WO2005115517A2 - Methodes de traitement du diabete - Google Patents

Methodes de traitement du diabete Download PDF

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Publication number
WO2005115517A2
WO2005115517A2 PCT/US2005/014395 US2005014395W WO2005115517A2 WO 2005115517 A2 WO2005115517 A2 WO 2005115517A2 US 2005014395 W US2005014395 W US 2005014395W WO 2005115517 A2 WO2005115517 A2 WO 2005115517A2
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Prior art keywords
cells
diabetes
subject
activity
cell
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PCT/US2005/014395
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English (en)
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WO2005115517A3 (fr
Inventor
Diane J. Mathis
Christophe O. Benoist
Laurent Poirot
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Joslin Diabetes Center, Inc.
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Publication of WO2005115517A2 publication Critical patent/WO2005115517A2/fr
Publication of WO2005115517A3 publication Critical patent/WO2005115517A3/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Type-I diabetes is an autoimmune disease characterized by immune system attack on the insulin-producing ⁇ cells of the pancreatic islets, resulting in the loss of glucose homeostasis and ultimately hyperglycemia (Tisch and McDevitt, Cell 85:291-297 (1996)).
  • TID Type-I diabetes
  • compositions and methods for staging, diagnosing and/or treating TID are described herein, which compositions and methods can include evaluating and or modulating NK cells.
  • the invention features methods of treating a subject, e.g., preventing the development of Type 1 diabetes in the subject, by administering to the subject a therapeutically effective amount of an agent that selectively reduces the number and/or activity of NK cells.
  • a therapeutically effective amount is an amount sufficient to treat (e.g., to delay or prevent the onset of, or reduce the penetrance or severity of, or to treat in the early stages of) an autoimmune disease (e.g., Type I diabetes, lupus, scleroderma, multiple sclerosis, Crohn's disease, chronic active hepatitis, rheumatoid arthritis, Graves' disease, myasthenia gravis, myositis, antiphospholipid syndrome (APS), Sj ⁇ gren's syndrome, uveitis, polymyositis, Raynaud's phenomenon, and demyelinating neuropathies).
  • an autoimmune disease e.g., Type I diabetes, lupus, scleroderma, multiple sclerosis, Crohn's disease, chronic active hepatitis, rheumatoid arthritis, Graves' disease, myasthenia gravis, myositis, antiphospholipid syndrome (APS), Sj ⁇ gren
  • the methods can also include identifying a subject a risk for onset of Type I diabetes, e.g., a subject having insulitis (but, e.g., no overt diabetes), or a subject with a family history of Type I diabetes.
  • the subject is identified as having recent onset diabetes, e.g., the subject has had overt diabetes for less than one year, e.g., less than 6 months, less than 3 months, less than 6 weeks, less than 4 weeks, 2 weeks, or less.
  • the number and/or activity of NK cells is reduced by at least 10%, preferably at least 20%, 30%, 40%), 50% or more.
  • the number or activity of NK cells is reduced 60%, 70%, 80% or more.
  • the agent that selectively reduces the number and/or activity of NK cells binds an NK cell polypeptide, e.g., an NK cell surface antigen, e.g., an NK receptor (NKR).
  • the agent binds (and preferably blocks the activity of) a stimulatory NKR.
  • the agent is an NKR-specific antibody, e.g., an anti-NKR monospecific antibody (e.g., a monoclonal antibody, a chimeric antibody, a humanized antibody, a single chain antibody, a single domain antibody, and antigen binding fragments thereof).
  • Exemplary antibodies bind to an NKR such as NK1.1 , Ly-49, NKp46, NKp30, NKp44 or NKG2D.
  • the agent that selectively reduces the number and/or activity of NK cells is a soluble NK receptor (e.g., a stimulatory NK receptor described herein which includes the ligand binding site but lacks a transmembrane domain), or a fusion thereof, e.g., a soluble NK receptor/Fc fusion protein.
  • the agent that selectively reduces the number and/or activity of NK cells is a ligand for an inhibitory NKR, e.g., an MHC class I molecule, e.g., an HLA-C or HLA-E molecule or multimer thereof.
  • the subject is a mammal, e.g., a human, e.g., a pediatric patient at risk for early onset Type I diabetes.
  • the subject is not a rat.
  • the subject has insulitis.
  • the methods can include evaluating the subject for insulitis, e.g., before, during, or after the administration of the agent as described herein.
  • the subject is evaluated for the presence of beta cell auto-antibodies and/or pancreatic inflammation as a proxy for insulitis.
  • the method includes evaluating the subject for one or more of: weight, insulin levels, insulin metabolism, glucose levels, and insulitis.
  • the evaluation can be performed before, during, and/or after the administration of the agent as described herein. For example, the evaluation can be performed at least 1 day, 2 days, 4, 7, 14, 21, 30 or more days before and/or after the administration. In some embodiments, the evaluation is repeated one, two, or more times.
  • the agent is administered in combination with another agent, e.g., insulin, insulin peptides or other ⁇ cell proteins.
  • the agent e.g., an NK cell blocking antibody
  • the cell can be autologous, allogeneic, or xenogeneic, but is preferably autologous.
  • the agent can be administered, e.g., orally, intravenously, percutaneously, subcutaneously, or injected or implanted into a chosen site, e.g., in a pancreatic tissue of the subject.
  • the agent can be modified, e.g., to increase circulatory half-life, increase cellular uptake, improve distribution to target tissues (e.g., pancreatic tissue), decrease clearance and/or decrease immunogenicity, e.g., as known in the art and described herein.
  • the administration of an agent that reduces NK cell levels and/or activity can be initiated, e.g., when the subject begins to show signs of insulitis or TID; when insulitis or TID is diagnosed; at the time a treatment for insulitis or TID is begun or begins to exert its effects; or generally, as is needed to maintain health, e.g., in a subject determined to be at risk for TID.
  • the period over which the agent is administered can be long term, e.g., for six months or more or a year or more, or short term, e.g., for less than a year, six months, one month, two weeks or less.
  • the invention features methods for evaluating a subject for risk, status, aggressiveness, progression, and/or severity of an autoimmune disorder, e.g., an autoimmune disorder described herein, e.g., Type I diabetes.
  • the method includes identifying a human subject having, or at risk for, an autoimmune disorder (e.g., a subject having insulitis); evaluating the number, frequency and/or activity of NK cells in a biological sample from the subject, e.g., blood or a pancreatic tissue sample; and correlating the number, frequency, and/or activity of NK cells in the sample with a level of risk, prognosis, and or diagnosis.
  • an autoimmune disorder e.g., a subject having insulitis
  • a biological sample from the subject e.g., blood or a pancreatic tissue sample
  • correlating the number, frequency, and/or activity of NK cells in the sample with a level of risk, prognosis, and or diagnosis.
  • a higher number, frequency or activity of NK cells in the sample compared to a control is correlated with a greater risk, aggressiveness, progression, and/or severity of an autoimmune disorder, such as Type I diabetes.
  • the evaluating step comprises performing one or more of: an immunoassay, FACS analysis, genomics analysis or proteomics analysis on the sample.
  • the methods include evaluating, e.g., monitoring, the subject for onset of the autoimmune disorder (e.g., onset of Type I diabetes). The methods can also include selecting a course of treatment for the subject based on the result of the evaluation.
  • correlating means identifying the level or activity of NK cells as a risk, prognostic or diagnostic factor for onset of Type I diabetes, e.g., providing a print material or computer readable medium, e.g., an informational, diagnostic, marketing or instructional print material or computer readable medium, e.g., to the subject or to a health care provider, identifying the alteration as a risk or diagnostic factor for Type I diabetes.
  • the methods include diagnosing a subject as being at risk for Type I diabetes.
  • the method includes prescribing or beginning a treatment for TID in the subject.
  • the method includes performing a second diagnostic test, e.g., evaluating one or more of: insulin metabolism, glucose metabolism.
  • the subject is preferably a human, e.g., a human with insulitis or a family history of diabetes.
  • the biological sample can be, e.g., a cell sample, tissue sample, or at least partially isolated molecules, e.g., nucleic acids, e.g., genomic DNA, cDNA, mRNA, and/or proteins derived from the subject.
  • the methods include contacting a biological sample, e.g., a pancreatic tissue sample, with an agent capable of detecting NK cells, such that the presence of NK cell-specific nucleic acid or protein is detected in the biological sample.
  • the agent capable of detecting NK cells is a nucleic acid probe capable of hybridizing to an NK specific nucleic acid, e.g., an NKR nucleic acid, or an antibody capable of binding to an NK specific protein, such as an NKR.
  • the evaluation is used to choose a course of treatment.
  • the invention features methods for identifying agents that can be used to treat an autoimmune disease, e.g., an agent that reduces one or more of: progression from insulitis to TID, severity or aggressiveness of insulitis lesions, and/or time or severity of onset of TID.
  • the methods include evaluating a test compound for the ability to reduce the number, frequency and/or activity of NK cells, wherein the ability of the test compound to reduce the number, frequency and or activity of NK cells is correlated with the ability to reduce progression from insulitis to Type I diabetes.
  • the methods can include correlating the effect of an agent on NK cells with a predicted effect of the agent on a mammal, e.g., a human, e.g., by providing (e.g., to the government, a health care provider, insurance company or patient) informational, marketing or instructional material, e.g., print material or computer readable material (e.g., a label or email), related to the test compound or its use, identifying the agent as a possible or predicted treatment in a mammal, e.g., a human.
  • a mammal e.g., a human
  • the methods can include identifying a test compound as a therapeutic agent, e.g., as a treatment or lead compound for, e.g., reducing TID onset, e.g., in humans, if it reduces NK cell levels or activity, e.g., in pancreatic tissue.
  • the identification can be in the form of informational, marketing or instructional material, e.g., as described herein.
  • the methods include correlating a value for reducing NK levels and/or activity with ability to reducing TID onset, e.g., generating a dataset of the correlation.
  • the methods include evaluating, e.g., quantitatively or qualitatively measuring, an effect of a test compound on NK cell activity, e.g., by evaluating NK cell lytic activity and/or NK cell levels. Evaluating the effect of a test compound on NK cell activity can include administering the test compound to an experimental mammal, e.g., an animal model for TID, e.g., a NOD mouse.
  • the evaluation includes entering a value for the evaluation, e.g., into a database or other record.
  • the subject is an experimental animal.
  • the animal can be, e.g., wild-type or a transgenic experimental animal, e.g., a NOD mouse.
  • the subject can also be a human, e.g., a human in a clinical trial or a patient.
  • the evaluating step includes administering a test compound, e.g., a test compound identified as a candidate therapeutic compound, that has a selected effect on NK levels and/or activity by an in vitro method as known in the art or described herein, to the subject and evaluating a parameter of NK cell levels and/or activity.
  • a test compound that decreases NK cell levels and/or activity in the subject can be considered a therapeutic agent.
  • the identifying step includes: (a) providing a test compound to an NK cell, e.g., by contacting the cell with the test compound; (b) evaluating the ability of the test compound to modulate NK cell activity and/or levels; and (c) selecting a test compound that decreases NK cell activity and/or levels as a candidate therapeutic compound, useful in a method described herein.
  • the test compound is not limiting and can be, e.g., a nucleic acid (e.g., an antisense, siRNA, aptamer, or ribozyme), a polypeptide (e.g., an antibody or antigen-binding fragment thereof), a peptide fragment, a peptidomimetic, or a small molecule (e.g., a small organic or inorganic molecule with a molecular weight of less than 2000 daltons).
  • the test compound is a member of a combinatorial library, e.g., a peptide or organic combinatorial library, or a natural product library.
  • a plurality of test compounds e.g., library members
  • the test compounds of the plurality e.g., library
  • the methods include two evaluating steps, e.g., first evaluating a test compound in a first system, e.g., an in vitro cell or tissue system, and then evaluating the test compound in a second system, e.g., a second in vitro cell or tissue system, or in vitro in a non-human animal.
  • the method includes two evaluating steps in the same type of system, e.g., the agent is re-evaluated in a non-human animal after a first evaluation in the same or a different non-human animal.
  • the two evaluations can be separated by any length of time, e.g., days, weeks, months or years.
  • the invention features computer readable records encoded with (a) a subject identifier, e.g., a patient identifier, (b) one or more results from an evaluation of the subject, e.g., a diagnostic evaluation described herein, e.g., the level or activity of NK cells in the subject, and optionally (c) a value for or related to a disease state, e.g., a value correlated with disease status or risk with regard to Type I diabetes, e.g., insulitis.
  • the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level and/or activity of NK cells in a sample, and a descriptor of the sample.
  • the descriptor of the sample can be an identifier of the sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment).
  • the data record further includes values representing the level and/or activity of genes other than NK cell markers (e.g., other genes associated with TID or other genes on an array).
  • the data records can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database of the Oracle or Sybase database environments).
  • the invention also includes a method of communicating information about a subject, e.g., by transmitting information, e.g., transmitting a computer readable record described herein, e.g., over a computer network.
  • the invention features methods for providing information, e.g., for making a decision with regard to the treatment of a subject having, or at risk for, a disorder described herein.
  • the method includes (a) evaluating a level and/or activity of NK cells; optionally (b) providing a value for the level and/or activity of NK cells; optionally (c) comparing the provided value with a reference value, e.g., a control or non-disease state reference, or a disease state reference; and (d) supplying information, e.g., information for making a decision on or related to the treatment of the subject, based, e.g., on the relationship of the provided value to the reference value.
  • the provided value relates to an NK cell activity described herein, e.g., NK lytic activity described herein.
  • the decision is whether to administer a preselected treatment.
  • the decision is whether a party, e.g., an insurance company, HMO, or other entity, will pay for all or part of a preselected treatment.
  • FIG. 1A is pair of line graphs showing live CD45 + cells and CD4 + BDC2.5- clonotype + cells were sorted from the pancreas of 23 to 25-day old BDC2.5/NOD or BDC2.5/B6.H-2 87 mice for microarray analysis.
  • the left panel shows a cumulative number of up- or down-regulated genes in BDC2.5/B6.H-2 87 vs.
  • BDC2.5/NOD pancreatic CD45 + cells for the indicated relative variation.
  • the grey line indicates the level of "background" variation as measured from a randomized data set.
  • the right panel shows a cumulative number of up-regulated genes in BDC2.5/B6.H-2 g7 vs.
  • FIG. IB is a panel of four log-scale plots of expression values in pancreatic CD45 + cells with highlights on genes characteristic of the indicated cell type.
  • FIG. 1C is a dot plot of microarray expression value of NK-specific genes in B6.H-2 8 purified NK cells versus CD45 + pancreatic infiltrate. NK-specific genes are upregulated in BDC2.5/B6.H-2 87 pancreatic infiltrate.
  • FIG. 2 A is a set of two scatter plots, a line graph, and a dot plot showing that NK frequency co ⁇ elates with pancreatic infiltrate aggressivity.
  • NK cell were identified as DX5 + CD3 " by cytofluorimetric analysis; their frequency was measured in the pancreas of 20 to 24- day old BDC2.5/ NOD and BDC2.5/B6.H-2 87 mice.
  • One representative scatter plot from each mouse type is shown as well as values for all individual mice tested (p ⁇ 0.03) (line graph). Spleen values are shown for control.
  • FIG. 2B is a set of two scatter plots, a line graph, and a dot plot showing that NK cell frequency in 16 to 22-day old anti-CTLA4 treated vs. untreated BDC2.5/NOD mice (pO.Ol).
  • FIG. 3 is a line graph showing that NK cells are early participants of an aggressive pancreatic lesion.
  • BDC2.5/NOD mice were treated with anti-CTLA4 Ab at day 10, 12, 14.
  • the number of NK cells and T cells was measured in the pancreas of each mouse from day 12 to day 22.
  • the average number of NK cells (and T cells respectively) found in the insulitis-free pancreas of a young NOD mice is 16 (50) at day 12, 38 (267) at day 15, 165 (1120) at day 18.
  • FIGs. 5 A and 5B show strain-specific expression of NK-specific genes. Fig.
  • FIG. 5 A is a line graphs showing relative variation (fold change B6.H-2 g7 /NOD) in expression values of NK-specific genes are plotted for splenic NK cells and CD45 + pancreatic leukocytes.
  • Fig. 5B is a table showing microarray data for NK-specific gene expression in the pancreatic infiltrate of BDC2.5/NOD and B6.H-2 87 as well as in splenic NOD and B6.H-2 87 NK cells. The genes were sorted by chromosomal position. Fold changes (B6.H-2 g7 /NOD) are indicated in each tissue. The specificity of the probes used in the affymetrix MuAv2 chip was confirmed.
  • FIG. 6A is a set of three bar graphs, each with a photograph of a gel, that shows wide differences in NK surface receptor expression between NOD and B6.H-2g7 mice.
  • FIG. 6B is a set of 24 scatter plots of the results of cytofluorimetric analysis of NK surface receptors on NOD, NOD.NK1.1 and B6.H-2g7 splenocytes.
  • Bold numbers indicate the average percentage of NK cells (+/- standard deviation) positive for the indicated surface molecule (when gated on DX5+ CD3-). Numbers in parenthesis indicate the mean fluorescence intensity on the X-axis of the designated population.
  • the monoclonal antibody used to stain Ly49AB6 (clone Al) has been showed to cross-react with Ly49ANOD and partially to Ly49PNOD (Silver et al., J. Immunol. 165: 1771-1781 (2000)).
  • the preferential expression of Ly49P by NOD NK cells might be reflected by the slight but clear staining observed on Ly49A- NOD NK cells versus Ly49A- B6.H-2g7 NK cells.
  • NK cells have been unexpectedly identified as important players in diabetes progression.
  • a high degree of genetic variation in the expression and epitope profile of NK-cell-specific genes was evident when diabetes-resistant and -susceptible mouse strains were compared.
  • the proportion and numbers of NK cells in pancreatic islet infiltrates, and the timing of their entry, correlated with the aggressivity of the lesion.
  • reduction or depletion of NK cell function significantly reduced the speed and penetrance of diabetes onset. Therefore, the invention includes, inter alia, methods of diagnosing and delaying the onset of diabetes by administering an agent that reduces NK cells numbers or activity.
  • TCR T-Cell Receptor
  • NK Cells As shown by the experiments described herein, NK cells have now unexpectedly been identified as important players in progression from insulitis to diabetes. An important role for NK cells in setting the tone of insulitis comes as a surprise because their impact on autoimmune diabetes seems to have been little studied, there being only scant mention in the literature (Ellerman et al., Diabetologia 36:596-601 (1993)). NK cells have been observed in the islet infiltrate of NOD mice (Miyazaki et al., Clin. Exp. Immunol.
  • NOD mice have been reported to exhibit low NK cell activity (Carnaud et al., J. Immunol. 166:2404-2411 (2001); Kataoka et al., Diabetes 32:247-253 (1983); Shultz et al., J. Immunol. 154:180-191 (1995); Poulton et al., Int. Immunol. 13:887-896 (2001)), such that a disease-promoting role has seemed counterintuitive.
  • NK cells natural killer gene complex
  • IFN interferon
  • NK cells can "help" both CD4+ and CD8+ T cell responses in infectious contexts (Mailliard et al., J. Immunol. 171 :2366- 2373 (2003); Vankayalapati et al., J. Immunol. 172: 130-137 (2004)). It may be instructive to compare the variations in gene expression profiles observed here with those seen in a kinetic analysis of cyclophosphamide-induced diabetes in BDC2.5/NOD mice. In that case, no changes in NK-cell-specific transcripts were detected.
  • IFN ⁇ -dominated response may be a commonality of aggressive end-stages, but the means of arriving to it may differ, sometimes promoted by NK cells, sometimes by cytotoxic agents.
  • NK cells might have a previously under-appreciated general function in promoting autoimmune diseases, quite opposed to the protective role previously emphasized (Baxter and Smyth, Autoimmunity 35:1-14 (2002)).
  • genetic variation or costimulatory blockade the stable insulitis of BDC2.5 NOD mice was perturbed, resulting in NK-cell-promoted ⁇ -cell destruction. While not wishing to be bound by any theory, it is reasonable to suggest that genes of the B6.H-2g7 background control NK cell activity.
  • CTLA-4 has been strongly implicated in the generation and action of T cells with regulatory potential, and we have found that an equilibrium between regulatory and effector T cells is also important in maintaining the stable insulitis of BDC2.5/NOD mice (Herman et al., J. Exp. Med. 199(11): 1479-1489 (2004)).
  • a regulatory T cell imbalance resulting from CTLA-4 blockade might promote an early recruitment of NK cells.
  • the anti -CTLA-4 mAb might affect the NK cells, themselves. Indeed, although implicitly assumed, it has never been formally demonstrated that the induction of diabetes by anti-CTLA-4 mAb is via an effect on T cells.
  • CTLA-4 has a dampening influence on NK cells, and that the diabetogenic effect of blocking CTLA-4 reflects release from that inhibition.
  • the NKR repertoire expressed by NK cells is highly variegated, each cell expressing a different subset of NKRs, likely resulting from stochastic regulatory processes (Raulet et al., Annu. Rev. Immunol. 19:291-330 (2001)).
  • many differences were found in the expression of NKC genes when splenic NK cells of BDC2.5/NOD mice were compared with those of BDC2.5/B6.H-2g7 animals.
  • NK cells The most influential of these regions was located proximally on chromosome 7.
  • Several immunologically relevant genes map to this region, some of which are of particular interest because they encode proteins of critical importance in NK cell biology (DAP12 and DAP10, Flt3L).
  • DAP12 and DAP10, Flt3L proteins of critical importance in NK cell biology
  • An important role for NK cells in unleashing an autoimmune lesion's potential for destruction has been identified.
  • Variance in the NOD and B6 genomes in the expression of NK receptors has been found. While not wishing to be bound by theory, one might propose that the very rapid progression to diabetes in a subset of pediatric human patients, who present with severe hyperglycemia in the first years of age, might also reflect an influence of NK cells and of the equally extensive NKR polymorphisms in humans.
  • NK cell activity is generally believed to be regulated by the opposing activity of stimulatory and inhibitory receptors.
  • NK cell binding to a target cell triggers its lytic activity through the stimulatory receptors.
  • Activation of NK cells can be shut down by inhibitory receptors, which deliver a negative signal upon NK cell binding to MHC class I molecules on target cells.
  • strategies for reducing the number or activity of NK cells can include blocking stimulatory receptor activity, or inducing inhibitory receptor activity.
  • monospecific (e.g., monoclonal) antibodies against stimulatory NK receptors e.g., NKp46 (Pessino et al., J. Exp. Med. 188:953-960 (1998)); NKp30 (Pende et al., J. Exp. Med. 190(10): 1505-1516 (1999)); NKp44 (Cantoni et al., J. Exp. Med., 189:787- 796 (1999)) and NKG2D (Houchins et al., J. Exp.
  • NK coreceptors e.g., 2B4, NTB-A, DNAM-1 and NKp80
  • NK mediated lytic activity see, e.g., Moretta et al., Annu. Rev. Immunol. 19: 197-223 (2001)
  • An antibody that inhibits an agent that activates NK cells e.g., natural killer stimulatory factor (NKSF) (see, e.g., U.S. 2004/0044186) can also be used.
  • NKSF natural killer stimulatory factor
  • Such antibodies can be modified, e.g., to be made more suitable for therapeutic use.
  • the antibody is humanized, chimeric, or a single chain biosynthetic antibody based on the monoclonal antigen binding site.
  • a compound that blocks NK cell activity or levels can be a soluble NKR (e.g., a truncated NKR, such as a stimulatory NKR including the ligand binding site but lacking a transmembrane domain) or fusion thereof, e.g., a soluble NKR/Fc fusion protein (see, e.g., Ashkenazi et al., Curr. Opin. Immunol. 9(2): 195-200 (1997)).
  • Agents that inhibit or reduce NK cell numbers or activity also include ligands that bind and activate an NKR, e.g., bind an inhibitory NKR, e.g., an MHC class I molecule, e.g. a classical or non-classical MHC class I molecule, or multimer thereof, e.g., an HLA-E (the ligand for CD94/NKG2 NK cell receptors, see, e.g., Sasaki et al., J. Immunol. 163(11):6301 - 6305 (1999)) or HLA-C molecule or multimer thereof.
  • an HLA-E tetramer e.g., coupled to a toxin (e.g., biotinylated HLA-E molecules linked via streptavidin).
  • NK Cells The invention is based, at least in part, on the observation that the level and activity of NK cells can be used to evaluate progression and/or aggressiveness of insulitis, and/or risk of TID.
  • a variety of methods can be used to determine the presence and/or level of NK cells. In general, these methods include contacting a biological sample (e.g., blood or a pancreatic biopsy) with an agent that selectively binds to NK cells (e.g., an antibody that selectively binds to an NK cell receptor (NKR)).
  • an agent that selectively binds to NK cells e.g., an antibody that selectively binds to an NK cell receptor (NKR)
  • Exemplary antibodies useful to detect NK cells include antibodies to NK1.1, CD56, CD57, CD16, CD161, CD94, and combinations thereof.
  • NK cells are CD3-; therefore, non-reactivity to CD3 antibody can be used in combination with one or more of the above NK markers to detect NK cells.
  • Such antibodies are commercially available. Methods of making and using polyclonal and monoclonal antibodies are described, e.g., in Harlow et al., Using Antibodies: A Laboratorv Manual: Portable Protocol I. Cold Spring Harbor Laboratorv (December 1, 1998).
  • modified antibodies and antibody fragments e.g., chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof, e.g., Fab', Fab, F(ab')2 fragments); or biosynthetic antibodies (e.g., single chain antibodies, single domain antibodies (DABs), Fv, single chain Fv (scFv), and the like), are known in the art and can be found, e.g., in Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Springer Verlag (December 15, 2000; 1st edition).
  • the antibody bears a detectable label.
  • labeled with regard to a probe or antibody, includes direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody, e.g., by reactivity with a detectable substance.
  • the antibody can be labeled with, e.g., a fluorescent tag, e.g., GFP, a radioactive tag, a myc tag, or a HIS tag.
  • a number of detection methods known in the art and described herein can be used to detect NK cells in a biological sample, e.g., blood or a pancreatic biopsy, in vitro as well as in vivo.
  • In vitro techniques for detection include flow cytometry, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.
  • In vivo techniques for detection include introducing into a subject a labeled antibody.
  • an antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the sample is labeled, e.g., biotinylated and then contacted with an antibody, e.g., an antibody positioned on an antibody array.
  • NK cells in the sample can be detected, e.g., using avidin coupled to a fluorescent label.
  • the presence and/or level of NK cells can also be evaluated by contacting a biological sample cells with a compound or an agent capable of detecting the nucleic acid, e.g., mRNA, that encodes an NK cell-specific marker or expressed gene, such that the presence of the nucleic acid is detected.
  • a compound or an agent capable of detecting the nucleic acid e.g., mRNA
  • the level of mRNA e.g., in a cell or sample, can be determined both by in situ and by in vitro methods, and can be used to detect and quantify NK cells in a sample.
  • Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • nucleic acid molecule e.g., a probe
  • the nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA expressed by NK cells.
  • the probe can be disposed on an address of an a ⁇ ay, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.
  • mRNA or cDNA is immobilized on a surface and contacted with one or more probes, for example by running isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probes are immobilized on a surface and the mRNA or cDNA is contacted with the probes, for example, in a two-dimensional gene chip array described below.
  • a skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the gene.
  • the level of mRNA in a sample that is encoded by a gene can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis, U.S.
  • Patent No. 4,683,202 ligase chain reaction (Barany, Proc. Natl. Acad. Sci. U.S.A. 88: 189-193 (1991)), self sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. U.S.A. 87: 1874-1878 (1990)), transcriptional amplification system (Kwoh et al., Proc. Natl. Acad. Sci. U.S.A. 86: 1 173- 1 177 (1989)), Q-Beta Replicase (Lizardi et al., Bio/Technology 6: 1197 (1988)), rolling circle replication (Lizardi et al., U.S.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to regions of a gene (e.g., to the plus and minus strands, respectively, or vice-versa) and can be used to amplify a region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length.
  • a cell or tissue can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the gene being analyzed.
  • the methods include contacting a control sample, e.g., cells not from the pancreas, e.g., cells from spleen, thymus, skeletal muscle, liver, colon, brain, kidney, lung, stomach, testis, or colon, with a compound or agent capable of detecting mRNA, or genomic DNA, and comparing the presence of the mRNA or genomic DNA in the control sample with the presence of NK mRNA in the test sample.
  • serial analysis of gene expression as described in U.S. Patent No. 5,695,937, is used to detect transcript levels of NK markers.
  • An agent that blocks NK cells be administered systemically or locally.
  • Intravenous, subcutaneous or intramuscular administration with an agent described herein, e.g., an antibody, is one route of administration.
  • Local injection or implantation of a controlled- release device, e.g., into or near the pancreas, can also be used.
  • a compound or agent can be administered via the oral route or the parenteral route, including subcutaneously, intraperitoneally, intramuscularly, intravenously or other route.
  • a compound or agent can be administered by injection or implantation.
  • a cell can be contacted extracellularly or intracellularly, e.g., by microinjection or transfection.
  • the compound or agent can be applied and removed immediately, applied and not removed, and/or repeatedly applied with constant, increasing or decreasing frequency and/or at increasing or decreasing doses or concentrations. More than one route of administration can be used simultaneously, e.g., parenteral in association with oral administration.
  • parenteral dosage forms include aqueous solutions of the active agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically acceptable excipient.
  • Solubilizing agents such as cyclodextrins, or other solubilizing agents known to those in the art, can be utilized as pharmaceutical excipients.
  • Intracellular administration can also be achieved using, e.g., liposomes or other cross- membrane delivery systems as are known in the art.
  • compositions can also be formulated into dosage forms for other routes of administration utilizing conventional methods.
  • a pharmaceutical composition can be formulated, for example, in dosage forms for oral administration as a powder or granule, or in a capsule, a tablet (each including timed release and sustained release formulations), or a gel seal, with optional pharmaceutical carriers suitable for preparing solid compositions, such as vehicles (e.g., starch, glucose, fruit sugar, sucrose, gelatin and the like), lubricants (e.g., magnesium stearate), disintegrators (e.g., starch and crystalline cellulose), and binders (e.g., lactose, mannitol, starch and gum arabic).
  • vehicles e.g., starch, glucose, fruit sugar, sucrose, gelatin and the like
  • lubricants e.g., magnesium stearate
  • disintegrators e.g., starch and crystalline cellulose
  • binders e.g., lactose, mann
  • solvents e.g., distilled water for injection
  • stabilizers e.g., sodium edetate
  • isotonizing agents e.g., sodium chloride, glycerine and mannitol
  • pH adjusting agents e.g., hydrochloric acid, citric acid and sodium hydroxide
  • suspending agents e.g., methyl cellulose
  • Pharmacokinetic Properties and Therapeutic Activity Modifications can be made to an agent described herein that result in pharmacokinetic properties of the protein which are desirable for use in protein therapy. For example, such modifications can result in longer circulatory half-life, an increase in cellular uptake, improved distribution to targeted tissues, a decrease in clearance and/or a decrease of immunogenicity.
  • a therapeutic protein described herein e.g., an anti-NKR antibody or ligand
  • Expression system For agents comprising recombinant proteins, the choice of expression system can influence pharmacokinetic characteristics.
  • a protein or other agent can be chemically altered to enhance the pharmacokinetic properties while maintaining activity.
  • the agent can be covalently linked to a variety of moieties, altering the molecular size and charge of the protein and consequently its pharmacokinetic characteristics.
  • the moieties are preferably non-toxic and biocompatible.
  • poly-ethylene glycol (PEG) can be covalently attached to the protein or agent (PEGylation).
  • PEG is a class of polymers comprised of repeating ethylene oxide subunits with terminal hydroxyl groups.
  • a variety of PEG molecules are known and/or commercially available (See, e.g., Sigma-Aldrich catalog).
  • PEG molecules are available in various lengths, molecular weights, and substitution patterns, and may be linear or branched.
  • PEG is attached via an activated terminal hydroxyl group; preferably, the hydroxyl group is activated as an ester, carbonate, aldehyde or tresylate.
  • the activated hydroxyl reacts with nucleophilic groups on the protein, forming a linkage between the protein and PEG.
  • the nucleophilic group is the amino group of a lysine or the N-terminus of the protein.
  • One or multiple chains of PEG may be attached.
  • the choice of site(s) and functionality of the linkage of PEGylation and the choice of PEG molecule can be optimized to achieve the desired pharmacokinetic properties.
  • PEGylation can increase stability, decrease immunogenicity by steric masking of epitopes, and improve half-life by decreasing glomerular filtration.
  • PEGylation See, e.g., PolyCethylene glycol: Chemistry and Biological Applications, Harris and Zalipsky, eds., ACS Symposium Series, No. 680, 1997; Harris et al., Clinical Pharmacokinetics 40:7, 485-563 (2001)).
  • therapeutic proteins See, e.g., PolyCethylene glycol: Chemistry and Biological Applications, Harris and Zalipsky, eds., ACS Symposium Series, No. 680, 1997; Harris et al., Clinical Pharmacokinetics 40:7, 485-563 (2001)).
  • PEG constructs include Adagen (PEG-ADA) and Oncospar " (Pegylated asparaginase).
  • the protein or agent can be similarly linked to oxidized dextrans via an amino group. (See Sheffield, Current Drug Targets — Cardiovas. & Haemat. Dis. 1(1): 1 -22 (2001)).
  • conjugation of arginine oligomers to cyclosporin A facilitates topical delivery (Rothbard et al., Nat. Med. 6(1 1):1253-1257 (2000)).
  • a therapeutic protein or agent can be chemically linked to another protein.
  • the therapeutic protein can be cross-linked to a carrier protein to form a larger molecular weight complex with longer circulatory half-life and improved cellular uptake.
  • the carrier protein can be a serum protein, such as albumin.
  • the therapeutic protein can be attached to one or more albumin molecules via a bifunctional cross-linking reagent.
  • the cross-linking reagent may be homo- or heterofunctional.
  • the therapeutic protein can cross-link with itself to form a homodimer, trimer, or higher analog. Again, either heterobifunctional or homobifunctional cross-linking reagents can be used to form the dimers or trimers. (See, e.g., Stykowski et al., Proc. Natl. Acad. Sci.
  • the formulation of the agent can include a carrier.
  • the carrier can be a colloidal system.
  • the colloidal system can be liposome, a phospholipid bilayer vehicle.
  • the agent e.g., a protein
  • the agent is encapsulated in a liposome while maintaining structural and functional integrity.
  • there are a variety of methods to prepare liposomes See Lichtenberg et al., Methods Biochem. Anal. 33:337-462 (1988); Liposome Technology.
  • Liposomes can be prepared from an assortment of phospholipids varying in size and substitution, and may also contain additional components with low toxicity, such as cholesterol.
  • the liposome can be formulated and isolated in a variety of shapes and sizes.
  • moieties can be attached to the surface of the liposome to further enhance the pharmacokinetic properties of the carrier.
  • the moieties can be attached to phospholipid or cholesterol molecules, and the percentage of the moiety incorporated on the surface may be adjusted for optimal liposome stability and pharmacokinetic characteristics.
  • One embodiment comprises a liposome with poly-ethylene glycol (PEG) added to the surface.
  • PEG poly-ethylene glycol
  • Liposomal formulations can delay clearance and increase cellular uptake, e.g., of agents that act intracellularly. (See Reddy, Annals of Pharmacotherapy, 34(7/8):915-923 (2000)).
  • the canier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix.
  • the therapeutic protein can be embedded in the polymer matrix while maintaining structural and functional integrity.
  • the polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly(I-hydroxy) acids.
  • polymers examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
  • the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA).
  • PVA poly-lactic acid
  • PGLA copoly lactic/glycolic acid
  • the polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Annals of Pharmacotherapy, 34(7/8): 915-923 (2000)).
  • a polymer formulation for human growth hormone (hGH) has been used in clinical trials.
  • An agent comprising a nucleic acid as described herein, e.g., an antisense nucleic acid, or a polypeptide-encoding nucleic acid can be incorporated into a gene construct.
  • Expression constructs of such components may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the component gene to cells in vivo.
  • Approaches include insertion of a selected sequence in a viral vector, e.g., recombinant retrovirus, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus- 1 , or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or calcium phosphate precipitation carried out in vitro or in vivo.
  • a viral vector containing a selected nucleic acid sequence, e.g., a cDNA or portion thereof. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
  • molecules encoded within the viral vector are typically expressed efficiently in cells which have taken up viral vector nucleic acid.
  • Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • a replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel et al.
  • retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include *Crip, *Cre, *2 and *Am.
  • Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis et al., Science 230: 1395-1398 (1985); Danos and Mulligan, Proc. Natl. Acad. Sci. U.S.A.
  • Another viral nucleic acid delivery system useful in the present invention utilizes adenovirus-derived vectors.
  • the genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434 (1991); and Rosenfeld et al., Cell 68: 143-155 (1992).
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are known to those skilled in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992), supra).
  • the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).
  • Yet another viral vector system useful for delivery of the subject gene is the adeno- associated virus (AAV).
  • AAV adeno- associated virus
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • another virus such as an adenovirus or a herpes virus
  • It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al, J.
  • AAV vector such as that described in Tratschin et al, Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al, Proc. Natl. Acad. Sci. U.S.A. 81 :6466-6470 (1984); Tratschin et al, Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al, Mol.
  • non-viral methods can also be employed to cause expression of an nucleic acid agent described herein (e.g., a NK cell marker polypeptide encoding nucleic acid) in the tissue of a subject.
  • an nucleic acid agent described herein e.g., a NK cell marker polypeptide encoding nucleic acid
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non- viral nucleic acid delivery systems of the present invention rely on endocytic pathways for the uptake of the subject gene by the targeted cell.
  • exemplary nucleic acid delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
  • Other embodiments include plasmid injection systems such as are described in Meuli et al, J. Invest. Dermatol. 116(1): 131-135 (2001); Cohen et al. Gene Ther. 7(22): 1896-1905 (2000); or Tarn et al. Gene Ther. 7(21): 1867-1874 (2000).
  • a nucleic acid as described herein can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. No Shinkei Geka 20:547-551 (1992); PCT publication WO 91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • the nucleic acid delivery systems described herein can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g. Chen et al, PNAS 91 :3054-3057 (1994)).
  • pharmaceutical preparations can consist essentially of nucleic acid in an acceptable diluent, or can comprise a slow release matrix in which the nucleic acid is imbedded.
  • the diagnostic assays described herein generally involve evaluating NK cells in the subject, e.g., in a pancreatic tissue, or in the blood.
  • Various art-recognized methods are available for evaluating the presence, numbers, and/or activity of NK cells or specific components thereof.
  • the method can include evaluating either the level of an NKR and/or an activity (e.g., lytic activity) of an NK cell.
  • Techniques for detection of NK cells re known in the art and include, inter alia: antibody based assays such as enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.
  • the level in the subject is compared to the level and/or activity in a control, e.g., the level and/or activity in a tissue from a non-disease subject.
  • Another method of evaluating NK cells in a subject is to determine the presence of an NK specific marker, or a gene that encodes an NK specific marker. The method includes one or more of the following: detecting, in a sample from the subject, the presence of a gene encoding an NK cell marker; detecting, in a sample from the subject, the expression of the gene, at the protein level, e.g., detecting a level of an NK cell marker protein.
  • the method includes contacting a sample from the subject with an antibody to an NK cell marker, or a nucleic acid which hybridizes specifically with the gene.
  • the level and/or presence of the NK specific marker gene or protein can then be compared to a reference, e.g., a control from an individual known to have diabetes or to be at risk for developing diabetes, e.g., a positive control, or a control from a normal, healthy individual, e.g., a negative control.
  • the level and/or presence is compared to a series of references that represent increasing severity, risk, risk of progression, or other clinical parameter.
  • the presence, level, or absence of an NK cell marker (protein or nucleic acid) in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes an NK cell marker such that the presence of the protein or nucleic acid is detected in the biological sample.
  • a biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject, e.g., urine.
  • Preferred biological samples are serum or urine.
  • the level of expression of an NK cell marker can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the an NK cell marker gene; measuring the amount of protein encoded by an NK cell marker; or measuring the activity of the protein encoded by the gene.
  • the level of mRNA corresponding to an NK cell marker in a cell can be determined both by in situ and by in vitro formats. Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • nucleic acid molecule that can hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA or genomic DNA of an NK cell marker.
  • the probe can be disposed on an address of an anay, e.g., an anay described below. Other suitable probes for use in the diagnostic assays are described herein.
  • mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below.
  • a skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the gene.
  • the level of mRNA in a sample that is encoded by a gene can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. U.S.A. 88: 189-193 (1991)), self sustained sequence replication (Guatelli et al, Proc. Natl. Acad. Sci. U.S.A. 87: 1874-1878 (1990)), transcriptional amplification system (Kwoh et al, Proc. Natl. Acad. Sci. U.S.A.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the gene being analyzed.
  • the methods further contacting a control sample with a compound or agent capable of detecting mRNA, or genomic DNA, and comparing the presence of the mRNA or genomic DNA in the control sample with the presence of an NK cell marker mRNA or genomic DNA in the test sample.
  • serial analysis of gene expression as described in U.S. Patent No. 5,695,937, is used to detect transcript levels of an NK cell marker.
  • a variety of methods can be used to determine the level of an NK cell marker. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • An intact antibody, or a fragment thereof e.g., Fab or F(ab')2
  • the term "labeled,” with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.
  • the detection methods can be used to detect an NK cell marker, e.g., in a biological sample in vitro as well as in vivo.
  • In vitro techniques for detection include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.
  • In vivo techniques for detection of include introducing into a subject a labeled antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an antibody positioned on an antibody array.
  • the sample can be detected, e.g., with avidin coupled to a fluorescent label.
  • the methods further include contacting the control sample with a compound or agent capable of detecting an NK cell marker, and comparing the presence of an NK cell marker in the control sample with the presence of the protein in the test sample.
  • the invention also includes kits for detecting the presence of an NK cell marker in a biological sample.
  • the kit can include a compound or agent capable of detecting an NK cell marker protein (e.g., an antibody) or mRNA (e.g., a nucleic acid probe); and a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to evaluate a subject, e.g., for risk or predisposition to a pigmentation related disorder.
  • the diagnostic methods described herein can identify subjects having, or at risk of progressing to, Type I diabetes.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent to treat Type I diabetes.
  • Example 1 Gene Expression Profiles at the Onset of Respectful and Aggressive Insulitis This example shows that aggressive pancreatic infiltrates have an increased frequency of NK cells. An islet infiltrate can follow either of two courses: respectful or aggressive. To elucidate the elements underlying this dichotomous behavior, a microarray analysis of gene expression was performed on early-infiltrating cells in young BDC2.5 TCR tg mice on the NOD and B6.H-2g7 genetic backgrounds, which present with innocuous and destructive insulitis, respectively (Gonzalez et al. Immunity 7:873-883 (1997)) (Fig 1A).
  • the autoimmune T cells themselves were sorted as a CD4+ population expressing high levels of the BDC2.5 clonotype.
  • a bulk CD45+ population was isolated, which includes a variety of hematopoietic cell-types that enter the islets along with the T cells (eg B cells, monocytes, NK cells).
  • RNA was prepared from the sorted populations, amplified using a technique that permits the use of very small cell numbers while still preserving data validity, and converted to labeled probe for hybridization to Affymetrix Mu74Av2 chips. Two to three independent experiments were performed for each cell population.
  • the raw data were processed with the RMA algorithm for probe-level normalization (Irizarry et al, Biostatistics 4:249-264 (2003)), and composite expression values were calculated for each of the genes on the chip, averaging without outlier elimination using custom-developed S+ scripts for this and subsequent analyses.
  • a gene-wise p-value was derived by Welch's t-test.
  • a conservative false-positive rate was estimated from a randomized data-set generated by shuffling expression values between categories.
  • Several of the key gene expression differentials were validated by RT-PCR and flow cytometry (see, e.g., Figs. 2 and 5).
  • NK receptors NK receptors
  • McMahon and Raulet Curr. Opin. Immunol. 13:465-470 (2001)
  • Fig. 1C the microarray expression values of the NK genes in the CD45+ cells isolated from pancreatic infiltrates and parallel values generated from sorted splenic NK cells
  • Example 2 The Fraction of NK Cells Correlates with Aggressivity This example shows that the proportion of NK cells in a pancreatic infiltrate positively correlates with aggressivity of the infiltrate.
  • the microarray data was validated by measuring the proportion of NK cells in the insulitic lesions of BDC2.5/NOD and BDC2.5/B6.H-2g7 mice by flow cytometry. NK cells were identified as CD49b(DX5)+CD3- lymphocytes. There were significantly more NK cells in the infiltrates of BDC2.5/B6.H-2g7 than of BDC2.5/NOD animals (p ⁇ 0.03, Fig 2A, left).
  • NK cells were recruited to the pancreas only because of a pre-existing inflammatory environment, and are merely a marker of an aggressive attack.
  • NK cells were a significant component of the infiltrate as soon as it could be detected, from day 14 onwards. If anything, the ratio of NK to T cells decreased slightly after this point. This indicated that NK cells are very early players in the insulitis of BDC2.5 mice, not late-comers to an already established inflammatory lesion, and thus can be used to detect the early stages of disease.
  • Example 3 NK Cells are Important for Islet Cell Destruction This example shows that NK cell activity is involved in progression to diabetes.
  • NK cell activity is involved in progression to diabetes.
  • An anti-Asialo GMl polyclonal antibody was first employed, a reagent classically used to deplete NK cells, administered at the same time as the anti-CTLA-4 mAb. This antibody led to a significant decrease (p ⁇ 0.03, Cox proportional hazards) in diabetes incidence versus mice treated with a polyclonal rabbit IgG (Fig. 4A).
  • the NKC haplotype of NOD mice carries an inactive Nkrpcl gene and so their NK cells lack the NK1.1 epitope.
  • mice All of the intercross mice were treated with an NK-depleting anti-NKl .1 mAb PK136 administered at the same time as the diabetes-inducing anti-CTLA-4 mAb, monitored development of diabetes, and later determined which mice carried the relevant Nkrpl-c gene. A clear reduction in the induction of diabetes was seen in those animals that expressed the NK1.1 epitope compared to NKl .1 -negative controls (p ⁇ 0.03) (Fig. 4B). The pancreas of animals that proved resistant to diabetes was examined by histology and typical pictures of respectful insulitis were seen.
  • the microarray analysis was extended by examining gene expression profiles of purified splenic NK cells from juvenile NOD and B6.H-2g7 mice.
  • a comparison of these datasets with those derived from CD45+ participants in the pancreatic infiltrate was expected to reveal which of the differences were intrinsic to NK cells, and thus possibly causative, and which might represent differential expression provoked by the aggressive insulitis.
  • NK-cell-specific genes were analyzed to determine whether the differences were a primary manifestation of the genes themselves or a secondary effect specified by genetic loci located elsewhere.
  • MHC is of the same haplotype in all three lines, an indirect influence of MHC ligands could already be ruled out.
  • Ly49 A/P The surface staining for Ly49 A/P confirmed the microarray and RT-PCR data (Fig. 6 and see legend), Ly49P being a rare gene upregulated in NOD relative to B6.H-2 g7 NK cells.
  • the staining patterns also indicated that the polymorphism in NK receptor expression co ⁇ elated with the NKC, rather than with the rest of the genome.
  • Expression of 2B4 was an exception in that staining levels on NOD and NOD.NK1.1 splenic cells were concordant, which is expected because 2B4 is the only gene in the panel encoded outside the NKC.
  • the NOD NKC encodes a set of NK receptor genes with expression patterns very different from those of the B6.H-2g7 NKC, a variation far more extensive than previously recognized. This variability is on par with the extensive polymorphism of the human NKC (McQueen and Parham, Cun. Opin. Immunol. 14:615-621 (2002)). It seems very unlikely that the outcome of the blocking experiments reflected effects on other NKl .1 -positive cells, in particular CD4 + NK-T cells. This T cell subset has been associated with diabetes protection rather than exacerbation in both the NOD and BDC2.5 models of diabetes (Carnaud et al, J. Immunol.
  • Sorted cells were washed and lysed in 500ml Trizol (Invitrogen) for RNA isolation.
  • Trizol Invitrogen
  • two rounds of amplification were performed using Message Amp aRNA kit (Ambion); during the second in vitro transcription, biotinylated ribonucleotides were incorporated using Bioanay Highyield T7 labeling kit (Enzo Life Sciences).
  • 10 to 14 mg of biotinylated amplified RNA was fragmented and hybridized to Affymetrix chips Mu74Av2. Quantitative real-time PCR assays were performed using SYBR Green (Applied Biosystems; see Supplemental Experimental Details). Microarray data processing.
  • Fig. 1 Cell-specific gene lists (Fig. 1) are shown as Supplemental Material. Diabetes induction via anti-CTLA-4 mAb treatment. Purified anti-CTLA4 mAb (clone UC10-4F10-1 1) was purchased from Bio Express, Inc. (West Riverside, NH). Diabetes was induced in BDC2.5/NOD mice by injection of 150 ⁇ g of antibody on days 10, 12 and 14, and monitoring for hyperglycemia from 16 to 30 days of age (daily urine glucose, positive reads confirmed by blood glucose > 250 mg/dl). NK cell depletion.

Abstract

L'invention concerne des méthodes destinées à diagnostiquer ou traiter le diabète de type 1 et/ou à en prévenir l'apparition.
PCT/US2005/014395 2004-04-28 2005-04-27 Methodes de traitement du diabete WO2005115517A2 (fr)

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WO2006070286A2 (fr) 2004-12-28 2006-07-06 Innate Pharma S.A. Anticorps monoclonaux contre le nkg2a
EP2476705A1 (fr) 2004-12-28 2012-07-18 Innate Pharma Anticorps monoclonaux contre NKG2A
EP3026063A1 (fr) 2004-12-28 2016-06-01 Innate Pharma S.A. Anticorps monoclonaux contre nkg2a
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US10526409B2 (en) 2006-12-21 2020-01-07 Novo Nordisk A/S Antibodies against human NKG2D and uses thereof
US7879985B2 (en) 2007-12-14 2011-02-01 Novo Nordisk A/S Antibodies against human NKG2D and uses thereof
EP2769993A1 (fr) 2007-12-14 2014-08-27 Novo Nordisk A/S Anticorps contre NKG2D humain et usages correspondants

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