent.

[0037] In another embodiment, the present invention features a substrate on which HPVLP is placed, for example, a high signal-to-noise HPVLP substrate.

[0038] In another embodiment, the present invention features a kit comprising at least one reagent for performing an assay to identify HPVLP and anti-JCV antibody levels in a sample such as a biological sample.

[0039] In other embodiments, the present invention relates to a solution comprising HPVLP particles essentially consisting of VP1-containing particles, which contain pentamers larger in size than VP1 pentamers (capsomeres), for example, about 5, 10, 20, 30, 40, 50, 60, 70, or 72, or about 360 VP1 molecules.

[0040] Another aspect of the present invention is a method for preparing a solution of HPVLP, which includes removing VP1-containing particles having a size of VP1 pentamer or less from the solution.

[0041] The methods disclosed herein are, at least in part, based on the finding that other properties, such as anti-JCV antibody titer and affinity / binding activity, may indicate a risk for patients developing progressive multifocal leukoencephalopathy (PML).

[0042] Accordingly, in another aspect, the present invention features a method for assessing the risk of a patient developing PML, comprising: obtaining knowledge of the JC virus (JCV) antibody titer (e.g., determined as described herein and expressed as normalized optical density (nOD) or index) or affinity / binding activity (e.g., determined as described herein and expressed as an inhibition percentage in the assay confirmation step) in a patient sample; and optionally, comparing the obtained value(s) with a reference substance disclosed herein to assess the risk.

[0043] In one embodiment, the anti-JCV antibody titer or inhibition percentage is determined in a biological sample from the patient, such as blood (serum or plasma) or a CSF sample. If a function of the titer and / or inhibition percentage, or both, is determined to be below a predetermined level, the patient is determined to be at a lower risk of developing PML. If a function of the titer and / or inhibition percentage, or both, is determined to be at or above a predetermined level, the patient is determined to be at a higher risk of developing PML.

[0044] This method can further determine whether determining the anti-JCV antibody titer or inhibition percentage in a patient's sample requires removing the biological sample from the patient's body or analyzing a sample from the patient, or whether the patient should be given immunosuppressive therapy or other therapies if it is determined that the patient is at a lower risk of developing PML.

[0045] In one embodiment, the anti-JCV antibody titer or inhibition percentage is determined in one or more biological samples from the patient, such as one or more blood (serum or plasma) or CSF samples.

[0046] In one embodiment, the subjects are multiple sclerosis patients who have already received treatment with, for example, an anti-VLA-4 antibody, such as natalizumab.

[0047] In one embodiment, the patient is determined to have a lower risk of developing PML, and is further administered anti-VLA-4 therapy, such as an anti-VLA-4 antibody like natalizumab, or a fragment thereof (such as its antigen-binding fragment).

[0048] In one embodiment, a patient is determined to be at higher risk of developing PML, and is identified as someone who should receive alternative therapy. For example, the patient should discontinue receiving anti-VLA-4 antibody therapy, such as natalizumab, and receive alternative therapy, such as an alternative approved multiple sclerosis (MS) therapy such as Avonex®. In another embodiment, a patient is determined to be at higher risk of developing PML, and is administered anti-VLA-4 antibody therapy, such as natalizumab.

[0049] In one embodiment, a patient is determined to be at higher risk of developing PML based on their anti-JCV antibody titer or inhibition percentage, and is identified as a person who should undergo further testing to determine their risk of developing PML.

[0050] In one embodiment, a patient is determined to be at a lower risk of PML if (i) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be less than 0.5, or (ii) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 0.5 but less than 3.0, and the inhibition percentage is 70% or 60% or less. A patient is determined to be at a moderate risk of PML if (i) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 0.5 but less than 1.5, and the inhibition percentage is determined to be less than 70%. A patient is determined to be at a higher risk of PML if (i) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 0.5, and the inhibition percentage is determined to be greater than 70%, or (ii) the patient has shown a twofold increase in the index, nOD, or titer since the previous test. The inhibition percentage of anti-JCV antibodies can be measured, for example, by (i) contacting a biological sample from a subject with HPVLP in solution under conditions suitable for the binding of anti-JCV antibodies in the sample to HPVLP; (ii) separating the JCV antibodies bound to HPVLP from the solution to create a secondary sample; (iii) contacting the secondary sample with HPVLP under the same conditions as in (i); and (iv) detecting the level of anti-JCV antibody binding to HPVLP in the secondary sample.

[0051] In one embodiment, the anti-JCV antibody titer is measured, for example, by (i) contacting a biological sample with HPVLP under conditions suitable for the binding of anti-JCV antibodies in the sample to HPVLP, (ii) detecting the level of anti-JCV antibody binding in the sample to HPVLP, and (iii) correlating the detected level with a reference set. The reference set can be selected to exhibit a false-negative rate not exceeding a predetermined amount, such as 3%. In another embodiment, the anti-JCV antibody titer is measured using the VIDAS® assay (bioMerieux). It is measured using commercial platforms such as the solution phase method or other alternative platforms such as the lateral flow method.

[0052] In one embodiment, the assay demonstrates that the biological sample does not contain JCV antibody, and the assay then further includes (iv) contacting a portion of the biological sample from the subject with HPVLP in solution prior to step (i) (the HPVLP in step (i) is bound to a solid substrate to provide a secondary sample), (v) contacting the secondary sample with HPVLP under the same conditions as in (i), (vi) detecting the level of anti-JCV antibody binding to HPVLP in the secondary sample, and (vii) comparing the detected level of anti-JCV antibody in the secondary sample with the level of binding in the biological sample when incubated with a solution without HPVLP. A decrease in the detected level in the sample pre-incubated with HPVLP compared to the sample incubated in solution indicates that the sample is positive for anti-JCV antibody, and no change in the detected level indicates that anti-JCV antibody is not present above background levels in the sample.

[0053] In one embodiment, the assay shows that the biological sample contains JCV antibodies, and the patient is determined to be at higher risk of PML.

[0054] In another embodiment, a patient is determined to have a lower risk of PML if (i) the anti-JCV antibody titer indicated by the index value or nOD is determined to be less than 0.5, or (ii) the anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 0.5 but less than 3.0, and the inhibition percentage is 70% or less. A patient is determined to have a higher risk of PML if (i) the anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 3, and the inhibition percentage is determined to be greater than 70%, or (ii) the patient shows a twofold increase in the index, nOD, or titer from the previous test.

[0055] In one embodiment, the risk of PML is determined using only an index value (nOD) or only an inhibition percentage. For example, in one embodiment, a patient is determined to have a lower risk of PML if the anti-JCV antibody titer indicated by the index value or nOD is determined to be less than 0.5, a patient is determined to have a higher risk if the anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 0.5 but less than 1.5, or an even higher risk if the patient is determined to have an anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 1.5.

[0056] In one embodiment, the assay shows that the biological sample does not contain JCV antibodies above background levels, and the patient is determined to be at lower risk of PML.

[0057] In another embodiment, the present invention features a method for evaluating or testing an assay procedure. The anti-JCV antibody assay can be re-evaluated for effectiveness at predetermined intervals, such as every six months or annually. In one example proficiency assay, a population of samples, e.g., 30, 40, or 50 serum samples and 30, 40, or 50 plasma samples, are provided for evaluation by the currently optimized method and preceding previous-generation methods, etc. The agreement between the results is assessed, and if the agreement is found to be, for example, greater than 90% or 95%, the performance of the assay may be determined to be acceptable. In one embodiment, the consistency of assay performance over time is assessed using a panel of samples containing, for example, 90, 100, or 150 or more samples having a known anti-JCV antibody status. The agreement between the results is assessed, and if the agreement is found to be, for example, greater than 90% or 95%, the performance of the assay may be determined to be acceptable. The panel of samples is patient serum, available in sufficient quantities to create a sample bank.

[0058] In one embodiment, an entity, for example, a healthcare provider, obtains information from an anti-JCV antibody assay described herein and, in response to that information, administers a treatment described herein to a patient, for example, a patient with MS.

[0059] In another embodiment, the JCV assay described herein is performed on a patient, and the patient is then treated based on the results of the assay, for example, a patient with MS is treated.

[0060] Patient anti-JCV antibody titers or inhibition percentages can be re-evaluated at regular intervals, such as every 3 months, 6 months, or 12 months, or at longer intervals or more frequently. An increase observed in antibody titer or inhibition percentage may indicate an increased risk of developing PML in patients. For example, a twofold or threefold increase in antibody titer (nOD or index) may indicate an increased risk of PML. Patients receiving anti-VLA-4 therapy such as natalizumab may discontinue anti-VLA-4 therapy and, optionally, initiate alternative therapy with other medications, such as non-anti-VLA-4 therapy or other immunosuppressants besides natalizumab. Increases in titer may appear differently in patients with high baseline titers compared to patients with low baseline titers (e.g., within a narrower range of titers).

[0061] In one embodiment, patients receiving an anti-VLA-4 antibody, such as natalizumab, can be monitored for anti-JCV antibody titers and / or inhibition percentages at intervals of, for example, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 30, and 40 months.

[0062] In one embodiment, the patient is not re-evaluated for the presence of JCV antibodies, or for anti-JCV antibody titers or inhibition percentages, within one, two, or three weeks after receiving plasmapheresis. In another embodiment, the patient is not re-evaluated for the presence of JCV antibodies, or for anti-JCV antibody titers or inhibition percentages, within one, two, or three weeks after receiving intravenous immunoglobulin (IVIG) therapy.

[0063] Anti-JCV antibody titers may be measured using nOD or index values.

[0064] The patient evaluations described herein can be performed before administration of anti-VLA-4 therapy or after the patient has initiated anti-VLA-4 therapy.

[0065] In one embodiment, the patient is determined to be at lower risk of PML by an assay described herein, and the patient is administered anti-VLA-4 therapy. In another embodiment, the patient is determined to be at higher risk of PML, and the patient is administered anti-VLA-4 therapy, such as an anti-VLA-4 antibody such as natalizumab. In yet another embodiment, the patient is determined to be at higher risk of PML, and the patient is administered a treatment other than anti-VLA-4 therapy, such as interferon, glatiramer acetate, or corticosteroids.

[0066] In one embodiment, it is determined that the patient has an increased risk of PML, and therefore the patient discontinues receiving anti-VLA-4 therapy.

[0067] Patients can be monitored at regular intervals, for example, every three months, every six months, annually, or more or less frequently, for a decrease in anti-JCV antibody titer or a decrease in JCV antibody inhibition percentage. A decrease in anti-JCV antibody titer or a decrease in JCV antibody inhibition percentage may indicate that the patient has a reduced risk of developing PML.

[0068] In one embodiment, it is determined that the anti-JCV antibody titer or JCV antibody inhibition percentage has decreased to below a predetermined level, even after being elevated, and the patient is then determined to be administered anti-VLA-4 therapy or to be a candidate for receiving anti-VLA-4 therapy. If the patient has previously received anti-VLA-4 therapy, the patient's anti-VLA-4 therapy may be reinstated. After reinstating anti-VLA-4 therapy, the patient is evaluated every six months or annually for the decrease in JCV antibody titer or inhibition percentage.

[0069] In one embodiment, if a patient is determined to be at higher risk of PML, for example, if a patient is determined to have an anti-JCV antibody titer greater than 0.5, for example greater than 1.0 or less than 1.5 as measured by an nOD, the patient is not tested again for JCV status. For example, the patient may discontinue treatment with anti-VLA-4 therapy such as natalizumab and is not tested again for anti-JCV antibody status.

[0070] In one embodiment, a method for evaluating a patient as described herein, such as for determining an anti-JCV antibody titer or inhibition percentage, may further include assessing other measurements of risk predictors. For example, a method for evaluating a patient may further include (a) determining whether the patient has received long-term treatment with anti-VLA-4 therapy (e.g., longer than 24 months), or (b) determining whether the patient has received a specified non-anti-VLA-4 immunosuppressive therapy (e.g., mitoxantrone or other therapies in the last 2, 3, or 5 years or at any point in the patient's lifetime). For patients with anti-JCV antibody titers or inhibition percentages above the default level but who have not used designated immunosuppressants and have never received long-term anti-VLA-4 therapy, the relative risk of PML is less relative to that of patients with anti-JCV antibody titers or inhibition percentages above the default level and who have used designated immunosuppressants and received long-term anti-VLA-4 therapy, and less relative to that of patients with anti-JCV antibody titers or inhibition percentages below the default level and who have used designated immunosuppressants or received long-term anti-VLA-4 therapy.

[0071] In one embodiment, the patient has previously received anti-VLA-4 therapy, such as natalizumab, and in another embodiment, the patient is administered anti-VLA-4 therapy based on an evaluation, such as an evaluation of anti-JCV antibody titer or inhibition percentage. For example, as a result of the evaluation, the patient may be classified as a candidate for anti-VLA-4 therapy. In one embodiment, the patient classified as a candidate for anti-VLA-4 therapy is further administered the treatment.

[0072] In some embodiments, the factors to be included in the hierarchical model are the patient's age or sex.

[0073] The methods described herein can incorporate one or more factors into the evaluation of a patient. In other embodiments, the present invention features a method for evaluating a patient as a candidate for receiving treatment with, for example, anti-VLA-4 therapy.

[0074] This method includes, for example, obtaining or determining the JC virus (JCV) antibody titer and inhibition percentage in a biological sample from a patient, by a method described herein. If the antibody titer or inhibition percentage is determined to be below a predetermined level, the patient may be classified as suitable for treatment in a first therapy category, such as anti-VLA-4 therapy, e.g., natalizumab. If the antibody titer or inhibition percentage is determined to be at or above a predetermined level, the patient is classified as suitable for a second therapy category, e.g., interferon, glatiramer acetate, or corticosteroid. Obtaining the anti-JCV antibody titer and inhibition percentage in a patient sample may include removing the biological sample from the patient's body or analyzing the sample from the patient. The evaluation method may also include administering a therapy from the first category (e.g., natalizumab) or the second category (e.g., interferon, glatiramer acetate, or corticosteroid), etc., to the patient.

[0075] In another embodiment, a patient is determined to be at lower risk of PML if (i) the anti-JCV antibody titer indicated by the index value or nOD is determined to be less than 0.5, or (ii) the anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 0.5 and less than 3.0, and the inhibition percentage is 70% or less. A patient is determined to be at higher risk of PML if (i) the anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 1.5, and the inhibition percentage is determined to be greater than 70%, or (ii) the patient shows a twofold increase in the index, nOD, or titer from the previous test. A patient is determined to be at moderate risk of PML if the anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 0.5 and less than 1.5, and the inhibition percentage is determined to be greater than 70%.

[0076] As discussed above, methods for evaluating patients can incorporate more than one consideration or factor. Therefore, methods for evaluating patients can be: (aa) To determine whether a patient has ever received long-term treatment with anti-VLA-4 therapy (e.g., longer than 24 months), and in embodiments, to provide a prior anti-VLA-4 therapy exposure classification, or (bb) Determine whether the patient has ever received a designated non-anti-VLA-4 immunosuppressive therapy (for example, in the last two, three, or five years or at any point in the patient's lifetime), and in embodiments, further include providing a prior immunosuppressant exposure classification.

[0077] Typically, patients with anti-JCV antibody titers or inhibition percentages above the predefined level but who have not used designated immunosuppressants and have never received long-term anti-VLA-4 therapy are classified as having a lower relative risk of developing PML than patients with anti-JCV antibody titers or inhibition percentages below the predefined level and who have used designated immunosuppressants or received long-term anti-VLA-4 therapy, which is a lower relative risk than patients with anti-JCV antibody titers or inhibition percentages below the predefined level and who have used designated immunosuppressants or received long-term anti-VLA-4 therapy.

[0078] In one embodiment, the patient has previously received anti-VLA-4 therapy. In another embodiment, the method includes administering anti-VLA-4 therapy, such as natalizumab, to the patient.

[0079] In one embodiment, a patient is classified as a candidate for anti-VLA-4 therapy, and the patient is further administered anti-VLA-4 therapy.

[0080] Patients who have received anti-VLA-4 therapy such as natalizumab for 24 months or less, have no prior history of immunosuppressive therapy (other than anti-VLA-4 therapy), and have tested negative for JCV exposure (e.g., negative for JCV antibodies) typically have the lowest risk of developing PML. Conversely, patients who have received anti-VLA-4 therapy for longer than 24 months, have a prior history of immunosuppressive therapy (other than anti-VLA-4 therapy), and have tested positive for JCV exposure (e.g., positive for JCV antibodies) typically have the highest risk of developing PML.

[0081] A patient's risk level for PML can be assessed by evaluating one, two, or all three of the identified risk factors. For example, a patient with a negative test result for anti-JCV antibody titer, such as a patient with multiple sclerosis (MS), may be determined to have a lower risk of PML. Patients at lower risk of PML may have a risk level of less than approximately 0.2 / 1000 patients, for example, 0.11 / 1000 or less.

[0082] In one embodiment, patients who have received anti-VLA-4 therapy such as natalizumab for 24 months or less (e.g., 23 months, 22 months, 20 months, 15 months, 12 months, 6 months, or 1 month or less) and who have not previously received immunosuppressive therapy, such as patients with MS, may be determined to have a lower risk of PML. For example, a patient may be determined to have a PML risk of approximately 0.54 / 1000 patients. The patient may then be determined to be a candidate for further treatment with anti-VLA-4 therapy such as natalizumab.

[0083] In one embodiment, patients who have received anti-VLA-4 therapy such as natalizumab for a longer period than 24 months, such as approximately 25 to 48 months or longer (e.g., 26, 28, 30, 36, 40, or 48 months or longer), and who have not previously received immunosuppressive therapy, may be determined to be at higher risk of PML, or may be classified as having a higher risk of PML. Patients at higher risk of PML may have a risk of approximately 3.7 / 1000 patients or more, for example, approximately 1.37 / 1000 patients. These patients may therefore be determined to be candidates for further treatment with anti-VLA-4 therapy such as natalizumab.

[0084] In one embodiment, patients who have received anti-VLA-4 therapy such as natalizumab for 24 months or less (e.g., 24 months, 22 months, 20 months, 15 months, 12 months, 6 months, or 1 month or less) and are determined to be negative for anti-JCV antibodies or JCV nucleic acids may be determined to have a lower risk of PML, or may be classified as having a lower risk of PML. For example, a patient may be determined to have a risk of 0.2 / 1000 patients or less. The patient may therefore be determined to be a candidate for further treatment with anti-VLA-4 therapy such as natalizumab.

[0085] In one embodiment, patients who have not previously received immunosuppressant therapy (other than anti-VLA-4 therapy) and are determined to be negative for JCV are determined to have a lower risk of PML, e.g., less than 0.2 / 1000 patients. These patients may then be determined to be candidates for further treatment with anti-VLA-4 therapy such as natalizumab.

[0086] In one embodiment, patients who have received anti-VLA-4 therapy such as natalizumab for longer than 24 months (e.g., 25, 26, 28, 30, 35, 38, 40, 48 months, or longer) and who have previously received immunosuppressive therapy other than anti-VLA-4 therapy may be determined to be at higher risk of PML. Patients at higher risk of PML may have a risk of approximately 0.37 / 1000 or higher, for example, approximately 4.3 / 1000 patients. Patients may then be determined not to be candidates for further treatment with anti-VLA-4 therapy such as natalizumab, or they may be determined to be candidates for treatment with anti-VLA-4 therapy with more frequent monitoring. For example, patients at higher risk of PML who accept treatment with anti-VLA-4 therapy may receive more frequent monitoring for the development of PML than patients at lower risk of PML.

[0087] In one embodiment, a patient who has received anti-VLA-4 therapy such as natalizumab for 24 months or less (e.g., 24 months, 22 months, 20 months, 15 months, 12 months, 6 months, or 1 month or less) and has previously received immunosuppressive therapy other than anti-VLA-4 therapy may be determined to be at higher risk of PML. For example, a patient may be determined to have a PML risk of 0.66 / 1000 patients. The patient may then be determined not to be a candidate for further treatment with anti-VLA-4 therapy such as natalizumab, or to be a candidate for treatment with anti-VLA-4 therapy with more frequent monitoring. For example, a patient at higher risk of PML who accepts treatment with anti-VLA-4 therapy may accept more frequent monitoring for the development of PML than a patient at lower risk of PML.

[0088] In one embodiment, patients who have received anti-VLA-4 therapy such as natalizumab for longer than 24 months (e.g., 25–48 months, or longer, such as 26, 30, 36, 42, or 48 months) and are determined to be positive for JCV are determined to be at higher risk of PML. Patients may then be determined not to be candidates for further treatment with anti-VLA-4 therapy, or they may be determined to be candidates for treatment with anti-VLA-4 therapy with more frequent monitoring.

[0089] In one embodiment, a patient who has received anti-VLA-4 therapy such as natalizumab for longer than 24 months (e.g., 25-48 months, or longer, such as 26, 30, 36, 42, or 48 months), has not received prior treatment with immunosuppressants (other than anti-VLA-4 therapy), is determined to be JCV positive, and is determined to be at higher risk of PML. For example, a patient may be determined to have a PML risk of 4 / 1000 patients. The patient may then be determined not to be a candidate for further treatment with anti-VLA-4 therapy, or may be determined to be a candidate for treatment with anti-VLA-4 therapy with more frequent monitoring.

[0090] In one embodiment, a patient who has previously received immunosuppressant therapy other than anti-VLA-4 therapy and is determined to be positive for anti-JCV antibodies or JCV nucleic acid, such as a patient with MS, may be determined to be at higher risk of PML. The patient may then be determined not to be a candidate for further treatment with anti-VLA-4 therapy, or may be determined to be a candidate for treatment with anti-VLA-4 therapy with more frequent monitoring.

[0091] In one embodiment, a patient who has previously received immunosuppressant therapy other than anti-VLA-4 therapy, has been determined to be positive for anti-JCV antibody or JCV nucleic acid, and has received anti-VLA-4 therapy such as natalizumab for longer than 24 months (e.g., 25-48 months, or longer, such as 26, 30, 36, 42, or 48 months) may be determined to be at higher risk of PML. For example, a patient may be determined to have a risk of 9.8 / 1000 patients. The patient may then be determined not to be a candidate for further treatment with anti-VLA-4 therapy, or may be determined to be a candidate for treatment with anti-VLA-4 therapy with more frequent monitoring.

[0092] In one embodiment, a patient who has received anti-VLA-4 therapy such as natalizumab for 24 months or less (e.g., for 24 months, 22 months, 20 months, 15 months, 12 months, 6 months, or 1 month or less), has received prior treatment with immunosuppressants other than anti-VLA-4 therapy, and is determined to be positive for JCV may be determined to be at higher risk of PML. For example, a patient may be determined to have a PML risk of 4.5 / 1000 patients. The patient may then be determined not to be a candidate for further treatment with anti-VLA-4 therapy, or may be determined to be a candidate for treatment with anti-VLA-4 therapy with more frequent monitoring.

[0093] In one embodiment, a patient who has received anti-VLA-4 therapy such as natalizumab for 24 months or less (e.g., for 24 months, 22 months, 20 months, 15 months, 12 months, 6 months, or 1 month or less), has not received prior treatment with immunosuppressants (other than anti-VLA-4 therapy), and is determined to be positive for JCV may be determined to be at higher risk of PML. For example, a patient may be determined to have a PML risk of 0.35 / 1000 patients. The patient may then be determined not to be a candidate for further treatment with anti-VLA-4 therapy, or may be determined to be a candidate for treatment with anti-VLA-4 therapy with more frequent monitoring.

[0094] Patients determined to have a lower risk of developing PML may be determined to have a risk of approximately 0.54 / 1000 or less, for example, 0.25 / 1000 or less, 0.2 / 1000 or less, 0.19 / 1000, 0.15 / 1000 or less, 0.11 / 1000 or less, 0.1 / 1000 or less, for example, 0.3 / 1000, 0.25 / 1000, 0.2 / 1000, 0.19 / 1000, 0.15 / 1000, 0.11 / 1000, or 0.1 / 1000, or a lower risk. Patients determined to be at higher risk of PML may be determined to be at higher risk, such as approximately 0.54 / 1000 or higher, for example, approximately 0.55 / 1000, approximately 0.60 / 1000, approximately 0.66 / 1000, approximately 1.2 / 1000, approximately 1.37 / 1000, approximately 2.0 / 1000, approximately 2.5 / 1000, approximately 3.0 / 1000, approximately 4.3 / 1000, approximately 5.0 / 1000, approximately 7.8 / 1000, approximately 8.0 / 1000. For example, patients determined to be at higher risk of PML may be determined to be at higher risk of 0.3 / 1000, 0.35 / 1000, 0.5 / 1000, 0.66 / 1000, 1.2 / 1000, 1.37 / 1000, 2.0 / 1000, 2.5 / 1000, 3.0 / 1000, 4.3 / 1000, 5.0 / 1000, 7.8 / 1000, 8.0 / 1000, or higher.

[0095] In one embodiment, patients who have received prior treatment with anti-VLA-4 therapy for longer than 24 months, have not received prior treatment with immunosuppressants other than anti-VLA-4 therapy, and are determined to be JCV-negative are determined to have a lower risk of developing PML and are therefore preferred candidates for further treatment with anti-VLA-4 therapy such as natalizumab. However, due to having received anti-VLA-4 therapy for longer than 24 months, the risk assessment may include a recommendation for more frequent monitoring of patients for adverse events, such as symptoms that may indicate the development of PML.

[0096] Enhanced patient monitoring for PML development may include increased frequency of testing to identify the presence of JCV, such as increased testing with anti-JCV antibody assays or nucleic acid-based assays. Enhanced monitoring may also include MRI scans to identify brain lesions attributable to PML.

[0097] In one embodiment, patients with anti-JCV antibodies below a pre-selected threshold have undetectable levels of anti-JCV antibodies.

[0098] In one embodiment, the patient has previously received anti-VLA-4 therapy, while in another embodiment, the patient has not previously received anti-VLA-4 therapy.

[0099] In yet another embodiment, the patient is classified as a candidate for anti-VLA-4 therapy, and anti-VLA-4 therapy, such as natalizumab, is administered to the patient.

[0100] In one embodiment, making a determination, for example, whether a patient is negative for JCV, requires providing a biological sample from the patient (e.g., obtaining or receiving it) and performing an immunoassay, such as an ELISA assay, to detect JCV antibodies in the sample. In another embodiment, making a determination, for example, whether a patient is negative for JCV, requires providing a biological sample from the patient and performing an assay, such as a PCR-based assay, to detect JCV nucleic acid in the sample.

[0101] If a patient is classified as a candidate for anti-VLA-4 therapy, the patient may be further administered anti-VLA-4 therapy. Patients classified as candidates for anti-VLA-4 therapy are determined to have a lower risk of developing PML, for example, less than approximately 0.2 / 1000 patients, for example, 0.3 / 1000 patients, or 0.2 / 1000 patients, or 0.19 / 1000 patients, or 0.11 / 1000 patients. For example, a patient with a lower risk of PML may have a risk of 0.2 / 1000 or less.

[0102] Patients who are not classified as candidates for anti-VLA-4 therapy, or who are determined to be candidates for anti-VLA-4 therapy through enhanced monitoring for the development of PML, are determined to have a higher risk of developing PML, e.g., a risk of approximately 0.37 / 1000 patients or higher. For example, patients determined to have a higher risk of PML may have a risk of 0.37 / 1000, 0.35 / 1000, 0.66 / 1000, 1.2 / 1000, 1.37 / 1000, 2.5 / 1000, 4.3 / 1000, or 7.8 / 1000 patients.

[0103] In one embodiment, the aforementioned immunosuppressant exposure classification, if selected, is one of the following: A positive previous immunosuppressant exposure classification corresponds to having received non-anti-VLA-4 immunosuppressant therapy within a pre-selected time frame, for example, within 1, 3, or 5 years, or during the patient's lifetime, and A negative previous immunosuppressant exposure classification, corresponding to not having received non-anti-VLA-4 immunosuppressive therapy within a pre-selected time frame, for example, within 1, 3, or 5 years, or throughout the patient's lifetime.

[0104] In one embodiment, the aforementioned VLA-4 therapy exposure classification, if selected, is one of the following: A positive prior VLA-4 therapy exposure classification corresponds to having received anti-VLA-4 therapy for a predetermined period beyond a set time, for example, 1, 2, 3, or 5 years or more, and A negative prior VLA-4 therapy exposure classification corresponding to having received anti-VLA-4 therapy for a pre-selected period of less than a certain duration, e.g., less than 6 months, 1, 2, 3, or 5 years.

[0105] In one embodiment, the method includes providing a therapeutic suitability classification, which may be selected from, for example, one of the following: Positive therapeutic suitability classifications correlate with the patient's suitability for anti-VLA-4 therapy (the positive therapeutic suitability classifications can be further subdivided into positive therapeutic suitability classifications that involve various warnings or needs for monitoring, such as increased monitoring for the onset of PML), and A negative treatment suitability classification correlates with patient suitability for anti-VLA-4 therapy, accompanied by various warnings or needs for increased monitoring, such as patient unsuitability for anti-VLA-4 therapy or the development of PML.

[0106] A positive treatment suitability classification correlates with a lower risk of developing PML, while a negative treatment suitability classification correlates with a higher risk of developing PML. A lower risk of developing PML typically corresponds to a risk of less than 0.2 / 1000 patients, while a higher risk of developing PML corresponds to a risk of 0.37 / 1000 or greater.

[0107] If a patient is assigned a low exposure classification and a negative JCV status classification, the patient is assigned a modified positive therapeutic suitability classification, such as one in which monitoring for the development of PML is advised or required.

[0108] If a patient is assigned a negative prior immunosuppressant exposure classification and a negative anti-JCV antibody status classification, the patient may be assigned a modified positive therapeutic suitability classification, such as one advising or requiring monitoring for the development of PML.

[0109] If a patient is assigned a low exposure classification, a negative pre-immunosuppressant exposure classification, and a negative JCV antibody classification, the patient is assigned a positive treatment suitability classification.

[0110] In one embodiment, the patient is assigned a positive treatment suitability classification and is further administered anti-VLA-4 therapy, such as natalizumab.

[0111] In one embodiment, a method for evaluating patients, for example, for evaluating the risk of patients developing PML, is also provided. This method is (aaa) Determining whether a patient is negative or positive for JCV by determining whether the level of anti-JCV antibody is below or above a pre-selected threshold, for example, by a method disclosed herein, (bbb) Determining whether the patient has received anti-VLA-4 therapy for a period exceeding a pre-selected period (e.g., longer than 24 months), or for a period less than a pre-selected period (e.g., 24 months or less), or whether the patient has never received anti-VLA-4 therapy during a pre-selected period (e.g., the last 2, 3, or 5 years), or in their lifetime. (ccc) This includes determining whether the patient has not received non-anti-VLA-4 immunosuppressive therapy for a pre-selected period of time, or whether the patient has received non-anti-VLA-4 immunosuppressive therapy for a pre-selected period of time (specified time) (for example, in the last 1, 2, 3, 4, 5, or 10 years, or at any point in the patient's lifetime), and evaluating the patient in response to that determination.

[0112] In one embodiment, in response to a determination that the patient is negative for JCV, it is determined that the patient is at a lower risk of developing PML.

[0113] In one embodiment, in response to a determination that the patient is positive for JCV, it is determined that the patient is at higher risk of PML.

[0114] In some embodiments, the determination, for example, that a patient is negative for JCV, includes or requires taking a sample from the patient's body or analyzing a sample from the patient, or the method further requires administering a treatment to the patient. The treatment may be, for example, an anti-VLA-4 therapy (e.g., an anti-VLA-4 antibody) for patients at lower risk, or an alternative (non-anti-VLA-4) therapy, for example, interferon, glatiramer acetate, or a corticosteroid, for patients at lower risk.

[0115] In one embodiment, a method for complying with instructions is provided. The instructions may appear, for example, on a package insert required by a government, such as on a package mandated by the FDA (Food and Drug Administration) or EMA (European Medicines Agency), and provide guidance on the use of anti-VLA-4 therapy. The method for complying with instructions may include, optionally, receiving the instructions, obtaining the results of the evaluation methods described herein, providing recommendations for therapy to the patient in response to the obtained results, and optionally, further administering the therapy to the patient. The instructions may specify evaluation methods described herein that are essential for the safe administration of the therapy. The therapy may be an anti-VLA-4 therapy, such as natalizumab.

[0116] A method for evaluating a patient is provided, which requires providing a kit for collecting or transporting patient samples to a healthcare provider, receiving patient samples from a healthcare provider, and performing the method claimed herein.

[0117] Methods for treating patients are also provided. These methods require obtaining results from patient or sample evaluation methods described herein and administering a treatment to the patient in response to the obtained results. The treatment may be an anti-VLA-4 therapy such as natalizumab. For example, administering a treatment, such as a treatment described herein, to the patient in response to the results of steps (a) and (b), steps (a), (b), and (c), steps (a), (b), (c), and (d), step (c), or steps (c) and (d).

[0118] A computer-based method is also provided that allows for reimbursement of costs related to anti-VLA-4 therapy. The party eligible for reimbursement may be a third-party payer, such as an insurance company or a government agency. The method may include (a) obtaining the results of the patient assessment method described herein and recording the results on a computer-readable medium; (b) obtaining evidence of the administration of anti-VLA-4 therapy to the patient and recording the evidence on a computer-readable medium; and (c) allowing or providing reimbursement to the party if the results are consistent with the administration of anti-VLA-4 therapy.

[0119] In one embodiment, a method is provided for selecting or classifying a patient as a candidate for treatment with anti-VLA-4 therapy, such as natalizumab. For example, the method may include determining whether the patient has previously received anti-VLA-4 therapy for 24 months or less, for example, 1–24 months, 2–20 months, 5–15 months, or 10–12 months, or whether the patient has not previously received treatment with an immunosuppressant, and assessing the anti-JCV antibody titer or inhibition percentage. In one embodiment, the assessment involves analyzing a sample from the patient. The sample may be, for example, a blood, plasma, serum, urine, or cerebrospinal fluid sample. If the assessment indicates that the patient is positive for JCV, for example, positive for anti-JCV antibody or JCV nucleic acid, the patient is not selected or classified as a candidate for treatment with anti-VLA-4 therapy. If the assessment indicates that the patient is negative for JCV, for example, negative for anti-JCV antibodies or JCV nucleic acid, the patient is selected or classified as a candidate for receiving anti-VLA-4 therapy.

[0120] Assays for the presence of anti-JCV antibodies can be performed using immunoassays such as ELISA. Assays for JCV nucleic acids can be performed using methods such as PCR assays or next-generation sequencing (NGS).

[0121] Patients determined to be at lower risk of PML may be further administered anti-VLA-4 therapy, such as natalizumab. Patients determined to be at higher risk of PML may be further administered alternatives to anti-VLA-4 therapy, such as interferon, glatiramer acetate, corticosteroids, or TNF agonists. In one embodiment, patients determined to be at higher risk of PML may be further administered anti-VLA-4 therapy and may be required to accept increased frequency testing for PML, and if the patient is initially determined to be JCV-negative, they may also be required to accept increased frequency testing for JCV.

[0122] In one embodiment, a method is provided for determining a patient's risk to PML. The method comprises (a) determining whether the patient has previously received anti-VLA-4 therapy (e.g., natalizumab) for 24 months or less, or whether the patient has previously received treatment with an immunosuppressant, and (b) assessing the patient's anti-JCV antibody status (the assessment step includes analyzing a sample from the patient). If the assessment indicates that the patient is JCV-negative, the patient is determined to be at lower risk to PML. If the assessment indicates that the patient is JCV-positive, the patient is determined to be at higher risk to PML.

[0123] In another embodiment, a method for treating a patient is provided. The treatment method includes, for example, determining the patient's prior exposure to anti-VLA-4 therapy and determining whether the patient has previously received treatment with immunosuppressants. Optionally, the patient's status for JCV can also be determined.

[0124] If it is determined that a patient has previously received anti-VLA-4 therapy for 24 months or less and has not previously received immunosuppressant therapy, the patient is determined to be at lower risk of PML and will be administered anti-VLA-4 therapy. If it is determined that a patient has previously received natalizumab for longer than 24 months (e.g., 25 months or longer) and has not previously received immunosuppressant therapy, the patient is determined to be at higher risk of PML and will be administered an alternative to anti-VLA-4 therapy, such as interferon, corticosteroids, statins, or TNF antagonists.

[0125] Determining a patient's prior exposure to anti-VLA-4 therapy or immunosuppressants may involve asking the patient or their caregiver, such as a physician, nurse, parent, or other caregiver. In some cases, determining a patient's prior exposure may involve accessing information in a database, such as a medical record database.

[0126] A method for determining a patient's risk to PML is also provided. This method includes determining the patient's prior exposure to anti-VLA-4 therapy and whether the patient has previously received treatment with immunosuppressants. Optionally, the patient's anti-JCV antibody status may also be determined. If it is determined that the patient has received anti-VLA-4 therapy for 24 months or less and has not previously received treatment with immunosuppressants, the patient is determined to be at lower risk of PML. If it is determined that the patient has received anti-VLA-4 therapy for longer than 24 months and has not previously received treatment with immunosuppressants, the patient is determined to be at higher risk of PML. Patients determined to be at lower risk of PML may be further administered anti-VLA-4 therapy, such as natalizumab. Conversely, patients determined to be at higher risk of PML may be further administered alternatives to anti-VLA-4 therapy, such as interferon, corticosteroids, statins, or TNF antagonists.

[0127] In one embodiment, the patient's JCV status is also determined, and if the patient is determined to be JCV-negative, the patient is determined to be at lower risk of PML than if the patient were determined to be JCV-positive.

[0128] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by experts in the art to which the present invention pertains. Methods and substances similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and substances are listed below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In the event of any conflict, this specification shall prevail, including definitions. In addition, substances, methods, and examples are illustrative only and are not intended to be limiting.

[0129] The present invention also provides, for example, the following items: (Item 1) A method for evaluating the level of anti-JCV antibody in a sample, (a) Forming a first reaction mixture comprising a first aliquot of the sample and a substrate on which the HPVLP is placed, (b) detecting the level of anti-JCV antibody bound to the substrate on which HPVLP is placed by detecting a labeled detection reagent bound to the anti-JCV antibody bound to the substrate, (i) 20 ngs to 60 ngs of HPVP are placed on the substrate, and (ii) the sample-to-substrate ratio is 1:50 to 1:30. Either one or both of these conditions are met. A method for evaluating the level of anti-JCV antibody in a sample. (Item 2) The method according to item 1, wherein the sample, for example, serum, is diluted, for example, about 100 times in a buffer before contact with the substrate. (Item 3) Detection is performed using the method described in item 1, for example, enzyme-labeled antibodies, such as enzyme-labeled IgG or HRP-labeled IgG. (Item 4) The method according to item 3, wherein the detection reagent, for example, HRP-labeled IgG, is added at a concentration of at least 0.01 μg / mL, 0.02 μg / mL, 0.03 μg / mL, 0.04 μg / mL, 0.05 μg / mL, 0.06 μg / mL, or 0.08 μg / mL. (Item 5) (c) A second reaction mixture is formed containing a second aliquot of the sample and a solution phase of HPVLP, and the level of unbound anti-JCV antibody in the second reaction mixture is detected by detecting anti-JCV antibody capable of binding to the substrate on which the HPVLP is placed, (d) optionally, a third reaction mixture containing a third aliquot is formed under conditions in which the anti-JCV antibody in the sample is not bound by HPLP or other antigens, and the level of unbound anti-JCV antibody in the third reaction mixture is detected by detecting anti-JCV antibody that can bind to a substrate on which HPLP is placed, etc. The method described in item 1, which provides values ​​for interference. (Item 6) The method of item 5, comprising comparing the detected level in the second reaction mixture with the detected level in the third reaction mixture. (Item 7) The method of item 6, wherein the comparison comprises determining the level to which the presence of HPVLP in the second reaction mixture inhibits the level of unbound anti-JCV antibody in the second reaction mixture compared to the level of unbound antibody in the third reaction mixture. (Item 8) The method according to item 7, wherein the inhibition is less than 45%, and optionally, the sample is classified as negative. (Item 9) The method according to item 7, wherein the inhibition is 45% or more, and the sample is classified as positive. (Item 10) The method according to item 5, wherein the method is performed in response to the level of anti-JCV antibody detected in step b of item 1, for example, an nOD value of less than 0.2 and less than 0.4, and the user proceeds to steps c and d. (Item 11) The method according to item 5, wherein the sample, for example, serum, is diluted, for example, in a buffer solution, for example, by about 100 times, before forming the second reaction mixture. (Item 12) The method according to item 5, wherein the sample, for example, serum, is diluted, for example, in a buffer solution, for example, by about 100 times, before forming the third reaction mixture. (Item 13) Detection of one or both of the second and third reaction mixtures is performed using an enzyme-labeled antibody, e.g., enzyme-labeled IgG, e.g., HRP-labeled IgG, according to the method of item 5. (Item 14) The method according to item 3, wherein detection of one or both of the second and third reaction mixtures is performed using HRP-labeled IgG added at a concentration of at least 0.01 μg / mL, 0.02 μg / mL, 0.03 μg / mL, 0.04 μg / mL, 0.05 μg / mL, 0.06 μg / mL, or 0.08 μg / mL. (Item 15) The method according to item 1, further comprising, in step b, evaluating a standard substance having a score of approximately 1, e.g., a cutoff calibration sample. (Item 16) The method according to item 1, further comprising, in step b, evaluating a standard substance having a score of approximately 1.3, for example, a positive control. (Item 17) The method according to item 1, further comprising, in step b, evaluating a standard substance having a score of approximately 0.1, for example, a negative control. (Item 18) A kit containing the substrates listed in item 1. (Item 19) The substrate is provided on a multiwall plate, for example, a 96-well plate, as described in item 18 of the kit. (Item 20) The kit described in item 18 further comprises one or more of the following: HPVLP in solution; a sample of human serum, a JCV cutoff calibration sample, an anti-JCV antibody positive control, and a JCV negative control; and a reagent for detecting a complex containing an anti-JCV antibody bound to the JCV antigen. (Item 21) The kit described in item 20, wherein the detection reagent is one or more of TMB (tetramethylbenzidine), washing buffer, and stop reagent. (Item 22) A method for evaluating the risk of developing progressive multifocal leukoencephalopathy (PML) in patients, wherein the method is: The assay for confirming anti-JCV antibodies in a biological sample from the aforementioned patient includes determining the JC virus (JCV) antibody titer, expressed as nOD, an index, or other units, or other characteristics such as affinity or binding activity, expressed as an inhibition percentage. If it is determined that the function of the titer and / or inhibition percentage, or both, is above or below a predetermined level, the patient is determined to have a lower risk of developing PML. If it is determined that the function of the titer and / or inhibition percentage, or both, is above or below a predetermined level, the patient is determined to have a moderate risk of developing PML. If the function of the titer and / or inhibition percentage, or both, is determined to be at or above a predetermined level, the patient is determined to be at a higher risk of developing PML, and further, (i) Determining the anti-JCV antibody titer or inhibition percentage in the patient's sample includes taking a biological sample from the patient's body or analyzing a sample from the patient, or (ii) If it is determined that the patient is at a lower risk of developing PML, the treatment is administered to the patient. (Item 23) The patient is determined to be at a lower risk of developing PML, and the patient is administered anti-VLA-4 therapy, as described in item 22. (Item 24) The aforementioned patient, (i) The anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be less than 0.5, or (ii) The method of item 22, wherein the anti-JCV antibody titer, as indicated by an index value or nOD, is determined to be greater than 0.5 and less than 3.0, and the inhibition percentage is determined to be 70% or less, thereby determining that there is a lower risk of PML. (Item 25) The aforementioned patient, (i) The anti-JCV antibody titer indicated by the index value or nOD is determined to be greater than 0.5 and less than 1.5, and (ii) The method of item 22, wherein if the inhibitory percentage value is determined to be greater than 70%, it is determined to have a moderate risk of PML. (Item 26) The aforementioned patient, (i) The anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 3, and the inhibition percentage is determined to be greater than 70%, or (ii) The method of item 22, wherein if the patient shows a twofold increase in an index, nOD, or titer from the previous test, it is determined that the patient is at higher risk of PML. (Item 27) The inhibition percentage of anti-JCV antibodies is, (i) Contacting a biological sample from the subject with highly purified VP1 particles (HPVLP) in a solution under conditions suitable for the binding of anti-JCV antibodies in the sample to the HPLP, (ii) Separating the JCV antibody bound to HPVLP from the solution to prepare a secondary sample, (iii) Contacting the secondary sample with HPVLP under the same conditions as in (i), (iv) The method of item 26, which is measured by detecting the level of anti-JCV antibody binding to HPVLP in the secondary sample. (Item 28) The aforementioned anti-JCV antibody titer is (i) The biological sample is brought into contact with HPVLP under conditions suitable for the binding of the anti-JCV antibody in the sample to the HPVLP, (ii) To detect the level of anti-JCV antibody binding to HPVLP in the sample, (iii) The method of item 22, wherein the detected level is measured by an assay comprising correlating it with a reference set, the reference set being selected to exhibit a false negative rate not exceeding 3%. (Item 29) The aforementioned anti-JCV antibody titer was determined using the VIDAS® assay (bioMerieux). The method described in item 22, which is measured by use. (Item 30) The assay shows that the biological sample does not contain JCV antibodies. (iv) Contacting a portion of the biological sample from the subject with HPVLP in solution before step (i), wherein the HPVLP in step (i) is bound to a solid substrate and thereby provides a secondary sample. (v) The secondary sample is brought into contact with HPVLP under the same conditions as in (i), (vi) To detect the level of anti-JCV antibody binding to HPVLP in the secondary sample, (vii) The method of item 28, further comprising comparing the detected level of anti-JCV antibody in the secondary sample with the level of binding in the biological sample when incubated with the solution without HPVLP, wherein a decrease in the detected level in the sample pre-incubated with HPVLP compared to the sample incubated with the solution indicates that the sample is positive for anti-JCV antibody, and no change in the detected level indicates that no anti-JCV antibody is present in the sample. (Item 31) The assay according to item 28, wherein the assay shows that the biological sample contains JCV antibodies, and the patient is determined to be at higher risk of PML. (Item 32) The assay according to item 30, wherein the assay shows that the biological sample contains JCV antibodies, and the patient is determined to be at higher risk of PML. (Item 33) The aforementioned patient, (i) The anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be less than 0.5, or (ii) The method of item 30, wherein the anti-JCV antibody titer, as indicated by an index value or nOD, is determined to be greater than 0.5 and less than 3.0, and the inhibition percentage is determined to be 70% or less, thereby determining that there is a lower risk of PML. (Item 34) The aforementioned patient, (i) The anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 3, and the inhibition percentage is determined to be greater than 70%, or (ii) The method of item 30, wherein if the patient shows a twofold increase in an index, nOD, or titer from the previous test, it is determined that the patient is at higher risk of PML. (Item 35) The method according to item 30, wherein the assay shows that the biological sample does not contain JCV antibodies, and it is determined that the patient is at lower risk of PML. (Item 36) The anti-JCV antibody titer or inhibition percentage is retested at intervals of 6 or 12 months, as described in item 22. (Item 37) The method according to item 36, wherein an increase in antibody titer or inhibition percentage indicates an increased risk in the patient to develop PML. (Item 38) A more than twofold increase in anti-JCV antibody titer indicates an increased risk of developing PML, as described in item 36. (Item 39) The aforementioned assessment is performed according to the method described in item 22, which occurs before the administration of anti-VLA-4 therapy. (Item 40) The assessment described above is the method described in item 22, which occurs after the patient initiates anti-VLA-4 therapy. (Item 41) The patient is determined to be at lower risk of PML, and the patient is administered anti-VLA-4 therapy, as described in item 22. (Item 42) The patient is determined to be at higher risk of PML, and the patient is administered anti-VLA-4 therapy, as described in item 22. (Item 43) The aforementioned anti-VLA-4 therapy is natalizumab, as described in item 22. (Item 44) The method according to item 37, wherein the patient is determined to have an increased risk of PML, and the patient further discontinues receiving the anti-VLA-4 therapy. (Item 45) The patient is determined to be at higher risk of PML, and the patient is administered an alternative to anti-VLA-4 therapy, such as interferon, glatiramer acetate, or a corticosteroid, as described in item 22. (Item 46) The patient is monitored at regular intervals for a decrease in anti-JCV antibody titer or a decrease in JCV antibody inhibition percentage, and a decrease in anti-JCV antibody titer or JCV antibody inhibition percentage indicates that the patient has a reduced risk of developing PML, as described in item 44. (Item 47) The method according to item 44, wherein the JCV titer or JCV antibody inhibition percentage is determined to have decreased to below a predetermined level, and the patient's anti-VLA-4 therapy is resumed. (Item 48) The patient is evaluated every six months or annually for a decrease in the antibody titer of the JCV antibody or a decrease in the inhibition percentage, as described in item 46. (Item 49) (a) Determine whether the patient has ever received long-term treatment with anti-VLA-4 therapy (for example, for longer than 24 months), or (b) further comprising determining whether the patient has ever received a designated non-anti-VLA-4 immunosuppressive therapy (for example, in the last two, three, or five years, or at any point in the patient's lifetime), The method according to item 22, wherein the relative risk of PML for patients having an anti-JCV antibody titer or inhibition percentage above a predetermined level but who have not used a designated immunosuppressant and have never had long-term treatment with anti-VLA-4 therapy is less than the relative risk for patients having an anti-JCV antibody titer or inhibition percentage above a predetermined level and who have used a designated immunosuppressant and have had long-term treatment with anti-VLA-4 therapy, and the relative risk for patients having an anti-JCV antibody titer or inhibition percentage below a predetermined level and who have used a designated immunosuppressant or have had long-term treatment with anti-VLA-4 therapy. (Item 50) The patient in question has previously received anti-VLA-4 therapy, as described in item 49. (Item 51) The method according to item 49, further comprising administering anti-VLA-4 therapy to the patient. (Item 52) The aforementioned anti-VLA-4 therapy is natalizumab therapy, as described in item 51. (Item 53) The method according to item 49, wherein the patient is classified as a candidate for anti-VLA-4 therapy, and the patient is further administered the anti-VLA-4 therapy. (Item 54) A method for evaluating a patient as a candidate for treatment with, for example, anti-VLA-4 therapy, wherein the method is: This includes obtaining the JC virus (JCV) antibody titer and inhibition percentage in a biological sample from the patient. If it is determined that the antibody titer or inhibition percentage is below a predetermined level, the patient is classified as suitable for treatment in the first therapy category. If it is determined that the antibody titer or inhibition percentage is at or above a predetermined level, the patient is classified as suitable for the second therapy category. Furthermore, (i) Obtaining the anti-JCV antibody titer and inhibition percentage in the patient's sample includes taking a biological sample from the patient's body or analyzing a sample from the patient, or (ii) The method further comprises administering to the patient a treatment from the first category or the second category, A method for evaluating the patient thereby. (Item 55) The first therapy category is anti-VLA-4 therapy, for example, anti-VLA-4 antibody therapy, for example, natalizumab, as described in item 54. (Item 56) The method according to item 54, wherein the second therapeutic category is interferon, glatiramer acetate, or corticosteroid. (Item 57) The patient is classified as suitable for treatment in the first therapy category, and the patient is further administered natalizumab, as described in item 54. (Item 58) The patient is classified as suitable for treatment in the second therapy category, and the patient is further administered interferon, glatiramer acetate, or a corticosteroid, as described in item 54. (Item 59) The aforementioned patient, (i) The anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be less than 0.5, or (ii) The method of item 54, wherein the anti-JCV antibody titer, as indicated by an index value or nOD, is determined to be greater than 0.5 and less than 3.0, and the inhibition percentage is determined to be 70% or less, thereby determining that there is a lower risk of PML. (Item 60) The aforementioned patient, (i) The anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 3, and the inhibition percentage is determined to be greater than 70%, or (ii) The method of item 54, wherein the patient is determined to be at higher risk of PML if the patient shows a twofold increase in an index, nOD, or titer from the previous test. (Item 61) (a) Determine whether the patient has ever received long-term treatment with anti-VLA-4 therapy (for example, for longer than 24 months), or (b) further comprising determining whether the patient has ever received a designated non-anti-VLA-4 immunosuppressive therapy (for example, in the last two, three, or five years, or at any point in the patient's lifetime), The method according to item 33, wherein a patient having an anti-JCV antibody titer or inhibition percentage above a predetermined level but without the use of a designated immunosuppressant and without long-term treatment with anti-VLA-4 therapy is classified as having a lower risk of developing PML than the relative risk of a patient having an anti-JCV antibody titer or inhibition percentage below a predetermined level and with the use of a designated immunosuppressant or long-term treatment with anti-VLA-4 therapy, which is the relative risk of a patient having an anti-JCV antibody titer or inhibition percentage above a predetermined level and with the use of a designated immunosuppressant or long-term treatment with anti-VLA-4 therapy. (Item 62) The patient has previously received anti-VLA-4 therapy, as described in item 61. (Item 63) The method according to item 61, further comprising administering anti-VLA-4 therapy to the patient. (Item 64) The aforementioned anti-VLA-4 therapy is natalizumab therapy, as described in item 63. (Item 65) The patient is classified as a candidate for anti-VLA-4 therapy, and the patient is further administered the anti-VLA-4 therapy, as described in item 61. (Item 66) A method for evaluating the risk of developing progressive multifocal leukoencephalopathy (PML) in patients, wherein the method is: (a) To determine whether the patient is negative or positive for exposure to the JC virus (JCV), (b) Determining whether the patient has ever received long-term treatment with anti-VLA-4 therapy (for example, for longer than 24 months), or (c) Determining whether the patient has ever received the specified non-anti-VLA-4 immunosuppressive therapy (for example, in the last two, three, or five years, or at any point in the patient's lifetime), This includes determining, in response to a determination that the patient is negative for exposure to JCV, that the patient has a relatively low risk of developing PML, or determining, in response to a determination that the patient is positive for JCV, that the patient has a relatively higher risk of developing PML. The relative risk of PML in patients who have been exposed to JCV but have not used the designated immunosuppressant and have not had long-term anti-VLA-4 treatment is less than the relative risk in patients who have been exposed to JCV and have used the designated immunosuppressant and have had long-term anti-VLA-4 treatment, or less than the relative risk in patients who have been exposed to JCV and have used the designated immunosuppressant or have had long-term anti-VLA-4 treatment. (Item 67) Furthermore, the method according to item 66, provided that (i) determining that the patient is negative for JCV includes taking a sample from the patient's body or analyzing a sample from the patient, or (ii) the method further includes administering a treatment to the patient, for example, an anti-VLA-4 therapy, for example, an anti-VLA-4 antibody, or an alternative treatment, for example, interferon, glatiramer acetate, or a corticosteroid. (Item 68) The patient has previously received anti-VLA-4 therapy, as described in item 66. (Item 69) The method according to item 66, further comprising administering anti-VLA-4 therapy to the patient. (Item 70) The anti-VLA-4 therapy is natalizumab therapy, as described in item 69. (Item 71) The method according to item 66, wherein determining whether the patient is negative for JCV comprises providing a biological sample from the patient and performing an immunoassay to detect JCV antibodies in the sample. (Item 72) The aforementioned immunoassay is an ELISA assay, as described in item 71. (Item 73) The immunoassay method described above is the method according to item 71, which detects a JCV antibody that is an IgG isotype, IgM isotype, IgA isotype, or IgE isotype. (Item 74) The sample is stored at 2-8°C for 1-14 days before performing the immunoassay, according to the method described in item 71. (Item 75) The method according to item 66, wherein determining whether the patient is negative for JCV comprises providing a biological sample from the patient and performing an assay to detect JCV nucleic acid in the sample. (Item 76) The method according to item 66, wherein the patient is classified as a candidate for anti-VLA-4 therapy, and the patient is further administered the anti-VLA-4 therapy. (Item 77) Patients at lower risk of developing PML have a risk of less than approximately 0.3 / 1000, for example, 0.11 / 1000 or less, as described in item 66. (Item 78) Patients at higher risk of developing PML are those with a risk of approximately 0.3 / 1000 or greater, for example, 0.35 / 1000, 1.2 / 1000, 2.5 / 1000, or 7.8 / 1000, as described in item 66. (Item 79) The method according to item 66, wherein the patient is positive for JCV, has received anti-VLA-4 therapy for less than a pre-selected period of time, has not received non-anti-VLA-4 immunosuppressive therapy for a pre-selected period of time, and in response to that determination, the patient is determined to be at risk of developing PML at approximately 0.35 / 1000. (Item 80) The method according to item 66, wherein the patient is positive for JCV, has received anti-VLA-4 therapy for less than a predetermined period, has received non-anti-VLA-4 immunosuppressive therapy for a predetermined period, and in response to that determination, the patient is determined to be at risk of developing PML at approximately 1.2 / 1000. (Item 81) The method according to item 66, wherein the patient is positive for JCV, has received anti-VLA-4 therapy for a predetermined period of time, has received non-anti-VLA-4 immunosuppressive therapy for a predetermined period of time, and in response to that determination, the patient is determined to be at risk of developing PML at a rate of approximately 7.8 / 1000. (Item 82) The method according to item 66, wherein the patient is positive for JCV, has received anti-VLA-4 therapy for a predetermined period of time, has not received non-anti-VLA-4 immunosuppressive therapy for a predetermined period of time, and in response to that determination, the patient is determined to be at risk of developing PML at a rate of approximately 2.5 / 1000. (Item 83) The method according to item 66, wherein the patient is determined to be JCV antibody positive, and to have a JCV antibody titer of 0.5 or greater than 1.5 as indicated by an index value or nOD, and an inhibition percentage value greater than 70%, and the patient is further determined to be at moderate risk of PML. (Item 84) The method according to item 66, wherein the patient is determined to be JCV antibody positive and to have a JCV antibody titer greater than 1.5 as indicated by an index value or nOD, and an inhibition percentage value greater than 70%, and the patient is further determined to be at higher risk of PML. (Item 85) The method of item 66, wherein the patient is determined to be JCV antibody positive and to have an antibody titer of less than 3 as indicated by an index value and an inhibition percentage value of 70% or less, and the patient is further determined to have a lower risk of PML. Details of one or more embodiments of the present invention are described in the accompanying drawings and the description below. Other features, subjects, and advantages of the present invention will become apparent from the description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0130] [Figure 1] Graphs illustrating the incidence of natalizumab-related PML based on cumulative treatment duration (Figure 1A) and 12-month treatment interval duration (Figure 1B). [Figure 2] A schematic diagram illustrating the approximate incidence of PML, stratified by the use of immunosuppressants and the duration of natalizumab treatment. [Figure 3] A schematic diagram illustrating the approximate incidence of PML, stratified by anti-JCV antibody serological status, prior use of immunosuppressants, and duration of natalizumab treatment. [Figure 4] A graph illustrating the susceptibility analysis estimates for PML development in anti-JCV antibody-positive patients, stratified by prior immunosuppressant use (with or without) and duration of natalizumab treatment (1–24 months (Figure 4A) or 25–48 months (Figure 4B)). Baseline = Baseline case scenario. [Figure 5] A graph illustrating the nOD and titer for Patient 1. [Figure 6] A graph illustrating the noOD and titer for patient 2. [Figure 7]A graph illustrating the nOD and titer for each of Patient 3. [Figure 8] A graph illustrating the nOD and titer for each of Patient 4. [Figure 9-1] A graph illustrating the noOD and titer for each of Patient 5. [Figure 9-2] A graph illustrating the noOD and titer for each of the six patients. [Figure 10] A graph illustrating the noOD and titer for each of the seven patients. [Figure 11] A scatter plot illustrating the collected indicator (x-axis) and inhibition percentage (y-axis) data for a group of MS patients. [Figure 12] A scatter plot illustrating the collected indicator (x-axis) and inhibition percentage (y-axis) data for a group of MS patients. [Figure 13] A scatter plot illustrating the determined index values ​​for a group of MS patients. [Modes for carrying out the invention]

[0131] The present invention is based, at least in part, on the discovery of novel and improved methods for assessing a patient's risk to PML, which include assessing anti-JCV antibody titers or inhibition percentages. The present invention is based, at least in part, on the discovery that anti-JCV antibody titers and antibody inhibition percentages may indicate a patient's risk of developing progressive multifocal leukoencephalopathy (PML).

[0132] The applicant has also developed an optimized assay for determining the level of anti-JCV antibody titers in a biological sample, and a method for qualitatively assaying the antibody by determining the inhibition percentage value and using this information to determine the patient's risk of developing PML. The assay comprises (a) forming a first reaction mixture containing a first aliquot of the sample and a substrate on which HPVLP is placed (the VLP particles are present in an amount of 0.04 μg and at a concentration of 0.4 μg / mL); (b) detecting the level of anti-JCV antibody bound to the HPVLP placed on the substrate by a labeled secondary detection reagent bound to the anti-JCV antibody bound to the substrate, such as detecting an enzyme-labeled anti-IgG antibody; (c) forming a second reaction mixture containing a second aliquot of the sample having a solution phase HPVLP provided at a concentration of, for example, 0.4 μg / mL, and a second aliquot of the sample provided at a dilution of, for example, 1:100 or 1:101; and (d) a negative control solution that does not contain HPVLP, and in the negative control solution For example, the method includes: (e) forming a third reaction mixture containing a third aliquot of a sample diluted 1:100, 1:101, or 1:110; (f) detecting the levels of unbound anti-JCV antibodies in the second and third reaction mixtures, for example, by detecting JCVs that can bind to the substrate on which the HPVLP is placed (on which the HPVLP is present); (g) providing a first value corresponding to the level of anti-JCV antibody binding to the HPVLP placed on the substrate in the first aliquot of the sample, and a second value corresponding to the level of unbound anti-JCV antibodies in the second reaction mixture, for example, the level of anti-JCV antibody binding to the HPVLP placed on the substrate from the second reaction mixture; and (g) optionally comparing the first and second antibody levels.

[0133] The applicant also found that patients have a lower risk of developing PML if (i) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be less than 0.5, or (ii) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 0.5 but less than 3.0, and the inhibition percentage is determined to be 70% or less. Patients have a higher risk of PML if (i) the anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 3, and the inhibition percentage is determined to be greater than 70%, or (ii) the patient shows a twofold increase in the index, nOD, or titer since the previous test.

[0134] Patients can be monitored at regular intervals, such as every 6 or 12 months, for changes in anti-JCV antibody titer or inhibition percentage. If the results of a subsequent assay show that the patient still has an anti-JCV antibody titer of less than 0.5 nOD and an inhibition percentage of less than 70%, the patient may be determined to still be at lower risk of developing PML. If a subsequent assay shows that the patient's antibody titer has increased 2-3 times from the initial assay, the patient may be determined to be at increased or higher risk of developing PML. The applicant observed that patients diagnosed with PML tended to demonstrate a 2-3 times increase in antibody titer and nOD in the 6 months prior to diagnosis.

[0135] A patient is at higher risk of PML if (i) their anti-JCV antibody titer, as indicated by the index value or nOD, is determined to be greater than 3 and their inhibition percentage is determined to be greater than 70%, or (ii) the patient shows a twofold increase in the index, nOD, or titer since the previous test.

[0136] Patients meeting these criteria may optionally be determined not to be candidates for anti-VLA-4 therapy, such as anti-VLA-4 antibodies, e.g., natalizumab, or the patient may be further assessed for other risk factors for developing PML. These risk factors include whether the patient has previously received anti-VLA-4 therapy such as natalizumab, and for how long, as well as whether the patient has previously received immunosuppressive therapy other than anti-VLA-4 therapy, and for how long. The patient's risk of PML is a combination of each of these factors.

[0137] Antibody titers can be measured by "nOD" or "index." "nOD" is the normalized optical density value for detecting anti-JCV antibodies in tests such as ELISA. The "index" value is obtained by dividing the optical density value of the sample by the optical density of the positive control in immunoassays such as ELISA.

[0138] The applicant previously found that patients who had received anti-VLA-4 therapy, such as natalizumab, for 24 months or less and had no prior history of immunosuppressive therapy were at lower risk of developing PML than patients who did not meet these two criteria. Furthermore, patients at the lowest risk were those who met these two criteria and were also JCV-negative, e.g., those who did not test positive for anti-JCV antibodies or JCV nucleic acids, e.g., JCV DNA. It was previously unknown that each of these three risk factors ((i) the amount of time the patient had previously received anti-VLA-4 therapy, (ii) whether the patient had previously received immunosuppressive drugs other than anti-VLA-4 therapy, and (iii) JCV status) independently contributed to the patient's risk of PML. The present invention as described herein can generally be used in patients treated with VLA-4 inhibitors. The ability to identify distinct subpopulations of patients at PML risk will enable better characterization of risk than previous methods (i.e., overall PML risk) and should assist healthcare professionals and patients in making more informed benefit-risk treatment decisions. These risk assessment criteria are described in U.S. Provisional Applications 61 / 491,810, filed May 31, 2011, and 61 / 508584, filed July 15, 2011, both common to assignees. The contents of each of these provisional applications are incorporated herein by reference in their entirety. The risk criteria described herein, directed to anti-JCV antibody titers (e.g., measured by nOD or index levels) and inhibition percentages, may be considered in combination with the risk factors described in the aforementioned common provisional applications to assignees.

[0139] Methods for determining PML risk may require obtaining one, two, or all three of the following: JCV classification for the patient (e.g., anti-JCV antibody titer and inhibition percentage as measured by nOD or index levels, etc.), prior anti-VLA-4 therapy history for the patient, and prior immunosuppressive therapy history (other than anti-VLA-4 therapy) for the patient. In response to these classifications, the patient may be assigned a treatment suitability classification. Patients determined to have a low risk of developing PML may be assigned a positive treatment classification, while patients determined to have a higher relative risk of developing PML may be assigned a negative treatment classification. Patients with a positive treatment classification may accept recommendations for further treatment or for initiating treatment with anti-VLA-4 therapy. Patients with a negative treatment classification may accept recommendations for discontinuing anti-VLA-4 therapy, initiating non-anti-VLA-4 therapy, or continuing or initiating anti-VLA-4 therapy with increased monitoring for signs and symptoms of PML.

[0140] For example, if one or more factors indicate that a patient may be at risk of increased PML, such as prior treatment with anti-VLA-4 therapy for longer than 24 months, e.g., 25 months or longer, or prior treatment with immunosuppressants other than anti-VLA-4 therapy, then a recommendation for further treatment with anti-VLA-4 therapy may be accompanied by further instructions or necessity for the patient to receive further or enhanced monitoring.

[0141] It may be determined that a patient has previously received anti-VLA-4 therapy or other immunosuppressive therapy through self-reporting by the patient or through information (oral or written) provided by a parent, physician, medical assistant, nurse, or other healthcare provider. Information can also be obtained through databases such as medical databases or clinical trial databases.

[0142] Prior immunosuppressive therapies other than anti-VLA-4 therapy that may indicate an increased risk of PML may include prior treatment with one or more antitumor agents, immunosuppressants, or immunomodulators, such as beta-interferon or glatiramer acetate. Examples of immunosuppressants include, for example, mitoxantrone, methotrexate, azathioprine, cyclophosphamide, and mycophenolates, anti-CD20 therapy (e.g., rituximab), anti-CD11a therapy (e.g., efalizumab), or mycophenolate mofetil. Prior treatment with other immunosuppressive therapies listed below may also be predicted to increase the risk of PML in patients after further administration of anti-VLA-4 therapy. Generally, the decision to use a prior immunosuppressant is a specified use, which may be any prior use of an immunosuppressant other than a VLA-4 inhibitor (e.g., anti-VLA-4 antibody), or prior use within a specified period, e.g., within the past 1, 2, 3, 5, or 10 years prior to the assessment of PML risk.

[0143] Determining JCV status refers to determining whether a patient has been exposed to JCV, and therefore includes direct methods (e.g., detecting JCV protein or JCV DNA) and indirect methods (e.g., detecting antibodies against JCV in a patient sample) for determining exposure. Assays for determining JCV status may include assays for detecting JCV nucleic acids (e.g., DNA or RNA), JCV serum prevalence, or anti-JCV antibodies in biological samples such as plasma, serum, blood, or urine samples, or in peripheral blood mononuclear cells (PBMCs) or cerebrospinal fluid samples. JCV nucleic acids can be detected using methods known in the art, for example, by amplification methods, such as polymerase chain reaction (PCR), or by next-generation sequencing (NGS) methods. JCV serum prevalence can be assayed using methods known in the art, such as hemoagulant inhibition (HI) assays. JCV antibodies can be detected by immunoassays, such as ELISA assays. In one embodiment, the JCV antibody can be detected by the method described in International Application PCT / US2011 / 20832, which utilizes HPVLP under conditions suitable for anti-JCV antibody binding to detect the level of anti-JCV antibody binding in a biological sample. The method for determining the JCV status also includes a method for determining the anti-JCV antibody titer and inhibition percentage. Detection of the anti-JCV antibody titer and inhibition percentage typically involves a two-step antibody detection assay described in International Application PCT / US2011 / 20832.

[0144] If the presence of JCV, for example, JCV antibodies, proteins, peptides, or nucleic acids, is identified in a biological sample from a patient, the patient is determined to be "JCV positive." A positive JCV classification corresponds to the presence of JCV antibodies in the biological sample, for example, JCV antibodies above a pre-selected threshold. The pre-selected threshold is typically a qualitative value, such as a "detectable" amount of antibody, according to a specific assay, such as an immunoassay.

[0145] The method described herein for determining the risk of PML may be useful in any human subject, including subjects considering treatment with immunomodulatory agents, such as anti-VLA-4 therapy (e.g., natalizumab), anti-CD20 therapy (e.g., rituximab), anti-CD11a therapy (e.g., efalizumab), or mycophenolate mofetil, subjects currently being treated with immunomodulatory agents, or subjects who have discontinued immunomodulatory agent treatment. The method may be useful in other individuals who may be susceptible to the effects of PML, such as individuals with lymphoproliferative disorders such as multiple myeloma or lymphoma; individuals infected with human immunodeficiency virus (HIV); or individuals with acquired immunodeficiency syndrome (AIDS), hematological malignancies, or autoimmune diseases such as systemic lupus erythematosus (SLE), inflammatory bowel diseases such as Crohn's disease (CD) or ulcerative colitis, multiple sclerosis (MS), or arthritis, such as rheumatoid arthritis (RA). Risk assessment methods may also be useful for recipients of immunosuppressants or immunomodulatory therapies, such as transplant patients. Examples of immunosuppressants or immunomodulatory therapies include natalizumab, rituximab, efalizumab, and mycophenolate mofetil. This method is based on Piccinni et al.'s "Stronger association of drug-induced progressive multifocal leukoencephalopathy (PML) with biological immunomodulating agents”Eur.J.Clin.Pharmacol. 66 The information disclosed in :199-206, 2010, which may be useful for assessing risks in subjects with disabilities or those being treated with drugs, is incorporated herein by reference.

[0146] definition As used herein, “HPVLP” refers to highly purified VLPs (“virus-like particles”) that consist primarily of the VP1 protein. The “HPVLP” featured in the present invention consists mainly of the main capsid protein “VP1,” which may be naturally occurring or recombinant VP1 from the polyomavirus JC virus (JCV). HPVLP may consist, for example, at least one pentameric subunit of VP1, more than one pentameric subunit, up to 72 pentameric subunits, or more than that number of pentameric subunits. The HPVLP of the present invention can bind to antibodies against naturally occurring, intact JC virus. In some embodiments, HPVLP includes a second and optionally third polypeptide, e.g., at least one VP2 or VP3 polypeptide, which is a minor capsid protein of the JC virus. VP2 or VP3 may be recombinant or naturally occurring or naturally derived polypeptides.

[0147] Such “highly purified” particles contain more than one VP1 pentamer, for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 72 VP1 pentamers, or less than 100 VP1 pentamers. Such highly purified particles can be obtained, for example, by a method involving double filtration. For example, in one embodiment, the preparation of highly purified VLPs is obtained by purifying the particles at least twice by centrifugation, for example, through a sucrose cushion. In other embodiments, HPVLPs are prepared using chromatography. Generally, HPVLP preparations can be identified by their activity in an ELISA assay using a predetermined control sample. In some cases, such a control sample is a negative control and / or control sample containing low levels of JCV antibody.

[0148] As used herein, “high signal-to-noise HPVLP substrate” is a substrate on which HPVLP is placed. It can be used to assess the level of free HPVLP (i.e., HPVLP not bound to an antigen or other target) in a sample. The concentration of HPVLP on the substrate is such that, when measuring the amount of anti-JCV antibody present, it provides a signal-to-noise ratio of 10–30, 15–30, 15–25, or 18–22. In embodiments, the signal-to-noise ratio is at least 10, 15, 18, or 20. In embodiments, the signal-to-noise ratio is about 10, 15, 18, or 20. The signal-to-noise ratio can be determined by a sample that gives an optical density of 1.0, e.g., a calibration control. In one embodiment, the HPVLP is placed on the substrate in wells of a 96-well plate, with 0.5 mL, 0.8 mL, and 1.0 mL of 0.4 μg / mL HPVLP in each well. HPVLP is provided in concentrations resulting from lyophilizing 1.2 mL, 1.2 mL, or 1.5 mL. In one embodiment, HPVLP is provided in concentrations resulting from lyophilizing 1.0 mL of 0.4 μg / mL HPVLP per well of a 96-well plate, which corresponds to 30 ng to 50 ng (e.g., 40 ng) of HPVLP per well. In another embodiment, HPVLP is provided in concentrations resulting from lyophilizing 0.05 mL to 0.35 mL or 0.1 mL to 0.2 mL of 0.4 μg / mL HPVLP per well of a 96-well plate. The amount of HPVLP placed on the substrate, or the conditions under which the placement is achieved, can be varied as long as the desired signal-to-noise ratio is obtained.

[0149] The signal-to-noise ratio is calculated by comparing the optical density value of the negative control with that of the calibration sample control to determine the dynamic range of signal intensity in the assay.

[0150] In one embodiment, the sample is diluted approximately 100 times, with a cutoff for negative scores being 45% or less, and a cutoff for positive scores being greater than 45%. In another embodiment, the dilution is other than 100 times but less than 200 times. For example, dilutions are 50 to 150 times, 75 to 125 times, or 85 to 115 times. In yet another embodiment, the dilution is 150 times, 125 times, 100 times, or less than 75 times. In yet another embodiment, if the dilution is other than 100 times (e.g., 200 times, 400 times, 500 times, 800 times, or more than 1,000,000 times), the cutoff or other parameters are adjusted so that the sample obtains the same score (positive or negative) that the sample would obtain if the dilution were 100 times, with a cutoff for negatives being less than 45% and a cutoff for positives being 45% or more.

[0151] Anti-JCV antibody detection assay. The assay is performed by adding a biological sample to a substrate coated with HPVLP and detecting the antibody using methods known in the art. Generally, a solid-based platform such as a microtiter plate (e.g., a 96-well plate) is used, but other forms known in the art may also be used. In some embodiments, the biological sample is diluted before use in the assay.

[0152] In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). More broadly, the method typically involves coating a substrate with a capture antigen such as HPVLP, incubating a sample containing a bound antibody that is guided to capture the reagent, washing to remove nonspecifically bound species, and detecting the bound immunocomplexes by, for example, a chromogenic or chemiluminescent assay. Chromogenic substrates produce a colored final product, which can be detected and measured visually or using a spectrophotometer. Chemiluminescent substrates generate light, which can be measured using a luminometer.

[0153] Coating plates with HPVLP generally involves incubating a solid substrate (such as the wells of a microtiter plate) in a solution of HPVLP at a suitable concentration (e.g., 0.4 μg / mL) for either overnight or for a specified number of hours. HPVLP may contain VP1 as the sole JCV virus component, or it may be heterogeneous particles containing at least one of VP2 or VP3 per particle, or at least one of each of VP2 and VP3 per particle. After coating with HPVLP, the plate wells are washed. The substrate is then "coated" with a nonspecific protein that is antigenically neutral with respect to the sample under test. Suitable coating materials are known in the art and include solutions of bovine serum albumin (BSA), casein, sugars, or powdered milk. The plates are then dried and may be stored for a fixed period of time, such as one day, one month, or one year, before proceeding to the next step in the assay.

[0154] The sample or reference material is incubated on the prepared substrate under conditions effective for enabling complex formation (HPVLP / JCV antibody), thus forming a bound complex. Detection of the bound complex is performed using a labeled antibody capable of binding to a human antibody. Generally, the labeled antibody can detect human IgG or human IgG and IgM. In some cases, the assay may be performed using a secondary or tertiary detection method.

[0155] The reference sample may be a sample of the same biological material (e.g., plasma, serum, urine, or CSF) isolated from an individual known to be infected with the JC virus (urine-positive (uro-positive)) based on the presence of JCV DNA in the individual's urine. Using the reference sample, an assay cut point is established where the false negative rate of the assay does not exceed 1% to 3%.

[0156] "Conditions effective for enabling complex formation" generally means conditions under which the reagent is diluted to reduce background and provide readouts of results within a specified range. In non-limiting examples, the diluent may include a solution containing BSA, phosphate-buffered saline (PBS), or PBS containing Tween.

[0157] "Preferred" conditions also include conditions that are at a temperature sufficient to allow effective bonding and / or for a certain period of time. Incubation is typically at a temperature of approximately 25°C to 27°C for about 1-2 hours or 1-4 hours, or at approximately 4°C overnight. However, those skilled in the art will understand that other conditions may be preferred.

[0158] Generally, one or more washes are performed during the incubation of the assay. Suitable wash solutions include diluent buffers (e.g., PBS or PBS / Tween) or borate buffers.

[0159] Generally, the detection of antibodies bound to HPVLP is performed using methods well known in the art. Generally, such methods are based on the detection of labels or markers, such as radioactive tags, fluorescent tags, biological tags, or enzyme tags. U.S. patents relating to the use of such labels include, for example, U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Generally, the detection of anti-JCV antibody binding is performed using a labeled secondary antibody. Generally, the secondary antibody is specific to the detection of human IgG. Quantification is achieved, for example, by measuring the degree of dye produced using a visible spectrum spectrophotometer.

[0160] In one embodiment, the assay is performed in a clinic by a healthcare provider, such as a physician, nurse, or technician, who works at the facility where the biological sample is obtained from the patient. In another embodiment, the biological sample obtained from the patient is transported to another facility where the assay is performed, such as a third-party facility. In the latter case, the assay results may be reported back to the healthcare provider by mail, electronically (e.g., via facsimile or email), or through a form or the like, which may be submitted via an online database. In one embodiment, the assay results (including screening assays and optionally confirmatory assays) may be stored in a database and accessible by the healthcare provider via the World Wide Web, etc.

[0161] Secondary testing. In some cases, for example, when the level of anti-JCV antibody in the sample falls into an "ambiguous interval" or "uncertain interval," for example, when it is determined that the certainty regarding the presence or absence of anti-JCV antibody is limited (for example, when it is determined that the nOD value is greater than 0.2 and less than 0.4), a secondary test of the sample (also referred to herein as a "confirmatory assay") is used. Two aliquots of the biological sample are used for the secondary test. The first aliquot is prepared by precubating the sample in the presence of assay buffer in solution for a certain period of time (e.g., 30 minutes, 1 hour, or longer, such as overnight) before use in the assay. The second aliquot is prepared by precubating the sample in the presence of HPVLP in solution for a certain period of time (e.g., 30 minutes, 1 hour, or longer, such as) before use in the assay. The two aliquots are then used in the HPVLP assay described herein to assign the sample to be anti-JCV antibody positive or antibody negative. If the assay result for an aliquot incubated with HPVLP shows a value less than 45% inhibition (i.e., the "cut point"), the sample is interpreted as negative for the presence of JCV-specific antibodies. If the assay result shows a value of 45% inhibition or greater, the sample contains JCV-specific antibodies and is therefore interpreted as antibody-positive.

[0162] The assays featured in the present invention that utilize a secondary test are also referred to herein as the “two-step test” or “two-step assay.” An earlier version of the two-step assay is described in the Common Assignees International Application No. PCT / US2011 / 020832, which is incorporated herein by reference in its entirety.

[0163] Substrate and solution-based methods. Any suitable solid substrate can be used for the HPVLP assay format. In some embodiments, the substrate is a microtiter plate (e.g., a 96-well plate) slide, beads, or column. The substrate may be suitable for solution-based methods such as colorimetric or chemiluminescent detection, or proximal ligation.

[0164] Cutpoint. This invention provides an analytical method that utilizes a “cutpoint” to reduce false negative and false positive rates. The cutpoint is established based on data from an HPVLP assay (e.g., for detecting JCV antibodies in a biological sample) and is averaged, for example, between a dual test sample and multiple repeat samples (e.g., at least two, at least four, or at least eight repeat samples of a control sample). The cutpoint can also be statistically determined using a large panel of non-PML and PML samples.

[0165] In one version of the assay according to the present invention, the results from the initial HPVLP screening assay, e.g., an ELISA assay, are used to classify the test sample as having or not having JCV-specific antibodies. Otherwise, if the sample does not fall into one of these two classifications, the sample is subjected to a supplemental confirmatory assay. For example, in the HPVLP ELISA assay featured in the present invention, below an established level (e.g., nOD), 450 Samples yielding results <0.2) will be classified as lacking JCV-specific antibodies, and in ELISA, they will exceed established levels (e.g., nOD).450 >0.4) Samples that provide results will be classified as positive for JCV-specific antibodies. Samples that do not clearly fall into one of these classifications (e.g., 0.2) <OD 450 <0.4) The sample can be tested in a confirmatory assay.

[0166] In one embodiment, the confirmatory assay requires a pre-incubation step, where the test sample is pre-incubated with a buffer (or other suitable solution) control or with HPVLP (in buffer or other suitable solution) before analysis in HPVLP ELISA, as further detailed below, to adsorb JCV-specific antibodies. If, after pre-incubation with HPVLP, the reaction in the primary assay is reduced by less than 45% compared to the buffer control, the sample is interpreted as negative for the presence of JCV-specific antibodies. If the result shows a reduction of 45% or more in the reaction in the primary assay compared to the buffer control after pre-incubation with HPVLP, the sample is interpreted as containing JCV-specific antibodies. In some embodiments, only the confirmatory assay is performed.

[0167] VP1. The use of HPVLP in assays for JCV antibodies can improve assay accuracy and is useful in assays suitable for analytical and diagnostic purposes. VP1 for use in producing HPVLP can be generated using methods known in the art and may be either naturally occurring VP1 or recombinantly produced VP1, e.g., VP1 from the JC virus. Generally, the VP1 used is VP1 from the MAD1 strain of JCV. In some embodiments, the VP1 used in the assay includes VP1 from one or more JCV strains, e.g., one or more of strains 1A, 1B, 2A, 2B, 3, 4, and 7. After preparation of VP1, e.g., recombinantly synthesized VP1, the VP1 for use in the assays described herein is then further purified by standard biochemical methods, including density gradient / ultracentrifugation, or by a series of chemical precipitation steps, such as concentration / dialysis and ion exchange chromatography. The purification method typically includes a step to remove smaller proteins, including monomeric VP1 polypeptide or pentameric VP1. The removal of these smaller particles can be carried out in, for example, one or two steps (e.g., a first filtration step to remove VP1 monomers, followed by a second filtration step to remove pentameric VP1 particles). Such biochemical purification methods are known to those skilled in the art. Examples 1 and 7 provide two different methods for purifying JCV VP1-VLP.

[0168] HPVLP preparations (HPVLP) according to one aspect of the present invention do not contain significant amounts of VP1 monomers (e.g., they are purified to remove monomers). HPVLP preparations according to another aspect of the present invention do not contain significant amounts of VP1 molecules, in a composition of VP1 pentamers or smaller sizes (including monomers). HPVLPs can be prepared from recombinant VP1 or naturally occurring VP1 (e.g., isolated from a virus or viral capsid). In some embodiments, further JCV components, such as non-major coat proteins from the JC virus, e.g., one or both of VP2 or VP3, are included in the HPVLP particles or associated with the substrate.

[0169] In some cases, recombinantly expressed VP1 may not assemble into a pentamer or HPVLP that resembles a naturally occurring viral capsid; for example, recombinantly expressed VP1 may assemble into a tube or other non-spherical shape. Therefore, the present invention relates to a method for producing HPVLP that is substantially spherical in shape. The present invention includes an HPVLP preparation in which at least about 10%, about 15%, about 20%, about 25%, about 50%, about 60%, about 65%, about 70%, about 80%, about 90%, about 95%, or about 99% of the HPVLP in the preparation resemble a naturally occurring JCV capsid (e.g., having an icosahedral or substantially spherical configuration). In some embodiments, the HPVLP preparation contains at least 10%, at least 15%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the HPVLP in the preparation is similar to the naturally occurring JCV capsid. Such a method may include expressing the viral protein under conditions resulting in such a preparation, and / or isolating and purifying the expressed viral protein as described herein in order to produce such a preparation.

[0170] Method for producing HPVLP. HPVLP can be produced, for example, by transforming a baculovirus with a vector expressing the VP1 gene, such as the VP1 gene from the JC virus. Using the baculovirus, a cell culture such as an insect cell culture (e.g., SF9 cells) or a mammalian cell culture is infected so that the cells express the VP1 protein. HPVLP is isolated by lysing the cells and purifying the particles through a series of centrifugation and ultrafiltration steps. Generally, purification is carried out using methods such as sucrose cushion sedimentation, isodense ultracentrifugation, and broad ultrafiltration, or other methods known to those skilled in the art. In certain embodiments, purification would involve centrifugation of the particles twice through a sucrose cushion. In alternative purification methods, the cells are lysed and the particles are isolated by a series of precipitation and concentration / dialysis steps, along with a final ion exchange step. In yet another alternative method, HPVLP is purified by chromatography without using a centrifugation step.

[0171] Purity can be assessed using any suitable technique known in the art, such as analytical ultracentrifugation, electron microscopy, PAGE analysis, mass spectrometry, protein concentration, or activity in ELISA using control serum. Insufficiently purified VLPs result in high background, leading to falsely high calculations of anti-JCV antibody levels or exposure rates.

[0172] In some embodiments, HPVLP contains VP1 as the sole JC virus protein.

[0173] In some embodiments, HPVLPs are heterogeneous particles and therefore contain the VP1 protein and at least one of the minor coat proteins of the JC virus, such as VP2 or VP3. In another embodiment, HPVLPs contain the VP1, VP2, and VP3 proteins. HPVLPs containing VP1 and VP2 can be produced using methods known in the art, for example, by transforming baculoviruses with nucleic acids containing the VP1 and VP2 genes under the control of the same or different promoters. Cell cultures are infected with baculoviruses, and the cells express VP1 and VP2, forming HPVLPs containing both types of proteins. In one embodiment, the VP1 and VP2 genes are located on different DNA molecules, and these DNA molecules are transformed into different baculoviruses, which are then used to transfect cells in the same culture. The cells express the VP1 and VP2 proteins, forming HPVLPs containing both types of proteins. In some cases, heterogeneous HPVLPs may contain, for example, one or two VP2 polypeptides for every five VP1 polypeptides. Generally, HPVLPs will contain more VP1 polypeptides than VP2 polypeptides, similar to naturally occurring JC viruses.

[0174] HPVLPs containing both VP1 and VP3 or both VP1 and VP2 molecules can be produced, for example, by transforming baculoviruses with nucleic acids containing the VP1 and VP3 genes or the VP1 and VP2 genes, respectively, under the control of the same or different promoters. Cell cultures are infected with baculoviruses, and the cells express VP1 and VP3 or VP1 and VP2, forming HPVLPs containing both types of proteins. In some embodiments, the VP1 and VP3 or VP1 and VP2 gene genes reside on different DNA molecules, and these DNA molecules are transformed into different baculoviruses, which are then used to transfect cells in the same culture. The cells express the VP1 and VP3 proteins or the VP1 and VP2 genes, respectively, forming HPVLPs containing both types of proteins. HPVLP particles can be isolated from such preparations using methods known in the art, such as those used to isolate JCV capsids.

[0175] Typically, a VP1 pentamer in heterogeneous HPVLP would contain, for example, five VP1 polypeptides, as well as one VP3 polypeptide and / or one VP2 polypeptide, depending on whether the VP3 gene or the VP2 gene was used to construct the construct. Typically, there would be more VP1 polypeptides than VP3 or VP2 polypeptides in HPVLP. In some embodiments, VP2 or VP3 are from polyomaviruses other than JC virus, such as BK virus polypeptides.

[0176] HPVLP containing all three VP1, VP2, and VP3 molecules can be produced by transforming baculoviruses with nucleic acids (e.g., circular DNA, e.g., less than 5.5 kb) containing the VP1, VP2, and VP3 genes, under the control of the same or different promoters. Cell cultures, such as mammalian cell cultures, are infected with baculoviruses, and these cells express the VP1, VP2, and VP3 proteins. HPVLP containing all three types of proteins is formed as a result. In one embodiment, the VP1, as well as either or both of the VP2 and VP3 genes, reside on different DNA molecules, and these DNA molecules are transformed into the same or different baculoviruses, which are then used to transfect cells in the same or separate cultures. The cells express the VP1, VP2, and VP3 proteins, and HPVLP containing both types of proteins is formed. Heterogeneous HPVLP may contain, for example, five VP1 polypeptides, as well as one each of VP2 and VP3 polypeptides, although their ratios may vary within the preparation. Typically, HPVLP will contain more VP1 polypeptides than VP2 and VP3 polypeptides.

[0177] In some embodiments, HPVLPs are larger in size than VP1 pentamers. Larger size means that the mass of protein contained in the HPVLP particles is greater than that of a pentamer containing only VP1.

[0178] In other embodiments, a method for preparing a solution of HPVLPs may include removing particles that are VP1 pentamers or smaller in size (e.g., VP1 monomers or small VP1-containing particles) from the solution. This purification step can be performed using methods such as centrifugation and size exclusion chromatography. In some embodiments, other methods known in the art, such as ion exchange chromatography, can be used in the preparation of HPVLPs larger than VP1 pentamers. Generally, an HPVLP preparation suitable for use in an assay would contain at least 20% HPVLP, at least 25% HPVLP, at least 40% HPVLP, at least 60% HPVLP, at least 65% HPVLP, at least 70% HPVLP, at least 80% HPVLP, at least 85% HPVLP, at least 90% HPVLP, at least 95% HPVLP, or at least 99% HPVLP compared to non-HPVLP particles (e.g., only the percentage of pentamers compared to aggregates containing VP1 monomers and fewer than five VP1 molecules).

[0179] Methods for evaluating samples and / or subjects. When used herein, a method for evaluating or analyzing a biological sample of a subject or subject includes one or more of the following: performing an analysis of the sample, requesting an analysis of the sample, requesting results from an analysis of the sample, or receiving results from an analysis of the sample. (Generally used herein, determination (or making a determination), analysis, or evaluation (or making an evaluation) may include one or both of performing an underlying method or receiving data from another person who has performed an underlying method.)

[0180] The analysis or evaluation requires some kind of transformation of the substance, such as a biological substance or assay component. For example, a biological sample can be evaluated for the presence of anti-JCV antibodies, the anti-JCV antibody titer, and the percentage of JCV antibody inhibition. The evaluation can be performed before, after, or concurrently with the patient receiving treatment for MS, etc. The evaluation is based at least in part on the analysis of a sample from the subject, such as blood, plasma, serum, urine, or CSF sample. The presence of anti-JCV antibodies can be determined by contact with a specific binder, such as a JCV protein such as VP1. The binder may be a JCV protein, such as VP1 in particle form, such as HPVLP.

[0181] In one embodiment, the assay for detecting the presence of anti-JCV antibodies is a two-step assay, as described herein. The assay utilizes HPVLP under conditions suitable for binding to anti-JCV antibodies. The assay can detect any isotype of anti-JCV antibodies (including IgG, IgM, IgA, and IgE). The assay is also highly sensitive and can detect anti-JCV antibodies at concentrations of, for example, 2.0 μg / mL or less, e.g., 1.5 μg / mL or less, 1.25 μg / mL or less, 1.0 μg / mL or less, 0.5 μg / mL or less, 50 ng / mL or less, 10 ng / mL or less, 5 ng / mL or less, 1.7 ng / mL or less, or 1 ng / mL or less.

[0182] In one embodiment, the sample is analyzed for the level of JCV nucleic acid present in the sample. For example, the nucleic acid can be isolated from the sample and used for PCR amplification or next-generation (Nex-Gen) sequencing techniques. In one embodiment, the crude lysate of the biological sample is subjected to amplification methods such as PCR, and the amplified product is analyzed by one or more of the following methods to determine whether and how much JCV DNA or RNA is present in the sample: electrophoresis, restriction fragment mapping, hybridization, or sequencing.

[0183] Biological samples can be taken from patients and analyzed.

[0184] In some embodiments, patient samples, such as serum or plasma or whole blood samples or CSF, can be stored before testing for JCV, for example, for JCV antibodies or JCV nucleic acids. Patient samples, such as those containing JCV antibodies or JCV nucleic acids, can be stored for 1 to 21 days, for example, 1 to 14 days or 1 to 7 days, or longer (e.g., 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, or longer); for 1 to 6 weeks, for example, 1 to 3 weeks or 1 to 2 weeks, or longer (e.g., up to 1 week, up to 2 weeks, up to 3 weeks, up to 6 weeks, or longer); or for 1 to 6 months, for example, 1 to 3 months or 1 to 2 months, or longer (e.g., up to 1 month, up to 2 months, up to 3 months, up to 6 months, or longer). The sample can be stored, for example, frozen (e.g., at -80°C to -20°C), at ambient temperature (18°C to 25°C) at 2 to 8°C, or at a warmer temperature, for example, 37°C.

[0185] When used herein, the terms “acquire” or “obtain” mean to acquire a physical entity or value, such as a patient condition, such as prior exposure to anti-VLA-4 therapy or other immunosuppressants, or a JCV condition, by “directly acquiring” or “indirectly acquiring” that physical entity or value, such as a numerical value. “Directly acquiring” means performing a process (e.g., performing a synthesis or analytical method) to obtain the physical entity or value. “Indirectly acquiring” means 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 involves performing a process that includes a physical change in a physical substance, such as a starting material. Examples of changes include producing a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more distinct entities in a mixture, and performing a chemical reaction that includes cleaving or forming covalent or non-covalent bonds. Directly obtaining a value includes performing a process that involves a physical change in a sample or another material, for example, performing an analytical process that involves a physical change in a substance, for example, a sample, analyte, or reagent (sometimes referred to herein as “physical analysis”), and performing an analytical method, for example, a method comprising one or more of the following: separating or purifying a substance, for example, an analyte, or a fragment or other derivative thereof from another material; combining an analyte, or a fragment or other derivative thereof with another material, for example, a buffer, solvent, or reactant; or altering the structure of an analyte, or a fragment or other derivative thereof, for example, by cleaving or forming a covalent or non-covalent bond between a first atom and a second atom of the analyte; or altering the structure of a reagent, or a fragment or other derivative thereof, for example, by cleaving or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent.

[0186] Determining a patient's condition (e.g., JCV status) or activity level, and determining whether that condition has a pre-selected relationship with a reference criterion, involves one or more of analyzing a sample, requesting an analysis of a sample, requesting results from an analysis of a sample, or receiving results from an analysis of a sample. (Generally, analysis may include performing a basic method (e.g., an immunoassay) or receiving data from another person who has performed a basic method.)

[0187] Anti-VLA-4 therapy. Anti-VLA-4 therapy is a molecule that blocks VLA-4 activity, such as a small molecule compound or a protein biologic (e.g., an antibody or a fragment thereof, such as an antigen-binding fragment). A molecule that is an anti-VLA-4 therapy is a VLA-4 antagonist. A VLA-4 antagonist includes any compound that inhibits VLA-4 integrin from binding to its ligand and / or receptor. Anti-VLA-4 therapy may be an antibody (e.g., natalizumab (TYSABRI®)) or a fragment thereof, or a soluble form of the ligand. Soluble forms of ligand proteins for α4 integrin include soluble VCAM-I or fibronectin peptides, VCAM-I fusion proteins, or bifunctional VCAM-I / Ig fusion proteins. For example, a soluble form or fragment of a VLA-4 ligand may be administered to bind to VLA-4, and in some cases, compete for the VLA-4 binding site on the cell, thereby producing an effect similar to that of administering an antagonist such as an anti-VLA-4 antibody. For example, a soluble VLA-4 integrin mutant that binds to a VLA-4 ligand but does not induce integrin-dependent signaling is suitable for use in the described method. Such a mutant can act as a competitive inhibitor of wild-type integrin protein and is considered an "antagonist." Other suitable antagonists are "small molecules."

[0188] A “small molecule” is a drug that mimics the action of a peptide that interferes with VLA-4 / ligand interactions, for example, by binding to VLA-4 and blocking its interaction with a VLA-4 ligand (e.g., VCAM-I or fibronectin), or by binding to a VLA-4 ligand and preventing the ligand from interacting with VLA-4. One example of a small molecule is an oligosaccharide that mimics the binding domain of a VLA-4 ligand (e.g., fibronectin or VCAM-I) and binds to the ligand-binding domain of VLA-4. (All incorporated herein by reference, Devlin et al., Science) 249 :400-406 (1990), Scott and Smith, Science 249 See 386-390 (1990) and U.S. Patent No. 4,833,092 (Geysen).

[0189] "Small molecules" are chemical compounds, such as organic compounds, or small peptides, or larger peptide-containing organic compounds or non-peptidic organic compounds. "Small molecules" are not intended to include antibodies or antibody fragments. The molecular weight of small molecules is generally less than 2000 daltons, but this number is not intended as an absolute upper limit for molecular weight.

[0190] Combination therapy or alternative to anti-VLA-4 therapy. In some embodiments, when it is determined that the patient is at higher risk of PML, anti-VLA-4 therapy, such as natalizumab, is administered in combination with a second agent, or an alternative therapy is administered instead of anti-VLA-4 therapy.

[0191] Non-limiting examples of second-line agents for treating multiple sclerosis in combination with anti-VLA-4 therapy, or alternative agents for use in place of anti-VLA-4 therapy, include fumarates such as dimethyl fumarate; sphingosine 1-phosphate (S1P) antagonists such as the S1B blocking antibody sphingomab; interferons such as human interferon beta-1a (e.g., AVONEX® or Rebif®) and interferon β-1b (BETASERON®, human interferon β substituted at position 17; Berlex / Chiron); and glatiramer acetate (also known as copolymer 1, Cop-1; COPAXONE®, Teva Pharmaceutical). Industries, Inc.); Anti-CD20 antibodies, such as Rituxan® (rituximab), or their fragments (including their antigen-binding fragments), or antibodies or their fragments that compete with or bind to duplicate epitopes by rituximab; Mitoxantrone (NOVANTRONE®, Lederle); Chemotherapy agents such as clabribine (LEUSTATIN®), azathioprine (IMURAN®), cyclophosphamide (CYTOXAN®), cyclosporine-A, methotrexate, 4-aminopyridine, and tizanidine; Corticosteroids such as methylprednisolone (MEDRONE®, Pfizer) or prednisone; CTLA4 Examples include: Ig; alemtuzumab (MabCAMPATH®) or daclizumab (an antibody that binds to CD25); statins; and TNF antagonists.

[0192] Glutameric acetates are proteins formed from random chains of amino acids (glutamic acid, lysine, alanine, and tyrosine (hence GLATiramer)). Glutameric acetates can be synthesized using N-carboxyamino acid anhydrides in a solution of these amino acids, in a ratio of approximately 5 parts alanine to 3 parts lysine, 1.5 parts glutamic acid, and 1 part tyrosine.

[0193] Further second agents, or agents to be used as an alternative to anti-VLA-4 therapy, include antibodies or antagonists of other human cytokines or growth factors, such as TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF. Still other exemplary second agents include antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, and CD90, or their ligands. For example, daclizumab is an anti-CD25 antibody that can induce remission of multiple sclerosis.

[0194] Other exemplary antibodies still include antibodies that provide the activity of the drugs described herein, such as antibodies that engage with interferon receptors, for example, interferon-beta receptors. Typically, in realizations where the second drug includes an antibody, it binds to a target protein other than VLA-4 or α4 integrin, or to an epitope on VLA-4 other than that recognized by natalizumab.

[0195] Other further exemplary second agents include FK506, rapamycin, mycophenolate mofetil, leflunomide, nonsteroidal anti-inflammatory drugs (NSAIDs), such as phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents, complement inhibitors, adrenergic agonists, agents that interfere with signaling by pro-inflammatory cytokines as described herein, T-cell signaling inhibitors such as IL-1β-converting enzyme inhibitors (e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and their derivatives as described herein, and anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13, and TGF).

[0196] In some embodiments, a second agent may also be used to treat one or more symptoms or side effects of MS. Such agents include, for example, amantadine, baclofen, papaverine, meclizine, hydroxyzine, sulfamethoxazole, ciprofloxacin, docusate, pemoline, dantrolene, desmopressin, dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam, isonaiazid, vardenafil, nitrofurantoin, cytoplasmic viscous viscous agent, alprostadil, gabapentin, nortriptyline, paroxetine, propantheline bromide, modafinil, fluoxetine, phenazopyridine, methylprednisolone, carbamazepine, imipramine, diazepam, sildenafil, bupropion, and sertraline. Many secondary drugs, being small molecules, have molecular weights ranging from 150 to 5000 daltons.

[0197] Examples of TNF antagonists include chimeric antibodies, humanized antibodies, human antibodies, or antibodies produced in vitro against TNF (e.g., human TNFα), such as D2E7 (human TNFα antibody, U.S. Patent No. 6,258,562; BASF), CDP-571 / CDP-870 / BAY-10-3356 (humanized anti-TNFα antibody; Celltech / Pharmacia), and cA2 (chimeric anti-TNFα antibody; REMICADE®, Centocor) (or their antigen-binding fragments); anti-TNF antibody fragments (e.g., CPD870); soluble fragments of the TNF receptor, such as p55 or p75 human TNF receptor or its derivatives, such as 75kDTNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL®); Immunex; e.g., Arthritis & Rheumatism Examples include 37:S295,1994, J.Invest.Med.44:235A,1996), p55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein (LENERCEPT®)); enzyme antagonists, e.g., TNFα-convertase (TACE) inhibitors (e.g., alpha-sulfonylhydroxamic acid derivatives, WO01 / 55112, and N-hydroxyformamide TACE inhibitors GW3333, -005, or -022); and TNF-bp / s-TNFR (soluble TNF-binding protein, e.g., Arthritis & Rheumatism 39:S284,1996, Amer.J.Physiol.- Heart and Circulatory Physiology 268:37-42,1995).

[0198] In one embodiment, the anti-VLA-4 therapy and the second agent are provided as a co-formulation, and the co-formulation is administered to a subject. For example, it is further possible to separately administer a single dose of the anti-VLA-4 therapy formulation followed by a single dose of the formulation containing the second agent at least 24 hours before or after administration of the co-formulation. In another embodiment, the anti-VLA-4 therapy and the second agent are provided as separate formulations, and the administering step includes sequentially administering the anti-VLA-4 therapy and the second agent. The sequential administrations can be provided on the same day (e.g., within 1 hour of each other, or at least 3, 6, or 12 hours apart) or on different days.

[0199] Each of the anti-VLA-4 therapy and the second agent can be administered at separate times as multiple doses. The anti-VLA-4 therapy and the second agent are typically each administered according to a regimen. The regimen for one or both can have a certain periodicity. The regimen for the anti-VLA-4 therapy can have a different periodicity from the regimen for the second agent, for example, one can be administered more frequently than the other. In one embodiment, one of the anti-VLA-4 therapy and the second agent is administered once a week and the other is administered once a month. In another embodiment, one of the anti-VLA-4 therapy and the second agent is administered continuously, for example, over a period exceeding 30 minutes but less than 1, 2, 4, or 12 hours, and the other is administered as a bolus. The anti-VLA-4 therapy and the second agent can be administered by any suitable method, for example, subcutaneously, intramuscularly, or intravenously.

[0200] In some embodiments, each of the anti-VLA-4 therapy and the second agent is administered at the same dose because each is formulated as a monotherapy. In other embodiments, the anti-VLA-4 therapy is administered at a dosage that is the amount required for efficacy when administered alone, or a lower amount. Similarly, the second agent can be administered at a dosage that is the amount required for efficacy when administered alone or a lower amount.

[0201] Reagents for performing a kit anti-JCV antibody assay can be provided in the form of a kit. Except for patient samples, some or all of the substances required for the assay can be provided in the kit. The kit can include a substrate, such as a plate having wells coated with a JCV antigen substrate, such as HPVLP. The plate can be, for example, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate. The plate provided in the kit can be pre-coated with a JCV VLP antigen such as 0.4 μg / mL. In one embodiment, the kit includes substances and reagents for use with a high-throughput system, such as an SPR (solid-phase receptor) chip for use with the Merieux system.

[0202] The kit may also include a JCV antigen, such as lyophilized or solution-based HPVLP, for use with the assay confirmation step and the like. In one embodiment, the kit includes a JCV cut-off calibration sample, an anti-JCV antibody positive control, and a JCV negative control, which are serum samples such as human serum. Solutions containing JCV antigen and serum can include preservatives such as sodium azide, for example, 0.05%, 0.1%, 1.5%, and 2% sodium azide. In one embodiment, the kit featured in the present invention can include one or more reagents for detecting a complex containing an anti-JCV antibody bound to an antigen such as HPVLP. The complex for detecting the reagent can include, for example, a JCV complex, a casein sample, a detectable reagent such as TMB (tetramethylbenzidine), a washing buffer, and a stop reagent.

[0203] The JCV substrate may be an anti-human antibody, such as an enzyme-labeled anti-human antibody. In one embodiment, the JCV complex is an affinity-purified, peroxidase-labeled donkey anti-human antibody. In another embodiment, the casein solution contains casein, a surfactant, and a non-azide preservative (e.g., phosphate-buffered saline (PBS)) in a buffer. In yet another embodiment, the TMB substrate solution contains TMB and hydrogen peroxide in a buffer. In yet another embodiment, the kit includes a wash buffer, which may contain a surfactant in PBS having a non-azide preservative, for example. The stop reagent may be an acid, such as sulfuric acid (e.g., 1 M sulfuric acid).

[0204] The solutions provided in the kit can be supplied at a concentration level that requires dilution before use. HPVLP for use in solutions that bind to anti-JCV antibodies in biological samples, such as in the confirmation step of a two-step assay, can be supplied at concentrations of 2 mg / mL, 1.5 mg / mL, 1 mg / mL, and 0.5 mg / mL for use at concentrations of 10 μg / mL, 5 μg / mL, 1 μg / mL, 0.8 μg / mL, 0.4 μg / mL, and 0.2 μg / mL, respectively. Wash buffers can be supplied at concentrations of 10-fold, for example. JCV substrates (e.g., affinity-purified and peroxidase-labeled donkey anti-human antibodies) can be supplied at concentrations of 1 mg / mL, 0.8 mg / mL, or 0.6 mg / mL for dilutions of 1:40,000, 1:30,000, 1:20,000, or 1:20,000, respectively, before use in anti-JCV antibody detection assays.

[0205] Materials for sealing reaction mixtures, such as sealing tape, can also be included in the kit.

[0206] Reporting of results. The results of the risk assessment analysis can be reported to the treatment center, healthcare provider, or insurance provider, etc. In one embodiment, the results of the risk assessment are stored in a database.

[0207] In one embodiment, informational materials are provided for performing and interpreting a risk assessment. The informational materials may provide guidance on where to report the assessment results, such as to a treatment center, healthcare provider, or database provider. The informational materials may be provided in a kit or packet and may include a form for reporting the assessment results, including each branch of the assessment (information on prior treatment with anti-VLA-4 therapy, prior treatment with immunosuppressants, and JCV status), as well as address and contact information for where to send such form or other relevant information; or it may include a URL (Uniform Resource Locator) address for reporting the results in an online database or online application (e.g., "app"). In another embodiment, the informational materials may include guidance on whether a patient should accept treatment with anti-VLA-4 therapy, depending on the patient's risk of PML according to the results of the risk assessment.

[0208] The kit or packet may also include instructions and items for collecting or transporting patient samples to or from healthcare providers, or for receiving samples from healthcare providers, or for performing the evaluation methods described herein. For example, in addition to instructional information, the kit or packet featured in the present invention may include one or more swabs or scrapers, or containers (e.g., cups, test tubes, ampoules, or bags) for collecting, storing, and transporting biological samples. The kit or packet may also each contain supplements for performing immunoassays or sequencing assays for the detection of JCV antibodies or nucleic acids.

[0209] A kit may include one or more containers for reagents required for an assay, such as a JCV detection assay. Reagents may be provided at concentrations suitable for use in the assay, or with instructions for dilution for use in the assay. In some embodiments, the kit includes separate containers, dividers, or compartments and informational materials for each assay component. For example, assay components may be contained in bottles or vials, and informational materials may be contained in plastic sleeves or packets. In other embodiments, the separate elements of the kit are contained in a single, undivided container. For example, assay reagents are contained in bottles or vials, with informational materials attached in the form of labels. In some embodiments, the kit includes multiple (e.g., packs) individual containers, each containing one or more unit forms of assay components (e.g., for use in a single assay). For example, the kit includes multiple ampoules, foil packets, or blister packs, each containing a single unit of assay reagent for use in a screening or confirmation assay. The containers of the kit may be airtight and / or waterproof. The container can be labeled for use.

[0210] The informational materials in a kit or packet are not limited in form. Often, the informational materials, such as instructions, are provided as printed materials, such as printed text, drawings, and / or photographs, such as labels or printed sheets. However, the informational materials may also be provided in other forms, such as computer-readable materials, video recordings, or audio recordings. In another embodiment, the informational materials in a kit are contact information, such as a physical address, email address, website, or telephone number, where the user of the kit or packet can obtain substantial information on how to find the information required for risk assessment analysis, such as where and how to identify the treatments administered to the subject and how to perform assays to determine the patient's JCV status. The informational materials may also be provided in any combination of forms.

[0211] In some embodiments, the biological sample is provided to an assay provider, e.g., a service provider (e.g., a third-party facility) or healthcare provider, who evaluates the sample in the assay and provides readouts. For example, in one embodiment, the assay provider receives a biological sample from a subject, such as a plasma, blood, or serum sample, evaluates the sample using the assay described herein, and determines whether the sample contains JCV antibodies or nucleic acids. In some embodiments, the assay provider, e.g., a service provider or healthcare provider, can further determine, e.g., by contacting a healthcare provider or database service provider, the amount of previous anti-VLA-4 therapy the patient has received, or whether the patient has previously received treatment with an immunomodulator. The assay provider can further determine whether the subject is not a candidate to receive treatment with an anti-VLA-4 therapy such as natalizumab, or whether the subject is a candidate to receive treatment with an immunomodulator, or whether the subject may be a candidate to have enhanced monitoring compared to subjects who are determined to have a negative JCV status (e.g., negative test results for JCV nucleic acids or anti-JCV antibodies). For example, a candidate who has previously received anti-VLA-4 therapy for 24 months or less, has not previously received immunosuppressant therapy, and is determined to be JCV-positive may be selected as a candidate for further anti-VLA-4 therapy, but this comes with a recommendation for more frequent monitoring of the patient for adverse events, such as symptoms that may indicate the development of PML.

[0212] In one embodiment, the assay provider assesses the PML risk as described herein and determines that the subject is a candidate for treatment with anti-VLA-4 therapy such as natalizumab. In one embodiment, the assay provider notifies the healthcare provider that the subject is a candidate for treatment with anti-VLA-4 therapy, and the candidate is administered anti-VLA-4 therapy. For example, the assay provider may determine that the patient is at lower risk of PML and then notify the healthcare provider of the lower risk determination and that the subject is a candidate for treatment with anti-VLA-4 therapy.

[0213] In another example, the assay provider determines that a patient is at higher risk of PML and subsequently notifies the healthcare provider of this higher risk determination and recommends that the patient is a candidate for treatment with anti-VLA-4 therapy, but should undergo increased testing for PML and optionally JCV status. In one embodiment, the assay provider notifies the healthcare provider that the patient is at higher risk of PML and therefore should accept alternatives to anti-VLA-4 therapy, or that the patient is a candidate for accepting anti-VLA-4 therapy with increased testing for PML and optionally JCV status.

[0214] Assay providers may provide the results of risk assessments, and optionally, conclusions regarding one or more of the following: diagnosis, prognosis, or appropriate treatment options, to healthcare providers, patients, or insurance companies in any preferred format, such as by mail, electronically, or through an online database. Information collected and provided by assay providers may be stored in a database.

[0215] In one embodiment, a healthcare provider or insurance provider or another entity recommends, for example, that the patient or a second healthcare provider undergo a risk assessment for PML as described herein.

[0216] The PML risk stratification tool is useful as one component in making individual benefit-risk treatment decisions for patients who are taking or considering taking VLA4 inhibitors or other medications known to increase the risk of developing PML. Quantifying a patient's PML risk can be used, for example, in benefit-risk analysis.

[0217] Headings, such as (a), (b), (i), etc., are provided solely to facilitate reading of this specification and the claims. The use of headings in this specification and the claims does not require that the steps or elements be in alphabetical or numerical order, or in the order in which they are presented.

[0218] The present invention is further illustrated by the following embodiments, but should not be construed as further limiting. [Examples]

[0219] Example 1. The risk of PML in MS patients was quantified for the first time using two established risk factors and anti-JCV antibody status determined by a proprietary two-step VP1 VLP-based ELISA. method Patients, samples, and data collection Due to the low incidence of PML, data on natalizumab-treated PML patients were collected from multiple sources, including post-marketing data from Biogen Idec's global natalizumab safety database and clinical trials as of March 4, 2011. Since data on prior immunosuppressant use were not available for all patients exposed to natalizumab, the proportion of patients with and without prior immunosuppressant use in the TYSABRI® Global Observational Program in Safety (TYGRIS; NCT00477113, NCT00483847) was used as an estimate for the overall natalizumab-treated population. TYGRIS is an observational cohort study designed to obtain long-term safety data in natalizumab-treated MS patients in a clinical practice setting. Assessment of anti-JCV antibody prevalence in the general MS population was based on single baseline plasma or serum samples collected from patients from four sources, including ongoing or completed natalizumab clinical studies (AFFIRM (Polman et al., N.Engl.J.Med.354:899-910,2006, STRATIFY-1 (NCT01070823), TYGRIS - USA, and independent MS registries in Sweden (available online at msreg.net / cms / sv / home (accessed February 3, 2011))). A clinical plan was developed for a large-scale collection of serum and plasma samples obtained before PML diagnosis, including both clinical trial and post-marketing cases.

[0220] Identifying the duration of natalizumab treatment as a risk factor for PML. The estimated PML incidence since natalizumab's reintroduction to the market was calculated based on post-marketing exposure to natalizumab up to February 28, 2011, and the number of confirmed PML cases as of March 2011. The PML incidence for each time frame (cumulative duration or 12-month treatment interval) was calculated by dividing the number of patients who developed PML during that time frame by the total number of patients who have been exposed to natalizumab over that time period.

[0221] Identification of prior immunosuppressive drug use as a risk factor for PML The immunosuppressive treatment histories of natalizumab-treated MS patients who developed PML in post-marketing settings and clinical trials were obtained from Biogen Idec's global natalizumab safety database as of November 2, 2010 and compared with data obtained from TYGRIS. This cut-off date for prior immunosuppressive drug use was chosen (versus March 4, 2011 for all other data) because it was anticipated that incorporating this into the labeling as a risk factor (Europe, December 2010) (package insert, TYSABRI® (natalizumab), Biogen Idec. Weston, MA, July 2010, product summary, TYSABRI® (natalizumab), Biogen Idec. Weston, MA, December 13, 2010) would be confounded.

[0222] Identification of anti-JCV antibody status as a risk factor for PML The overall prevalence of anti-JCV antibodies in the general MS population was determined using an independent two-step VP1 VLP-based ELISA as previously described (Gorelik et al., Ann. Neurology, 2010). The prevalence of anti-JCV antibodies in MS patients with PML when pre-PML serum or plasma samples were available prior to diagnosis was also determined using this assay and compared with the overall prevalence in the general MS population.

[0223] Estimation of the incidence of PML by anti-JCV antibody serostatus The incidence of PML in anti-JCV antibody-positive and anti-JCV antibody-negative patients was estimated using the overall global incidence of PML, the incidence after 25–48 infusions (the point in time after the most significant increase in PML incidence in this analysis), the prevalence of anti-JCV antibody in the general MS population, and the number of MS patients with PML for whom pre-PML samples with anti-JCV antibody-positive test results prior to diagnosis were available. Estimated incidences of PML in anti-JCV antibody-positive and anti-JCV antibody-negative patients were compared using a one-sided Fisher's exact test. To provide a conservative estimate of PML incidence in anti-JCV antibody-negative patients, susceptibility analysis was performed to assess the impact of hypothetical anti-JCV antibody-negative PML cases on this estimate. Furthermore, susceptibility analysis was performed to assess the statistical certainty of this estimate by varying the number of anti-JCV antibody-positive PML cases.

[0224] Quantification of PML risk: prior use of immunosuppressants, duration of natalizumab treatment, and positivity of anti-JCV antibodies. Risk factor algorithms were developed to estimate the incidence of natalizumab-associated PML in patients with or without certain risk factors for natalizumab-associated PML and anti-JCV antibody serological status. These algorithms estimated the risk of PML based on prior immunosuppressant use (with or without), duration of natalizumab treatment (1–24 months and 25–48 months), and anti-JCV antibody status. These risk algorithms were based on PML incidence data based on the duration of natalizumab treatment (1–24 or 25–48 months), as well as estimations of prior immunosuppressant use in natalizumab-treated patients from TYGRIS and in patients with PML. In addition, the estimated overall morbidity of anti-JCV antibodies in the general MS population was used to attribute the incidence of PML associated with serological status to the three-factor risk algorithm, assuming that all confirmed cases of natalizumab-associated PML were anti-JCV antibody positive prior to diagnosis. A sensitivity analysis was performed to quantify the effect of varying the estimates used to develop this three-factor algorithm, based on the highest and lowest observed values.

[0225] result Identifying the duration of natalizumab treatment as a risk factor for PML. Globally, 102 confirmed cases of PML as of March 4, 2011, were identified. Overall, the risk of PML increased with increasing treatment duration (Figure 1A), with the greatest increase in risk occurring after two years of therapy, peaking at 1.68 cases per 1,000 patients at three years (Figure 1B). Data beyond four years were limited.

[0226] Identifying the use of immunosuppressants as a risk factor for PML. Prior immunosuppressant use was more common in natalizumab-treated patients who developed PML compared to patients enrolled in TYGRIS, which represented the overall patient population receiving natalizumab. 45 percent of natalizumab-treated PML patients had received one or more immunosuppressive therapies prior to initiating natalizumab treatment, compared to 20.3% (13.9% in the US and 23.6% in Europe) in natalizumab-treated patients from TYGRIS. The most common prior immunosuppressants used in both the natalizumab-treated PML population and TYGRIS included mitoxantrone, methotrexate, cyclophosphamide, azathioprine, and mycophenolate. No specific patterns were observed in the type of immunosuppressant, duration of use, or the break period between discontinuation of immunosuppressants and initiation of natalizumab (Table 1). [Table 1]

[0227] Quantification of PML risk: Duration of natalizumab treatment and prior use of immunosuppressants When patients were stratified by duration of natalizumab treatment (1–24 or 25–48 months) and prior use of immunosuppressants (with or without), four distinctly different subgroups were identified in terms of PML incidence (Figure 2). Three of these subgroups had an estimated PML incidence of less than or approximately 1 in 1000. The lowest PML risk was 0.19 in 1000 (95% confidence interval: 0.10–0.33) in patients treated with natalizumab for 1–24 months and who had not received prior immunosuppressive therapy. A fourth subgroup, including patients with both of these risk factors for PML, had the highest risk, with an estimated PML incidence of 4.3 in 1000 (95% confidence interval: 2.9–6.2).

[0228] Identifying anti-JCV antibody status as a risk factor for PML. AFFIRM, TYGRIS-US, STRATIFY-1, and Swedish MS Baseline samples for anti-JCV antibody testing were available from 5,896 patients from the registry. Demographics, including duration of natalizumab treatment and prior immunosuppressant use, were similar across these data sources where available (Table 2). The overall anti-JCV antibody prevalence in the general MS population assessed in this study was 55% (95% confidence interval: 54–56%). [Table 2]

[0229] In the TYGRIS-US dataset, age, sex, and prior immunosuppressant information were available for 1451 out of 1480 patients. In the STRATIFY-1 dataset, prior immunosuppressant information was available for 988 out of 1096 patients. In the Swedish MS dataset, age information was available for 2464 out of 2497 patients, and sex information was available for 2494 out of 2497 patients. Prior immunosuppressant use was not available in Swedish MS patients (not applicable).

[0230] One or more pre-PML samples were obtained from 25 natalizumab-treated MS patients 6.5–187 months prior to PML diagnosis. As shown in Supplementary Table 1, these 25 patients had clinical characteristics similar to 102 confirmed PML cases worldwide, indicating no apparent selection bias. All patients from whom multiple pre-PML samples were available had positive anti-JCV antibody test results at all time points, including samples collected before the initiation of natalizumab treatment. The 100% (25 out of 25) anti-JCV antibody positivity rate in natalizumab-treated MS PML patients prior to PML diagnosis was significantly different from the expected 55% positivity rate observed in the general MS population (p<0.0001), demonstrating the ability of anti-JCV antibody status to serve as a further PML risk stratification tool. [Table 3]

[0231] The onset date of MS was unknown for 5 patients in the anti-JCV antibody-positive group and for 38 patients in the overall PML group. The use of immunosuppressants was unknown for 1 patient in the anti-JCV antibody-positive group and for 9 patients in the overall PML group.

[0232] Estimation of PML incidence based on anti-JCV antibody serological status The incidence of PML in patients who were positive for anti-JCV antibodies was estimated to be approximately twice that of the overall natalizumab-treated population (Table 4). To estimate the overall incidence of PML due to anti-JCV antibody status, the following method was used: Based on 25 natalizumab-treated MS patients for whom pre-PML samples were available, it was estimated that these 25 MS patients with PML came from approximately 20,276 patients receiving natalizumab treatment, based on the overall PML rate of 1.23 patients per 1000 (Figure 1). Assuming that 55% of these 20,276 patients were anti-JCV antibody positive (i.e., 11,152 patients) and 45% were anti-JCV antibody negative (i.e., 9,124 patients), the incidence of PML in anti-JCV antibody positive patients was estimated to be 2.24 cases per 1,000 treated patients (=1,000 × 25 / 11,152), similar to the rate estimated in the literature (Tyler, Ann. Neurol. 68:271-274, 2010), with a 95% confidence interval of 1.45 to 3.31. Conversely, the estimated incidence of PML in anti-JCV antibody negative patients was 0 cases per 1,000 patients (95% confidence interval: 0 to 0.40), which was significantly different from the estimated incidence in anti-JCV antibody positive patients. p<0.0001. [Table 4]

[0233] The effect of natalizumab treatment duration on this estimation was assessed in the analysis using the PML incidence rate 25–48 months after natalizumab treatment (the treatment duration at which the increase in incidence was most pronounced). In anti-JCV antibody-positive patients, the risk of PML after 25–48 months of treatment was 3.97 per 1,000 (95% confidence interval: 2.36–6.27), while in anti-JCV antibody-negative patients, it was 0 per 1,000 (95% confidence interval: 0–0.99), Table 3.

[0234] At the time of this document, the incidence of PML in anti-JCV antibody-negative patients could not be fully confirmed because no PML cases had tested negative for anti-JCV antibodies before diagnosis. Therefore, to estimate the incidence of PML in anti-JCV antibody-negative patients, a susceptibility analysis was performed, assuming that hypothetical cases of PML occurred in anti-JCV antibody-negative patients, thus allowing for a conservative estimation decision that the proportion is likely to be lower. This analysis demonstrated an estimated incidence of PML in anti-JCV antibody-negative patients that was at least 20 times lower than in anti-JCV antibody-positive patients, p<0.0001 (Table 3).

[0235] A sensitivity analysis of the effect of increasing the number of anti-JCV antibody-positive PML patients for whom pre-PML samples were available, based on the results of anti-JCV antibody-positive testing, demonstrated that the statistical certainty regarding the increased risk of PML in anti-JCV antibody-positive patients was not improved beyond 25 available pre-PML samples (Table 5). [Table 5]

[0236] Quantification of PML risk: duration of natalizumab treatment, prior use of immunosuppressants, and anti-JCV antibody status. A combined quantitative PML risk algorithm was developed for natalizumab-treated MS patients based on the duration of natalizumab treatment, prior use of immunosuppressants, and anti-JCV antibody status (Figure 3). Since JCV exposure is a requirement for PML, patients who were anti-JCV antibody-negative represented the lowest risk subgroup in the PML risk stratification algorithm, with an estimated risk of less than 0.11 per 1000 (95% confidence interval: 0–0.59), based on conservative estimates determined in susceptibility analysis. Conversely, the highest risk group consisted of patients who were anti-JCV antibody-positive, had prior immunosuppressant use, and had been treated with natalizumab for 25–28 months. This algorithm assumed that all 102 confirmed cases of PML were anti-JCV antibody-positive prior to PML diagnosis. For the subgroup with higher risk (patients with all three risk factors), the estimated PML risk was approximately 7.8 per 1000 (95% confidence interval: 5.2–11.3). For patients who were anti-JCV antibody positive and had not previously used immunosuppressants, the PML risk was consistent with the overall natalizumab-treated population risk at a similar time point (Figures 1A and 1B). Sensitivity analysis of the effects of variability in the estimates used to develop this algorithm yielded minimum and maximum values ​​that were generally consistent with the original estimates seen in baseline case scenarios (see Figures 4A and 4B). The risk in anti-JCV antibody-negative patients was determined by assuming that hypothetical cases of PML occurred in anti-JCV antibody-negative patients.

[0237] This analysis varied the prevalence of anti-JCV antibodies in the general MS population from 48% to 59% (as seen in TYGRIS-US) and 59% (as seen in an independent Swedish registry), the use of prior immunosuppressants in the natalizumab-treated MS population from 14% to 24% (based on US and European estimates in TYGRIS, respectively), and the estimated 25-48 month natalizumab exposure from 35% to 45% (based on the current estimate of 40% and a 10% increase over the past year). The plots represent point estimates and 95% confidence intervals for each scenario (baseline case, minimum, and maximum). In general, the baseline case scenario was relatively consistent with the minimum and maximum estimates.

[0238] Since JCV infection is required for PML to develop, we determined that the lowest risk of PML was at least 20 times lower in patients who were anti-JCV antibody-negative (less than 0.11 cases per 1000 natalizumab-treated patients, regardless of other risk factors) than in patients who were anti-JCV antibody-positive (95% confidence interval 0-0.59). p<0.0001. To date, there have been no reported cases of PML in anti-JCV antibody-negative patients, but because the anti-JCV antibody assay has an estimated analysis false-negative rate of 2.5-3.2%, the true risk of developing PML cannot be zero. 1、19 Conversely, the risk of PML was highest in patients with all three risk factors (25–48 months of natalizumab treatment, prior use of immunosuppressants, and positive anti-JCV antibody status), with an estimated incidence of 7.8 cases per 1000 patients (95% confidence interval: 5.2–11.3).

[0239] Example 2. To demonstrate the robustness of this method, the anti-JCV antibody ELISA was validated in a clinical laboratory. A novel two-step enzyme-linked immunosorbent assay (ELISA) for detecting anti-JCV antibodies in human serum or plasma has recently been described (see PCT / US2011 / 020832). Key features of the assay include both direct binding and competing components in solution; the use of well-characterized JC virus-like particle (VLP) preparations; the incorporation of appropriate quality control (QC) samples; statistical determination of assay cut points using numerous longitudinally collected clinical samples; normalization of assay signals; and detection of multiple isotypes of anti-JCV antibodies (including IgG, IgM, IgA, and IgE).

[0240] To demonstrate the robustness of this method, the anti-JCV antibody ELISA was validated in three clinical laboratories. Analytical validation was performed by evaluating intra-assay and inter-assay precision, analytical specificity and sensitivity, matrix interference, robustness, and reagent capability.

[0241] The stability of anti-JCV antibodies in serum and plasma samples was demonstrated using assay QC samples prepared from pooled human serum, as well as serum and plasma samples from individual donors. Anti-JCV antibodies were shown to be stable in serum or plasma for 14 days when stored at both ambient (18–25°C) and 2–8°C, through six freeze / thaw cycles. Furthermore, the stability of anti-JCV antibodies in whole blood stored for 7 days at 2–8°C, 7 days at ambient (18–25°C), and 3 days at 37°C, respectively, before processing, was also demonstrated using both serum and plasma collection tubes. The stability of JC VLPs was demonstrated for 18 months at 2–8°C, through four freeze / thaw cycles.

[0242] Analytical validation demonstrated that the assay was sensitive, specific, and precise. Assay sensitivity was estimated at 1.7 ng / mL using a humanized anti-JCV monoclonal antibody control and at 1.25 μg / mL using purified polyclonal antibodies from anti-JCV antibody-positive serum. Susceptibility for detecting JCV infection was estimated at 97.5%. The assay's specificity in distinguishing JCV-specific antibodies from antibodies directed towards the related polyomavirus, BK virus, was also demonstrated. Mean intra-assay and inter-assay accuracies were approximately 6.4% and 12.2% for the screening step, and 2.6% and 5.3% for the confirmation step. The results obtained for plasma and serum were highly consistent, and assay robustness was demonstrated by highly agreed results from three laboratories that tested a panel of 100 blinded samples.

[0243] Example 3. The improved two-step JCV assay (second-generation assay) provides more accurate results than the original assay (first-generation assay). The two-step anti-JCV antibody assay was modified according to the optimization round. The new assay differs from the first assay in at least the following ways: Unlike the 1 μg / mL used in the first-generation assay, HPVLP is used at a substrate concentration of 0.4 μg / mL on the plate in the first step and in the solution in the confirmation assay. Unlike the 1:200 dilution used in first-generation assays, patient serum is diluted 1:101 before being applied to HPVLP on a plate in the first step of the assay, or to HPVLP in solution in the confirmatory assay. The secondary reagent (anti-human IgG) that is conjugated with HRP is typically diluted 1:20,000 (however, readjustment may be necessary for a new lot to match the signal to a previous lot), and the incubation time with the conjugate is only 30 minutes. In the first-generation assay, the same reagent was diluted 1:80,000, and the incubation time was 60 minutes. The binding reaction is assayed by incubating the HRP substrate TMB for 20 minutes ± 2 minutes, although in the first generation, the TMB incubation was 20 minutes ± 5 minutes. • In the confirmatory assay, 10 μL of the sample was added to 1 mL of confirmatory buffer (1:101 dilution), and the reaction proceeded for 10–20 minutes. In the first-generation assay, a 2x concentration sample (1:100 dilution) and HPVLP (2 μg / mL) were mixed in equal proportions and then incubated for 60 minutes. • The cutoff calibration sample (CO) is prepared to have an nOD reactivity index of approximately 1.0, and the positive control (PC) is prepared to have an nOD reactivity index of approximately 1.3. CO and PC are prepared by mixing anti-JCV antibody-positive serum and anti-JCV antibody-negative serum. For the negative control (NC), which is typically in-vitre negative serum, the reactivity index target is approximately 0.1. Qualitatively, the controls are derived from different pools of human serum, but they are derived from assay target concentrations and are similar to first-generation control levels.

[0244] The results of the JCV second-generation clinical agreement studies are summarized in Table 6 below. All first-generation trials were conducted at the Focus Diagnostics reference laboratory (Cypress, CA), and the trial sites for the second-generation assays included Denver (total = 275; TYSABRI®-based = 149; naive = 126), New York (total = 275; TYSABRI®-based = 136; naive = 139), and Focus Diagnostics (total = 262; TYSABRI®-based = 95; naive = 167). The agreement percentage is expressed as (second-generation / first-generation), lower and upper limits of the 96% confidence interval (95%Cl: LB vs. UB). [Table 6]

[0245] Example 4. Patient risk of PML can be classified using anti-JCV antibody status. We hypothesized that patients positive for anti-JCV antibodies could be further stratified for the risk of developing PML based on anti-JCV antibody titer (nOD or index) and anti-JCV antibody binding activity / affinity (inhibition%). This hypothesis stemmed from the observation that patients with anti-JCV antibody titers and inhibition percentages below a predetermined level ("clinical cutpoint") were at lower risk of developing PML compared to the overall anti-JCV antibody-positive population. To determine the relative risk of PML suggested by the antibody titer and inhibition percentage status, anti-JCV antibody titers and inhibition percentages could be measured before initiating TYSABRI® (natalizumab) or while the patient was already receiving TYSABRI®.

[0246] To determine the relative risk of PML as suggested by antibody titers and inhibition percentages in combination with other risk factors, existing data from two different anti-JCV antibody assays ("Generation I" and "Generation II") were collected and analyzed. Existing data included anti-JCV antibody titer information, expressed as "nOD" or "indicator."

[0247] In the first-generation assay, 22% (77 / 356) of anti-JCV antibody-positive patients had an nOD of ≥1.0 (C-1801), and 34% (13 / 38) of anti-JCV antibody-positive PML patients had an nOD of ≥1.0 (C-1801). Therefore, approximately 1.5 times more PML patients had an nOD of ≥1.0 compared to non-PML patients. This corresponds to a 2-3 times risk ratio associated with an nOD of ≥1.0.

[0248] Six percent of TYSABRI® non-PML patients were also observed to have more than a twofold change in anti-JCV antibody titer (nOD) in longitudinal samples collected over more than two years (C-1801). However, the majority of PML patients with longitudinal samples collected at signaling time points (more than one year before PML diagnosis, within six months of PML diagnosis, and at the time of PML diagnosis) showed more than a twofold increase in anti-JCV antibody titer (nOD). This suggests that patients who do not show a significant change in JCV titer over time are at a lower risk of developing PML.

[0249] Exemplary nOD and antibody titers are provided in Figures 5A–10B. Patient data are summarized in Table 7.

[0250] The graph of the statistical analysis is shown in Figure 11.

[0251] Statistical analysis showed that, regarding inhibition percentage, approximately 17% of antibody-positive samples had an inhibition percentage of less than 0.502, and approximately 0% of PML samples had an inhibition percentage of less than 0.502. Approximately 30% of antibody-positive samples had less than 70% inhibition, and approximately 0% of PML samples (with an index of less than 3.0) had less than 70% inhibition. [Table 7]

[0252] Example 5. An analytically validated anti-JCV antibody assay was introduced into clinical practice to stratify MS patients into those at higher or lower risk of PML. The objective of the study below was to assess changes in anti-JCV antibody titers before and after the initiation of natalizumab treatment.

[0253] The anti-JCV antibody assay (Gorelik et al., Ann. Neurol. 2010) was applied to samples from Swedish MS patients treated with natalizumab, including five PML-positive patients. Normalized OD (nOD) values ​​of the anti-JCV antibody assay were tested before and after natalizumab treatment. Positive samples were diluted 1:3 in the dilution step to determine the titer level. The same proportion of patients were also tested for antibodies against nuclear human cytomegalovirus (CMV) antigen (Schmitz et al., J. Clin. Microbiol. 1977) and recombinant varicella-zoster virus (VZV) glycoprotein E antigen (Thomsson, J. Virol. Methods 2011). [Table 8]

[0254] Following the initiation of natalizumab treatment, anti-JCV antibody levels remained relatively stable, and a gradual decline in nOD levels was observed in anti-JCV-positive patients. A significant decline in anti-JCV antibody levels (nOD) was observed when patients received natalizumab treatment (n=471), but not during preceding interferon-beta therapy (n=210). This suggests a potential effect of natalizumab therapy on anti-JCV antibody levels, without significantly affecting serological status or seropositivity (before: 56%, after: 55%).

[0255] After initiation of natalizumab treatment, anti-VZV(OD) ​​(n=715) levels decreased slightly, but anti-CMV (n=502) antibody levels did not decrease.

[0256] Table 9 below summarizes the observed changes in serum anti-JCV antibody (nOD) levels in five patients who developed PML. Serum anti-JCV antibody (nOD) levels increased at the time of PML diagnosis compared to baseline levels. [Table 9]

[0257] From this study, we concluded that natalizumab therapy may result in a gradual decrease in anti-JCV antibody levels (nOD) without affecting the JCV seropositivity rate. Notably, only 5% of the anti-JCV-positive reference population showed a change in nOD levels (ΔnOD) above 0.151 (95th percentile) compared to baseline, which was observed at diagnosis in all five Swedish cases of PML. Therefore, investigation of increases in anti-JCV antibody levels during natalizumab therapy prior to PML diagnosis is justified in the context of PML risk stratification.

[0258] Example 6. Use of a clinical cutoff distinct from the analytical cutoff to identify high-risk and low-risk groups among anti-JCV antibody-positive patients. Using results from stratified Study I, we determined clinical cutoffs, distinct from analytical cutoffs, to define high-risk and low-risk groups among anti-JCV antibody-positive patients. Therefore, a patient's PML risk would primarily be based on their baseline anti-JCV antibody titer level. Second-generation anti-JCV antibody assays were used in this study.

[0259] TYSABRI® evaluated non-PML patients (stratified group I, n=1044) and PML patients (more than 6 months prior to PML diagnosis (n=38)) (Figure 12). In the second-generation assay, 17% of anti-JCV antibody-positive patients had titers (indicators) below the lowest titer (indicator) observed in samples from PML patients collected more than 6 months prior to PML diagnosis, suggesting that these patients may have a lower risk of developing PML (similar to anti-JCV antibody-negative patients). In contrast to only 13% of PML patients whose samples were collected more than 6 months prior to PML diagnosis, 50% of anti-JCV antibody-positive patients had titers (index) below index 1.5. Furthermore, only 4.4% of PML patients with no known prior immunosuppressant drug use had samples with an index below 1.5, suggesting that these patients may have been at lower risk of developing PML compared to patients with high anti-JCV antibody titers (nOD or index).

[0260] Since no PML patients had an index score of less than 0.5, patients with an nOD score of less than 0.5 (109 / 1044 (10.4% of total samples) or 109 / 549 (20% of anti-JCV antibody-positive patients)) were determined to be in the lowest PML risk group (possibly as low as anti-JCV antibody-negative patients). Patients with an index score greater than 0.5 but less than 1.5 were determined to be in the lower risk group, as 50% of non-PML anti-JCV antibody-positive patients and only 13% of PML patients had samples in this range. Furthermore, only 4% of PML patients who were not known to have received prior immunosuppressive therapy had samples with an index score greater than 1.5 (Figure 13). Patients with an index score greater than 1.5 in the anti-JCV antibody-positive population (271 / 549 (50%)) were determined to be at higher risk for PML. 47 percent of patients were anti-JCV antibody-negative.

[0261] After PML diagnosis, patients are subjected to immunoadsorption (IA) or plasma exchange (PLEX) to remove circulating natalizumab and restore immune function. Anti-JCV antibody levels are rapidly restored to pre-treatment levels in these patients.

[0262] Example 7. A proposed statistical method for assigning stratified risk in the improved two-step anti-JCV assay described herein to multiple sclerosis (MS) patients receiving TYSABRI® treatment who have already tested positive for anti-JCV antibodies. This study evaluates two alternative strategies (represented as strategies 1 and 2) from the STRATIFY-II (American Academy of Neurology (AAN) Meeting, April 21-28, 2012, abstract S041.002) for assigning PML risk to anti-JCV seropositivity. Strategy 1, the more conservative of the two methods, refines one of the nonparametric bivariate tolerances provided in the attached report. Strategy 2, whose statistical methods are outlined below, should assign a higher proportion of future anti-JCV seropositivity to a lower risk of developing PML compared to strategy 1.

[0263] Strategy 2 devises a lower concurrent tolerance around the fitted equation, measuring inhibition % versus index for PML patient samples. Strategy 1 constructs a low risk region based on inhibition % / index measurements from two-step anti-JCV assays in STRATA (Ann. Neurol., 68:295-303, 2010) and STRATIFY-I (Ann. Neurol., 70:742-750, 2011) patients (almost all of whom are assumed to have a very low risk of developing PML), while Strategy 2 constructs a high risk region based on measurements collected from MS patients prior to the date of their PML diagnosis. Our limited population of PML samples may not represent the entire universe of Tysabri-treated MS patients infected with PML, but Strategy 2 assumes that the inhibition % vs. index relationship in these samples is representative of all pre-diagnostic PML. This assumption was statistically supported by anti-JCV PML data that showed the inhibition % vs. index relationship in parallel with that of STRATIFY-1+STRATA. This parallelism is utilized by Strategy 2 to model the PML inhibition % vs. index relationship.

[0264] The relationship between inhibition % and the index is first statistically modeled for a set of STRATIFY-1 / STRATA / PML samples. The fitted equation for inhibition % versus index will distinguish between PML and STRATIFY-1 / STRATA samples. A lower simultaneous 95% or 99% tolerance is then constructed around the constrained fitted equation for PML samples. Future anti-JCV seropositive cases whose inhibition % / index measurement falls within this tolerance will be assigned a higher risk of developing PML, but this tolerance should ensure that at least 95% (or 99%) of samples from pre-diagnosed PML patients are assigned a higher risk. Note that future samples with index measurements greater than 2.5 will be automatically assigned a higher risk of developing PML.

[0265] Statistical details.Apply the following mixed model (or some improvement) first to the combined set of second-generation anti-JCV STRATIFY-1+STRATA+PML sample inhibition % / index measurements using the SAS MIXED procedure.

Number

Number

[0266] Table 10 below provides the estimated percentage of anti-JCV antibody positives that would be classified as lower risk based on different nODs.

Table 10

[0267] Other embodiments are described in the claims.

Claims

1. A kit for evaluating the level of anti-JCV antibody in a sample, A substrate on which highly purified virus-like particles (HPVLPs) are placed; HPVLP in solution and; Samples for JCV cutoff calibration; Compared to an anti-JCV antibody-positive control; A JCV-negative control, which is a human serum sample, and a JCV-negative control; A reagent for detecting a complex containing an anti-JCV antibody bound to the HPVLP disposed on the substrate, A kit comprising 20 ngs to 60 ngs of HPVLP disposed on the substrate, and / or a sample-to-substrate ratio of 1:50 to 1:

30.

2. The kit according to claim 1, wherein the JCV cutoff calibration sample is adjusted to have a normalized optical density (nOD) of 1.0, the anti-JCV antibody positive control is adjusted to have an nOD of 1.3, and the JCV negative control is adjusted to have an nOD of 0.

1.

3. The kit according to claim 1, wherein the substrate is a multiwell plate.

4. The kit according to claim 3, wherein the substrate is a 96-well plate.

5. The kit according to claim 1, wherein the JCV-negative control includes anti-JCV antibody-negative serum.

6. The kit according to claim 1, wherein the cutoff calibration sample and the positive control each comprise a mixture of serum positive for anti-JCV antibody and serum negative for anti-JCV antibody.

7. The kit according to claim 1, wherein 30 ngs to 50 ngs of HPVLP are arranged on the substrate.

8. The kit according to claim 7, wherein 40 ngs of HPVLP are disposed on the substrate.

9. The kit according to claim 1, wherein the HPVLP in the solution is present at a concentration of 0.4 μg / mL.

10. The kit according to claim 1, which provides a signal-to-noise ratio of 10 to 30 in concentration of HPVLP disposed on the substrate.

11. The kit according to claim 1, wherein the reagent for detecting the complex, which includes an anti-JCV antibody bound to the HPVLP disposed on the substrate, comprises one or more of the following: a labeled JCV complex, a casein solution, a detectable reagent, a washing buffer, and a stop reagent.

12. The kit according to claim 11, wherein the JCV complex is an anti-human IgG that is complexed with horseradish peroxidase (HRP).

13. The kit according to claim 11, wherein the JCV complex is an affinity-purified and peroxidase-conjugated donkey anti-human antibody.

14. The kit according to any one of claims 11 to 13, wherein the detectable reagent comprises tetramethylbenzidine (TMB).

15. The kit according to claim 14, wherein the detectable reagent comprises TMB and hydrogen peroxide in a buffer.

16. The kit according to claim 11, wherein the washing buffer comprises phosphate-buffered saline (PBS), PBS / Tween, or borate buffer.

17. The kit according to claim 16, wherein the washing buffer comprises PBS.

18. The kit according to claim 16 or 17, wherein the washing buffer further comprises a surfactant together with a non-azide preservative.

19. The kit according to claim 11, wherein the stopping reagent contains an acid.

20. The kit according to claim 19, wherein the acid is sulfuric acid.

21. The kit according to claim 20, wherein the sulfuric acid is 1 M sulfuric acid.

22. The kit according to claim 11, wherein the casein solution comprises casein, a surfactant, and a non-azide preservative in a buffer.

23. The kit according to claim 22, wherein the buffer solution comprises phosphate-buffered saline.

24. The kit according to claim 1, wherein the HPVLP in solution, the JCV cutoff calibration sample, the anti-JCV antibody positive control, and the JCV negative control each further contain a preservative.

25. The kit according to claim 24, wherein the preservative comprises sodium azide.

26. The kit according to claim 1, wherein the reagent for detecting the complex, which includes an anti-JCV antibody bound to the HPVLP disposed on the substrate, comprises one or more of the following: anti-human IgG that is complexed with horseradish peroxidase (HRP), tetramethylbenzidine (TMB), a washing buffer, and a stop reagent.

27. The kit according to claim 26, wherein the washing buffer comprises phosphate-buffered saline (PBS), PBS / Tween, or borate buffer.

28. The kit according to claim 27, wherein the washing buffer comprises PBS.

29. The kit according to claim 27 or 28, wherein the washing buffer further comprises a surfactant together with a non-azide preservative.

30. The kit according to any one of claims 26 to 29, wherein the stopping reagent contains an acid.

31. The kit according to claim 30, wherein the acid is sulfuric acid.

32. The kit according to claim 31, wherein the sulfuric acid is 1 M sulfuric acid.

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