WO2011071039A1

WO2011071039A1 - Identification of hla-dr4 epitope and use for treatment of arthritis

Info

Publication number: WO2011071039A1
Application number: PCT/JP2010/071901
Authority: WO
Inventors: 宏文庄田; 圭志藤尾; 一彦山本
Original assignee: 国立大学法人東京大学
Priority date: 2009-12-07
Filing date: 2010-12-07
Publication date: 2011-06-16
Also published as: JP2011116719A

Abstract

Provided are novel means for determining the amino acid sequence structure of a specific antigen polypeptide (epitope) presented by RA-sensitive MHC class II molecules, such as HLA-DR4 or HLA-DR1, with respect to CD4-positive T cells, and for preventing or treating disease associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis, using this type of epitope. It is clarified that a polypeptide which is formed from a total of 12 to 20 amino acid residues and includes a specific amino acid sequence motif comprising the amino acid sequence X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (Seq. ID No. 1) is an epitope presented by HLA-DR4 or HLA-DR1.

Description

Identification of HLA-DR4 epitope and its application to arthritis treatment

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. .

Rheumatoid arthritis (RA) is an inflammatory disease characterized by inflammation and proliferation of the synovial membrane, destruction of bone and cartilage, and eventually joint deformation. Although the cause of this disease has not been fully elucidated, it is considered to be one of the autoimmune diseases caused by abnormalities in immunity caused by the stimulation of environmental factors added to the inherited constitution.

Current RA treatment guidelines recommend the use of disease-modifying anti-rheumatic drugs (DMARDs) and biologicals as early as possible, and at the same time non-steroidal anti-inflammatory as symptomatic treatment It is common practice to use analgesics (NSAIDs). However, in these treatment methods, it is known that about 30% of patients cannot obtain a sufficient improvement effect even when using a therapeutic agent, and diseases such as antigen-specific immunosuppression that suppress only the cause of the disease Development of a treatment method that can treat the cause of the disease itself is desired.

It is known that 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. (Nielen MM et al., Arthritis Rheum., 2004, 50, pp 380-386). It was also found that experimental arthritis is hated when anti-CCP antibody is experimentally administered (KuhnuhKA et al., J. Clin. Invest., 2006, 116, 961-973). Anti-CCP antibodies have such characteristics and are used as one of the diagnostic markers for RA. However, it is known that there is a problem that the sensitivity is insufficient for early detection of RA individuals.

In recent years, analysis of the genetic background causing RA has progressed, and HLA (human leukocyte antigen Human leukocyte) is one of the MHC class II molecules that has the function of presenting antigen to CD4 positive T cells and inducing activation. It has been shown that when the serotype of antigen (HLA) -DR is HLA-DR4 or HLA-DR1, there is a tendency to develop RA. This suggests that CD4-positive T cells are involved in the onset of RA. And HLA-DRB1 * 0401 (Europe and America), HLA-DRB1 * 0405 (Asia), HLA-DRB1 * 0101 (Israel), which are specific genotypes of HLA-DR β chain, 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.

On the other hand, the presence of immunoglobulin heavy chain binding protein (Binding protein of immunoglobulin heavy chain: BiP) is known as a protein known to be associated with RA. This BiP is a molecule belonging to the 78 kDa heat shock protein (HSP) 70 family and is overexpressed in the synovium of RA patients. This antibody to BiP is markedly increased in RA patients compared to symptomatic individuals. Specifically, anti-BiP antibodies appear in 60 to 70% of RA patients, and T cell proliferative responses to BiP also occur. (Blass S et al., Arthritis Rheum., 2001, 44, pp 761-771). However, it is not known by what mechanism BiP is related to rheumatoid arthritis.

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.

In the present invention, 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. By clarifying that it is an epitope presented by -DR4 or HLA-DR1, it was clarified that the above-mentioned problems can be solved.

That is, 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.

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 . Figure 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. 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. FIG.

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. As a result, 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.

In one embodiment of the invention, 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.

In this formula, 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), Ser (S), Val (V), Phe (F), Lys (K), or Met (M) Is;
X9 is selected from Glu (E), Ala (A), Thr (T), Ser (S), Asn (N), Val (V), or Pro (P);
X10 is selected from Asp (D), Met (M), Val (V), Leu (L), or Ala (A);
X11 is selected from Ser (S), Thr (T), Gln (Q), Tyr (Y), Lys (K), Asn (N), Leu (L), or Trp (W); and X12 is , Asp (D), Gly (G), Ser (S), Gln (Q), Val (V), His (H), or Ala (A).

Here, X4 is preferably Phe (F) or Val (V); X7 is preferably Val (V); and X10 is preferably Asp (D). X1 X2 X3-Phe-X5 X6-Val-X8 X9-Asp-X11 X12 or X1 X2 X3-Val-X5 X6-Val-X8 X9 if there are preferred amino acids at the three amino acid sites X4, X7 and X10 It has an amino acid sequence structural motif of -Asp-X11 X12.

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.

The present invention, in one aspect, 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. For example, 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.

Utilizing such characteristics, 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. A pharmaceutical composition for preventing or treating a disease associated with HLA-DR4 or HLA-DR1, including rheumatoid arthritis, comprising a nucleic acid construct for expressing the polypeptide in a cell. As previously mentioned, 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.

In another embodiment, 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. With this vaccine composition, as described above, in addition to 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. By forming an antibody against this polypeptide in vivo, binding between BiP and HLA-DR4 or HLA-DR1 in patients with diseases related to HLA-DR4 or HLA-DR1 such as RA Inhibiting and blocking the onset cascade of diseases associated with HLA-DR4 or HLA-DR1, such as RA, resulting from binding of BiP to HLA-DR4 or HLA-DR1.

Here, 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. For example, 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.

In addition, 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. In this case, if 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. For example, a lentivirus (Invitrogen) preferable for use in gene therapy can be used. For example, in the case of a vector, the target 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.

In the present invention, based on the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1), a nucleic acid encoding a polypeptide containing the amino acid sequence can be prepared. For example, when MKP V QK V LE D SD (SEQ ID NO: 21) is used as the amino acid sequence of X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1), a polypeptide having this sequence Nucleic acid encoding includes a nucleotide sequence of atgaagcccg tccagaaagt gttggaagat tctgat (SEQ ID NO: 20) or a degenerate nucleotide sequence thereof. Further, X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1) RKP F QS V IA D TG as the amino acid sequence of (SEQ ID NO: 23) When using the polypeptide having the sequence Nucleic acid encoding includes a nucleotide sequence of cgcaagccgt tccagtcggt gatcgctgac accggc (SEQ ID NO: 22) or a degenerate nucleotide sequence thereof. In the present invention, 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 にて 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. Therefore, in the present invention, 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.

Examples of 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. Examples of 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 In this example, an experiment was conducted for the purpose of searching for an epitope of HLA-DR4.

First, 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 μl in a 96-well cell culture plate (Corning). After culturing with 10 μM 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.

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.

The amount of thymidine incorporation in the sample without the antigen polypeptide was 1, and the relative value of thymidine incorporation in the sample with the antigen polypeptide was measured as the stimulation index (SI) (ie, stimulation index (SI) = epitope-stimulated PBMC). Thymidine incorporation / Thymidine incorporation of PBMC without stimulation). Data for each polypeptide derived from each of BiP and mycHSP70 is shown in FIG. The figure shows the average (n = 15) stimulus index (S.I.). As a result, among the 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.

Next, a peptide binding assay was performed to determine the strength with which the B22 polypeptide and D21 polypeptide obtained by the above method can bind to the HLA-DR4 molecule. 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. for 18 hours, anti-HLA− at a concentration of 10 μg / mL in advance After binding to a 96-well plate (Nunc) coated with DR antibody (LB3.1, transferred from CCATCC), it was reacted with streptavidin-alkaline phosphatase reagent (GEcareHealthcare), and phenolphthalein phosphate (MBL) Using the luminometer (BioRad), the intensity of fluorescence emitted from the biotinylated HA-derived peptide 306-318 (SEQ ID NO: 24) that still binds to the HLA-DR4 molecule after competition is used. Measured.

The data show the competitive polypeptide concentration that reduces the fluorescence intensity of the positive control by 50% as 50% inhibition concentration (IC50). The result is shown in figure 2. As shown in Figure 2, 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. In addition, although the 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. In contrast, it was revealed that the polypeptide in the region overlapping with the B22 polypeptide, the B21 polypeptide, has a very weak binding property with respect to the binding to the HLA-DR4 molecule. This suggests that 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.

Of the B22 polypeptide (Figure 3A; RST MKPVQKVLEDSD LKKSD, SEQ ID NO: 6) and D21 polypeptide (Figure 3B; DRT RKPFQSVIADTG ISVSE, SEQ ID NO: 13), the amino acid at the center of the polypeptide (indicated by underline) ) Is substituted with alanine one amino acid at a time, and each alanine substitution-added polypeptide is added to the culture solution at 6 nM or 30 nM. By the method, 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. In this figure, 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, and FIG. 3B shows the results of alanine substitution of D21 polypeptide.

From these results, in the case of B22 polypeptide, the two valines (V) and aspartic acid (D) indicated by the arrows in FIG. 3A play an important role for binding to the HLA-DR4 molecule, In the case of D21 polypeptide, it is clear that phenylalanine (F), valine (V) and aspartic acid (D) indicated by arrows in Fig. 3B play an important role for binding to HLA-DR4 molecule became. It was revealed that the position of the important amino acid on the B22 polypeptide and the position of the important amino acid on the D21 polypeptide correspond to the positions when sequence alignment was performed (FIG. 5).

Next, how the binding capacity of the epitope polypeptide to the HLA-DR4 molecule changes when the number of amino acids of the polypeptide, which was 20 amino acids in the screening stage of FIGS. 1 and 2, is shortened to 15 amino acids. Was measured by the same method as in FIG.

Specifically, the following polypeptide in which a total of 5 amino acids have been removed from both ends of the B22 polypeptide (SEQ ID NO: 6):
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)
In addition, the following polypeptide with a total of 5 amino acids removed from both ends of the D21 polypeptide (SEQ ID NO: 13):
D21A polypeptide: DRTRKPFQSVIADTG (SEQ ID NO: 14)
D21B polypeptide: RTRKPFQSVIADTGI (SEQ ID NO: 15)
D21C polypeptide: TRKPFQSVIADTGIS (SEQ ID NO: 16)
D21D polypeptide: RKPFQSVIADTGISV (SEQ ID NO: 17)
D21E polypeptide: KPFQSVIADTGISVS (SEQ ID NO: 18)
D21F polypeptide: PFQSVIADTGISVSE (SEQ ID NO: 19)
Were prepared.

Except for adding each of these polypeptides in the amount of 30 nM to the culture solution, the binding force of each polypeptide to the HLA-DR4 molecule was determined using the biotinylated HA-derived peptide. The intensity of fluorescent color development from 306-318 (SEQ ID NO: 24) was measured in a test tube. The obtained data is shown in FIG. In this figure, the data is shown as fluorescence intensity. 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.

These results show that the fluorescence intensity is higher for B22 polypeptide in the case of B22E polypeptide and B22F polypeptide and in the case of D21 polypeptide for D21E polypeptide and D21F polypeptide (ie, HLA-DR4). It became clear that the binding force with the molecule was weakened.

Therefore, 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 In this example, 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 μg (0.1 μmL) 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 μg / 50 μL together with an equal amount of complete Freundage band on day 7 (D7). For the control group, the same volume (0.1 mL) of PBS was administered instead of the polypeptide solution. Thereafter, from the start of administration of the polypeptide to 24 to 32 days (D24 to D32), 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. An outline of the dosing schedule and the results of these experiments are shown in FIG. As a result, both the mean arthritis score and the mean arthritis incidence (%) in the experimental group B22 polypeptide administration group and the D21 polypeptide administration group were significantly reduced compared to the negative control PBS administration group. Indicated.

Next, on the 35th day (D35) after the start of the experiment, the mice were sacrificed, and CD4 positive T cells were separated from the mouse spleen using a mouse CD4 + isolation kit (Miltenyi Biotech). 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 μM, and bovine type II collagen was added at a concentration of 10 μg / 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. When cells were cultured under the condition of addition of B22 polypeptide or D21 polypeptide, the proliferation activity of CD4 + T cells was not significantly observed under any condition, and B22 polypeptide and D21 polypeptide were not observed under the condition of collagen immunization. It was considered that the proliferation response of CD4-positive T cells to the peptide did not occur.

Next, 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 μL 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 μg / 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). These results are shown in FIG. As a result, when immunized after taking B22 polypeptide and D21 polypeptide in advance, the appearance of antibodies against immunizing antigens can be suppressed, and anti-CCP antibodies that are autoantibodies that are also involved in the pathology of arthritis Appearance could also be suppressed, and it became clear that oral administration of these B22 polypeptides and D21 polypeptides can broadly suppress B-cell immune abnormalities.

Example 4: Therapeutic effect of HLA-DR4 epitope on arthritis In this example, 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) were subcutaneously injected with 100 μg (50 μL) bovine type II collagen together with 50 μL complete Freundage band (D1). From the day of arthritis onset, 100 μg of B22 polypeptide or D21 polypeptide dissolved in 100 μL of PBS was orally administered (orally) for 5 consecutive days. For the control group, PBS of the same volume of 100 μL was administered instead of the polypeptide solution.

Thereafter, the progress of mice that developed arthritis was observed for each group, and the average arthritis score after the onset of arthritis was shown in a graph. An outline of the dosing schedule and the results of these experiments are shown in FIG. As a result, it was shown that the mean arthritis score in the experimental group B22 polypeptide administration group and the D21 polypeptide administration group was significantly reduced as compared with the negative administration PBS administration group.

Next, for the purpose of confirming the physiological mechanism in the body, the reason why the mean arthritis score significantly decreased in the B22 polypeptide administration group and the D21 polypeptide administration group, respectively, In this experimental group, what kind of immune system cells were stimulated was examined.

Specifically, in the same experiment, on the 14th day after the onset of arthritis (after the start of polypeptide administration), the mice were sacrificed and the inguinal lymph nodes that belong to the ankle joint were collected. Stain lymph node cells with anti-mouse Foxp3-PE staining kit (eBioscience) using anti-mouse CD4 antibody, anti-mouse CD25 antibody and anti-mouse Foxp3 antibody (eBioscience), then use FACS Vantage (Becton Dickinson) First, after gating CD4 + cells, CD4 + cells were measured for CD25 + and / or Foxp3 positivity using anti-CD25 antibody and anti-Foxp3 antibody (FIG. 10a). As a result of such FACS, the abundance ratio of CD4 + CD25 + Foxp3 + regulatory T cells relative to the total number of CD4 + T cells was clarified (FIG. 10b). From these results, oral administration of B22 polypeptide and D21 polypeptide induced proliferation of regulatory T cells rather than helper T cells in lymphoid organs, and as a result, arthritis was suppressed in vivo It was shown that.

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.

Spleen cells of DBA / 1J mice are stained with anti-mouse CD4 antibody and anti-mouse CD25 antibody (eBioscience), and then CD4 + CD25 + T cells (Figure 11a) and CD4 + CD25-T cells by FACS Vantage (Becton Dickinson) (Figure 11b) was isolated. These cells were stained by reacting with Carboxyfluorescein Succinimidyl Ester (CFSE) (Dojindo) 10 μg / mL at 37 ° C for 30 minutes, and then each cell group 2 × 10 ⁴ cells / 0.2 mL RPMI1640 with 1 × 10 ⁵ cells / mL antigen-presenting cells (spleen cells) treated with 1300 rad of radiation and B22 polypeptide, D21 polypeptide, or HA polypeptide (antigen added during culture) (Gibco) + 10% fetal bovine serum + mouse IL-2 100 U / mL (R6D systems) cultured at 37 ° C, 5% CO ₂ for 96 hours, and then CFSE brightness was observed again by FACS Vantage . In the control group, no antigen added during culture was added. The results are shown in FIG.

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.

In 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.

From these results, it was found that both B22 polypeptide and D21 polypeptide are epitopes recognized by CD4 + CD25 + regulatory T cells. At the same time, both B22 polypeptide and D21 polypeptide were found to have an effect of selectively proliferating CD4 + CD25 + regulatory T cells. Furthermore, it has been found that 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: 6: B22 polypeptide SEQ ID NO: 7: B22A polypeptide SEQ ID NO: 8: B22B polypeptide SEQ ID NO: 9: B22C polypeptide SEQ ID NO: 10: B22D polypeptide SEQ ID NO: 11 : B22E polypeptide SEQ ID NO: 12: B22F polypeptide SEQ ID NO: 13: D21 polypeptide SEQ ID NO: 14: D21A polypeptide SEQ ID NO: 15: D21B polypeptide SEQ ID NO: 16: D21C polypeptide SEQ ID NO: 17: D21D polypeptide SEQ ID NO: 18: D21E polypeptide SEQ ID NO: 19: D21F polypeptide SEQ ID NO: 20: Nucleotide sequence encoding a partial peptide of human BiP-derived B22 polypeptide SEQ ID NO: 21: Partial peptide of B22 polypeptide derived from human BiP SEQ ID NO: 22: Nucleotide sequence encoding partial peptide of D21 polypeptide derived from Mycobacterium tuberculosis mycHSP70 SEQ ID NO: 23: Mycobacterium tuberculosis D21 port derived from mycHSP70 Peptides partial peptide SEQ ID NO: 24: influenza hemagglutinin (HA) derived peptide SEQ ID NO: 25: B21 polypeptide

Claims

Amino acid sequence consisting of the following formula:
X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 (SEQ ID NO: 1)
Where X1 is selected from M or R;
X2 is K,
X3 is P and X4 is selected from F, V, W, Y, I, L, or M;
X5 is selected from Q, R, V, I, K, or L;
X6 is selected from K, S, I, M, L, F, or Y, but not D or E,
X7 is selected from V, D, M, S, E, H, I, or T;
X8 is selected from L, I, T, P, S, V, F, K, or M;
X9 is selected from E, A, T, S, N, V, or P;
X10 is selected from D, M, V, L, or A;
X11 is selected from S, T, Q, Y, K, N, L, or W;
X12 is a polypeptide comprising a total length of 12 to 20 amino acid residues, including selected from D, G, S, Q, V, H, or A.
2. The polypeptide according to claim 1, wherein X4 is F or V.
The polypeptide according to claim 1 or 2, wherein X7 is V.
The polypeptide according to any one of claims 1 to 3, wherein X10 is D.
The polypeptide according to any one of claims 1 to 4, wherein the amino acid sequence of SEQ ID NO: 1 is MKPVQKVLEDSD (SEQ ID NO: 21) or RKPFQSVIADTG (SEQ ID NO: 23).
A polypeptide for preventing or treating a disease associated with HLA-DR4 or HLA-DR1, comprising the polypeptide according to any one of claims 1 to 5 or a nucleic acid construct for expressing the polypeptide in a cell. Pharmaceutical composition.
If the disease associated with HLA-DR4 or HLA-DR1 is rheumatoid arthritis, idiopathic juvenile arthritic polyarthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), or multiple 7. A pharmaceutical composition according to claim 6, selected from sclerosis.
A vaccine composition for preventing a disease associated with HLA-DR4 or HLA-DR1, comprising the polypeptide according to any one of claims 1 to 5 or a nucleic acid construct for expressing the polypeptide in a cell. object.
If the disease associated with HLA-DR4 or HLA-DR1 is rheumatoid arthritis, idiopathic juvenile arthritic polyarthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), or multiple 9. A vaccine composition according to claim 8, selected from sclerosis.