CA2225145A1 - In vivo diabetes test - Google Patents
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- CA2225145A1 CA2225145A1 CA002225145A CA2225145A CA2225145A1 CA 2225145 A1 CA2225145 A1 CA 2225145A1 CA 002225145 A CA002225145 A CA 002225145A CA 2225145 A CA2225145 A CA 2225145A CA 2225145 A1 CA2225145 A1 CA 2225145A1
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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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- 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
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- A—HUMAN NECESSITIES
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- 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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- 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
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- G—PHYSICS
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- 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
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Abstract
The invention concerns the use of substances which react together to prepare an agent for the diagnosis of cell-transmitted diseases or a predisposition towards such diseases, the agent being applied intradermally and the diagnosis being made on the basis of the appearance or absence of a positive reaction at the point of application.
Description
CA 0222~14~ 1997-12-18 11957P W0/WWjumy In vivo diabetes test Description The present invention concerns the use of autoreactive substances to produce an agent for the diagnosis of cell-mediated diseases or a predisposition to cell-mediated diseases which influence the immune system.
The elucidation of the molecular relationships in the development of auto-immune diseases such as rheumatoid arthritis and juvenile diabetes (IDDM) has advanced rapidly during recent years and has now revealed concrete applications for the early diagnosis and a causal therapy of these diseases.
It has now been established that, in addition to a genetic disposition, environmental factors also play a role in the development of these diseases. At the level of genetic risk factors only a few alleles of the MHC
class II antigens are, for example in the case of IDDM, closely associated with this disease. Hence it is possible to define a risk group for IDDM by analysing these alleles (cf. e.g. Thomson et al., Am. J. Hum.
Genet. 43 (1988), 799-816 or Todd et al., Nature 329 (1987), 599-604).
The environmental factors involved in the development of IDDM are probably exogenous immunogenically active peptide sequences. ~_ral antigens among others have been discussed in this connection which have partial homologies to endogenous structures. Under special CA 0222~14~ 1997-12-18 circumstances, in particular in the postnatal phase, antigens that are taken up through the food such as bovine serum albumin can induce an immune response which, due to homologies to endogenous structures, can initiate an autoaggressive process.
The progressive destruction of the pancreatic ,B cells by cytotoxic lymphocytes is typical for the course of the disease in IDDM. This process begins a long time before an impairment of glucose metabolism becomes apparent.
When manifestations of diabetes can be detected already over 80 % of the ,~ cells are destroyed. It would therefore be extremely important to be able to detect these autoaggressive T cells at an early stage in persons at risk ln order to be able to provide the affected individuals with a causal therapy.
It has nowadays been established that the destruction of endogenous tissue in auto-immune diseases initially progresses very slowly. In the initial stage of this process the autoaggressive T cells probably only recognize one or a few autoantigens. Publications by Kaufman et al. (Nature 368 (1993), 69-72) and Tisch et al. (Nature 368 (1993), 72-78) on an animal model (NOD
mouse) of type I diabetes have shown that in the case of spontaneously occurring diabetes in this mouse strain the initial T cell-mediated auto-immune reaction is directed towards glutamic acid decarboxylase. Initially only 1 to 2 epitopes at the C-terminus of glutamic acid decarboxylase (GAD) are recognized in the NOD mouse in this process. As described above changes in glucose metabolism cannot yet be detected at this stage whereas in contrast a perinsulitis is already detectable. It is not until the disease progresses further that the spectrum of the peptides of GAD that are recognized by CA 0222~14~ 1997-12-18 the autoaggressive T cells becomes broader. Once the diabetes is manifest it is also possible to detect pre-activated T cells against other islet cell antigens e.g.
peripherin, heat shock protein HSP 65 and carboxypeptidase H.
There is evidence that also in humans the immune response towards GAD is causally related to the development of type I diabetes. Hence for example auto-antibodies to GAD can be detected in about 80 % of pre-diabetics although the aetiological role of these auto-antibodies is estimated to be low. Rather it is assumed that in type I diabetes there is a progressive destruction of the pancreatic ~-cells by T lymphocytes.
These T lymphocytes directed towards 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). T lymphocytes found by these groups reacted with a peptide fragment of the GAD
67 kd molecule comprising amino acids 208 to 404.
EP-A-0 519 469 discloses auto-immunely reacting polypeptides from the human GAD 65 kd molecule. These polypeptides have the amino acid sequence:
X-P-E-V-K-(T or E)-K-Z
in which 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.
The European Patent Application No. 95 100 764.0 also proposes autoreactive peptide sequences from human GAD
65 as well as a pharmaceutical composition containing CA 0222~14~ 1997-12-18 these peptides. It also describes the use of this pharmaceutical composition to produce an agent for the diagnosis of diseases or a predisposition to diseases which influence the immune system or of tumour diseases or a predisposition to tumour diseases.
One of the objects of the present invention was to provide a diagnostic method for cell-mediated diseases of the immune system, in particular auto-immune diseases, which on the one hand causes as little discomfort as possible for the patient and on the other hand enables a rapid and reliable diagnosis.
This object is achieved by the use of autoreactive substances for the production of an agent for the diagnosis of cell-mediated diseases or a predisposition to cell-mediated diseases wherein the agent is administered intradermally or intracutaneously and the diagnosis is made on the basis of the occurrence or absence of a positive reaction at the site of application.
The diagnostic method according to the invention differs from the presently available methods on the market that are used for a general test for 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 state of the immune system of the test person. Cell-mediated diseases of the immune system are not diagnosed.
In another widely used test such as the prick test used to determine allergic reactions, the antigens are not CA 0222~14~ 1997-12-18 administered intradermally. A positive reaction which is not T cell-mediated can be detected within four hours and is characterized by the appearance of a superficial swelling but not by the appearance of a nodule.
The present invention provides a method for the in vivo detection of the occurrence of a cell-mediated immune reaction towards autoreactive substances. This cell-mediated immune reaction indicates the presence of specific diseases of the immune system, in particular auto-immune diseases, or a predisposition to such diseases. Furthermore the cell-mediated immune reaction can also indicate the presence of tumour diseases or a predisposition thereto.
The autoreactive substances or auto-antigens that are 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. Further suitable autoreactive substances are specific immunogenic epitopes, fragments, analogues, mimetics or derivatives which are for example produced by chemical modification as well as mixtures of the aforementioned substances.
The autoreactive substances can be administered as such or in a carrier-bound form e.g. in the form of lipopeptides. The autoreactive substances can be administered alone or together with additional substances such as accessory stimulating components or adjuvants. The auto-antigens can be produced from natural sources, by recombinant DNA technology or by synthetic methods e.g. by peptide solid phase synthesis.
CA 0222~14~ 1997-12-18 In addition to autoreactive substances in the sense of endogenous antigens, it is also possible to use those autoreactive substances which, although being foreign antigens, are immunologically related to auto-antigens.
Examples of such foreign antigens are peptides which have a homology to autoreactive peptide sequences from endogenous proteins. Specific examples are a peptide from bovine ~-casein A with the sequence L-V-Y-P-G-P-I-P-N (aa 60-68) which has a homology region to the endogenous glucose transporter GLUT-2 in the region of the sequence Q-I-G-P-G-P-I-P-W (aa 412-420) and a peptide from the Coxsackie virus protein P2-C with the sequence K-V-K-I-L-P-E-V-K-E-K-H-E (aa 33-45) which has a homology to glutamate decarboxylase in the region of the sequence R-F-K-M-F-P-E-V-K-E-K-G-M (aa 155-167).
The method according to the invention comprises the intradermal administration of the respective auto-antigens and the determination of the occurrence or absence of a local positive cellular reaction at the site of administration. This positive reaction is determined at a time which is characteristic for a T
cell-mediated immune response preferably at a time which is at least 24 hours after the administration particularly preferably during a period of 24 h to 1 week after the administration and most preferably during a period of 40 to 80 h after the administration.
The positive reaction preferably comprises the appearance of a nodule at the site of administration and can be determined qualitatively by visual observation or/and by palpation and also quantitatively e.g. by ultrasound or photographic methods.
CA 0222~14~ 1997-12-18 On the other hand it is also possible to determine the products of a positive reaction e.g. cytokines such as y interferon which are released at the site of administration by infiltrating cells e.g. leucocytes.
The method according to the invention is particularly suitable for the diagnosis of auto-immune diseases or a predisposition to auto-immune diseases.
Examples of auto-immune diseases which can be diagnosed by the method according to the invention are diabetes mellitus in particular type I diabetes (IDDM), multiple sclerosis, rheumatoid arthritis, Basedow disease, ankylosing spondylitis, acute anterior uveitis, Good-pasture syndrome, myasthenia gravis, systemic lupus erythematodes, pemphigus vulgaris, immune thyreoiditis, sclerodermia, Crohn's disease, Sjogren syndrome, Reiter's syndrome, inflammatory bowel disease or Graves' disease. The method according to the invention is particularly preferably used to diagnose T cell-mediated diseases such as IDDM, rheumatoid arthritis or multiple sclerosis and the method according to the invention is most preferably used to diagnose IDDM.
Autoreactive substances that are for example used to diagnose IDDM are glutamic acid decarboxylase (GAD) 65 kd or 67 kd, tyrosine phosphatase (38 kd antigen), carboxypeptidase H, insuline, heat shock protein, 38 kd insulin secretory granule protein or parts thereof.
Human GAD 65 kd or peptide partial sequences thereof are particularly preferably used as the autoreactive substance.
Analogous diagnostic applications are, however, also CA 0222~14~ 1997-12-18 possible for other auto-immune diseases. Examples of such auto-immune diseases are multiple sclerosis in which reactive T cells e.g. against myelin basic protein or the proteolipid protein can be determined, rheumatoid arthritis in which reactive T cells e.g. against collagen type II, cytokeratins, dnaJ protein from E.
coli and Hsp 65 can be determined and Basedow disease in which reactive T cells e.g. against thyroid peroxidase can be determined. In the case of myasthenia gravis it is possible to determine reactive T cells against the acetylcholine receptor or other relevant autoreactive substances. In the case of lupus erythematodes there are reactive T cells against HSP 90 and in the case of Graves disease against the TSH receptor.
In general a diagnostic application is possible for all diseases which influence the immune system such as e.g.
also in the case of arteriosclerosis. In this case the disease has been proven to be associated with an immune response against the heat shock protein Hsp 65 (Xu et al., _ancet 341, 8840 tl993), 255-259).
A further application is the diagnostic detection of T
cells which react with tumour antigens. Examples of this are 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). These T
cells can be already detected at a stage in which the tumour is not yet detectable by conventional methods because the cell mass is still too small. Furthermore specifically reacting T cells could also be used to monitor an anti-tumour vaccination.
CA 0222~14~ 1997-12-18 In addition it is also possible to use peptides, peptide derivatives or molecules which bind analogously as autoreactive substances. For the diagnosis of IDDM it is preferable to use one or several peptides from GAD in particular from human GAD 65 kd or peptide derivatives or peptide mimetics derived therefrom.
For the diagnosis of IDDM peptides or peptide derivatives are particularly preferably used which comprise:
(a) the amino acid sequence (I) D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R, (b) the amino acid sequence (II) S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K, (c) the amino acid sequence (III) N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T, (d) the amino acid sequence (IV) T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G, (e) the amino acid sequence (V) P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W, (f) the amino acid sequence (VI) T-Y-E-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R, (g) the amino acid sequence (VII) F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I, CA 0222~14~ 1997-12-18 (h) the amino acid sequence (VIII) G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A, (i) the amino acid sequence (IX) E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K, (j) one of the amino acid sequences shown in fig. 1 or 2, (k) partial regions of the amino acid sequences shown in (a) to (i) with a length of at least 6 amino acids or/and (1) amino acid sequences which have an essentially equivalent specificity or/and affinity of binding to MHC molecules as that of the amino acid sequences shown in (a) to (j).
The amino acid sequences (I) to (VII) correspond to the amino acid residues 86-105 (I), 246-265 (II), 146-165 (III), 166-186 (IV), 176-195 (V), 206-225 (VI) and 556-575 (VII) of human GAD 65 kd. The amino acid sequences I-VII are stated in the sequence protocols SEQ ID NO. 1-7.
The amino acid sequence (VIII) corresponds to the amino acid residues 266-290 of human GAD 65 kd and the amino acid sequence (IX) corresponds to the amino acid residues 306-325 of human GAD 65 kd. The amino acid sequences shown in figs. 1 and 2 are also partial sequences of human GAD 65 kd. The amino acid sequences of figs. 1 and 2 are given in the sequence protocols SEQ ID NO. 8-28. The amino acid sequences (VIII) and CA 0222~14~ 1997-12-18 (IX) are given in the sequence protocols SEQ ID NO. 29 and 30.
It was surprisingly found that peptides which contain the aforementioned regions of human GAD 65 react specifically with T cell subpopulations that have been isolated from recently discovered type I diabetics.
Hence the peptides according to the invention are early autoepitopes the use of which enables a very early diagnosis to type I diabetes.
The amino acid sequences (I) to (IX) are partial regions from the 65 kd isoform of human glutamic acid decarboxylase (GAD) the complete amino acid sequence of which was described by Bu et al. (Proc. Nat. Acad. Sci.
USA 89 (1992), 2115 ff.). The amino acid sequences (I) to (IX) were found by setting up T cell lines from the peripheral blood of type I diabetics and subsequently stimulating them in vitro with GAD from porcine brain or with recombinant human GAD and testing these T cell lines in a proliferation assay with synthetic peptide sequences which were derived from the human GAD
sequence.
The peptides can be produced by known synthetic processes using chemical methods or they can be produced by genetic engineering by cloning and expressing a DNA
sequence coding for these peptides in a suitable host cell in particular E. coli.
Peptides are also preferred which have partial regions of the specified amino acid sequences (I) to (IX) or of the amino acid sequences shown in figs. 1 and 2 which have a length of at least 6 amino acids, preferably of CA 0222~14~ 1997-12-18 at least 8 amino acids, particularly preferably of at least 10 amino acids and most preferably of at least 15 amino acids. The minimum length of a peptide according to the invention is determined by its ability to recognize a MHC molecule, to bind specifically to it and to react with the corresponding T cell receptor.
The maximum length of the sections of a peptide according to the invention which are derived from GAD
and bind to MHC is preferably 100 amino acids, particularly preferably 50 amino acids and most preferably 25 amino acids.
In addition to the aforementioned peptides it is also possible to use peptides which have an essentially equivalent specificity or/and affinity of binding to MHC
molecules as the aforementioned sequences and which are preferably derived from the aforementioned amino acid sequences by substitution, deletion or insertion of individual amino acid residues or short sections of amino acid residues or modified substances which bind in an analogous manner.
In particular the present invention also concerns peptide variants whose sequence does not fully correspond to that of the aforementioned amino acid sequences but instead only have the same or closely related "anchor positions". In this connection the term "anchor position" means an amino acid residue that is essential for binding to an MHC molecule in particular to a MHC molecule of the classes DR3, DR4 or DQ. The anchor position for the DRB10401 binding motif is stated for example in Hammer et al., Cell 74 (1993), 197-203.
Such anchor positions are conserved in peptides CA 0222~14~ 1997-12-18 according to the invention or optionally replaced by amino acid residues with chemically very 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 aforementioned specific peptides for their ability to bind to MHC molecules. A characteristic of peptides according to the invention is that they have an essentially equivalent specificity or/and affinity of binding to MHC molecules as 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 of at least 60 % to the initial peptides or partial sequences thereof.
Examples of variants of the specified peptides are the corresponding homologous peptide sections from human GAD
67, the complete amino acid sequence of which has also been described by Bu et al., supra.
The term "essentially equivalent specificity or/and affinity of binding to MHC molecules" also includes a binding specificity or/and affinity that is improved in comparison to the amino acid sequences (I) to (VIII) or to the amino acid sequences shown in figures 1 and 2 which is found especially in the case of shortened peptides which have a length of preferably 8 to 15 amino acids.
The present invention also concerns peptide derivatives.
This term includes peptides in which one or several amino acids have been derivatized by a chemical CA 0222~14~ 1997-12-18 reaction. Examples of peptide derivatives according to the invention are in particular those molecules in which the backbone or/and reactive amino acid side groups e.g.
free amino groups, free carboxyl groups ortand free hydroxyl groups have been derivatized. Specific examples of derivatives of amino groups are sulfonamides or carboxamides, thiourethane derivatives and ammonium salts e.g. hydrochlorides. Examples of carboxyl group derivatives are salts, esters and amides. Examples of hydroxyl group derivatives are O-acyl or 0-alkyl derivatives. The term peptide derivative according to the present invention also includes those peptides in which one or several amino acids are substituted by naturally occurring or non-naturally occurring amino acid homologues of the 20 "standard" amino acids.
Examples of such homologues are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, ~-alanine and 4-amino butyric acid.
Additional suitable peptide derivatives are complexes or covalent bonds between peptides and adjuvant components which are administered together with the autoantigen e.g. lipids. In this case the peptides can also be used as lipopeptides.
The present invention also encompasses polypeptides in which the MHC-binding peptide section is a component of a larger polypeptide unit wherein the linkage between the MHC-binding peptide and the remainder of the polypeptide unit preferably has a pre-determined breaking point e.g. a protease cleavage site.
The invention also concerns peptide-mimetic substances which have an essentially equivalent specificity or/and CA 0222~14~ 1997-12-18 affinity of binding to MHC molecules as the aforementioned peptide or peptide derivatives. Peptide-mimetic substances or peptide-mimetics are compounds which can replace peptides with regard to their interaction with the MHC molecules and, compared to the native peptides, have a higher metabolic stability, improved bioavailability and longer duration of action.
Methods for the preparation 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., Kontakte (Darmstadt) (1993) (2), 48-56. Reference is made to the disclosure of these literature references with regard to the preparation of peptide-mimetic substances according to the invention.
A further suitable autoreactive substance is a complex which contains at least one peptide, peptide derivative or peptide-mimetic and at least one MHC molecule or a peptide-binding derivative of an MHC molecule. In this complex a peptide, peptide derivative or peptide mimetic with a binding constant of preferably at least 10-7 l/mol particularly preferably in the range of 10-8 - 10-9 l/mol is bound to a MHC molecule or a peptide-binding derivative of a MHC molecule. Alternatively the peptide, peptide derivative or peptide mimetic can also be covalently coupled to the MHC molecule e.g. via a photolinker or a covalent genetic peptide-MHC fusion.
Such a peptide-MHC fusion protein preferably contains a HLA-DR beta chain and an autoreactive peptide which is genetically fused thereto. The complex particularly preferably contains a MHC class II molecule or a peptide-binding derivative thereof.
The nucleotide sequences of the genes coding for MHC
.. . .. ..
CA 0222~l4~ l997- l2- l8 class II molecules are published in Corell et al., (Mol.
Immunol. 28 (1991), 533-543). Reference is herewith made to the contents of this publication.
The term "peptide-binding derivative of a MHC molecule"
includes fragments of MHC molecules which are prepared by proteolytic cleavage of native MHC molecules or by recombinant DNA techniques and which have essentially retained their peptide-binding properties. This term is also understood to include fusion proteins which contain further polypeptide components in addition to a MHC part that is responsible for the peptide binding.
The peptide-MHC complexes are preferably prepared 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 for example be prepared by unfolding native MHC molecules in order to dissociate bound peptides and refolding the empty MHC molecules (see Dornmair and McConnell, Proc. Natl. Acad. Sci. USA
87 (1990), 4134-4138 and WO91/14701). Other methods for the preparation of complexes of peptides and MHC
molecules are described in the European Patent application No. 95 100 764Ø Reference is herewith made to this disclosure.
The invention also concerns the use of a composition which contains an autoreactive substance e.g. 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 common pharmaceutical additives in a diagnostic method which comprises the intradermal administration of CA 0222~14~ 1997-12-18 the composition. The composition can in addition contain an accessory stimulating component e.g. cytokines such as IL-2 and IL-4 or/and the surface antigen B7 (Wyss-Coray et al., Eur. J. Immunol. 23 (1993), 2175-2180;
Freeman et al., Science 262 (1993), 909-911) which can bind to the surface molecule CD-28 on a T cell. The presence of the accessory stimulating component can improve or/and modify the diagnostic effect of the compositlon.
Furthermore it is also preferable to carry out additional determinations with positive or/and negative controls in the method according to the invention. One can for example use a non-reactive polypeptide such as human serum albumin or a preparation that only contains additives, e.g. the buffer, as the negative control.
Purified protein derivative (PPD) from the culture medium in which tubercle bacteria have been cultured (commercially available for example from Statens Serum Institute, Copenhagen Denmark) or tetanus-toxoid which is known to cause a strong T cell stimulation in many people can for example be used as a positive control antigen.
In certain embodiments of the invention it is also preferable to administer the autoreactive substances together with an adjuvant. Examples of suitable adjuvants are aluminium hydroxide, incomplete Freund's adjuvant, aluminium phosphate and lipids which are suitable for coupling to peptides (lipopeptides).
The autoreactive substances can be administered by known methods in which for example a normal hypodermic syringe, e.g. with a size 26G needle, is used. The CA 0222~14~ 1997-12-18 .
composition containing the autoreactive substance is preferably administered intracutaneously in a volume of 20 ~l to 500 ~1, preferably 50 ~l to 200 ~l at a suitable site of the body which is most suitable for the autoantigen presented to the cells of the immune system.
For example to diagnose IDDM it can be injected into the forearm (front side).
The concentration of the autoantigen in the composition can be varied over wide ranges depending on the autoantigens used in each case. Concentrations of 1 to 100 ~g/ml in particular of 5 to 20 ~g/ml are preferably used.
Furthermore it is also possible to carry out the administration in a device which comprises one chamber for holding a preparation containing autoreactive substances, one chamber for holding a preparation for the positive control and one chamber for holding a preparation for the negative control. Such a device can for example contain 2 to 10 chambers for holding preparations containing autoreactive substances. This device can be used in a simple manner to test several autoreactive substances simultaneously.
A further subject matter of the present invention is the determination of a specific T cell subpopulation in which the sample containing T cells which is preferably derived from a tissue sample from the area of the site of administration is contacted with an autoreactive substance and the reaction of T cells with the substance is determined. A specific reaction of T cells with the autoantigen can for example be detected by an increased T cell proliferation which can be measured by the CA 0222~14~ 1997-12-18 incorporation of radioactivity. On the other hand the reaction of T cells can also be determined directly by using a labelled autoantigen e.g. in a complex form with a soluble MHC molecule. In this embodiment the autoreactive substance is preferably used with a fluorescent labelling group that is coupled thereto. The evaluation can for example be carried out by FACS
analysis in which the T cells are contacted with a first fluorescent marker that is coupled to a T cell specific antibody and then contacted with the autoantigen which is coupled to a second fluorescent marker and the presence of double-labelled cells is determined by fluorographic analysis. In this manner a T cell subpopulation is determined which is characterized by its reactivity with an autoantigen. The method can optionally also include a selection for pre-activated T
cells e.g. a selective enrichment of IL-2 receptor-positive T cells by incubation with IL-2 or/and by incubation with IL-2 receptor antibodies and subsequent separation of the antibody-binding cells for example using immuno-magnetic methods. On the other hand the preactivated cells can be first selected after contact of the T cells with the autoantigen.
In a modification of this method it is also possible to determine the ratio of preactivated autoreactive T
cells, i.e. T cells with the IL-2 receptor as a surface marker, to non-activated autoreactive T cells i.e. T
cells without the IL-2 receptor.
A further subject matter of the present invention is the isolation of T cell subpopulations which react with an autoantigen. In such a method a tissue sample containing T cells which is derived from the area of the site of administration is contacted with an autoantigen, the T
CA 0222~14~ 1997-12-18 cells reacting with the autoantigen are identified and they are optionally separated from other T cells. In this case it is also possible to select for preactivated T cells, i.e. T cells containing the IL-2 receptor, before or/and after contact of the T cells with the autoantigen. In such a method one can use the autoantigen for example in the form of a complex with an MHC molecule, in an immobilized form on a carrier which simplifies the separation of the positively reacting T
cell population from other T cells. T cell lines can be established from the T cell subpopulations isolated in this manner by restimulation. These autoreactive T cell lines can then be used to immunize patients.
In the diagnostic method for identifying specific T cell subpopulations it is also possible to use an anti-idiotypic antibody instead of the autoantigens which imitates the action of the MHC peptide complex. Such antibodies can be readily obtained by using a T cell subpopulation which is specific for a particular antigen as an immunogen for producing an antibody te.g. in a mouse) or by firstly producing a first antibody to the autoantigen and then an anti-idiotypic antibody to the first antibody.
Specific T cell subpopulations can also be identified by identifying the products of these in vivo activated T
cells instead of isolating the T cells. This can be achieved by collecting a tissue sample (e.g. fine needle aspirate) and determining the cytokines that are contained therein. Alternatively the cytokines can also be determined in situ by applying a plaster for a long time in which antibodies to various cytokines are immobilized. The cytokines are detected by a conventional solid phase immunoassay method using enzyme CA 0222~14~ 1997-12-18 substrates which yield an insoluble coloured product which is either read off directly or using a scanner.
Finally the invention concerns a reagent kit for the in vivo diagnosis of cell-mediated diseases or a predisposition thereto comprising:
(a) at least one autoreactive substance in the form of a pharmaceutically acceptable composition, (b) optionally at least one control substance in the form of a pharmaceutically acceptable composltlon (c) device for the intradermal administration of the autoreactive substance and the control substance and (d) optionally device for the diagnostic evaluation of the test result.
The compositions for the autoreactive substance and the control substance preferably contain adjuvants and optionally other common pharmaceutical carrier substances, diluents and additives.
It is intended to further elucidate the invention by the following examples in conjunction with figures 1 and 2 and the sequence protocols SEQ ID NO. 1-30.
Fig. l shows autoreactive amino acid sequences from human GAD 65 kd, ... ..
CA 0222~14~ 1997-12-18 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 -2 8 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).
EXAMPLE 1 (comparative example) In vitro investi~ation Peripheral venous blood was collected from eight patients that had recently contracted IDDM and from twelve healthy persons. The peripheral blood lymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation and were used for an in vitro stimulation with:
a) recombinant human GAD 65 kd as the test antigen;
b) purified protein derivative (PPD) as a positive control antigen;
c) human serum albumin (HSA) as a negative control antigen.
1 x 105 peripheral blood lymphocytes (PBL) were incubated for five days at 37~C and 5 ~ C02 in a well of CA 0222~14~ 1997-12-18 a 96-well round bottom plate 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/l 2-mercaptoethanol) in the presence of the aforementioned antigens or in medium alone.
After 5 days 20 ~l RPMI 1640 medium containing 0.5 ~Ci 3H thymidine was added per well. The incubation was continued for 16 hours and then the cells were harvested. The incorporation of thymidine was measured by liquid scintillation counting. The results are stated as a stimulation index (SI, i.e. cpm in the presence of the antigen divided by cpm in the absence of the antigen i.e. medium alone).
The proliferation reactions of lymphocytes from IDDM
patients and control persons towards the individual antigens are summarized in tables 1 and 2.
S.I.=cpm PBL stimulated/
cpm PBL non-stimulated IDDM base value PPD rhGAD 65 HSA
patients cpm 10 ~g/ml 10 ~g/ml 10 ~g/ml 1 730 17.9 2.6 1.1 2 833 16.7 8.8 1.2 3 639 23 6.6 0.9 4 328 28.9 0.8 2.9 1211 10.6 1.9 0.5 6 1041 11.9 1.8 2.2 7 520 19.8 7.2 1.0 8 412 2.7 8.3 0.8 CA 0222~14~ 1997-12-18 S.I.=cpm PBL stimulated/
cpm PBL non-stimulated healthy base value PPD rhGAD 65 HSA
persons cpm 10 ~g/ml 10 ~g/ml 10 ~g/ml 1 1108 25 1.8 0.7 2 1035 2.1 3.0 0.5 3 390 6.5 2.7 1.1 4 578 6.2 0.9 1.2 243 1.3 0.9 1.9 6 375 6.3 2.5 2.6 7 648 12.5 4.2 0.8 8 730 20.6 4.2 0.5 9 542 0.8 0.9 2.0 gl5 0.5 2.8 2.8 11 567 90.6 3.5 2.3 12 1247 12.5 1.0 0.6 A statistical analysis of the investigation showed that the proliferation of PBL from recently diagnosed IDDM
patients with rhGAD 65 kd could be regarded as positive with an SI of more than 4.7 (mean of the control persons + 2 SD).
Hence an in vitro T cell reaction to rhGAD 65 kd was observed in 50 % of the IDDM patients (patients 2, 3, 7 and 8). Antibodies to GAD were found in patients 1, 2, 4 and 8 but not in patients 3 and 5. Patients 6 and 7 were not tested for antibodies to GAD. The control persons did not react to GAD 65 kd.
CA 0222~14~ 1997-12-18 The diabetics and control persons exhibited no statistically significant difference at all in their reaction to the controls HSA and PPD.
In vivo investigation 100 ~l rhGAD 65 kd at a concentration of 10 ~g/ml was injected intradermally into the right forearm of the eight IDDM patients from example 1.
The antigen was diluted in physiological saline pH 7.3 containing 1 % HSA. The same volume (100 ~l) HSA at 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.
For the in vivo diagnosis the formation of a nodule (specific increase of the derma thickness) at the site of administration was regarded as a positive reaction.
The diagnosis was made 48 h after the administration.
The results were confirmed and quantified by ultrasound measurements in the area of the administration site. No side reactions were observed.
The results of this investigation are shown in table 3.
IDDM PPD rhGAD 65 HSA time of patients 10 ~/ml 10 ~g/ml 10 ~g/ml evaluation 1 + ++ - 48h 2 n.t. ++ - 48h 3 n.t. + - 48h 4 - - - 48h _ 48h 6 - - - 48h 7 n.t. +++ - 48h 8 + + - 48h Five of the eight tested IDDM patients reacted positively to rhGAD 65.
CA 0222~14~ 1997-12-18 SEQUENCE PROTOCOL
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Boehringer Mannheim GmbH
(B) ROAD: Sandhofer Str. 112-132 (C) CITY: Mannheim (E) COUNTRY: DE
(F) POSTAL CODE (ZIP): 68305 (ii) TITLE OF THE INVENTION: In vivo diabetes test (iii) NUMBER OF SEQUENCES: 30 (iv) COMPUTER-READABLE FORM:
(A) DATA CARRIER: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, version #1.30 (EPA) (vi) DATA OF ORIGINAL APPLICATION:
(A) APPLICATION NUMBER: DE 19526561.0 (B) DATE OF APPLICATION: 20-July-1995 (2) INFORMATION FOR SEQ ID NO: 1:
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The elucidation of the molecular relationships in the development of auto-immune diseases such as rheumatoid arthritis and juvenile diabetes (IDDM) has advanced rapidly during recent years and has now revealed concrete applications for the early diagnosis and a causal therapy of these diseases.
It has now been established that, in addition to a genetic disposition, environmental factors also play a role in the development of these diseases. At the level of genetic risk factors only a few alleles of the MHC
class II antigens are, for example in the case of IDDM, closely associated with this disease. Hence it is possible to define a risk group for IDDM by analysing these alleles (cf. e.g. Thomson et al., Am. J. Hum.
Genet. 43 (1988), 799-816 or Todd et al., Nature 329 (1987), 599-604).
The environmental factors involved in the development of IDDM are probably exogenous immunogenically active peptide sequences. ~_ral antigens among others have been discussed in this connection which have partial homologies to endogenous structures. Under special CA 0222~14~ 1997-12-18 circumstances, in particular in the postnatal phase, antigens that are taken up through the food such as bovine serum albumin can induce an immune response which, due to homologies to endogenous structures, can initiate an autoaggressive process.
The progressive destruction of the pancreatic ,B cells by cytotoxic lymphocytes is typical for the course of the disease in IDDM. This process begins a long time before an impairment of glucose metabolism becomes apparent.
When manifestations of diabetes can be detected already over 80 % of the ,~ cells are destroyed. It would therefore be extremely important to be able to detect these autoaggressive T cells at an early stage in persons at risk ln order to be able to provide the affected individuals with a causal therapy.
It has nowadays been established that the destruction of endogenous tissue in auto-immune diseases initially progresses very slowly. In the initial stage of this process the autoaggressive T cells probably only recognize one or a few autoantigens. Publications by Kaufman et al. (Nature 368 (1993), 69-72) and Tisch et al. (Nature 368 (1993), 72-78) on an animal model (NOD
mouse) of type I diabetes have shown that in the case of spontaneously occurring diabetes in this mouse strain the initial T cell-mediated auto-immune reaction is directed towards glutamic acid decarboxylase. Initially only 1 to 2 epitopes at the C-terminus of glutamic acid decarboxylase (GAD) are recognized in the NOD mouse in this process. As described above changes in glucose metabolism cannot yet be detected at this stage whereas in contrast a perinsulitis is already detectable. It is not until the disease progresses further that the spectrum of the peptides of GAD that are recognized by CA 0222~14~ 1997-12-18 the autoaggressive T cells becomes broader. Once the diabetes is manifest it is also possible to detect pre-activated T cells against other islet cell antigens e.g.
peripherin, heat shock protein HSP 65 and carboxypeptidase H.
There is evidence that also in humans the immune response towards GAD is causally related to the development of type I diabetes. Hence for example auto-antibodies to GAD can be detected in about 80 % of pre-diabetics although the aetiological role of these auto-antibodies is estimated to be low. Rather it is assumed that in type I diabetes there is a progressive destruction of the pancreatic ~-cells by T lymphocytes.
These T lymphocytes directed towards 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). T lymphocytes found by these groups reacted with a peptide fragment of the GAD
67 kd molecule comprising amino acids 208 to 404.
EP-A-0 519 469 discloses auto-immunely reacting polypeptides from the human GAD 65 kd molecule. These polypeptides have the amino acid sequence:
X-P-E-V-K-(T or E)-K-Z
in which 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.
The European Patent Application No. 95 100 764.0 also proposes autoreactive peptide sequences from human GAD
65 as well as a pharmaceutical composition containing CA 0222~14~ 1997-12-18 these peptides. It also describes the use of this pharmaceutical composition to produce an agent for the diagnosis of diseases or a predisposition to diseases which influence the immune system or of tumour diseases or a predisposition to tumour diseases.
One of the objects of the present invention was to provide a diagnostic method for cell-mediated diseases of the immune system, in particular auto-immune diseases, which on the one hand causes as little discomfort as possible for the patient and on the other hand enables a rapid and reliable diagnosis.
This object is achieved by the use of autoreactive substances for the production of an agent for the diagnosis of cell-mediated diseases or a predisposition to cell-mediated diseases wherein the agent is administered intradermally or intracutaneously and the diagnosis is made on the basis of the occurrence or absence of a positive reaction at the site of application.
The diagnostic method according to the invention differs from the presently available methods on the market that are used for a general test for 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 state of the immune system of the test person. Cell-mediated diseases of the immune system are not diagnosed.
In another widely used test such as the prick test used to determine allergic reactions, the antigens are not CA 0222~14~ 1997-12-18 administered intradermally. A positive reaction which is not T cell-mediated can be detected within four hours and is characterized by the appearance of a superficial swelling but not by the appearance of a nodule.
The present invention provides a method for the in vivo detection of the occurrence of a cell-mediated immune reaction towards autoreactive substances. This cell-mediated immune reaction indicates the presence of specific diseases of the immune system, in particular auto-immune diseases, or a predisposition to such diseases. Furthermore the cell-mediated immune reaction can also indicate the presence of tumour diseases or a predisposition thereto.
The autoreactive substances or auto-antigens that are 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. Further suitable autoreactive substances are specific immunogenic epitopes, fragments, analogues, mimetics or derivatives which are for example produced by chemical modification as well as mixtures of the aforementioned substances.
The autoreactive substances can be administered as such or in a carrier-bound form e.g. in the form of lipopeptides. The autoreactive substances can be administered alone or together with additional substances such as accessory stimulating components or adjuvants. The auto-antigens can be produced from natural sources, by recombinant DNA technology or by synthetic methods e.g. by peptide solid phase synthesis.
CA 0222~14~ 1997-12-18 In addition to autoreactive substances in the sense of endogenous antigens, it is also possible to use those autoreactive substances which, although being foreign antigens, are immunologically related to auto-antigens.
Examples of such foreign antigens are peptides which have a homology to autoreactive peptide sequences from endogenous proteins. Specific examples are a peptide from bovine ~-casein A with the sequence L-V-Y-P-G-P-I-P-N (aa 60-68) which has a homology region to the endogenous glucose transporter GLUT-2 in the region of the sequence Q-I-G-P-G-P-I-P-W (aa 412-420) and a peptide from the Coxsackie virus protein P2-C with the sequence K-V-K-I-L-P-E-V-K-E-K-H-E (aa 33-45) which has a homology to glutamate decarboxylase in the region of the sequence R-F-K-M-F-P-E-V-K-E-K-G-M (aa 155-167).
The method according to the invention comprises the intradermal administration of the respective auto-antigens and the determination of the occurrence or absence of a local positive cellular reaction at the site of administration. This positive reaction is determined at a time which is characteristic for a T
cell-mediated immune response preferably at a time which is at least 24 hours after the administration particularly preferably during a period of 24 h to 1 week after the administration and most preferably during a period of 40 to 80 h after the administration.
The positive reaction preferably comprises the appearance of a nodule at the site of administration and can be determined qualitatively by visual observation or/and by palpation and also quantitatively e.g. by ultrasound or photographic methods.
CA 0222~14~ 1997-12-18 On the other hand it is also possible to determine the products of a positive reaction e.g. cytokines such as y interferon which are released at the site of administration by infiltrating cells e.g. leucocytes.
The method according to the invention is particularly suitable for the diagnosis of auto-immune diseases or a predisposition to auto-immune diseases.
Examples of auto-immune diseases which can be diagnosed by the method according to the invention are diabetes mellitus in particular type I diabetes (IDDM), multiple sclerosis, rheumatoid arthritis, Basedow disease, ankylosing spondylitis, acute anterior uveitis, Good-pasture syndrome, myasthenia gravis, systemic lupus erythematodes, pemphigus vulgaris, immune thyreoiditis, sclerodermia, Crohn's disease, Sjogren syndrome, Reiter's syndrome, inflammatory bowel disease or Graves' disease. The method according to the invention is particularly preferably used to diagnose T cell-mediated diseases such as IDDM, rheumatoid arthritis or multiple sclerosis and the method according to the invention is most preferably used to diagnose IDDM.
Autoreactive substances that are for example used to diagnose IDDM are glutamic acid decarboxylase (GAD) 65 kd or 67 kd, tyrosine phosphatase (38 kd antigen), carboxypeptidase H, insuline, heat shock protein, 38 kd insulin secretory granule protein or parts thereof.
Human GAD 65 kd or peptide partial sequences thereof are particularly preferably used as the autoreactive substance.
Analogous diagnostic applications are, however, also CA 0222~14~ 1997-12-18 possible for other auto-immune diseases. Examples of such auto-immune diseases are multiple sclerosis in which reactive T cells e.g. against myelin basic protein or the proteolipid protein can be determined, rheumatoid arthritis in which reactive T cells e.g. against collagen type II, cytokeratins, dnaJ protein from E.
coli and Hsp 65 can be determined and Basedow disease in which reactive T cells e.g. against thyroid peroxidase can be determined. In the case of myasthenia gravis it is possible to determine reactive T cells against the acetylcholine receptor or other relevant autoreactive substances. In the case of lupus erythematodes there are reactive T cells against HSP 90 and in the case of Graves disease against the TSH receptor.
In general a diagnostic application is possible for all diseases which influence the immune system such as e.g.
also in the case of arteriosclerosis. In this case the disease has been proven to be associated with an immune response against the heat shock protein Hsp 65 (Xu et al., _ancet 341, 8840 tl993), 255-259).
A further application is the diagnostic detection of T
cells which react with tumour antigens. Examples of this are 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). These T
cells can be already detected at a stage in which the tumour is not yet detectable by conventional methods because the cell mass is still too small. Furthermore specifically reacting T cells could also be used to monitor an anti-tumour vaccination.
CA 0222~14~ 1997-12-18 In addition it is also possible to use peptides, peptide derivatives or molecules which bind analogously as autoreactive substances. For the diagnosis of IDDM it is preferable to use one or several peptides from GAD in particular from human GAD 65 kd or peptide derivatives or peptide mimetics derived therefrom.
For the diagnosis of IDDM peptides or peptide derivatives are particularly preferably used which comprise:
(a) the amino acid sequence (I) D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R, (b) the amino acid sequence (II) S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K, (c) the amino acid sequence (III) N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T, (d) the amino acid sequence (IV) T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G, (e) the amino acid sequence (V) P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W, (f) the amino acid sequence (VI) T-Y-E-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R, (g) the amino acid sequence (VII) F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I, CA 0222~14~ 1997-12-18 (h) the amino acid sequence (VIII) G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A, (i) the amino acid sequence (IX) E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K, (j) one of the amino acid sequences shown in fig. 1 or 2, (k) partial regions of the amino acid sequences shown in (a) to (i) with a length of at least 6 amino acids or/and (1) amino acid sequences which have an essentially equivalent specificity or/and affinity of binding to MHC molecules as that of the amino acid sequences shown in (a) to (j).
The amino acid sequences (I) to (VII) correspond to the amino acid residues 86-105 (I), 246-265 (II), 146-165 (III), 166-186 (IV), 176-195 (V), 206-225 (VI) and 556-575 (VII) of human GAD 65 kd. The amino acid sequences I-VII are stated in the sequence protocols SEQ ID NO. 1-7.
The amino acid sequence (VIII) corresponds to the amino acid residues 266-290 of human GAD 65 kd and the amino acid sequence (IX) corresponds to the amino acid residues 306-325 of human GAD 65 kd. The amino acid sequences shown in figs. 1 and 2 are also partial sequences of human GAD 65 kd. The amino acid sequences of figs. 1 and 2 are given in the sequence protocols SEQ ID NO. 8-28. The amino acid sequences (VIII) and CA 0222~14~ 1997-12-18 (IX) are given in the sequence protocols SEQ ID NO. 29 and 30.
It was surprisingly found that peptides which contain the aforementioned regions of human GAD 65 react specifically with T cell subpopulations that have been isolated from recently discovered type I diabetics.
Hence the peptides according to the invention are early autoepitopes the use of which enables a very early diagnosis to type I diabetes.
The amino acid sequences (I) to (IX) are partial regions from the 65 kd isoform of human glutamic acid decarboxylase (GAD) the complete amino acid sequence of which was described by Bu et al. (Proc. Nat. Acad. Sci.
USA 89 (1992), 2115 ff.). The amino acid sequences (I) to (IX) were found by setting up T cell lines from the peripheral blood of type I diabetics and subsequently stimulating them in vitro with GAD from porcine brain or with recombinant human GAD and testing these T cell lines in a proliferation assay with synthetic peptide sequences which were derived from the human GAD
sequence.
The peptides can be produced by known synthetic processes using chemical methods or they can be produced by genetic engineering by cloning and expressing a DNA
sequence coding for these peptides in a suitable host cell in particular E. coli.
Peptides are also preferred which have partial regions of the specified amino acid sequences (I) to (IX) or of the amino acid sequences shown in figs. 1 and 2 which have a length of at least 6 amino acids, preferably of CA 0222~14~ 1997-12-18 at least 8 amino acids, particularly preferably of at least 10 amino acids and most preferably of at least 15 amino acids. The minimum length of a peptide according to the invention is determined by its ability to recognize a MHC molecule, to bind specifically to it and to react with the corresponding T cell receptor.
The maximum length of the sections of a peptide according to the invention which are derived from GAD
and bind to MHC is preferably 100 amino acids, particularly preferably 50 amino acids and most preferably 25 amino acids.
In addition to the aforementioned peptides it is also possible to use peptides which have an essentially equivalent specificity or/and affinity of binding to MHC
molecules as the aforementioned sequences and which are preferably derived from the aforementioned amino acid sequences by substitution, deletion or insertion of individual amino acid residues or short sections of amino acid residues or modified substances which bind in an analogous manner.
In particular the present invention also concerns peptide variants whose sequence does not fully correspond to that of the aforementioned amino acid sequences but instead only have the same or closely related "anchor positions". In this connection the term "anchor position" means an amino acid residue that is essential for binding to an MHC molecule in particular to a MHC molecule of the classes DR3, DR4 or DQ. The anchor position for the DRB10401 binding motif is stated for example in Hammer et al., Cell 74 (1993), 197-203.
Such anchor positions are conserved in peptides CA 0222~14~ 1997-12-18 according to the invention or optionally replaced by amino acid residues with chemically very 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 aforementioned specific peptides for their ability to bind to MHC molecules. A characteristic of peptides according to the invention is that they have an essentially equivalent specificity or/and affinity of binding to MHC molecules as 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 of at least 60 % to the initial peptides or partial sequences thereof.
Examples of variants of the specified peptides are the corresponding homologous peptide sections from human GAD
67, the complete amino acid sequence of which has also been described by Bu et al., supra.
The term "essentially equivalent specificity or/and affinity of binding to MHC molecules" also includes a binding specificity or/and affinity that is improved in comparison to the amino acid sequences (I) to (VIII) or to the amino acid sequences shown in figures 1 and 2 which is found especially in the case of shortened peptides which have a length of preferably 8 to 15 amino acids.
The present invention also concerns peptide derivatives.
This term includes peptides in which one or several amino acids have been derivatized by a chemical CA 0222~14~ 1997-12-18 reaction. Examples of peptide derivatives according to the invention are in particular those molecules in which the backbone or/and reactive amino acid side groups e.g.
free amino groups, free carboxyl groups ortand free hydroxyl groups have been derivatized. Specific examples of derivatives of amino groups are sulfonamides or carboxamides, thiourethane derivatives and ammonium salts e.g. hydrochlorides. Examples of carboxyl group derivatives are salts, esters and amides. Examples of hydroxyl group derivatives are O-acyl or 0-alkyl derivatives. The term peptide derivative according to the present invention also includes those peptides in which one or several amino acids are substituted by naturally occurring or non-naturally occurring amino acid homologues of the 20 "standard" amino acids.
Examples of such homologues are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, ~-alanine and 4-amino butyric acid.
Additional suitable peptide derivatives are complexes or covalent bonds between peptides and adjuvant components which are administered together with the autoantigen e.g. lipids. In this case the peptides can also be used as lipopeptides.
The present invention also encompasses polypeptides in which the MHC-binding peptide section is a component of a larger polypeptide unit wherein the linkage between the MHC-binding peptide and the remainder of the polypeptide unit preferably has a pre-determined breaking point e.g. a protease cleavage site.
The invention also concerns peptide-mimetic substances which have an essentially equivalent specificity or/and CA 0222~14~ 1997-12-18 affinity of binding to MHC molecules as the aforementioned peptide or peptide derivatives. Peptide-mimetic substances or peptide-mimetics are compounds which can replace peptides with regard to their interaction with the MHC molecules and, compared to the native peptides, have a higher metabolic stability, improved bioavailability and longer duration of action.
Methods for the preparation 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., Kontakte (Darmstadt) (1993) (2), 48-56. Reference is made to the disclosure of these literature references with regard to the preparation of peptide-mimetic substances according to the invention.
A further suitable autoreactive substance is a complex which contains at least one peptide, peptide derivative or peptide-mimetic and at least one MHC molecule or a peptide-binding derivative of an MHC molecule. In this complex a peptide, peptide derivative or peptide mimetic with a binding constant of preferably at least 10-7 l/mol particularly preferably in the range of 10-8 - 10-9 l/mol is bound to a MHC molecule or a peptide-binding derivative of a MHC molecule. Alternatively the peptide, peptide derivative or peptide mimetic can also be covalently coupled to the MHC molecule e.g. via a photolinker or a covalent genetic peptide-MHC fusion.
Such a peptide-MHC fusion protein preferably contains a HLA-DR beta chain and an autoreactive peptide which is genetically fused thereto. The complex particularly preferably contains a MHC class II molecule or a peptide-binding derivative thereof.
The nucleotide sequences of the genes coding for MHC
.. . .. ..
CA 0222~l4~ l997- l2- l8 class II molecules are published in Corell et al., (Mol.
Immunol. 28 (1991), 533-543). Reference is herewith made to the contents of this publication.
The term "peptide-binding derivative of a MHC molecule"
includes fragments of MHC molecules which are prepared by proteolytic cleavage of native MHC molecules or by recombinant DNA techniques and which have essentially retained their peptide-binding properties. This term is also understood to include fusion proteins which contain further polypeptide components in addition to a MHC part that is responsible for the peptide binding.
The peptide-MHC complexes are preferably prepared 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 for example be prepared by unfolding native MHC molecules in order to dissociate bound peptides and refolding the empty MHC molecules (see Dornmair and McConnell, Proc. Natl. Acad. Sci. USA
87 (1990), 4134-4138 and WO91/14701). Other methods for the preparation of complexes of peptides and MHC
molecules are described in the European Patent application No. 95 100 764Ø Reference is herewith made to this disclosure.
The invention also concerns the use of a composition which contains an autoreactive substance e.g. 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 common pharmaceutical additives in a diagnostic method which comprises the intradermal administration of CA 0222~14~ 1997-12-18 the composition. The composition can in addition contain an accessory stimulating component e.g. cytokines such as IL-2 and IL-4 or/and the surface antigen B7 (Wyss-Coray et al., Eur. J. Immunol. 23 (1993), 2175-2180;
Freeman et al., Science 262 (1993), 909-911) which can bind to the surface molecule CD-28 on a T cell. The presence of the accessory stimulating component can improve or/and modify the diagnostic effect of the compositlon.
Furthermore it is also preferable to carry out additional determinations with positive or/and negative controls in the method according to the invention. One can for example use a non-reactive polypeptide such as human serum albumin or a preparation that only contains additives, e.g. the buffer, as the negative control.
Purified protein derivative (PPD) from the culture medium in which tubercle bacteria have been cultured (commercially available for example from Statens Serum Institute, Copenhagen Denmark) or tetanus-toxoid which is known to cause a strong T cell stimulation in many people can for example be used as a positive control antigen.
In certain embodiments of the invention it is also preferable to administer the autoreactive substances together with an adjuvant. Examples of suitable adjuvants are aluminium hydroxide, incomplete Freund's adjuvant, aluminium phosphate and lipids which are suitable for coupling to peptides (lipopeptides).
The autoreactive substances can be administered by known methods in which for example a normal hypodermic syringe, e.g. with a size 26G needle, is used. The CA 0222~14~ 1997-12-18 .
composition containing the autoreactive substance is preferably administered intracutaneously in a volume of 20 ~l to 500 ~1, preferably 50 ~l to 200 ~l at a suitable site of the body which is most suitable for the autoantigen presented to the cells of the immune system.
For example to diagnose IDDM it can be injected into the forearm (front side).
The concentration of the autoantigen in the composition can be varied over wide ranges depending on the autoantigens used in each case. Concentrations of 1 to 100 ~g/ml in particular of 5 to 20 ~g/ml are preferably used.
Furthermore it is also possible to carry out the administration in a device which comprises one chamber for holding a preparation containing autoreactive substances, one chamber for holding a preparation for the positive control and one chamber for holding a preparation for the negative control. Such a device can for example contain 2 to 10 chambers for holding preparations containing autoreactive substances. This device can be used in a simple manner to test several autoreactive substances simultaneously.
A further subject matter of the present invention is the determination of a specific T cell subpopulation in which the sample containing T cells which is preferably derived from a tissue sample from the area of the site of administration is contacted with an autoreactive substance and the reaction of T cells with the substance is determined. A specific reaction of T cells with the autoantigen can for example be detected by an increased T cell proliferation which can be measured by the CA 0222~14~ 1997-12-18 incorporation of radioactivity. On the other hand the reaction of T cells can also be determined directly by using a labelled autoantigen e.g. in a complex form with a soluble MHC molecule. In this embodiment the autoreactive substance is preferably used with a fluorescent labelling group that is coupled thereto. The evaluation can for example be carried out by FACS
analysis in which the T cells are contacted with a first fluorescent marker that is coupled to a T cell specific antibody and then contacted with the autoantigen which is coupled to a second fluorescent marker and the presence of double-labelled cells is determined by fluorographic analysis. In this manner a T cell subpopulation is determined which is characterized by its reactivity with an autoantigen. The method can optionally also include a selection for pre-activated T
cells e.g. a selective enrichment of IL-2 receptor-positive T cells by incubation with IL-2 or/and by incubation with IL-2 receptor antibodies and subsequent separation of the antibody-binding cells for example using immuno-magnetic methods. On the other hand the preactivated cells can be first selected after contact of the T cells with the autoantigen.
In a modification of this method it is also possible to determine the ratio of preactivated autoreactive T
cells, i.e. T cells with the IL-2 receptor as a surface marker, to non-activated autoreactive T cells i.e. T
cells without the IL-2 receptor.
A further subject matter of the present invention is the isolation of T cell subpopulations which react with an autoantigen. In such a method a tissue sample containing T cells which is derived from the area of the site of administration is contacted with an autoantigen, the T
CA 0222~14~ 1997-12-18 cells reacting with the autoantigen are identified and they are optionally separated from other T cells. In this case it is also possible to select for preactivated T cells, i.e. T cells containing the IL-2 receptor, before or/and after contact of the T cells with the autoantigen. In such a method one can use the autoantigen for example in the form of a complex with an MHC molecule, in an immobilized form on a carrier which simplifies the separation of the positively reacting T
cell population from other T cells. T cell lines can be established from the T cell subpopulations isolated in this manner by restimulation. These autoreactive T cell lines can then be used to immunize patients.
In the diagnostic method for identifying specific T cell subpopulations it is also possible to use an anti-idiotypic antibody instead of the autoantigens which imitates the action of the MHC peptide complex. Such antibodies can be readily obtained by using a T cell subpopulation which is specific for a particular antigen as an immunogen for producing an antibody te.g. in a mouse) or by firstly producing a first antibody to the autoantigen and then an anti-idiotypic antibody to the first antibody.
Specific T cell subpopulations can also be identified by identifying the products of these in vivo activated T
cells instead of isolating the T cells. This can be achieved by collecting a tissue sample (e.g. fine needle aspirate) and determining the cytokines that are contained therein. Alternatively the cytokines can also be determined in situ by applying a plaster for a long time in which antibodies to various cytokines are immobilized. The cytokines are detected by a conventional solid phase immunoassay method using enzyme CA 0222~14~ 1997-12-18 substrates which yield an insoluble coloured product which is either read off directly or using a scanner.
Finally the invention concerns a reagent kit for the in vivo diagnosis of cell-mediated diseases or a predisposition thereto comprising:
(a) at least one autoreactive substance in the form of a pharmaceutically acceptable composition, (b) optionally at least one control substance in the form of a pharmaceutically acceptable composltlon (c) device for the intradermal administration of the autoreactive substance and the control substance and (d) optionally device for the diagnostic evaluation of the test result.
The compositions for the autoreactive substance and the control substance preferably contain adjuvants and optionally other common pharmaceutical carrier substances, diluents and additives.
It is intended to further elucidate the invention by the following examples in conjunction with figures 1 and 2 and the sequence protocols SEQ ID NO. 1-30.
Fig. l shows autoreactive amino acid sequences from human GAD 65 kd, ... ..
CA 0222~14~ 1997-12-18 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 -2 8 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).
EXAMPLE 1 (comparative example) In vitro investi~ation Peripheral venous blood was collected from eight patients that had recently contracted IDDM and from twelve healthy persons. The peripheral blood lymphocytes were isolated by Ficoll-Hypaque density gradient centrifugation and were used for an in vitro stimulation with:
a) recombinant human GAD 65 kd as the test antigen;
b) purified protein derivative (PPD) as a positive control antigen;
c) human serum albumin (HSA) as a negative control antigen.
1 x 105 peripheral blood lymphocytes (PBL) were incubated for five days at 37~C and 5 ~ C02 in a well of CA 0222~14~ 1997-12-18 a 96-well round bottom plate 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/l 2-mercaptoethanol) in the presence of the aforementioned antigens or in medium alone.
After 5 days 20 ~l RPMI 1640 medium containing 0.5 ~Ci 3H thymidine was added per well. The incubation was continued for 16 hours and then the cells were harvested. The incorporation of thymidine was measured by liquid scintillation counting. The results are stated as a stimulation index (SI, i.e. cpm in the presence of the antigen divided by cpm in the absence of the antigen i.e. medium alone).
The proliferation reactions of lymphocytes from IDDM
patients and control persons towards the individual antigens are summarized in tables 1 and 2.
S.I.=cpm PBL stimulated/
cpm PBL non-stimulated IDDM base value PPD rhGAD 65 HSA
patients cpm 10 ~g/ml 10 ~g/ml 10 ~g/ml 1 730 17.9 2.6 1.1 2 833 16.7 8.8 1.2 3 639 23 6.6 0.9 4 328 28.9 0.8 2.9 1211 10.6 1.9 0.5 6 1041 11.9 1.8 2.2 7 520 19.8 7.2 1.0 8 412 2.7 8.3 0.8 CA 0222~14~ 1997-12-18 S.I.=cpm PBL stimulated/
cpm PBL non-stimulated healthy base value PPD rhGAD 65 HSA
persons cpm 10 ~g/ml 10 ~g/ml 10 ~g/ml 1 1108 25 1.8 0.7 2 1035 2.1 3.0 0.5 3 390 6.5 2.7 1.1 4 578 6.2 0.9 1.2 243 1.3 0.9 1.9 6 375 6.3 2.5 2.6 7 648 12.5 4.2 0.8 8 730 20.6 4.2 0.5 9 542 0.8 0.9 2.0 gl5 0.5 2.8 2.8 11 567 90.6 3.5 2.3 12 1247 12.5 1.0 0.6 A statistical analysis of the investigation showed that the proliferation of PBL from recently diagnosed IDDM
patients with rhGAD 65 kd could be regarded as positive with an SI of more than 4.7 (mean of the control persons + 2 SD).
Hence an in vitro T cell reaction to rhGAD 65 kd was observed in 50 % of the IDDM patients (patients 2, 3, 7 and 8). Antibodies to GAD were found in patients 1, 2, 4 and 8 but not in patients 3 and 5. Patients 6 and 7 were not tested for antibodies to GAD. The control persons did not react to GAD 65 kd.
CA 0222~14~ 1997-12-18 The diabetics and control persons exhibited no statistically significant difference at all in their reaction to the controls HSA and PPD.
In vivo investigation 100 ~l rhGAD 65 kd at a concentration of 10 ~g/ml was injected intradermally into the right forearm of the eight IDDM patients from example 1.
The antigen was diluted in physiological saline pH 7.3 containing 1 % HSA. The same volume (100 ~l) HSA at 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.
For the in vivo diagnosis the formation of a nodule (specific increase of the derma thickness) at the site of administration was regarded as a positive reaction.
The diagnosis was made 48 h after the administration.
The results were confirmed and quantified by ultrasound measurements in the area of the administration site. No side reactions were observed.
The results of this investigation are shown in table 3.
IDDM PPD rhGAD 65 HSA time of patients 10 ~/ml 10 ~g/ml 10 ~g/ml evaluation 1 + ++ - 48h 2 n.t. ++ - 48h 3 n.t. + - 48h 4 - - - 48h _ 48h 6 - - - 48h 7 n.t. +++ - 48h 8 + + - 48h Five of the eight tested IDDM patients reacted positively to rhGAD 65.
CA 0222~14~ 1997-12-18 SEQUENCE PROTOCOL
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Boehringer Mannheim GmbH
(B) ROAD: Sandhofer Str. 112-132 (C) CITY: Mannheim (E) COUNTRY: DE
(F) POSTAL CODE (ZIP): 68305 (ii) TITLE OF THE INVENTION: In vivo diabetes test (iii) NUMBER OF SEQUENCES: 30 (iv) COMPUTER-READABLE FORM:
(A) DATA CARRIER: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, version #1.30 (EPA) (vi) DATA OF ORIGINAL APPLICATION:
(A) APPLICATION NUMBER: DE 19526561.0 (B) DATE OF APPLICATION: 20-July-1995 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asp Val Asn Tyr Ala Phe Leu His Ala Thr Asp Leu Leu Pro Ala Cys Asp Gly Glu Arg (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear CA 0222~14~ 1997-12-18 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Asn Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro Glu Val Lys Glu Lys (2) INFORMATION FOR SEQ ID NO: 3:
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(A) LENGTH: 20 amino acids (B) TYPE: amino acid - (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Asn Trp Glu Leu Ala Asp Gln Pro Gln Asn Leu Glu Glu Ile Leu Met His Cys Gln Thr (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Thr Leu Lys Tyr Ala Ile Lys Thr Gly His Pro Arg Tyr Phe Asn Gln Leu Ser Thr Gly CA 0222~14~ 1997-12-18 (2) INFORMATION FOR SEQ ID NO: 5:
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Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Val Thr Leu Lys Lys Met Arg (2) INFORMATION FOR SEQ ID NO: 7:
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Phe Phe Arg Met Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp Phe Leu Ile (2) INFORMATION FOR SEQ ID NO: 8:
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tA) LENGTH: 14 amino acids (8) TYPE: amino acid (C) STRAND FORM:
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Ile Leu Ile Lys Cys Asp Glu Arg Gly Lys Met Ile Pro Ser (2) INFORMATION FOR SEQ ID NO: 9:
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Leu Gly Ile Gly Thr Asp Ser Val Ile Leu Ile Lys Cys Asp (2) INFORMATION FOR SEQ ID NO: 10:
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Leu Ala Phe Leu Gln Asp Val Met Asn Ile Leu Leu Gln Tyr 1 5 lO
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(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Val Ser Tyr Gln Pro Leu Gly Asp Lys Val Asn Phe Phe Arg 1 5 lO
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Leu Ala Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr Asn Met 1 5 lO
CA 0222~14~ 1997-12-18 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Leu Leu Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gly Gly (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Val Asn Tyr Ala Phe Leu His Ala Thr Asp Leu Leu Pro Ala (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Leu Leu Gln Tyr Val Val Lys Ser Phe Asp Arg Ser Thr Lys CA 0222~l4~ l997-l2-l8 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr l 5 lO
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids ~B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Leu Glu Tyr Val Thr Leu Lys Lys Met Arg Glu Ile Ile Gly (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asn Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro CA 0222~l4~ l997-l2-l8 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Lys Ile Trp Met His Val Asp Ala Ala Trp Gly Gly Gly Leu l 5 10 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Trp Gly Gly Gly Leu Leu Met Ser Arg Lys His Lys Trp Lys l 5 10 (2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Glu Gly Tyr Glu Met Val Phe Asp Gly Lys Pro Gln His Thr l 5 10 ....
CA 0222~14~ 1997-12-18 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu Asp Met Val Gly (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids ~B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Trp Leu Thr Ser Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu 1 5 lO
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val 1 5 lO
CA 0222~l4~ l997-l2-l8 (2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
Leu Val Ser Ala Thr Ala Gly Thr Thr Val Tyr Gly Ala Phe (2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Tyr Ile Pro Pro Ser Leu Arg Thr Leu Glu Asp Asn Glu Glu (2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp Phe l 5 10 . . .
CA 0222~14~ 1997-12-18 (2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Gly Met Ala Ala Leu Pro Arg Leu Ile Ala Phe Thr Ser Glu His Ser His Phe Ser Leu Lys Lys Gly Ala Ala (2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRAND FORM:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Glu Arg Gly Lys Met Ile Pro Ser Asp Leu Glu Arg Arg Ile Leu Glu Ala Lys Gln Ly~
Claims (28)
1. Use of autoreactive substances to produce an agent for the diagnosis of cell-mediated diseases or a predisposition to cell-mediated diseases, wherein the agent is administered intradermally and the diagnosis is based on the occurrence or absence of a positive reaction at the site of application.
2. Use as claimed in claim 1 for the diagnosis of auto-immune diseases or a predisposition to auto-immune diseases.
3. Use as claimed in claim 1 for the diagnosis of tumour diseases or a predisposition to tumour diseases.
4. Use as claimed in claims 1 to 3, wherein nucleic acids, lipids, saccharides, proteins, polypeptides as well as complexes, immunogenic epitopes, fragments, analogues, mimetics or derivatives thereof as well as mixtures thereof are used as autoreactive substances optionally in a carrier-bound form.
5. Use as claimed in claim4, wherein peptides, peptide derivatives or peptide-mimetics are used as autoreactive substances optionally in the form of complexes with MHC molecules or peptide-binding derivatives thereof.
6. Use as claimed in one of the claims 1 to 5 for the diagnosis of diabetes mellitus, multiple sclerosis, rheumatoid arthritis, Basedow disease, ankylosing spondylitis, acute anterior uveitis, Good-pasture syndrome, myasthenia gravis, systemic lupus erythematodes, pemphigus vulgaris, immune thyreoiditis, sclerodermia, Crohn's disease, Sjögren syndrome, Reiter's disease, inflammatory bowel disease or Graves' disease.
7. Use as claimed in claim 6 for the diagnosis of type I diabetes (IDDM).
8. Use as claimed in claim 7, wherein glutamic acid decarboxylase (GAD) or parts thereof are used as autoreactive substances.
9. Use as claimed in claim 7 or 8, wherein human GAD 65 kd is used as the autoreactive substance.
10. Use as claimed in claim 7, wherein one or several peptides from GAD or peptide derivatives or peptide-mimetics derived therefrom are used as the autoreactive substance optionally in the form of complexes with MHC molecules or peptide-binding derivatives thereof.
11. Use as claimed in claim 10, wherein peptides or peptide derivatives are used which comprise:
(a) the amino acid sequence (I) D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R, (b) the amino acid sequence (II) S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K, (c) the amino acid sequence (III) N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T, (d) the amino acid sequence (IV) T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G, (e) the amino acid sequence (V) P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W, (f) the amino acid sequence (VI) T-Y-E-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R, (g) the amino acid sequence (VII) F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I, (h) the amino acid sequence (VIII) G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A, (i) the amino acid sequence (IX) E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K, (j) one of the amino acid sequences shown in fig. 1 or 2, (k) partial regions of the amino acid sequences shown in (a) to (i) with a length of at least 6 amino acids or/and (l) amino acid sequences which have an essentially equivalent specificity or/and affinity of binding to MHC molecules as that of the amino acid sequences shown in (a) to (j).
(a) the amino acid sequence (I) D-V-N-Y-A-F-L-H-A-T-D-L-L-P-A-C-D-G-E-R, (b) the amino acid sequence (II) S-N-M-Y-A-M-M-I-A-R-F-K-M-F-P-E-V-K-E-K, (c) the amino acid sequence (III) N-W-E-L-A-D-Q-P-Q-N-L-E-E-I-L-M-H-C-Q-T, (d) the amino acid sequence (IV) T-L-K-Y-A-I-K-T-G-H-P-R-Y-F-N-Q-L-S-T-G, (e) the amino acid sequence (V) P-R-Y-F-N-Q-L-S-T-G-L-D-M-V-G-L-A-A-D-W, (f) the amino acid sequence (VI) T-Y-E-I-A-P-V-F-V-L-L-E-Y-V-T-L-K-K-M-R, (g) the amino acid sequence (VII) F-F-R-M-V-I-S-N-P-A-A-T-H-Q-D-I-D-F-L-I, (h) the amino acid sequence (VIII) G-M-A-A-L-P-R-L-I-A-F-T-S-E-H-S-H-F-S-L-K-K-G-A-A, (i) the amino acid sequence (IX) E-R-G-K-M-I-P-S-D-L-E-R-R-I-L-E-A-K-Q-K, (j) one of the amino acid sequences shown in fig. 1 or 2, (k) partial regions of the amino acid sequences shown in (a) to (i) with a length of at least 6 amino acids or/and (l) amino acid sequences which have an essentially equivalent specificity or/and affinity of binding to MHC molecules as that of the amino acid sequences shown in (a) to (j).
12. Use as claimed in one of the claims 5, 10 or 11, wherein the peptides or peptide derivatives have a length of at least 8 amino acids.
13. Use as claimed in claim 12, wherein the peptides or peptide derivatives have a length of at least 10 amino acids.
14. Use as claimed in one of the claims 5, 10 or 11, wherein the peptides or peptide derivatives have a length of up to 25 amino acids.
15. Use as claimed in one of the previous claims, wherein the positive reaction is determined at a time point of at least 24 h after the administration.
16. Use as claimed in claim 15, wherein the positive reaction is determined within a time period of 24 h to 1 week after the administration.
17. Use as claimed in one of the previous claims, wherein a positive reaction is determined by the occurrence of a nodule at the site of administration.
18. Use as claimed in claim 16, wherein the positive reaction is determined by visual observation of the site of administration or/and palpitation of the respective skin area.
19. Use as claimed in claim 16, wherein the positive reaction is determined quantitatively preferably by ultrasound or by photographic methods.
20. Use as claimed in one of the previous claims, wherein a positive reaction is determined by detecting substances which are released by infiltrating cells at the site of administration.
21. Use as claimed in claim 20, wherein cytokines are determined.
22. Use as claimed in one of the previous claims, wherein additional determinations are carried out with positive or/and negative controls.
23. Use as claimed in one of the previous claims, wherein the autoreactive substances are administered together with an adjuvant.
24. Use as claimed in one of the previous claims, wherein the administration is carried out in a device which comprises at least one chamber for holding a preparation of autoreactive substances, a chamber for holding a preparation for the positive control and a chamber for holding a preparation for the negative control.
25. Use as claimed in one of the previous claims additionally comprising the determination or isolation of a specific autoreactive T cell subpopulation in which a tissue sample from the area of the site of administration is contacted with an autoreactive substance, the T cells that react with the autoreactive substance are identified and they are optionally separated from other T cells.
26. Method for the in vivo diagnosis of cell-mediated diseases or a predisposition to cell-mediated diseases, wherein an autoreactive substance is administered intradermally and the diagnosis is based on the occurrence or absence of a positive reaction at the site of application.
27. Reagent kit for the in vivo diagnosis of cell-mediated diseases or a predisposition thereto comprising:
(a) at least one autoreactive substance in the form of a pharmaceutically acceptable composition, (b) optionally at least one control substance in the form of a pharmaceutically acceptable composition (c) device for the intradermal administration of the autoreactive substance and the control substance and (d) optional device for the diagnostic evaluation of the test result.
(a) at least one autoreactive substance in the form of a pharmaceutically acceptable composition, (b) optionally at least one control substance in the form of a pharmaceutically acceptable composition (c) device for the intradermal administration of the autoreactive substance and the control substance and (d) optional device for the diagnostic evaluation of the test result.
28. Reagent kit as claimed in claim 27, wherein the compositions for the autoreactive substance and the control substance contain adjuvants.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19526561.0 | 1995-07-20 | ||
| DE19526561A DE19526561A1 (en) | 1995-07-20 | 1995-07-20 | In vivo diabetes test |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2225145A1 true CA2225145A1 (en) | 1997-02-06 |
Family
ID=7767366
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002225145A Abandoned CA2225145A1 (en) | 1995-07-20 | 1996-07-19 | In vivo diabetes test |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0839058A2 (en) |
| JP (1) | JPH11509538A (en) |
| KR (1) | KR19990035757A (en) |
| CN (1) | CN1191493A (en) |
| AU (1) | AU6658296A (en) |
| CA (1) | CA2225145A1 (en) |
| DE (1) | DE19526561A1 (en) |
| IL (1) | IL122816A0 (en) |
| NO (1) | NO980252D0 (en) |
| WO (1) | WO1997003704A2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997036618A1 (en) * | 1996-04-01 | 1997-10-09 | Chugai Seiyaku Kabushiki Kaisha | Diagnostic composition for diseases accompanied by autoimmune reactions caused by 65k-glutamic acid decarboxylase |
| 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 |
| KR100681339B1 (en) * | 2004-12-23 | 2007-02-15 | 최성환 | A briquet boiler |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3626054A (en) * | 1967-10-25 | 1971-12-07 | Bard Hamilton Co Inc | Lupus erythematosus skin test |
| US5674978A (en) * | 1990-09-21 | 1997-10-07 | The Regents Of The University Of California | Peptides derived from glutamic acid decarboxylase |
| CA2140591A1 (en) * | 1994-01-20 | 1995-07-21 | Josef Endl | Antigen-specific, activated t lymphocytes, detection and use |
| DE19525784A1 (en) * | 1995-07-14 | 1997-01-16 | Boehringer Mannheim Gmbh | Autoreactive peptides from human glutamine decarboxylase (GAD) |
-
1995
- 1995-07-20 DE DE19526561A patent/DE19526561A1/en not_active Withdrawn
-
1996
- 1996-07-19 CA CA002225145A patent/CA2225145A1/en not_active Abandoned
- 1996-07-19 KR KR1019980700413A patent/KR19990035757A/en not_active Application Discontinuation
- 1996-07-19 EP EP96926371A patent/EP0839058A2/en not_active Withdrawn
- 1996-07-19 WO PCT/EP1996/003192 patent/WO1997003704A2/en not_active Application Discontinuation
- 1996-07-19 CN CN96195689A patent/CN1191493A/en active Pending
- 1996-07-19 IL IL12281696A patent/IL122816A0/en unknown
- 1996-07-19 AU AU66582/96A patent/AU6658296A/en not_active Abandoned
- 1996-07-19 JP JP9506304A patent/JPH11509538A/en active Pending
-
1998
- 1998-01-20 NO NO980252A patent/NO980252D0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH11509538A (en) | 1999-08-24 |
| WO1997003704A2 (en) | 1997-02-06 |
| IL122816A0 (en) | 1998-08-16 |
| KR19990035757A (en) | 1999-05-25 |
| AU6658296A (en) | 1997-02-18 |
| WO1997003704A3 (en) | 1997-06-05 |
| DE19526561A1 (en) | 1997-01-23 |
| NO980252L (en) | 1998-01-20 |
| CN1191493A (en) | 1998-08-26 |
| EP0839058A2 (en) | 1998-05-06 |
| NO980252D0 (en) | 1998-01-20 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |