WO1997003704A2 - In-vivo-diabetes-test - Google Patents
In-vivo-diabetes-test Download PDFInfo
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- WO1997003704A2 WO1997003704A2 PCT/EP1996/003192 EP9603192W WO9703704A2 WO 1997003704 A2 WO1997003704 A2 WO 1997003704A2 EP 9603192 W EP9603192 W EP 9603192W WO 9703704 A2 WO9703704 A2 WO 9703704A2
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- amino acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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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
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- 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
- the present invention relates to the use of autoreactive substances for the production of an agent for the diagnosis of cellular mediated diseases or a predisposition to cellular mediated diseases which influence the immune system.
- IDDM The environmental factors involved in the formation of IDDM are probably exogenous peptide sequences which act as immunogens.
- viral antigens which have partial homologies to the body's own structures, are discussed.
- antigens such as bovine serum albumin, which are ingested through food can induce an immune response which is due to can start an auto-aggressive process from homologies to the body's own structures.
- pancreatic ⁇ cells The progressive destruction of the pancreatic ⁇ cells by cytotoxic lymphocytes is typical of the course of the disease in IDDM. This process started long before a recognizable disturbance of the glucose metabolism. If there is a noticeable manifestation of diabetes, over 80% of the ß cells have already been destroyed. It would therefore be extremely important to detect these autoaggressive T cells at an early stage in those at risk in order to be able to provide the affected individuals with causal therapy.
- T lymphocytes directed against GAD have already been detected by several research groups (Harrison et al., J. Clin. Invest. 89 (1992), 1161; Honeyman et al., J. Exp. Med. 177 (1993), 535 ). The T lymphocytes found by these groups reacted with a peptide fragment of the GAD 67 kd molecule consisting of amino acids 208 to 404.
- EP-A-0 519469 discloses autoimmune polypeptides from the human GAD 65 kd molecule. These polypeptides have the amino sequence:
- X is an optional sequence selected from 1 to 10 amino acids and Z is an optional sequence selected from 1 to 8 amino acids.
- European patent application No. 95 100 764.0 also proposes autoreactive peptide sequences from human GAD 65 and a pharmaceutical composition containing these peptides.
- the use of this pharmaceutical composition for the production of an agent for the diagnosis of diseases or a predisposition for diseases which influence the immune system, or of tumor diseases or a predisposition for tumor diseases is further described.
- One object on which the present invention was based was to provide a diagnostic method for cellularly mediated diseases of the immune system, in particular autoimmune diseases, which on the one hand is associated with the least possible inconvenience for the patient and on the other hand enables rapid and reliable diagnosis.
- This object is achieved by the use of auto-active substances for the production of an agent for the diagnosis of cell-mediated diseases or a predisposition for cell-mediated diseases, the agent being applied intradermally or intracutaneously and the diagnosis based on the occurrence or absence of one positive reaction at the application site.
- the diagnostic method of the invention differs from methods currently on the market which are used for a general test of cell-mediated immunity in that these tests use foreign antigens to which the patient has already been exposed, e.g. purified protein derivative (PPD), tetanus toxoid, candida etc. These tests are used to determine the general condition of the test subject's immune system. A diagnosis of cellular mediated diseases of the immune system is not made.
- PPD purified protein derivative
- tetanus toxoid candida etc.
- the antigens are not administered intradermally.
- a positive reaction that is not T cell mediated can be detected within four hours and is characterized by the appearance of a superficial swelling, not by the appearance of a knot.
- a method is provided with which the occurrence of a cell-mediated immune reaction against autoreactive substances can be detected in vivo.
- This cell-mediated immune response indicates the presence of specific diseases of the immune system, in particular autoimmune diseases, or a predisposition to such diseases.
- the cell-mediated immune reaction can also indicate the presence of tumor diseases or a predisposition therefor.
- the autoreactive substances or autoantigens suitable for the method according to the invention are natural, synthetic or recombinant substances such as nucleic acids, lipids, saccharides, polypeptides, proteins, peptides and complexes thereof, e.g. Complexes of peptides with polypeptides, e.g. MHC molecules or peptide binding derivatives.
- Also suitable as autoreactive substances are specific immunogenic epitopes, fragments, analogs, mimetics or derivatives, which e.g. are generated by chemical modification, and mixtures of the above substances.
- the autoreactive substances can be used as such or in a carrier-bound form, e.g. in the form of lipopeptides.
- the autoreactive substances can be used alone or together with other substances, e.g. accessory stimulating components or adjuvants are administered.
- the autoantigens can come from natural sources, by recombinant DNA technology or by synthetic methods, e.g. by peptide solid phase synthesis.
- autoreactive substances in the sense of the body's own antigens, those autoreactive substances are also used which are foreign antigens but are immunologically related to autoantigens.
- examples of such external anti genes are peptides that have homology to autoreactive peptide sequences from the body's own proteins.
- Specific examples include a peptide from bovine ⁇ -casein A with the sequence LVYPGPIPN (AS 60-68), which has a homology region to the body's own glucose transporter GLUT-2 in the region of the sequence OIGPGPIPW (AS 412-420), and a peptide from the Coxsackie Virus protein P2-C with the sequence KVKILPEVKEKHE (AS 33-45), which has homology to glutamate decarboxylase in the region of the sequence RFKMFPEVKEKGM (AS 155-167).
- the method according to the invention comprises the intradermal administration of the autoantigens in question and the determination of the occurrence or absence of a local positive cellular reaction at the application site.
- This positive reaction is determined at a point in time which is characteristic of a T-cell-mediated immune response, preferably at a point in time of at least 24 hours after the application, particularly preferably in a period of 24 hours to 1 week after the application and most preferably in one Period of 40 to 80 h after application.
- the positive reaction preferably comprises the appearance of a knot at the application site and can be qualitatively determined by visual observation or / and by scanning, but also quantitatively, e.g. by ultrasound or photographic methods.
- products of a positive reaction for example cytokines, such as ⁇ -interferon, can also be determined, which are released by infiltrating cells, for example leukocytes, at the application site.
- cytokines such as ⁇ -interferon
- the method according to the invention is particularly suitable for the diagnosis of autoimmune diseases or a predisposition for autoimmune diseases.
- autoimmune diseases examples include diabetes mellitus, especially type I diabetes (IDDM), multiple sclerosis, rheumatoid arthritis, Graves' disease, ankylosing spondylitis, acute anterior uveitis, goodpasture syndrome, myasthenia gravis, systemic Lupus erythematosus, pemphigus vulgaris, immune thyroiditis, scleroderma, Crohn's disease, Sjogren's syndrome, Reiter's disease, inflammatory bowel disease or Graves disease.
- IDDM type I diabetes
- multiple sclerosis rheumatoid arthritis
- Graves' disease ankylosing spondylitis
- acute anterior uveitis goodpasture syndrome
- myasthenia gravis systemic Lupus erythematosus
- pemphigus vulgaris immune thyroiditis
- scleroderma scleroderma
- Sjogren's syndrome Reiter's disease
- Reiter's disease
- autoreactive substances used to diagnose IDDM are glutamic acid decarboxylase (GAD) 65 kd or 67 kd, tyrosine phosphatase (38 kd antigen), carboxypeptidase H, insulin, heat shock protein, 38 kd insulin secretory granule protein or Parts of it.
- GAD 65 kd or partial peptide sequences thereof are particularly preferably used as the autoreactive substance.
- autoimmune diseases are multiple sclerosis, where reactive T cells can be determined, for example, against the myelin basic protein or the proteolipid protein, rheumatoid arthritis, where reactive T cells, for example against type II collagen, cytokeratins, dnaJ protein from E. coli and Hsp 65 can be determined, and Graves' disease, where reactive T cells can be determined eg against thyroid peroxidase. at Myasthenia gravis, reactive T cells against the acetylcholine receptor or other relevant autoreactive substances can be determined. In lupus erythematosus there are reactive T cells against HSP 90 and in Graves disease against the TSH receptor.
- T cells that react to tumor antigens.
- T cells against a melanoma-associated antigen MAGE 1 which were isolated from melanoma patients (van der Bruggen et al., Science 254 (1991), 1643-1647).
- MAGE 1 T cells against a melanoma-associated antigen MAGE 1
- T cells can already be detected at a stage in which the tumor cannot yet be detected using conventional methods due to a cell mass that is still too low.
- the detection of specifically reacting T cells could also be used to monitor the course of an anti-tumor vaccination.
- peptides, peptide derivatives or analog binding molecules can also be used as autoreactive substances.
- One or more peptides from GAD, in particular from human GAD 65 kd or peptide derivatives or peptide mimetics derived therefrom are preferably used for the diagnosis of IDDM.
- peptides or peptide derivatives for the diagnosis of IDDM, comprising: (a) the amino acid sequence (I) DVNYAFLHATDLLPACDGER,
- amino acid sequences (I) to (VII) correspond to amino acid residues 86-105 (I), 246-265 (II), 146-165 (III), 166-185 (IV), 176-195 (V), 206-225 (VI) and 556-575 (VII) the human GAD 65 kd.
- the amino acid sequences I-VII are in the sequence listing SEQ ID NO. 1-7 specified.
- the amino acid sequence (VIII) corresponds to the amino acid residues 266-290 of the human GAD 65 kd and the amino acid sequence (IX) corresponds to the amino acid residues 306-325 of the human GAD 65 kd.
- the amino acid sequences shown in Figures 1 and 2 are also partial sequences of the human GAD 65 kd.
- the amino acid sequences of Figs. 1 and 2 are in the sequence listing SEQ ID NO. 8-28 indicated.
- the amino acid sequences (VIII) and (IX) are in the sequence listing SEQ ID NO. 29 and 30 indicated.
- peptides containing the above ranges of human GAD 65 showed a specific reaction with T cell subpopulations isolated from newly discovered Type I diabetics.
- the peptides according to the invention are thus early autoepitopes, the use of which enables very early diagnosis of type I diabetes.
- amino acid sequences (I) to (IX) are partial regions from the 65 kD isoform of human glutamic acid decarboxylase
- amino acid sequences (I) to (IX) were determined by applying T cell lines from the peripheral blood of Type I diabetics and subsequent in vitro stimulation with GAD from porcine brain or recombinant human GAD and testing of these T cell lines in a proliferation assay using synthetic peptide sequences that were derived from the human GAD sequence.
- the peptides can be generated by known synthetic methods using chemical methods or by cloning and expression of a DNA sequence coding for these peptides in a suitable host cell, in particular E. coli, in a genetic engineering manner.
- the minimum length of a peptide according to the invention is determined by its ability to recognize an MHC molecule, to bind with it specifically and to react with the corresponding T cell receptor.
- the maximum length of the GAD-derived and MHC-binding sections in a peptide according to the invention is preferably 100 amino acids, particularly preferably 50 amino acids and most preferably 25 amino acids.
- peptides which show essentially equivalent specificity and / or affinity for binding to MHC molecules, such as the sequences mentioned above, and which preferably by substitution, deletion or insertion of individual amino acid residues or short sections of Amino acid residues from the Above-mentioned amino acid sequences are derived or analog binding alien substances.
- the present invention also relates to peptide variants which do not completely match in their sequence with the abovementioned amino acid sequences, but only have the same or closely related "anchor positions".
- anchor position in this context means an amino acid residue that is essential for binding to an MHC molecule, in particular to an MHC molecule of classes DR3, DR4 or DQ.
- the anchor positions for the DRB10401 binding motif are e.g. in Hammer et al. , Cell 74 (1993), 197-203.
- Such anchor positions are preserved in peptides according to the invention or, if appropriate, replaced by amino acid residues with chemically closely related side chains (e.g. alanine by valine, leucine by isoleucine and vice versa).
- the anchor positions in the peptides according to the invention can be determined in a simple manner by testing variants of the specific peptides specified above for their ability to bind to MHC molecules.
- Peptides according to the invention are characterized in that they show an essentially equivalent specificity and / or affinity for binding to MHC molecules like the aforementioned peptides.
- the peptides derived from these peptides preferably have a sequence homology of at least 30%, particularly preferably of at least 50% and most preferably at least 60% with the starting peptides or partial sequences thereof.
- variants of the specifically stated peptides are the corresponding homologous peptide sections from the human GAD 67, the complete amino acid sequence of which was also described by Bu et al. , supra.
- substantially equivalent specificity and / or affinity for binding to MHC molecules also includes an improved binding specificity or / and affinity compared to the amino acid sequences (I) to (VIII) or the amino acid sequences shown in FIGS. 1 and 2, which is found in particular in shortened peptides which preferably have a length of 8 to 15 amino acids.
- the present invention also includes peptide derivatives.
- This term encompasses peptides in which one or more amino acids have been derivatized by a chemical reaction.
- peptide derivatives according to the invention are, in particular, those molecules in which the backbone and / or reactive amino acid side groups, e.g. free amino groups, free carboxyl groups and / or free hydroxyl groups have been derivatized.
- Specific examples of derivatives of amino groups are sulfonic acid or carboxamides, thiourethane derivatives and ammonium salts, e.g. Hydrochloride.
- Examples of carboxyl group derivatives are salts, esters and amides.
- hydroxyl group derivatives are O-acyl or O-alkyl derivatives.
- peptide derivative according to the present invention also includes those peptides in which one or more amino acids are replaced by naturally occurring or non-naturally occurring amino acid homologs of the 20 "standard” amino acids.
- homologs are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, ⁇ -alanine and 4-aminobutyric acid.
- Suitable peptide derivatives are complexes or covalent connections between peptides and adjuvant components which are administered together with the autoantigen, e.g. Lipids.
- the peptides can be used as lipopeptides.
- the present invention also covers polypeptides in which the MHC-binding peptide section is part of a larger polypeptide unit, the Connection of MHC-binding peptide and the rest of the polypeptide unit preferably has a predetermined breaking point, for example a protease cleavage site.
- the invention also relates to peptide-mimetic substances which show an essentially equivalent specificity and / or affinity for binding to MHC molecules, such as the aforementioned peptides or peptide derivatives.
- Peptide mimetic substances or peptide mimetics are compounds which can replace peptides in their interaction with the MHC molecules and, compared to the native peptides, can have increased metabolic stability, better bioavailability and a longer duration of action.
- Methods for the production of peptide mimetics are described in Giannis and Kolter, Angew. Chem. 105 (1993), 1303-1326, Lee et al. , Bull. Chem. Soc. Jpn. 66 (1993), 2006-2010 and Dorsch et al., Contacts (Darmstadt) (1993) (2), 48-56.
- the production of peptide-mimetic substances according to the invention reference is made to the disclosure of these references.
- an autoreactive substance is a complex which comprises at least one peptide, peptide derivative or peptide mimetic and at least one MHC molecule or a peptide-binding derivative of an MHC molecule.
- this complex is a peptide, peptide derivative or peptide mimetic with a binding constant of preferably at least 10 " 7 1 / mol, particularly preferably in the range of 10 " 8 -IO " 9 1 / mol, to an MHC molecule or
- the peptide, peptide derivative or peptide mimetic can also be covalently coupled to the MHC molecule, for example via a photolinker or as a covalent genetic peptide-MHC fusion.
- Fusion protein preferably contains an HLA-DR beta chain and an autoreactive peptide fused to it genetically, particularly preferred the complex contains an MHC class II molecule or a peptide binding derivative thereof.
- peptide-binding derivative of an MHC molecule encompasses fragments of MHC molecules which have been produced by proteolytic cleavage of native MHC molecules or by recombinant DNA techniques and have essentially retained their peptide-binding properties. This term also means fusion proteins which, in addition to an MHC portion responsible for peptide binding, also contain other polypeptide components.
- the peptide-MHC complexes are preferably produced by associating peptide-free MHC molecules or MHC molecule derivatives with the peptides, peptide derivatives or peptide mimetics according to the invention.
- Peptide-free MHC molecules can be produced, for example, by unfolding native MHC molecules in order to dissociate bound peptides and refolding the empty MHC molecules (see Dornmair and McConnell, Proc. Natl. Acad. Sei. USA 87: 4134-4138 (1990) and WO91 / 14701). Further methods for the production of complexes from peptides and MHC molecules are described in European Patent Application No. 95 100 764.0. Reference is hereby made to this disclosure.
- the invention also relates to the use of a composition comprising an autoreactive substance, for example a protein, polypeptide, peptide, peptide derivative, peptide mimetic or / and a peptide-MHC complex, as the active component optionally in combination with adjuvants or other pharmaceutically customary additives, in a diagnostic method which comprises the intradermal application of the composition.
- the composition can also contain an accessory stimulating component, for example cytokines such as IL-2 and IL-4 or / and the surface antigen B7 (Wyss-Coray et al., Eur. J. Immunol.
- negative controls can be a non-reactive polypeptide such as human serum albumin or just additives, e.g. use the preparation containing the buffer.
- a positive control antigen for example, purified protein derivative (PPD) from the culture medium in which tubercle bacteria are grown (commercially available, for example from Statens Serum Institute, Copenhagen, Denmark) or tetanus toxoid, of which one can be used it is known that they induce strong T cell stimulation in many people.
- PPD purified protein derivative
- the autoreactive substances are applied together with an adjuvant.
- suitable adjuvants are aluminum hydroxide, incomplete Freund's adjuvant, aluminum phosphate and lipids (lipopeptides) suitable for coupling peptides.
- the autoreactive substances can be applied by methods known per se, for example using a normal injection syringe, for example with a size 26G needle.
- the composition containing the autoreactive substance is preferably administered intracutaneously in a volume of 20 .mu.l to 500 .mu.l, preferably 50 .mu.l to 200 .mu.l at a suitable location in the body, which is best suited for the autoantigen presented to the cells of the immune system.
- the application can take place in the forearm (front), for example.
- the concentration of the autoantigen in the composition can vary within wide limits depending on the autoantigens used in each case. Concentrations from 1 to 100 ⁇ g / ml, in particular from 5 to 20 ⁇ g / ml, are preferably used.
- a device which comprises at least one chamber for receiving a preparation with autoreactive substances, one chamber for receiving a preparation for the positive control and one chamber for receiving a preparation for the negative control.
- a device can contain, for example, 2 to 10 chambers for receiving preparations with autoreactive substances. With this device, several autoreactive substances can be tested simultaneously in a simple manner.
- Another object of the present invention is the determination of a specific T cell subpopulation, wherein a sample containing T cells, which preferably comes from a tissue sample from the area of the application site, is brought into contact with an autoreactive substance and the reaction of T - cells with the substance determined.
- a specific reaction of T cells with the autoantigen can be demonstrated, for example, by an increased T cell proliferation, which can be measured by the incorporation of radioactivity.
- the reaction of T cells can also be determined directly by using a labeled autoantigen, for example in complex form with a soluble MHC molecule.
- the autoreactive substance is preferably used with a fluorescent label group coupled thereto.
- the evaluation can be carried out, for example, by FACS analysis, the T cells being in contact with a first fluorescence marker, which is coupled to a T cell-specific antibody, and then with the autoantigen, which is coupled with a second fluorescence marker brought and the presence of double-labeled cells is determined by fluorographic analysis. In this way, a T cell subpopulation is determined, which is characterized by its reactivity with an autoantigen.
- the method can optionally also be a selection for preactivated T cells, for example a selective enrichment of IL-2 receptor positive T cells by incubation with IL-2 or / and by incubation with IL-2 receptor antibody and subsequent separation of the antibody-binding cells, for example by immunomagnetic methods.
- the selection for preactivated cells can only be made after the T cells have come into contact with the autoantigen.
- the ratio of pre-activated autoreactive T cells, i.e. T cells with the IL-2 receptor as surface marker to non-activated autoreactive T cells, i.e. T cells without the IL-2 receptor can be determined.
- Another object of the present invention is the isolation of T cell subpopulations that react with an autoantigen.
- a tissue sample containing T cells which comes from the area of the application site, is brought into contact with an autoantigen
- the T cells reacting with autoantigen are identified and, if necessary, separated from other T cells.
- a selection for preactivated T cells ie T cells with the IL-2 receptor
- the autoantigen for example in the form of a complex with an MHC molecule, can be used in immobilized form on a support, which simplifies the separation of the positively reacting T cell population from other T cells.
- T cell lines can be created from the T cell subpopulations isolated in this way by restimulation. These autoreactive T cell lines can then be used to immunize patients.
- an anti-idiotypic antibody can be used instead of the autoantigens, which mimics the action of the MHC-peptide complex.
- Such antibodies can be readily obtained by using a T cell subpopulation specific to a particular antigen as an immunogen to produce an antibody (e.g. in a mouse) or by first using a first antibody against the autoantigen and then an anti-idiotypic antibody against the first antibody is generated.
- the products of these in vivo activated T cells can also be identified. This can be done by taking a tissue sample (eg fine needle aspirate) and determining the cytokines it contains. Alternatively, the cytokine determination can also be carried out in situ by placing a longer one Patch in which antibodies against various cytokines are immobilized. The detection of the cytokines is carried out according to common methods of a solid phase immunoassay using enzyme substrates which provide an insoluble colored product which can be read either without further help or using a scanner.
- tissue sample eg fine needle aspirate
- the cytokine determination can also be carried out in situ by placing a longer one Patch in which antibodies against various cytokines are immobilized.
- the detection of the cytokines is carried out according to common methods of a solid phase immunoassay using enzyme substrates which provide an insoluble colored product which can be read either without further help or using a scanner.
- the invention relates to a reagent kit for in vivo diagnosis of cell-mediated diseases or a predisposition therefor, comprising:
- compositions for the autoreactive substance and the control substance preferably contain adjuvants and optionally other conventional pharmaceutical carriers, diluents and additives.
- Fig. 1 shows autoreactive amino acid sequences from human GAD 65 kd
- Fig. 2 shows further autoreactive amino acid sequences from human GAD 65 kd
- SEQ ID NO. 1-7 show autoreactive amino acid sequences from human GAD (I) - (VII),
- SEQ ID NO. 8-11 show autoreactive amino acid sequences from human GAD according to FIG. 1,
- SEQ ID NO. 12-28 show autoreactive amino acid sequences from human GAD according to FIG. 2,
- SEQ ID NO. 29 and 30 show autoreactive amino acid sequences from human GAD (VIII) and (IX).
- Peripheral venous blood was collected from eight recently ill IDDM patients and twelve healthy individuals.
- the peripheral blood lymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation and used for in vitro stimulation with:
- PPD purified protein derivative
- HSA human serum albumin
- peripheral blood lymphocytes were in a well of a 96 well round bottom plate for 5 days at 37 ° C and 5% CO 2 in 100 ⁇ l of a culture medium (RPMI 1640 medium, 5% human serum, 2 mmol / l glutamine, 10 U / ml penicillin, 10 ⁇ g / ml streptomycin and 50 nmol / 1 2-mercaptoethanol) in the presence of the above-mentioned antigens or incubated in medium alone.
- a culture medium RPMI 1640 medium, 5% human serum, 2 mmol / l glutamine, 10 U / ml penicillin, 10 ⁇ g / ml streptomycin and 50 nmol / 1 2-mercaptoethanol
- a stimulation index i.e. cpm in the presence of the antigen divided by cpm in the absence of the antigen, i.e. (medium alone).
- the eight IDDM patients from Example 1 were injected with 100 ⁇ l rhGAD 65 kd at a concentration of 10 ⁇ g / ml intradermally into the right forearm.
- the antigen was diluted in physiological saline pH 7.3 and 1% HSA.
- An equal volume (100 ⁇ l) of HSA with a concentration of 10 ⁇ g / ml was injected into the right upper arm as a negative control.
- PPD was used as a positive control antigen in 5 of the 18 IDDM patients.
- HSA was used as a negative control in all patients.
- the formation of a knot (specific increase in derma thickness) at the application site was regarded as a positive reaction.
- the diagnosis was made 48 hours after the application. The results were confirmed and quantified by ultrasound measurements in the area of the application site. No side reactions were observed.
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IL12281696A IL122816A0 (en) | 1995-07-20 | 1996-07-19 | In vivo diabetes test |
AU66582/96A AU6658296A (en) | 1995-07-20 | 1996-07-19 | In vivo diabetes test |
JP9506304A JPH11509538A (ja) | 1995-07-20 | 1996-07-19 | In vivo 糖尿病試験 |
EP96926371A EP0839058A2 (de) | 1995-07-20 | 1996-07-19 | In-vivo-diabetes-test |
NO980252A NO980252D0 (no) | 1995-07-20 | 1998-01-20 | In vivo diabetes-test |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE19526561.0 | 1995-07-20 | ||
DE19526561A DE19526561A1 (de) | 1995-07-20 | 1995-07-20 | In-vivo-Diabetes-Test |
Publications (2)
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WO1997003704A2 true WO1997003704A2 (de) | 1997-02-06 |
WO1997003704A3 WO1997003704A3 (de) | 1997-06-05 |
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ID=7767366
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PCT/EP1996/003192 WO1997003704A2 (de) | 1995-07-20 | 1996-07-19 | In-vivo-diabetes-test |
Country Status (10)
Country | Link |
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EP (1) | EP0839058A2 (de) |
JP (1) | JPH11509538A (de) |
KR (1) | KR19990035757A (de) |
CN (1) | CN1191493A (de) |
AU (1) | AU6658296A (de) |
CA (1) | CA2225145A1 (de) |
DE (1) | DE19526561A1 (de) |
IL (1) | IL122816A0 (de) |
NO (1) | NO980252D0 (de) |
WO (1) | WO1997003704A2 (de) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6750201B1 (en) * | 1997-10-17 | 2004-06-15 | The Trustees Of The University Of Pennsylvania | Compositions and methods for promoting internalization and degradation of urokinase-type plasminogen activator |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997036618A1 (fr) * | 1996-04-01 | 1997-10-09 | Chugai Seiyaku Kabushiki Kaisha | Composition diagnostique pour maladies s'accompagnant de reactions auto-immunes declenchees par la decarboxylase d'acide 65k-glutamique |
KR100681339B1 (ko) * | 2004-12-23 | 2007-02-15 | 최성환 | 연탄보일러 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE1805257A1 (de) * | 1967-10-25 | 1969-05-14 | Bard Hamilton Company Inc | Diagnostische Zubereitung und Verfahren zu deren Herstellung |
WO1995007992A2 (en) * | 1993-09-17 | 1995-03-23 | The Regents Of The University Of California | Cloned glutamic acid decarboxylase |
EP0665289A2 (de) * | 1994-01-20 | 1995-08-02 | Roche Diagnostics GmbH | Autoimmunreaktion hervorrufende GAD65 Peptide |
WO1997004085A1 (de) * | 1995-07-14 | 1997-02-06 | Boehringer Mannheim Gmbh | Autoreaktive peptide aus der humanen glutaminsäure-decarboxylase (gad) |
-
1995
- 1995-07-20 DE DE19526561A patent/DE19526561A1/de not_active Withdrawn
-
1996
- 1996-07-19 EP EP96926371A patent/EP0839058A2/de not_active Withdrawn
- 1996-07-19 CN CN96195689A patent/CN1191493A/zh active Pending
- 1996-07-19 WO PCT/EP1996/003192 patent/WO1997003704A2/de not_active Application Discontinuation
- 1996-07-19 JP JP9506304A patent/JPH11509538A/ja active Pending
- 1996-07-19 KR KR1019980700413A patent/KR19990035757A/ko not_active Application Discontinuation
- 1996-07-19 IL IL12281696A patent/IL122816A0/xx unknown
- 1996-07-19 CA CA002225145A patent/CA2225145A1/en not_active Abandoned
- 1996-07-19 AU AU66582/96A patent/AU6658296A/en not_active Abandoned
-
1998
- 1998-01-20 NO NO980252A patent/NO980252D0/no not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE1805257A1 (de) * | 1967-10-25 | 1969-05-14 | Bard Hamilton Company Inc | Diagnostische Zubereitung und Verfahren zu deren Herstellung |
WO1995007992A2 (en) * | 1993-09-17 | 1995-03-23 | The Regents Of The University Of California | Cloned glutamic acid decarboxylase |
EP0665289A2 (de) * | 1994-01-20 | 1995-08-02 | Roche Diagnostics GmbH | Autoimmunreaktion hervorrufende GAD65 Peptide |
WO1997004085A1 (de) * | 1995-07-14 | 1997-02-06 | Boehringer Mannheim Gmbh | Autoreaktive peptide aus der humanen glutaminsäure-decarboxylase (gad) |
Non-Patent Citations (4)
Title |
---|
CHEMICAL ABSTRACTS, vol. 118, no. 23, 7.Juni 1993 Columbus, Ohio, US; abstract no. 231816, MAUCH, LUDWIG ET AL: "Characterization of a linear epitope within the human pancreatic 64-kDa glutamic acid decarboxylase and its autoimmune recognition by sera from insulin-dependent diabetes mellitus patients" XP002029840 & EUR. J. BIOCHEM. (1993), 212(2), 597-603 CODEN: EJBCAI;ISSN: 0014-2956, 1993, * |
DATABASE EMBASE ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL AN=7900706, XP002029570 & ACTA BIOQUIM. CLIN. LATINOAM., Bd. 12, Nr. 4, 1978, Seiten 339-344, ZANOTTI-CAVAZZONI V. ET AL.: "CORRELATION OF SKIN TESTS WITH AUTO-ANTIGEN AND SKIN TESTS WITH DNCB AND TUBERCULIN." * |
DATABASE MEDLINE US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US AN=6702734, XP002029569 & ACTA ALLERGOLICA, Bd. 22, Nr. 1, 1967, Seiten 57-60, PETRANYI J. ET AL.: "INTRACUTANEOUS FLUORESCENT ANTIGLOBULIN TEST IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS." * |
PROC. NATL. ACAD. SCI. U. S. A. (1990), 87(15), 5783-7 CODEN: PNASA6;ISSN: 0027-8424, 1990, XP002029568 MCELRATH, M. JULIANA ET AL: "Cutaneous response to recombinant interleukin 2 in human immunodeficiency virus 1-seropositive individuals" * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6750201B1 (en) * | 1997-10-17 | 2004-06-15 | The Trustees Of The University Of Pennsylvania | Compositions and methods for promoting internalization and degradation of urokinase-type plasminogen activator |
US7247611B2 (en) | 1997-10-17 | 2007-07-24 | The Trustees Of The University Of Pennsylvania | Compositions and methods for promoting internalization and degradation of urokinase-type plasminogen activator |
Also Published As
Publication number | Publication date |
---|---|
IL122816A0 (en) | 1998-08-16 |
AU6658296A (en) | 1997-02-18 |
NO980252L (no) | 1998-01-20 |
DE19526561A1 (de) | 1997-01-23 |
KR19990035757A (ko) | 1999-05-25 |
JPH11509538A (ja) | 1999-08-24 |
NO980252D0 (no) | 1998-01-20 |
CA2225145A1 (en) | 1997-02-06 |
EP0839058A2 (de) | 1998-05-06 |
CN1191493A (zh) | 1998-08-26 |
WO1997003704A3 (de) | 1997-06-05 |
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