JPH07508503A - Early antigen for autoimmune diabetes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Early antigens in autoimmune diabetes This research is a national National Institute of Health The research was supported in part by grants from have certain rights.

technical field The present invention is useful for the diagnosis and treatment of type I diabetes. Concerning factors. More specifically, the present invention relates to T-cell proliferation associated with disease induction. and markers in β1sletcells that mediate activation. related.

Background technology Type T diabetes is known to be a chronic autoimmune disease that appears early in life. . This condition, also called insulin-dependent diabetes mellitus (IDDM), is a genetic disease. Although conceivable, nonfamilial forms also occur. ■Genetic control of type diabetes The properties of ic control have been studied in detail. major histocompatibility genes It is clear that genes associated with the MMC complex (MMC) are involved in the genetic transmission of diseases. (Wicker, L. S. et al., J. Exp. Med , (1987) 165:1639-1654). Some groups are human H The amino acid at position 57 of the β-chain of the LA-DQ class II MHC-encoded glycoprotein showed a link between the absence of sparachic acid residues and the occurrence of disease (Parhd, J. A, et al, Nature (1987) Danmatan: 599Acad Sc i USA (1988) 85:8111-8115).

The most commonly used model for human type I diabetes is non-obese diabetes ( NOD) mouse model. The mouse NOD strain was first exposed to M. This model The diseases that appear in humans are similar to those found in humans. disease symptoms Long before the onset of symptoms, islet development is primarily caused by mononuclear cells derived from T-cells. There is a sexual infiltration that leads to the destruction of insulin-producing B-cells. The onset of disease was as long as 8 weeks, which also occurred in a similar BB rat model system (see below). Ru. NOD disease is controlled by at least three genes, one of which is MHC and In both cases, insulin-producing β-cells in pancreatic islets are selectively destroyed during the disease process, and other insulin-producing β-cells are destroyed. internal and external pressure/islets, α-cells, such as those producing glucagon or somatostatin Not affected. Destruction of β-cells is partially mediated by T-cell proliferation and induction In the phase, effectors of β-cell destruction, such as cytotoxic antibodies, natural killer - Cells, macrophages and lymphokines Immunol (1990) 8 : 617-679).

Thus, the pathogenesis of type I diabetes is generally due to a common induction of both normal and autoimmune responses. phase, which consists of MHC class II associated cells present on antigen-presenting cells (APCs). Helper CD due to peptide occupying T-cell antigen receptor (TCR) 41T--includes initial activation of cells. This activation causes the secretion of lymphokines, which This time, effector β-cells, cytotoxic T-cells, natural killer cells, Activates macrophages etc. In fact, in a murine model system, anti-CD4 When CD4”7923 cells are removed from the circulation using an antibody (GKl, 5), type I It has been shown that the development of diabetes is prevented (Schizuru, J.S., et al. I, 5science (1988) 240:659 662).

■The involvement of T-cells in the progression of type diabetes suggests that spleen cells from NOD mice were can be used to transfer autoimmune diabetes to non-susceptible strains (Wicker, L, S. , et al., Diabetes (1986) 35: 855-860) , and autoreactive 7923 spheres in NOD mice. can cause diabetes (Reich).

E.-P. et al., Diabetes (1989) 38:1647-1 651). Pancreatic islet-specific T-cell clones also H askins, et al, Proc NatlAcad Sci US A (1989) 86:8000-8004 and Nakano, N. et al. , J Exp Med (1991) 173: 1091-1097. was created.

NOD? Lehuen, A. et al., JImmuno l (1990) 144: 2147-2151 is humoral in NOD strain Humoral anomalies are identified from the onset of diabetes and islet bundles. It showed that it would be separated. Nevertheless, IDDM = autoimmune disease present in infected patients. Identifying antigens associated with type I diabetes by identifying proteins that bind to autoantibodies Attempts have been made. (and all these antigens are present at the effector level) and may or may not accompany the progression of the condition.

Baekkeskov, S. et al., 5science (1984) 4:1348-1350, the BB club was maintained for as long as 8 weeks before the onset of IDDM. The 64’kd We show that antibodies against islet cell proteins exist and that antibodies are directed to pancreatic β-cells. It was suggested that it could be used to predict the immune response to. then predicted The autoantigen responsible for this is glutamate decarboxylase (CAD), which has an inhibitory This group is involved in the synthesis of the neurotransmitter γ-aminobutyric acid (GABA). The loop connects the 65kd heat shock protein (hsp65) of Cross-reactive molecules interact with diabetes-associated autoantibodies and immune responses in the NOD mouse model. It was shown by the reactivity of 1579. These authors reported that drugs administered in asinibant Proteins tend to be immunogenic, but the soluble form of the protein is a tolerogen. induce diabetes or vaccinate against diabetes using the principle of suggested that the hsp65 antigen could be used to actually immunogenic or showed that administering a non-immunogenic form of hsp65 gave results consistent with this principle. It was reported that the

In an unpublished manuscript, Ca5tano, L. et al. These antibodies were tested by screening a rat islet λgtl1 expression library. as a probe to identify colonies that produce proteins immunoreactive with It is described that it is used. This colony is carboquine peptidase-H (Enkef). The insert encoding a 136 amino acid fragment of It was found that it contains.

Furthermore, the serum used for library screening was a membrane-form carboxypetrope. It was shown that it reacts with an antigen of 52 kd, which corresponds to the molecular weight of Putidase-H.

64kd GABA carbokinlase that reacts with autoantigens associated with type 1 diabetes , hSp65 antigen, and 52kd carboxypeptidase-H, as well as insulin It is known that autoantibodies against this condition are also present in patients affected by this condition.

VonVliet human T-cell clone specific for insulinoma cell antigens et al, Eur J Immunol (1989) 19: 213- Manufactured by 216. These T-cell clones use lasotoin as an antigen. Using thrinoma membranes, cell lines were derived from peripheral blood mononuclear cells of patients with recent onset of diabetes. Manufactured by producing. In addition, T-cell clones from type I diabetic patients was purified 5000-fold and was shown to have monomers with a molecular weight of 38 kd. was shown to respond to the integral membrane components of insulin secretory granules (Roep, B. , O, et al., Nature (1990) 34th: 632-634).

All of the above antigens appear to be associated with the Nifector stage of type 1 diabetes. Before All of the antigens listed above are associated with the early activation of CD4'' T helper cell induction phase. It was not shown that These antigens are found after the onset of IDDM symptoms. Ru.

Thus, in these extensive studies, the Only antigens evidenced by factors were identified. The present invention aims to prevent the occurrence of this condition. Always provide the antigen corresponding to the initial stage. These antigens are useful for diagnostics and immunotherapy. Useful in design.

Disclosure of the invention Here, antigens related to the early T-cell induction phase that lead to the onset of type II diabetes were identified. Ta. These antigens are useful in early diagnosis of disease manifestations and in immunotherapy of susceptible patients. It can also be used in law.

In one aspect, the invention relates to the use of mammalian, particularly human or murine pancreatic islet β-cells (p in the cytosol and/or membrane of ancreatic 1slet β-cell) Antigens with a molecular weight in the range of 30 to 60 kd that are normally present, as well as antigens specific to this antigen. antibodies or T-cells immunoreactive with the target. Human protein with this activity The molecular weight determined by HEPC is approximately 37kd, 41kd and 51kd. The molecular weight determined by gel electrophoresis of the murine protein is approximately 36 kd, 4 2kd and 55kd. In other aspects, the invention provides antibodies that respond to the antigen. type diabetes by evaluating the patient for the presence or absence of body or T-cells The purpose is a method for diagnosing susceptibility to. In other aspects, the invention provides antigen The peptide subunits of the present invention can be used alone or in combination with immunomodulatory agents. by making the patient insensitive to the antigen and blocking the immune response to the antigen. The purpose of this study is to develop a method for preventing the onset of type 2 diabetes.

Brief description of the drawing Figure 1 shows the results of T-cell proliferation assays on various insulinoma extracts. This is a bar graph.

Figure 2 is a bar graph showing the effect of the presence and absence of antigen-presenting cells on T-cell proliferation. It is f.

Figure 3 shows that insulinoma membrane proteins inhibit the proliferation of T~ cells from NOD mice. One-dimensional gel electrophoresis of the fractions assayed for their ability to activate nitrocellulose shows the space plot.

Figure 4 shows that Fuzumi insulinoma extract was used in N○D mice of various ages. High performance electrophoresis chromatography (H The elution pattern of PEC) is shown.

Figure 5 shows murine insulinoma whole cells assayed using NOD mouse T-cells. The HPEC elution pattern of the cell extract is shown.

Figure 6 shows T-cell proliferation stimulation between murine insulinoma membrane and cytosolic fractions. The distribution of activity is shown.

Figure 7 shows the ability to stimulate proliferation of T-cells obtained from 30-day-old NOD mice. Showing the results of two-dimensional gel electrophoresis of a murine insulinoma extract assayed using .

Figure 8 shows the HP of human islet whole cell extracts assayed by NOD splenic cell responses. The EC elution pattern is shown.

FIGS. 9A and 9B show the results using T-cell clones LN-7 and LN*CM, respectively. Figure 2 shows analysis of antigen-containing extracts separated by molecular weight distribution.

Figures 10A and IOB show reference standard mice and T-cell clone LN-7 administered. Figure 2 shows a stained pancreata from a mouse.

Mode of implementation of the invention The present invention provides antigens associated with early events associated with the lag phase of type I diabetes.

Opportunity for initial screening of patients developing disease while the antigen of the present invention remains intact and the opportunity to interfere with and prevent its expression and/or perpetuation.

The antigen of the present invention is, for example, derived from pancreatic islet β-cells of mammals such as rats or humans. derived cell lines. Interspecies cross-reactivity of related antigens (cros s5pecies reactivity) was demonstrated, and the reactivity of murine and human antigens was demonstrated. There appears to be significant homology between them. Thus lies, pigs, sheep, cats, etc. It is likely that similar antigens exist in β-islets of other mammals and can be isolated.

The antigen may be isolated from islet β-cells or their derived cell lines, or related neuroblastoma cells or other neuronal cells. It can be obtained from either the membrane fraction or the cytosolic fraction of endocrine cells. antigen is present in both patients who are insusceptible to type I diabetes and those who are susceptible. follow The cells used as a source of antigen need not be derived from infected cells.

Antigens are initially obtained by extraction and fractionation of native material from β-cells or their derived cell lines. However, it is easier to use recombinant methods from coding DNA. It can be prepared. Having the native protein in purified and isolated form This has made it possible to design probes useful for recovering cDNAs encoding these antigens. Ru. Furthermore, for example, λgtllA can be obtained and transformed into E. coli. This expression library can be used in murine models for type I diabetes, such as NOD mice. Screening for the ability of expression products to affect the proliferation of T-cells obtained from be able to. Antibodies immunoreactive with the antigen can also be used to screen expression libraries. I can be there.

Preparation of antigen The antigen of the present invention is derived from pancreatic β-islet cells from a mammalian patient, particularly a human or murine patient, or It can be prepared by suitable extraction of cell lines derived therefrom. The antigen is I type diabetes - cells of the β-cells of both susceptible patients and those not susceptible to IDDM present in the plasma and/or membrane fractions. Furthermore, tumor cell lines derived from β-cells, e.g. For example, insulinoma can be used as a starting material. at least one of the antigens It has also been shown below that this is present in human neuroblastoma cell lines and human islets. Extraction is one Cells are homogenized in the presence of a suitable membrane buffer, generally at about pH 7-8, and centrifuged. to remove cellular material, collect the supernatant, and then centrifuge the supernatant at high speed to remove the membrane. The cytoplasm is obtained as a supernatant and the cytoplasm is obtained as a supernatant. whole cell extract can also be used. A general method for the preparation of membranes is described by Fava and Co. hen, J. Biol Chem (1984) 25th, 2636-2645. It is listed.

The extract was then subjected to gel filtration, anion exchange chromatography and polyacrylic chromatography. Separation using commonly known methods including Midkel electrophoresis and Pond's standard method It is preferable to Collect the active fraction.

Both cells contained NOD mice [T-cells obtained from type diabetes-susceptible patients, or Its ability to influence the proliferation of T-cell lines derived from They can be assayed for activity by evaluation using assays. the One such assay uses labeled thymidine insertion as a measure of proliferation and is generally Spleens and lymph from pure (untreated) NOD mice Obtain T-cell samples from single cell suspensions from nodes or PBLs. Dead cells and red blood cells Cells were incubated in the gradient by Fico11 gradient centrifugation at 2500 rpm for 25 min at room temperature. Rotate and remove. This gives an enriched population of lymphocytes, antigen presenting cells (APCs) including. T-cells and APCs were placed in 96-well U-shaped plates (Costar). The cells should be plated at 0.25-0.5x106/well and tested at various concentrations. Incubate with samples for 72 hours at 37°C and 5% CO2. continue Add 1 μC1 of tritiated thymidine per well to the plate and incubate for an additional 16 hours. Incubate.

Next, a microcell harvester (Skat) was used. Harvest and count the cells using a 1000 µm (Ron). Increased absorption of labeled thymidine increases cell proliferation. Indicates an increase in reproduction. Instead of the above, cloned with APC added separately. T-cell lines obtained can be used in the assay.

The collected fractions can then be further purified using standard protein purification methods. can. A particularly useful method that actually yields pure proteins is two-dimensional Kell electrophoresis. It is dynamic. Both parts or extract 1% Triton X-100, 15% glycerin roll and 6% ampholine, adjust pH to 3-10. Next, O' Farr e]], P, H, J Biol Ch-4 Bi (1975) λ River 4007-4 Isoelectric focusing is performed according to the method of 021. For the one-dimensional separation obtained, continue Load each area onto a 10% 5DS-PAGE gel and , electrophoresis is performed according to Natu et al. (1970) 2λ Uni 680-685.

The identified fractions are subsequently purified and recovered in isolated form. They are amino acid sequences cDNA library prepared by determination of β-islet mRNA and reverse transcription of β-island mRNA. further characterization by recovery of the coding gene from the plant or from a gene library. can be done. DNA libraries can be prepared using standard methods; Probes designed based on the entire or partial amino acid sequence of the recovered antigen can be used for screening. The recovered DNA will now be transferred to other species. used as a probe for the recovery of DNA encoding the corresponding antigen in be able to.

In addition to recovering the antigen-encoding gene using a DNA probe, the starting cDNA The library can be prepared, for example, as an expression library in λgtll, The library is then screened using a method that detects the synthesized antigen. be able to. For screening libraries to detect produced antigens Two methods are particularly clever in this regard.

In one method, antibodies prepared against an isolated antigen are combined in a conventional manner. It can be used to screen libraries for In the second method, the library Type II diabetes-susceptible patients, including NOD mice, of each clone contained therein. by evaluating the ability to produce antigens that stimulate the proliferation of T-cells obtained from Can be cleaned.

Colonies that produce proteins that can stimulate this proliferation will have a stimulating antigen. Contains the encoding gene.

One or more clones containing DNA encoding the antigen are identified as described above. Once the DNA is isolated from the clone, the relevant inserts are sequenced and/or recovered. and can be used for subsequent antigen production. Sequenced DNA and/or Its degenerate encoded form can be partially or completely encoded for use in such expression systems. can be synthesized independently.

For recombinant production of antigens, the coding DNA is linked into a simple host-compatible expression system. conclude. The technology uses a variety of host systems and regulatory sequences that can act in the host systems. currently available in the medical field. Suitable hosts include prokaryotes such as E. coli. cells, as well as eukaryotic cells such as yeast, avian cells, insect cells, mammalian cervical cells and plants. Cells have been included, and more recently, whole plants or animals have also been used. expression system Methods for constructing and transforming suitable hosts using constructed systems are currently within the skill of the art. standard.

An antigen-encoding gene operably linked to regulatory sequences for production of the desired antigen. A recombinant host cell transformed with an expression system containing the gene is used to express the code DNA. The antigen is harvested from the culture using standard methods. separate the antigen The antigen can be subsequently recovered from the culture medium using a construct system that secretes the antigen, or Can be produced intracellularly, in which case it is necessary to lyse the host cell .

The isolation and characterization of the β-islet cell antigens of the present invention result in a linkage with the TCR on helper knee cells. provides sequence information regarding the identification of peptides involved in the interaction of These pep The tide segment is a contiguous sequence of at least about 7 amino acids that is present on the surface of antigen-presenting cells. associates with MHC class II glycoproteins present on the surface and interacts with TCR produces a complex. Overlapped regions of the sequenced antigen were analyzed as described above. by testing in the presence of antigen-presenting cells in a T-cell proliferation assay , related peptides can be systematically identified. of such screening The method is described in Hickling, J. et al., Eur J. Immun.

1 (submitted in 1991, in press). Next, MHC A modified protein that retains the ability to complex with Ras II glycoproteins but cannot react with TCR. Peptides with altered structures are incubated with T in the presence of known activators in this assay. - designed by evaluating the ability of these modified peptides to inhibit cell proliferation. can be done. Peptide modifications include extensions, deletions and substitutions, as well as combinations thereof. Includes matching. Peptides that inhibit proliferation are successful candidates.

assay The assay is directed against purified antigen, T-cell clones or antigen compositions Several forms of simple assays are described herein. one Generally all assays can be performed in microtiter wells, each well containing one 0.000-30.000 T cells, approximately 105-106 histocompatibility antigen-presenting cells, and antigen (in the case of crude extract this amounts to approximately 0.3-20 μg/ml). include. However, for relatively purified antigens, the required amount of antigen is much smaller; When a lung cell composition is used as a source of T cells, APCs are Contained in the composition. Alteration of the nature and quantity of T cells, histocompatible APCs and antigens It will be understood as a routine matter of optimization and experimental design. as a measure of proliferation If tritium absorption is used, 5% FC3, IOU/ml 1O-stre p and a suitable solution such as RPMT-1640 supplemented with 200 μM L-glutamine. culture medium or other art-understood medium suitable for T-cell proliferation. A simple solution is to incubate for 72 hours. Next, we compare it to cell culture. Add 1 μCi/well of tritiated thymidine and harvest cells after 6-16 hours. harvest and count.

Other methods of measuring T-cell proliferation and modifications of the above proposal are known in the art. It is.

Generation of T-cell clones Spleen or lymph node cells from 30-40 day old native female NOD mice were collected in vitro. Insulinoma (B23720, see below) antigen extract or radiation injection activated in thrinoma cells. Whole cell extracts or extractions from membranes or cytosol The output was used at a protein concentration of 10 μg/ml. In another case, 1% NMS, P e n-3t r e p, glutamine and Iμg/ml Leuko-A Irradiated insulinoma cells (5000 cells/ml) in RPMI medium containing can be used.

Spleen or lymph node cells used as a source of T-cells were incubated at 37°C and 5% C. Incubate for 3-4 days in O2, followed by washing and 10% Fe2P. en-3trep, glutamine and 15% rat Con-A supernatant or 20 U/ml in RPMI medium (complete medium) containing rMIL-2 (Genzyme) The mixture was resuspended and incubated for 5 days. T = Activation of cell line is 8-9 days cycle repeat 2-3 times (3-4 days with antigen in “complete” medium as described above, and 5 days in complete medium + 20 U/ml rIL-2).

Single cell cloning of T-cell lines was performed using 96-well plates (Costar flat bottom 1/2 flat bottom 1 to 2 m1 irradiated viable insulinoma cells) and 0.5x106 irradiated tiles It was carried out in complete medium in the presence of visceral cells (as APC). Upload the clone after 7 days It was reactivated with the antigen + APC described above. After 10-14 days, evaluate the wells for positive growth. I valued it. Transfer the growing T cell clone to a 24-well Co5tar plate; Expanded.

In assays based on labeled thymidine insertion, irradiated spleens with 0.5xlO' (200OR) Expand cells using 20,000 T cells with antigen-presenting cells I looked into it. Culture of T-cells and APC cells was performed using antigen extract (0.3-20 μg/mI) in triplicate, incubated for 72 hours, then 1μg/mI). Cs/well tritium-labeled thymidine (Amersham. I nc.) was added and harvested 16 hours later.

The inserted radioactivity was measured using a β-plate scintillation counter, and the results was expressed as the average cpm of inserted thymidine. The standard deviation is less than 10%. Ta. Many T-cell clones including LN7 and LN*CM T-cell clones Obtained.

T-cell clones were harvested from the wells, reactivated as above, and tested for specificity. Expanded for.

antibody production Antigens containing the protein antigens of the invention and related peptide fragments are used for standard immunization. Alternatively, the antigen or peptide subunit thereof can be administered to a mammalian patient to specifically immunize the patient with the antigen or its peptide subunits. Reactive antibodies can be produced. Peptide subunits can be isolated by conjugation to carriers. Methods of conferring immunogenicity on 1- are well known in the art. protein or injecting the carrier-conjugated peptide into a suitable agent, preferably in the presence of an adjuvant. The progress of immunization can be monitored by detecting antibody titers in plasma or serum. Wear. Standard EL ISA or other immunoassay using antigen as immunogen can be used to assess the amount of antibody.

Antiserum can be isolated from red blood cells and subsequently used as a polyclonal composition. or immortalize antibody-secreting cells from the immunized host using standard methods; secrete monoclonal antibodies immunospecific for the antigen or its subunits Cell lines can be obtained. Antibodies themselves can also be separated from other plasma proteins can. In addition to polyclonal and monoclonal compositions, FabSFab' Immunologically reactive and specific fragments such as and is included in the antibodies of the present invention. These antibodies or fragments are standard I using the law? Iu can be used.

Prognosis regarding the onset of type I diabetes Antibodies of the invention can be used for early detection of patients exhibiting type I diabetes. . Although the antigen itself is present in both normal and susceptible patients, IDDNi Only those who are present are helpers whose proliferation is activated by the presence of these antigens. produce cells. Thus, for one way to an early prognosis, which patients should be investigated? The T-cells obtained from the method of the present invention were used in the standard T-cell proliferation assay described above. Examine its response to antigen. patients whose T-cells proliferated in these assays. The person is showing signs of illness.

It is currently unknown whether these animals produce antibodies against the initial antigen of the invention. not present. The availability of these antigens allows us to detect the presence or absence of these antibodies. You'll be able to pin it down. that the patient produces antibodies before the onset of the condition Detection of the presence or absence of these antibodies using these antigens also It can be used as a prognostic tool. Testing for the presence or absence of these antibodies Standard immunoassays for the detection of I can be there.

treatment The antigen of the present invention can be used to prevent the onset of type I diabetes and to prevent the onset of the disease. It offers the possibility of novel treatments for prevention. Current tactics are highly invasive , generally using immune system suppressants. A milder treatment may be provided by the antigens of the invention. and it can be administered under non-immunogenic conditions, which conditions provide immunity to the antigen. rendering the patient unresponsive to the antigen without inducing an infectious response. Acceptable dosage of antigen or General methods for administration of the related peptides are known in the art. , introduction in the absence of adjuvant and administration in soluble form. antigen forms a complex with MHC class II glycoproteins on the surface of presenting cells and Utilization of the identified peptides to activate can also be used to induce non-responsiveness. , administration of small amounts of peptide is more suitable for this method.

Besides the known routes of subcutaneous, intravenous or peritoneal injection, the antigens or peptides of the invention can be Other modes of formulation suitable for peptide-containing compositions, including transmucosal and transdermal administration effects. It can also be administered by oral administration if properly formulated. can be administered. Injected as is by other than the injection route (by injection) Pharmaceutically acceptable excipients for introducing peptides or proteins into the bloodstream A suitable formulation containing the agent can be used with a pump that delivers the doses on the spot.

The antigen of the invention can be used alone or with anti-CD4 antibodies or other CD4 blocking agents. can be administered in conjunction with other immunomodulators. This method of imparting resistance is National Patent Nos. 4.681,760 and 4. Disclosed in No. 904゜481 . In this method, the antigen and anti-CD4 antibody or immunoreactive fragment are concomitantly Administer. “Concomitant” administration means that the anti-CD4 component is directed against the antigen by helper cells. means within a time frame in which a response can be prevented. The nature of “incidental” in this sense is The contents of this patent are incorporated herein by reference. This will be the contents of the statement.

Finally, listed as antagonists capable of binding to MHC class II glycoproteins. using a modified peptide that produces a complex that interacts with T helper cells but does not interact with T helper cells. Other treatments can also be used. The mode of administration and formulation for these peptides is Same as above.

The antigens of the present invention provide a relatively mild form of interference to prevent the onset of IDDM. Again, the prognostic method given is a standard that is universally applied to infants and/or children. It can be used as a cleaning means. The assay methods described herein are Requires only a small amount of blood sample, which provides a relatively non-invasive screening . the test is positive because of the ability of the T-cell or antibody to respond to the antigen of the invention The patient can subsequently be treated as described above to prevent disease progression.

The following examples are for illustrative purposes and are not intended to limit the invention.

Example 1 Simply stated, a confluent cell layer is regenerated in the absence of calcium and magnesium ions. Washed three times with phosphate buffered saline. Aspirate any remaining liquid after the final wash. , 20mM HEPES, pH 7, 5, 1, 5mM Mg Cl 2.1mM Membrane buffer consisting of EGTA, 1mM PMSF and 1μg/ml leupeptin added. Scrape the cells into the buffer, homogenize (dounce), and Modify the modulate in 5 orval S31 centrifuge at 2500 rpm at 4°C. Centrifuged for 10 minutes. I threw away the pellets. Use the supernatant as a “whole cell extract” Alternatively, separate the membrane from the cytosol by centrifuging at 48,000 x g for 30 min. Ta. The membrane-containing pellet was suspended in 20mM HEPES and frozen at -70°C. Ta.

Cytosol was also retained for testing.

- Ma cell line B23720, glucagonoma pancreatic α-cell line and human neuroblastoma cells System 5Y5Y (Goya, L,, et al,, Neurochem Res ( 1991) 16:113 116).

Furthermore, App l i ed Bi for the cytosolic fraction of insulinoma cells. high performance electrophoresis chromatography using HPEC23OA system, Inc. Tography (HPEC) was performed. (The extract is derived from the membrane along with the cytoplasm. HPEC was performed on 400 μg of total protein. cormorant. ) For HPEC, extract was added to 7.5mM tris-phosphate pH 7,5,0 , 25% SDS, adjusted to a final 1 degree of 15% glycerol, followed by 10% 5DS-tris phosphate tube gel (tubege I) (3,5xlO cm) and electrophoresis was performed in a tris-phosphate buffer system. Tan Proteins were eluted from the bottom of the cell into 7.5mM tris-HCI, pH 7.5. , assayed for protein concentration, 12.5% SDS polyacrylamide gel Analyzed above. 80% of the protein was recovered in both of these sections.

Fractions that induced a proliferative response in the assay described below were collected.

These fractions correspond to a molecular weight range of 30-60 kd and are shown in Figure 1 as C-pool. It is labeled I. C-Pool I and α-cells and human neuroblastoma cells The corresponding pool obtained from the “cytosolic” extract (also containing some membrane parts) (Fig. (labeled α-pool and NB-pool, respectively) in T-cell expansion. Comparisons were made using a proliferation assay in which T-cells were aged between 3 and 30 days and were not activated. It was prepared from the gamma of unpr.jmed female NOD mice. Single cell V suspension The T-cell composition was prepared as described above, in this case from the glenium and lymph nodes. was obtained and cleared by removing dead cells and red blood cells using F1coll gradient centrifugation.

The resulting composition contains the full range of T-cell types and antigen presenting cells.

As described above, lymphocyte compositions in 6th instars were collected in 96-well U-shaped biplate cells. Plated at 025-0.5x106 cells per well. Lymphocytes are undifferentiated Separately (unprocessed) from the lungs, as a source of B-cells and APCs, and as a source of macrophages. Contained. For groups of 6 instars, the spleen cells gave a pool of 3-8 mice.

Wells were incubated with the protein fraction (10 μg/ml). reaction The mixture was incubated for 72 hours at 37°C in 5% CO2, followed by plating. Give 1 μCi of tritiated thymidine per well and incubate for an additional 16 hours. I bet. Cells were harvested and counted.

The results are shown in Figure 1. As shown in the figure, RPMI medium alone was used under both conditions. did not activate thymidine uptake in this assay, and neither extract pool B Proliferation of T-cells induced by the above method from ALB/c or C57B1 mice It was not activated. This indicates that the response is specific to T-cells from NOD mice. and Additionally, pools from alpha cells show no activation. these mice ■Does not exhibit symptoms similar to type diabetes. For activation of T-cells from NOD mice Susceptibility was investigated at various ages from 8 to 28 days.

As shown in Figure 1, C derived from B23720 murine insulinoma (β islet) - Pool I is from very young mice as well as from mice with a relatively high content of T-cells. It was active against. Human neuroblastoma-matched pool grows in all instars was activated. However, α-pool was unable to activate NOD cell proliferation; It was shown to be a β-cell specific antigen. Reference standard using PHA as inducer is expected for T-cells from all NOD, BALB/C and C57B1/6 strains. showed a certain degree of activation.

The T-cell assay described above was performed by removing antigen-presenting cells from the test NOD cell composition. and various conditions in the presence and absence of APC, as well as in the presence of immobilized APC. Modified by evaluating the effect of extracts.

For this T-cell composition, a single cell suspension of flax cells from NOD mice was Passed over a Robin Lab column (Julius et al. al., Eur J Immunol (1973) 3:645), T-cells was enriched to deplete B-cells, plasma cells, and antigen-presenting cells. Color cells Incubate for 45 minutes at 37°C in complete medium (RPMI, 10% FC3). incubated followed by slow washing with copious amounts of medium. Average yield of 15-25% were obtained and the resulting cells did not contain effective APC. Experimented in the presence of APC In the case of samples, APC was prepared from irradiated (4000R) NOD spleen glistens; Fixed APC was obtained by treating APC with 0.1% glutaraldehyde for 60 seconds. It was prepared as follows.

The presence of β-islet cytosolic extract or human neuroblastoma cell cytosol as shown in Figure 2 The presence of antigen-presenting cells is required in order to elicit a response under from α-cells The extract did not elicit a significant response both in the presence and absence of APC. antigen When using whole cell extract as a source, incubate with APC or antigen. After only 4 hours, fixed APCs were treated with insulinoma extract or human neuroblastoma 5. It was necessary for the activation of T-cell proliferation by Y5Y extract.

As shown by the above results, human antigen (derived from neuroblastoma cell line) is NOD Cross-species cross-reactive with T-cell compositions and derived from both β-islet and human cell lines. The antigen presented requires the presence of antigen-presenting cells to be effective in stimulating thymidine absorption. do.

5DS-PAG for membrane protein extracted from B23720 insulinoma Size separation was performed using E with a total load of 2 mg per gel. Elution pattern The plot is shown in Figure 3 as a nitrocellulose plot. Shown in Figure 3 As expected, separate peaks were observed at molecular weights 37.8, 41.9 and 55, and 1 The small peak at 08.7 appears to be twice the 55 kd peak. Shown in Figure 3 The elution pattern was similar to that of gamma-induced T-cells from 30-40 day old NOD mice. The thymidine absorption is expressed as counts per minute observed for thymidine absorption.

Samples from mouse insulinoma B23720 prepared as described in Example 1. High performance electrophoresis chromatography performed as described in Example 1 for the cytosolic fraction. fee (HPEC) was performed. The results are shown in Figure 4.

Elution patterns assayed for T-cells derived from NOD mice of various ages. shows. As can be seen in Figure 4, the proliferative response for the various fractions ranged from 17 to 54 It increases steadily over days.

Whole cell extracts of rat insulinomas were also subjected to HPEC under the above conditions. Figure 5 was prepared from the pancreas of 40-day-old female NOD mice using the method described above. Lysis determined by the proliferative response of the insulinoma-specific T-cell line N0D-F2O. Shows the separation pattern. Three peaks of activity are shown.

As is clear from the above results, the antigen is a cytosolic extract of insulinoma , appears to be present in purported whole cell extracts and membrane extracts. Figure 6 shows N Relative activity of antigen in each extract evaluated using 0D-F2OT-cell line proliferation Show comparison. As shown in Figure 6, the majority of the antigen appears to be present in the membrane. .

Two-dimensional electrical I performed electrophoresis. Sample 1% Tr i tonX-100, 15% glycerol and 6% amphorine, adjusted to pH 3-10. - After a long period of time, apply the gel to the Using the method described in Example 1, remove 10% SDS phage from the glass tube. Whole cell extracts of islets were obtained. H performed on these extracts using the method described above. PEC gave the elution profile shown in Figure 8, with a molecular weight of 3 for the corresponding human antigen. It was confirmed that the range was 0 to 60 kd. The elution profile in Figure 8 is 40 days old. was determined using fresh pancreatic cells from female NOD mice.

As shown in Figure 8, human islet extracts are approximately 37kd, 41kd It contains three separate peaks, one at and one at approximately 51 kd.

Using a panel of T-cell clones prepared as described above, three protein The different properties of texture were studied. First, selection on insulinoma whole cell extracts A T-cell line established from lymph node cells of 30-day-old female NOD mice , exhibiting T-cell activity similar to that of unactivated NOD lymphocytes, i.e. There are three peaks of response corresponding to three antigen peaks in the 30-60 kd region. . However, after two cycles of antigen retrieval, T- Cell lines showed differences in the antigens to which they responded. T-cell clone LN*CM clone LN7 responds to an antigen of approximately 51 kcl (Figure 9A), and clone LN7 responds to an antigen of approximately 37 kd. (Figure 9B). Thus, each peak of T-cell activity is a multimer or degradation product. (corresponding to separate proteins).

Example 3 To distinguish the antigen of the invention from other proteins obtained from islets, 10 μg 7 ml of test antigen and female murine NOD gills prepared in RPMI-based medium. Use a culture of 1-3xlO' cells per well in a single-cell suspension of cells as described above. A standard thymidine insertion assay was used. The results are shown in Table 1.

Table 1 whole cell extract Beta insulinoma 31553±1862 Alpha glucagonoma 1 012±38 islands (NOD mouse) 13416±2178 islands 1-) 2903 2±2090 pancreatic hormone Rat insulin 899±105 Uno Insulin 958±87 C-1 peptide (mouse) 854±119 C-1 peptide (rat) 997 ±68 Glucacon 1002±95 Somatostatin 765±74 recombinant protein hsp65 1078:! :329 hsp70 932±119 Carboxypeptidase-H559±121PM-1669±98 CAD-6599±25 The data in Table 1 shows that islets from β-insleema, NOD mice and islets from humans This shows that thymidine insertion into lung cells can be successfully activated. α-glucago Norma extracts are not successful. Insulin, C-peptide, glucagon and soma Other pancreatic hormones, including tosuftin, are also inactive. heat shock protein (hs p)65, hsp70. Carboquinodase H, PM-1, CAD-65, CA D-67 and belliferin are also inactive. Both of these proteins are highly extractable. It is not possible to show the activation effect of the product.

The proteins listed in Table 1 as recombinant proteins were prepared as follows.

cDNA encoding human hsp65 was incubated for 2 hours at 42°C. PC using polyA'' RNA isolated from a human EBV-transformed B cell line. Cloned by H. Reverse transcription followed by hsp55-coding fragment Primer 5° -CGGGGATCCGCCAAAGATGTAAAAATTT C; GTGCAGATGCC and 5°-GTccTcGAGTTAGAAcAT Amplified by PCR using GccAccTcccATACCACCTCC ( 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute). human Carboxyhebutidase-H, PM-1 (gift from G. Eisinbarth) ) and cD encoding human hsp70 (ATCC/clone pH2,3) NA encodes a protein labeled with six histidine residues at the N-terminus A synthetic NA fragment encoding six histidine residues was inserted into pT. The expression vector constructed by inserting into the polylinker of the rc99A expression vector vector pTrc99A (Aman.

E., et al., Gene (1988) June 301-315) (H15 6) was cloned into. The plasmid construct was transformed into E, coli Tgl (supE) hsd△5lac-proAB)F' [traD36proAB+lacIg The cells were transformed into lacZΔ M15 cells and the protein was transformed by adding IPTG to the medium. protein expression was induced. Bacteria 100mM Tris pH 8.0.6M Remove insoluble materials by dissolving in GuHC1 and centrifuging at 40 kg for 30 minutes. Removed. N1-NTA-agarose (Qiagen , Chatswo rth, CA) to purify the recombinant protein; Dialyzed against PBS. Using BCA assay (Pi e r c e) Protein concentration was determined. Mouse CAD-65 expressed in a baculovirus system, CAD-67 and belliferin were obtained from Dr. Roland Ti5ch (H, Mc David David Iab, 5tanford).

The T-cell line LN-7 was activated with insulinoma antigen and 3 days later, 2-5x10 Activated cells were injected intraperitoneally into 3-week-old female NOD mice. After 4 weeks, the pancreas was removed. The cells were removed, fixed in formalin buffer, and stained with hematoxylin and eosin.

Pancreas from age-matched female NOD mice was used as a reference standard.

The results of this experiment are shown in FIGS. 10A and IOB. Figure 10A shows the reference standard; 10B shows a mouse injected with LN-7T cells. The injected mouse is the reference standard. showed promotion of destructive insulitis compared to standard. Using LN*CMT cell clones Similar results were obtained.

′ ′ ′ 3 J Y '(-Moha 4 〆mi →; T B (co =]) N 11 Ding / Mi,! Yohi 69 and SYNd0 B 23720 Insulinoma Differentiated Cell 71 Extract Antigen against NOD Liver Cell proliferative response B 23720 Cytoplasmic I-(P E C fraction tNia5Co■ FIG. 5 (Yubetsu cold fall 4 Miko (tncb) y <÷064・ Fig, IOA Continuation of front page (51) Int, C1,6 identification symbol Internal office reference number C12N 1/21 8 828-4B C12P 21100 C9282-48GOIN 33153 D 7055 -2J//(A61K 39100 39:395) (72) Inventor Papowski, Lisa San Francisco Jerseyist, California 94114, United States leet 165 ei I (72) Inventor: Gelber, Kohaba Mountain View Deer Woods, California 94040, United States coat

Claims (14)

[Claims] 1. Monomers that can be isolated from pancreatic islet β-cells and range from approximately 30 to 60 kd. has a molecular weight that stimulates the proliferation of NOD murine T-cells in the presence of antigen-presenting cells. The antigen in purified and isolated form. 2. can be isolated from human islet β-cell membranes and determined by HPEC. has a molecular weight of about 37 kd, 41 kd or 51 kd, or Murine pancreatic islet β-can be isolated from cell membranes and determined by gel electrophoresis The antigen according to claim 1, having a molecular weight of about 36 kd, 42 kd or 55 kd. . 3. Binds to MHC class II-encoded glycoproteins on antigen presenting cells (APCs) to form a complex, which is recognized by NOD murine T-cells. A peptide fragment of the protein antigen according to claim 1, characterized in that Yeah binds to MHC class II-encoded glycoproteins on APC to form a complex; A modified form thereof, characterized in that the complex is recognized by NOD murine T-cells. Status. 4. A purified and isolated protein encoding a protein antigen according to claim 1. recombinant DNA in the form of contains code DNA, and can express the code DNA when contained in a host cell. A recombinant protein encoding the protein antigen according to claim 1 contained in an expression system comprising Eh DNA. 5. A recombinant host cell comprising an expression system according to claim 4. 6. Cultivating the cell according to claim 5 under conditions that allow expression of the coding DNA, isolation from islet β-cells, including recovering proteins from cell culture; , monomer molecular weight in the range of 30-60 kd, and the presence of antigen-presenting cells. Treatment of type I diabetes, which can stimulate the proliferation of NOD murine T-cells under A method for producing proteins useful in cancer and diagnosis. 7. in purified, isolated form or substantially free of red blood cells contained in the composition or substantially free of plasma proteins; specifically immunoreactive with the antigen according to claim 1, which is a monoclonal antibody. Antibodies or immunologically reactive fragments thereof. 8. T-cells obtained from a patient are subjected to claim 1 under the conditions of a T-cell proliferation assay. contacting the T-cell with the antigen described and determining the ability of the T-cell to proliferate in the presence of the antigen; Patients whose T-cells proliferate under these conditions are considered to be exhibiting type I diabetes. 1. A method for identifying a patient exhibiting type I diabetes, including determining the type I diabetes. 9. Serum or plasma obtained from a patient is treated with an antibody immunoreactive with the antigen according to claim 1. contact with the antigen according to claim 1 under conditions in which the body forms a complex, and the complex is detecting the presence or absence of the complex and providing serum or plasma capable of forming said complex. Type I diabetes, including identifying patients with type I diabetes as having type I diabetes. patient identification method. 10. Non-immunogenic forms for use in preventing the onset or progression of type I diabetes The antigen according to claim 1 or the peptide according to claim 3. 11. In combination with an immunomodulator for use in preventing the onset or progression of type I diabetes The antigen according to claim 1 or claim 3 in a non-immunogenic form, combined Peptides described in. 12. Antigen according to claim 1 in non-immunogenic form as active ingredient, or claim The peptide according to Scope 3 of the above is mixed with a pharmaceutically acceptable excipient, A drug useful in preventing the onset or progression of type I diabetes, optionally further comprising an immunomodulatory agent. Pharmaceutical composition for use. 13. The immunomodulator comprises an anti-CD4 antibody or an immunoreactive fragment thereof. The composition according to claim 12, characterized in that: 14. A cell containing red blood cells enriched with T-cells responsive to the antigen according to claim 1. No composition.