EP0874901A2 - Reactifs diagnostiques associes au diabete - Google Patents

Reactifs diagnostiques associes au diabete

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Publication number
EP0874901A2
EP0874901A2 EP96941561A EP96941561A EP0874901A2 EP 0874901 A2 EP0874901 A2 EP 0874901A2 EP 96941561 A EP96941561 A EP 96941561A EP 96941561 A EP96941561 A EP 96941561A EP 0874901 A2 EP0874901 A2 EP 0874901A2
Authority
EP
European Patent Office
Prior art keywords
iar
leu
polypeptide
sequence
iddm
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96941561A
Other languages
German (de)
English (en)
Inventor
Catherine Jane Pallen
Lin Cui
Wei-Ping Yu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Molecular and Cell Biology
Original Assignee
Institute of Molecular and Cell Biology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9526036.0A external-priority patent/GB9526036D0/en
Priority claimed from GBGB9605710.4A external-priority patent/GB9605710D0/en
Priority claimed from GBGB9620265.0A external-priority patent/GB9620265D0/en
Application filed by Institute of Molecular and Cell Biology filed Critical Institute of Molecular and Cell Biology
Publication of EP0874901A2 publication Critical patent/EP0874901A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the present invention relates to an autoantigen associated with type I diabetes (Insulin Dependent Diabetes Mellitus (IDDM)) .
  • IDDM Insulin Dependent Diabetes Mellitus
  • This autoantigen is useful in the diagnosis of IDDM.
  • IDDM is an autoimmune disease. It is caused by the autoimmune system-mediated destruction of pancreatic islet beta-cells. This leads to an imbalance in the levels of the hormones insulin and glucagon which disrupts glucose metabolism, leading to the well-known symptoms of diabetes, such as high glucose levels in urine. As IDDM is an autoimmune disease, autoantigens can often be detected in the blood of individuals suffering from IDDM.
  • IA-2 Human IA-2 (hIA-2) was originally cloned in a truncated form, and given the name ICA-512 (Islet Cell Antigen 512) (Rabin et al (1994) J. Immunol. 152, 3183) . Subsequently, the complete DNA and amino acid sequences were determined and the protein was given the name IA-2 (Lan et al (1994) DNA and Cell Biology vol 13, No 5, pp 505-514) .
  • ICA-512 Islet Cell Antigen 512
  • a mouse homologue (mIA-2) is also known (Lu et al (1994) Biophys. Biochem. Res. Commun. vol 204, No 2) . Its cDNA shows 92% identity to that of hIA-2. Two possible rat homologues have also been identified. The first of these is known as PTPLP (Kambayashi et al (1995) Biochem. J. 306, 331-335) and has 79.3% identity to hIA-2 at the amino acid level. The second has a sequence identified from a series of overlapping clones (Passini et al (1995), Proc. Natl. Acad. Sci. USA 92, 9412-9416) . This DNA shows 93% homology to the corresponding mIA-2 sequence and 86% homology to the corresponding hIA-2 sequence.
  • hIA-2 is the 40kDA autoantigen associated with IDDM.
  • Other putative IDDM-associated autoantigens inc.ude glutamic acid decarboxylase (GAD) and the islet cell autoantigens (ICA) known as ICA-12, ICA-13, ICA-208, ICA- 302, ICA-313, ICA-123, ICA-525 and ICA-505 EP-A-0, 383, 129; EP-A-0, 569,800
  • GAD isot alpha-12
  • ICA-208 islet cell autoantigens
  • ICA-505 islet cell autoantigens
  • Some of these IDDM-associated antoantigens have been proposed as diagnostic reagents for screening patients for IDDM, notably GAD, ICA-512 and ICA-12 (EP-A-0 569, 800) .
  • IAR Islet Cell Antigen-Related
  • IAR-PTP Islet Cell Related Protein Tyrosine Phosphatase
  • the IAR sequence identified by the inventors shows around 70% nucleotide and amino acid sequence homology to the IA-2 sequence in the intracellular region. This provides further evidence that IAR is an IDDM-associated autoantigen. In the extracellular region, the nucleotide homology to IA-2 is about 35% and the amino acid sequence homology is about 20%.
  • IAR protein tyrosine phosphatase
  • PTP protein tyrosine phosphatase
  • IAR-PTP Islet Cell Antigen-Related Protein Tyrosine Phosphatase
  • IAR has three characteristics of an IDDM-associated autoantigen: (i) a characteristic structure, in terms of organisation into intra- and extra- cellular domains and the sequence of its intracellular domain; (ii) PTP activity; and (iii) a characteristic tissue distribution. IAR is also reactive with IDDM patient sera. A study with a group of IDDM patients shows that recombinant IAR is precipitated by autoantibodies present in more than 50% of the serum samples while IAR does not react with any sera from control patients.
  • IAR is distinct from previously isolated autoantigens; in particular, its sequence identity to hIA-2, mIA-2 and PTPLP is too low for it to be any of these proteins, especially as it has only 20% homology to hIA-2 at the amino acid level in the extracellular region. Further, IAR and IA-2 can distinguish different populations of IDDM autoantibodies.
  • IAR Ab IAR Ab
  • IA-2 Ab IA-2 antibodies
  • Antibodies to IAR and IA-2 measured by radiobinding assay, were correlated in recent-onset IDDM patients. Some individuals had IAR Ab in the absence of IA-2 Ab, and others had IA-2 Ab in the absence of IAR Ab. Therefore, IAR and IA-2 can distinguish different populations of IDDM autoantibodies since they identify overlapping but non-identical sets of IDDM patients.
  • the diagnostic sensitivity for IDDM was similar for IAR Ab and IA-2 Ab. Accordingly, IAR is clearly a useful predictive tool for the diagnosis of IDDM, either as an alternative IA-2 or in combination with IA-2.
  • IAR Ab are signi icantly associated with progression to IDDM while IA-2 Ab were not. This is significant because it establishes that IAR Ab are likely to be better predictors of IDDM than IA-2 Ab in first degree relatives of IDDM sufferers, who are a high risk group for contracting IDDM. Therefore, the IAR of the invention is likely to be effective in predicting the onset of IDDM in high-risk groups such as first-degree relatives. Further, the inventors have found that the cytoplasmic (intracellular) domain of IAR is itself a useful predictor of IDDM. It is likely to be as useful as, or more useful than, complete IAR.
  • EST brain Expressed Sequence Tag
  • This is EST03250 (Adams et al; Nature Genetics Vol. 4, July 1993) .
  • the length of the EST is 313 nucleotides. It was identified during the partial sequencing of over 3400 ESTs from human brain cDNA, in an attempt to demonstrate the diversity of RNA transcripts present in brain cells. IAR of course differs in terms of sequence from this EST because it has a different, longer, sequence. Also, it is unknown whether or not the EST encodes a functional autoantigen.
  • IAR sequences of the invention can be used in the diagnosis of IDDM, for example to screen for or predict the onset, presence or development of IDDM.
  • IDDM individuals who have, or are predisposed to develop, IDDM can be expected to have autoantibodies to the autoantigenic IAR in their blood.
  • blood, serum or other samples can be taken and contacted with the IAR of the invention.
  • the anti-IAR autoantibodies will recognise the exogenous IAR and this recognition can be detected, for example by detecting the IAR/anti-IAR complex thus formed.
  • the present invention provides:
  • a nucleic acid encoding a polypeptide that has the properties of an Insulin-dependent diabetes mellitus (IDDM) -associated autoantigen which nucleic acid comprises: (a) the coding sequence of SEQ. ID. No. 1, 10 or 12 and/or the sequence complementary thereto;
  • determining whether or not said recognition occurs a method of producing a polypeptide as defined above which comprises culturing a cell as defined above under conditions which permit the expression of the polypeptide; and recovering the polypeptide; use of a polypeptide as defined above in diagnosing IDDM; a diagnostic test kit for diagnosing IDDM which comprises a polypeptide as defined above, and means for determining whether or not the polypeptide is recognised by autoantibodies that recognise IAR.
  • Human multiple tissue Northern blots are from Clontech and the source of the mRNA in each lane is indicated at the bottom of the figure.
  • the r75 probe was labelled with 32 P- dCTP (Amersham) using the High Prime DNA Labeling Kit (Boehringer Mannheim) .
  • Hybridizations were carried out at 65°C overnight in 0.5M sodium phosphate (pH 6.8) , 7% SDS, 15% forma ide, ImM EDTA. After washing 3 times with 50mM sodium phosphate (pH 6.8) , 1% SDS for 40 min at 65°C, the blots were exposed to film at -70°C.
  • Figure 2 Schematic depiction of IAR clones and predicted protein product.
  • IAR protein product aligned with the IA-2 protein.
  • the predicted partial IAR polypeptide (top) translated from and aligned with the clones shown in A is depicted in alignment with the IA-2 protein (Lan et al . , 1994) .
  • the % amino acid similarity between the intracellular regions of IAR and IA-2 is shown, as is that: between the partial extracellular region of IAR and the corresponding portion of IA-2.
  • TM transmembrane region.
  • Human multiple tissue Northern blots (different membranes from those used for Fig.l) are from Clontech and the source of the mRNA in each lane is indicated at the bottom of the figure.
  • the IA-2 probe (nucleotides 1590-1906, coding for a portion of the extracellular region, the transmembrane region, and a small portion of the intracellular region; and non-homologous to IAR sequence) was labelled and hybridization carried out as described in the legend to Fig. 1. The position of the IA-2 transcript is indicated by the arrow at the right of the figure.
  • the ⁇ -actin signal (position indicated by the parentheses to the right of each panel) results from a previous hybrization with a / 3-actin probe, and could not be completely stripped from the membranes before reprobing with IA-2.
  • the positions of molecular size markers (in kb) are shown to the left of each blot.
  • the -actin signal (position indicated by the parentheses to the right of each panel) results from a previous hybrization with a -actin probe, and could not be completely stripped from the membranes before reprobing with IAR.
  • the positions of molecular size markers (in kb) are shown to the left of each blot.
  • FIG. 3 Comparison of IAR and IA-2 transcripts in human brain and pancreas.
  • the indicated lanes from the Northern analyses of IA-2 (Fig. 3) and IAR expression (Fig.4) are shown together here for the purpose of direct comparison.
  • the same membrane was hybridized with the IA-2 probe, then stripped and rehybridized with the IAR probe as described in the legends to Figs. 3 and 4.
  • Figure 6. Purification and phosphatase activity of the intracellular regions of IAR and IA-2.
  • IA-2 open arrow
  • IAR polypeptides closed arrows obtained after expression, affinity purification and protease cleavage of GST-fusion proteins.
  • Lane 1 IA-2 obtained from expression of pGEX-3C-IA-2 and cleavage with 3C protease;
  • lane 2 IA-2 obtained from expression of pGEX- KG-IA-2 and cleavage with thrombin;
  • lane 3 IAR obtained from expression of pGEX-3C-IAR and cleavage with 3C protease;
  • IAR obtained from expression of pGEX-KG- IAR and cleavage with thrombin.
  • the positions of molecular size markers (kD) are shown to the right of the panel.
  • IAR Phosphatase activity of IAR.
  • the IAR was produced as n lane 3 of panel A. IAR was added to a reaction mixture containing 50mM sodium acetate (pH 4.5), 0.5 mg/ml BSA, 0.5mM dithiothreitol and 5mM para-nitrophenyl phosphate (PNPP) and incubated at 30°C. At the times indicated, aliquots of the reaction (each containing 16 ⁇ g of purified IAR as measured by Bradford analysis) were removed and mixed with 13% KHP0 4 to terminate the reaction. The absorbance of the stopped reaction was monitored at 405 nm and is a measure of the amount of para-nitrophenol product formed by dephosphorylation of PNPP. All reactions were performed in duplicate and the absorbances averaged. Non- enzymatic hydrolysis of PNPP was accounted for by performing appropriate control reactions without added IAR, and this absorbance was subtracted from that attained in the presence of enzyme.
  • IA-2 Phosphatase activity of IA-2.
  • the IA-2 was produced as in lane 1 of panel A, and phosphatase assays carried out as described above for IAR except that reactions contained 2 ⁇ _M PNPP. Each reaction aliquot removed at the indicated times contained 20 ⁇ g of purified IA-2 as measured by Bradford analysis.
  • IAR polypeptide amino acids 646-1015
  • Lane 1 molecular size markers (sizes in kDa shown at left)
  • lane 2 IAR after cleavage with 3C protease.
  • the two uppermost bands in lane 2 are bacterial proteins which are often present after purification of various GST-fusion proteins, including IAR(C945S) .
  • the purified IAR was quantitated by densitometric scanning of Coomassie Blue-stained IAR protein band on SDS-PAGE (indicated by arrow in A) alongside known amounts of protein standards.
  • the invention provides nucleic acids encoding the IDDM-associated autoantigen known as IAR or IAR-PTP and other related polypeptides having the properties of IDDM- associated antigens.
  • Nucleic acids of the invention include the partial IAR-PTP cDNA having the sequence shown in SEQ ID No. 1, the complete cDNA sequence (SEQ ID. No 10) and the cDNA sequence of the cytoplasmic region of the IAR
  • nucleic acids of SEQ ID Nos. 1 and 10 are preferred nucleic acids of the invention.
  • the nucleic acid of the invention includes a sequence encoding the cytoplasmic domain of I ⁇ , also known as the intracellular domain (SEQ ID No. 12) .
  • the DNA sequence encoding the cytoplasmic domain is given in SEQ ID No. 12, along with the encoded ammo acid sequence of the cytoplasmic domain (SEQ ID NO. 13) .
  • the nucleotide sequence of SEQ ID NO. 12 corresponds to the nucleotides which encode ammo acids 646 to 1015 of SEQ 3D NO.11.
  • the sequence of the invention may consist of the sequence encoding the cytoplasmic domain or it may include other IAR sequences or non-IAR sequences at one or both ends.
  • nucleic acids are included within the scope of the invention.
  • Nucleic acids of the invention may have substantially the sequence of SEQ ID No. 1, 10 or 12.
  • Nucleic acids of the invention may consist essentially of the sequence of SEQ ID No. 1, 10 or 12.
  • the nucleic acid sequences of the present invention are preferably DNA, for example, cDNA or genomic DNA, though they may be RNA. It will be appreciated by those of skill in the art that, in RNA sequences according to the invention, the T residues shown in SEQ ID No. 1, 10 or 12 will be replaced by U. Further, the invention provides both single-stranded and double-stranded nucleic acids.
  • the nucleic acids of the present invention are not limited to the nucleic acids of SEQ. ID. No. 1, 10 and 12.
  • the nucleic acids of the invention include nucleic acids having sequences that are closely related to these sequences; these nucleic acids encode polypeptides of the invention.
  • nucleic acids of the invention may, for example, be prepared by altering that of SEQ ID No. 1, 10 or 12 by any conventional method, or isolated from any organism or made synthetically. Such alterations, isolations or syntheses may be performed by any suitable method, for example by the methods of Sambrook et al : (Molecular Cloning: A Laboratory Manual; 1989) .
  • nucleic acids of the invention may include substitutions, deletions, insertions, or extensions that distinguish them from the nucleic acid of SEQ. ID. No. 1, 10 or 12 as long as these do not destroy the ability of the nucleic acid to encode a polypeptide that functions as an IDDM-associated autoantigen.
  • a substitution, deletion or insertion may suitably involve one or more nucleotide positions.
  • nucleic acid sequences of the invention may differ from those of SEQ ID No. 1, 10 or 12 at any number of nucleotide positions, for example as a result of deletion, substitution, insertion or extension, as long as they encode a polypeptide of the invention.
  • Sequences of the invention may, for example, differ from that of SEQ ID No. 1, 10 or 12 at 1, 2, 3, 4, 5, 6 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50 or 50 to 100 nucleotide positions.
  • sequences of the invention include sequences that are capable of selective hybridisation to either strand of the sequence of SEQ. ID. No. 1, 10 or 12 and that encode a polypeptide of the invention.
  • sequences capable of selectively hybridizing to the DNA of SEQ. ID. No. 1, 10 or 12 will generally be up to 80%, preferably at least 80%, more preferably at least 90%, at least 95% or 99% homologous to the DNA of SEQ. ID. No. 1, 10 or 12.
  • homology will preferably apply over a region of at least 20, preferably at least 50, for instance 100, 500 or 1000 or more contiguous nucleotides.
  • Such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al (1989) : Molecular Cloning: A Laboratory Manual) .
  • suitable conditions include 0.2 x SSC at 60°C.
  • suitable conditions include 2 x SSC at 60°C.
  • sequences that differ from those defined above and because of the degeneracy of the genetic code encode the same polypeptide of the invention.
  • the invention provides degenerate variants of the sequence of SEQ ID No
  • nucleic sequences of the invention may be of any length as long as they encode a polypeptide of the invention.
  • a nucleic acid sequence according to the invention will typically comprise one or more sequences including one or more of the antigenic epitopes of IAR-PTP.
  • a nucleic acid sequence according to the invention may be a contiguous fragment of the sequence of SEQ. ID. No. 1, 10 or 12 or a sequence that is related to it in any of the ways described above.
  • nucleic acids of the invention may comprise DNA sequences that are not contiguous in the sequence of SEQ. ID. No. 1, 10 or 12. These sequences may be fragments of the sequence of SEQ. ID. No.
  • Nucleic acid sequences of the invention will preferably comprise at least 50 bases or base pairs, for example 50 to 100, 100 to 500, 500 to 1000, or 1000 to 2000 bases or base pairs.
  • SEQ. ID. No. 1 is a partial cDNA sequence of IAR-PTP.
  • the invention also includes sequences extended at either the 3' end or the 5' end, or both ends, by further nucleic acid sequence. 5' extensions are preferred.
  • This sequence may be of any nature and of any suitable length.
  • an extension may comprise up to 10, up to 20, up to 50, up to 100, up to 200, up to 500, up to 1000, up to 1500, up to 2000, up to 2500 or up to 3000 or more bases or base pairs.
  • an extension may comprise the sequence that is contiguous with the sequence of SEQ. ID. No. 1 in the complete IAR-PTP cDNA (SEQ ID. No. 10) .
  • nucleic acids of the invention include complete IAR-PTP cDNAs and fragments thereof which include SEQ. ID. No. 1, particularly human cDNAs and fragments. Also, the invention provides nucleic acids encoding fusion proteins comprising polypeptides of the invention.
  • a particularly preferred nucleic acid of the invention has the sequence of SEQ ID No. 10, the complete IAR cDNA sequence.
  • Another particularly preferred nucleic acid of the invention has the sequence of the cytoplasmic domain of IAR, SEQ ID NO. 12.
  • the extension or extensions could have any other sequence.
  • the sequence of SEQ. ID. No. 1, 10 or 12 may be interrupted in one or more places by non- coding sequence.
  • the non-coding sequence may be introns that occur in the IAR-PTP genomic DNA.
  • Further preferred nucleic acids of the invention are thus IAR-PTP genomic DNAs, and fragments thereof, that include the sequence of SEQ ID No. 1, 10 or 12, particularly human genomic DNAs and fragments thereof.
  • Three preferred polypeptides of the inventicn have the sequences shown in SEQ. ID. No. 2, (partial amino acid sequence of IAR) , SEQ. ID. No. 11 (complete amino acid sequence of IAR) and 13 (amino acid sequence of cytoplasmic domain of IAR) .
  • SEQ ID No. 13 corresponds to amino acids 646 to 1015 of SEQ ID No. 11.
  • the polypeptides of the invention are not limited to the polypeptides of SEQ. ID. No. 2, 11 and 13. Rather, the polypeptides of the invention also include polypeptides with sequences closely related to those of SEQ. ID. No. 2, 11 and 13 that have suitable properties. Thus, the polypeptides of the invention typically have substantially the sequence of SEQ ID No. 2, 11 or 13.
  • Polypeptides of the invention may have a sequence which consists essentially of the sequence of SEQ ID No. 2 , 11 or 13.
  • Polypeptides of the invention may have a sequence which is substantially the sequence of SEQ. ID. No. 2, 11 or 13.
  • polypeptides of the invention are encoded by the nucleic acids of the invention, as described herein.
  • the polypeptides of the invention, IAR-PTP and closely related polypeptides are IDDM-associated autoantigens, or have the properties of IDDM-related autoantigens.
  • the polypeptides of the invention are typically capable of provoking an autoimmune response in individuals who have, or are predisposed to have, or are likely to develop IDDM.
  • the polypeptides of the invention may provoke the production of IDDM-associated autoantibodies in such individuals. Also, or alternatively, they may provoke other elements of the autoimmune response.
  • polypeptides of the invention are typically recognised by autoantibodies that already present in the sera of such individuals, and are capable of recognising the natural IAR-PTP which contributes to the autoimmune response in these individuals.
  • polypeptides of the invention may have PTP activity.
  • Polypeptides of the invention having sequences related to the sequence may be prepared by altering the polypeptide of SEQ. ID. No. 2, 11 or 13 by any conventional method, or isolated from any organism or made synthetically. Such alterations, isolations or syntheses may be performed by any conventional method, for example by the methods of Sambrook et al (Molecular cloning: A Laboratory Manual; 1989) .
  • polypeptides related to those of SEQ. ID. No. 2, 11 and 13 may be prepared by modifying DNA sequences as shown in SEQ. ID. No. 1, 10 or 12 and expressing them recombinantly.
  • Polypeptides of the invention may include substitutions, deletions, insertions, or extensions that distinguish them from the polypeptide of SEQ. ID. No. 2, 11 or 13 as long as these do not destroy the ability of the polypeptide to function as an IDDM-associated autoantigen.
  • a substitution, deletion or insertion may suitably involve one or more amino acids, typically from one to five, one to ten or one to twenty amino acids.
  • a substitution, deletion or insertion may involve one, two, three, four, five, eight, ten, fifteen, or twenty amino acids.
  • Polypeptides of the invention are substantially homologous to that of SEQ. ID. No. 2, 11 or 13.
  • a polypeptide of the invention has up to 80%, preferably at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the polypeptide of SEQ. ID. No. 2, 11 or 13.
  • sequences of the invention should be preserved in a sequence of the invention. Such sequences will generally be similar in charge, hydrophobicity and size to that of SEQ. ID. No. 2, 11 or 13. Thus, substitution will typically be conservative. Examples of conservative substitutions that do not greatly affect the physicochemical nature of amino acid sequences are those in which an amino acid from one of the following groups is substituted by a different amino acid from the same group: H, R and K I, L, V and M A, G, S and T D, E, P and N. As far as extensions are concerned, a sequence of one or more amino acids may be provided at either or both of the C- and N- termini of the sequence of SEQ. ID. No. 2, 11 or 13 or a sequence related to it in any of the ways defined herein.
  • An extension may comprise up to 5, up to 10, up to 20, up to 50, up to 100, up to 200, up to 300, up to 400, up to 500 or more amino acids.
  • an extension may comprise one, two, three, four, five, up to ten, up to 20, up to 30, up to 50, up to 100, up to 200 or more amino acids.
  • extensions may include amino acid sequences which are found in the native complete IAR-PTP protein.
  • preferred polypeptides of the invention include complete IAR-PTP proteins and polypeptide fragments thereof that include the amino acid sequence of SEQ. ID. No. 2, particularly human proteins and fragments thereof.
  • the invention provides fusion proteins including polypeptides of the invention.
  • a particularly preferred polypeptide of the invention has the sequence of SEQ. ID. No. 11, the sequence encoded by the complete IAR cDNA (SEQ. ID. No. 10) .
  • a further particularly preferred polypeptide has the sequence of SEQ ID NO. 13, which is the sequence of the cytoplasmic domain of IAR .
  • a polypeptide of the invention may be subjected to one or more chemical modifications, such as glycosylation, sulphation, COOH-amidation or acylation.
  • a polypeptide of the invention may comprise multiple copies of the sequence of SEQ. ID. No. 2, 11 or 13 or a sequence related to it in any of the ways defined herein.
  • a polypeptide of the invention may be of any length as long as it has the ability to function as an IDDM- associated autoantigen.
  • polypeptides of the invention preferably comprise one or more epitopes of IAR- PTP and therefore retain the ability to provoke an autoimmune response and/or be recognised by preexisting IDDM autoantibodies.
  • Such polypeptides of the invention could be considerably smaller than the polypeptide of SEQ. ID. No. 2.
  • Polypeptides according to the invention may include one or more fragments of the sequence of SEQ. ID. No. 2, 11 or 13 or fragments that are related to SEQ. ID. No. 2 in any of the ways described above. Such fragments typically comprise at least 10, for example 10 to 20 or 20 to 50, amino acids.
  • polypeptides of the invention may comprise amino acid sequences that are not contiguous in SEQ. ID. No. 2, 11 or 13. These amino acid sequences may be identical to parts of the native amino acid sequence or related to such parts in any of the ways described herein.
  • polypeptides of the invention may comprise two or more epitopes of IAR-PTP, optionally separated by IAR sequence or non-IAR sequence.
  • Polypeptides according to the invention preferably comprise at least 10 amino acids, for example 10 to 20, 20 to 50, 50 to 100, 100 to 200, or 200 to 500 or 500 to 1000 amino acids.
  • Polypeptides and nucleic acids according to the invention may be substantially isolated, isolated, purified or substantially purified. For example, they may be present in preparations, for example solutions, vvhich have undergone one or more purification steps. Optionally, they may be completely pure. They may be present in preparations, for example solutions, which, apart from the nucleic acid and/or polypeptide of the invention consist essentially of solvents and/or biologically inert carriers.
  • Polypeptides of the invention in substantially purified form will generally comprise the polypeptide in a preparation in which more than 90%, eg. up to 95£, up to 98% or up to 99% of the peptide material in the preparation is that of a polypeptide or polypeptides according to the invention.
  • nucleic acids and polypeptides of the invention were originally derived from the human genome.
  • nucleic acid sequences and/or polypeptides of the invention may also be obtained from other eukaryotic genomes, especially other mammalian genomes. They may be obtained either by conventional cloning techniques or by probing genomic or cDNA libraries with nucleic acid sequences according to the invention. This can be done by any conventional method, such as the methods of Sambrook et al (Molecular Cloning: A Laboratory Manual; 1989) .
  • the complete cDNA and/or genomic DNA sequences of human IAR-PTP can be determined in this way, and the complete amino acid sequence of IAR-PTP can be determined.
  • a nucleic acid sequence according to the invention may be included within a vector, suitably a replicable vector or an expression vector, for instance a replicable expression vector.
  • a replicable vector typically comprises an origin of replication so that the vector can be replicated in a host cell such as a bacterial host cell or a yeast host cell.
  • a suitable vector will also typically comprise the following elements, usually in a 5' to 3 ' arrangement: a promoter for the directing expression of the nucleic acid sequence of the invention and optionally a regulator of the promoter, a translational start codon, a nucleic acid sequence according to the invention.
  • the vector may also contain one or more selectable marker genes, for example an ampicillin resistance gene for the identification of bacterial transformants or a marker gene that allows selection of yeast or other eukaryotic transformants.
  • the vector may also comprise an enhancer for the promoter.
  • the vector may also comprise a polyadenylation signal operably linked 3' to the nucleic acid of the invention.
  • the vector may also comprise a transcriptional terminator 3' to the sequence encoding the polypeptide of the invention.
  • the vector may also comprise one or more introns or other coding sequences 3' to the nucleic acid sequence of the invention.
  • the intron or introns may be from the human genome (the organism from which the sequences of the invention were originally derived) or the host organism which is to be transformed with the vector or from another eukaryotic organism.
  • the nucleic acid sequence of the invention is operably linked to a promoter capable of expressing the sequence.
  • "Operably linked” refers to a juxtaposition wherein the promoter and the nucleic acid sequence encoding the polypeptide of the invention are in a positional relationship permitting the coding sequence to be expressed under the control of the promoter.
  • elements such as non-coding sequence 3' or 5' to the coding sequence. These elements may be native either to the human genome or, for example, to the organism from which the promoter sequence is derived. Alternatively, the said element or elements may be native to neither the organism from which the promoter sequence is derived nor to the human genome. Such sequences can be included in the vector if they enhance or do not impair the correct control of the coding sequence by the promoter.
  • An expression vector may be of any type.
  • the vector may be in linear or circular form.
  • it may be a plasmid or viral vector.
  • suitable vectors comprising nucleic acid sequences encoding polypeptides of the invention starting with widely available vectors which will be modified by genetic engineering techniques such as those described by Sambrook et al (Molecular Cloning: A Laboratory Manual; 1989) .
  • any promoter capable of directing expression of a sequence of the invention may be operably linked to the nucleic acid sequence of the invention.
  • suitable promoters include yeast: promoters, mammalian promoters, viral promoters and bacterial promoters. Such promoters may be constitutive cr regulable.
  • Some suitable promoters include the cytomegalovirus (CMV) early promoter, the SV40 promoter, the mouse mammary tumour virus promoter, the human elongation factor 1 ⁇ -P promoter (EF-l ⁇ -P) , the SR ⁇ promoter and the mouse metallothienein gene I (mMTl) promoter.
  • CMV cytomegalovirus
  • SV40 promoter the SV40 promoter
  • the mouse mammary tumour virus promoter the human elongation factor 1 ⁇ -P promoter
  • EF-l ⁇ -P human elongation factor 1 ⁇ -P promoter
  • SR ⁇ promoter mouse metallothienein gene I
  • nucleic acid sequences according to the invention will be inserted into such vectors in a sense orientation.
  • nucleic acid sequences according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production antisense RNA.
  • Antisense RNA may also be produced by synthetic means. Such antisense RNA may be used in a method of controlling the levels of the polypeptide of SEQ. ID. No. 2 or 11 or 13 or a protein encoded by a related nucleic acid sequence in a cell. Such a protein is, for example, a complete IAR-PTP protein.
  • antisense sequences of the invention can form the basis of methods of gene therapy aimed at suppressing the autoantigenicity of IAR-PTP.
  • Vectors according to the invention may be used in vitro, for example for the production of RNA hybridisabl €: to cDNA, of the invention. Such vectors may be used to transfect or transform a host cell. Depending on the type of vector, they may be used as cloning vectors to amplify DNA sequences according to the invention or to express this DNA in a host cell.
  • a further embodiment of the invention provides host cells harbouring one or more vectors and/or nucleic acids of the invention.
  • such cells are transformed or transfected with the vectors for the replication and/or expression of nucleic acid sequences according to the invention, including the DNA of SEQ. ID. No. 1, 10 and 12.
  • the cells will be chosen to be compatible with the vector and may for example be bacterial cells, mammalian, insect or yeast cells.
  • the cells may be transformed or transfected by any suitable method, such as the methods disclosed by Sambrook et al (Molecular Cloning: A Laboratory Manual; 1989) .
  • vectors comprising nucleic acid sequences according to the invention may be packaged into infectious viral particles, such as retroviral particles.
  • the constructs may also be introduced by electroporation, calcium phosphate precipitation, biolistic methods or by contacting naked nucleic acid vectors with the cells in solution.
  • the nucleic acid may be DNA or RNA, preferably DNA, it may be single-stranded or double-stranded.
  • the vectors with which the host cells are transformed or transfected may be of any suitable type.
  • the vectors may be able to effect integration of nucleic acid sequences of the invention into the host cell genome or they may remain free in the host cell.
  • the vectors will be expression vectors, such as a retroviral vector or a DNA expression vector as defined herein.
  • the vector used for transformation construct may be a plasmid vector.
  • the transformed or transfected cells of the invention can be used in a process of production of polypeptides of the invention.
  • Such processes will typically comprise transforming or transfecting host cells with vectors comprising nucleic acid sequences according to the invention and expressing the nucleic acid sequence in these cells.
  • the nucleic acid sequence will be operably linked to a promoter capable of directing its expression in the host cell.
  • a promoter capable of directing its expression in the host cell.
  • such a promoter will be a "strong" promoter capable of achieving high levels of expression in the host cell. It may be desirable to overexpress the polypeptide according to the invention in the host cell.
  • Suitable host cells for this purpose include bacterial cells, for example E. coli cells; yeast cells; mammalian cells, for example CHO cells; and insect cells, preferably in combination with a baculovirus expression system.
  • Eukaryotic cells such as mammalian, insect and yeast cells are preferred in situations where glycosylated polypeptide products are desired.
  • the thus produced polypeptide of the invention may be recovered by any suitable method known in the art..
  • the thus recovered polypeptide may be purified or partly purified by any suitable method, for example a method according to Sambrook et al (Molecular Cloning: A Laboratory Manual) .
  • the nucleic acid sequences of the invention may be used to prepare probes and primers. These will be useful in the isolation of nucleic acid sequences having sequences similar to that of SEQ. ID. No. 1, 10 or 12, which may encode polypeptides of the invention.
  • Such probes and primers may be of any suitable length, desirably from 10 to 100, for example from 10 to 20, 20 to 50, 50 to 100 bases in length.
  • the probes and primers will correspond exactly to part of the sequence of SEQ. ID. No. 1, 10 or 12 over their entire length, although they may vary from this sequence in any of the ways described above.
  • Such probes and/or primers may be used, in particular to isolate complete IAR-PTP cDNA and/or genomic DNA sequences from the human genome; or from the genomes of other organisms .
  • the present invention also provides antibodies, preferably monoclonal antibodies, to the polypeptides of the invention. Such antibodies may be useful in methods of diagnosing IDDM, as described herein.
  • Antibodies of the invention include fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, Fab' and Fab' 2 fragments, as well as single chain antibodies.
  • the antibodies may be produced by any suitable method known in the art, such as the methods of Sambrook et al (Molecular Cloning: A Laboratory Manual; 1989) .
  • they may be prepared by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technology, for example by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter, as described above.
  • suitable host cells include bacterial (for example E. coli) , yeast, insect and mammalian cells.
  • the invention also provides nucleic acid sequences encoding antibodies of the invention, vectors comprising such sequences, cells harbouring such vectors or sequences, and methods for expressing antibodies of the invention by recombinant techniques.
  • Polyclonal antibodies may also be raised, using conventional techniques in the art, using the polypeptides of the invention.
  • polyclonal antibodies may be raised by inoculating a host animal, such as a rat, mouse or rabbit, with a polypeptide of the invention, and recovering the immune serum.
  • Antibodies according to the invention may be useful in methods of the invention, as described herein, for the diagnosis of IDDM.
  • polypeptides of the invention can be used in methods of diagnosing any aspect of IDDM.
  • they may be used in diagnosing the onset of IDDM, the presence of IDDM, or predisposition to IDDM, or in monitoring the development or progress of IDDM, ⁇ tnd in methods of predicting the possible future occurrence of IDDM.
  • individuals who have, or are predisposed to develop, IDDM can be expected to have autoantibodies to the autoantigenic IAR-PTP.
  • the IAR polypeptides of the invention are useful in methods carried out on individuals having a high likelihood of developing IDDM, for example, relations, especially close relations, more especially first: degree relations (parents and siblings), of known IDDM sufferers.
  • the IAR polypeptides of the invention are particularly useful in determining the likelihood that a close relation of an IDDM sufferer will contract IDDM. Close relations include, for example, parents, grandparents, siblings, children, grandchildren, cousins, uncles, aunts, nieces, grand aunts and uncles and grand nephews.
  • samples of biological fluid suspected of containing such autoantibodies can be taken from individuals and contacted with polypeptides of the invention.
  • the anti-IAR autoantibodies if present, will recognise the polypeptides of the invention and this recognition can be detected, for example by detecting the IAR/anti-IAR complex thus formed.
  • the methods of the invention typically comprise a step of determining whether or not recognition of the polypeptide of the invention by autoantibodies occurs in the future. Similarly, if no such recognition occurs, the test suggests that the individual does not have IDDM or is unlikely to develop it in the future.
  • Recognition of polypeptides of the invention by antibodies present in such a sample correlates with IDDM or a potential for developing IDDM.
  • the methods of the invention are thus typically methods of assaying for the presence or absence of autoantibodies that recognise IAR. They may also quantitate the amount or concentration of such autoantibodies in a sample.
  • diagnostic methods of the invention typically comprise:
  • the sample will comprise a biological fluid, preferably blood, plasma or serum.
  • the sample may be of any size.
  • the sample is contacted with a polypeptide of the invention and the presence or absence of immunoreactivity between them is determined.
  • a second IDDM- related autoantigen may be used.
  • the invention provides methods, as described above, which further comprise, in addition to steps (a) and (b) , contacting the sample with at least one further IDDM-related autoantigen, preferably an islet cell antigen (ICA) . This may be done at the same time, or at a different time to the contacting with the polypeptide of the invention.
  • ICA islet cell antigen
  • autoantigens recognise the further autoantigen or autoantigens.
  • the same sample or a different sample from the same individual may be contacted with the polypeptide of the invention and the further IDDM-associated autoantigen.
  • a further autoantigen may be, for example, hIA-2, GAD, ICA-512, ICA-12, ICA-13, ICA-208, ICA- 302, ICA-313, ICA-525 or ICA-505; or a polypeptide that functions as one of these further autoantigens.
  • Preferred further autoantigens include: IA-2, preferably hIA-2; ICA-512; and GAD; and antigenic fragments thereof.
  • Preferred combinations of autoantigens therefore! include: a polypeptide of the invention and one, two or all three of IA-2, GAD and ICA-512.
  • Further IDDM-associated autoantigens may be used, as appropriate, in any of the techniques described below.
  • Any conventional technique may be used to determine whether or not polypeptides of the invention are recognised by autoantibodies. This also applies to determining whether or not any further autoantigens which may be used are recognised by autoantibodies.
  • suitable immunoassay formats employ a combination of solid phase or immobilized reagents and labelled reagents whereby the association of the label with the solid phase is a function of the presence or absence of recognition of the polypeptide of the invention cr a further autoantigen.
  • a solid phase reagent comprises a binding substance such as a polypeptide of the invention, an anti-antibody (e.g., anti-IgG) , or other immunobinder or other binding agent according to the' assay protocol involved, bound or attached, covalently or non-covalently, to a solid phase matrix or in an otherwise' immobilized form.
  • Suitable solid phase matrices are conventional in the art and include such matrices as microtiter plate wells, test tubes and other test containers or vessels, test strips, beads, and particles such as plastic microparticles and latexes.
  • a solid phase reagent comprises more than one type of polypeptide of the present invention and/or a polypeptide of the invention and one or more further autoantigens, it will be recognised that each reagent can be physically separated or isolable from the others or two or more can be mixed or otherwise associated in an undifferentiated manner with the solid phase.
  • reagents can be immobilised in separate wells of a microtiter plate or can occupy the sane wells; or, where the solid phase is in the form of particles, each individual particle can have attached only a single reagent or can have attached two or more reagents.
  • useful labelled reagents comprise a binding substance such as a polypeptide of the invention, a further autoantigen, an anti-antibody (e.g., anti-IgG), or other immunobinder or other binding agent according to the assay protocol involved, which is chemically coupled with a detectable chemical moiety.
  • Useful labels are conventional in the art and include fluorescers, chemiluminescers, radioisotopes, and enzymes.
  • Enzyme labels are particularly useful and are generally selected from alkaline phosphatase, peroxidase, and -galactosidase. Enzyme labels are readily detectable by addition of a corresponding chromogenic substrate and detecting the resulting colour or fluorescent response.
  • One particular immunoassay format that can be applied to the present method employs an immobilized form of the polypeptides of the invention, and optionally an immobilized form of a further autoantigen, as an immunoreagent. A test sample is incubated with the solid phase reagent and preferentially washed to remove unbound material. A labelled antibody reagent is then added.
  • Such antibody reagents can be specific for a particular class of immunoglobulin, e.g., IgG, IgM, IgA, etc., or can be a mixture of conjugates so that all immunoglobulin types are detectable.
  • Islet cell antibodies of interest are generally of the IgG isotype and thus anti-IgG would normally be employed.
  • the solid phase is washed to remove unbound labelled antibody reagent and the label activity remaining on the solid phase is measured qualitatively or quantitatively.
  • a variation of this protocol uses a ligand-modified form of the polypeptide of the invention, and optionally a ligand-modified form of a further autoantigen with immobilization to the solid phase being accomplished by using a solid phase bearing a binding partner to the ligand.
  • a solid phase bearing a binding partner to the ligand For example, biotin or a hapten (e.g., fluroscein) can be used as the ligand and can be rendered immobilized by contact with a solid phase form of avidin or anti-hapten antibody, respectively.
  • the addition of the solid phase binding partner can occur at any convenient time in the assay, such as prior to contact of sample with the ligand- antigen(s) or thereafter.
  • Another immunoassay format that can be applied to the present method employs an immobilized form of an antibody reagent.
  • Antibody specific for the desired ICA immunoglobulin type to be detected e.g., IgG
  • cr a mixture of antibodies against different IgG isotypes
  • Resulting islet cell antibody that has become bound to the solid phase antibody reagent can then be detected in any suitable manner. For instance, one can add labelled forms of one or more types of polypeptide of the invention and/or of other IDDM autoantigens either as individually labelled reagents or as a combined labelled reagent.
  • the previously described variation of using a ligand-modified form of the solid phase reagent, in this case, antibody, with immobilization to the solid phase being accomplished by using a solid phase bearing a binding partner to the ligand can also be used.
  • a competitive immunoassay format is also useful.
  • Immobilized polypeptide of the invention is employed along with a labelled form of islet cell antibodies, for example antibodies of the invention as described herein.
  • Labelled antibody and ICA from the test sample are allowed to compete for binding to the polypeptide of the invention either simultaneously or sequentially, e.g, by exposing the solid phase polypeptide of the invention first to the sample and thereafter to the labelled antibody reagent.
  • the polypeptide of the invention can be immobilized directly to the solid phase or through a ligand-binding partner bridge as described above, with the immobilization step being performed at any convenient time in the assay, including as the last step.
  • Latex or particle agglutination methods are also suitable.
  • Particles are coated or covalently coupled with the polypeptides of the invention, and optionally with one or more further IDDM-associated autoantigens.
  • the particles are then incubated with the test sample and resulting agglutination of the particles due to the formation of ICA-antibody linkages between particles is detected. Detection can be accomplished, for example, by visual observation (a slide agglutination format) or quantitated by measuring turbidity change with a spectrophotometer or nephelometer. A variation is based on inhibition of particle agglutination.
  • Each particle reagent comprises one or more monoclonal antibodies corresponding specifically with one or more particular polypeptide of the invention and optionally with one or more further IDDM-associated autoantigens, respectively.
  • an agglutinator reagent is prepared comprising multiple antigens, e.g., a water soluble polymer backbone to which are attached multiples of one or more types of polypeptides of the invention, and optionally one or more further autoantigens.
  • the absence of autoantibodies in the sample results in agglutination of the particles by formation of bridges formed by the agglutinator reagent.
  • autoantibodies When autoantibodies are present, they bind to the agglutinator and thereby prevents or inhibits agglutination.
  • any suitable quantity of polypeptides of the invention and/or of the further autoantigens may be used.
  • a quantity of from 1 x 10 "12 g to 1 x IO "6 g of a polypeptide of the invention and optionally of one or more further autoantigens as defined herein may be used.
  • quantities of from 1 x 10 "10 g to 1 x 10 "7 g may be used, with quantities of from 10 "9 g to 10 "7 g being more preferred.
  • quantities of from 10 to 50 ng, for example from 20 ng to 30ng may be used (1 x 10 ⁇ 8 g to 5 x 10 ⁇ 8 g) .
  • Such quantities of polypeptides of the invention and optionally of further autoantigens as defined herein may, for example, be applied to a given microtiter well in a microtiter plate or to any other solid phase matrix.
  • Diagnostic methods of the invention are typically performed in vi tro or ex vivo although, where appropriate, in vivo methods are contemplated.
  • the methods of the invention have a number cf clinical applications related to IDDM.
  • the methods of the invention are useful in screening of patients from the general population, particularly juveniles, wherein a positive test result (i.e., a finding that a patient's blood contains antibodies that bind to a polypeptide of the invention) indicates that such a patient is at risk of developing IDDM, that is, that such a patient may have a pre-IDDM condition.
  • the present method is applicable to the screening of first degree relatives (i.e., siblings and children) of individuals alre:ady diagnosed as having IDDM.
  • Patients with positive test results from the methods of the invention could be examined in more detail, for example by means of metabolic and genetic testing, to determine whether glucose intolerance has already occurred, or whether additional risk factors are present.
  • Other applications of the present method include the testing of a patient already diagnosed with diabetes for the purpose of determining if IDDM is involved, for instance in the case of a Type II diabetic who may also experience the onset of IDDM.
  • the methods of the invention are useful in monitoring of diabetic therapy.
  • it is possible to monitor the effectiveness of any type of treatment of IDDM, for example gene therapy, immunosuppressant therapy or immune therapy.
  • it is also possible to monitor the effects of drug therapy, the impact of diet on IDDM, and the effect of other factors on IDDM.
  • the invention further provides medical uses of the polypeptides of the invention. Accordingly, the invention provides polypeptides of the invention for use in methods of treatment of the human or animal body, especially the treatment of IDDM; and for use in methods of diagnosis of IDDM. In methods of treatment of IDDM, polypeptides of the invention may, for instance, be used to saturate an individual's immune response, blocking the individual's autoimmune response to natural IAR-PTP. The invention also provides polypeptides of the invention for use in methods of treatment of the human or animal body, particularly for IDDM, in combination with one or more further autoantigens as described herein.
  • the invention provides the use of polypeptides of the invention in the manufacture of medicaments for the treatment of the human or animal body, especially the treatment of IDDM; and for use in methods of diagnosis of IDDM, as described herein.
  • medicaments may comprise a further IDDM-associated autoantigens as described herein, or may be administered in combination with such a further autoantigen.
  • compositions comprising a polypeptide of the invention, and optionally a further autoantigen as defined herein; and a pharmaceutically acceptable carrier.
  • the invention further provides diagnostic test kits comprising polypeptides and/or antibodies of the invention, optionally together with one or more further IDDM-associated autoantigens, as described herein.
  • test kits Any suitable quantity of these components may be present in the test kits.
  • Other components may also be included in the kits, for example buffers and/or other solutions; labels; tubes, plates, membranes, membrane sticks or other items of equipment, for example for immobilising reagents; and/or anti-human antibodies for monitoring immunoreactivity.
  • Test kits of the invention typically further comprise means for determining whether or not the polypeptides of the invention are recognised by autoantibodies.
  • Test kits comprising one or more further autoantigens typically include means for determining whether or not the further autoantigen or autoantigens are recognised by autoantibodies.
  • Test kits according to the invention may, for example, enable the use of methods according to the invention. Thus, they may comprise, as means for determining whether or no: the polypeptides of the invention, and optionally one or more further IDDM-associated autoantigens as described herein, are recognised by autoantibodies, any reaigents suitable for putting into practice the techniques described above.
  • the components may be in any suitable form; for example they may be packaged in any suitable container.
  • the components may be intended or separate, for example sequential, use; or for concurrent use.
  • the components may be in any suitable form, for example they may be in sterile solutions, buffered solutions or sterile and buffered solutions. They may be labelled or unlabelled.
  • the components may be in substantially purified, purified, isolated or substantially isolated form.
  • Primers corresponding to conserved amino acid sequences (DYINA (SEQ. ID. No. 14) and VHCSAGV (SEQ. ID No. 15) of the catalytic domain of PTPases were designed (with additional EcoRI sites added) and used to amplify a human colon carcinoma cell (SW480) cDNA library (Clontech, HL3014b) using PCR.
  • the primers used had the following sequences: 5' -GGGAATTCNGAYTAYATHAAYGC-3 ' (SEQ ID No. 3) and 5' -GGGAATTCACNCCNGCRCTRCARTGNAC-3' (SEQ ID No. 4) .
  • the PCR was carried out as follows: 94°C x 5 mm >(94°C x 40 sec >50°C x 30 sec >72°C x 90 sec) x 30 >72°C x 5 min- -- > 4°C.
  • the major amplified fragment of ⁇ 460bp was subcloned into the pGEM-T vector (Promega) and 94 clones were sequenced.
  • r75 was identical over a 312bp portion of its sequence to a human brain EST sequence (# 03250) [Adams et al . , 1993, Nature Genet. 4, 256-267] which was noted in this paper as having 80.6% similarity to ICA512) .
  • human multiple Northern blots (Clontech) were probed with 32 P-labelled r75 (Fig. 1) . Two major transcripts of about 3.7kb and 5.5kb were observed. The highest levels of expression were detected in brain and pancreas, followed by prostate and testis, with lower expression detectable in a few other tissues, including colon.
  • Each fragment was subcloned into Bluescript SK(+) and sequenced across both strands using either primers to the vector or synthesized primers hybridizing to the insert in conjunction with the Sequenase Version 2.0 Sequencing Kit (United States Biochemical Corporation) .
  • the C3 clone contained a coding sequence missing initiation and termination signals, and was significantly smaller than than the 3.7 and 5.5kb RNA transcrip>ts detected by Northern blotting with r75 (Fig.l) , suggesting that it was incomplete at both the 5' and 3' ends.
  • the 3' end of the clone was obtained by 3' RACE using the Marathon cDNA Amplification Kit (Clontech) .
  • the first-strand cDNA was reverse transcribed from human brain polyA * mRNA (Clontech) using the cDNA synthesis primer provided with the kit.
  • the gene-specific primer used for 3 '-RACE was 5'- CCTGCCTCCTCAGGCGGAGCAAGA-3' (SEQ ID No. 9) , corresponding to nucleotides 1389-1412 of SEQ ID No.l.
  • PCR was carried out at 94°C x 3 min >(94°C x 40 sec >68°C x 4 min) x 30-
  • the combined sequences of C3 and 3-7 comprise a partial cDNA encoding a novel putative receptor-like PTPase which we term IAR (for islet cell antigen-related) (Fig.2) .
  • the predicted protein fragment (SEQ. ID. No.2) translated by the only possible open reading frame comprises an incomplete N-terminal extracellular region, a 21 amino acid transmembrane-spanning region, and a 379 amino acid intracellular region.
  • pancreas library was rescreened with a 300 bp probe close to the 5' end of C3. No IA-2 clones were found. Of the positive clones, clone Bll appeared to have the longest 5' extension to C3 and was selected for further sequencing along both strands . The 3' end of Bll was identical to C3 sequence over a length of about 2 kb, but the 5' ends of Bll and C3 were different (Fig. 2C) . Bll had a unique 5' sequence of 412 bp, followed by a 54 bp sequence that is found in the opposite orientation in C3. The sequence of C3 found 5' to this inverted region was shorter and different from that of Bll.
  • Template cDNA was prepared from human brain polyA * mRNA (using the Marathon cDNA amplification Kit (Clontech) using random primers or a primer (P5, with the sequence 5'-
  • CGTGTGGGCCACATAGGTCAGGATGCTCTCGGAGAA-3' (SEQ. ID. No. 20) ) corresponding to shared B11/C3 sequence located about 170 bp 3 ' to the invert.
  • the 5' RACE used a primer to the ligated adaptor sequence at the ends of the cDNA and an IAR specific reverse primer (Pll with the sequence
  • the complete cDNA (SEQ. ID. No. 10) is predicted to encode a receptor-like PTP of 1015 amino acids (SEQ. ID. No. 11) .
  • This protein has: about 43% overall sequence identity to the putative PTP IA- 2 (Lan et al (1994) ; Rabin et al (1994)) , a protein identified as an islet cell auto-antigen in IDDM, and another PTP-like molecule called PTPLP ( (Kambayashi et al (1995)) .
  • the novel PTP as islet cell antigen-related PTP or IAR PTP.
  • the IAR extracellular region does not contain FN-III or Ig-like repeats, but has the adhesion recognition peptide sequence RDGS (amino acids 372-375) .
  • RDGS adhesion recognition peptide sequence
  • the catalytic domain of IAR has conserved sequences typical of other PTPases, although the active site is unusual in having an aspartate residue at position 947 (IVHCSDGAGRTG (SEQ. ID. No. 16)) in place of a conserved alanine residue.
  • the IAR probe comprised nucleotides 790-1197 of SEQ. ID. No.l (corresponding to a part of the predicted extracellular region)
  • the IA-2 probe comprised nucleotides 1665-1906 of IA-2 cDNA (Lan et al . , 1994), corresponding to a part of the extracellular region, the transmembrane region, and a small portion of the intracellular region.
  • These probes were labelled with 32 P as described in the legends to Figs. 3 and 4.
  • a set of human multiple tissue Northern blots (Clontech) was probed first with the IA-2 probe (Fig.3) , and later stripped and reprobed with the IAR probe (Fig. 4) .
  • the two probes recognize distinct transcripts in terms of size and tissue specificity.
  • the IA-2 probe detects a single transcript of about 3.9kb (Lan et al . , 1994)
  • the IAR probe detects two transcripts of about 3.7 and 5.5kb. It is possible that the 3.7kb transcript has a 3' untranslated sequence corresponding to that of the 3-7 clone, while the larger 5.5kb transcript represents an alternative 3' untranslated sequence corresponding to that in the longer 3-10 clone.
  • IA-2 The highest expression of IA-2 is detected in brain, followed by spinal cord and pancreas, with low levels expressed in small intestine and adrenal gland.
  • the highest expression of IAR is in pancreas and brain, followed by trachea, prostate, stomach and spinal cord, with low levels detectable in small intestine and adrenal gland.
  • Figure 5 shows an alignment of the IAR and IA-2 transcripts detected in brain and pancreas. It is obvious that the IA-2 transcript is larger than the smallest 3.7kb IAR transcript, and furthermore the 3.7kb IAR transcript is much more highly expressed in pancreas than brain while the 3.9kb IA-2 transcript is more highly expressed in brain than pancreas. Thus IAR and IA-2 appear to be encoded by distinct genes .
  • the intracellular region of IAR was amplified by PCR using the 3-7 clone as template.
  • the primers used had the sequences 5' -GGGCTCGAGTCTAGACAGGCTGAAGGAGAAGCTCTC-3 ' (SEQ ID No. 5) (corresponding to nucleotides 1517-1538 of SEQ. ID. No.l, with added Xbal and Xhol sites) and 5'- GGGGAATTCCATGGTTATAATAGAAGACACACA-3' (SEQ ID No. 6) (corresponding to nucleotides 2704-2723 of SEQ. ID. No.l, with added EcoRI and Ncol sites) .
  • PCR was carried out at 94°C x 5 min >(94°C x 40 sec >60°C x 30 sec >72°C x 2 min) x 10 >72°C x 5 min >4°C using the Expand Long PCR
  • Each primer was used in combination with one of the primers (of SEQ. ID. No. 5 or 6) (used to amplify the intracellular region of IAR) to generate two overlapping DNA fragments corresponding to nucleotides 1993-2904 and 2878-3197 of IAR. These fragments were mixed and used as template for a PCR reaction with the primers of SEQ. ID. No. 5 and 6.
  • the above plasmids were transformed into E. coli (DH5 ⁇ F' ) .
  • the recombinant bacterial host was cultured and GST-IAR expression induced with 0.15mM IPTG.
  • the GST-IAR was purified from bacterial lysates by standard methods (Smith and Johnson, 1988) .
  • the IAR was cleaved from GST using thrombin or 3C protease as appropriate (Smith and Johnson, 1988; Walker et al . , 1994), and the purified IAR quantitated by SDS-PAGE followed by densitometric scanning alongside known amounts of standards as described (Wang and Pallen, 1991) .
  • the purified 40kD form of the IAR intracellular region was analyzed for phosphatase activity. Low but detectable phosphatase activity towards the substrate para-nitrophenyl phosphate was measured (Fig. 6B) . Since the related molecule IA-2 reportedly has no detectable phosphatase activity (Rabin et al . , 1994; Lu et al. , 1994) , we examined this in side-by-side experiments with IAR.
  • the intracellular region of IA-2 was amplified by PCR using the D2 clone as template.
  • the primers used had the sequences 5' -GGGCTCGAGTCTAGAGCGGCAGCAAGACAAGGAG-3 ' (SEQ IE No. 7) (corresponding to nucleotides 1885-1903 of IA-2 (Lan et al . , 1994) , with added Xbal and Xhol sites) and 5' -GGGGAATTCGAGCTCGCATGCCCAAGAGGTGGC-3' (SEQ ID No. 8)
  • Recombinant bacterial GST-IA-2 protein was produced from both the above plasmids, cleaved, purified and quantitated as described above for IAR.
  • the type of protease used for cleavage from GST did not affect the size of the IA-2 produced, with the purified IA-2 migrating on SDS-PAGE in both cases as a major band with an apparent size of 44kD (Fig.6A, lanes 1 and 2) .
  • the IAR and IA-2 proteins differ in that IAR appears to possess an internal thrombin cleavage site which is absent in IA-2.
  • the intracellular region of IAR (amino acids 646-1015) was expressed as a GST-fusion protein and purified following 3C protease cleavage from GST.
  • the IAR migrated on SDS-PAGE as a major protein band of about 41 kDa (Fig. 7A, lane 2) , in accord with the predicted size of 41.7 kDa.
  • the purified IAR possessed low but detectable phosphatase activity towards pNPP.
  • the IAR is active over ⁇ i narrow pH range, with optimal activity at pH 4.5, and is essentialLy inactive at pH 5.5 (Fig.7B) .
  • IAR catalyzes the time- dependent dephosphorylation of pNPP (Fig.
  • Beads were washed once with TBST-BSA, once with high salt wash buffer (0.5% Triton X-100, 200mM NaCl, 50mM NaH,POfact) , twice in PBS plus 0.5% Triton X-100 and once in 0.5% Triton X-100, 4.17mM Tris, 192mM glycine, pH 7.4. Precipitated proteins were resolved by 10% SDS-PAGE. Gels were dried and exposed to a phosphorimager, and bands were quantified by densitometry after subtraction of local background.
  • IAR and IA-2 can distinguish different autoantibody populations in a subset of IDDM patients.
  • IAR Ab and IA-2 Ab in these sera has allowed us direct comparison of the abilities of these assays to predict IDDM, using identical assay formats and standardised reference ranges. In total, 33 of these subjects have developed IDDM. IAR and IA-2 antibodies were measured in 115 subjects with ICA > . 20 and/or IAA _> 100, who have been followed for 3.52 years (mean, range 0.30- 7.28 years) . Of this group, 18 progressed to IDEM during follow-up over 2.28 years (range 0.3-4.52 years) .
  • cDNA Complementary DNA encoding full length human IAR was cloned into the pBluescript vector under control of the T3 promoter; cDNA encoding amino acids 646-1015 of IAR, representing the intracellular region was inserted into the pGEM3 vector under control of the SP6 promoter and cDNA encoding full- length IA-2 was inserted into the pSP64 poly A vector under control of the SP6 promoter.
  • Recombinant protein was synthesised using the Promega TNT reticulocyte lysate kit (Promega, Madison, Wis., USA) , in the presence of b S methionine, according to the manufacturers' instructions, using the appropriate RNA polymerase. Two ⁇ g DNA template was used in all in vi tro translation and transcription reactions, which proceeded for two hours at 30°C.
  • the transcription/translation product (50 ⁇ l) was diluted into 12ml TBST-BSA buffer (50mM Tris-HCl pH 7.2, 150mM NaCl, 1% Tween 20, ImM L-methionine, 0.1% BSA), and 50 ⁇ l of this dilute lysate was added to 5 ⁇ l test serum and incubated overnight at 4°C in a 96-well plate (well volune 2ml, Beckman, Palo Alto, CA, USA) . Fifty ⁇ l of protein A Sepharose suspension (50mg/ml dry weight) in TBST-BSA was added and the plate was shaken on a plate shaker for lh at 4°C.
  • TBST-BSA buffer 50mM Tris-HCl pH 7.2, 150mM NaCl, 1% Tween 20, ImM L-methionine, 0.1% BSA
  • the negative control consisted of pooled normal serum and the positive control was strongly IAR and IA-2 positive plasmapheresis fluid from a patient with stiff-man syndrome and diabetes.
  • the inter assay coefficient of variation of the IAR antibody assay was 5.8% for a sample with a mean value of 57.3 units, and 12.5% for the IA-2 Ab assay, using a sample with a mean value of 96.8 units.
  • ICA were measured by indirect immunofluorescence on frozen sections of blood group O human pancreas (Bonifacio et al (1990)) and assigned a value in JDF units by dilution and comparison with standard sera. This assay has been standardised in all Immunology of Diabetes and Proficiency Workshops. Insulin autoantibodies were measured by a competitive fluid phase radiobinding assay (Vardi et al (1989) ) .
  • IVGTT Intravenous glucose tolerance test
  • IVGTT Islet Cell Antibody Registry Users Study
  • the partial (intracellular domain) and full-length IAR clones were compared for recognition by IDDM sera, to determine whether additional reactivity was present on the extracellular domain of the full-length IAR molecule that was not present in the intracellular (cytoplasmic) domain.
  • There was a strong relationship between levels of antibodies to full- length IAR, and antibodies to the intracellular domain (r 94.9%, p ⁇ 0.0001) .
  • Use of a standardised specificity of 97.9% resulted in a sensitivity of 58.1% for antibodies to the cytoplasmic domain and 48.8% for antibodies to the full-length molecule. Because of the strong relationship between antibody levels and the similar assay sensitivities, the intracellular fragment was used as the antigen in the remaining experiments.
  • IAR Ab and IA-2 Ab were measured in 53 recent-onset IDDM and 144 healthy control subjects.
  • the reference range for each assay was adjusted to give a specififcity of 97.9% for each assay, which was 0.5 units for IAR Ab and 3.0 units for IA-2 Ab.
  • the corresponding sensitivities were 56.6% for IAR Ab and 62.3% for IA-2 Ab (Table 2) .
  • Sensitivities and specificities of combinations of IAR Ab and IA-2 Ab, and IAR Ab or IA-2 Ab, using the reference ranges described above, are also shown in Table 2.
  • IAR Ab In some cases there were marked differences between levels of IAR Ab and IA-2 Ab, for example there were six sera with IA-2 Ab levels greater than 50 units and IAR Ab less than 5 units, and one with IAR Ab of 25 units and IA-2 Ab of 2.9 units.
  • IAR and IA-2 antibody positivity and combinations of these antibodies are summarised in Table 2.
  • IAR Ab were detectable in nine (50.0%) and IA-2 Ab in 10 (55.6%) 2.6 ⁇ 1.8 . (mean, range 0.3-6.3) years prior to diagnosis, using the threshold for positivity defined above.
  • 21 (21.6%) had IAR. antibodies
  • 31 (31.9%) had IA-2 antibodies.
  • IA-2 Ab positivity did not significantly influence IDDM-free survival.
  • the combination of either IAR or IA-2 antibodies did not significantly affect IDDM-free survival, but the presence of both antibodies had a significant effect (p ⁇ 0.0005) .
  • IA-2 Ab positivity did not affect IDDM-free survival when these standardised thresholds of positivity were used, the use of higher thresholds for IA-2 Ab positivity resulted in significant differences. For example, when nine units was used as the threshold, IA-2 Ab positivity was significantly associated with progression to IDDM (p ⁇ 0.0005, log-rank test) .
  • IAR Ab were associated with markers of high risk of IDDM, in relatives with ICA or IAA.
  • Insulin autoantibodies Ziegler et al (1989)
  • impaired intravenous glucose tolerance Vardi et al (1991)
  • IAR Ab are markers of ⁇ cell destruction.
  • IAR Ab have similar diagnostic sensitivity for recent-onset IDDM to IA- 2 Ab, but are better predictors of IDDM in our high-risk subjects As preclinical intervention therapies m IDDM are critically reliant on effective screening tests to define high-risk subjects, IAR Ab may well have an important role IDDM screening, either alone or m combination with other humoral markers of IDDM
  • IAR Ab IA-2 Ab IAR Ab or IAR Ab or & IA-2 Ab.
  • IA-2 Ab IAR Ab or IAR Ab or & IA-2 Ab.
  • Positivity threshold (units) ⁇ 0.5 ⁇ 3.0
  • SEQ. ID. Nos. 1 and 2 partial cDNA sequence of IAR-PTP (SEQ ID.
  • GGT GTG GAC AGA CAC CAT CTG ATG GCG GCC CTC AGT GCC TAT GCT GCC
  • CAG GAG GCC CTC AGC GAG GGC CTG CAA TTG GAG GTC CAG CCT TCC GAG
  • Glu Glu Ala Arg Gly Tyr lie Val Thr Asp Arg Asp Pro Leu Arg Pro 370 375 380
  • GAG GAA GGA AGG CGG CTG GTG GAG GAC GCC CGC CTC CTG CAG GTG
  • Lys lie Leu Gin Thr Gly Val Gly Ser Lys Ser Lys Leu Lys Phe Leu 450 455 460
  • Gly Pro lie Pro Ser Pro Ser Ala Arg Ser Ser Ala Ser Ser Trp Ser 565 570 575
  • CCTCAGGGGC 5 C ⁇ GGGG ⁇ C CCCCTACCCC ACGGATCTTG TCAGGA ⁇ TC .5 TGATCTGACT
  • SEQ. ID. Nos. 10 and 11 Complete nucleotide (SEQ ID No. 10) and amino acid (SEQ ID NO. 11) sequence of IAR.
  • the signal peptide is underlined ana the transmemtuantr region ⁇ r double-underlined.
  • An extracellular RGDS sequence and .- potential s:Lf of N- linked glycosylation are m bold.
  • Tl ⁇ " active site or the phosphatase is found at ammo acids 942-954.
  • GCT GCC CTG GCC AAC GCC CTC CGA CGC CAC CTG CCC TTC CTG GAG GCC
  • SEQ ID Nos 12 and 13 Nucleotide (SEQ ID No. 12) and amino acid (SEQ ID NO. 13) sequences of the cytoplasmic (intracellular) domain of IAR.
  • SEQ ID NO. 12 corresponds to the nucleotides of SEQ ID No. 10 which encode amino acids 646 to 1015 of SEQ ID No. 11.
  • SEQ ID No. 13 corresponds to amino acids 646 to 1015 of SEQ ID No. 11.

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Abstract

Acide nucléique codant un polypeptide possédant les propriétés d'un auto-antigène associé au diabète sucré insulinodépendant (IDDM), et comprenant: (a) la séquence codante de SEQ. ID. No. 1, 10 ou 11, et/ou la séquence complémentaire à cette dernière; (b) une séquence qui s'hybride avec une séquence telle que définie en (a); (c) une séquence résultant du code génétique dégénéré d'une séquence d'acide nucléique telle que définie en (a) ou (b); et (d) une séquence possédant une homologie d'au moins 80 % avec une séquence telle que définie en (a), (b) ou (c). L'invention se rapporte également aux polypeptides codés par de tels acides nucléiques, ainsi qu'à des méthodes diagnostiques faisant intervenir de tels polypeptides.
EP96941561A 1995-12-20 1996-12-20 Reactifs diagnostiques associes au diabete Withdrawn EP0874901A2 (fr)

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GB9526036 1995-12-20
GBGB9526036.0A GB9526036D0 (en) 1995-12-20 1995-12-20 Diagnostic reagents relating to diabetes
GB9605710 1996-03-19
GBGB9605710.4A GB9605710D0 (en) 1996-03-19 1996-03-19 Diagnostic reagents relating to diabetes
GBGB9620265.0A GB9620265D0 (en) 1996-09-27 1996-09-27 Diagnostic reagents relating to diabetes
GB9620265 1996-09-27
PCT/CA1996/000867 WO1997022694A2 (fr) 1995-12-20 1996-12-20 Reactifs diagnostiques associes au diabete

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IL134137A0 (en) * 1997-07-22 2001-04-30 Novimmune Sa Methods for diagnosis and theraphy of autoimmune diseases, such as insulin dependent diabetes mellitus, involving retroviral superantigens
EP0893691A1 (fr) * 1997-07-23 1999-01-27 Mach, Bernard François, Prof. Procédés pour le diagnostic et la thérapie des maladies autoimmunes associées avec super-antigènes rétroviraux, en particulier le diabète mellitus insulinodépendant
US6355244B1 (en) 1997-11-17 2002-03-12 University Of Kentucky Research Foundation Methods and compositions for the treatment of psoriasis
WO2002084249A2 (fr) * 2001-04-10 2002-10-24 The Board Of Trustees Of The Leland Stanford Junior University Utilisation therapeutique et diagnostique de profils de specificite d'anticorps
US7635559B2 (en) 2003-12-24 2009-12-22 Samsung Electronics Co., Ltd. Polynucleotide associated with a type II diabetes mellitus comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for analyzing polynucleotide using the same
KR100695147B1 (ko) 2005-02-15 2007-03-14 삼성전자주식회사 다중좌 마커를 이용한 2형 당뇨병의 진단방법, 2형당뇨병과 연관된 마커를 포함하는 폴리뉴클레오티드 및마이크로어레이

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NL9001083A (nl) * 1990-05-04 1991-12-02 Rijksuniversiteit Beta-cel antigeen.
WO1997007211A1 (fr) * 1995-08-11 1997-02-27 THE GOVERNMENT OF THE UNITED STATES, represented by THE SECRETARY DEPARTMENT OF HEALTH AND HUMAN SERVICES Substances et procedes pour la detection et le traitement du diabete insulino-dependant

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