Classifications

• • 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
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
  • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

• This invention relates to methods for detecting autoimmune diseases and pre-clinical autoimmune diseases.
• IDDM insulin dependent diabetes mellitus
• Peptide fragments are provided for use in these methods.
• Figures Figure 1 1A: Serum IgG anti-BSA Antibody Concentrations at Diagnosis of Insulin-Dependent Diabetes in 142 Children (diagonal hatching) and 79 Normal Children (horizontal hatching) .
• Figure IB shows distribution of serum IgA anti-BSA Antibody Concentrations and
• Figure 1C shows serum IgM anti-BSA Antibody Concentrations, both after normalisation by smoothing.
• Figure 2 Measurements of total and ABBOS-Specific Anti- BSA Antibodies in 17 Diabetic Patients.
• Panel 2A shows IgG, panel 2B IgA and panel 2C IgM.
• the hatched bars represent patient anti-BSA levels, the white bars the levels remaining after removal of ABBOS-specific antibodies.
• the horizontal bar indicates the mean anti- BSA concentrations in 17 normal children (upper line) and the concentrations after removal of anti-ABBOS antibodies in the same sera.
• Figure 3 shows distribution of moderately- (horizontal hatching) , highly (diagonal hatching) , and very highly elevated (vertical hatching) IgG-anti-bovine serum albumin antibodies in 40 diabetic children as determined by particle concentration fluoroimmunoassay (PCFIA) .
• PCFIA particle concentration fluoroimmunoassay
• 3B and 3D PCFIA standard curve for a serum pool (from diabetic children) containing (3B) 12.3 KfU/ ⁇ l IgG- and (3D) 4.2 KfU/ ⁇ l IgA-anti-BSA antibodies: binding competition with increasing amounts of BSA and ovalbumin/Tween-20.
• 3C shows IgG anti-BSA standard curve for enzyme im unoassay (EIA) .
• 3E shows IgA anti-BSA standard curve for enzyme immunoassay (EIA) .
• 3F shows binding competition with increasing amounts of free ovalbumin/Tween-20 for IgG-( ⁇ ) and IgA-(___>) anti-BSA antibodies, as well as with increasing amounts of free BSA for IgG-(D) and IgA- ( ⁇ ) anti-BSA antibodies.
• KfU kilo fluorescence units.
• Figure 4 Mean levels ( ⁇ SEM) of anti-BSA antibodies in Type 1 diabetic- and matched control children as detected by particle concentration fluoroimmunoassay (PCFIA, Figures 4A and 4B) and enzyme immunoassay (EIA, Figures 4C and 4D) . Difference between diabetic and control children: PCFIA:IgG, p ⁇ 0.0001; IgA, p ⁇ 0.001. EIA: IgA, p ⁇ 0.01.
• FIG. 5 Correlation between the levels of anti-BSA antibodies as determined by enzyme immunoassay (EIA) and particle concentration fluoroimmunoassay (PCFIA) in diabetic- and control children. Shaded areas represent BSA-antibody levels considered as "non-elevated”: (vertical band) negative for BSA antibodies by PCFIA, (horizontal band) negative for BSA antibodies by EIA) .
• the measurement of autoimmune disease-associated antibody levels specific for proteins or protein fragments derived from common dietary sources as well as measurement of T lymphocyte sensitization to such fragments by the methods in the invention provide a unique and new clinical and investigational tool for
• a method for detecting IDDM or pre-clinical IDDM by measuring serum antibodies to bovine serum albumin (BSA) by a particle concentration fluoroimmunoassay (PCFIA) technique as described in Examples 1 and 3.
• BSA bovine serum albumin
• PCFIA particle concentration fluoroimmunoassay
• Particle concentration fluoroimmunoassay detected elevated IgG-anti-bovine serum albumin antibodies in all diabetic children, enzyme immunoassay in 25% (p ⁇ 0.0001). Fluoroimmunoassay detected elevated levels in 2.2% and enzyme immunoassay in 10% of control children (p ⁇ 0.002). Elevated IgA-anti-bovine serum albumin antibodies in patients were slightly more often detected by fluoroimmunoassay than by enzyme immunoassay, while in control children enzyme immunoassays detected elevated levels three times more often (p ⁇ 0.01).
• the inventors' findings demonstrate that these assay techniques detected distinct subsets of anti-bovine serum albumin antibodies with little (IgG) or some (IgA) overlap.
• fluoroimmunoassay procedures antigen:antibody binding occurs within 1-2 min while hours are allowed in an enzyme immunoassay.
• Antibodies with high on-off binding rates typical for immune response following hyperimmunization are therefore measured preferentially by particle concentration fluoroimmunoassay and it is these antibodies which appear to be associated with diabetes.
• the PCFIA assay described above may be performed using particle-bound BSA-peptides such as particle-bound ABBOS instead of particle-bound BSA.
• the ABBOS peptide can be used to detect up to 90% of IDDM-associated anti-BSA antibodies even when modified at the C-terminus as described in Example 4.
• peptides may be fragments of the natural BSA protein or synthetic peptides prepared by a suitable technique. Such techniques will be known to those skilled in the art and include chemical synthesis and recombinant techniques.
• a method for detecting in IDDM patients and in pre-clinical IDDM subjects sensitised T lymphocytes which specifically recognise and proliferate in response to peptide fragments of BSA, including ABBOS and CS2267.
• This detection method can be used to detect
• IDDM pre-clinical IDDM and also to detect reactivation of the ⁇ cell-attacking immune process in patients after jScell or islet transplantation.
• a peptide is provided, CS2267, which binds specifically to the sensitised T lymphocytes present in IDDM and pre-clinical IDDM patients.
• immunotoxins By coupling a peptide such as CS2267 to toxic compounds, immunotoxins can be prepared which are directed to and can destroy the specific T lymphocytes which mediate ⁇ cell destruction in IDDM, providing a means of arresting the disease process.
• a peptide such as CS2267
• Blood samples were obtained from 142 Finnish children (83 males, 59 females, mean ( ⁇ SD) age 8.4 ⁇ 4.3 years) with newly diagnosed insulin-dependent diabetes mellitus. Fifty patients had diabetic ketoacidosis, 48 diabetic ketosis only, and the remainder hypoglycemia alone. All were continuously dependent on at least one daily injection of human insulin and had increasing insulin dependence after diagnosis. We also studied 79 age-, sex- and region-matched children admitted for minor surgery (42 males, 37 females, mean age 8.413.1 yr) , and from 300 adult Toronto blood donors. Blood samples were obtained from the patients before the first insulin injection and 3 to 4 months later and, in a random subset of 44 patients, 1 to 2 years later. The serum samples from the two groups of children were sent coded to Toronto.
• Clinical assessment included history and measurements of insulin and islet cell autoantibodies identified either by indirect immunofluorescence or complement fixation test . Sample volumes were insufficient for full titration of the earliest samples, islet cell antibody results are therefore expressed as positive or negative. The HLA-A, -B, -C, DR.Dw haplotypes of all patients were determined as described (17).
• Anti-BSA antibodies were measured by particle concentration fluoro-immuno assay (PCFIA ) as described in Dosch et al, (21) .
• PCFIA particle concentration fluoro-immuno assay
• test serum dilutions (1:100-1:1,000) were added to microwells containing 20 ⁇ l of 1:20 diluted BSA-coated icrospheres (initial 2.5% weight/volume) .
• Up to ten plates were inserted into the Screen-Machine for programmed phase separations, washings and addition (100 ng/well) of affinity purified, custom BSA-free fluorescein-conjugated goat anti-human IgG, IgA, and IgM (Fc-fragment specific, BioCan, Mississauga, Ontario, Canada) . Drying and precipitation of serum protein was avoided by short (1 min) incubation, phase separation and washing procedures at low (5mm Hg) vacuum pressure.
• a calibrated pool of serum from diabetic patents was used as the standard in each plate.
• This standard contained 12.3 kilofuorescence Units (KfU) IgG anti-BSA antibodies per microliter, 4.2KfU/ ⁇ l IgA and 4.0 KfU/ ⁇ l IgM anti-BSA antibodies.
• the anti-BSA assays had a sensitivity of 1.0(IgG, IgA) and 10.0 (IgM) ng/ml, the intraassay-and interassay coefficients of variation were 8.9% and 9.8%, respectively.
• Addition of BSA but not ovalbumin blocked antibody binding in a dose-dependent fashion.
• Anti-BSA antibody concentrations exceeding the mean plus 2 SD in the 79 normal children were defined as elevated.
• Antibody concentrations as expressed as kilofluorescence units per microliter relative to the standard serum pool. The results were analyzed using Chi-square statistics, parametric one-way analysis of variance, and Student's unpaired t-test for normally distributed values. The distribution of anti- BSA concentrations was normal for each isotype. In the case of skewed distribution, the Mann-Whitney-U test and Spearman's rank-correlation test were used(r,) . Anti-BSA antibody concentrations after diagnosis were evaluated using a paired test.
• Anti-BSA Antibodies The serum IgG anti-BSA antibody concentrations in the diabetic patients were considerably higher than those in the normal children ( Figure 1) , the mean concentration being almost seven fold higher (Table, 1,P ⁇ 0.001, Table 2). The elevated serum IgG anti-BSA concentrations in the diabetic patients did not reflect generalized immune responses against nutritional antigens, since the patients and the normal children had similar serum concentrations of IgG antibodies to the major cow milk proteins casein and ⁇ - lactoglobulin (Table 1) .
• the mean serum concentration of IgA anti-BSA antibodies was higher in the diabetic patients than in the normal children (P ⁇ 0.0001, Table 1), but the values overlapped ( Figure 1) .
• the mean serum concentration of IgM anti-BSA antibodies in the diabetic patients were slightly lower than those in the normal children (P ⁇ 0.05, Table 1). Less than 1 percent of patients had elevanted concentrations compared with 8 percent of normal children (P ⁇ 0.01, Table 2, Figure 1).
• Solid phase ABBOS and solid phase ABRAS were prepared by binding the biotinylated peptide to streptavidin coupled to carboxylated polystyrene microspheres, as described by Dosch et al. (1988) above. 20 ⁇ l (0.5% w/v) of ABBOS-or ABRAS- conjugated microspheres were mixed with 3 ⁇ l patient or normal serum in a final volume of 0.3 or 3 ml. After incubation at 4°C for 15 minutes, the mixtures were centrifuged and 20 ⁇ l of the supernatant was used for measurement of residual anti-BSA antibodies.
• Anti-ABBOS Antibodies Measurement of anti-BSA antibodies before (Fig. 2, black bars) and after exposure of serum to solid phase ABBOS peptide (white bars) was done to determine the proporation of anti-BSA antibodies that specifically bound the ABBOS region of BSA.
• Anti-BSA Antibodies and Disease Markers No relationships were found between the concentration of anti-BSA or anti-ABBOS antibodies and the severity of disease presentation (blood glucose, HbAj, serum C-peptide concentrations) , the duration of symptoms before diagnosis, or the severity of diabetic ketosis or acidosis. The specificity, concentrations and isotype distribution of the antibodies were similar in multiplex and simplex families.
• Niether anti-BSA concentrations, isotype diversity nor specifity were associated with the presence or absence of islet cell- or insulin autoantibodies.
• HLA-DR3/4 or -Dw3/4 initially had more severe diabetes (higher blood glucose and HbAj and lower serum C-peptide concentrations) than those negative for such haplotype combinations, but the frequencies or concentrations of insulin- and islet cell autoantibodies were similar in both haplotype groups.
• concentrations of BSA/ABBOS antibodies were comparable among the diabetic children with or without HLA-DR3/4 or -Dw3/4, as well as in those with -DR3/x, -DR4/x and -Dw4/x.
• Solid phase ABBOS was also used to assay serum anti-ABBOS antibodies as described above for use of solid phase BSA (BSA coated microspheres) .
• ABBOS anti-BSA/ABBOS antibodies
• addition of up to 1000 and 100 ⁇ g of BSA or ABBOS peptide respectively did not alter the results of assays for islet-cell antibodies and insulin autoantibodies in the serum of all 15 diabetic patients tested.
• Anti-BSA antibodies in patient and control sera were measured by PCFIA as described in Example 1.
• the serum samples assayed by PCFIA as described above were also assayed by an enzyme linked immunosorbent technique for anti-BSA antibodies, a conventional three- layer solid phase procedure modified from Tainio et al. (25) .
• the method employs polystyrene Microstrip wells (Labsystems, Helsinki, Finland) that are processed in an automatic EIA analyser (Auto-EIA II; Labsystems, Helsinki, Finland) , which can process up to three plates (66 samples) per day.
• the plates were coated with 2 ⁇ g/ml (100 ⁇ l) BSA (A-4378; Sigma) in 0.1 mol/1 PBS-5 mol/lNaN 3 (pH 7.4) overnight at room temperature. After washing with PBS-NaN 3 , the wells were saturated with 1% gelatin-PBS-NaN 3 for 1 h at 37°C and stored at +4°C until used.
• Serum samples were diluted 1:40 in 0.5% gelatin-PBS-NaN 3 prepared in 0.05% Tween-20. Three replicates of lOO ⁇ l of serum dilutions were plated, two in coated wells and one in a non-coated well. After a 60 min incubation at 37°C wells were washed (4x) . Diluted alkaline phosphatase conjugated anti-human IgG- or -IgA (cat. no. 67806 and 67808; Or-ion Diagnostica, Espoo, Finland) was added and incubated for 60 min followed by four washes as before.
• Intra-assay and inter-assay variations of the assay were 9.3% and 15.8%, respectively.
• serial dilutions of a BSA-antibody positive standard were run on each plate (Fig. 3C) and the results expressed as percent binding of the standard serum.
• Values for both IgG- and IgA were skewed despite log- transformation, and thus the limit for positivity was set at the 90th percentile of the values in control subjects. This limit was selected after examining a series of cut ⁇ off values as giving the highest sensitivity with acceptable specificity. Sera having an absorbance of 3.9% for IgG and 14.2% for IgA of the standard were considered as positive.
• the 40 sera from diabetic children contained a range of elevated IgG-anti-BSA PCFIA antibodies; however, only 25% were found positive by EIA (Table 4, p ⁇ 0.0001). Conversely, IgG-anti-BSA 1 ⁇ antibodies in control subjects were elevated more frequently than those detected by PCFIA (Table 4, p ⁇ 0.0002). Elevated IgA-anti-BSA antibodies in diabetic children were found in 50% and 42% (p NS) , however only 3.3% and 10% of control children were positive in PCFIA and EIA, respectively (Table 4, p ⁇ 0.01). These results demonstrated that PCFIA but not EIA preferentially detects disease-associated BSA antibodies in children with Type 1 diabetes. In contrast, EIA shows preference for detection of antibodies more prevalent in the general population. Neither procedure detected all BSA antibodies.
• BSA antibody levels in diabetic children are shown in Figure 4. There was a significant difference in the levels of both IgG-, and IgA-anti-BSA antibodies between diabetic and control children when determined by PCFIA (p ⁇ 0.0001 and P ⁇ 0.001). In contrast, the levels of IgG-anti-BSA ⁇ antibodies were roughly similar in diabetic and control children. IgA-anti-BSA ⁇ antibodies were higher in diabetic children, but the difference (p ⁇ 0.01) was less prominent than in PCFIA (p ⁇ 0.001).
• PCFIA detected BSA antibodies in children with diabetes, whereas only a few non-diabetic children were positive. In contrast, only a small proportion of the antibodies were disease-associated in EIA, and levels were elevated more often in non-diabetic subjects. With only one out the 18 controls positive for IgG- or IgA- anti-BSA EtA elevated by PCFIA as well, the dichotomy was clear between antibody subsets detected in either procedure. Interestingly, the single elevated IgG and IgA values detected in both procedures derived from the same serum sample, suggesting that the host determines the choice of antibody species utilized in the common immune response to dietary BSA.
• the BSA molecule consists of 608 amino acids and there are several areas where the sequence differs from human serum albumin. One of those is the described ABBOS (pre-BSA position 153-169 (19)).
• An antigen such as BSA has several epitopes which can induce a wide spectrum of high and low affinity antibodies.
• Our results are very pronounced of the observation that insulin-autoantibodies (IAA) detected by EIA are poorly disease-associated (27) and have a low predictive value compared to fluid-phase radiobinding assays (RIA) (28-30) .
• EIA has been characterized by low sensitivity and an unacceptably high rate of false positives (27) , similar to the results obtained in this study.
• EIA detects mainly low affinity IAA that have high binding capacity
• RIA detects high affinity, low binding capacity and strongly disease-associated subset(s) of IAA (30) .
• Example 1 The samples in this study were obtained as a subset of the blood samples employed in Example 1 which included over 90% of all families with a newly diagnosed child with IDDM ("INDEX CASE") over a period of several years (the study is still ongoing) . Blood samples were taken from siblings of index ceases at the time of diagnosis for the latter and every six-twelve months later. Over 700 families were enrolled and 19 siblings of index cases turned diabetic so far. Our samples represent a subset of 11 of these converters and one hundred siblings which appear healthy (at most 1-2 of these are expected to convert to diabetes) . Of all these children there were 1-6 samples available taken at the above interval. Serum IgG anti-BSA antibody levels were measured as described in Example 1.
• Table 6 shows IgG anti-BSA antibody levels present in the very first sample taken from each subject (i.e. when the 'index case' was diagnosed). Diabetes developed in these children 1-5 years later.
• Table 7 shows IgG anti-BSA antibody levels in a random subset of 100 healthy siblings to the index cases.
• Elevated levels are defined as levels above the Mean +2 Standard Deviations (SD) found in normal controls.
• SD Standard Deviations
• Venous blood samples were obtained with informed consent from patients with recent onset IDDM and from healthy controls.
• T lymphocytes were prepared from the blood samples and T cell proliferation in response to BSA and to various synthetic peptide fragments of -BSA was assayed as described in Dosch et al. (22), but wi €h substitution of serum free media.
• Synthetic peptides were generated on a Pharmacia peptide synthesiser (Pharmacia LTD, Montreal, Quebec) following the manufacturers' recommendations and purified by conventional HPLC techniques.
• ABBOS ac-Phe-Lys-Ala-Asp-Glu-Lys-Lys-Phe-Trp-Gly-Lys-
• ABRAS ac-Gln-Glu-Asn-Pro-Thr-Ser-Phe-Leu-Cys-His-Tyr- Leu-His-Glu-Val-Ala-Lys-Lys-NHj
• CS2270 ac-Tyr-Ala-Asn-Lys-Tyr-Gly-Val-NH 2 (YANKYGV)
• CS2268 ao-Lys-Phe-Trp-Gly-Lys-Tyr-NH 2 (KFWGKY)
• CS2267 ac-Glu-Phe-Lys-Ala-Asp-Glu-Lys-Lys-NH 2 (EFKADEKIO
• CS2240 ac-Ile-Glu-Thr-Met-Arg-Glu-Lys-Val-Leu-Thr—Cys-
• heparinized blood is diluted 1:1 with serum-free HL/1 medium and mononuclear cells are purified on Ficoll-Hypaque gradients, washed in HL/1, counted and 2xl0 5 are added to 96 well microculture plates in a volume of 200 ⁇ l HL/1.
• Control cultures receive either no or a supplement of the pan-T cell mitogen Phytohemagglutinin (l ⁇ g) .
• BSA (0.01-10 ⁇ g) or other test proteins, or 0.1- l ⁇ g of a synthetic peptide are added to triplicate test cultures.
• IDDM patients were found to have sensitized T lymphocytes which specifically recognize and proliferate in response to peptide CS 2267 while normal controls lacked such sensitized T lymphocytes. Subjects with pre- clinical IDDM as assessed by currently available indicators showed the same response as IDDM patients.
• the present inventors Having established the predictive power of anti-ABBOS and anti-BSA immunity, the present inventors have also developed an in vitro system to detect T lymphocytes which initiate and sustain this immunity until final ⁇ cell demise.
• Diabetes Res. Clin. Pract. 14 (Suppl. 1) : 89A. 28. Kuglin, B., Kolb, H., Greenbaum, C. , Maclaren, N.K.,

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