WO2011071039A1 - Identification de l'epitope hla-dr4 et utilisation pour le traitement de l'arthrite - Google Patents

Identification de l'epitope hla-dr4 et utilisation pour le traitement de l'arthrite Download PDF

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WO2011071039A1
WO2011071039A1 PCT/JP2010/071901 JP2010071901W WO2011071039A1 WO 2011071039 A1 WO2011071039 A1 WO 2011071039A1 JP 2010071901 W JP2010071901 W JP 2010071901W WO 2011071039 A1 WO2011071039 A1 WO 2011071039A1
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polypeptide
hla
seq
amino acid
cells
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宏文 庄田
圭志 藤尾
一彦 山本
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国立大学法人東京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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 present invention relates to identifying unknown epitopes of HLA-DR4 and using the identified epitopes to treat or prevent diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis. .
  • RA Rheumatoid arthritis
  • DMARDs disease-modifying anti-rheumatic drugs
  • NSAIDs analgesics
  • an antibody against a cyclic citrullinated peptide (Cyclic Citrullinated Peptide: CCP) and an anti-CCP antibody are produced in an individual who has developed RA.
  • This anti-CCP antibody is an autoantibody highly specific for RA, and if the presence of anti-CCP antibody in the blood is positive, diagnoses that the individual has rheumatoid arthritis with an accuracy of about 97% It is known that it can be.
  • This anti-CCP antibody has already appeared not only in individuals with RA, but also in individuals before the onset of RA, and it is known that the positive rate of anti-CCP antibodies increases as the onset approaches.
  • HLA human leukocyte antigen Human leukocyte
  • HLA-DRB1 * 0401 European and America
  • HLA-DRB1 * 0405 Asia
  • HLA-DRB1 * 0101 Israel
  • HLA-DRB1 A positive correlation was shown between the prevalence and the onset of RA. It is assumed that a specific antigen-derived polypeptide (epitope) is presented by RA-sensitive MHC class II molecules such as HLA-DR4 or HLA-DR1 and is recognized by CD4-positive T cells, resulting in immune abnormalities and developing RA However, it is not known at all what epitope these HLA-DR4 or HLA-DR1 recognizes and presents.
  • BiP immunoglobulin heavy chain binding protein
  • HSP heat shock protein
  • the present invention elucidates the amino acid sequence structure of a specific antigen polypeptide (epitope) presented to CD4-positive T cells by RA-sensitive MHC class II molecules such as HLA-DR4 or HLA-DR1, and The purpose is to develop a new means for preventing or treating diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis using various epitopes.
  • a polypeptide comprising a specific amino acid sequence motif consisting of the amino acid sequence: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) and having a total length of 12-20 amino acid residues is HLA.
  • the present invention provides an amino acid sequence consisting of the following formula: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) It was shown that the above-mentioned problems can be solved by providing a polypeptide comprising 12 to 20 amino acid residues in total.
  • the present invention also prevents or treats diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis, by administering a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 thus obtained. Clarified that it can be treated, and showed that it can provide a pharmaceutical composition for preventing or treating diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis containing this polypeptide .
  • the invention further reveals that administration of a polypeptide as described above or a nucleic acid construct for expressing the polypeptide in a cell can produce immune tolerance against the polypeptide, It was shown that a vaccine composition for preventing diseases associated with HLA-DR4 or HLA-DR1 including rheumatoid arthritis including a nucleic acid construct for expressing the polypeptide in a cell can be provided.
  • a polypeptide having such an amino acid sequence By administering mucosal sensitization by orally administering a polypeptide having such an amino acid sequence, it can exert an effect of preventing or treating diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis. it can. Moreover, such a polypeptide can also be used as a vaccine composition for preventing diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis.
  • FIG. 1 is a graph showing the proliferation response of RA peripheral blood mononuclear cells to a 20 amino acid polypeptide obtained from human BiP of the HSP70 family and mycobacterium mycHSP70.
  • Figure 2 shows that BiP-derived B22 polypeptide and mycHSP70-derived D21 polypeptide that caused significant RA peripheral blood mononuclear cell proliferation strongly inhibited the binding of positive control polypeptide to HLA-DR4 molecule in vitro.
  • FIG. 3 shows that these polypeptides have a significantly high binding ability to HLA-DR4.
  • FIG. 3 shows amino acids that play an important role in the ability of B22 polypeptide (FIG. 3A) and D21 polypeptide (FIG. 3B) to bind to HLA-DR4.
  • FIG. 4 is a view showing a region of amino acids that are largely involved in binding to HLA-DR4 among 20 amino acids of B22 polypeptide (FIG. 4A) and D21 polypeptide (FIG. 4B).
  • FIG. 5 is a diagram showing an outline of the sequence of an epitope that binds to HLA-DR4.
  • FIG. 6 is a diagram showing the preventive effect on the onset of arthritis when oral immune tolerance is induced by oral administration of B22 polypeptide or D21 polypeptide prior to bovine type II collagen immunization for arthritis induction .
  • FIG. 7 shows the proliferative response of CD4 + T cells to type II collagen and B22 polypeptide stimulation when oral immune tolerance was induced by administering B22 or D21 polypeptide prior to type II collagen immunization. It is a figure which shows falling.
  • FIG. 8 is a graph showing that autoantibodies decrease when oral immunization is performed by administering B22 polypeptide or D21 polypeptide prior to type II collagen immunization.
  • FIG. 9 is a graph showing the therapeutic effect on arthritis exacerbation when B22 polypeptide or D21 polypeptide is administered after the onset of arthritis due to type II collagen immunization.
  • FIG. 8 is a graph showing that autoantibodies decrease when oral immunization is performed by administering B22 polypeptide or D21 polypeptide prior to type II collagen immunization.
  • FIG. 9 is a graph showing the therapeutic effect on arthritis exacerbation when B22 polypeptide or D21 polypeptide is administered after the onset of arthritis due to type II
  • FIG. 10 is a graph showing an increase in regulatory T cells in regional lymph nodes when B22 polypeptide or D21 polypeptide is administered after the onset of arthritis due to type II collagen immunization.
  • Figure 11 shows that when CD4 + CD25 + T cells and CD4 + CD25-T cells of DBA / 1J mice were cultured with B22 polypeptide or D21 polypeptide, CD4 + CD25 + T cells reacted and proliferated.
  • the inventors of the present invention have revealed that a partial polypeptide of an immunoglobulin heavy chain binding protein (BiP) is presented as an antigen by strongly binding to an RA-sensitive MHC class II molecule such as HLA-DR4 or HLA-DR1.
  • HLA-DR4 or HLA-DR1 etc. revealed the amino acid sequence structure of a specific antigen polypeptide (epitope) presented to CD4-positive T cells, and rheumatoid arthritis using such epitope
  • the present invention has been completed on the basis of the development of a new means for the prevention or treatment of diseases related to HLA-DR4 or HLA-DR1, including the above.
  • an amino acid sequence consisting of the following formula: X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) And a polypeptide consisting of a total length of 12 to 20 amino acid residues.
  • X1 to X12 each define an amino acid, and each is defined as follows: X1 is selected from Met (M) or Arg (R) X2 is Lys (K) X3 is Pro (P) and X4 is selected from Phe (F), Val (V), Trp (W), Tyr (Y), Ile (I), Leu (L), or Met (M); X5 is selected from Gln (Q), Arg (R), Val (V), Ile (I), Lys (K), or Leu (L); X6 is selected from Lys (K), Ser (S), Ile (I), Met (M), Leu (L), Phe (F), or Tyr (Y), provided that Asp (D) or Not Glu (E); X7 is selected from Val (V), Asp (D), Met (M), Ser (S), Glu (E), His (H), Ile (I), or Thr (T); X8 is selected from Leu (L), Ile (I), Thr (T), Pro (P),
  • X4 is preferably Phe (F) or Val (V);
  • X7 is preferably Val (V); and
  • X10 is preferably Asp (D).
  • the polypeptide defined by SEQ ID NO: 1 includes a partial peptide of human immunoglobulin heavy chain binding protein (BiP, GenBank AFF13605 (AF188611.1), SEQ ID NO: 2 and 3), MKP V QK V LE D MycHSP70 (also called DNAK, the amino acid sequence of GenBank CAD93221, equivalent to nucleotides 7764-79741 of the nucleotide sequence of GenBank BX248335.1, which is a homologous polypeptide of SD (SEQ ID NO: 21) or BiP Mycobacterium tuberculosis , SEQ ID NO: partial peptide of 4 and 5), RKP F QS V IA D TG (SEQ ID NO: contains 23), include polypeptides consisting of full-length 12-20 amino acid residues (where underlined The subtracted amino acids correspond to X4, X7 and X10, respectively).
  • polypeptides having these specific amino acid sequences could actually bind to HLA-DR4 or HLA-DR1. Therefore, based on these specific amino acid sequences, the amino acid sequence of a polypeptide capable of binding to HLA-DR4 is converted into a known epitope sequence prediction algorithm (Hammer J, et al. J Exp med 1994; 180: 2353 -8), the amino acid candidates that can be used as X1 to X12 of SEQ ID NO: 1 are defined by these specific amino acid sequences even when the amino acids defined above are used. It was shown to have the same activity as the polypeptide.
  • Polypeptides defined in this way can bind to RA-sensitive MHC class II molecules such as HLA-DR4 or HLA-DR1, and as a result of that binding, they bind to CD4 positive T cells.
  • Peptides can be presented as antigens. That is, the defined polypeptide can function as an epitope of an RA-sensitive MHC class II molecule such as HLA-DR4 or HLA-DR1.
  • Diseases known to be associated with HLA-DR4 or HLA-DR1 include rheumatoid arthritis (RA), polyarthritis of idiopathic juvenile arthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin Dependent diabetes (type 1 diabetes) or multiple sclerosis is known. All of these diseases are thought to occur as a result of HLA-DR4 or HLA-DR1 being stimulated by an epitope, resulting in activation of CD4 + T cells.
  • RA rheumatoid arthritis
  • the present invention uses a polypeptide comprising an amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) as defined herein, in vivo.
  • Immunological tolerance can be generated without generating an autoimmune response to the peptide. Any method commonly used in the art may be used as a method for generating immune tolerance to the polypeptide.
  • the polypeptide to be used as an antigen is orally ingested to the antigen.
  • Intravenous administration in the form presented to dendritic cells having an inhibitory function can be used.
  • the present invention in one aspect, comprises a polypeptide comprising the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) as defined herein.
  • diseases associated with HLA-DR4 or HLA-DR1 include idiopathic juvenile arthritic polyarthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), In addition, there are multiple sclerosis, etc., and by using the pharmaceutical composition provided here, diseases associated with HLA-DR4 or HLA-DR1 include rheumatoid arthritis and idiopathic juvenile arthritis. Diseases such as arthritic, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), or multiple sclerosis can be treated.
  • the present invention provides a polypeptide comprising the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) as defined herein or Provided is a vaccine composition for preventing diseases associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis, comprising a nucleic acid construct for expression.
  • rheumatoid arthritis polyarthritis type of idiopathic juvenile arthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), or multiple Diseases such as sclerosis can also be prevented.
  • the affected site when administering a polypeptide containing the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) prepared in vitro, for example, in the case of rheumatoid arthritis, the affected site (for example, any method and route of administration that can deliver the polypeptide to each joint) can be used.
  • the polypeptide of interest is encapsulated in a lipid such as a liposome or fused to an antennapedia sequence.
  • a cell-permeable synthetic polypeptide to which modifications such as these are added can be administered intravenously or intramuscularly.
  • a nucleic acid encoding a polypeptide containing the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) is introduced into the cell to produce the desired polypeptide in the cell be able to.
  • a construct for expressing a polypeptide containing the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) in a cell is intended for gene therapy, Any construct such as a virus or an expression vector may be used.
  • a lentivirus preferable for use in gene therapy can be used.
  • the target vector in the case of a vector, can be encapsulated in a lipid membrane such as a liposome, or in the case of a virus, it can be administered intravenously or intramuscularly.
  • a nucleic acid encoding a polypeptide containing the amino acid sequence can be prepared.
  • Nucleic acid encoding includes a nucleotide sequence of atgaagcccg tccagaaagt gttggaagat tctgat (SEQ ID NO: 20) or a degenerate nucleotide sequence thereof.
  • Nucleic acid encoding includes a nucleotide sequence of cgcaagccgt tccagtcggt gatcgctgac accggc (SEQ ID NO: 22) or a degenerate nucleotide sequence thereof.
  • a nucleic acid encoding a polypeptide having an amino acid sequence other than that can also be used.
  • Each of these nucleic acids encodes a polypeptide containing the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: ⁇ 1). Or X1 X2 ⁇ ⁇ X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) by transfecting the cells in vitro or in vivo. The containing polypeptide can be expressed.
  • a recombinant expression vector containing these nucleic acids in an expressible manner or a recombinant virus containing these nucleic acids in an expressible manner can also be provided.
  • Such recombinant expression vectors or recombinant viruses can be made using methods well known in the art and are used for the production of the polypeptides of the invention in vitro or in rheumatoid arthritis. Can be used in gene therapy for diseases associated with HLA-DR4 or HLA-DR1.
  • vectors that can be used to prepare the recombinant expression vector of the present invention include pREP9 vector (Invitrogen), pCDNA3.0 vector (Invitrogen), and pCDNA3.1 vector (Invitrogen). It is not limited to.
  • viruses that can be used to produce the recombinant expression virus of the present invention include, but are not limited to, adenovirus, adeno-associated virus, retrovirus (Invitrogen), and lentivirus.
  • Example 1 Search for HLA-DR4 epitope
  • an experiment was conducted for the purpose of searching for an epitope of HLA-DR4.
  • venous blood was collected from 15 HLA-DR4-positive rheumatoid arthritis patients, and peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation using Ficoll-Paque, and RPMI1640 (Gibco) + 10% human serum was collected.
  • a cell suspension having a concentration of 1 ⁇ 10 5 cells / 0.1 mL / well was prepared and seeded at 100 ⁇ l in a 96-well cell culture plate (Corning). After culturing with 10 ⁇ M of the antigen culture polypeptide under culture conditions of 37 ° C., 5% CO 2 and humidified conditions for 3 hours, 3 [H] -thymidine was added, and uptake was measured 18 hours later. Proliferation of peripheral blood mononuclear cells (PBMC) in response to 70-derived peptides was examined.
  • PBMC peripheral blood mononuclear cells
  • the added polypeptide is based on GenBank AF188611.1 (SEQ ID NO: 2) for human BiP and nucleotides 77864-79741 of the nucleotide sequence of GenBank BX248335.1 for mycbacterium HSP70 mycHSP70 (DNAK) Based on (SEQ ID NO: 4), each amino acid sequence (SEQ ID NO: 3 (GenBank AFF13605) for BiP, SEQ ID NO: 5 (GenBank CAD93221) for mycHSP70) has an overlap of 5 amino acids. Those obtained by dividing the C-terminal to the N-terminal in 20 amino acid increments were numbered 1 to 42 for human BiP and 1 to 43 for mycHSP70, respectively.
  • SI stimulation index
  • FIG. The figure shows the average (n 15) stimulus index (S.I.).
  • polypeptides derived from human BiP the polypeptide that induced the strongest thymidine incorporation was the most of the B22 polypeptide (RSTMKPVQKVLEDSDLKKSD, SEQ ID NO: 6) and the polypeptide derived from Mycobacterium HSP70 (mycHSP70).
  • Polypeptides that induced strong thymidine incorporation were designated as D21 polypeptides (DRTRKPFQSVIADTGISVSE, SEQ ID NO: X 13), respectively.
  • HLA-DR4 molecule synthesized in vitro is biotinylated influenza hemagglutinin (HA) -derived peptide 306-318 (amino acid sequence PKYVKQNTLKLAT, SEQ ID NO: 24) 30 nM, which is known to bind strongly to HLA-DR4 molecule HA-derived peptide 306-318 (positive control), 10 nM, 30 nM, 100) nM, 300 nM, 1000 nM concentrations of B22 or D21 polypeptide (test group), and B22 polypeptide is 5 amino acids Reaction with B21 polypeptide (NMDLFRSTMKPVQKVLEDSD, SEQ ID NO: 25, negative control) with shifted sequence in PBS + 0.05% octyl-glucoside at 37 ° C.
  • HA hemagglutinin
  • SEQ ID NO: 24 amino acid sequence
  • the data show the competitive polypeptide concentration that reduces the fluorescence intensity of the positive control by 50% as 50% inhibition concentration (IC50).
  • IC50 50% inhibition concentration
  • B22 polypeptide shows strong binding with HLA-DR4 molecules and binds to HLA-DR4 molecules with approximately the same strength compared to HA used as a positive control. It was shown that.
  • D21 polypeptide was weaker than the B22 polypeptide, it was shown to bind to the HLA-DR4 molecule with a strength similar to that of the B22 polypeptide.
  • the polypeptide in the region overlapping with the B22 polypeptide has a very weak binding property with respect to the binding to the HLA-DR4 molecule.
  • the BiP protein-derived B22 polypeptide (RSTMKPVQKVLEDSDLKKSD, SEQ ID NO: 6) and the mycHSP70 protein-derived D21 polypeptide (DRTRKPFQSVIADTGISVSE, SEQ ID NO: 13) can function as epitopes of the HLA-DR4 molecule. Indicated.
  • Example 2 Sequence analysis of HLA-DR4 epitope The purpose of this example is to search for functionally important amino acids or regions of B22 and D21 polypeptides identified as epitopes of HLA-DR4. The experiment was conducted.
  • the binding strength of each polypeptide to the HLA-DR4 molecule was measured in vitro as the intensity of fluorescence development from the biotinylated HA-derived peptide 306-318 (SEQ ID NO: 24).
  • the obtained data is shown in FIG.
  • the data is shown as fluorescence intensity, that is, the higher the fluorescence intensity, the hindered binding between the influenza HA-derived peptide 306-318 and the HLA-DR4 molecule, that is, the binding force with the HLA-DR4 molecule is weak, it is conceivable that.
  • FIG. 3A shows the results of alanine substitution of B22 polypeptide
  • FIG. 3B shows the results of alanine substitution of D21 polypeptide.
  • B22A polypeptide RSTMKPVQKVLEDSD (SEQ ID NO: 7)
  • B22B polypeptide STMKPVQKVLEDSDL (SEQ ID NO: 8)
  • B22C polypeptide TMKPVQKVLEDSDLK (SEQ ID NO: 9)
  • B22D polypeptide MKPVQKVLEDSDLKK (SEQ ID NO: 10)
  • B22E polypeptide: KPVQKVLEDSDLKKS SEQ ID NO: 11
  • B22F polypeptide PVQKVLEDSDLKKSD (SEQ ID NO: 12)
  • the following polypeptide with a total of 5 amino acids removed from both ends of the D21 polypeptide SEQ ID NO: 13
  • D21A polypeptide D21A polypeptide: DRTRKPFQSVIADTG (SEQ ID NO: 14)
  • D21A polypeptide D21A polypeptide: DRTRKPFQSVIADTG (SEQ ID NO: 14)
  • Fig. 4A shows the results of experiments using a 30 nM B22 polypeptide shortened polypeptide (B22A polypeptide to B22F polypeptide), and Fig. 4B shows a 30 nM D21 polypeptide truncated polypeptide (D21A). The results of experiments using (polypeptide to D21F polypeptide) are shown.
  • the key peptide region on the B22 polypeptide (MKPVQKVLEDSD, SEQ ID NO: 21) and the key peptide region on the D21 polypeptide (RKPFQSVIADTG, SEQ ID NO: 23) corresponded to the sequence alignment. It became clear that it was a position (Fig. 5).
  • FIG. 5 summarizes the results of FIGS. 1 to 4 described above and shows details of the B22 polypeptide and D21 polypeptide sequences.
  • Example 3 Immunological tolerance inducing ability of HLA-DR4 epitope
  • B22 and D21 polypeptides identified as epitopes of HLA-DR4 have a prophylactic effect in vivo in rheumatoid arthritis induction models. An experiment was conducted with the aim of investigating whether or not it could be demonstrated.
  • DBA / 1J mice (6 weeks old, Japanese SLC, 8 mice in each group) were orally administered with 100 ⁇ g (0.1 ⁇ mL) of B22 polypeptide or D21 polypeptide in PBS for 5 consecutive days (D1-D5) After that, arthritis induction treatment was performed by subcutaneously injecting bovine type II collagen 100 ⁇ g / 50 ⁇ L together with an equal amount of complete Freundage band on day 7 (D7).
  • the same volume (0.1 mL) of PBS was administered instead of the polypeptide solution.
  • the arthritis score and the occurrence of arthritis were investigated for these mice, and the average arthritis score and the average arthritis incidence (%) were calculated.
  • mice were sacrificed, and CD4 positive T cells were separated from the mouse spleen using a mouse CD4 + isolation kit (Miltenyi Biotech).
  • CD4 + T cells After preparing these CD4 + T cells to a concentration of 5 ⁇ 10 5 cells / mL, antigen-presenting cells (spleen cells) treated with 1300 rad of radiation (2.5 ⁇ 10 6 cells / mL) and the antigens indicated 3 [H] -thymidine was added after incubation for 96 hours in RPMI 1640 (Gibco) + 10% fetal bovine serum under conditions of 37 ° C. and 5% CO 2 , and uptake was measured 18 hours later.
  • the polypeptide concentration was 10 ⁇ M
  • bovine type II collagen was added at a concentration of 10 ⁇ g / mL. The results of this experiment are shown in FIG.
  • FIG. 7 shows a graph of thymidine incorporation (C.P.M.) (left) and a graph of the relative amount of thymidine incorporation (Stimulatory index: S.I.) compared to the sample without antigen (right).
  • the CD4 + D T cells in the B22 polypeptide and D21 polypeptide group were significantly suppressed in the proliferation response to type II collagen compared to the PBS group. This result means that the internal use of B22 polypeptide and D21 polypeptide was able to suppress CD4-positive T cell-induced abnormal immune conditions that cause arthritis.
  • the anti-bovine type II collagen antibody titer and the anti-CCP antibody titer, which is an autoantibody, in mouse serum 35 days after the start of the experiment were measured by ELISA.
  • Serum was prepared from 20 ⁇ L of blood collected from the tail vein of mice.
  • Anti-bovine type II collagen antibody titer in serum was determined by adsorbing bovine type II collagen (Chondrex) to a 96-well plate (Nunc) at a concentration of 10 ⁇ g / mL, then adding 100-fold serum, and binding IgG antibody.
  • Anti-mouse-IgG antibody-HRP (Zymed) was bound as a secondary antibody, developed with TMB solution (KPL), and measured with a luminometer (BioRad) at an absorbance of 450 nM.
  • the anti-CCP antibody titer in the serum was measured at an absorbance of 570 nM with a luminometer (BioRad) after reacting the serum 10 times using the MESACUP-CCP test (MBL).
  • Example 4 Therapeutic effect of HLA-DR4 epitope on arthritis
  • B22 and D21 polypeptides identified as epitopes of HLA-DR4 are therapeutically treated in vivo in rheumatoid arthritis models actually induced An experiment was conducted with the aim of investigating whether or not a sufficient effect could be exhibited.
  • DBA / 1J mice female 6 weeks old, Japan SLC, 7 mice in each group
  • 100 ⁇ g of B22 polypeptide or D21 polypeptide dissolved in 100 ⁇ L of PBS was orally administered (orally) for 5 consecutive days.
  • PBS of the same volume of 100 ⁇ L was administered instead of the polypeptide solution.
  • mice were sacrificed and the inguinal lymph nodes that belong to the ankle joint were collected.
  • FACS Vantage Becton Dickinson
  • Example 5 B22 polypeptide and D21 polypeptide as epitopes of regulatory T cells This example shows what T cell population is stimulated by oral administration of B22 polypeptide and D21 polypeptide. For the purpose of clarifying in more detail, the proliferative activity of various T cell subsets upon stimulation with B22 and D21 polypeptides was examined.
  • CFSE Once CFSE is taken up into cells, it is characterized by a decrease in CFSE content per cell as cell division progresses.Thus, in proliferating cells, CFSE per cell Each time splitting occurs, the brightness decreases, and the FACS graph shifts to the left. Therefore, as compared with the control group, when the graph shifts to the left side due to the addition of the antigen during culture, it indicates that the cells are proliferating due to the effect of the addition of the antigen.
  • FIG. 11a FOXP3-positive CD4 + CD25 + T cells are shifted to the left by the stimulation of B22 polypeptide and D21 polypeptide, and CD4 + CD25 + FOXP3 + T cells are B22 polypeptide and D21 polypeptide. It was shown that the cells proliferated in response to peptide stimulation ( ⁇ in FIG. 11a). On the other hand, FIG. 11b) showed that CD4 + CD25-T cells did not proliferate significantly upon stimulation with B22 and D21 polypeptides, regardless of whether they were FOXP3-positive or negative.
  • both B22 polypeptide and D21 polypeptide are epitopes recognized by CD4 + CD25 + regulatory T cells.
  • both B22 polypeptide and D21 polypeptide were found to have an effect of selectively proliferating CD4 + CD25 + regulatory T cells.
  • B22 and D21 polypeptides can treat diseases associated with HLA-DR4 or HLA-DR1 through inducing proliferation of CD4 + CD25 + regulatory T cells.
  • SEQ ID NO: 1 Partial peptide of epitope polypeptide for HLA-DR4 molecule
  • SEQ ID NO: 2 Nucleotide sequence of human-derived BiP
  • SEQ ID NO: 3 Amino acid sequence of human-derived BiP (SEQ ID NO: 2 nucleotide sequence) (1-1917 of them are CDS)
  • SEQ ID NO: 4 Nucleotide sequence of mycHSP70 derived from Mycobacterium tuberculosis
  • SEQ ID NO: 5 Amino acid sequence of mycHSP70 derived from Mycobacterium tuberculosis (1 to 1878 of the above nucleotide sequences were used as CDS) thing)
  • SEQ ID NO: 9 B22C polypeptide
  • SEQ ID NO: 10 B22D polypeptide
  • SEQ ID NO: 11 B22

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Abstract

L'invention concerne de nouveaux moyens permettant de déterminer la structure de la séquence d'acides aminés d'un polypeptide d'antigène spécifique (épitope) présentée par des molécules du CMH de classe II sensibles à RA, par exemple HLA-DR4 ou HLA-DR1, par rapport à des lymphocytes T CD4 positifs, et de prévenir ou de traiter des maladies associées à HLA-DR4 ou HLA-DR1, y compris la polyarthrite rhumatoïde, à l'aide de ce type d'épitope. Plus précisément, un polypeptide, formé d'un total de 12 à 20 résidus d'acides aminés, comporte un motif de séquence d'acide aminé spécifique comprenant la séquence d'acide aminé X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (Seq. ID No. 1), est une épitope présentéEepar HLA-DR4 ou HLA-DR1.
PCT/JP2010/071901 2009-12-07 2010-12-07 Identification de l'epitope hla-dr4 et utilisation pour le traitement de l'arthrite WO2011071039A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003523167A (ja) * 1998-10-09 2003-08-05 キングス カレッジ ロンドン 炎症疾患の治療
EP1332760A1 (fr) * 2002-02-04 2003-08-06 Academisch Ziekenhuis Leiden Nouveaux épitopes de la maladie coeliaque et de maladies autoimmunes; méthodes de détection de ces épitopes et de nouveaux composés alimentaires non-antigéniques
JP2009525727A (ja) * 2006-02-02 2009-07-16 ザ ジェネラル ホスピタル コーポレイション 操作された抗体−ストレスタンパク質融合体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003523167A (ja) * 1998-10-09 2003-08-05 キングス カレッジ ロンドン 炎症疾患の治療
EP1332760A1 (fr) * 2002-02-04 2003-08-06 Academisch Ziekenhuis Leiden Nouveaux épitopes de la maladie coeliaque et de maladies autoimmunes; méthodes de détection de ces épitopes et de nouveaux composés alimentaires non-antigéniques
JP2009525727A (ja) * 2006-02-02 2009-07-16 ザ ジェネラル ホスピタル コーポレイション 操作された抗体−ストレスタンパク質融合体

Non-Patent Citations (2)

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KEISHI FUJIO ET AL.: "Kansetsu Rheumatism ni Kanren suru Jiko Kogen ni Taisuru T Saibo Men'eki Oto no Kaiseki ni Kansuru Kenkyu", KOSEI RODO KAGAKU KENKYUHI HOJOKIN MEN'EKI ALLERGY SHIKKAN YOBO · CHIRYO KENKYU JIGYO KANSETSU RHEUMATISM NO SOKI SHINDAN NI YORU HASSHO OYOBI JUSHOKA YOBO, 23 March 2009 (2009-03-23), pages 83 - 84 *
TING J. ET AL.: "Human gene encoding the 78,000-dalton glucose-regulated protein and its pseudogene: structure, conservation, and regulation", DNA, vol. 7, no. 4, 1988, pages 275 - 286, XP000877141 *

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