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WO1998008943A1 - Peptides de hla-drb1 ayant une affinite de liaison specifique pour les molecules de hla-dq et utiles dans la prevention et le traitement de la polyarthrite rhumatoide - Google Patents

Peptides de hla-drb1 ayant une affinite de liaison specifique pour les molecules de hla-dq et utiles dans la prevention et le traitement de la polyarthrite rhumatoide Download PDF

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Publication number
WO1998008943A1
WO1998008943A1 PCT/US1996/014041 US9614041W WO9808943A1 WO 1998008943 A1 WO1998008943 A1 WO 1998008943A1 US 9614041 W US9614041 W US 9614041W WO 9808943 A1 WO9808943 A1 WO 9808943A1
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Prior art keywords
hla
peptide
mice
drbl
rheumatoid arthritis
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S. Harvinder Luthra
S. Chella David
Eric Zanelli
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Priority to AU72355/96A priority Critical patent/AU7235596A/en
Priority to PCT/US1996/014041 priority patent/WO1998008943A1/fr
Publication of WO1998008943A1 publication Critical patent/WO1998008943A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases
    • 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
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • RA Rheumatoid arthritis
  • HLA human leukocyte antigen
  • HLA-class I and class II molecules The primary function of HLA-class I and class II molecules is binding and presentation of processed antigenic peptides to T cells bearing receptors specific for particular HLA-peptide complexes.
  • the presentation event plays a pivotal role in shaping the cellular immune repertoire and in shaping the nature and scope of the immune response against a given antigen.
  • Collagen-induced arthritis is an experimental model of autoimmune polyarthritis that has numerous similarities to human RA. David, APMIS 98: 575- 84 (1990) .
  • the histopathologic lesions of CIA resemble those seen in RA with synovial proliferation that progresses to pannus formation, with subsequent marginal bone erosions and cartilage destruction.
  • Radiographs of joints affected by CIA often show erosive changes similar to those seen in human RA, and progressive arthritis often results in joint deformity and destruction similar to that seen with RA.
  • anti-collagen antibodies develop in the CIA model.
  • mice are injected intradermally with either homologous or heterologous species type II collagen with complete or incomplete Freund's adjuvant.
  • the immune response is detected by the presence of delayed type hypersensitivity (DTH) to type II collagen as measured by skin test or ear thickness measurements.
  • DTH response peaks at day 10 after injection.
  • Antibodies to type II collagen are detected in the peripheral blood 2 weeks after injection and IgG peak titers are reached at about 4 weeks. X-ray evidence of joint involvement is apparent by 4-6 weeks with the onset of clinically apparent arthritis by 7-9 weeks.
  • mice studies using congenic and recombinant mice have narrowed down the gene(s) controlling CIA susceptibility to the MHC class H-2A molecules, such that only mice with the H-2 q , H-2 , H-2 w3 or H-2 w17 haplotypes are susceptible to the disease.
  • Wooley et al . J . Exp . Med. 154: 688-700 (1981); Wooley et al . , Transplant . Proc. 157: 180-85 (1983); Holmdahl et al . , Proc . natl. Acad. Sci. USA 86: 9475-79 (1989).
  • Type II collagen (CII) peptide 250-270 by the H-2A q molecule is probably the most important event in disease induction.
  • the H-2E molecule is not functional because of mutations in both the Ea and Eb genes. Begovich et al . , J. Immunol. 144: 1957-64 (1990).
  • mouse CIA is associated with polymorphisms in the H-2Ab locus (human DQB1 equivalent) .
  • HLA-DRBl locus mouse H-2Eb equivalent
  • mouse CIA is associated with polymorphisms in the H-2Ab locus (human DQB1 equivalent) .
  • the invention includes a transgenic mouse susceptible to collagen-induced arthritis, comprising a human HLA-DQ transgene representing an HLA-DQ allele associated with susceptibility to rheumatoid arthritis in humans.
  • the transgenic mouse is deficient in functional mouse H-2 class II molecules.
  • the transgene can be, for example, an HLA-DQ8 transgene.
  • the term "transgene” means an exogenous gene introduced into a mouse through human intervention, e.g., by microinjection into a fertilized egg or by other methods known to those of average skill in the art .
  • the term includes copies of the exogenous gene present in descendants of the mouse into which the exogenous gene was originally introduced.
  • the term "transgenic mouse” includes the original mouse into which the exogenous gene was introduced, as well as descendants of the original mouse so long as such descendants carry the transgene.
  • the invention further includes a method for identifying peptides potentially effective for prevention or treatment of human rheumatoid arthritis.
  • the method involves a) providing a transgenic mouse susceptible to collagen-induced arthritis, the mouse comprising a human HLA-DQ transgene representing an HLA-DQ allele associated with susceptibility to rheumatoid arthritis in humans, and further being deficient in functional mouse H-2 class II molecules; b) administering to the transgenic mouse a test peptide; c) after administering the test peptide, exposing lymph node cells taken from the transgenic mouse to the test peptide in vitro; and d) identifying the test peptide as potentially effective for prevention or treatment of rheumatoid arthritis if the peptide induces a proliferative response in the lymph node cells.
  • a method for identifying peptides potentially effective for prevention or treatment of human rheumatoid arthritis may comprise a) providing a test group of transgenic mice susceptible to collagen- induced arthritis, each of the mice comprising a human HLA-DQ transgene representing an HLA-DQ allele associated with susceptibility to rheumatoid arthritis in humans, and each of the mice being deficient in functional mouse H-2 class II molecules; b) providing a control group of such transgenic mice; c) administering to the test group of transgenic mice a test peptide; and d) identifying the peptide as potentially effective for prevention or treatment of rheumatoid arthritis if the test group mice exhibit reduced susceptibility to collagen- induced arthritis compared to the control group mice.
  • reduced susceptibility is meant herein that the mice exhibit a delayed onset of collagen- induced arthritis, or that the arthritis symptoms are reduced in severity, compared to the control group mice .
  • the test peptide can be an HLA-DRBl peptide, for example an HLA-DR HV3 peptide.
  • the HLA-DR HV3 peptide may comprise amino acids 67-74 of the HLA DR protein.
  • the invention further includes a method for preventing or treating rheumatoid arthritis in a patient having a rheumatoid arthritis-susceptible HLA-DQ, DR haplotype, comprising administering to the patient an HLA-DRBl peptide having specific binding affinity for HLA-DQ molecules expressed in the patient .
  • the HLA-DRBl peptide may be an HLA-DR HV3 peptide, and may comprise, for example, amino acids 67-74 of the HLA DR protein.
  • the peptides can be administered either orally or parentally, and may be coupled to blocking agents or to carrier proteins to facilitate survival of the relevant peptide motif after administration to the patient.
  • the invention also includes a pharmaceutical composition for administration to a patient having a rheumatoid arthritis-susceptible HLA-DQ, DR haplotype, comprising a pharmaceutically acceptable diluent, for example a sterile, physiological saline solution, and an HLA-DRBl peptide, the peptide having specific binding affinity for HLA-DQ molecules express in the patient.
  • a pharmaceutically acceptable diluent for example a sterile, physiological saline solution
  • HLA-DRBl peptide the peptide having specific binding affinity for HLA-DQ molecules express in the patient.
  • the peptide can be an HLA-DR HV3 peptide, and may comprise amino acids 67-74 of the HLA DR protein.
  • the invention further includes an article of manufacture, comprising packaging material and an HLA- DRBl peptide within the packaging material.
  • the HLA-DRBl peptide has specific binding affinity for HLA-DQ molecules expressed in a rheumatoid arthritis patient.
  • the packaging material further comprises a label or package insert indicating that the HLA-DRBl peptide may be administered to a patient for treatment or prevention of rheumatoid arthritis.
  • Fig. 1.1 and Fig. 1.2 depicts DRB1 * 1502 expression on the surface of peripheral blood lymphocytes of DRB1 * 1502 transgene positive and negative B10.RQB3 mice, using the DRBl-specific L227 monoclonal antibody.
  • Fig. 2 depicts the results of a T cell proliferation assay against different concentrations of HII 250-270 peptide, expressed in ⁇ cpm (mean cpm in experimental wells - mean cpm in control wells) .
  • Fig. 3 depicts results of a T cell proliferation assay against DRBl peptides. Results are expressed in ⁇ cpm (mean cpm in experimental wells - mean cpm in control wells) . Values shown in the Figure are ⁇ cpm of one representative experiment.
  • FIG. 4 is a schematic illustration of the generation of HLA-DQ8 + ,H-2A (3 ° mice.
  • Fig. 5.1 - Fig. 5.7 depicts an analysis of HLA- DQ8, urine MHC and CD4 expression in transgenic HLA- DQ8 + ,H-2A 3 ° mice.
  • Peripheral blood lymphocytes from HLA- DQ8 + ,H-2A originate° mice (#1), BIO (#2), HLA-DQ8 " , H-2 ⁇ ° (#3) and B10.E ⁇ k (#4) animals were analyzed by flow cytometry for surface expression of the molecules HLA-DQ8 (Fig. 5.1), H-2A ⁇ b (Fig. 5.3), H-E ⁇ b (Fig. 5.4), H-2A (S b (Fig. 5.5) and H-2D b (Fig. 5.6).
  • the level of CD4 + cells in PBL was similarly analyzed (Fig. 5.2).
  • Fig. 6 is a graphical representation of measurements of type II collagen antibody in HLA-DQ8 + ,H- 2A mice. Data from transgenic HLA-DQ8 + ,H-2A ⁇ ° mice, negative littermate HLA-DQ8 " ,H-2A /3 ° animals as well as control H-2A and B10.T(6R) mice are presented.
  • Fig. 7.1 - Fig. 7-9 presents photographs of clinical and histological presentation of collagen arthritis in HLA-DQ8 + , H-2A mice.
  • Figs. 7.1-7.3 illustrate the appearance of a normal rear paw from a bovine CII immunized HLA-DQ8 " , H-2A ⁇ ° mouse (Fig. 7.1) contrasted with arthritic paws from an HLA-DQ8*, H-2A /3 ° animal (Fig. 7.2) and a positive control B10.T(6R) mouse (Fig. 7.3).
  • Figs. 7.4-7.9 represent cross sections of the hind foot from an arthritis-resistant HLA-DQ8 , H-2A 0 ° mouse (Figs.
  • Figs. 7.4 and 7.7 show normal cartilage and synovial lining while Figs. 7.5, 7.6, 7.8 and 7.9 show regions of mononuclear cell infiltration of the synoviu with pannus formation and cartilage and subchondral bone erosions.
  • the - 8 /A - magnifications of each section are noted in the lower right hand corner.
  • Figs. 7.7, 7.8 and 7.9 are higher magnifications of the boxed areas in Figs. 7.4, 7.5 and 7.6, respectively.
  • Fig. 8 depicts the polymorphic residues at positions 67, 70, 71 and 74 of the HV3 region of HLA-DRBl molecules.
  • the region 65-79 covering this polymorphic region constitutes the seven DRBl HV3 peptides used in the present study. They are referred to herein, from top to bottom, as Dwl4, Dw2 , Dw21, Dw4 , Dwl3, DwlO and Dw8.1 , respectively.
  • Fig. 9.1 - Fig. 9.2 depicts the response of HLA- DQ8,H-2A 3 ° mice to DwlO HV3 peptide (65-79).
  • Fig. 9.1 summarizes data demonstrating that the response is dose- dependant.
  • Fig. 9.2 summarizes data demonstrating that the response is HLA-DQ-restricted and driven by CD4 + CD8- negative T cells. The values are expressed in ⁇ cpm (mean cpm in experimental wells - mean cpm in control wells without peptide) .
  • Fig. 10 depicts the results of a T cell proliferation assay using cells from HLA-DQ8 + , H-2A 0 0 mice immunized with the seven DRBl HV 3 peptides listed in Fig. 8.
  • H-2E (HLA-DR equivalent) molecules in fact play a role in CIA (i.e., to further explore the apparent paradox described above)
  • the present inventors performed experiments with mice from the B10.RQB3 (Ab q , Aa q , Eb°, Ea k ) strain.
  • This strain of mice expresses the H-2A molecule and possesses a functional Ea k gene, but does not have an intact H-2E molecule since the E ⁇ chain is absent due to a nonfunctional Eb b gene.
  • These mice are CIA susceptible due to the predisposing H-2A q molecule.
  • Transgenic mice were - 10 - generated using the entire Eb d gene, and mated with B10.RQB3 mice resulting in expression of a functional E? d /Ec ⁇ molecule on the cell surface. These mice showed a significant decrease in both incidence and severity of CIA.
  • the protective effect of the E ⁇ d molecule on CIA was confirmed using a second line of transgenic mice generated with a 10.2 Kb DNA construct containing only 200 bp of promoter region. No significant difference was found in the V ⁇ usage between Eb d+ and Eb d" littermates, thus ruling out specific V?-bearing T-cell deletions as an explanation for this protective phenomenon.
  • B10.Q (H-2A q ) mice were immunized with peptides 65-79 covering the HV3 region of the E ⁇ ⁇ or E ⁇ s molecules. Lymph node cells from the immunized animals proliferated in response to these peptides. Similar results using B10.RQB3 mice confirmed the presentation of E ⁇ d and E ⁇ s peptides by H-2A q molecules. Conversely, E3 ,k and ES p peptides failed to induce T-cell proliferation using B10.RQB3 lymph node cells. Therefore, a correlation exists between T-cell proliferation to HV3 peptides 65-79 and protection of the corresponding H-2E molecules against CIA.
  • the H-2A r molecule not only presents an arthritogenic CII peptide to induce autoreactivity, but also fails to present a potentially protective E ⁇ peptide in these animals.
  • HV3 regions of protective forms of mouse E ⁇ molecules might, themselves, constitute antigenic peptides capable of binding to CIA-susceptible forms of the mouse H-2A molecule, thereby mediating the protective effect.
  • Transgene-positive DRB1 * 1502 mice displayed a significant reduction in the incidence and severity of arthritis. T cells from the transgene-positive mice were dramatically less responsive to an arthritogenic type II collagen peptide than were T cells from control mice. In addition, the clinical reduction in arthritis incidence and severity correlated with the T cell proliferative response of the BIO .RQB3-DRB1 * 1502 mice against a self- - 12 - derived DRBl peptide from the third hypervariable region (HV3) .
  • HV3 hypervariable region
  • mice were directed to expression of an RA-associated human HLA-DQ8 molecule in class II -deficient mice.
  • Transgenes encoding the ⁇ and ⁇ molecules of the RA-associated HLA-DQ8 molecule were introduced into mouse class II deficient H-2Ag° mice. These mice were then evaluated for susceptibility to CIA.
  • the HLA-DQ8 + ,H-2A E ° mice displayed good expression of the human HLA-DQ8 molecule while no surface expression of endogenous urine class II molecule could be detected.
  • HLA-DQ8 molecule induced selection of CD4 + T cells expressing a normal repertoire of V ⁇ T cell receptors (TCR) .
  • Immunization of the HLA-DQ8*, H-2A ⁇ ° mice with bovine type II collagen (CII) induced a strong antibody response that was cross-reactive to homologous mouse CII.
  • the anti-CII response was arthritogenic; a severe polyarthritis developed in a majority of the HLA-DQ8 + ,H-2A ⁇ ° mice that was indistinguishable from the polyarthritis seen in the arthritis-susceptible BIO .T(6R) (H-2A q ) controls.
  • HLA-DQ8 t ,H-2A ⁇ ° mice generated in this study represent a novel model to study autoimmune arthritis, and provide useful vehicles to assess the therapeutic efficacy of human HLA class II blocking agents in modulation of a pathogenic in vivo immune response .
  • the present inventors investigated the antigenicity of peptides from the HV3 regions of RA- associated and non-associated human DRBl molecules in the above-described HLA-DQ8 , H-2Ag° transgenic mice (see EXAMPLE 3, below) .
  • some of the peptides are carried by HLA haplotypes associated with RA predisposition, while others are carried by HLA haplotypes that have not been associated with RA.
  • the HLA-DQ8 transgenic mice were immunized with the synthesized peptides. Strikingly, only DRBl HV3 peptides derived from RA-associated DRBl allelic chains, i.e., Dw4 , Dwl3 and Dwl4 peptides, failed to induce proliferation in vitro of T cells from the HLA-DQ8 transgenic mice. The data therefore confirm a correlation between an ability of T cells from HLA-DQ8,H- 2Ag° mice to proliferate against DRBl HV3 peptides and the non-association of the corresponding HLA-DR subtypes with RA predisposition. - 14 -
  • the Dwl3 haplotype can carry either HLA-DQ8 (DQB1 * 0302) or HLA-DQ7 (DQB1 * 0301) alleles.
  • Most of the studies associating the Dwl3 subtype with low incidence of arthritis have failed to indicate which DQ allele was linked to the DRB1 * 0403, Bl * 0406, Bl * 0407 or Bl * 04011 alleles.
  • HLA-DQ8 is predominant (Gao and Serjeanston, Hum . Immunol .
  • HLA class II-derived peptides constitute a major fraction of naturally processed peptides bound to HLA class II molecules.
  • Vogt et al . J. Immunol. 153: 1665- 73 (1994); Chicz et al . , Int . Immunol . 6: 1639-49 (1994); Hunt et al., Science 256: 1817-20 (1992); Urban et al . , Chem. Immunol. 57: 197-234 (1993).
  • Dw2 , Dw ⁇ .l, DwlO and Dwl2 HV3 peptides are naturally processed antigenic peptides, it is likely that they fit into the groove of the HLA-DQ8 molecule.
  • DRBl peptides may act as "endogenous competitors" that modulate the binding of a second antigenic peptide, for example potential autoantigens, to DQ molecules.
  • This model is compatible with both the mechanism of peptide exchange proposed by Adorini et al . , Nature 342: 800-03 (1989), as well as a model where peptide binding to a class II molecule is dependent on a two-peptide, class II -intermediate stage.
  • De Kroon and McConnell J. Immunol. 152: 609-19 (1994).
  • HLA-DRBl peptides To identify human peptides effective or potentially effective for prevention or treatment of rheumatoid arthritis, selected peptides representing - 16 - portions of HLA-DRBl molecules (HLA-DRBl peptides) are synthesized and purified according to standard methods. These peptides are then tested for binding to HLA-DQ molecules through use of mice possessing functional human HLA-DQ transgenes but lacking functional mouse H-2 class II molecules. For example, the HLA-DQ8,H-2A ⁇ ° mice described above and in the following Examples may be administered a preparation of peptide comprising 10- lOOO ⁇ g, preferably about lOO ⁇ g, of peptide in a saline solution and Complete Freund's adjuvant.
  • Administration can be by injection into the tail or by other routes of administration known to the average skilled artisan (e.g., into the rear footpads).
  • lymph node cells are isolated and challenged in vitro with varying concentrations of the corresponding peptide.
  • Those peptides inducing a significant dose-dependent lymphocyte proliferative response that is HLA-DQ- restricted are selected for further study.
  • the selected peptides are administered to transgenic mice as described above, i.e., mice possessing functional human HLA-DQ transgenes but lacking functional mouse H-2 class II molecules.
  • peptides can be dissolved in an appropriate saline solution and administered intravenously to HLA-DQ8 ,H-2A ⁇ ° mice.
  • an adjuvant is not included in the administered injectate.
  • the transgenic mice are administered type II collagen and adjuvant under conditions that would normally lead to development of CIA.
  • the selected peptides are administered prior to, concomitant with, or after administration of the type II collagen, and the mice are monitored for symptoms of CIA. Peptides that engender a delayed onset or a reduced severity of CIA are targeted for further study in a clinical setting. - 17 -
  • the peptides are administered to the transgenic mice by an oral route.
  • orally administered peptides enter from the intestinal lumen into lymphatics and are carried to draining mesenteric lymph nodes, where various immune interactions occur.
  • Some ingested peptides may be transported via M cells into Peyer's patches, where they are able to engender responses from both T and B lymphocytes.
  • Lymphocytes that are activated in mesenteric lymph nodes may migrate to the lamina intestinal.
  • Lymphocytes stimulated in Peyer's patches may also migrate to the lamina limbal, or into mesenteric lymph nodes and, finally, into the general circulation.
  • orally administered peptides may gain access to, and engender responses in, the mucosal immune system and the rest of the immune system. See, e.g., Abbas et al . , Cellular and Molecular Immunology, 2nd Ed., W.B. Saunders Company 1994, pages 233-235; Chen et al . , Nature 376: 177-180 (1995).
  • the peptides are dissolved in saline, without adjuvant, and fed to selected mice (e.g., HLA-DQ8,H-2Ag° mice), preferably by gastric intubation using a 21G ball tipped animal feeding needle.
  • mice may be fed with various concentrations of peptide and for various lengths of time.
  • the mice are fed with a selected amount, for example 100 ⁇ l (1 mg/ml) , of peptide every other day for a period of 20 days. This is followed, two days after the last peptide dose, by immunization with type II collagen to induce CIA.
  • the animals may be first immunized to induce arthritis on day 0, prior to peptide administration.
  • Mice are then orally fed peptide from about 20 to about 45 days post -immunization with a selected amount, for example 100 ⁇ g, of peptide every other day.
  • Various other combinations of peptide and - 18 - type II collagen administration protocols may be used as appropriate to particular peptide preparations and mouse strains. Such routine variations in protocol will be apparent to the average skilled artisan.
  • mice Following administration of the relevant peptides and the type II collagen, the mice are monitored two to three times per week for onset and severity of arthritis. Generally this monitoring is performed for a period of from about 3 to about 16 weeks. Severity of arthritis is assessed by monitoring joint involvement; this can be manifested, for example, in redness or swelling in the paws or toes, severe swelling or joint deformity, and joint ankylosis. The symptoms can be worked into a grading system of severity that is applicable to individual limbs. The scores for each limb can be summed to provide a severity score for each animal. Arthritis incidence and severity can be compared between experimental groups using appropriate statistical analyses known to the skilled artisan, for example the X 2 test with Yates' correction and the non-parametric Mann- Whitney U test.
  • Human HLA-DRBl peptides demonstrating anti-CIA activity in the transgenic mice are useful for treatment of, and testing in, human patients.
  • the peptides can be administered to human patients either by oral administration or parenteral administration (e.g., intravenous injection) .
  • the peptides generally are administered without an adjuvant.
  • the peptides are administered in an appropriate physiological saline solution, although any appropriate carrier solution known to the skilled artisan may be used for administration .
  • the peptides can be coupled, at either or both of the amino- and carboxyl- terminal residues, with a blocking agent in order to - 19 - facilitate survival of the relevant peptide motif in vivo.
  • a blocking agent in order to - 19 - facilitate survival of the relevant peptide motif in vivo.
  • Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino- and/or carboxyl -terminal residues of the HLA-DRBl peptide to be administered.
  • blocking agents such as pyroglutamic acid or other molecules known to those of average skill in the art may be attached to the amino- and/or carboxyl-terminal residues of the HLA-DRBl peptide.
  • the peptides can be coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
  • the peptides may be administered to presymptomatic patients at risk for developing rheumatoid arthritis to delay or prevent the onset of clinical disease.
  • patients can include, without limitation, individuals having HLA-DR and HLA-DQ haplotypes associated with susceptibility to rheumatoid arthritis as described herein.
  • the peptides may be administered to patients displaying clinical symptoms of rheumatoid arthritis to delay or prevent continued deterioration, or to effect improvement in the existing condition.
  • the peptides administered to human patients have specific binding affinity for HLA-DQ molecules expressed in the patients.
  • the peptides are derived from the HV3 regions of human HLA-DRBl molecules.
  • Such peptides may consist of or include amino acids 67-74 of the HLA DR HV3 region.
  • the administered peptides may be a single species of peptide, or may include a "cocktail" of various peptides identified as useful for prevention or amelioration of rheumatoid arthritis in particular patients. - 20 -
  • mice The mice employed in these studies were bred and maintained in the pathogen- free Immunogenetics Mouse Colony of the Mayo Clinic.
  • DRB1 * 1502 transgenic mice were generated by microinjecting a linearized 34 Kb DNA fragment containing the entire DRB1 * 1502 gene, isolated from the HLA-homozygous B cell line AKIBA (DR2, Dwl2, DQw6) (see Kawai et al . , J. Immunol . 142: 312-17 (1989)) into (SWR x B10.M)F 2 fertilized eggs.
  • DRB1 * 1502 positive founders were identified by polymerase chain reaction using the primers (5 ' -C (CT) TAAGAGGGAGTGTCATTTCTTC3 ' ) (SEQ ID NO : 1) and (5'TGTGAAGCTCTC(AC) (AC) CAACCCC-3 ' ) (SEQ ID NO: 2), located in the second DRBl exon.
  • the DRB1 * 1502 transgene was introduced into the B10.RQB3 (Aa q Ab q Eb q Ea k ) mice by crossing with the founder mouse. Those offspring of this cross who were positive for the transgene were backcrossed for ten generations.
  • the B10.RQB3 mice express the CIA susceptible H-2A q molecule but lack H-2E expression since their Eb q gene is mutated.
  • the DRB1 * 1502 molecule is expressed on the cell surface by pairing of the DRB1 * 1502 molecule with the Ea k molecule, which is highly homologous to the DR ⁇ molecule.
  • Peripheral blood lymphocytes were isolated by Ficoll separation, washed and incubated with the DRBl-specific monoclonal antibody L227 (Grumet et al., J . Immunol . 125: 2785-89 (1980)) for 30 min. at 4°C. - 21 -
  • BIO .RQB3-DRB1 * 1502 mice along with congenic B10.RQB3 controls were immunized with a cold emulsion of bovine type II (BII) collagen in complete Freund's adjuvant (CFA) , as previously described.
  • BII bovine type II
  • CFA complete Freund's adjuvant
  • mice Eight to twelve week-old mice were intradermally injected with 100 ⁇ g of the emulsion in the root of the tail and then monitored for the onset and development of arthritis from the third to the ninth week postimmunization.
  • An arthritic score for each mouse was obtained by summing the score in each paw.
  • the possible severity as measured by the arthritic score ranged from 0 to 12. Animals were followed for the onset and severity of arthritis from the third to the ninth week postimmunization. Incidence and severity of arthritis did not change after the ninth week postinjection. Arthritis score was calculated at the end of the study considering only arthritic mice. - 22 -
  • DRB1 * 1502 65-79: K-D-I-L-E-Q-A-R-A-A-V-D-T-Y-C (SEQ ID NO: 3)
  • DRB1 * 1601 65-79: K-D-F-L-E-D-R-R-A-A-V-D-T-Y-C
  • HII 250-270 An immunodominant peptide (HII 250-270) of human type II collagen was similarly synthesized as previously described by Krco et al . , cited supra . This peptide has been previously recognized as a T cell epitope in arthritis susceptible H-2 q mice. Khare et al . , FASEB J. 8: A967 (1994).
  • the sequence of HII 250-270 is as follows: G-P-K-G-Q-T-G-K-P-G-I-A-G-F-K-G-E-Q-G-P-K (SEQ ID NO: 5) . T cell proliferation assay.
  • B10.RQB3-DRB1 * 1502 positive and negative littermates were injected with 200 ⁇ g of HII 250-270 peptide emulsified in CFA. Ten days later, draining lymph nodes were removed and tested in vitro for lymphocyte proliferation assay. One hundred ⁇ l of cells at a concentration of lxl0 7 /ml were cultured for - 23 -
  • RQB3-DRB1 * 1502 positive and negative littermates and BIO.RIII (H-2 ) mice were immunized with peptides covering the region 65-79 of the DRB1 * 1502 or DRB1 * 1601 molecules. Seven days later, cells from draining lymph nodes were removed and tested in vitro against the same peptides, as described in Khare et al . , cited supra.
  • Statistical analysis Arthritis incidence between B10.RQB3-DRB1 * 1502 mice and B10.RQB3 controls was compared using Chi square test with Yates' correction. The Student's t-test was employed to analyze antibody levels.
  • the HrLA -DRBl * 1502 (DR2Dwl2 ) mol ecul e protects mi ce f om arthri ti s .
  • the DRB1 * 1502 chain was able to pair with the E ⁇ k chain as measured by cell surface expression since DR ⁇ and E ⁇ chains are highly homologous (Grummet et al . , J. Immunol. 125: 2785-89 (1980); see also Fig. 1.1 - Fig. 1.2) . This is in agreement with results previously published by Lawrence et al . , Cell 58: 583-94 (1989), showing that an HLA-DRa transgene can restore H-2E expression.
  • the percentage of PBLs expressing the DRBl chain was 41.4£3.2% for BIO .RQB3-DRB1 * 1502 mice versus 3.0 ⁇ 0.9% for the negative littermate.
  • mice were immunized with 100 ⁇ g of BII in Complete Fruend's adjuvant on day 0 and monitored regularly for the onset and development of CIA. t Determined at 9 week postimmunization. The mean severity of arthritis was calculated using arthritic mice only. t Mice were bled at 5 week postimmunization and the level of antibody against BII and Mil determined by a standard ELISA. Data are presented as the mean OD at 1:100 dilution. Arthritis incidence between groups was compared using ⁇ 7 - test with Yates' correction. Antibody levels were studied using the Student's IT test.
  • E ⁇ k E ⁇ molecules such as E ⁇ p , E ⁇ b or E ⁇ k does not lead to protection in CIA (data not shown) .
  • this protection is not related to expression of the E ⁇ k molecule.
  • V ⁇ -specific T cell receptor (TCR) repertoire between the BIO .RQB3-DRB1 * 1502 and the B10.RQB3 controls (data not shown) .
  • TCR V ⁇ -specific T cell receptor
  • Lymphoyroliferative response to type II collagen peptide 250 -270 Both T and B cells are involved in pathogenesis of CIA. As disclosed above, the autoantibody response to Mil in the B10. RQB3-DRB1 * 1502 mice was significantly reduced. Next, T cell response against a T cell epitope of type II collagen was tested both in B10.RQB3-DRB1 * 1502 positive and negative littermates. B10.RQB3-DRB1 * 1502 positive and negative littermates were immunized with the immunodominant arthritogenic peptide HII 250-270.
  • T cells from B10.RQB3-DRB1 * 1502 and B10.RQB3 control mice were challenged in vitro with various concentrations of HII 250-270, a dramatic reduction in the T cell response of DRB1 * 1502 transgene positive animals, only, was observed, especially at lower concentrations of peptides (Fig. 2) .
  • T cell proliferation against PPD a constituent of CFA
  • proliferation against LPS and Con A was similar using transgene positive and negative littermates (data not shown) . Therefore, it may be concluded that the impaired response to HII 250-270 in DRB1 * 1502 mice is antigen-specific and is related to protection against arthritis.
  • RQB3-DRB1 * 1502 positive or BIO.RIII animals were immunized with 200 ⁇ g of the DRB1 * 1502 (DR2Dwl2) peptide (65-79) (KDILEQARAAVDTYC) (SEQ ID NO: 3) or 200 ⁇ g of the DRBl * 1601(DR2Dw21) peptide (65-79) (KDFLEDRRAAVDTYC) (SEQ ID NO: 4) .
  • DRB1 * 1601 peptide induced only a weak proliferation in all the mice tested.
  • BIO.RIII mice showed only a weak response against these peptides, indicating that the binding of DRB1 * 1502 peptide to H-2A molecules is haplotype-dependent .
  • Lymph node cells were challenged . in vitro without peptide (negative control: mean cpm ⁇ 5,000), with Con A (mean cpm>100,000) or with specific peptides (100 ⁇ g/ml) . Both groups of animals showed a strong proliferative response when challenged in vitro with the DRB1 * 1502 peptide (65-79) (Fig. 3) .
  • mice All mice used in this study were bred and maintained as described in EXAMPLE 1. Derivation of the HLA-DQ8 + ,H-2A ⁇ ° line was achieved as described in Fig. 4.
  • Transgenic B10.M-DQ8 mice were generated by microinjection of HLA-DQA1 and DQB1 genes into fertilized eggs. Specifically, genomic HLA-DQA1 * 0301 and HLA- DQB1 * 0302 genes were isolated from cosmid clones HllA and X10A, respectively. The cosmid clones HllA and X10A were derived from the human B cell line Priess (DR4+, DQ8+, DP3/4+) and are described in Okada et al .
  • cosmid clones were provided by J.L. Strominger.
  • Clone HllA contains a 30 kilobase (Kb) DNA insert containing the DQAl"0301 gene, and the DQBl'0302 gene with a truncated promoter, while clone X10A contains a 38 Kb DNA insert containing in its center the DQB1 * 0302 gene (Okada et al . , Proc. Natl. Acad. Sci. USA 82: 3410-3414 (1985)).
  • the inserts were released by Sal I restriction enzyme digestion.
  • the resulting constructs including the coding sequences with associated native regulatory sequences present in the respective cosmid clones, were microinjected separately into (CBA/J x B10.M)F 2 and (SJL x SWR) F 2 fertilized eggs respectively.
  • General microinjection procedures were substantially as described in Wei et al . , In: Transgenic Mice and Mutants in MHC - 28 -
  • mice Viable embryos deriving from microinjected eggs were reimplanted into the oviducts of pseudopregnant foster mothers. DQAl-positive and DQBl-positive founders were identified and mated to B10.M mice. Resulting offspring were crossed and back-crossed to produce the transgenic B10.M-DQ8 mice (HLA-DQA1 * 0301/HLA-DQB1 * 0302) used for further manipulation.
  • Mouse class II deficient H-2A ⁇ ° mice (Cosgrove et al . , Cell 66: 1051-1066 (1991)) were provided by Drs . Diane Mathis and Christopher Benoist. Analogous class II deficient mice may be obtained commercially from GenPharm International, Mountain View, California, USA. Transgene positive founders and subsequent mice were identified by polymerase chain reaction, using standard procedures, with primers 5'-
  • Transgene positivity of the founders was also determined by Southern blot analysis of tail DNA with DQBl and DQAl cDNA probes using standard protocols (Sambrook et al . , Molecular Cloning: A laboratory Manual (Cold Spring Harbor Lab., Plainview, NY (1989). Segregation of the HLA-DQ8 transgenes was also monitored by flow cytometric analysis of PBL using the HLA-DQ a specific mAb IVD12 (see below) . Segregation of the mutant H-2A ⁇ ° gene was evaluated by flow cytometry by monitoring the expression of the H-2A f and H-2A b molecules using the mAbs 3F-12 (D. McKean, J. Immunol. 136: 2953 (1986) ) and AF6-120 (see below) , respectively. Mice of - 29 - both sexes were used in this study and were eight to twelve weeks of age at the start of the experiment.
  • Flow cvtometrv. Analysis of HLA-DQ8, urine class I, class II and CD4 expression in PBL was achieved as follows. Mice were bled via the tail artery and the white cell fraction isolated by centrifugation over a ficoll-hypaque gradient. After extensive washing in phosphate buffered saline containing 1% bovine serum albumin and 0.1% sodium azide (PBS/BSA) the cells were incubated with one of the following monoclonal antibodies: IVD12 , anti-HLA-DQ ⁇ (Giles et al . , J . Exp . Med.
  • mice were sacrificed and the peripheral lymph nodes were removed and homogenized to dislodge the cells .
  • the lymph node cells were then extensively washed with PBS/BSA and approximately 10 6 cells were incubated with one of the following V ⁇ TCR specific mAbs : KT4 , rat anti-V ⁇ 4
  • mice Twenty-eight days later, the animals received a booster injection of 100 ⁇ g bovine CII emulsified in Incomplete Freund's Adjuvant (IFA) . The mice were carefully monitored three to four times a week for the onset and progression of CIA from the beginning of the experiment until its termination at twelve weeks postimmunization. The severity of arthritis was evaluated as described in EXAMPLE 1.
  • IFA Incomplete Freund's Adjuvant
  • Anti-type II collagen ELISA The level of IgG antibody reactive against bovine and mouse CII was determined using a highly sensitive ELISA technique (Griffiths et al . , J . Immunol . 153: 2758-2768 (1984)). Briefly, day 35 sera from bovine CII immunized mice were diluted in PBS containing 0.05% Tween 20 and 0.2M NaCl (PNT) . Microtiter wells were coated with either bovine - 31 - or mouse CII dissolved in KP0 4 buffer pH 7.6 at 300 ⁇ l/well (20 ⁇ g/ml of CII) overnight at 4°C.
  • mice were sacrificed at the end of the experiment and histological sections of the hind limbs were prepared by the Pathology Department of the Mayo Clinic. Limbs were dissected, the joints decalcified three to four days and then embedded in paraffin blocks. Sections of approximately 6 ⁇ m thickness were cut for each joint at differing intervals, mounted and stained with hematoxylin and eosin before analysis .
  • HLA-D08 molecule in H-2A mice induces selection of CD4 * T Cells.
  • the RA-associated HLA-DQ8 molecule was introduced into mouse class II deficient H-2A ⁇ ° mice.
  • Fig. 4 illustrates the strategy to derive the HLA-DQ8 + ,H-2A ⁇ 0 line. Briefly, as described above, B10.M(H-2 f ) mice bearing trangenes - 32 - encoding the a and ⁇ genes of the HLA-DQ8 molecule (see above) were mated with H-2A /3 ° mice.
  • the offspring were screened for HLA-DQ8 expression by flow cytometric analysis of PBL using the HLA-DQ ⁇ specific mAb IVD12.
  • the HLA-DQ8 -positive (HLA-DQ8*) , H-2A /o progeny were intercrossed and segregation of the H-2A and H-2A gene was monitored via fluorescent analysis using the H-2A ⁇ b specific mAb 3F-12 and the H-2A b specific mAb AF6-120.
  • the offspring which typed as HLA-DQ8 ⁇ H-2A ⁇ 00 were selected and intercrossed to develop the HLA-DQ8", H-2A ⁇ ° and HLA-DQ8 -negative (HLA-DQ8-) ,H-2A ⁇ ° lines.
  • Fig. 5.1 shows that transgenic HLA-DQ8 4 , U-2A ⁇ ° mice expressed the HLA-DQ8 molecule on approximately 30-40% of the PBL population. In addition, expression of HLA-DQ8 was sufficient to induce a partial repopulation of the
  • CD4 + T cell subset in the periphery (Fig. 5.2) .
  • the level of CD4 + cells in PBL of HLA-DQ8 + , H-2A ⁇ ° animals ranged from 5 to 10%. In no instance, however, did the percentage of CD4 + cells approach the level of control B10 mice. Also, no staining for CD4 + PBL was detected in HLA-DQ8 " , H-2A /3 ° littermates.
  • the percentage of CD8 + cells in HLA-DQ8 + ,H- 2A /3 ° PBL was approximately two- to three-fold higher than B10 animals and the CD4:CD8 ratio was approximately 1:3 (data not shown) .
  • H-2A ⁇ b and H-2E /3 b chains in H-2A ⁇ ° mice Given the presence of intracytoplasmic H-2A ⁇ b and H-2E /3 b chains in H-2A ⁇ ° mice (Cosgrove et al . , cited supra , and Grusby et al . , Science 253:1417-1420 (1991)), it was possible that the restored expression of CD4 + cells in the HLA-DQ8 + ,H-2A ⁇ ° line was due to the formation of hybrid A ⁇ b -DQ8 ⁇ or DQ8 ⁇ -E ⁇ b molecules. To eliminate this possibility, PBL from HLA-DQ8 + ,H-2A /3 ° mice were analyzed for surface expression of the H-2A ⁇ b and H-2E /3 b molecule.
  • H-2A ⁇ b -specific mAb 7-16.17 did not result in detection of expression of H-2A ⁇ in HLA-DQ8 + ,H-2A ⁇ ° animals (Fig. 5.3) .
  • T cell receptor V ⁇ expression on CD4 ⁇ cells in HLA-DQ8*,H-2A acute° mice.
  • the presence of CD4 * cells in the periphery of HLA-DQ8 * , H-2A mice suggested that the HLA- DQ8 molecule induced the positive selection of CD4 + , T cell receptor (TCR) positive lymphocytes.
  • TCR T cell receptor
  • lymph node cells from HLA-DQ8 + , H-2A ⁇ ° animals were analyzed for the expression of V ⁇ TCR within the CD4 + subset.
  • HLA-DQ8 + , H-2A ⁇ ° mice did indeed express a variety of V ⁇ TCRs in the CD4 + population.
  • lymph node cells from B10.T(6R) mice which express the collagen arthritis susceptible H-2A q molecule (Wooley et al . , cited supra) , were also analyzed.
  • T Cell Receptor V a Expression in HLA-DQ8 + ,H-2A a ° Micet
  • t Lymph node cells from normal HLA-DQ8 + , H-2A ⁇ ° and B10.T(6R) mice were isolated and analyzed by flow cytometry for V ⁇ TCR expression in the CD4+ subset as detailed above.
  • HLA-DQ8 * H-2A ⁇ ° mice possessed the potential to mount a pathogenic immune response against CII. Therefore, HLA-DQ8 + ,H-2Ag° animals along with transgene negative littermates, positive control B10.T(6R) and negative control H-2A ⁇ ° mice were immunized with bovine CII in CFA and monitored for the generation of CII specific antibody (Ab) .
  • mice were immunized on day 0 with 100 ⁇ g bovine CII in CFA and boosted with 100 ⁇ g bovine CII in IFA on day 28. Sera were collected on day 35 and the level of IgG antibody specific for bovine and mouse CII determined by ELISA. The data were obtained using five to fifteen animals per group. The data revealed that HLA- DQ8 + ,H-2A ⁇ ° mice mounted a strong IgG Ab response against bovine CII (Fig. 6) .
  • the level of bovine CII Ab was - 36 - comparable to arthritis-susceptible B10.T(6R) controls and no CII reactivity was detected in sera from HLA-DQ8 ,H-2A 3 ° littermates or H-2A ⁇ ° animals. Moreover, HLA- DQ8 + ,H-2A /3 ° sera was highly crossreactive against mouse CII. Like the reactivity against bovine CII, the level of mouse CII reactive Ab was similar to B10.T(6R) sera and the extent of crossreactivity in both strains was greater than 50%.
  • HLA-DQ8 H-2A /) ° mice development of arthritis in HLA-DQ8 H-2A /) ° mice.
  • bovine CII immunized HLA-DQ8 + , H-2A 3 ° mice, HLA-DQ8 " , H-2Ap° littermates, B10.T(6R) animals and H-2A ⁇ ° mice were monitored for the onset and development of CIA.
  • experiment 1 HLA-DQ8 + ,H- 2A ⁇ ° and B10.T(6R) animals developed severe inflammation, swelling and joint deformity in afflicted limbs (Fig. 7.2 and Fig. 7.3).
  • t Mice were immunized with 100 ⁇ g bovine CII in CFA on Day 0 and boosted with 100 ⁇ g bovein CII in IFA on Day 28. All animals were monitored regularly for the onset and development of CIA until the termination of the experiment at 12 weeks post-immunization. t Mean arthritic score was calculated at the end of the study using arthritic animals only.
  • mice from a second HLA-DQ8 + ,H-2Ag° line that expresses the HLA-DQ8 molecule on approximately 15% of PBL were immunized with bovine CII and monitored for CIA.
  • a majority of HLA-DQ8 + ,H-2Ag° mice developed CIA (Table 3, experiment 2).
  • the onset of clinical arthritis was significantly earlier in the line compared to B10.T(6R) mice.
  • the severity of CIA was significantly greater in arthritic HLA-DQ8 + , H-2Ag° animals.
  • the seven DRBl peptides (65-79) of sequence Lys-Asp-X-Leu-Glu-X-X-Arg-Ala-X-Val-Asp-Thr-Tyr- Cys (SEQ ID NO: 10) , where amino acids X at positions 67, - 39 -
  • Fig. 8 70, 71 and 74 are listed in Fig. 8, were synthesized by the Peptide Core Facility at Mayo Foundation using an automated 430A peptide synthesizer (Applied Biosystems) and purified by high-pressure liquid chromatography. Amino acid compositions were confirmed by sequencing using the Edman's method.
  • T cell proliferation assay T cell proliferation assays were preformed as described (Krco et al . , Transplantation 54: 920-923 (1992)). For each peptide challenge, 100 ⁇ g of peptide emulsified in a saline solution and Complete Freund's adjuvant were injected subcutaneously into the tails of HLA-DQ8,H-2Ag 0 and transgene negative full siblings. For dose-response experiments, 100, 10, 1 or 0.1 ⁇ g/ml (final concentration) of peptide were added to the cells.
  • the percentage of CD4 + T cells in DQB1 * 0302.H-2A ⁇ ° mice at 6 week-old was approximately 11% compared to 29.5% in B10 animals (Table 4) .
  • the relative - 40 - percentages of specific V ⁇ -bearing CD4" T cells were comparable to normal BIO animals (Table 3) .
  • the possibilities of potential inter- isotypic H-2E ⁇ /DQAl * 0301 and H-2A ⁇ /DQBl * 0302 dimers were ruled out by showing the absence of staining on flow cytometry using mAbs 4D5 and 7- 16.17 (anti-H-2A ⁇ b -specific) , and Y-17 (anti-H-2E ⁇ b - specific) (data not shown) .
  • the DQB1 * 0302 chain is not expressed in the absence of DQA1 * 0301 molecule in single-transgenic mice (data not shown) . Therefore, in the absence of mouse class II molecules, the HLA-DQ8 molecule can positively select CD4 + T cells.
  • t Lymph node cells were removed and analyzed by flow cytometry as described above.
  • the frequency of CD4+/V ⁇ TCR positive cells was calculated from the gated CD4+ population shown in column 1. The data are presented as the mean percent positive cells ⁇ standard deviation of three animals per group.
  • Transgene-positive and negative HLA-DQ8 , H-2A /3 ° littermates were immunized with the DwlO HV3 peptide (65-79) (Fig. 8) .
  • lymph node cells were isolated and challenged in vi tro with varying concentrations of the HV3 peptide (Fig. 9.1) .
  • a very strong, dose-dependent lymphocyte proliferative response was observed in the HLA-DQ8, H-2A (3 ° animals, while transgene-negative littermates showed no response (Fig. 9.1).
  • the Dwl4 peptide possesses an amino acid sequence shared by several DR4 alleles, two out of three DR1 alleles and one of the DR6 alleles; all of them are carried by HLA haplotypes associated with RA predisposition (Nepom et al., Ann. Rev. Immunol. 9:493-525 (1991); Wordsworth et al., Proc. Natl. Acad. Sci. USA 86:10049-53 (1989); Oilier et al . , Rheum. Pis. Clin. North. Am. 18:741-59; and Willkens et al . , Arthritis Rheum. 34:43-47 (1991) (Fig. 8)) .
  • the Dw4 peptide is unique to the RA- associated DRB1 * 0401 allele, while the Dwl3 peptide is shared by several DR4 subtypes that have not been associated with RA, although this latter negative - 42/A - association is less clear (Oilier et al . , cited supra) .
  • Dw2 , Dw21 and Dw8.1 peptides correspond to motifs found in non-RA- associated DRBl alleles (Nepom et al . , cited supra ; Oilier et al., cited supra ; Gregerson et al . , Arthritis Rheum. 30: 1205-13; and Winchester et al . , Rheum. Pis. Clin. North. Am. 18 : 761-783 (1992) ) .
  • HLA-DQ8,H-2A jj ° animals were immunized with the seven DRBl HV3 peptides (65-79) (Fig. 8) .
  • DwlO induced the strongest proliferative response when lymph node cells were challenged in vi tro, followed by Dw2, Dw21 and Dw8.1 HV3 peptides (Fig. 10) .
  • T cells challenged with Dw4, Dwl3 and Dwl4 HV3 peptides failed to proliferate (Fig. 10) .
  • residues 67, 70, 71 and 74 of the HLA-DRBl chains effect a HLA-DQ8-restricted proliferative response against DRBl HV3 peptides (65-79) .
  • Leu (L) at position 67 found in peptides Dw4 , Dwl3 and Dwl4, seems to prevent a HLA-DQ8-restricted proliferative response
  • Asp (D) at position 70 in peptides DwlO, Dw21 and Dw8.1 seems to promote T cell proliferation (Figs. 8 and 10) .
  • Lys Asp lie Leu Glu Gin Ala Arg Ala Ala Val Asp Thr Tyr Cys 1 5 10 15

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Abstract

L'invention porte sur des souris transgéniques porteuses d'un transgène humain de HLA-DQ. Ces souris transgéniques ont une carence en molécules H-2 de la classe II. Ces souris génèrent des systèmes de modèles d'animaux qui permettent d'identifier des peptides utiles dans la prévention ou le traitement de la polyarthrite rhumatoïde. L'invention porte également sur des procédés et des substances de traitement de la polyarthrite rhumatoïde, ces procédés consistant à administrer des peptides ayant une affinité de liaison spécifique pour les molécules de HLA-DQ exprimées chez un patient atteint de polyarthrite rhumatoïde.
PCT/US1996/014041 1996-08-30 1996-08-30 Peptides de hla-drb1 ayant une affinite de liaison specifique pour les molecules de hla-dq et utiles dans la prevention et le traitement de la polyarthrite rhumatoide Ceased WO1998008943A1 (fr)

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PCT/US1996/014041 WO1998008943A1 (fr) 1996-08-30 1996-08-30 Peptides de hla-drb1 ayant une affinite de liaison specifique pour les molecules de hla-dq et utiles dans la prevention et le traitement de la polyarthrite rhumatoide

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* Cited by examiner, † Cited by third party
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Publication number Priority date Publication date Assignee Title
FR2827302A1 (fr) * 2001-07-13 2003-01-17 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations
WO2003006639A1 (fr) * 2001-07-13 2003-01-23 Genoway Cellule et animal transgenique modelisant la presentation antigenique humaine et leurs utilisations
WO2010075249A2 (fr) 2008-12-22 2010-07-01 Genentech, Inc. Méthode de traitement de la polyarthrite rhumatoïde avec des antagonistes de cellules b

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