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WO1993016725A1 - Antigene precoce pour diabete autoimmun - Google Patents

Antigene precoce pour diabete autoimmun Download PDF

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Publication number
WO1993016725A1
WO1993016725A1 PCT/US1993/001716 US9301716W WO9316725A1 WO 1993016725 A1 WO1993016725 A1 WO 1993016725A1 US 9301716 W US9301716 W US 9301716W WO 9316725 A1 WO9316725 A1 WO 9316725A1
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WIPO (PCT)
Prior art keywords
antigen
cells
diabetes
cell
protein
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PCT/US1993/001716
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English (en)
Inventor
C. Garrison Fathman
Lisa Paborsky
Cohava Gelber
Original Assignee
The Board Of Trustees Of The Leland Stanford Junior University
Immulogic Pharmaceutical Corporation
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Application filed by The Board Of Trustees Of The Leland Stanford Junior University, Immulogic Pharmaceutical Corporation filed Critical The Board Of Trustees Of The Leland Stanford Junior University
Priority to EP93907049A priority Critical patent/EP0627934A4/fr
Priority to JP5515082A priority patent/JPH07508503A/ja
Publication of WO1993016725A1 publication Critical patent/WO1993016725A1/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the insulin- producing ⁇ -cells of the pancreatic islets are selectively destroyed; other endocrine islet cells, ⁇ cells, such as those that produce glucagon or somatostatin are unaffected.
  • the destruction of the ⁇ - cells is mediated in part by T-cell proliferation, in an induction phase, resulting in the production of effectors of the 0-cell destruction, such as cytotoxic antibodies, natural killer cells, macrophage and lymphokines (Castano, L. et al., Ann Rev Immunol (1990) £:617-679) .
  • the onset of type I diabetes is believed to involve an induction phase common to both normal and autoimmune responses, which involves an initial activation of helper CD4 + T-cells by engagement of the T-cell antigen receptor (TCR) with MHC class II associated peptides presented on antigen-presenting cells (APC) .
  • TCR T-cell antigen receptor
  • APC antigen-presenting cells
  • This activation results in secretion of ly phokines which in turn activate the effector ⁇ -cells, cytotoxic T-cells, natural killer cells, macrophage, and the like.
  • GK1.5 anti-CD4 antibody
  • hsp65 65 kd heat shock protein
  • T-cell clones which are specific for insulinoma cell antigens have been prepared by Von Vliet et al., Eur J Immunol (1989) 19_:213-216. These T-cell clones were prepared using rat insulinoma membranes as antigen, and generating the cell lines from peripheral blood mononuclear cells of patients with recent onset of the disease. In addition, it has been shown that T-cell clones from a type I diabetes patient respond to an integral membrane component of the insulin secretory granules which has been purified 5000-fold and shown to have a monomer of molecular weight 38 kd (Roep, B.O. et al., Nature (1990) 145.:632-634) .
  • the present invention provides antigens which are responsible for the very early inductive stages in the development of this condition. These antigens are useful in diagnosis and design of immunotherapy.
  • Antigens associated with the initial T-cell induction phase leading to the onset of type I diabetes have now been identified. These antigens are useful for the early diagnosis of the development of the disease and also can be used in immunotherapy of susceptible subjects.
  • the invention is directed to an antigen having a molecular weight in the range of 30-60kd normally present in mammalian, particularly human or murine, pancreatic islet 0-cell cytosol and/or membranes, and to antibodies or T-cells specifically immunoreactive with this antigen.
  • Human proteins with this activity have molecular weights of about 37 kd, 41 kd and 51 kd, as determined by HEPC; murine proteins have molecular weights of about 36 kd, 42 kd and 55 kd as determined by gel electrophoresis.
  • the invention is directed to methods to diagnose susceptibility to type I diabetes by assessing subjects for the presence or absence of antibodies or T-cells responsive to this antigen.
  • the invention is directed to methods to prevent the onset of type I diabetes by rendering subjects unresponsive to the antigen of the invention and by blocking immune response to the antigen using peptide subunits thereof alone or in combination with immune modulating agents.
  • Figure 1 is a bar graph showing the results of a T-cell proliferation assay with respect to various insulinoma extracts.
  • Figure 3 shows a nitrocellulose blot of one-dimensional gel electrophoresis of insulinoma membrane proteins assayed for the ability of these fractions to stimulate the proliferation of T-cells from NOD mice.
  • Figure 6 shows the distribution of T-cell proliferation stimulation activity between membrane and cytosol fractions of a murine insulinoma.
  • Figure 7 shows the results of two-dimensional gel electrophoresis of a murine insulinoma extract as assayed for the ability to stimulate proliferation of T-cells obtained from 30 day old NOD mice.
  • Figures 9A and 9B show the analysis of antigen- containing extracts separated by molecular weight distribution using the T-cell clones LN-7 and LN*CM respectively.
  • Figures 10A and 10B show stained pancreata from a control mouse and from a mouse administered the T-cell clone LN-7.
  • the invention provides antigens associated with early events connected with the inductive phase of type I diabetes.
  • the antigens of the invention offer the opportunity for early screening of individuals developing the disease and offer the opportunity for intervention to prevent its development and/or perpetuation.
  • the antigens of the invention are obtainable from mammalian, such as the exemplified murine or human, pancreatic islet ⁇ -cells or cell lines derived therefrom. Cross species reactivity of the relevant antigens has been demonstrated, and there appears to be significant homology between the murine and human antigens. Thus similar antigens are believed present and isolatable from ⁇ -islets of other mammals, such as bovine, porcine, ovine, feline and the like.
  • the antigens may be obtained from either the membrane fraction or the cytosol fraction of islet ⁇ - cells or their derived cell lines, or the related neuroblastoma or other neuroendocrine cells.
  • the antigens are present in both non-susceptible individuals and in individuals susceptible to type I diabetes. Therefore, the cells used as a source of the antigens need not be derived from affected subjects.
  • the antigens may initially be prepared by extraction and fractionation of the native material from the 0-cells or their derived cell lines, but may more conveniently be prepared using recombinant techniques from the encoding DNA. Provision of the native protein in purified and isolated form permits the design of probes useful for retrieval of the cDNA encoding these antigens.
  • expression libraries obtained in, for example, ⁇ gtll and transformed into iL. coli can be screened for the ability of the expression products to effect proliferation of T-cells obtained from murine models for type I diabetes, such as NOD mice.
  • Antibodies immunoreactive with the antigens may also be used to screen expression libraries.
  • Extraction is generally conducted by homogenizing the 5 cells in the presence of appropriate membrane buffer at about pH 7-8; removing the cellular materials by centrifugation, recovering the supernatant, and then centrifuging the supernatant at high speed to obtain the membranes, as the pellet and the cytosol as the
  • the fractions may be assayed for activity by
  • T-cell proliferation assay utilizes labeled thymidine incorporation
  • the T-cell preparation is obtained from single- cell suspensions from spleen, lymph nodes or PBL taken from naive (untreated) NOD mice. Dead cells and red
  • blood cells are removed by Ficoll gradient centrifugation by spinning in the gradient for 25 minutes at 2500 rpm at room temperature. This provides an enriched lymphocyte population and contains antigen presenting cells (APC) .
  • the T-cells and APC are plated at 0.25-0.5 x 10 6 /well in
  • fractions or extracts are adjusted toll Triton X-100,.15% glycerol and 6% a pholines, pH 3-10.
  • Isoelectric focusing is then performed according to the method of O'Farrell, P.H. J Biol Chem (1975) 250:4007-4021.
  • the resulting one-dimensional separation is then followed by loading each region.onto a 10% SDS-PAGE gel and electrophoresis conducted according to Laemmli, U.K. Nature (1970) 227:680-685.
  • the fractions identified are then recovered in purified and isolated form. They can be further characterized by determination of amino acid sequence and by retrieval of the encoding gene from cDNA libraries prepared by reverse transcription of ⁇ -islet mRNA or from a genetic library.
  • the DNA libraries are prepared using standard techniques, and can be screened using probes designed on the basis of total or partial amino acid sequence of the recovered antigen.
  • the recovered DNA can, in turn, be used as a probe to recover DNA encoding the corresponding antigens in other species.
  • the starting cDNA library may be prepared as an expression library in, for example, ⁇ gtll, and the library then screened using techniques which detect the synthesized antigen.
  • Two means for screening the library to detect the antigen produced are particularly preferred.
  • antibodies prepared against the isolated antigen can be used in screening the library in conventional techniques.
  • the library may be screened by assessing the ability of each of the clones contained therein to produce an antigen which stimulates proliferation of T-cells obtained from type I diabetes-susceptible individuals, including NOD mice. Those colonies that produce proteins capable of stimulating this proliferation contain the gene encoding the stimulatory antigen.
  • the encoding DNA is ligated into expression systems compatible with a convenient host.
  • host systems and control sequences operable in said host are now available in the art.
  • Suitable hosts include prokaryotes such as £_ • . coli and eucaryotes. such as yeast, avian cells, insect cells, mammalian cells, and plant cells; more recently, whole plant or animal organisms have also been used.
  • Techniques for constructing expression systems and transforming appropriate hosts with the constructed systems are by now standard in the art.
  • the reco binant host cells transformed with an expression system containing the gene encoding the antigen operably linked to control sequences are cultured under conditions which permit the expression of the encoding DNA, and the antigen is recovered from the culture using standard procedures. Construction systems may be employed which result in secretion of the antigen, which can then be recovered from the medium, or the antigen may be produced intracellularly, which will necessitate lysing the host cells.
  • the ⁇ -islet cell antigens of the invention when isolated and characterized, provide sequence information for identification of peptides associated with interaction with the TCR on the helper T-cells.
  • These peptide segments are contiguous sequences of at least about 7 amino acids that are ' associated with the MHC class II glycoprotein present on the surface of antigen-presenting cells, resulting in a complex that interacts with the TCR.
  • the relevant peptides can be systematically identified by testing overlapping regions of the sequenced antigen in the presence of antigen- presenting cells in T-cell proliferation assays conducted as described above. Techniques for such a screen are included in the report by Hickling, J.K. et al., Eur J Immunol (submitted 1991, in press) .
  • Peptides with modified structures can then be designed which retain their ability to complex with the MHC class II glycoprotein but fail to effect reaction with the TCR by assessing the ability of these modified peptides to inhibit the T-cell proliferation in the presence of known activators in this assay.
  • Peptide modifications include extensions, deletions and substitutions, and combinations thereof. Peptides that inhibit proliferation are successful candidates. Assay
  • a convenient assay is described herein, depending on whether the assay is directed to a purified antigen, a T-cell clone, or an antigen preparation.
  • all assays may be performed in microtiter wells wherein each well contains 10,000-30,000 T cells, about 10 5 -10 6 histocompatible antigen-presenting cells, and the antigen (for crude extracts this amounts to about 0.3-20 ⁇ g/ml) .
  • the antigen for more purified antigen, less antigen would be required; for splenocyte preparations used as a source of T cells, the APCs are already contained in the original preparation. Variations of the nature and amounts of T cells, histocompatible APCs, and antigen will be understood as routine matters of optimization and experimental design.
  • insulinoma B23720, see below
  • antigen extracts or irradiated insulinoma cells Either whole cell extracts or the extracts from membranes or cytosol were used at a protein concentration of lO ⁇ g/ml.
  • irradiated insulinoma cells 5000 cells/ml in RPMI medium containing 1% NMS, Pen-Strep, glutamine and I ⁇ g/ml Leuko-A. can be used.
  • the spleen or lymph node cells used as a source of T-cells were incubated at 37°C, 5% C0 2 for 3-4 days, then washed and resuspended in RPMI medium containing 10% FCS Pen-Strep, glutamine and 15% rat Con-A supernatant or 20 U/ml rMIL-2 (Genzyme) (complete medium) , and incubated for 5 days. Stimulation of the T-cell lines was repeated 2-3 times in cycles of 8-9 days: (3-4 days with the antigen in the above "complete” medium and 5 days in complete medium + rIL-2 at 20 U/ml without antigen) .
  • Single cell cloning of the T-cell line was performed in complete medium in the presence of 10 ⁇ g/ml antigen (or 5000/ml live irradiated insulinoma cells) with 0.5x10 ⁇ irradiated spleen cells (as APC) in 96 well plate (Costar flat bottom 1/2 area) . Seven days later the clones were restimulated again with the antigen + APC as above. 10-14 days afterwards, the wells were scored for positive growth. Growing T cell clones were transferred to 24 well Costar plates and expanded.
  • the cells were tested for proliferation using 20,000 T cells with 0.5 x 10 6 irradiated splenic (2000 R) antigen-presenting cells in an assay based on labeled thymidine incorporation.
  • Cultures of the T-cells and APC cells were set up in triplicate in the presence of antigen extracts (0.3-20 ⁇ g/ml) and incubated for 72 hours, then pulsed with 1 ⁇ Cs/well of tritium-labeled thymidine (Amersham-, Inc.) and harvested 16 hours later.
  • the incorporated radioactivity was determined using a ⁇ - plate scintillation counter and results expressed as mean cpm of incorporated thymidine. Standard deviations were less than 10%.
  • a number of T-cell clones were obtained, including LN7 and LN*CM T-cell clones. T-cell clones were picked from the wells, restimulated as above, and expanded for specificity tests.
  • the antigens including protein antigens of the invention and the relevant peptide fragments can be administered to mammalian subjects in standard immunization protocols to prepare antibodies specifically immunoreactive with the antigen or peptide subunit thereof.
  • Techniques for conferring immunogenicity on peptide subunits by conjugation to carriers is well known in the art.
  • the protein or carrier-coupled peptide is injected into a suitable subject, preferably in the presence of adjuvants, and the progress of immunization can be monitored by detection of antibody titers in the plasma or serum. Standard ELISA or other immunoassays may be used with the immunogen as antigen to assess the levels of antibodies.
  • the antisera are separated from the red blood cells and can then be used as polyclonal preparations or antibody-secreting cells from the immunized host may be immortalized using standard techniques to obtain cell lines that secrete monoclonal antibodies immunospecific for the antigens or subunit thereof.
  • the antibodies per se may also be separated from other plasma proteins.
  • immunologically reactive and specific fragments such as Fab, Fab', and the like, are also useful in immunoassay techniques and are included in the antibodies of the invention. These antibodies or fragments may be purified using standard techniques.
  • the antigens of the invention can be used in early detection of subjects who are developing type I diabetes.
  • the antigens themselves are present in both normal and susceptible individuals; however, only those individuals developing IDDM produce helper T-cells whose proliferation is stimulated by the presence of these antigens.
  • T-cells obtained from the subject to be tested are used in the standard T-cell proliferation assays described hereinabove to test for their response to the antigens of the invention. Subjects whose T-cells proliferate in these assays are developing the disease.
  • Use of the identified peptides that form complexes with MHC class II glycoprotein on the surface of antigen presenting cells and activate T-cells may also be employed for inducing unresponsiveness, and administration of small peptides may be more amenable to this technique.
  • the antigen or peptides of the invention may be administered using ot'her modes of formulation suitable for peptide- containing compositions including transmucosal and transdermal forms of administration, and when properly formulated, by oral dosage.
  • suitable formulations that include pharmaceutically acceptable excipients for introducing intact peptides or proteins to the bloodstream by other than injection routes (as well as by injection) can be found in Remington's Pharmaceutical Sciences, (latest edition) Easton, PA.
  • pumps which provide the active ingredient in situ may be used.
  • the antigens of the invention may be administered alone or in concert with anti-CD4 antibodies or other CD4 blockers and/or other immune modulating substances.
  • This approach to conferring tolerance is disclosed in U.S. Patents 4,681,760 and 4,904,481.
  • the antigen and the anti-CD4 antibodies or immunoreactive fragments are administered concomitantly.
  • concomitant administration is meant within a time frame which permits the anti-CD4 component to block the helper T-cell response to the antigen.
  • the nature of "concomitant” in this sense is described in the above- referenced U.S. patents, incorporated herein by reference.
  • the methods of prognosis may be employed as a screening tool applied universally to infants and/or children.
  • the assay methods described herein require only a small blood sample; this provides a relatively noninvasive screen. Individuals who test positive by virtue of the ability of their T-cells or antibodies to respond to the antigens of the invention can then be treated as described above to prevent the progression of the disease.
  • the cells were scraped into the buffer and homogenized (Dounce) and the hqmogenate was centrifuged at 2500 rpm in a Sorval S31 Centrifuge for 10 minutes at 4°C. The pellet was discarded. The supernatant was used as a "whole cell extract" or centrifuged for 30 minutes at 48,000 xg to separate the membrane from the cytosol. The membrane- containing pellet was suspended in 20 mM HEP ⁇ S and frozen at -70°C. The cytosol supernatant was also retained for testing.
  • the extracts are adjusted to a final concentration of 7.5 mM tris-phosphate pH 7.5, 0.25% SDS, 15% glycerol, and then loaded onto a 10% SDS-tris phosphate tube gel (3.5 x 10 cm) and electrophoresed in a tris-phosphate buffer system.
  • the proteins were eluted from the bottom of the gel into 7.5 mM tris-HCI, pH 7.5; assayed for protein concentration and analyzed on a 12.5% SDS polyacrylamide gel. 80% of the protein was recovered in these fractions. These fractions that induced the proliferative response in the assay described below were pooled.
  • C-pool I these fractions corresponded to a molecular weight range of 30- 60 kd and are labeled C-pool I in Figure 1.
  • the C-pool I and the corresponding pools obtained from the "cytosol" extracts (also containing some membrane portion) of the ⁇ or-cells and human neuroblastoma cells (labeled ⁇ -pool and NB-pool in Figure 1, respectively) were compared using the T-cell proliferative assay wherein T-cells were prepared from the spleens of unprimed NOD female mice of 3-30 days of age.
  • the T-cell preparation was obtained as described hereinabove wherein single cell suspensions were prepared, in this case from spleen and lymph nodes, and clarified by removing dead cells and red blood cells using Ficoll gradient centrifugation.
  • the resulting preparation contains a full range of T-cell types, as well as antigen presenting cells.
  • the lymphocyte preparation at each age was plated at 0.25-.5 x 10 6 cells per well in 96 well U-shaped plates.
  • the lymphocytes were obtained from unfractionated (untreated) spleen and contained B- cells and macrophages as a source for APC.
  • the splenocytes for each age group represented a pool of 3-8 mice.
  • the wells were incubated with the protein fractions (10 ⁇ g/ml) .
  • the reaction mixtures were incubated for 72 hours at 37°C at 5% C0 2 and the plates were then pulsed with 1 ⁇ Ci of tritiated thymidine per well and incubated for an additional 16 hours.
  • the cells were harvested and counted.
  • the human neuroblastoma-corresponding pool stimulated proliferation at all ages.
  • the ⁇ -pool was not capable of stimulating NOD T-cell proliferation, showing a ⁇ -cell-specific antigen.
  • Controls using PHA as an inducer showed the expected levels of stimulation of T- cells from all of NOD, BALB/c and C57B1/6 strains.
  • the T-cell assay described above was modified by removing antigen-presenting cells from the test NOD T-cell preparation and assessing the effect of the various extracts in the presence and absence of APC as well as in the presence of fixed APC.
  • a single-cell suspension of spleen cells from NOD mice was passed over a nylon wool (Robin Lab) column (Julius et al., Eur J Immunol (1973) 2:645) to enrich for T-cells and deplete B-cells, plasma cells and accessory cells.
  • the cells were incubated in the column for 45 min at 37°C in complete medium (RPMI, 10% FCS) and then washed slowly with a large volume of medium. An average of 15-25% yield was obtained, and the resulting cells contained no effective APC.
  • the APC were prepared from irradiated (4000 R) NOD spleen cells; fixed APC were prepared by treating APC with 0.1% glutaraldehyde for 60 sec.
  • human antigen (derived from the neuroblastoma line) is cross- species-reactive with the NOD T-cell preparation, and the antigen derived from both ⁇ islets and the human cell line requires the presence of antigen-presenting cells in order to be effective in stimulating thymidine uptake.
  • A. Murine Insulinoma The membrane proteins extracted from B23720 insulinoma were subjected to size separation using SDS- PAGE, using 2 mg total load per gel. The elution pattern is shown as a nitrocellulose blot in Figure 3. As shown in Figure 3, discrete peaks at molecular weights 37.8, 41.9 and 55 were observed; it is believed that the small peak at 108.7 is a multiple of the 55 kd peak. The elution pattern shown in Figure 3 is presented in terms of the counts per minute observed of thymidine uptake by the spleen-derived T-cells from 30-40 day old NOD mice. The cytosol fraction from mouse insulinoma
  • Example 2 B23720 prepared as described in Example 1 was subjected to high performance electrophoretic chromatography (HPEC) , conducted as described in Example 1. These results are shown in Figure 4. Elution patterns assayed with respect to T-cells derived from NOD mice of various ages is shown. As seen in Figure 4, the proliferation response for the various fractions increases steadily over a period of 17-54 days.
  • HPEC high performance electrophoretic chromatography
  • FIG. 5 shows the elution pattern as determined by the proliferative response of the insulinoma-specific T-cell line NOD-F40 prepared from the spleen of a 40 day old female NOD mouse using the method described hereinabove. Three peaks of activity are shown.
  • the antigen appears to be present in what are purportedly cytosol extracts, whole-cell extracts and membrane extracts of the murine insulinoma.
  • Figure 6 shows a comparison of the relative activities of the antigen in each extract as assessed using NOD-F40 T-cell line proliferation. As shown in Figure 6, the majority of the antigen appears to be present in the membrane.
  • the membrane fraction of the insulinoma prepared as described in Example 1 was subjected to two- dimensional electrophoresis.
  • the samples were adjusted to 1% Triton X-100, 15% glycerol and 6% ampholines, pH 3- 10.
  • Isoelectric focusing in one dimension was performed according to O'Farrell, P.H., J Biol Chem (1975) 250:4007-4021. and after this procedure, the gels were removed from their glass tubes and loaded onto a 10% SDS phage electrophoresed according to Laemmli, U.K., Nature (1970) 227:680-685. The results are shown in Figure 7.
  • T-cell lines established from lymph node cells of 30-day-old NOD female mice, selected on whole cell extracts of the insulinoma, exhibited a T-cell reactivity similar to that of unprimed NOD lymphocytes -- that is, there were three peaks of response corresponding to the three antigen peaks in the 30-60 kd region.
  • T-cell lines cloned by limiting dilution showed differences in the antigen to which they responded.
  • T-cell clone LN*CM responded to the antigen of approximately 51 kd ( Figure 9A)
  • the clone LN7 responded to the antigen of approximately 37 kd ( Figure 9B) .
  • the individual peaks of T-cell activity presumably correspond to distinct proteins as opposed to multimers or degradation products.
  • Example 3 To distinguish the antigens of the invention from other proteins obtainable from islets, the standard thymidine incorporation assay described above was used, with the test antigens at 10 ⁇ g/ml and cultures of 1-3 x 10 5 cells per well of single cell female murine NOD splenocyte suspensions prepared in the RPMI-based medium. The results are shown in Table 1. TABLE 1.
  • the proteins listed in Table 1 as recombinant proteins were prepared as follows.
  • the cDNA encoding human hsp65 was cloned by PCR using polyA + RNA isolated from human EBV-transformed B cell line, post-incubation at 42°C for 2 hr.
  • the hsp65-encoding fragment was amplified by PCR using the primers 5' -CGGGGATCCGCC-AAAGATGTAAAATTTGGTGCAGATGCC and 5'-GTCCTCGAGTTAGAACATGCCACCTCCCATACCACCTCC (30 cycles of 30 sec at 94°C, 30 sec at 55°C and 1 in at 72°C) .
  • coIi-Tgl (supE hsd ⁇ slac- proAB)F' [traD36proAB+lacIqlacZ ⁇ M15] and protein expression was induced by addition of IPTG to the culture medium.
  • Bacteria were lysed in 100 mM Tris pH 8.0, 6M GuHCl, and insoluble material was removed by centrifugation at 40 Kg for 30 min.
  • Recombinant proteins were purified using Ni-NTA-agarose (Qiagen, Chatsworth, CA) in the presence of 6M GuHCl and dialyzed against PBS. Protein concentration was determined using BCA assay (Pierce).
  • Mouse GAD-65, GAD-67 and peripherin expressed in baculovirus system were gifts from Dr. Roland Tisch (H. McDevitt lab, Stanford) .
  • the T-cell line LN-7 was stimulated with insulinoma antigen, and three days later 2-5 x 10° stimulated cells were injected intraperitoneally into 3- week-old NOD female mice. Four weeks later, the pancreata were removed, fixed in formalin buffer, and stained with hematoxylin and eosin. Pancreata from age- matched NOD female mice were used as controls.
  • Figure 10A represents the control
  • Figure 10B represents the mouse injected with LN-7 T cells.
  • the injected mice showed acceleration of destructive insulitis, as compared to controls. Similar results were obtained with LN*CM T cell clones.

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Abstract

L'invention se rapporte à des antigènes associés à des cellules ilôts β qui activent les populations de lymphocytes T en induisant le diabète de type I. Ces antigènes peuvent être utilisés dans le diagnostic et la thérapie du diabète de type I.
PCT/US1993/001716 1992-02-27 1993-02-25 Antigene precoce pour diabete autoimmun WO1993016725A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP93907049A EP0627934A4 (fr) 1992-02-27 1993-02-25 Antigene precoce pour diabete autoimmun.
JP5515082A JPH07508503A (ja) 1992-02-27 1993-02-25 自己免疫性糖尿病のための初期抗原（ｅａｒｌｙ　ａｎｔｉｇｅｎ）

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US84287492A 1992-02-27 1992-02-27
US07/842,874 1992-02-27

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US9603948B2 (en) 2012-10-11 2017-03-28 Uti Limited Partnership Methods and compositions for treating multiple sclerosis and related disorders
US10124045B2 (en) 2013-11-04 2018-11-13 Uti Limited Partnership Methods and compositions for sustained immunotherapy
US10485882B2 (en) 2015-05-06 2019-11-26 Uti Limited Partnership Nanoparticle compositions for sustained therapy
US10988516B2 (en) 2012-03-26 2021-04-27 Uti Limited Partnership Methods and compositions for treating inflammation
US12397038B2 (en) 2017-11-29 2025-08-26 Uti Limited Partnership Ubiquitous antigens for treatment of autoimmune or inflammatory diseases

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US10988516B2 (en) 2012-03-26 2021-04-27 Uti Limited Partnership Methods and compositions for treating inflammation
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EP0627934A4 (fr) 1997-01-08
EP0627934A1 (fr) 1994-12-14
AU3778993A (en) 1993-09-13
JPH07508503A (ja) 1995-09-21
CA2130997A1 (fr) 1993-09-02

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