WO2011019670A1 - Procédés de détection de réponses à des thérapies utilisant les niveaux de perforine - Google Patents

Procédés de détection de réponses à des thérapies utilisant les niveaux de perforine Download PDF

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Publication number
WO2011019670A1
WO2011019670A1 PCT/US2010/044921 US2010044921W WO2011019670A1 WO 2011019670 A1 WO2011019670 A1 WO 2011019670A1 US 2010044921 W US2010044921 W US 2010044921W WO 2011019670 A1 WO2011019670 A1 WO 2011019670A1
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Prior art keywords
perforin
sample
cells
level
expression
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PCT/US2010/044921
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English (en)
Inventor
Ramesh C. Nayak
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Nayak Ramesh C
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Priority to EP10808602.6A priority Critical patent/EP2464741A4/fr
Priority to CA2770680A priority patent/CA2770680A1/fr
Publication of WO2011019670A1 publication Critical patent/WO2011019670A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • NK ceils Natural Killer cells
  • MS Multiple Sclerosis
  • gialiramer acetate e.g., Copaxone®
  • the molecular mechanism of the NK cell functional deficiency remains unknown, as does the mechanism by which glatiramer acetate (e.g., Copaxone®) restores NK cell function.
  • Perforin is a channel/pore forming protein that is produced by NK ceils and creates a channel between the NK cell and the target cell through which it transfers proteins such as granzymes. Granzymes trigger processes such as apoptosis that cause the death of the target cell.
  • a response e.g., positive response
  • a therapy such as glatiramer acetate (e.g., Copaxone®) therapy.
  • a percentage of perforin* NK cells e.g., CD16+/perforin+ cells
  • a level of perforin may be measured in a sample from a patient (e.g., multiple sclerosis patient) and compared to the values for normal samples.
  • peforin levels may be relied on to diagnose a disease or condition (e.g., multiple sclerosis), or perforin levels may be relied on to monitor progression of a disease or condition, for example after a treatment or therapy.
  • perforin levels may be used to help detect impending clinical relapse in a patient.
  • the present invention features methods of detecting a positive response to a therapy.
  • the method may comprise obtaining a sample from a patient undergoing the therapy; and either (i) detecting a percentage of perforin+ natural killer (NK) ceils in the sample wherein a positive response to the therapy is detected if the percentage of perforin ⁇ NK cells is greater than or equal to about 75% of total NK cells; or (ii) detecting a level of perforin expression in the sample, wherein a positive response to the therapy is detected if the level of perforin expression in the sample is within a level of perforin expression in a normal sample pius or minus 25%.
  • NK natural killer
  • a positive response to the therapy is detected if the percentage of perforin* NK cells is greater than or equal to about 51 % of total NK ceils. In some embodiments, a positive response to the therapy is detected if the percentage of perforin+ NK ceils is greater than or equal to about 65% of totai NK cells. In some embodiments, a positive response to the therapy is detected if the percentage of perforin ⁇ NK cells is greater than or equal to about 85% of totai NK ceils. Sn some embodiments, a positive response to the therapy is detected if the level of perforin expression in the sample is within a level of perforin expression in a normal sample pius or minus about 50%.
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within a level of perforin expression in a normal sample plus or minus about 40%. In some embodiments, a positive response to the therapy is detected if the level of perforin expression in the sample is within a level of perforin expression in a normal sample pius or minus about 10%. f 00071 The present invention also features methods of diagnosing a disease or condition.
  • the method may comprise obtaining a sampie from a patient; and either (i) detecting a level of perforin expression in the sample, wherein the disease or condition is detected if the level of perforin expression in the sample is less than a level of perforin expression in a normal sample minus about 25%; or (ii) detecting a percentage of perforin+ NK ceils in the sample, wherein the disease or condition is detected if the percentage of perforin* NK cells is less than about 75% of total NK ceils. in some embodiments, the disease or condition is detected if the level of perforin expression in the sample is less than a level of perforin expression in a normal sampie minus about 30%.
  • the disease or condition is detected if the level of perforin expression in the sampie is less than a level of perforin expression in a normal sample minus about 50%. in some embodiments, the disease or condition is detected if the percentage of perforin* NK cells is less than or equal to about 70% of total NK cells. Sn some embodiments, the disease or condition is detected if the percentage of perforin* NK cells is less than or equal to about 50% of total NK cells.
  • the present invention also features methods of detecting impending clinical relapse.
  • the method may comprise obtaining a sample from a patient; and either (i) detecting a levei of perforin expression in the sample, wherein an impending clinical relapse is detected if the level of perforin expression in the sample is less than a level of perforin expression in a normal sample minus about 25%; or (ii) detecting a percentage of perforin* NK cells in the sample, wherein an impending clinical reiapse is detected if the percentage of perforin* NK cells is less than about 75% of total NK cells.
  • an impending clinical relapse is detected if the ievel of perforin expression in the sample is iess than a level of perforin expression in a normal sample minus about 40%. in some embodiments, an impending clinical relapse is detected if the percentage of perforin* NK cells is less than or equal to about 80% of total NK cells. jO ⁇ llf in some embodiments, the therapy is glatiramer acetate.
  • the sample is a blood sample (e.g., a sample of peripheral blood mononuclear cells (PBMCs)). Sn some embodiments, the NK cells are CD18+ ceils, CD56+ cells, or CD57+ cells.
  • detecting a level of perforin expression in the sample includes either (i) perforin RNA expression is measured; (ii) perforin protein expression is measured; or (iii) both perforin RNA expression and perforin protein expression are measured.
  • FIG. 1 shows the percent of CD16+/perforin+ cells in normal (non-MS) patients.
  • the normal patients were not undergoing giatiramer acetate (Copaxone ⁇ ) treatments. Percentages in FIG. 1 were: 86.60%, 83.33%, 88.55%, 85.33%, 80.24%, 91.14%, 91.27%, 76.12%, 88.23%, 85.81 %.
  • normal individuals as in individuals not suffering from any type of neurological disease or condition
  • 9 of the 10 samples passed this criteria. It is possible that the diminished level of perforin in sample 2-038 is a result of an unknown immune response reason.
  • FIG. 2 shows the percent of CD16+/perforin+ ceils in MS patients that were not undergoing giatiramer acetate (Copaxone®) treatments. Percentages in FIG. 2 were: 72.89%, 65.82%, 72.64%, 64.61 %. Without wishing to limit the present invention to any theory or mechanism, it is believed that peforin levels in MS patients not undergoing giatiramer acetate (Copaxone ⁇ ) treatment (and possibly MS patients not showing a positive response to giatiramer acetate treatment) are diminished, as in less than about 75% of CD16+ cells are CD16+/perforin+. In FIG. 2, all samples (4 out of 4) passed this criteria.
  • FSG. 3 shows the percent of CD16+/perforin+ cells in MS patients that were undergoing giatiramer acetate (Copaxone®) treatments. Percentages in FIG. 3 were: 66.40%, 87.66%. 93.02%, 81.56%, 86.71%, 83.94%, 82.66%, 73.46%, 86.98%. Without wishing to limit the present invention to any theory or mechanism, it is believed that perforin levels should be restored to normal (as in about 75% or more of CD16+ ceils are CD16+/ ⁇ erforin+), In FIG. 3, 7 of the 9 samples passed this criteria. Two samples (e.g., "non-responders") were below about 75%.
  • FIG. 4 shows the percent of CD16+/perforin+ cells in stroke patients.
  • the stroke patients were not undergoing glatiramer acetate (Copaxone®) treatments. Percentages in FIG. 4 were: 80.33%, 71.64%, 78.59%, 81.51 %, 80.38%, 82.08%, 70.23%, 85.66%, 82.23%, 81.97%.
  • perforin ievels should be normal (as in about 75% or more of CD16+ cells are CD16+/perforin+) in stroke patients (and possibly in patients suffering from other neurological disease). In FIG. 4, 8 of the 10 samples passed this criteria.
  • FSG. 5 shows perforin expression in CD 16 lymphocytes in relapsing/remitting multiple sclerosis (RRMS) patients.
  • Lower levels of CD16+/Perforin+ cells were found in untreated RRMS patients (NoTX) as compared to normal human subjects (NHS).
  • RRMS patients treated with glatiramer acetate (e.g., Copaxone®) (CoP) have a similar frequency of CD16+/Perforin+ cells to normal human subjects (NHS).
  • NK ceils had diminished ievels of perforin (as determined by percent of CD16+/perforin+ cells) in multiple scierosis (MS) patients not treated with giatirarner acetate (Copaxone®), as compared to glatiramer acetate-treated MS patients that had more normal ievels of perforin (percent of CD16+/perforin+ cells) in their CD16+ NK ceils.
  • Therapeutic failure is typically seen by the appearance of neurological symptoms and/or the appearance of lesions by magnetic resonance imaging (MR!).
  • the present invention features methods of detecting a positive response to a particular therapy (e.g., glatiramer acetate therapy) using perforin levels, e.g., expression, (e.g., in NK cells).
  • perforin levels in NK cells may be used to monitor patients for either a positive response to glatiramer acetate (Copaxone ⁇ ) or for therapeutic failure of glatiramer acetate (Copaxone®).
  • the results may help determine an appropriate treatment regimen for MS patients (e.g., alternative drugs may be more appropriate for patients that don ' t respond to glatiramer acetate) and/or may increase the speed at which therapeutic failure of glatiramer acetate
  • the present invention also features methods of detecting an impending clinical relapse with detection of perforin levels (e.g., in NK cells).
  • the present invention also features methods of diagnosing diseases and/or conditions with detection of performing levels (e.g., in NK cells).
  • the present invention is not limited to detecting perforin levels.
  • other markers may be used in combination with (or substituting) perforin.
  • An example of another marker includes but is not limited to leptin receptor.
  • percentages of leptin receptor* NK cells may be detected in combination with percentages of perforin ⁇ NK cells.
  • three markers are simultaneously detected, for example a NK marker (e.g., CD16), perforin, and leptin receptor.
  • the present invention features methods of detecting a positive response to a particular therapy using perforin expression (e.g., in NK cells).
  • perforin expression e.g., in NK cells.
  • the therapy is glatiramer acetate (Copaxone®) therapy.
  • the method comprises obtaining a sample from a patient undergoing the therapy, e.g., glatiramer acetate (Copaxone®) therapy.
  • the sample may comprise a blood sample, for example PBMCs.
  • the sample is not limited to blood, for example in some embodiments, the sample comprises, cerebrospinal fluid (CSF), central nervous system tissues, synovia! fluid, cystic fluid, lymph fluid, ascites, pleura! effusion, interstitial fluid, ocular fluids, vitreal fluid, the like, or a combination thereof, in some embodiments, the patient is a multiple sclerosis patient or a patient suspected of having multiple sclerosis.
  • CSF cerebrospinal fluid
  • the patient is a multiple sclerosis patient or a patient suspected of having multiple sclerosis.
  • measurements may be made on samples (e.g.. NK ceils) that have been enriched or purified from biological fluids using any avaiiabie method.
  • Methods may include adsorption/non-adsorption to plastic surfaces (e.g., p!astic dishes), adsorption/non-adsorption to antibody coated surfaces (e.g., antibody-coated piastic dishes, antibody-coated beads (magnetic or non-magnetic)), and/or centrifugation through gradient media among other methods (e.g., resetting).
  • the method comprises detecting a percentage of perforin* NK cells (e.g., CD16+/perforin+ ceils) in the sample.
  • the percentage of perforin+ NK cells e.g., CD16+/perforin+ cells
  • a positive response to the therapy is detected if the majority of NK ceils (e.g., CD16* cells) are also perforin*, for example if the percentage of perforin+ NK ceils (e.g., CD16+/perforin+ ceils) is greater than or equal to about 51 % (of the total NK cells (e.g., CD16* ceils), both perforin- NK cells and perforin+ NK ceils).
  • a positive response to the therapy is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ ceils) is greater than or equal to about 55% (of the tota!
  • NK ceils e.g., CD18+ cells
  • perforin- NK ceils both perforin- NK ceils and perforin* NK cells.
  • a positive response to the therapy is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ celis) is greater than or equal to about 60% (of the total NK celis (e.g., CDI 6+ ceils), both perforin- NK ceils and perforin* NK cells).
  • a positive response to the therapy is detected if the percentage of perforin* NK celis (e.g., CD16*/perforin* celis) is greater than or equal to about 85% (of the total NK ceils (e.g., CD16+ celis), both perforin- NK celis and perforin* NK cells).
  • a positive response to the therapy is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells) is greater than or equal to about 70% (of the total NK cells (e.g., CD16+ ceils), both perforin- NK cells and perforin+ NK cells).
  • a positive response to the therapy is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ ceils) is greater than or equai to about 75% (of the total NK cells (e.g., CD18+ celis), both perforin- NK cells and perforin* NK cells).
  • a positive response to the therapy is detected if the percentage of perforin* NK ceils (e.g., CD16+/perforin+ ceils) is greater than or equai to about 80% of the totai CD16+ celis.
  • a positive response to the therapy is detected if the percentage of perforin* NK celis (e.g...).
  • CD16+/perforin+ celis is greater than or equai to about 85%.
  • a positive response to the therapy is detected if the percentage of perforin* NK celis (e.g., CD16+/perforin+ ceils) is greater than or equal to about 90%.
  • the present invention is not limited to using CD18 to identify NK ceils (e.g., percentage of CD16+/perforin+ cells detected).
  • Other markers may be used.
  • CD16* may be substituted with another NK ceil markers including but not limited to CD57 and CD56.
  • the percentage of CD56+/perforin+ ceils is measured.
  • the percentage of CD57+/perforin+ celis is measured.
  • a positive response to the therapy is detected if the majority of CD58+ celis (or CD57+ cells) are also perforin +, for example if the percentage of CD56+/perforin+ ceils (or
  • CD57+/perforin+ cells is greater than or equai to 51 %. in some embodiments, a positive response to the therapy is detected if the percentage of CD56+/perforin+ ceils (or CD57+/perforin+ cells) is greater than or equal to 55%. In some
  • a positive response to the therapy is detected if the percentage of CD56+/perforin+ celis (or CD57*/perforin* cells) is greater than or equal to 80%. is greater than or equal to 85%, is greater than or equal to 70%, is greater than or equai to 75%, is greater than or equai to 80%, is greater than or equal to 85%, is greater than or equai to 90%, etc.
  • the method comprises detecting a level of perforin in the sample.
  • the levei of perforin may be a measurement of RNA expression or a measurement of protein expression, for example.
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 25% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 25%.
  • a positive response is detected if the level of perforin expression in the sample is between about 75 units to 125 units (e.g., plus or minus 25% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 80 of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 60%. For example, if the level of perforin in a norma!
  • a positive response is detected if the level of perforin expression in the sample is between about 40 units to 160 units (e.g., plus or minus 60% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 50% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 50%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is detected if the level of perforin expression in the sample is between about 50 units to 150 units (e.g., plus or minus 50% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 40% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 40%. For example, if the level of perforin in a norma! sample is about 100 units, a positive response is detected if the level of perforin expression in the sample is between about 60 units to 140 units (e.g., plus or minus 40% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 30% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 30%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is detected if the level of perforin expression in the sample is between about 70 units to 130 units (e.g., plus or minus 30% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 20% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 20%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is detected if the level of perforin expression in the sample is between about 80 units to 120 units (e.g., plus or minus 20% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 10% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 10%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is detected if the level of perforin expression in the sample is between about 90 units to 110 units (e.g., plus or minus 10% the level of perforin in the norma! sample). In some embodiments, a positive response to the therapy is detected if the level of perforin expression in the sample is within about within about 5% of a level of perforin expression in a norma!
  • a positive response is detected if the level of perforin expression in the sample is between about 95 units to 105 units (e.g., plus or minus 5% the level of perforin in the normal sample).
  • a positive response to the therapy is detected if the level of perforin expression in the sample is within about 1 % of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 1 %.
  • the level of perforin in a norma! sample is about 100 units, a positive response is detected if the level of perforin expression in the sample is between about 99 units to 101 units (e.g., plus or minus 1 % the level of perforin in the normal sample).
  • the present invention is not limited to detecting a positive response to glatiramer acetate (Copaxone®) therapy.
  • the present invention may be used to detect a response to any drug used to treat multiple sclerosis, any drug or compound that may increase perforin levels, any drug or compound that may decrease perforin levels, natalizaumab (Tysabri ⁇ ), interferon beta-1a (Avonex®, R ⁇ bif®), interferon beia-1 b (Betaseron®).
  • antidepressants e.g., selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, paroxetine), chernofberapeutic drugs/compounds, the like, or a combination thereof.
  • the present invention is used to detect a response to glatiramer acetate (Copaxone®) or a chemotherapeuiic drug in cancer patients.
  • the methods of the present invention may also be used for drug design and/or drug screening.
  • a compound and/or a library of compounds may be screened for the ability to upregulate or downregulate perforin levels.
  • Upregulation of perforin may be a desirable effect in some diseases or conditions, for example in autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, Graves Disease, Sjogren's Syndrome, etc.) and/or other diseases such as cancer.
  • downregulation of perforin may be a desirable effect. For example, high levels of perforin during the first trimester of pregnancy may contribute to a spontaneous abortion. Thus, it may be advantageous to screen drugs for their ability to
  • the present invention is not limited to using flow cytometry to measure perforin expression.
  • Alternative methods are well known to one of ordinary skill in the art.
  • Alternative methods include, for example, ELISA, immunological techniques, real time PCR, western blot, the like, or a combination thereof.
  • the present methods may be used to monitor patient compliance with drug therapy.
  • a physician seeing that the result was below the threshold for therapeutic efficacy would then question the patient to determine whether the patient was complying with the dosing regime and if satisfied that the patient was in compliance would regard it as a treatment failure and prescribe alternative drug therapy.
  • the present invention further comprises a method comprises administering an appropriate therapy (e.g., glatiramer acetate (Copaxone®) therapy) to a patient (e.g., and later obtaining a sample from the patient undergoing the therapy).
  • the method may further comprise determining a positive response to the therapy via either (i) detecting a percentage of perforin* natural killer (NK) cells in the sample; or (ii) detecting a level of perforin expression in the sample.
  • an appropriate therapy e.g., glatiramer acetate (Copaxone®) therapy
  • a patient e.g., and later obtaining a sample from the patient undergoing the therapy.
  • the method may further comprise determining a positive response to the therapy via either (i) detecting a percentage of perforin* natural killer (NK) cells in the sample; or (ii) detecting a level of perforin expression in the sample.
  • NK natural killer
  • a positive response to the therapy is determined if the majority of NK cells (e.g., CD16* cells) are also perforin*, for example if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells) is greater than or equal to about 51 % (of the total NK cells (e.g., CD16+ cells), both perforin- NK cells and perforin* NK cells).
  • NK cells e.g., CD16* cells
  • perforin* NK cells e.g., CD16+/perforin+ cells
  • a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD16*/perforin* cells) is greater than or equal to about 55% (of the total NK cells (e.g., CD16+ ceils), both perforin- NK ceils and perforin* NK cells).
  • perforin* NK cells e.g., CD16*/perforin* cells
  • a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD18+/perforin+ cells) is greater than or equal to about 80% (of the total NK cells (e.g., CD16+ cells), both perforin- NK cells and perforin* NK cells), in some embodiments, a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD16*/perforin* cells) is greater than or equal to about 65% (of the total NK cells (e.g., CDI 6+ ceils), both perforin- NK ceils and perforin* NK cells).
  • the percentage of perforin* NK cells e.g., CD18+/perforin+ cells
  • a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells) is greater than or equal to about 70% (of the total NK cells (e.g., CD16+ cells), both perforin- NK cells and perforin* NK cells), in some embodiments, a positive response to the therapy is determined if the percentage of perforin* NK ceils (e.g., CD16*/perforin+ cells) is greater than or equal to about 75% (of the total NK cells (e.g., CD18+ cells), both perforin- NK ceils and perforin* NK cells).
  • the percentage of perforin* NK cells e.g., CD16+/perforin+ cells
  • a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells) is greater than or equal to about 80% of the total CD 16* cells. In some embodiments, a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD16+/perforin+ ceils) is greater than or equal to about 85%. in some embodiments, a positive response to the therapy is determined if the percentage of perforin* NK cells (e.g., CD16*/perforin* cells) is greater than or equal to about 90%.
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 25% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 25%. For example, if the level of perforin in a norma! sample is about 100 units, a positive response is determined if the level of perforin expression in the sample is between about 75 units to 125 units (e.g., plus or minus 25% the level of perforin in the normal sample).
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 80 of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 60%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is determined if the level of perforin expression in the sample is between about 40 units to 180 units (e.g., plus or minus 60% the level of perforin in the norma! sample). ⁇ n some embodiments, a positive response to the therapy is determined if the level of perforin expression in the sample is within about 50% of a level of perform expression in a norma!
  • a positive response is determined if the level of perforin expression in the sample is between about 50 units to 150 units (e.g., plus or minus 50% the level of perform in the norma! sample).
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 40% of a level of perforin expression in a normal sample, as in a level of perform expression in a norma! sample plus or minus about 40%.
  • a positive response is determined if the level of perforin expression in the sample is between about 80 units to 140 units (e.g., plus or minus 40% the level of perforin in the norma! sample).
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 30% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 30%. For example, if the level of perforin in a norma!
  • a positive response is determined if the level of perforin expression in the sample is between about 70 units to 130 units (e.g., plus or minus 30% the level of perform in the norma! sample).
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 20% of a level of perforin expression in a normal sample, as in a level of perforin expression in a normal sample plus or minus about 20%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is determined if the level of perforin expression in the sample is between about 80 units to 120 units (e.g., plus or minus 20% the level of perforin in the norma! sample).
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 10% of a level of perforin expression in a norma! sample, as in a level of perforin expression in a normal sample plus or minus about 10%. For example, if the level of perforin in a normal sample is about 100 units, a positive response is determined if the level of perforin expression in the sample is between about 90 units to 110 units (e.g., plus or minus 10% the level of perforin in the normal sample). Sn some embodiments, a positive response to the therapy is determined if the level of perforin expression in the sample is within about within about 5% of a leve! of perforin expression in a norma!
  • a positive response is determined if the level of perforin expression in the sample is between about 95 units to 105 units (e.g., plus or minus 5% the leve! of perforin in the norma! sample).
  • a positive response to the therapy is determined if the level of perforin expression in the sample is within about 1 % of a level of perforin expression in a norma! sample, as in a leve! of perforin expression in a norma! sample plus or minus about 1 %.
  • a positive response is determined if the leve! of perforin expression in the sample is between about 99 units to 101 units (e.g., plus or minus 1 % the level of perforin in the norma! sample).
  • the methods of the present invention are used to detect a disease/condition such as but not limited to a demyelinating disease (e.g., multiple sclerosis).
  • a demyelinating disease e.g., multiple sclerosis.
  • the methods of the present invention may be a part of a diagnostic panel.
  • the present invention features methods of diagnosing a disease or condition using perforin expression (e.g., in NK cells).
  • the disease or condition may include but is not limited to demyelinating diseases such as multiple sclerosis.
  • the method comprises obtaining a sample from a patient (suspected of having the disease or condition).
  • the method may further comprise detecting a percentage of perforin* NK cells (e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.) and/or a level of perforin expression (e.g., protein, RNA, etc.) in the sample.
  • a percentage of perforin* NK cells e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.
  • a level of perforin expression e.g., protein, RNA, etc.
  • the disease or condition is detected if the level of perforin expression in the sample is less than about 25% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 25%.
  • the disease or condition may be detected if the level of perforin expression in the sample is less than about 75 units (e.g., less than the level of perforin in the normal sample minus 25%).
  • the disease or condition is detected if the level of perforin expression in the sample is less than about 30% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 30%.
  • the disease or condition may be detected if the level of perforin expression in the sample is less than about 70 units (e.g., less than the level of perforin in the normal sample minus 30%).
  • the disease or condition is detected if the level of perforin expression in the sample is less than about 35% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 35%.
  • the disease or condition may be detected if the level of perforin expression in the sample is less than about 85 units (e.g., less than the level of perforin in the normal sample minus 35%).
  • the disease or condition is detected if the level of perforin expression in the sample is less than about 40% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 40%.
  • the level of perforin in a normal sample is about 100 units
  • the disease or condition may be detected if the level of perforin expression in the sample is less than about 80 units (e.g., less than the level of perforin in the normal sample minus 40%).
  • the disease or condition is detected if the level of perforin expression in the sample is less than about 50% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 50%.
  • the disease or condition may be detected if the level of perforin expression in the sample is less than about 50 units (e.g., less than the level of perforin in the normal sample minus 50%).
  • the disease or condition is detected if the percentage of perforin* NK ceils (e.g., CD16+/perforin+ ceils, CD56*/perforin* ceils,
  • CD57*/perforin* cells, etc. is less than about 80% of the total NK cell population (e.g., perforin* NK ceils and perforin- NK cells),
  • the disease or condition is detected if the percentage of perforin* NK ceils (e.g., CD16+/perforin+ ceils, CD5 ⁇ +/perforin+ cells, CD57+/perforin+ cells, etc.) is less than about 75% of the total NK ceil population (e.g., perforin* NK ceils and perforin- NK cells).
  • the disease or condition is detected if the percentage of perforin* NK ceils (e.g., CD16+/perforin* cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.) is less than about 70% of the total NK ceil population (e.g., perforin* NK ceils and perforin- NK ceils).
  • perforin* NK ceils e.g., CD16+/perforin* cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.
  • the disease or condition is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ ceils, CD56+/perforin+ cells, CD57*/perforin+ cells, etc.) is less than about 65% of the total NK cell population (e.g., perforin* NK cells and perforin- NK ceils).
  • perforin* NK cells e.g., CD16+/perforin+ ceils, CD56+/perforin+ cells, CD57*/perforin+ cells, etc.
  • the disease or condition is detected if the percentage of perforin* NK ceils (e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.) is less than about 80% of the total NK ceil population (e.g., perforin* NK ceils and perforin- NK ceils).
  • perforin* NK ceils e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.
  • the disease or condition is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.) is less than about 55% of the total NK cell population (e.g., perforin* NK cells and perforin- NK ceils).
  • perforin* NK cells e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.
  • the disease or condition includes some viral infections, for example human immunodeficiency virus (HIV) as HIV has been associated with reduced NK cell activity.
  • the disease or condition includes some parasitic infections, for example amoebic diseases, trypanosomiasis, malaria, and the like.
  • Some metastatic diseases have been associated with suppressed NK eel! activity.
  • the present invention may be used to diagnose a presence of a cancer, e.g. lung cancer, pancreatic cancer, breast cancer, stomach cancer, metastatic cancers, leukemia, and/or the iike.
  • the methods of the present invention are used to monitor progression of a disease/condition such as but not limited to a demyelinating disease (e.g., multiple sclerosis). Progression may be monitored following treatment with a particular therapy (e.g., glatiramer acetate).
  • the method may comprise obtaining a sample from a patient having the disease or condition and detecting a percentage of perforin+ NK ceils (e.g., CD18+/ ⁇ erforin+ ceils, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.) and/or a level of perforin expression in the sample, for example after the patient has undergone a treatment or therapy.
  • the percentage of perforin+ NK ceils e.g., CD16+/perforin+ cells, CD58+/perforin+ ceils,
  • CD57+7perforin+- ceils, etc. and/or the level of perforin expression may then be compared to the patient's percentage of perforin* NK cells (e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.) and/or his/her level of perforin expression before the treatment or therapy was begun.
  • perforin* NK cells e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells, etc.
  • the disease or condition to be monitored includes some viral infections, for example human immunodeficiency virus (HSV) as HiV has been associated with reduced NK cell activity.
  • HSV human immunodeficiency virus
  • the disease or condition includes some parasitic infections, for example amoebic diseases, trypanosomiasis, malaria, and the like.
  • the present invention may be used to monitor the progression of a cancer, e.g., lung cancer, pancreatic cancer, breast cancer, stomach cancer, metastatic cancers, leukemia, and/or the iike (as some cancers have been associated with suppressed NK cell activity).
  • a cancer e.g., lung cancer, pancreatic cancer, breast cancer, stomach cancer, metastatic cancers, leukemia, and/or the iike (as some cancers have been associated with suppressed NK cell activity).
  • the present invention may be used to monitor the remission of cancer (e.g., monitor levels of perforin wherein "normal" levels of perforin may be indicative of remission and diminished levels of perforin may be an indication of a recurrence of a cancer).
  • the present invention may be used to monitor progression of a cancer after a patient undergoes a particular treatment (e.g., chemotherapeutic agent, giatiramer acetate, etc.).
  • the term "about” refers to plus or minus 10% of the referenced number.
  • an embodiment wherein the percentage of CD18+/perforin+ cells is about 80% includes a percentage that is between 72 and
  • the present invention also features methods for detecting impending clinical relapse (e.g., an exacerbation, breakthrough disease activity, etc.).
  • the method comprises obtaining a sample from a patient (e.g., an MS patient, etc).
  • the sample may comprise a blood sample, for example PBMCs.
  • the sample is not limited to blood, for example in some embodiments, the sample comprises, cerebrospinal fluid (CSF), central nervous system tissues, synovial fluid, cystic fluid, lymph fluid, ascites, pleura! effusion, interstitial fluid, ocular fluids, vitreal fluid, the like, or a combination thereof.
  • CSF cerebrospinal fluid
  • measurements may be made on samples (e.g., NK cells) that have been enriched or purified from biological fluids using any available method.
  • Methods may include adsorption/non-adsorption to plastic surfaces (e.g., plastic dishes), adsorption/non-adsorption to antibody coated surfaces (e.g., antibody-coated plastic dishes, antibody-coated beads (magnetic or non-magnetic)), and/or centrifugation through gradient media among other methods (e.g., rosetting).
  • the method comprises detecting a percentage of perforin+ natural killer (NK) cells in the sample, for example CD18+/perforin+ cells, CD56+/perforin+ cells, CD57+/p ⁇ rforin+ cells, etc.
  • NK perforin+ natural killer
  • the present invention is not limited to the aforementioned markers for NK cells.
  • the percentage of perforin* NK cells may be detected with flow cytometry or other methods.
  • an impending clinical relapse is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells, CD56*/perforin* cells, CD57*/perforin* cells) is less than about 80% of the total NK cells (both perforin* NK cells and perforin- NK cells).
  • an impending clinical relapse is detected if the percentage of perforin* NK cells (e.g., CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells) is less than about 75%.
  • an impending clinical relapse is detected if the percentage of perforin* NK cells (e.g., CD16*/perforin+ cells, CD56+/perforin+ cells,
  • CD57+/perforin+ cells are less than about 85%.
  • an impending clinical relapse is detected if the percentage of perforin* NK cells (e.g.,
  • CD16+/perforin+ cells, CD56+/perforin+ cells, CD57+/perforin+ cells) is less than about 50%.
  • the patient's percentage of perforin* NK cells is measured (e.g., on a first date), and that percentage is compared to a percentage of perforin* NK cells (e.g., CD16+/perforin+ cells, CD58+/perforin+ cells, CD57+/perforin+ cells) measured on a prior date (e.g., a second date, the second date being earlier than the first date).
  • a prior date e.g., a second date, the second date being earlier than the first date.
  • an impending clinical relapse may be detected if the percentage of perforin* NK cells measured on the first date is less than the percentage of perforin* NK cells measured on the second date, for example is less than about 90% of the percentage of perforin* NK cells measured on the second date, is less than about 85% of the percentage of perforin* NK cells measured on the second date, is less than about 80% of the percentage of perforin* NK cells measured on the second date, is less than about 75% of the percentage of perforin* NK cells measured on the second date, is less than about 70% of the percentage of perforin* NK cells measured on the second date, is less than about 65% of the percentage of perforin* NK cells measured on the second date, etc.
  • the method comprises detecting a level of perforin in the sample.
  • the level of perforin may be a measurement of RNA expression or a measurement of protein expression, for example.
  • an impending clinical relapse is detected if the level of perforin expression in the sample is less than about 25% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 25%.
  • the level of perforin in a normal sample is about 100 units
  • an impending clinical relapse may be detected if the level of perforin expression in the sample is less than about 75 units (e.g., less than the level of perforin in the normal sample minus 25%).
  • an impending clinical relapse is detected if the level of perforin expression in the sample is less than about 30% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 30%. For example, if the level of perforin in a normal sample is about 100 units, an impending clinical relapse may be detected if the level of perforin expression in the sample is less than about 70 units (e.g., less than the level of perforin in the normal sample minus 30%).
  • an impending clinical relapse is detected if the level of perforin expression in the sample is less than about 35% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 35%. For example, if the level of perforin in a normal sample is about 100 units, an impending clinical relapse may be detected if the level of perforin expression in the sample is less than about 85 units (e.g., less than the level of perforin in the normal sample minus 35%).
  • an impending clinical relapse is detected if the level of perforin expression in the sample is less than about 40% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 40%. For example, if the level of perforin in a normal sample is about 100 units, an impending clinical relapse may be detected if the level of perforin expression in the sample is less than about 80 units (e.g., less than the level of perforin in the normal sample minus 40%).
  • an impending clinical relapse is detected if the level of perforin expression in the sample is less than about 50% of a level of perforin expression in a normal sample, as in less than a level of perforin expression in a normal sample minus about 50%. For example, if the levei of perforin in a normal sampie is about 100 units, an impending clinical relapse may be detected if the level of perforin expression in the sample is less than about 50 units (e.g., iess than the level of perforin in the normal sample minus 50%).
  • the patient's perforin level is measured (e.g., on a first date), and that ievel is compared to a levei of perforin measured on a prior date (e.g., a second date, the second date being earlier than the first date).
  • an impending clinical relapse may be detected if the perforin level measured on the first date is less than the perforin level measured on the second date, for example is less than about 90% of the perforin ievel measured on the second date, is iess than about 85% of the perforin ievel measured on the second date, is iess than about 80% of the perforin levei measured on the second date, is less than about 75% of the perforin level measured on the second date, is less than about 70% of the perforin ievel measured on the second date, is less than about 65% of the perforin levei measured on the second date, etc.
  • the present invention is not limited to using flow cytometry to measure perforin expression.
  • Alternative methods are well known to one of ordinary skill in the art.
  • Alternative methods include, for example, ELISA, immunological techniques, real time PCR, western blot, the like, or a combination thereof.
  • Perforin expression can be monitored at a certain frequency, for example every first length of time, for example patients may be tested for peforin expression every month, every two weeks, etc.
  • the first length of time may include but is not limited to about two weeks, about three weeks, about four weeks, about a month, about five weeks, etc.
  • the methods may be repeated after a second length of time (e.g., about 1 week, about 10 days, about 2 weeks, etc.) after the initial measurements.
  • the procedure is further repeated after a third length of time (e.g., about 1 week, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, etc.) after the second measurements.
  • Detecting impending clinical reiapse may heip a physician determine whether to treat a patient with a therapy (e.g., prior to the onset of impending symptoms).
  • kits comprise a means of detecting natural killer cells and a means of delecting perforin expressing cells. Detection of natural killer cells and perforin expressing ceils can be used for methods of detecting positive responses to a therapy, methods of monitoring progression of a disease or condition, methods of diagnosing a disease or condition, and/or methods of defecting an impending clinical reiapse.
  • the means of detecting natural killer cells include: detection of expression of NK cell marker mRNA by PCR and variants of the method (e.g., RT-PCR followed with qPCR), northern blot, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE; Fluorescence In Situ Hybridization, including variants such as Fiow-FISH, qFiSH and double fusion fish (D-FISH); flow cytometry.
  • SAGE serial analysis of gene expression
  • D-FISH double fusion fish
  • the means of detecting perforin expressing cells include: detection of perforin mRNA by PCR and variants of the method e.g., RT- PCR followed with qPCR), northern blot, tag based methods such as serial analysis of gene expression (SAGE) including variants such as LongSAGE and SuperSAGE; Fluorescence In Situ Hybridization, including variants such as Fiow-FSSH, qFSSH and double fusion fish (D-FISH); flow cytometry.
  • SAGE serial analysis of gene expression
  • D-FISH double fusion fish
  • the kit may be used for procedures including but not limited to flow cytometry.
  • the means of detecting natural killer ceils is an antibody to CD18, an antibody to CD58, or an antibody to CD57.
  • Other antibodies include killer-cell immunoglobulin-like receptors (KIRs), C-typ ⁇ lectin family receptors, leukocyte inhibitory receptors and natural cytotoxicity receptors.
  • the means of detecting perforin expressing cells is a perforin antibody.
  • the kit further comprises other standard reagents useful for procedures including but not limited to flow cytometry.
  • the kit further comprises PBS (phosphate buffered saline).
  • the kit further comprises a reagent to fix cell surface staining, in some embodiments, the kit further comprises a reagent permeab ⁇ ize cells. In some embodiments, the kit further comprises a reagent for intracellular staining.
  • the kit further comprises means for treating samples (e.g., blood samples, PBMC samples, etc.).
  • samples e.g., blood samples, PBMC samples, etc.
  • the kit further comprises a means of isolating PBMCs from a blood sample.
  • kits comprise a means of isolating natural killer cells from a sample and a means of detecting perforin expression in the natural killer cells.
  • isolation of NK ceils utilizes antibody coupled magnetic and non-magnetic beads.
  • Other means of isolating NK ceils from a sample may utilize antibody coated to plastics or other surfaces.
  • the specific means recited herein are non-limiting examples only. Other means may be employed in accordance with the present invention as would be known to one of ordinary skill in the art.
  • Detection of perforin expression in natural killer cells can be used for methods of detecting positive responses to a therapy, methods of monitoring progression of a disease or condition, methods of diagnosing a disease or condition, and/or methods of detecting an impending clinical relapse.
  • the kit may be used for procedures including but not limited to
  • the means of detecting perforin expression may include a perforin antibody.
  • the kit further comprises other standard reagents useful for procedures including but not limited to immunoassays (e.g., ELISA), RNA detection (e.g., real time PCR), western blots, the like, or a combination thereof.
  • immunoassays e.g., ELISA
  • RNA detection e.g., real time PCR
  • western blots the like, or a combination thereof.
  • kits further comprise a positive control sample, a negative control sample, or both a positive control sample and a negative control sample. In some embodiments, the kits further comprise a sample from a non-MS patient (e.g., for comparison of perforin expression levels, for example).
  • natural killer ceil may refer to a traditional natural killer cell or a natural killer T cell (e.g., NKT cell).
  • a method comprising detecting a percentage of perforin* natural killer cells in the sample includes detecting a percentage of perforin+ natural killer T cells in the sample.
  • Markers used to defect NKT cells may include but if not limited to CD3.
  • perforin levels are measured/detected in cells that express natural killer cell markers but not natural killer T ceil markers (e.g., CD3), and in some embodiments, perforin levels are measured/detected in cells that express natural killer cell marker and NKT ceil markers (e.g., CD3).
  • EXAMPLE 1 FLOW CYTOMETRY PROCEDURE FOR DETECTING
  • PBMCs peripheral blood mononuclear cells
  • CRTTM ceil preparation tube
  • PBS 1X phosphate buffered saline
  • FACS fluorescent-activated cell sorting
  • Fluorescence-activated cell sorting is a type of flow cytometry that sorts a mixture of biological ceils, one at a time, into separate containers based upon the specific light scattering and fluorescent characteristics of each ceil. It provides quantitative recording of fluorescent signals from individual cells as well as physical separation of ceils of particular interest. Generally, a current of a rapidly flowing stream of liquid carries a suspension of cells through a nozzle. The flow is selected such that there is a large separation between ceils relative to their diameter.
  • Vibrations at the tip of the nozzle cause the stream of cells to break into individual droplets, and the system is adjusted so that there is a low probability of more than one ceil being in a droplet.
  • a monochromatic laser beam illuminates the droplets, which are electronically monitored by fluorescent detectors.
  • the droplets that emit the proper fluorescent wavelengths are electrically charged between deflection plates in order to be sorted into collection tubes.
  • Samples are pooled from about 500 patients who do not experience any symptoms of multiple sclerosis (or other demye ⁇ nating diseases) and who do not test positive for multiple sclerosis as detected by MRS. From those pooled samples, the average level of the perforin (e.g., RNA expression, protein expression, percentage of CD16+/perforin+) can be quantified and then defined as being the normal level of perforin.
  • the average level of the perforin e.g., RNA expression, protein expression, percentage of CD16+/perforin+

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Abstract

L’invention concerne des procédés et des kits pour détecter des réponses positives à des thérapies, diagnostiquer une maladie ou un trouble et détecter des rechutes cliniques imminentes. Le procédé peut comprendre l’obtention d’un échantillon d’un patient et soit la détection d’un pourcentage de perforine + cellules tueuses naturelles (NK), soit la détection d’un niveau d’expression de perforine dans l’échantillon. Des réponses positives à la thérapie peuvent être détectées si le pourcentage de perforine + cellules NK est supérieur ou égal à environ 75 % du total des cellules NK ou si le niveau d’expression de perforine dans l’échantillon est dans la plage d’un niveau d’expression de perforine d’un échantillon normal +/- 25 %. La maladie ou le trouble ou la rechute clinique imminente peut être détecté si le niveau d’expression de perforine dans l’échantillon est inférieur à un niveau d’expression de perforine dans un échantillon normal moins environ 25 % ou si le pourcentage de perforine + cellules NK est inférieur à environ 75 % du total des cellules NK.
PCT/US2010/044921 2009-08-10 2010-08-09 Procédés de détection de réponses à des thérapies utilisant les niveaux de perforine WO2011019670A1 (fr)

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CA2770680A CA2770680A1 (fr) 2009-08-10 2010-08-09 Procedes de detection de reponses a des therapies utilisant les niveaux de perforine

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CA2770680A1 (fr) 2011-02-17
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