Anti-ILT7 antibody

Abstract

The present invention relates to anti-ILT7 antibodies. Antibodies capable of binding to IPC are obtained by using animal cells in which a cell membrane protein that binds to ILT7 is co-expressed as an immunogen. The antibodies of the present invention have high specificity, such that the antibodies can immunologically distinguish ILT7 from other ILT family molecules. The anti-ILT-7 antibodies of the present invention can bind to IPC and inhibit its activity. The activity of IPC can be inhibited by the anti-ILT-7 antibodies of the present invention, and interferon-related diseases can be treated or prevented. In the presence of IFNα, the expression of ILT7 in IPC can still be maintained. Therefore, in patients with various autoimmune diseases with increased IFNα production, an inhibitory effect of anti-ILT-7 antibodies on the activity of IPC can be expected.

Description

[0001] This application is a divisional application of the invention application with the application date of December 20, 2006, the Chinese application number of 201510296327.2, and the invention name of "Anti-ILT7 Antibody". Technical Field

[0002] The present invention relates to antibodies capable of binding to human ILT7. Background Art

[0003] Interferon α (IFNα: hereinafter "interferon" is represented by the abbreviation IFN) and interferon β (IFNβ) are known as type I IFNs, and this type of IFN has antiviral activity or antitumor activity. On the other hand, it has been reported that IFNα is involved in autoimmune diseases. For example, abnormal production of IFNα has been reported in the patients with the following autoimmune diseases. It has been suggested that the symptoms of autoimmune diseases can be alleviated by neutralizing IFNα.

[0004] Systemic lupus erythematosus (Shiozawa et al., Arthr. & Rheum. 35, 412, 1992)

[0005] Chronic rheumatism (Hopkins et al., Clin. Exp. Immunol. 73, 88, 1988)

[0006] There have been reported examples where the symptoms of autoimmune diseases appear or deteriorate after administration of recombinant IFNα2 or IFN (Wada et al., Am. J. Gastroenterol. 90, 136, l995; Perez et al., Am. J. Hematol. 49, 365, 1995; Wilson LE et al, Semin Arthritis. Rheum. 32, 163 - 173, 2002.).

[0007] Furthermore, it has also been suggested that IFNα can induce the differentiation of dendritic cells. Dendritic cells are also a type of antigen-presenting cells. Therefore, it is considered that the induction of dendritic cell differentiation is an important mechanism including autoimmune diseases. It has been reported that there is a deep connection between IFNα-induced dendritic cell differentiation and the onset of systemic lupus erythematosus (Blanco et al., Science, 16: 294, 1540 - 1543, 2001). Therefore, it has been pointed out that IFNα is closely related to antitumor activity and autoimmune diseases. Moreover, IFNα is closely related to the onset of psoriasis (Nestle FO et al., J. Exp. Med. 202, 135 - 143, 2005).

[0008] Cells that can produce large amounts of type I IFN upon viral infection are identified as interferon-producing cells (IPC). Very few IPCs are present in the blood. Researchers believe that only 1% or less of IPCs are present in peripheral blood lymphocytes. However, IPCs have a high ability to produce IFN. The ability of IPCs to produce IFN can reach, for example, 3000 pg / ml / 10 4 cells. That is to say, it can be said that although there are only a few cells, most of the IFNα or IFNβ produced in the blood upon viral infection is produced by IPCs.

[0009] On the other hand, IPCs are undifferentiated lymphoid dendritic cells, which are considered to be the precursor cells of dendritic cells. IPCs may refer to plasmacytoid dendritic cells. Upon viral stimulation, IPCs will differentiate into dendritic cells and induce T cells to produce IFNγ or IL-10. IPCs can also differentiate into dendritic cells upon stimulation with IL-3. The dendritic cells differentiated upon stimulation with IL-3 will induce T cells to produce Th2 cytokines (IL-4, IL-5, and IL-10). Therefore, IPCs have the property of differentiating into different dendritic cells under different stimulations.

[0010] Accordingly, IPCs have two types: IFN-producing cells and precursor cells of dendritic cells. Both types of cells play important roles in the immune system. In other words, IPCs are important cells that support the immune system in multiple aspects.

[0011] Non-Patent Document 1: Shiozawa et al., Arthr. & Rheum. 35, 412, 1992

[0012] Non-Patent Document 2: Hopkins et al., Clin. Exp. Immunol. 73, 88, 1988

[0013] Non-Patent Document 3: Wada et al., Am. J. Gastroenterol. 90, 136, 1995

[0014] Non-Patent Document 4: Parez et al., Am. J. Hematol. 49, 365, 1995

[0015] Non-Patent Document 5: Bianco et al., Science, 16: 294, 1540 - 1543, 2001

[0016] Non-patent document 6: Ju et al., Gene. 2004 Apr 28; 331: 159-64.

[0017] Non-patent document 7: Colonna M et al., Seminars in Immunology 12: 121-127, 2000.

[0018] Non-patent document 8: Nakajima H. et al., J. Immunology 162: 5-8.1999

[0019] Non-patent document 9: Wilson LE et al, Semin Arthritis. Rheum. 32, 163-173, 2002

[0020] Non-patent document 10: Nestle FO et al., J. Exp. Med. 202, 135-143, 2005

[0021] Patent Document 1: WO03 / 12061 (US Patent Published Application No. 2003-148316) Summary of the invention

[0022] [Problems to be solved by the present invention]

[0023] The object of the present invention is to provide an antibody that can bind to immunoglobulin-like transcript-7 (ILT7) and detect, identify or isolate IPC. Another object of the present invention is to modulate the activity of IPC.

[0024] In order to regulate the activity of humoral factors such as IFN, it is effective to administer antibodies that can recognize the factor. For example, attempts to treat autoimmune diseases by antibodies against interleukin (IL)-1 or IL-4 have been achieved (Guler et al., Arthritis Rheum., 44.S307, 2001). Further, it is envisioned that neutralizing antibodies can act as therapeutic preparations for autoimmune diseases like interferon (Stewart, TA. Cytokine Growth Factor Rev. 14; 139-154, 2003). It can be predicted that the same method as above is equally effective for IFN produced by IPC. However, such a method is based on suppressing the efficacy of the factor after the humoral factor is produced. If the production of the desired humoral factor can be directly controlled, a more obvious therapeutic effect can be obtained.

[0025] Antibodies that can recognize human IPCs have been reported. For example, the anti-BDCA-2 monoclonal antibody is a human IPC-specific monoclonal antibody (Dzionek A. et al. J. Immunol. 165: 6037-6046, 2000). The researchers found that the anti-BDCA-2 monoclonal antibody can effectively inhibit human IPCs from producing IFN (J. Exp. Med. 194: 1823-1834, 2001.). Moreover, it has also been reported that monoclonal antibodies that can recognize interferon-producing cells in mice can inhibit the production of interferon (Blood 2004 Jun 1; 103 / 11: 4201-4206. Epub 2003 Dec). It has been reported that monoclonal antibodies against murine plasmacytoid dendritic cells can cause a decrease in the number of dendritic cells (J. Immunol. 2003, 171: 6466-6477).

[0026] Similarly, it would be very useful if antibodies that can recognize human IPCs and regulate their activity could be provided. For example, the inventors of the present invention have shown that antibodies that can recognize Ly49Q specifically bind to murine IPCs. However, antibodies against Ly49Q do not interfere with the activity of murine IPCs (Blood, 1 April 2005, Vol. 105, No. 7, and pp. 2787-2792.; WO2004 / 13325). On the other hand, ILT7 is known to be a molecule specifically expressed in plasmacytoid dendritic cells (Ju XS et al. and Gene. 2004 Apr 28; 331: 159-64.; WO03 / 12061). However, no antibodies against ILT7 have been obtained. Therefore, the effect of antibodies on IPCs remains unknown.

[0027] ILT7 is a membrane protein containing immunoglobulin-like motifs. It has been reported that this molecule is one of the molecules expressed in cells of the myeloid or lymphoid systems (Colonna M et al., Seminars in Immunology 12: 121-127, 2000.). A group of molecules with a structure similar to that of ILT7 has been designated as the ILT family. The ILT family is also similar in structure and function to the killer cell inhibitory receptor (KIR). ILT7, like other molecules in the ILT family, has four C-type immunoglobulin-like domains. Researchers believe that ILT7, like ILT1, ILT1-like protein, ILT8, and LIR6a, etc., sends activating signals into cells. It has been confirmed that molecules belonging to the ILT family are expressed in hematopoietic cells (Young et al., Immunogenetics 53: 270-278, 2001; "The KIR Gene Cluster." Carrington, Mary and Norman, Paul. Bethesda (MD): National Library of Medicine (US), NCBI; 2003).

[0028] Thus, high expression of ILT7 in plasmacytoid dendritic cells (PDC) and low expression in monocyte-derived dendritic cells (MDDC) were detected by subtractive hybridization. ILT2 and ILT3 are expressed not only in PDC but also in DCs obtained from MDDC or CD34-positive cells. However, since the mRNA of ILT7 is specifically expressed in PDC, it was found that this mRNA can be used as a marker for PDC. In addition, it had been found at that time that the expression of ILT7 can be reduced by CpG stimulation (Ju XS et al. Gene. 2004 Apr 28; 331: 159-64.; WO03 / 12061).

[0029] The inventors of the present invention confirmed through research on human IPC that the expression of ILT7 is specifically enhanced in IPC. Thus, the inventors of the present invention attempted to prepare an ILT7 antibody and elucidate its function. For example, molecules constituting the ILT family such as ILT2 and ILT3 are highly conserved, particularly in the amino acid sequence of their extracellular domains (Figure 9). These ILT family molecules each exhibit a unique expression profile in different blood cells. Therefore, it is very important to obtain an antibody that can immunologically distinguish ILT7 from other ILT family molecules. However, in practice, it is difficult to prepare an antibody that specifically binds to human IPC using ILT7 as an immunogen due to the following obstacles.

[0030] Generally, a protein prepared by genetic recombination technology is used as an immunogen in order to obtain an antibody that can recognize a trace amount of protein obtained from a living tissue. The inventors of the present invention attempted to express human ILT7 based on the cDNA sequence of human ILT7 and the information on the amino acid sequence translated from this base sequence (GenBank Accession No. NM_012276). However, under conventional conditions, human ILT7 cannot be expressed as a recombinant.

[0031] In order to obtain an antibody against a protein, it is also often attempted to use a partial amino acid sequence of a natural protein as an immunogen. However, since the amino acid sequence homology in the ILT family is extremely high, there are almost no amino acid sequences specific to human ILT7. Moreover, in order for an antibody to recognize a molecule on the cell surface, it is necessary to select a region located on the cell surface, which is composed of parts that can be recognized by the antibody as antigenic epitopes. Therefore, researchers have recognized that it is unrealistic to use an amino acid sequence fragment as an immunogen to prepare an antibody specific for ILT7.

[0032] The inventors of the present invention clarified that under such conditions, an antibody that can bind to IPC can be obtained by using a specific immunogen. Furthermore, the inventors of the present invention found that the antibody obtained by this method can specifically recognize human IPC and further has the effect of regulating its activity, thus successfully completing the present invention. That is, the present invention relates to the following anti-ILT-7 antibody, its preparation method and its application.

[0033] [Effects of the Present Invention]

[0034] The present invention provides an immunogen that can be used to prepare an antibody capable of recognizing human ILT7 and a method for preparing an anti-human ILT-7 antibody using the immunogen. ILT7 is a membrane protein belonging to the ILT family. Specifically, the amino acid sequences of the extracellular regions of the ILT family are highly conserved. Therefore, it is very difficult to prepare an antibody capable of distinguishing ILT family members by conventional immunization methods. The inventors of the present invention have shown that an antibody capable of recognizing human ILT7 can be conveniently obtained using animal cells that co-express ILT7 and a cell membrane protein. The anti-ILT-7 antibody obtained by the method of the present invention has high specificity, and the antibody can distinguish human IPC from other cells expressing ILT family members.

[0035] In a preferred application example, the anti-human ILT-7 antibody provided by the present invention can bind to human IPC. In addition, the antibody of the present invention specifically recognizes human IPC. Therefore, the antibody can be used for detecting and isolating IPC. IPC is a cell that produces most type I interferons. Therefore, it is very important to detect and isolate IPC in the diagnosis and study of IPC-related diseases such as autoimmune diseases. Specifically, according to the findings of the inventors of the present invention, the expression of ILT7 in IPC is not reduced due to the presence of IFNα. In patients with autoimmune diseases, the expression of IFNα is often promoted. This means that the anti-ILT-7 antibody of the present invention can be used to detect and isolate IPC of patients with autoimmune diseases in which the expression of IFNα is promoted.

[0036] In a preferred application example, the anti-ILT-7 antibody provided by the present invention has the effect of regulating the activity of human IPC. Therefore, the anti-ILT-7 antibody of the present invention can be used to inhibit the activity of IPC. As described above, in the presence of IFNα, the expression of ILT7 in IPC does not decrease. Therefore, if the activity of IPC is inhibited using the antibody of the present invention, then a therapeutic effect of the antibody on patients with autoimmune diseases in which the expression of IFNα is promoted can be expected.

[0037] A small amount of IPC can produce a large amount of IFN. As many antibodies as IFN molecules are required to neutralize IFN. However, in the present invention, the activation of the producing cells is directly inhibited. Therefore, it can be expected that a potent IFN inhibitory effect can be obtained even using a small amount of antibody compared with neutralizing IFN using anti-IFN antibodies. In addition, in the case of continuous IFN production, it can be predicted that neutralization by IFN antibodies is a temporary inhibition. In the present invention, since the activity of IPC is inhibited, it can be expected that the inhibitory effect on IFN production can be effective for a long time.

[0038] The present invention relates to the following items.

[0039] 1. A monoclonal antibody capable of binding to the extracellular domain of human ILT7, or a fragment containing the antigen-binding region of the monoclonal antibody.

[0040] 2. The monoclonal antibody according to item 1 or the fragment containing the antigen-binding region of the monoclonal antibody, wherein the monoclonal antibody is capable of binding to human interferon-producing cells.

[0041] 3. A monoclonal antibody produced by hybridoma ILT7#11 with deposit number FERM BP-10704 or hybridoma ILT7#17 with deposit number FERM BP-10705, or a fragment containing the antigen-binding region of the monoclonal antibody.

[0042] 4. The monoclonal antibody according to item 1 or the fragment containing the antigen-binding region of the monoclonal antibody, wherein the monoclonal antibody contains the amino acid sequence of any one of the following i) to iii) as CDR1, CDR2, and CDR3 in the heavy chain variable region and the light chain variable region:

[0043] i) CDR1 of the heavy chain variable region: SDYAWN (SEQ ID NO: 58);

[0044] CDR2 of the heavy chain variable region: YISYSGSTSYNPSLKSR (SEQ ID NO: 59); and

[0045] CDR3 of the heavy chain variable region: SPPYYAMDY (SEQ ID NO: 60);

[0046] CDR1 of the light chain variable region: KASQDVGTAVA (SEQ ID NO: 61);

[0047] CDR2 of the light chain variable region: WASTRHT (SEQ ID NO: 62); and

[0048] CDR3 of the light chain variable region: QQYSSYPLT (SEQ ID NO: 63);

[0049] ii) CDR1 of the heavy chain variable region: SYWIH (SEQ ID NO: 64);

[0050] CDR2 of the heavy chain variable region: RIYPGTGSTYYNEKFKG (SEQ ID NO: 65); and

[0051] CDR3 of the heavy chain variable region: YPTYDWYFDV (SEQ ID NO: 66);

[0052] CDR1 of the light chain variable region: RASQSISNYLH (SEQ ID NO: 67);

[0053] CDR2 of the light chain variable region: YASQSIS (SEQ ID NO: 68);

[0054] CDR3 of the light chain variable region: QQSNSWPLT (SEQ ID NO: 69);

[0055] iii) CDR1 of the heavy chain variable region: SDYAWN (SEQ ID NO: 70);

[0056] CDR2 of the heavy chain variable region: YISYSGSTSYNPSLKSR (SEQ ID NO: 71);

[0057] CDR3 of the heavy chain variable region: ALPLPWFAY (SEQ ID NO: 72);

[0058] CDR1 of the light chain variable region: KASQDVGTAVA (SEQ ID NO: 73);

[0059] CDR2 of the light chain variable region: WASTRHT (SEQ ID NO: 74); and

[0060] CDR3 of the light chain variable region: QQYSSYPYT (SEQ ID NO: 75).

[0061] 5. The monoclonal antibody according to item 1 or a fragment containing the antigen-binding region of the monoclonal antibody, wherein the monoclonal antibody contains the mature sequence of the amino acid sequence selected from any one of the combinations of (a) to (c) below as the heavy chain variable region and the light chain variable region:

[0062] a) The heavy chain variable region of SEQ ID NO: 39 and the light chain variable region of SEQ ID NO: 41;

[0063] b) The heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO: 45; and

[0064] c) The heavy chain variable region of SEQ ID NO: 47 and the light chain variable region of SEQ ID NO: 49.

[0065] 6. A polynucleotide encoding the monoclonal antibody according to item 4 or 5 or a fragment containing the antigen-binding region of the monoclonal antibody.

[0066] 7. A vector containing the polynucleotide encoding the monoclonal antibody according to item 4 or 5 or a fragment containing the antigen-binding region of the monoclonal antibody.

[0067] 8. A transformed cell carrying the vector of item 7 in an expressible manner.

[0068] 9. A method for preparing the monoclonal antibody of item 4 or 5, or a fragment containing the antigen-binding region of the monoclonal antibody, the method comprising the steps of: culturing the transformed cell of item 8, and recovering the monoclonal antibody or the fragment containing the antigen-binding region of the monoclonal antibody from the culture.

[0069] 10. A hybridoma for producing the monoclonal antibody of item 1 or 2.

[0070] 11. Hybridoma ILT7#11 with deposit number FERM BP-10704 or hybridoma ILT7#17 with deposit number FERM BP-10705.

[0071] 12. A method for preparing a monoclonal antibody, the method comprising the steps of: culturing the hybridoma of item 11, and collecting the monoclonal antibody from the culture.

[0072] 13. A method for preparing a monoclonal antibody-producing cell, wherein the monoclonal antibody is capable of binding to the extracellular domain of human ILT7, the method comprising the following steps:

[0073] (1) Administering cells to an immunized animal, the cells expressing an exogenous protein containing the extracellular domain of human ILT7 and an exogenous molecule that binds to human ILT7; and

[0074] (2) Selecting the following antibody-producing cells from the antibody-producing cells of the immunized animal, the antibody-producing cells producing an antibody capable of binding to human ILT7.

[0075] 14. The method according to item 13, wherein the molecule that binds to human ILT7 is a cell membrane protein.

[0076] 15. The method according to item 14, wherein the cell membrane protein is the Fc receptor gamma chain.

[0077] 16. The method according to item 15, wherein the cell expressing human ILT7 and the molecule that binds to human ILT7 is a cell carrying the following (a) and (b) in an expressible manner:

[0078] (a) An exogenous polynucleotide encoding an amino acid sequence containing the extracellular domain of human ILT7; and

[0079] (b) An exogenous polynucleotide encoding the Fc receptor gamma chain.

[0080] 17. The method according to item 16, wherein the cell is an animal cell.

[0081] 18. The method according to item 17, wherein the cell is a human-derived cell.

[0082] 19. The method according to item 18, wherein the human cell is a 293T cell.

[0083] 20. The method according to item 13, further comprising the step of cloning the antibody-producing cells obtained by the method according to item 13.

[0084] 21. A method for preparing a monoclonal antibody capable of binding to the extracellular domain of human ILT7, the method comprising the steps of: culturing the antibody-producing cells obtained by the method according to item 8, and collecting the monoclonal antibody from the culture.

[0085] 22. A monoclonal antibody capable of recognizing human ILT7 or a fragment containing the antigen-binding region of the monoclonal antibody, the monoclonal antibody or fragment being obtainable by the following steps:

[0086] (1) Administering to an immunized animal cells that ectopically express a protein containing the extracellular domain of human ILT7 and a molecule that binds to human ILT7;

[0087] (2) Selecting from the antibody-producing cells of the immunized animal the following antibody-producing cells that produce an antibody capable of binding to human ILT7; and

[0088] (3) Culturing the antibody-producing cells selected in step (2) and recovering from the culture an antibody capable of recognizing human ILT7.

[0089] 23. An immunogen for preparing an antibody capable of binding to the extracellular domain of human ILT7, the immunogen comprising the following animal cells or their cell membrane components, the cells carrying in an ectopically expressible manner (a) a polynucleotide encoding an amino acid sequence containing the extracellular domain of human ILT7 and (b) a polynucleotide encoding the Fc receptor gamma chain.

[0090] 24. The immunogen according to item 23, wherein the animal cell is a human cell.

[0091] 25. A method for detecting interferon-producing cells, the method comprising the following steps:

[0092] Contacting a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing the antigen-binding region of the monoclonal antibody with a test cell; and

[0093] Detecting the monoclonal antibody or the fragment containing the antigen-binding region of the monoclonal antibody that has bound to the cell.

[0094] 26. A detection reagent for detecting interferon-producing cells, the detection reagent comprising a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing the antigen-binding region of the monoclonal antibody.

[0095] 27. A method for inhibiting the activity of interferon-producing cells, the method comprising the step of contacting interferon-producing cells with any of the following components:

[0096] (a) A monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of interferon-producing cells, or a fragment containing the antigen-binding region of the monoclonal antibody; and

[0097] (b) An immunoglobulin or a fragment containing the antigen-binding region of the immunoglobulin, in which the complementarity-determining regions of the monoclonal antibody of (a) are introduced.

[0098] 28. A method for inhibiting the activity of interferon-producing cells in a living body, the method comprising the step of administering to the living body any of the following components:

[0099] (a) A monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of interferon-producing cells, or a fragment containing the antigen-binding region of the monoclonal antibody;

[0100] (b) An immunoglobulin or a fragment containing the antigen-binding region of the immunoglobulin, in which the complementarity-determining regions of the monoclonal antibody of (a) are introduced; and

[0101] (c) A polynucleotide encoding the component of (a) or (b).

[0102] 29. The method according to item 27 or 28, wherein the activity of the interferon-producing cells is interferon-producing activity, or the survival of interferon-producing cells, or both.

[0103] 30. An inhibitor of interferon-producing cell activity, which comprises any of the following components as an active ingredient:

[0104] (a) A monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of interferon-producing cells, or a fragment containing the antigen-binding region of the monoclonal antibody;

[0105] (b) An immunoglobulin or a fragment containing the antigen-binding region of the immunoglobulin, in which the complementarity-determining regions of the monoclonal antibody of (a) are introduced; and

[0106] (c) A polynucleotide encoding the component of (a) or (b).

[0107] 31. The inhibitor of interferon-producing cell activity according to item 30, wherein the activity of the interferon-producing cells is interferon-producing activity, or the survival of interferon-producing cells, or both. <110> SBI BIOTECH CO., LTD. <120> Anti-ILT7 antibody <130> G2-A0501P <150> JP 2005-366465 <151> 2005-12-20 <160> 76 <170> PatentIn version 3.3 <210> 1 <211> 1577 <212> DNA <213> Human <220> <221> CDS <222> (24)..(1520) <220> <221> sig_peptide <222> (24)..(71) <220> <221> mat_peptide <222> (72)..(1520) <400> 1 cagggccagg aggaggagat gcc atg acc ctc att ctc aca agc ctg ctc ttc 53 Met Thr Leu Ile Leu Thr Ser Leu Leu Phe -15 -10 ttt ggg ctg agc ctg ggc ccc agg acc cgg gtg cag gca gaa aac cta 101 Phe Gly Leu Ser Leu Gly Pro Arg Thr Arg Val Gln Ala Glu Asn Leu -5 -1 1 5 10 ccc aaa ccc atc ctg tgg gcc gag cca ggt ccc gtg atc acc tgg cat 149 Pro Lys Pro Ile Leu Trp Ala Glu Pro Gly Pro Val Ile Thr Trp His 15 20 25 aac ccc gtg acc atc tgg tgt cag ggc acc ctg gag gcc cag ggg tac 197 Asn Pro Val Thr Ile Trp Cys Gln Gly Thr Leu Glu Ala Gln Gly Tyr 30 35 40 cgt ctg gat aaa gag gga aac tca atg tcg agg cac ata tta aaa aca 245 Arg Leu Asp Lys Glu Gly Asn Ser Met Ser Arg His Ile Leu Lys Thr 45 50 55 ctg gag tct gaa aac aag gtc aaa ctc tcc atc cca tcc atg atg tgg 293 Leu Glu Ser Glu Asn Lys Val Lys Leu Ser Ile Pro Ser Met Met Trp 60 65 70 gaa cat gca ggg cga tat cac tgt tac tat cag agc cct gca ggc tgg 341 Glu His Ala Gly Arg Tyr His Cys Tyr Tyr Gln Ser Pro Ala Gly Trp 75 80 85 90 tca gag ccc agc gac ccc ctg gag ctg gtg gtg aca gcc tac agc aga 389 Ser Glu Pro Ser Asp Pro Leu Glu Leu Val Val Thr Ala Tyr Ser Arg 95 100 105 ccc acc ctg tcc gca ctg cca agc cct gtg gtg acc tca gga gtg aac 437 Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly Val Asn 110 115 120 gtg acc ctc cgg tgt gcc tca cgg ctg gga ctg ggc agg ttc act ctg 485 Val Thr Leu Arg Cys Ala Ser Arg Leu Gly Leu Gly Arg Phe Thr Leu 125 130 135 att gag gaa gga gac cac agg ctc tcc tgg acc ctg aac tca cac caa 533 Ile Glu Glu Gly Asp His Arg Leu Ser Trp Thr Leu Asn Ser His Gln 140 145 150 cac aac cat gga aag ttc cag gcc ctg ttc ccc atg ggc ccc ctg acc 581 His Asn His Gly Lys Phe Gln Ala Leu Phe Pro Met Gly Pro Leu Thr 155 160 165 170 ttc agc aac agg ggt aca ttc aga tgc tac ggc tat gaa aac aac acc 629 Phe Ser Asn Arg Gly Thr Phe Arg Cys Tyr Gly Tyr Glu Asn Asn Thr 175 180 185 cca tac gtg tgg tcg gaa ccc agt gac ccc ctg cag cta ctg gtg tca 677 Pro Tyr Val Trp Ser Glu Pro Ser Asp Pro Leu Gln Leu Leu Val Ser 190 195 200 ggc gtg tct agg aag ccc tcc ctc ctg acc ctg cag ggc cct gtc gtg 725 Gly Val Ser Arg Lys Pro Ser Leu Leu Thr Leu Gln Gly Pro Val Val 205 210 215 acc ccc gga gag aat ctg acc ctc cag tgt ggc tct gat gtc ggc tac 773 Thr Pro Gly Glu Asn Leu Thr Leu Gln Cys Gly Ser Asp Val Gly Tyr 220 225 230 atc aga tac act ctg tac aag gag ggg gcc gat ggc ctc ccc cag cgc 821 Ile Arg Tyr Thr Leu Tyr Lys Glu Gly Ala Asp Gly Leu Pro Gln Arg 235 240 245 250 cct ggc cgg cag ccc cag gct ggg ctc tcc cag gcc aac ttc acc ctg 869 Pro Gly Arg Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu 255 260 265 agc cct gtg agc cgc tcc tac ggg ggc cag tac aga tgc tac ggc gca 917 Ser Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala 270 275 280 cac aac gtc tcc tcc gag tgg tcg gcc ccc agt gac ccc ctg gac atc 965 His Asn Val Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile 285 290 295 ctg atc gca gga cag atc tct gac aga ccc tcc ctc tca gtg cag ccg 1013 Leu Ile Ala Gly Gln Ile Ser Asp Arg Pro Ser Leu Ser Val Gln Pro 300 305 310 ggc ccc acg gtg acc tca gga gag aag gtg acc ctg ctg tgt cag tca 1061 Gly Pro Thr Val Thr Ser Gly Glu Lys Val Thr Leu Leu Cys Gln Ser 315 320 325 330 tgg gac ccg atg ttc act ttc ctt ctg acc aag gag ggg gca gcc cat 1109 Trp Asp Pro Met Phe Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala His 335 340 345 ccc ccg ttg cgt ctg aga tca atg tac gga gct cat aag tac cag gct 1157 Pro Pro Leu Arg Leu Arg Ser Met Tyr Gly Ala His Lys Tyr Gln Ala 350 355 360 gaa ttc ccc atg agt cct gtg acc tca gcc cac gcg ggg acc tac agg 1205 Glu Phe Pro Met Ser Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg 365 370 375 tgc tac ggc tca cgc agc tcc aac ccc tac ctg ctg tct cac ccc agt 1253 Cys Tyr Gly Ser Arg Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser 380 385 390 gag ccc ctg gag ctc gtg gtc tca gga gca act gag acc ctc aat cca 1301 Glu Pro Leu Glu Leu Val Val Ser Gly Ala Thr Glu Thr Leu Asn Pro 395 400 405 410 gca caa aag aag tca gat tcc aag act gcc cca cac ctc cag gat tac 1349 Ala Gln Lys Lys Ser Asp Ser Lys Thr Ala Pro His Leu Gln Asp Tyr 415 420 425 aca gtg gag aat ctc atc cgc atg ggt gtg gct ggc ttg gtc ctg ctg 1397 Thr Val Glu Asn Leu Ile Arg Met Gly Val Ala Gly Leu Val Leu Leu 430 435 440 ttc ctc ggg att ctg tta ttt gag gct cag cac agc cag aga agc ccc 1445 Phe Leu Gly Ile Leu Leu Phe Glu Ala Gln His Ser Gln Arg Ser Pro 445 450 455 cca agg tgc agc cag gag gca aac agc aga aag gac aat gca ccc ttc 1493 Pro Arg Cys Ser Gln Glu Ala Asn Ser Arg Lys Asp Asn Ala Pro Phe 460 465 470 aga gtg gtg gag cct tgg gaa cag atc tgatgatctg aggaggttct 1540 Arg Val Val Glu Pro Trp Glu Gln Ile 475 480 ggaagactgg ggcagcagtt ggggaagtgt ctgctga 1577 <210> 2 <211> 499 <212> PRT <213> Human <400> 2 Met Thr Leu Ile Leu Thr Ser Leu Leu Phe Phe Gly Leu Ser Leu Gly -15 -10 -5 -1 Pro Arg Thr Arg Val Gln Ala Glu Asn Leu Pro Lys Pro Ile Leu Trp 1 5 10 15 Ala Glu Pro Gly Pro Val Ile Thr Trp His Asn Pro Val Thr Ile Trp 20 25 30 Cys Gln Gly Thr Leu Glu Ala Gln Gly Tyr Arg Leu Asp Lys Glu Gly 35 40 45 Asn Ser Met Ser Arg His Ile Leu Lys Thr Leu Glu Ser Glu Asn Lys 50 55 60 Val Lys Leu Ser Ile Pro Ser Met Met Trp Glu His Ala Gly Arg Tyr 65 70 75 80 His Cys Tyr Tyr Gln Ser Pro Ala Gly Trp Ser Glu Pro Ser Asp Pro 85 90 95 Leu Glu Leu Val Val Thr Ala Tyr Ser Arg Pro Thr Leu Ser Ala Leu 100 105 110 Pro Ser Pro Val Val Thr Ser Gly Val Asn Val Thr Leu Arg Cys Ala 115 120 125 Ser Arg Leu Gly Leu Gly Arg Phe Thr Leu Ile Glu Glu Gly Asp His 130 135 140 Arg Leu Ser Trp Thr Leu Asn Ser His Gln His Asn His Gly Lys Phe 145 150 155 160 Gln Ala Leu Phe Pro Met Gly Pro Leu Thr Phe Ser Asn Arg Gly Thr 165 170 175 Phe Arg Cys Tyr Gly Tyr Glu Asn Asn Thr Pro Tyr Val Trp Ser Glu 180 185 190 Pro Ser Asp Pro Leu Gln Leu Leu Val Ser Gly Val Ser Arg Lys Pro 195 200 205 Ser Leu Leu Thr Leu Gln Gly Pro Val Val Thr Pro Gly Glu Asn Leu 210 215 220 Thr Leu Gln Cys Gly Ser Asp Val Gly Tyr Ile Arg Tyr Thr Leu Tyr 225 230 235 240 Lys Glu Gly Ala Asp Gly Leu Pro Gln Arg Pro Gly Arg Gln Pro Gln 245 250 255 Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Ser Pro Val Ser Arg Ser 260 265 270 Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Val Ser Ser Glu 275 280 285 Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly Gln Ile 290 295 300 Ser Asp Arg Pro Ser Leu Ser Val Gln Pro Gly Pro Thr Val Thr Ser 305 310 315 320 Gly Glu Lys Val Thr Leu Leu Cys Gln Ser Trp Asp Pro Met Phe Thr 325 330 335 Phe Leu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Arg Leu Arg 340 345 350 Ser Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro Met Ser Pro 355 360 365 Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Arg Ser 370 375 380 Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu Val 385 390 395 400 Val Ser Gly Ala Thr Glu Thr Leu Asn Pro Ala Gln Lys Lys Ser Asp 405 410 415 Ser Lys Thr Ala Pro His Leu Gln Asp Tyr Thr Val Glu Asn Leu Ile 420 425 430 Arg Met Gly Val Ala Gly Leu Val Leu Leu Phe Leu Gly Ile Leu Leu 435 440 445 Phe Glu Ala Gln His Ser Gln Arg Ser Pro Pro Arg Cys Ser Gln Glu 450 455 460 Ala Asn Ser Arg Lys Asp Asn Ala Pro Phe Arg Val Val Glu Pro Trp 465 470 475 480 Glu Gln Ile <210> 3 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 3 ctccaacccc tacctgctgt c 21 <210> 4 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 4 ttcccaaggc tccaccactc t 21 <210> 5 <211> 23 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 5 cctcaatcca gcacaaaaga agt 23 <210> 6 <211> 24 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 6 cggatgagat tctccactgt gtaa 24 <210> 7 <211> 18 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 7 ccacccatgg caaattcc 18 <210> 8 <211> 22 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 8 tgggatttcc attgatgaca ag 22 <210> 9 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 9 cagggccagg aggaggagat g 21 <210> 10 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 10 tcagcagaca cttccccaac t 21 <210> 11 <211> 105 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 11 ccgctcgaga tgaccctcat tctcacaagc ctgctcttct ttgggctgag cctgggcgat 60 tacaaggatg acgacgataa gcccaggacc cgggtgcagg cagaa 105 <210> 12 <211> 31 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 12 ctagactagt tcagatctgt tcccaaggct c 31 <210> 13 <211> 30 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 13 ccgctcgaga tgaccctcat tctcacaagc 30 <210> 14 <211> 55 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 14 ctagactagt tcacttatcg tcgtcatcct tgtaatcgat ctgttcccaa ggctc 55 <210> 15 <211> 313 <212> DNA <213> Human <220> <221> CDS <222> (7)..(267) <400> 15 cccaag atg att cca gca gtg gtc ttg ctc tta ctc ctt ttg gtt gaa 48 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu 1 5 10 caa gca gcg gcc ctg gga gag cct cag ctc tgc tat atc ctg gat gcc 96 Gln Ala Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala 15 20 25 30 atc ctg ttt ctg tat gga att gtc ctc acc ctc ctc tac tgt cga ctg 144 Ile Leu Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu 35 40 45 aag atc caa gtg cga aag gca gct ata acc agc tat gag aaa tca gat 192 Lys Ile Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp 50 55 60 ggt gtt tac acg ggc ctg agc acc agg aac cag gag act tac gag act 240 Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr 65 70 75 ctg aag cat gag aaa cca cca cag tag ctttagaata gatgcggtca 287 Leu Lys His Glu Lys Pro Pro Gln 80 85 tattcttctt tggcttctgg ttcttc 313 <210> 16 <211> 86 <212> PRT <213> Human <400> 16 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85 <210> 17 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 17 cccaagatga ttccagcagt g 21 <210> 18 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 18 ggaagaacca gaagccaaag a 21 <210> 19 <211> 30 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 19 ccgctcgaga tgattccagc agtggtcttg 30 <210> 20 <211> 61 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 20 ctagactagt ctacagatcc tcttcagaga tgagtttctg ctcctgtggt ggtttctcat 60 g 61 <210> 21 <211> 23 <212> PRT <213> Artificial <220> <223> Artificially synthesized peptide sequence <400> 21 Cys Ser Gln Glu Ala Asn Ser Arg Lys Asp Asn Ala Pro Phe Arg Val 1 5 10 15 Val Glu Pro Trp Glu Gln Ile 20 <210> 22 <211> 31 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 22 ccgctcgaga tgacccccat cctcacggtc c 31 <210> 23 <211> 55 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 23 ctagactagt tcacttatcg tcgtcatcct tgtaatccct cccggctgca tcttg 55 <210> 24 <211> 1425 <212> DNA <213> Human <220> <221> CDS <222> (1)..(1425) <400> 24 atg acc ccc atc ctc acg gtc ctg atc tgt ctc ggg ctg agt ctg ggc 48 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 ccc agg acc cac gtg cag gca ggg cac ctc ccc aag ccc acc ctc tgg 96 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 gct gag cca ggc tct gtg atc atc cag gga agt cct gtg acc ctc agg 144 Ala Glu Pro Gly Ser Val Ile Ile Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 tgt cag ggg agc ctt cag gct gag gag tac cat cta tat agg gaa aac 192 Cys Gln Gly Ser Leu Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn 50 55 60 aaa tca gca tcc tgg gtt aga cgg ata caa gag cct ggg aag aat ggc 240 Lys Ser Ala Ser Trp Val Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly 65 70 75 80 cag ttc ccc atc cca tcc atc acc tgg gaa cac gca ggg cgg tat cac 288 Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His 85 90 95 tgt cag tac tac agc cac aat cac tca tca gag tac agt gac ccc ctg 336 Cys Gln Tyr Tyr Ser His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu 100 105 110 gag ctg gtg gtg aca gga gcc tac agc aaa ccc acc ctc tca gct ctg 384 Glu Leu Val Val Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu 115 120 125 ccc agc cct gtg gtg acc tta gga ggg aac gtg acc ctc cag tgt gtc 432 Pro Ser Pro Val Val Thr Leu Gly Gly Asn Val Thr Leu Gln Cys Val 130 135 140 tca cag gtg gca ttt gac ggc ttc att ctg tgt aag gaa gga gaa gat 480 Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp 145 150 155 160 gaa cac cca caa cgc ctg aac tcc cat tcc cat gcc cgt ggg tgg tcc 528 Glu His Pro Gln Arg Leu Asn Ser His Ser His Ala Arg Gly Trp Ser 165 170 175 tgg gcc atc ttc tcc gtg ggc ccc gtg agc ccg agt cgc agg tgg tcg 576 Trp Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser 180 185 190 tac agg tgc tat gct tat gac tcg aac tct ccc tat gtg tgg tct cta 624 Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Val Trp Ser Leu 195 200 205 ccc agt gat ctc ctg gag ctc ctg gtc cca ggt gtt tct aag aag cca 672 Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro 210 215 220 tca ctc tca gtg cag cca ggt cct atg gtg gcc cct ggg gag agc ctg 720 Ser Leu Ser Val Gln Pro Gly Pro Met Val Ala Pro Gly Glu Ser Leu 225 230 235 240 acc ctc cag tgt gtc tct gat gtc ggc tac gac aga ttt gtt ctg tat 768 Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr 245 250 255 aag gag gga gaa cgt gac ttc ctc cag cgc cct ggt tgg cag ccc cag 816 Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro Gly Trp Gln Pro Gln 260 265 270 gct ggg ctc tcc cag gcc aac ttc acc ctg ggc cct gtg agc ccc tcc 864 Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Pro Ser 275 280 285 cac ggg ggc cag tac aga tgc tac agt gca cac aac ctc tcc tcc gag 912 His Gly Gly Gln Tyr Arg Cys Tyr Ser Ala His Asn Leu Ser Ser Glu 290 295 300 tgg tcg gcc ccc agt gac ccc ctg gac atc ctg atc aca gga cag ttc 960 Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Phe 305 310 315 320 tat gac aga ccc tct ctc tcg gtg cag ccg gtc ccc aca gta gcc cca 1008 Tyr Asp Arg Pro Ser Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro 325 330 335 gga aag aac gtg acc ctg ctg tgt cag tca cgg ggg cag ttc cac act 1056 Gly Lys Asn Val Thr Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr 340 345 350 ttc ctt ctg acc aag gag ggg gca ggc cat ccc cca ctg cat ctg aga 1104 Phe Leu Leu Thr Lys Glu Gly Ala Gly His Pro Pro Leu His Leu Arg 355 360 365 tca gag cac caa gct cag cag aac cag gct gaa ttc cgc atg ggt cct 1152 Ser Glu His Gln Ala Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro 370 375 380 gtg acc tca gcc cac gtg ggg acc tac aga tgc tac agc tca ctc agc 1200 Val Thr Ser Ala His Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser 385 390 395 400 tcc aac ccc tac ctg ctg tct ctc ccc agt gac ccc ctg gag ctc gtg 1248 Ser Asn Pro Tyr Leu Leu Ser Leu Pro Ser Asp Pro Leu Glu Leu Val 405 410 415 gtc tca gca tcc cta ggc caa cac ccc cag gat tac aca gtg gag aat 1296 Val Ser Ala Ser Leu Gly Gln His Pro Gln Asp Tyr Thr Val Glu Asn 420 425 430 ctc atc cgc atg ggt gtg gct ggc ttg gtc ctg gtg gtc ctc ggg att 1344 Leu Ile Arg Met Gly Val Ala Gly Leu Val Leu Val Val Leu Gly Ile 435 440 445 ctg cta ttt gag gct cag cac agc cag aga agc cta caa gat gca gcc 1392 Leu Leu Phe Glu Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala Ala 450 455 460 ggg agg gat tac aag gat gac gac gat aag tga 1425 Gly Arg Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 <210> 25 <211> 474 <212> PRT <213> Human <400> 25 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Ile Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Ser Leu Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn 50 55 60 Lys Ser Ala Ser Trp Val Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly 65 70 75 80 Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His 85 90 95 Cys Gln Tyr Tyr Ser His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu 100 105 110 Glu Leu Val Val Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu 115 120 125 Pro Ser Pro Val Val Thr Leu Gly Gly Asn Val Thr Leu Gln Cys Val 130 135 140 Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp 145 150 155 160 Glu His Pro Gln Arg Leu Asn Ser His Ser His Ala Arg Gly Trp Ser 165 170 175 Trp Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser 180 185 190 Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Val Trp Ser Leu 195 200 205 Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro 210 215 220 Ser Leu Ser Val Gln Pro Gly Pro Met Val Ala Pro Gly Glu Ser Leu 225 230 235 240 Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr 245 250 255 Lys Glu Gly Glu Arg Asp Phe Leu Gln Arg Pro Gly Trp Gln Pro Gln 260 265 270 Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Pro Ser 275 280 285 His Gly Gly Gln Tyr Arg Cys Tyr Ser Ala His Asn Leu Ser Ser Glu 290 295 300 Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Phe 305 310 315 320 Tyr Asp Arg Pro Ser Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro 325 330 335 Gly Lys Asn Val Thr Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr 340 345 350 Phe Leu Leu Thr Lys Glu Gly Ala Gly His Pro Pro Leu His Leu Arg 355 360 365 Ser Glu His Gln Ala Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro 370 375 380 Val Thr Ser Ala His Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser 385 390 395 400 Ser Asn Pro Tyr Leu Leu Ser Leu Pro Ser Asp Pro Leu Glu Leu Val 405 410 415 Val Ser Ala Ser Leu Gly Gln His Pro Gln Asp Tyr Thr Val Glu Asn 420 425 430 Leu Ile Arg Met Gly Val Ala Gly Leu Val Leu Val Val Leu Gly Ile 435 440 445 Leu Leu Phe Glu Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala Ala 450 455 460 Gly Arg Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 <210> 26 <211> 1953 <212> DNA <213> Human <220> <221> CDS <222> (1)..(1953) <400> 26 atg acc ccc atc ctc acg gtc ctg atc tgt ctc ggg ctg agt ctg ggc 48 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 ccc cgg acc cac gtg cag gca ggg cac ctc ccc aag ccc acc ctc tgg 96 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 gct gaa cca ggc tct gtg atc acc cag ggg agt cct gtg acc ctc agg 144 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 tgt cag ggg ggc cag gag acc cag gag tac cgt cta tat aga gaa aag 192 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 aaa aca gca ccc tgg att aca cgg atc cca cag gag ctt gtg aag aag 240 Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 70 75 80 ggc cag ttc ccc atc cca tcc atc acc tgg gaa cat gca ggg cgg tat 288 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 cgc tgt tac tat ggt agc gac act gca ggc cgc tca gag agc agt gac 336 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 ccc ctg gag ctg gtg gtg aca gga gcc tac atc aaa ccc acc ctc tca 384 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 gcc cag ccc agc ccc gtg gtg aac tca gga ggg aat gta acc ctc cag 432 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Thr Leu Gln 130 135 140 tgt gac tca cag gtg gca ttt gat ggc ttc att ctg tgt aag gaa gga 480 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly 145 150 155 160 gaa gat gaa cac cca caa tgc ctg aac tcc cag ccc cat gcc cgt ggg 528 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175 tcg tcc cgc gcc atc ttc tcc gtg ggc ccc gtg agc ccg agt cgc agg 576 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 tgg tgg tac agg tgc tat gct tat gac tcg aac tct ccc tat gag tgg 624 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 tct cta ccc agt gat ctc ctg gag ctc ctg gtc cta ggt gtt tct aag 672 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 aag cca tca ctc tca gtg cag cca ggt cct atc gtg gcc cct gag gag 720 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 acc ctg act ctg cag tgt ggc tct gat gct ggc tac aac aga ttt gtt 768 Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 ctg tat aag gac ggg gaa cgt gac ttc ctt cag ctc gct ggc gca cag 816 Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270 ccc cag gct ggg ctc tcc cag gcc aac ttc acc ctg ggc cct gtg agc 864 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 cgc tcc tac ggg ggc cag tac aga tgc tac ggt gca cac aac ctc tcc 912 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 tcc gag tgg tcg gcc ccc agc gac ccc ctg gac atc ctg atc gca gga 960 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 cag ttc tat gac aga gtc tcc ctc tcg gtg cag ccg ggc ccc acg gtg 1008 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 gcc tca gga gag aac gtg acc ctg ctg tgt cag tca cag gga tgg atg 1056 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 caa act ttc ctt ctg acc aag gag ggg gca gct gat gac cca tgg cgt 1104 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365 cta aga tca acg tac caa tct caa aaa tac cag gct gaa ttc ccc atg 1152 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 ggt cct gtg acc tca gcc cat gcg ggg acc tac agg tgc tac ggc tca 1200 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 cag agc tcc aaa ccc tac ctg ctg act cac ccc agt gac ccc ctg gag 1248 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 ctc gtg gtc tca gga ccg tct ggg ggc ccc agc tcc ccg aca aca ggc 1296 Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430 ccc acc tcc aca tct ggc cct gag gac cag ccc ctc acc ccc acc ggg 1344 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445 tcg gat ccc cag agt ggt ctg gga agg cac ctg ggg gtt gtg atc ggc 1392 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460 atc ttg gtg gcc gtc atc cta ctg ctc ctc ctc ctc ctc ctc ctc ttc 1440 Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 ctc atc ctc cga cat cga cgt cag ggc aaa cac tgg aca tcg acc cag 1488 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 aga aag gct gat ttc caa cat cct gca ggg gct gtg ggg cca gag ccc 1536 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 aca gac aga ggc ctg cag tgg agg tcc agc cca gct gcc gat gcc cag 1584 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 gaa gaa aac ctc tat gct gcc gtg aag cac aca cag cct gag gat ggg 1632 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540 gtg gag atg gac act cgg agc cca cac gat gaa gac ccc cag gca gtg 1680 Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val 545 550 555 560 acg tat gcc gag gtg aaa cac tcc aga cct agg aga gaa atg gcc tct 1728 Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565 570 575 cct cct tcc cca ctg tct ggg gaa ttc ctg gac aca aag gac aga cag 1776 Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590 gcg gaa gag gac agg cag atg gac act gag gct gct gca tct gaa gcc 1824 Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605 ccc cag gat gtg acc tac gcc cag ctg cac agc ttg acc ctt aga cgg 1872 Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620 aag gca act gag cct cct cca tcc cag gaa ggg ccc tct cca gct gtg 1920 Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val 625 630 635 640 ccc agc atc tac gcc act ctg gcc atc cac tag 1953 Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 645 650 <210> 27 <211> 650 <212> PRT <213> Human <400> 27 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Thr Leu Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met 340 345 350 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg 355 360 365 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540 Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val 545 550 555 560 Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565 570 575 Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln 580 585 590 Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala 595 600 605 Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg 610 615 620 Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val 625 630 635 640 Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 645 650 <210> 28 <211> 1347 <212> DNA <213> Human <220> <221> CDS <222> (1)..(1347) <400> 28 atg atc ccc acc ttc acg gct ctg ctc tgc ctc ggg ctg agt ctg ggc 48 Met Ile Pro Thr Phe Thr Ala Leu Leu Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 ccc agg acc gac atg cag gca ggg ccc ctc ccc aaa ccc acc ctc tgg 96 Pro Arg Thr Asp Met Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp 20 25 30 gct gag cca ggc tct gtg atc agc tgg ggg aac tct gtg acc atc tgg 144 Ala Glu Pro Gly Ser Val Ile Ser Trp Gly Asn Ser Val Thr Ile Trp 35 40 45 tgt cag ggg acc ctg gag gct cgg gag tac cgt ctg gat aaa gag gaa 192 Cys Gln Gly Thr Leu Glu Ala Arg Glu Tyr Arg Leu Asp Lys Glu Glu 50 55 60 agc cca gca ccc tgg gac aga cag aac cca ctg gag ccc aag aac aag 240 Ser Pro Ala Pro Trp Asp Arg Gln Asn Pro Leu Glu Pro Lys Asn Lys 65 70 75 80 gcc aga ttc tcc atc cca tcc atg aca gag gac tat gca ggg aga tac 288 Ala Arg Phe Ser Ile Pro Ser Met Thr Glu Asp Tyr Ala Gly Arg Tyr 85 90 95 cgc tgt tac tat cgc agc cct gta ggc tgg tca cag ccc agt gac ccc 336 Arg Cys Tyr Tyr Arg Ser Pro Val Gly Trp Ser Gln Pro Ser Asp Pro 100 105 110 ctg gag ctg gtg atg aca gga gcc tac agt aaa ccc acc ctt tca gcc 384 Leu Glu Leu Val Met Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala 115 120 125 ctg ccg agt cct ctt gtg acc tca gga aag agc gtg acc ctg ctg tgt 432 Leu Pro Ser Pro Leu Val Thr Ser Gly Lys Ser Val Thr Leu Leu Cys 130 135 140 CAG TCA CGG AGC CCA ATG GAC ACT TTC CTT CTG ATC AAG GAG CGG GCA 480 Gln Ser Arg Ser Pro Met Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala 145 150 155 160 GCC CAT CCC CTA CTG CAT CTG AGA TCA GAG CAC GGA GCT CAG CAG CAC 528 Ala His Pro Leu Leu His Leu Arg Ser Glu His Gly Ala Gln Gln His 165 170 175 CAG GCT GAA TTC CCC ATG AGT CCT GTG ACC TCA GTG CAC GGG GGG ACC 576 Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Val His Gly Gly Thr 180 185 190 TAC AGG TGC TTC AGC TCA CAC GGC TTC TCC CAC TAC CTG CTG TCA CAC 624 Tyr Arg Cys Phe Ser Ser His Gly Phe Ser His Tyr Leu Leu Ser His 195 200 205 CCC AGT GAC CCC CTG GAG CTC ATA GTC TCA GGA TCC TTG GAG GGT CCC 672 Pro Ser Asp Pro Leu Glu Leu Ile Val Ser Gly Ser Leu Glu Gly Pro 210 215 220 AGG CCC TCA CCC ACA AGG TCC GTC TCA ACA GCT GCA GGC CCT GAG GAC 720 Arg Pro Ser Pro Thr Arg Ser Val Ser Thr Ala Ala Gly Pro Glu Asp 225 230 235 240 cag ccc ctc atg cct aca ggg tca gtc ccc cac agt ggt ctg aga agg 768 Gln Pro Leu Met Pro Thr Gly Ser Val Pro His Ser Gly Leu Arg Arg 245 250 255 cac tgg gag gta ctg atc ggg gtc ttg gtg gtc tcc atc ctg ctt ctc 816 His Trp Glu Val Leu Ile Gly Val Leu Val Val Ser Ile Leu Leu Leu 260 265 270 tcc ctc ctc ctc ttc ctc ctc ctc caa cac tgg cgt cag gga aaa cac 864 Ser Leu Leu Leu Phe Leu Leu Leu Gln His Trp Arg Gln Gly Lys His 275 280 285 agg aca ttg gcc cag aga cag gct gat ttc caa cgt cct cca ggg gct 912 Arg Thr Leu Ala Gln Arg Gln Ala Asp Phe Gln Arg Pro Pro Gly Ala 290 295 300 gcc gag cca gag ccc aag gac ggg ggc cta cag agg agg tcc agc cca 960 Ala Glu Pro Glu Pro Lys Asp Gly Gly Leu Gln Arg Arg Ser Ser Pro 305 310 315 320 gct gct gac gtc cag gga gaa aac ttc tgt gct gcc gtg aag aac aca 1008 Ala Ala Asp Val Gln Gly Glu Asn Phe Cys Ala Ala Val Lys Asn Thr 325 330 335 cag cct gag gac ggg gtg gaa atg gac act cgg cag agc cca cac gat 1056 Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Gln Ser Pro His Asp 340 345 350 gaa gac ccc cag gca gtg acg tat gcc aag gtg aaa cac tcc aga cct 1104 Glu Asp Pro Gln Ala Val Thr Tyr Ala Lys Val Lys His Ser Arg Pro 355 360 365 agg aga gaa atg gcc tct cct ccc tcc cca ctg tct ggg gaa ttc ctg 1152 Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu 370 375 380 gac aca aag gac aga cag gca gaa gag gac aga cag atg gac act gag 1200 Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu 385 390 395 400 gct gct gca tct gaa gcc ccc cag gat gtg acc tac gcc cgg ctg cac 1248 Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Arg Leu His 405 410 415 agc ttt acc ctc aga cag aag gca act gag cct cct cca tcc cag gaa 1296 Ser Phe Thr Leu Arg Gln Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu 420 425 430 ggg gcc tct cca gct gag ccc agt gtc tat gcc act ctg gcc atc cac 1344 Gly Ala Ser Pro Ala Glu Pro Ser Val Tyr Ala Thr Leu Ala Ile His 435 440 445 taa 1347 <210> 29 <211> 448 <212> PRT <213> Human <400> 29 Met Ile Pro Thr Phe Thr Ala Leu Leu Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr Asp Met Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Ser Trp Gly Asn Ser Val Thr Ile Trp 35 40 45 Cys Gln Gly Thr Leu Glu Ala Arg Glu Tyr Arg Leu Asp Lys Glu Glu 50 55 60 Ser Pro Ala Pro Trp Asp Arg Gln Asn Pro Leu Glu Pro Lys Asn Lys 65 70 75 80 Ala Arg Phe Ser Ile Pro Ser Met Thr Glu Asp Tyr Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Arg Ser Pro Val Gly Trp Ser Gln Pro Ser Asp Pro 100 105 110 Leu Glu Leu Val Met Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala 115 120 125 Leu Pro Ser Pro Leu Val Thr Ser Gly Lys Ser Val Thr Leu Leu Cys 130 135 140 Gln Ser Arg Ser Pro Met Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala 145 150 155 160 Ala His Pro Leu Leu His Leu Arg Ser Glu His Gly Ala Gln Gln His 165 170 175 Gln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Val His Gly Gly Thr 180 185 190 Tyr Arg Cys Phe Ser Ser His Gly Phe Ser His Tyr Leu Leu Ser His 195 200 205 Pro Ser Asp Pro Leu Glu Leu Ile Val Ser Gly Ser Leu Glu Gly Pro 210 215 220 Arg Pro Ser Pro Thr Arg Ser Val Ser Thr Ala Ala Gly Pro Glu Asp 225 230 235 240 Gln Pro Leu Met Pro Thr Gly Ser Val Pro His Ser Gly Leu Arg Arg 245 250 255 His Trp Glu Val Leu Ile Gly Val Leu Val Val Ser Ile Leu Leu Leu 260 265 270 Ser Leu Leu Leu Phe Leu Leu Leu Gln His Trp Arg Gln Gly Lys His 275 280 285 Arg Thr Leu Ala Gln Arg Gln Ala Asp Phe Gln Arg Pro Pro Gly Ala 290 295 300 Ala Glu Pro Glu Pro Lys Asp Gly Gly Leu Gln Arg Arg Ser Ser Pro 305 310 315 320 Ala Ala Asp Val Gln Gly Glu Asn Phe Cys Ala Ala Val Lys Asn Thr 325 330 335 Gln Pro Glu Asp Gly Val Glu Met Asp Thr Arg Gln Ser Pro His Asp 340 345 350 Glu Asp Pro Gln Ala Val Thr Tyr Ala Lys Val Lys His Ser Arg Pro 355 360 365 Arg Arg Glu Met Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu 370 375 380 Asp Thr Lys Asp Arg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu 385 390 395 400 Ala Ala Ala Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Arg Leu His 405 410 415 Ser Phe Thr Leu Arg Gln Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu 420 425 430 Gly Ala Ser Pro Ala Glu Pro Ser Val Tyr Ala Thr Leu Ala Ile His 435 440 445 <210> 30 <211> 24 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 30 ccatagttcc attttacagt tacc 24 <210> 31 <211> 20 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 31 gggaccaagg gatagacaga 20 <210> 32 <211> 24 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 32 tccagagttc caggtcaagg tcac 24 <210> 33 <211> 20 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 33 gccagtggat agaccgatgg 20 <210> 34 <211> 36 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <220> <221> modified_base <222> (24)..(25) <223> I <220> <221> modified_base <222> (29)..(30) <223> I <220> <221> modified_base <222> (34)..(35) <223> I <400> 34 ggccacgcgt cgactagtac gggnngggnn gggnng 36 <210> 35 <211> 20 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 35 ggccacgcgt cgactagtac 20 <210> 36 <211> 24 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 36 ttcactgcca tcaatcttcc actt 24 <210> 37 <211> 21 <212> DNA <213> Artificial <220> <223> Artificially synthesized primer sequence <400> 37 gatggataca gttggtgcag c 21 <210> 38 <211> 408 <212> DNA <213> Mouse <220> <221> CDS <222> (1)..(408) <220> <221> sig_peptide <222> (1)..(54) <220> <221> mat_peptide <222> (55)..(408) <400> 38 atg aga gtg ctg att ctt ttg tgg ctg ttc aca gcc ttt cct ggt atc 48 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile -15 -10 -5 ctg tct gat gtg cag ctt cag gag tcg gga cct ggc ctg gtg aaa cct 96 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro -1 1 5 10 tct cag tct ctg tcc ctc acc tgc act gtc act ggc tac tca atc acc 144 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 15 20 25 30 agt gat tat gcc tgg aac tgg atc cgg cag ttt cca gga aac aaa ctg 192 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 gag tgg atg ggc tac ata agc tac agt ggt agc act agc tac aac cca 240 Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro 50 55 60 tct ctc aaa agt cga atc tct atc act cga gac aca tcc aag aac cag 288 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 ttc ttc ctg cag ttg aat tct gtg act act gag gac aca gcc aca tat 336 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 80 85 90 tac tgt gca aga tct ccc cct tac tat gct atg gac tac tgg ggt caa 384 Tyr Cys Ala Arg Ser Pro Pro Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 110 gga acc tca gtc acc gtc tcc tca 408 Gly Thr Ser Val Thr Val Ser Ser 115 <210> 39 <211> 136 <212> PRT <213> Mouse <400> 39 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile -15 -10 -5 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro -1 1 5 10 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 15 20 25 30 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro 50 55 60 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 80 85 90 Tyr Cys Ala Arg Ser Pro Pro Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 <210> 40 <211> 381 <212> DNA <213> Mouse <220> <221> CDS <222> (1)..(381) <220> <221> sig_peptide <222> (1)..(60) <220> <221> mat_peptide <222> (61)..(381) <400> 40 atg gag aca cat tct cag gtc ttt gta tac atg ttg ctg tgg ttg tct 48 Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser -20 -15 -10 -5 ggt gtt gaa gga gac att gtg atg acc cag tct cac aaa ttc atg tcc 96 Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser -1 1 5 10 aca tca gta gga gac agg gtc agc atc acc tgc aag gcc agt cag gat 144 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 gtg ggt act gct gta gcc tgg tat caa cag aaa cca ggg caa tct cct 192 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 30 35 40 aaa cta ctg att tac tgg gca tcc acc cgg cac act gga gtc cct gat 240 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 45 50 55 60 cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctc acc att agc 288 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 aat gtg cag tct gaa gac ttg gca gat tat ttc tgt cag caa tat agc 336 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser 80 85 90 agc tat cct ctc acg ttc ggt gct ggg acc aag ctg gag ctg aaa 381 Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 95 100 105 <210> 41 <211> 127 <212> PRT <213> Mouse <400> 41 Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser -20 -15 -10 -5 Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser -1 1 5 10 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 30 35 40 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 45 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser 80 85 90 Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 95 100 105 <210> 42 <211> 414 <212> DNA <213> Mouse <220> <221> CDS <222> (1)..(414) <220> <221> sig_peptide <222> (1)..(57) <220> <221> mat_peptide <222> (58)..(414) <400> 42 atg gga tgg agc tgg gtc ttt ctc ttc ctc ctg tca gga act gca ggt 48 Met Gly Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly -15 -10 -5 gtc cac tgc cag gtc cag ctg aag cag tct gga gct gag ctg gtg agg 96 Val His Cys Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg -1 1 5 10 cct ggg gct tca gtg aag ctg tcc tgc aag act tct gga tac atc ttc 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe 15 20 25 acc agc tac tgg att cac tgg gta aaa cag agg tct gga cag ggc ctt 192 Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu 30 35 40 45 gag tgg att gca agg att tat cct gga act ggt agt act tac tac aat 240 Glu Trp Ile Ala Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn 50 55 60 gag aag ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tcc agc 288 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 65 70 75 act gcc tac atg cag ctc agc agc ctg aaa tct gag gac tct gct gtc 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Lys Ser Glu Asp Ser Ala Val 80 85 90 tat ttc tgt gca aga tac cct acc tac gac tgg tac ttc gat gtc tgg 384 Tyr Phe Cys Ala Arg Tyr Pro Thr Tyr Asp Trp Tyr Phe Asp Val Trp 95 100 105 ggc gca ggg acc acg gtc acc gtc tcc tca 414 Gly Ala Gly Thr Thr Val Thr Val Ser Ser 110 115 <210> 43 <211> 138 <212> PRT <213> Mouse <400> 43 Met Gly Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly -15 -10 -5 Val His Cys Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg -1 1 5 10 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe 15 20 25 Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu 30 35 40 45 Glu Trp Ile Ala Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn 50 55 60 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 65 70 75 Thr Ala Tyr Met Gln Leu Ser Ser Leu Lys Ser Glu Asp Ser Ala Val 80 85 90 Tyr Phe Cys Ala Arg Tyr Pro Thr Tyr Asp Trp Tyr Phe Asp Val Trp 95 100 105 Gly Ala Gly Thr Thr Val Thr Val Ser Ser 110 115 <210> 44 <211> 381 <212> DNA <213> Mouse <220> <221> CDS <222> (1)..(381) <220> <221> sig_peptide <222> (1)..(60) <220> <221> mat_peptide <222> (61)..(381) <400> 44 atg gtt ttc aca cct cag att ctt gga ctt atg ctt ttc tgg att tca 48 Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser -20 -15 -10 -5 gcc tcc aga ggt gat att gtg cta act cag tct cca gcc acc ctg tct 96 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser -1 1 5 10 gtg act cca gga gat aga gtc agt ctt tcc tgc agg gcc agt caa agt 144 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 15 20 25 att agc aac tac cta cac tgg tat caa caa aaa tca cat gag tct cca 192 Ile Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 30 35 40 agg ctt ctc atc aag tat gct tcc cag tcc atc tct ggg atc ccc tcc 240 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 45 50 55 60 agg ttc agt ggc agt gga tca ggg aca gat ttc act ctc agt atc aac 288 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 65 70 75 agt gtg gag act gaa gat ttt gga atg tat ttc tgt caa cag agt aac 336 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 80 85 90 agc tgg ccg ctc acg ttc ggt gct ggg acc aag ctg gag ctg aaa 381 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 95 100 105 <210> 45 <211> 127 <212> PRT <213> Mouse <400> 45 Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser -20 -15 -10 -5 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser -1 1 5 10 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 15 20 25 Ile Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 30 35 40 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 65 70 75 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 80 85 90 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 95 100 105 <210> 46 <211> 408 <212> DNA <213> mouse <220> <221> CDS <222> (1)..(408) <220> <221> sig_peptide <222> (1)..(54) <220> <221> mat_peptide <222> (55)..(408) <400> 46 atg aga gtg ctg att ctt ttg tgg ctg ttc aca gcc ttt cct ggt atc 48 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile -15 -10 -5 ctg tct gat gtg cag ctt cag gag tcg gga cct ggc ctg gtg aaa cct 96 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro -1 1 5 10 tct cag tct ctg tcc ctc acc tgc act gtc act ggc tac tca atc acc 144 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 15 20 25 30 agt gat tat gcc tgg aac tgg atc cgg cag ttt cca gga aac aaa ctg 192 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 gag tgg atg ggc tac ata agc tac agt ggt agc act agc tac aac cca 240 Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro 50 55 60 tct ctc aaa agt cga atc tct atc act cga gac aca tcc aag aac cag 288 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 ttc ttc ctg cag ttg aat tct gtg act act gag gac aca gcc aca tat 336 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 80 85 90 tac tgt gca aga gcc ctc cca tta ccc tgg ttt gct tac tgg ggc caa 384 Tyr Cys Ala Arg Ala Leu Pro Leu Pro Trp Phe Ala Tyr Trp Gly Gln 95 100 105 110 ggg act ctg gtc act gtc tct gca 408 Gly Thr Leu Val Thr Val Ser Ala 115 <210> 47 <211> 136 <212> PRT <213> Mouse <400> 47 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile -15 -10 -5 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro -1 1 5 10 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 15 20 25 30 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro 50 55 60 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 80 85 90 Tyr Cys Ala Arg Ala Leu Pro Leu Pro Trp Phe Ala Tyr Trp Gly Gln 95 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 <210> 48 <211> 381 <212> DNA <213> Mouse <220> <221> CDS <222> (1)..(381) <220> <221> sig_peptide <222> (1)..(60) <220> <221> mat_peptide <222> (61)..(381) <400> 48 atg gag aca cat tct cag gtc ttt gta tac atg ttg ctg tgg ttg tct 48 Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser -20 -15 -10 -5 ggt gtt gaa gga gac att gtg atg acc cag tct cac aaa ttc atg tcc 96 Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser -1 1 5 10 aca tca gta gga gac agg gtc agc atc acc tgc aag gcc agt cag gat 144 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 gtg ggt act gct gta gcc tgg tat caa cag aaa cca ggg caa tct cct 192 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 30 35 40 aaa cta ctg att tac tgg gca tcc acc cgg cac act gga gtc cct gat 240 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 45 50 55 60 cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctc acc att agc 288 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 aat gtg cag tct gaa gac ttg gca gat tat ttc tgt cag caa tat agc 336 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser 80 85 90 agc tat cct tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa 381 Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 95 100 105 <210> 49 <211> 127 <212> PRT <213> Mouse <400> 49 Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser -20 -15 -10 -5 Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser -1 1 5 10 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 30 35 40 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 45 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser 80 85 90 Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 95 100 105 <210> 50 <211> 1401 <212> DNA <213> Artificial <220> <223> Artificially synthesized nucleotide sequence <220> <221> CDS <222> (1)..(1398) <220> <221> sig_peptide <222> (1)..(54) <220> <221> mat_peptide <222> (55)..(1398) <400> 50 atg aga gtg ctg att ctt ttg tgg ctg ttc aca gcc ttt cct ggt atc 48 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile -15 -10 -5 ctg tct gat gtg cag ctt cag gag tcg gga cct ggc ctg gtg aaa cct 96 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro -1 1 5 10 tct cag tct ctg tcc ctc acc tgc act gtc act ggc tac tca atc acc 144 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 15 20 25 30 agt gat tat gcc tgg aac tgg atc cgg cag ttt cca gga aac aaa ctg 192 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 gag tgg atg ggc tac ata agc tac agt ggt agc act agc tac aac cca 240 Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro 50 55 60 tct ctc aaa agt cga atc tct atc act cga gac aca tcc aag aac cag 288 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 ttc ttc ctg cag ttg aat tct gtg act act gag gac aca gcc aca tat 336 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 80 85 90 tac tgt gca aga tct ccc cct tac tat gct atg gac tac tgg ggt caa 384 Tyr Cys Ala Arg Ser Pro Pro Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 110 gga acc tca gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc 432 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc 480 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg 528 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc 576 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 160 165 170 cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc 624 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 175 180 185 190 tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag 672 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt gac 720 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga 768 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc 816 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 240 245 250 tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa 864 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 255 260 265 270 gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat 912 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac cgt 960 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag 1008 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag 1056 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 320 325 330 aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac 1104 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 335 340 345 350 acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg 1152 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg 1200 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg 1248 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac 1296 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 400 405 410 aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat 1344 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 415 420 425 430 gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg 1392 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 ggt aaa tga 1401 Gly Lys <210> 51 <211> 466 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 51 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile -15 -10 -5 Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro -1 1 5 10 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 15 20 25 30 Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 Glu Trp Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro 50 55 60 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 80 85 90 Tyr Cys Ala Arg Ser Pro Pro Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 95 100 105 110 Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 160 165 170 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 175 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 240 245 250 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 255 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 320 325 330 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 335 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 400 405 410 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 415 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys <210> 52 <211> 705 <212> DNA <213> Artificial <220> <223> Artificially synthesized nucleotide sequence <220> <221> CDS <222> (1)..(702) <220> <221> sig_peptide <222> (1)..(60) <220> <221> mat_peptide <222> (61)..(702) <400> 52 atg gag aca cat tct cag gtc ttt gta tac atg ttg ctg tgg ttg tct 48 Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser -20 -15 -10 -5 ggt gtt gaa gga gac att gtg atg acc cag tct cac aaa ttc atg tcc 96 Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser -1 1 5 10 aca tca gta gga gac agg gtc agc atc acc tgc aag gcc agt cag gat 144 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 gtg ggt act gct gta gcc tgg tat caa cag aaa cca ggg caa tct cct 192 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 30 35 40 aaa cta ctg att tac tgg gca tcc acc cgg cac act gga gtc cct gat 240 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 45 50 55 60 cgc ttc aca ggc agt gga tct ggg aca gat ttc act ctc acc att agc 288 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 aat gtg cag tct gaa gac ttg gca gat tat ttc tgt cag caa tat agc 336 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser 80 85 90 agc tat cct ctc acg ttc ggt gct ggg acc aag ctg gag ctg aaa cga 384 Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 95 100 105 act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag 432 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 110 115 120 ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat 480 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 125 130 135 140 ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg 528 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc acc 576 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 160 165 170 tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa 624 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 175 180 185 cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc 672 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 190 195 200 gtc aca aag agc ttc aac agg gga gag tgc tag 705 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 <210> 53 <211> 234 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 53 Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu Leu Trp Leu Ser -20 -15 -10 -5 Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser -1 1 5 10 Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp 15 20 25 Val Gly Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 30 35 40 Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp 45 50 55 60 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser 80 85 90 Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 95 100 105 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 110 115 120 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 125 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 160 165 170 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 175 180 185 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 190 195 200 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 <210> 54 <211> 1407 <212> DNA <213> Artificial <220> <223> Artificially synthesized nucleotide sequence <220> <221> CDS <222> (1)..(1404) <220> <221> sig_peptide <222> (1)..(57) <220> <221> mat_peptide <222> (55)..(1404) <400> 54 atg gga tgg agc tgg gtc ttt ctc ttc ctc ctg tca gga act gca ggt 48 Met Gly Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly -15 -10 -5 gtc cac tgc cag gtc cag ctg aag cag tct gga gct gag ctg gtg agg 96 Val His Cys Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg -1 1 5 10 cct ggg gct tca gtg aag ctg tcc tgc aag act tct gga tac atc ttc 144 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe 15 20 25 30 acc agc tac tgg att cac tgg gta aaa cag agg tct gga cag ggc ctt 192 Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu 35 40 45 gag tgg att gca agg att tat cct gga act ggt agt act tac tac aat 240 Glu Trp Ile Ala Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn 50 55 60 gag aag ttc aag ggc aag gcc aca ctg act gca gac aaa tcc tcc agc 288 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 65 70 75 act gcc tac atg cag ctc agc agc ctg aaa tct gag gac tct gct gtc 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Lys Ser Glu Asp Ser Ala Val 80 85 90 tat ttc tgt gca aga tac cct acc tac gac tgg tac ttc gat gtc tgg 384 Tyr Phe Cys Ala Arg Tyr Pro Thr Tyr Asp Trp Tyr Phe Asp Val Trp 95 100 105 110 ggc gca ggg acc acg gtc acc gtc tcc tca gcc tcc acc aag ggc cca 432 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca 480 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg 528 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg 576 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 160 165 170 gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 624 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 175 180 185 190 gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat 672 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 cac aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct 720 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa ctc ctg 768 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 816 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 240 245 250 atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 864 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 255 260 265 270 cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 912 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 960 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 1008 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 1056 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 320 325 330 atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 1104 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 335 340 345 350 gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 1152 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 1200 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 1248 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 1296 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 400 405 410 gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 1344 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 415 420 425 430 atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 1392 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 tct ccg ggt aaa tga 1407 Ser Pro Gly Lys 450 <210> 55 <211> 468 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 55 Met Gly Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly -15 -10 -5 Val His Cys Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg -1 1 5 10 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe 15 20 25 Thr Ser Tyr Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu 30 35 40 45 Glu Trp Ile Ala Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn 50 55 60 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 65 70 75 Thr Ala Tyr Met Gln Leu Ser Ser Leu Lys Ser Glu Asp Ser Ala Val 80 85 90 Tyr Phe Cys Ala Arg Tyr Pro Thr Tyr Asp Trp Tyr Phe Asp Val Trp 95 100 105 Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 110 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 160 165 170 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 175 180 185 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 190 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 240 245 250 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 255 260 265 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 270 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 320 325 330 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 335 340 345 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 350 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 400 405 410 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 415 420 425 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 430 435 440 445 Ser Pro Gly Lys <210> 56 <211> 705 <212> DNA <213> Artificial <220> <223> Artificially synthesized nucleotide sequence <220> <221> CDS <222> (1)..(702) <220> <221> sig_peptide <222> (1)..(60) <220> <221> mat_peptide <222> (61)..(702) <400> 56 atg gtt ttc aca cct cag att ctt gga ctt atg ctt ttc tgg att tca 48 Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser -20 -15 -10 -5 gcc tcc aga ggt gat att gtg cta act cag tct cca gcc acc ctg tct 96 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser -1 1 5 10 gtg act cca gga gat aga gtc agt ctt tcc tgc agg gcc agt caa agt 144 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 15 20 25 att agc aac tac cta cac tgg tat caa caa aaa tca cat gag tct cca 192 Ile Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 30 35 40 agg ctt ctc atc aag tat gct tcc cag tcc atc tct ggg atc ccc tcc 240 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 45 50 55 60 agg ttc agt ggc agt gga tca ggg aca gat ttc act ctc agt atc aac 288 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 65 70 75 agt gtg gag act gaa gat ttt gga atg tat ttc tgt caa cag agt aac 336 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 80 85 90 agc tgg ccg ctc acg ttc ggt gct ggg acc aag ctg gag ctg aaa cga 384 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 95 100 105 act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag 432 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 110 115 120 ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat 480 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 125 130 135 140 ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg 528 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc acc 576 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 160 165 170 tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa 624 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 175 180 185 cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc 672 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 190 195 200 gtc aca aag agc ttc aac agg gga gag tgc tag 705 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 <210> 57 <211> 234 <212> PRT <213> Artificial <220> <223> Synthetic construct <400> 57 Met Val Phe Thr Pro Gln Ile Leu Gly Leu Met Leu Phe Trp Ile Ser -20 -15 -10 -5 Ala Ser Arg Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser -1 1 5 10 Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser 15 20 25 Ile Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro 30 35 40 Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser 45 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn 65 70 75 Ser Val Glu Thr Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn 80 85 90 Ser Trp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 95 100 105 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 110 115 120 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 125 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 160 165 170 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 175 180 185 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 190 195 200 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 <210> 58 <211> 6 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(6) <223> CDR <400> 58 Ser Asp Tyr Ala Trp Asn 1 5 <210> 59 <211> 17 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(17) <223> CDR <400> 59 Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 Arg <210> 60 <211> 9 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR <400> 60 Ser Pro Pro Tyr Tyr Ala Met Asp Tyr 1 5 <210> 61 <211> 11 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(11) <223> CDR <400> 61 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 62 <211> 7 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR <400> 62 Trp Ala Ser Thr Arg His Thr 1 5 <210> 63 <211> 9 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR <400> 63 Gln Gln Tyr Ser Ser Tyr Pro Leu Thr 1 5 <210> 64 <211> 5 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(5) <223> CDR <400> 64 Ser Tyr Trp Ile His 1 5 <210> 65 <211> 17 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(17) <223> CDR <400> 65 Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 66 <211> 10 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(10) <223> CDR <400> 66 Tyr Pro Thr Tyr Asp Trp Tyr Phe Asp Val 1 5 10 <210> 67 <211> 11 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(11) <223> CDR <400> 67 Arg Ala Ser Gln Ser Ile Ser Asn Tyr Leu His 1 5 10 <210> 68 <211> 7 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR <400> 68 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 69 <211> 9 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR <400> 69 Gln Gln Ser Asn Ser Trp Pro Leu Thr 1 5 <210> 70 <211> 6 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(6) <223> CDR <400> 70 Ser Asp Tyr Ala Trp Asn 1 5 <210> 71 <211> 17 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(17) <223> CDR <400> 71 Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 Arg <210> 72 <211> 9 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR <400> 72 Ala Leu Pro Leu Pro Trp Phe Ala Tyr 1 5 <210> 73 <211> 11 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(11) <223> CDR <400> 73 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 74 <211> 7 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR <400> 74 Trp Ala Ser Thr Arg His Thr 1 5 <210> 75 <211> 9 <212> PRT <213> Mouse <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR <400> 75 Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr 1 5 <210> 76 <211> 4 <212> PRT <213> Artificial <220> <223> Artificially synthesized peptide sequence <220> <221> misc_feature <222> (2)..(3) <223> Xaa can be any natural amino acid <400> 76 Tyr Xaa Xaa Leu 1 Brief Description of the Drawings

[0108] Figure 1a It is a photograph for detecting the mRNA expression of the ILT7 gene by the RT-PCR method. This figure is the analysis result of the mRNA expression of the ILT7 gene in human immune cells.

[0109] Figure 1b It is a chart for detecting and comparing the mRNA expression of the ILT7 gene in various human tissues and cells by the quantitative PCR method. The horizontal axis shows the detected tissues and cells, and the vertical axis shows the expression level of ILT7, where the expression level of ILT7 is normalized according to the expression level of GAPDH.

[0110] Figure 2 It is a chart showing the protein structure of ILT7. Among them Figure 2 (a) shows the amino acid sequence of the ILT7 protein, and further shows the putative secretion signal sequence and transmembrane region in the figure; Figure 2 (b) shows a schematic diagram of the ILT7 protein encoded by the constructed expression vector.

[0111] Figure 3 It is a picture showing the result of introducing the ILT7 expression vector and the FcRγ expression vector into cells and detecting the expression of the ILT7 molecule on the cell surface by FCM. The horizontal axis represents the fluorescence intensity detected by the anti-FLAG antibody, that is, the expression intensity of the ILT7 molecule with the FLAG tag on the cell surface, while the vertical axis represents the number of cells.

[0112] Figure 4is a photograph showing molecular binding in cells transfected with an ILT7 expression vector and an FcRγ expression vector, analyzed by immunoprecipitation and Western blotting. The left panel shows pictures of the results of blotting the ILT7 molecule with an anti-FLAG antibody (upper picture) and of blotting the FcRγ molecule with an anti-myc antibody (lower picture) after immunoprecipitating the FcRγ molecule with an anti-myc antibody. Similarly, the right panel shows pictures of the results of blotting with an anti-FLAG antibody (upper picture) and an anti-myc antibody (lower picture) after immunoprecipitating the FcRγ molecule with an anti-FLAG antibody.

[0113] Figure 5 is a photograph showing detection of ILT7 molecule glycosylation by transfecting an ILT7 expression vector and an FcRγ expression vector into cells and treating with N-glycosidase. The left photograph shows the size of ILT7 without treatment with N-glycosidase, and the right photograph shows the size of ILT7 after treatment with N-glycosidase.

[0114] Figure 6a is a picture showing detection of the reactivity of the produced anti-ILT7 monoclonal antibody by FCM analysis. Figure 6a shows the results of binding of the anti-ILT-7 antibody to BDCA-2 positive IPCs, analyzed by double staining of human peripheral blood lymphocytes with an anti-ILT-7 antibody and an anti-BDCA-2 antibody. The vertical axis shows reactivity with the BDCA-2 antibody, and the horizontal axis shows reactivity of each of the various anti-ILT7 antibodies prepared.

[0115] Figure 6b is a picture showing detection of the reactivity of the prepared anti-ILT7 monoclonal antibody by FCM analysis. Figure 6b shows the results of detection of the binding ability of the anti-ILT-7 antibody to the ILT7 molecule, detected using 293T cells transfected with ILT7 and an FcRγ expression vector. The vertical axis shows reactivity with the anti-FLAG antibody, i.e., the expression intensity of the ILT7 molecule with a FLAG tag, and the horizontal axis shows reactivity of each of the anti-ILT7 antibodies.

[0116] Figure 7It is a picture that shows the reactivity of two clones in the prepared anti-ILT7 monoclonal antibody detected by FCM analysis towards human peripheral blood lymphocytes. The three pictures on the left show the results of #11, and the three pictures on the right show the results of #17. In the left graph, each axis labeled with ILT7 represents the reactivity of ILT7#11. Similarly, in the right graph, each axis labeled with ILT7 represents the reactivity of ILT7#17.

[0117] Figure 8 It is the detection result of the binding ability of the prepared anti-ILT7 monoclonal antibodies ILT7#11 and ILT7#17 to human lymphocytes and the comparison result of the corresponding binding ability with the anti-BDCA-2 antibody. The vertical axis shows the reactivity of the anti-CD123 antibody, and the horizontal axis shows the reactivity of each antibody. That is, each antibody binds to a part of CD123-positive cells. This figure shows the results obtained by analyzing the reactivity when lymphocytes are stimulated with CpG and IFNα.

[0118] Figure 9a It is a picture that shows the amino acid sequences of family molecules with high homology to the ILT7 molecule. It mainly shows the alignment of the amino acid sequences of each extracellular region; Figure 9b It is Figure 9a a continuation of the picture; Figure 9c It is

[0119] Figure 9b a continuation of the picture.

[0120] Figure 10 It is the detection result of the reactivity of the prepared anti-ILT7 monoclonal antibodies ILT7#11 and ILT7#17 against the ILT1 molecule, ILT2 molecule, and ILT3 molecule, which is detected using cells transfected with the expression vectors of these three molecules. The upper picture shows the reconfirmation of the reactivity results against cells co-expressing the ILT7 molecule with a FLAG tag and FcRγ. The lower picture shows the reactivity results against cells transfected with ILT1, ILT2, ILT3, and FcRγ (left picture: ILT7#11, right picture: ILT7#17). The horizontal axis shows the reactivity of each anti-ILT-7 antibody.

[0121] Figure 11 It is a picture that shows the effect of the prepared anti-ILT7 monoclonal antibodies ILT7#11 and ILT7#17 on the interferon production activity of human lymphocytes. In this picture, the horizontal axis shows the concentration of IFNα in the culture supernatant when human lymphocytes are stimulated with influenza virus, and the vertical axis shows the treated antibodies. The term "no infection" refers to the results of cells not stimulated with influenza virus.

[0122] Figure 12 It is a picture showing the CDC activities of the prepared anti-ILT7 monoclonal antibodies ILT7#37, ILT7#28 and ILT7#33. When using the anti-ILT7 monoclonal antibodies obtained from any hybridoma, when the antibody concentration is 0.1 μg / ml or higher, 80% or higher CDC activities are shown. In the examples of antibodies other than the anti-ILT7 monoclonal antibodies, no CDC activity against the target cells was observed.

[0123] Figure 13 It is a picture showing the internalization of the prepared anti-ILT7 monoclonal antibodies ILT7#17, ILT7#26, ILT7#37, ILT7#28 and ILT7#33 in the target cells. The fluorescence intensity of APC is an indicator of the amount of ILT7-anti-ILT-7 antibody immune complexes present on the cell surface before incubation, and this complex can be detected whether the ILT7-anti-ILT-7 antibody immune complexes are on the target cell surface or integrated into the cells after incubation. On the other hand, the fluorescence intensity of FITC is an indicator of the amount of ILT7-anti-ILT-7 antibody immune complexes remaining on the cell surface after incubation. That is to say, internalization reduces the fluorescence intensity of FITC.

[0124] Specific Embodiments of the Invention

[0125] It has been reported that human ILT7 (immunoglobulin-like transcript 7) is a molecule specifically expressed in plasmacytoid dendritic cells (Gene. 2004 Apr 28; 331: 159-64.; WO03 / 12061). Alternatively, it is known that human ILT7 can be used as a predictive indicator for predicting lymphoma (WO2005 / 24043). However, no method for preparing an antibody capable of recognizing human ILT7 has been found.

[0126] Human ILT7 consists of 499 amino acid residues shown in SEQ ID NO: 2, and this protein is a type I transmembrane protein having 4 immunoglobulin-like domains and a transmembrane region (445-466; positions 429 to 450 shown in SEQ ID NO: 2) in structure. Among the 444 amino acid residues including the N-terminus, 16 amino acid residues (positions -15 to -1 shown in SEQ ID NO: 2) constitute the signal sequence, and the extracellular domain is composed of amino acid residues from position 17 to position 444 (positions 1 to 428 shown in SEQ ID NO: 2). On the other hand, the C-terminal region is the intracellular region. Most of human ILT7 is the extracellular domain, and the intracellular domain is composed of 33 amino acid residues (from 467 to 499; positions 451 to 483 shown in SEQ ID NO: 2). No motif related to signaling has been predicted to appear in the intracellular domain. The full-length amino acid sequence of human ILT7 is shown in SEQ ID NO: 2, and the cDNA base sequence encoding this amino acid sequence is shown in SEQ ID NO: 1. Here, the coding region (72)..(1520) of the mature peptide shown in SEQ ID NO: 1 does not contain the stop and start codons. That is, the protein coding sequence containing the stop and start codons in SEQ ID NO: 1 is from 24 to 1523.

[0127] It is considered that ligand signals are transmitted to cells through the binding of human ILT7 to signal transduction molecules. For example, most of the γ chains of Fc receptors are present in cells. In addition, the intracellular domain contains an immunoreceptor tyrosine-based activation motif (ITAM) involved in signaling. ITAM is an amino acid sequence part that is common in adapter molecules associated with immunoreceptors such as Fc receptors. Motifs such as tyrosine phosphorylation targets like YxxL (SEQ ID NO: 76) are included in ITAM and the signal is transmitted through phosphorylation. Examples of signal transduction molecules known to contain ITAM in the intracellular domain include the γ chain of Fc receptors, CD3ζ, and DAP12 in addition to the γ chain of Fc receptors. No ligand capable of binding to human ILT7 has been found yet.

[0128] The inventors of the present invention confirmed the specific expression of ILT7 in human IPC through gene expression analysis. The inventors of the present invention believe that if an antibody capable of distinguishing human ILT7 from other immune molecules immunologically can be obtained, then this antibody can be used in research on IPC. However, there are many molecules with similar structures in the ILT family including ILT7. Molecules such as ILT1, ILT2, ILT3, ILT4, ILT5, ILT6 or LIR-8 contain highly homologous amino acid sequences, especially in their extracellular domains. Therefore, the inventors of the present invention believe that it is difficult to obtain an antibody capable of distinguishing these molecules by using a partial amino acid sequence constituting the extracellular domain as an immunogen. Thus, the inventors of the present invention used cells expressing human ILT7 as an immunogen to try to prepare an antibody against human ILT7.

[0129] However, the use of a conventional expression vector cannot result in the expression of the cDNA of human ILT7 in animal cells. It has been reported that the ILT1 molecule, which has a very similar structure to ILT7, is associated with the γ chain of the Fc receptor. That is, when cells expressing the γ chain of the Fc receptor such as RBL (rat basophilic leukemia) cells and P815 (mouse mastocytoma) cells are used as host cells, the expression of ILT1 on the cell surface can be observed. However, if ILT1 is forced to be expressed in cells such as 293 that originally do not express the γ chain of the Fc receptor, then the expression of this protein on the cell surface cannot be observed. On the other hand, research has shown that the expression of ILT1 on the cell surface can be confirmed when ILT1 is co-expressed with the γ chain of the Fc receptor (Nakajima H. et al., J. Immunology 162: 5-8, 1999). However, there is no information on the immunogen for preparing the ILT7 antibody.

[0130] For example, in this report, RBL cells transfected with the ILT1 gene were used as an immunogen to prepare the ILT1 antibody. The inventors of the present invention tried to prepare the ILT7 antibody using RBL cells transfected with the ILT7 gene in the same manner as above. However, even if ILT7 was forced to be expressed in RBL cells (P815), the expression of ILT7 on the cell surface was not observed, so these cells could not be used as an antigen.

[0131] The inventors of the present invention conducted dedicated research to obtain an antibody capable of recognizing human ILT7. Therefore, the inventors of the present invention discovered that a specified transformed cell can be used as an immunogen to prepare the required antibody, and completed the present invention. That is, the present invention relates to a monoclonal antibody capable of binding to the extracellular domain of human ILT7, and relates to a fragment containing its antigen-binding region.

[0132] In the present invention, human ILT7 is defined as a natural molecule expressed in human IPCs or a molecule immunologically equivalent to ILT7 expressed in human IPCs. In the present invention, the binding of an antibody to human ILT7 can be confirmed by, for example, the following methods.

[0133] - Confirmation based on reactivity with human cells:

[0134] According to the findings of the inventors of the present invention, specific expression of human ILT7 has been observed in human IPCs. Initially, human ILT7 was isolated as a gene expressed in plasmacytoid dendritic cells (Blood. 2002 100; 3295 - 3303, Gene. 2004 Apr 28; 331: 159 - 64.). In addition, it is also known that it can be used as a marker for plasmacytoid dendritic cells (WO03 / 12061). It is assumed that plasmacytoid dendritic cells and IPCs are essentially the same cell population or most of them are the same. Therefore, there is no contradiction between these reports and the findings of the inventors of the present invention.

[0135] Considering such an expression profile of human ILT7, first, an important property of an antibody capable of binding human ILT in the present invention is its binding activity to IPCs or at least a subset of plasmacytoid dendritic cells. Specific cell surface markers of each cell population can be used to determine whether a certain cell is an IPC or a plasmacytoid dendritic cell. For example, the binding of the antibody to be tested to the target cell can be confirmed by double staining using an antibody that can bind to the cell surface marker and the antibody whose binding activity needs to be detected. That is to say, the IPCs in the present invention include, for example, cells expressing BDCA2.

[0136] - Confirmation based on reactivity with transformed cells expressing the human ILT7 gene:

[0137] The inventors of the present invention found that when the expression of the human ILT7 gene is carried out under certain conditions, the immunological properties of ILT7 expressed in human IPCs can be reconstructed. Therefore, the reactivity against human ILT7 can also be confirmed based on the reactivity of the antibody against cells into which the gene encoding ILT7 has been artificially introduced. That is, the present invention relates to a monoclonal antibody or a fragment containing the antigen-binding region of the antibody, wherein the antibody contains an amino acid sequence having an extracellular domain and is capable of binding a molecule co-expressed with a signal transduction molecule. Here, the extracellular domain includes an amino acid sequence corresponding to positions 17 to 444 at the N-terminus (positions 1 to 428 in SEQ ID NO: 2) of the amino acid sequence shown in SEQ ID NO: 2.

[0138] For example, the immunological properties of ILT7 expressed in human IPC can be maintained in cells co-transfected with two vectors, namely an expression vector containing DNA encoding human ILT7 and an expression vector containing DNA encoding a signal transduction molecule. Therefore, in the present invention, transformed cells co-expressing human ILT7 and a signal transduction molecule are preferably used as cells for confirming the affinity of the antibody in the present invention for the extracellular domain of human ILT7. In the present invention, when using transformed cells to confirm the reactivity of an antibody, it is necessary to use non-transformed cells as a control. In addition, it is also important to use the same host cells expressing only the signal transduction molecule as a control to confirm that the binding ability of the antibody is not detected.

[0139] In the present invention, a molecule capable of inducing the surface expression of human ILT7 can be used as the co-expressed signal transduction molecule. The signal transduction molecule in the present invention can also be defined as: a molecule that can endow at least the extracellular domain of the ILT7 molecule with the immunological properties of natural human ILT7 in cells expressing ILT7. Here, the immunological properties of natural human ILT7 refer to the ability to be recognized by an antibody capable of binding to human IPC.

[0140] Specifically, it is preferred to use the γ chain of the Fc receptor or DAP12 as the signal transduction molecule. In the present invention, the γ chain of the Fc receptor is particularly preferably used as the signal transduction molecule. The γ chain of the Fc receptor is a molecule composed of the amino acid sequence shown in SEQ ID NO: 16. The signal transduction molecule can also be a fragment as long as the co-expressed human ILT7 is localized on the cell surface. As long as the co-expressed human ILT7 is localized on the cell surface, mutations or additions can be made to the amino acid sequence shown in SEQ ID NO: 16. That is, the present invention provides a method for preparing cells capable of producing a monoclonal antibody that can bind to the extracellular domain of human ILT7, the method comprising the following steps:

[0141] (1) Administering cells to an immunized animal, wherein the cells heterologously express a protein containing the extracellular domain of human ILT7 and a molecule containing the amino acid sequence as described in SEQ ID NO: 16; and

[0142] (2) Selecting antibody-producing cells that produce an antibody capable of binding to human ILT7 from the antibody-producing cells of the immunized animal.

[0143] Subsequently, as an antibody capable of binding to human ILT7 in the present invention, an antibody that can cross-react with a cell population in which no expression of other members of the ILT family other than ILT7 has been observed is preferably used. Specifically, as an antibody capable of binding to human ILT7 in the present invention, an antibody that can bind to a designated cell population in which no expression of other members of the ILT family other than ILT7 has been observed is preferably used, and this observation is carried out under the same conditions as those for confirming the binding to IPC. As already described, for example, ILT2 and ILT3 are expressed not only in PDC but also in DCs obtained from MDDC or CD34-positive cells (Gene. 2004 Apr 28; 331: 159-64.). On the other hand, since IPC differentiates into dendritic cells, the expression of ILT7 cannot be detected. Therefore, antibodies that cannot be detected to bind to DCs obtained from MDDC or CD34-positive cells under the conditions where binding to IPC can be confirmed are also included in the antibodies capable of binding to human ILT7 in the present invention.

[0144] The following expression patterns of other ILT family molecules have been reported (“The KIR Gene Cluster” Carrington, Mary and Norman, Paul. Bethesda (MD): National Library of Medicine (US), NCBI; 2003, Gene. 2004 Apr 28; 331: 159-64.). Therefore, antibodies that can bind to human IPC or PDC and cannot be confirmed to bind to the following cells are also included in the antibodies having specificity for ILT7:

[0145] ILT1; myeloid cells (monocytes, monocyte-derived DCs, macrophages);

[0146] ILT2; PDC, B cells, CD34-positive cells, DCs derived from CD34-positive cells, and DCs derived from monocytes;

[0147] ILT3; PDC and DCs;

[0148] ILT5; monocytes, DCs derived from CD34-positive cells, and DCs derived from monocytes; and

[0149] ILT8; monocytic lineage.

[0150] That is to say, monoclonal antibodies capable of binding to the extracellular domain of human ILT7 in the present invention preferably include monoclonal antibodies having the following immunological properties:

[0151] a) A monoclonal antibody that binds to human IPC;

[0152] b) Under the condition of binding to human IPC, the monoclonal antibody can be confirmed not to bind to one or more cells selected from the group consisting of monocytes, macrophages, B cells, CD34-positive cells, and dendritic cells derived from these cells.

[0153] In particular, as the monoclonal antibody of the present invention, preferably an antibody that cannot be confirmed to bind to monocytes, macrophages, B cells, CD34-positive cells, and dendritic cells derived from these cells under the condition of binding to human IPC.

[0154] Alternatively, in the present invention, the monoclonal antibody that can bind to the extracellular domain of human ILT7 preferably includes a monoclonal antibody having the following immunological properties:

[0155] c) A monoclonal antibody that can bind to a transformed cell co-transfected with two expression vectors, wherein the two expression vectors are an expression vector having a DNA encoding human ILT7 and an expression vector having a DNA encoding a signal transduction molecule;

[0156] d) Under the condition of binding to the co-transfected cell described in c), it may not be confirmed to bind to the host cell before transformation; or

[0157] The monoclonal antibody of the present invention includes a monoclonal antibody having the following immunological properties:

[0158] e) Under the condition of binding to the co-transfected cell described in c), it cannot be confirmed to bind to a host cell that only expresses a signal transduction molecule.

[0159] In the present invention, the fact that the monoclonal antibody against ILT7 does not cross-react with other molecules of the ILT family can be confirmed by using cells that are forced to express each other ILT family molecule. That is, for forced expression, the cDNA encoding the amino acid sequence of each ILT family molecule is introduced into a suitable host cell. The monoclonal antibody against ILT7 that needs to verify cross-reaction is added to the obtained host cell. Then, it can be confirmed that if the antibody binds to the cell, then the antibody can immunologically distinguish ILT7 from other ILT family molecules, and no expression of ILT family molecules other than ILT7 is observed in the cell. For example, in the following example, the fact that the monoclonal antibody against ILT7 obtained by the method of the present invention does not cross-react with ILT1, ILT2, and ILT3 was confirmed. Therefore, a preferred application example of the monoclonal antibody in the present invention is a monoclonal antibody that can bind to ILT7 and cannot detect the binding of the monoclonal antibody to ILT1, ILT2, and ILT3 under the same conditions.

[0160] Specifically, ILT2 and ILT3 are genes that have been shown to be expressed in IPC (Ju et al. Gene 331, 159-164, 2004). However, depending on the level of differentiation of IPC or certain conditions, each of these molecules may exhibit an inherent expression profile for each type of cell, where the certain conditions are such as stimulation with a virus or other cytokines. By using an antibody capable of immunologically distinguishing these ILT family molecules from ILT7, changes in ILT7 expression can be specifically measured.

[0161] The binding of a monoclonal antibody whose binding ability needs to be confirmed to various types of cells can be confirmed based on, for example, the principle of flow cytometry. To confirm the reactivity of an antibody based on the principle of flow cytometry, it is convenient to pre-label the antibody with a molecule or moiety that can generate a detectable signal. Fluorescent or luminescent labels are usually used. Based on the principle of flow cytometry, a fluorescence-activated cell sorter (FACS) can be used to analyze the binding of a fluorescently labeled antibody to cells. Using FACS, the binding of multiple antibodies to cells can be effectively confirmed.

[0162] Specifically, for example, it has previously been found that antibody A capable of identifying IPC and antibody B whose binding property to IPC needs to be analyzed react simultaneously with a cell population containing IPC. Antibody A and antibody B are pre-labeled with fluorescent signals capable of distinguishing these antibodies from each other. In an example where two signals are detected in the same cell population, it can be confirmed that these antibodies bind to the same cell population. In other words, antibody A and B have the same binding property. In an example where the antibodies bind to different cell populations, it is obvious that the two antibodies have different binding properties.

[0163] The preferred monoclonal antibody in the present invention includes monoclonal antibodies produced by hybridomas ILT7#11 or ILT7#17. The hybridomas ILT7#11 and ILT7#17 were deposited on October 21, 2005, at the Patent Microorganisms Depositary, National Institute of Advanced Industrial Science and Technology, with deposit numbers FERM BP-10704 and FERM BP-10705.

[0164] The specific deposit details are as follows:

[0165] (a) Name and address of the depositary institution

[0166] Name: Patent Microorganisms Depositary, National Institute of Advanced Industrial Science and Technology

[0167] Address: AIST Tsukuba Central 6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan (zip code 305-8566)

[0168] (b) Date of deposit: October 21, 2005

[0169] (c) Accession number: FERM BP-10704 (hybridoma ILT7#11)

[0170] (c) Accession number: FERM BP-10705 (hybridoma ILT7#17)

[0171] The monoclonal antibody of the present invention may also be a fragment containing its antigen-binding region. For example, antibody fragments containing its antigen-binding region obtained by enzymatic digestion of IgG can be used as the antibody in the present invention. Specifically, antibody fragments such as Fab and F(ab’)2 can be obtained by digestion with papain or pepsin. It has been well known that these antibody fragments can be used as antibody molecules with antigen affinity. Alternatively, antibodies constructed by genetic recombination can also be used as long as good antigen-binding activity can be maintained. Examples of antibodies constructed by genetic recombination include chimeric antibodies, CDR-grafted antibodies, single-chain Fv, diabodies, linear antibodies, and multispecific antibodies formed by antibody fragments. It has been well known that these antibodies can be obtained by using monoclonal antibodies or antibody-producing cells capable of producing such antibodies.

[0172] Certain transformed cells can be used as immunogens to obtain the monoclonal antibody of the present invention. That is to say, the present invention relates to a method for preparing cells, wherein the cells produce a monoclonal antibody capable of binding to the extracellular domain of human ILT7, and the method comprises the following steps:

[0173] (1) administering to an immunized animal cells that can express an exogenous protein containing the extracellular domain of human ILT7 and an exogenous molecule that binds to human ILT7; and

[0174] (2) selecting, from the antibody-producing cells of the immunized animal, antibody-producing cells that produce an antibody capable of binding to human ILT7.

[0175] Culturing the thus obtained antibody-producing cells or immortalized antibody-producing cells, and recovering the desired monoclonal antibody from the culture. Regarding methods for immortalizing antibody-producing cells, many methods are known.

[0176] In the method for preparing the monoclonal antibody of the present invention, examples of molecules that can be used include cell membrane proteins, where the molecules are used to prepare transformed cells that can serve as immunogens and can bind to human ILT7. Among them, the preferred cell membrane protein of the present invention is a signal transduction molecule located on the cell membrane. A "signal transduction molecule" refers to a molecule that binds to a protein having a receptor structure in the extracellular domain on the cell membrane and transduces the stimulation of ligand binding to the receptor into the cell. Examples of signal transduction molecules include the Fc receptor γ chain, DAP12, and molecules of the like. For example, the cell membrane protein preferably used in the present invention is the Fc receptor γ chain. The amino acid sequences of human DAP12 and the Fc receptor γ chain and the cDNA base sequences encoding these amino acid sequences are well-known. The base sequence of the human Fc receptor γ chain and the amino acid sequence encoded by this base sequence are shown in SEQ ID NO: 15 and 16, respectively.

[0177] In the present invention, the transformed cells used as immunogens can be obtained by preparing, for example, cells having the following (a) and (b):

[0178] (a) An exogenous polynucleotide encoding an amino acid sequence containing the extracellular domain of human ILT7; and

[0179] (b) An exogenous polynucleotide encoding the γ chain of the Fc receptor.

[0180] In the present invention, an exogenous polynucleotide refers to a polynucleotide artificially introduced into a host cell. When using human cells as host cells, human genes are introduced into human cells. In this combination, the artificially introduced polynucleotide is an exogenous polynucleotide. Therefore, the expression of the exogenous polynucleotide includes the ectopic expression of human ILT7 or the human Fc receptor γ chain.

[0181] Here, the "extracellular domain of human ILT7" refers to the amino acid sequence from the 17th to the 444th positions corresponding to its extracellular domain in the amino acid sequence of SEQ ID NO: 2 (shown as 1 to 428 in SEQ ID NO: 2). In the present invention, as the amino acid sequence containing the extracellular domain of human ILT7, preferably, for example, an amino acid sequence containing each region in the following order from the N-terminal side:

[0182] [Signal sequence + extracellular domain + transmembrane region + intracellular region]

[0183] Alternatively, the amino acid sequence of the extracellular domain of human ILT7 in the present invention also includes the amino acid sequence with a partially deleted intracellular region as described below.

[0184] [Signal sequence + extracellular domain + transmembrane region + a part of the intracellular region]

[0185] In addition, the amino acid sequence of the human ILT7 extracellular domain in the present invention also includes the structure with the intracellular region deleted as described below.

[0186] [Signal sequence + extracellular domain + transmembrane region]

[0187] In the above structure, the regions other than the extracellular domain can be amino acid sequences selected from the amino acid sequence shown in SEQ ID NO: 2, or can be combinations of other amino acid sequences having homology with these regions. For example, the amino acid sequences constituting the signal sequence, transmembrane region and intracellular region can be amino acid sequences of ILT family molecules other than ILT7. Or, it can be a combination of amino acid sequences of ILT families of other species other than humans. In addition, in the amino acid sequences constituting the regions other than the extracellular domain, mutations within the range of maintaining the functions of each region can be included. Or, other regions can be inserted between any regions. For example, an epitope tag such as FLAG can be inserted between the signal sequence and the extracellular domain. Specifically, the signal sequence will be removed by processing during the process of being transferred to the cell membrane surface after being translated into a protein. Therefore, any amino acid sequence that can induce the translated protein to pass through the cell membrane can be used as the signal sequence. More specifically, preferably the amino acid sequence of human ILT7 (SEQ ID NO: 2) is used as the amino acid sequence containing the human ILT7 extracellular domain.

[0188] Therefore, in the present invention, any base sequence encoding the amino acid sequence containing the above structure [signal sequence + extracellular domain + transmembrane region + intracellular region] can be used as the polynucleotide in the exogenous polynucleotide described in (a). For example, the amino acid sequence of SEQ ID NO: 2 is encoded by the base sequence described in SEQ ID NO: 1.

[0189] In the present invention, in order to obtain transformed cells that can be used as immunogens, an expression vector expressing the above (a) and (b) polynucleotides can be introduced into a suitable host cell. The (a) and (b) polynucleotides can be carried on one vector or different vectors. When different polynucleotides are carried on different vectors, the host cell is co-transfected with the two vectors.

[0190] In the present invention, preferred host cells include mammalian cells. Specific examples of host cells include cells derived from humans, monkeys, mice, or rats. Particularly preferred host cells are cells derived from humans. For example, in the present invention, 293T cells are preferably used as host cells derived from humans. 293T cells can be obtained from ATCC CRL-11268. Additionally, cells derived from immunized animals can also be used as host cells. When cells derived from immunized animals are used as immunogens, the immune response to the host cells is minimal. For this reason, antibodies against the extracellular domain of exogenously expressed ILT7 can be effectively obtained. Thus, for example, when mice are used as immunized animals, cells derived from mice can also be used as host cells.

[0191] The above polynucleotide can be introduced into cells by a vector that can be induced to express in host cells. Commercially available vectors that can be induced to express in mammalian cells can be used. Expression vectors such as pCMV-Script(R) vector, pSG5 vector (produced by Stratagene), pcDNA3.1 (produced by Invitrogen) can be used in the present invention.

[0192] If necessary, the transformed cells thus obtained can be administered to an immunized animal together with additional components such as adjuvants. Examples of adjuvants that can be used include Freund's complete adjuvant, etc. In an example where mice are used as immunized animals, the number of transformed cells that can be administered ranges from 10 4 to 10 9 , and more specifically, from 10 4 to 10 6 cells. Generally, multiple-dose administration involves administering the immunogen at regular time intervals until the antibody titer increases. For example, in the case of short-term immunization, the transformed cells are administered every 2 to 4 days, more specifically, every 3 days. After administering two or three times, antibody-producing cells can be recovered. Alternatively, it can be administered once a week and antibody-producing cells can be recovered after administering five or six times.

[0193] In the present invention, the recovered antibody-producing cells are cloned to obtain monoclonal antibodies. For cloning, it is preferred to immortalize the antibody-producing cells. For example, cell fusion methods represented by the hybridoma method, or transformation using Epstein-Barr virus (EBV) can be used as methods for immortalizing antibody-producing cells.

[0194] As an antibody-producing cell, a single cell can produce one antibody. Therefore, by establishing a cell population derived from a single cell (i.e., cloning), monoclonal antibodies can be obtained. The hybridoma method is a method of fusing antibody-producing cells with a suitable cell line, immortalizing them, and then cloning them. Techniques such as the limiting dilution method can be used to clone immortalized antibody-producing cells. Many cell lines are known to be usable for the hybridoma method. These cell lines perform well in immortalizing lymphocytes and have various genetic markers required for selecting fused cells. In addition, when it is necessary to obtain antibody-producing cells, cell lines lacking the ability to produce antibodies can also be used.

[0195] For example, in the mouse or rat cell fusion method, mouse myelomas P3x63Ag8.653 (ATCC CRL-1580) and P3x63Ag8U.1 (ATCC CRL-1597) are widely used as cell lines. Usually, hybridomas are prepared by fusing cells of the same species, however, monoclonal antibodies can also be obtained from heterohybridomas of closely related different species.

[0196] Specific protocols for cell fusion are well-known. That is, antibody-producing cells of an immunized animal are mixed with a suitable fusion partner to perform cell fusion. Examples of usable antibody-producing cells include spleen cells, lymphocytes collected from lymph nodes, and peripheral blood B cells. As the fusion partner, various cell lines described above can be used. The polyethylene glycol method and the electrofusion method can be used to perform cell fusion.

[0197] Thereafter, based on the selection markers of the fused cells, the successfully fused cells are selected. For example, when using a HAT-sensitive cell line for cell fusion, the cells growing in HAT medium are selected as the successfully fused cells. In addition, it has been confirmed that the antibodies produced by the selected cells have the required reactivity.

[0198] Each hybridoma is screened based on antibody reactivity. That is, the antibodies produced by hybridomas capable of binding to human ILT7 can be selected by the method described above. Preferably, the selected hybridomas are first subcloned and then the required antibodies are finally confirmed, and the confirmed antibodies are selected as the monoclonal antibodies produced by the hybridomas of the present invention.

[0199] Specifically, the desired hybridomas can be selected based on reactivity against human cells or transformed cells expressing the human ILT7 gene. Antibodies capable of binding to cells can be detected based on the principle of immunoassay. For example, ELISA using cells as antigens can be used to detect the desired antibodies. Specifically, the culture supernatant of hybridomas is added to a support on which the immunogen or transformed cells are immobilized. In this example, the desired antibody is contained in the culture supernatant, and the cells immobilized on the support will recruit the antibody. Then, the solid phase is separated from the culture supernatant and, if necessary, washed. Thereafter, the antibody recruited on the solid phase can be detected. Antibodies can be detected using antibodies capable of recognizing antibodies. For example, mouse antibodies can be detected by anti-mouse immunoglobulin antibodies. If the antibody capable of recognizing the antibody is labeled, such detection can be easily performed. Examples of available labels include enzymes, fluorescent dyes, luminescent dyes, and the like.

[0200] On the other hand, microparticles and the inner wall of a microplate can be used as supports for immobilizing cells. Cells can be immobilized on the surface of microparticles made of plastic or a container by physical adsorption. Examples of supports available for immobilizing cells include beads made of polystyrene and reactors.

[0201] In the selection of hybridomas, sometimes it can be predicted that antibodies are produced against the host cells of the transformed cells used as immunogens rather than against ILT7. For example, as shown in the examples, in the case of using human cells as immunogens and mice as immunized animals, it can be expected that human cells are recognized as foreign substances and antibodies that bind to them are produced. In the present invention, it is desired to obtain antibodies capable of recognizing human ILT7. Therefore, it is not necessary to obtain antibodies capable of recognizing human cell antigens other than human ILT7. In the screening, in order to remove hybridomas capable of producing such antibodies, unwanted antibodies can be absorbed before confirming antibody reactivity.

[0202] Unwanted antibodies can be absorbed by antigens that bind to the presumed existing antibodies. Specifically, for example, antibodies capable of recognizing human cell antigens other than human ILT7 can be absorbed by cells that cannot detect human ILT7 expression. In the present invention, preferably, the host cells used as immunogens are used as antigens for absorbing unwanted antibodies. Alternatively, host cells that do not express the extracellular domain of human ILT7 but express molecules that bind to ILT7 can be used as antigens for absorbing antibodies.

[0203] For a monoclonal antibody whose antigen-binding activity has been confirmed, if necessary, its actual effect on IPC activity can be confirmed. Its effect on IPC can be confirmed by methods such as those described below.

[0204] As the monoclonal antibody of the present invention, hybridomas that produce the monoclonal antibody are cultured and the monoclonal antibody of the present invention is recovered from the resulting culture. The hybridomas can be cultured in vivo or in vitro. In the case of in vitro culture, the hybridomas can be cultured using a known medium such as RPMI1640. The immunoglobulins secreted by the hybridomas will accumulate in the culture supernatant. Therefore, if desired, the monoclonal antibody of the present invention can be obtained by collecting and purifying the culture supernatant. Not adding serum to the medium makes the purification of immunoglobulins easier. However, for the purpose of rapidly expanding the hybridomas and increasing the antibody production, 10% fetal bovine serum can be added to the medium.

[0205] The hybridomas can also be cultured in vivo. Specifically, intraperitoneal culture can be achieved by inoculating the hybridomas into the peritoneal cavity of nude mice. The monoclonal antibodies will accumulate in the ascites. Therefore, if the ascites is obtained and purified as needed, the desired monoclonal antibody can be prepared. The obtained monoclonal antibody can be appropriately modified or processed according to the intended use.

[0206] The monoclonal antibody of the present invention can be expressed by obtaining cDNA encoding the antigen-binding region of the antibody from the hybridoma and inserting it into a suitable expression vector. Techniques for obtaining cDNA encoding the variable region of the antibody and expressing the cDNA in a suitable host cell are known. In addition, methods for obtaining chimeric antibodies by linking the variable region containing the antigen-binding region to the constant region are also known.

[0207] Preferred monoclonal antibodies in the invention include monoclonal antibodies produced by hybridoma #11 (deposit number: FERM BP-10704), hybridoma #17 (deposit number: FERM BP-10705), or hybridoma #37. The amino acid sequences of the variable regions constituting these monoclonal antibodies and the cDNA base sequences encoding the amino acid sequences are shown below. Therefore, for example, chimeric antibodies formed by linking these variable regions to the constant regions of other immunoglobulins are preferred in the present invention. In the amino acid sequences described in the sequence listing, the amino acid sequence from position 1 to the C-terminus constitutes the mature protein. That is to say, for each amino acid sequence, the continuous amino acid sequence from position 1 to the C-terminus is the mature sequence of each amino acid sequence. On the other hand, the amino acid sequence represented by the value from the N-terminus to -1 is the signal sequence.

[0208]

[0209] For example, murine (variable region)-human (constant region) chimeric antibodies are prepared by separately linking these variable region genes to the human IgG1 heavy chain constant region and the human Igκ light chain constant region. The amino acid sequence of such chimeric antibodies and the base sequence encoding the antibody are described below, respectively. The chimeric antibodies represented by these sequences show a preferred embodiment of the construct of the anti-ILT7 monoclonal antibody of the present invention. In the amino acid sequence of the following chimeric antibodies, the amino acid sequence from the N-terminus to -1 corresponds to the signal sequence, and the amino acid sequence from 1 to the C-terminus corresponds to the mature protein. That is, chimeric antibodies containing heavy and light chains are preferred in the present invention, wherein the chimeric antibody contains the amino acid sequence from 1 to the C-terminus of each amino acid sequence.

[0210]

[0211] In addition, the antigen-binding activity of monoclonal antibodies can also be transplanted onto other immunoglobulins. The variable region of an immunoglobulin contains complementarity-determining regions (CDRs) and framework regions. The antigen-binding property of each immunoglobulin is determined by the CDRs, and the framework maintains the structure of the antigen-binding region. The amino acid sequences of the CDRs have extremely rich diversity, while the amino acid sequences of the framework part are highly conserved. It is known that the transplantation of antigen-binding activity can be achieved by integrating the amino acid sequences constituting the CDRs into the framework region of other immunoglobulin molecules. A method for transplanting the antigen-binding property of different immunoglobulins onto human immunoglobulins has been established. In the present invention, the "antigen-binding region" can include the CDRs transplanted into the framework region. Therefore, the "fragment containing the antigen-binding region" of a specified monoclonal antibody includes a fragment of a human immunoglobulin containing the variable region, onto which the CDRs of the monoclonal antibody are transplanted. For example, the amino acid sequences of the above variable regions respectively contain the following amino acid sequences (SEQ ID NO) as CDRs.

[0212]

[0213] Based on the information of the base sequences encoding the above amino acid sequences and the base sequences encoding the framework (FR) of human immunoglobulins, primers can be designed and cDNA can be amplified, wherein the base sequence of the cDNA is formed by linking these two base sequences. The operation for each framework is repeated, and a variable region formed by linking murine CDR1, CDR2, and CDR3 to the human FR can be constructed. In addition, when the base sequences encoding the constant regions of human immunoglobulins are linked as needed, humanized antibodies with the constant regions can be obtained.

[0214] As a chimeric antibody containing the above variable regions or a humanized antibody transplanted with the variable regions composed of CDRs, the antibody of the present invention preferably contains an antibody having a constant region derived from IgG or IgM. The inventors of the present invention confirmed that the monoclonal antibody against ILT7 showed CDC activity against ILT7-expressing cells. Therefore, an antibody having a constant region derived from IgG or IgM shows cytotoxicity against ILT7-expressing cells due to its CDC activity. Such an antibody can be used to inhibit the number of ILT7-expressing cells such as IPC.

[0215] The chimeric antibody or humanized antibody capable of recognizing ILT7 provided by the present invention can be prepared by genetic engineering by using a polynucleotide encoding the chimeric antibody or humanized antibody capable of recognizing ILT7. For example, the polynucleotides of the base sequences described in the following SEQ ID NOs and the polynucleotides encoding the amino acid sequences can be used as the polynucleotides encoding variable regions #11 or #17, wherein the amino acid sequences are the mature proteins constituting each amino acid sequence. Each continuous amino acid sequence from 1 to the C-terminus of each amino acid sequence corresponds to a mature protein. In an example where each mature protein is expressed as an isolated protein, it is preferable to place a secretion signal at the N-terminus of each amino acid sequence. For example, when such a protein is expressed in animal cells, the amino acid sequence from the N-terminus to -1 in the amino acid sequences shown in these SEQ ID NOs can be used as a signal sequence. Alternatively, these variable regions can be secreted as mature proteins by using any signal sequence capable of ensuring immunoglobulin secretion.

[0216] #11 SEQ ID NO: 50 (base sequence) SEQ ID NO: 52 (base sequence)

[0217] #17 SEQ ID NO: 54 (base sequence) SEQ ID NO: 56 (base sequence)

[0218] In the same method as described above, for the polynucleotide encoding the humanized antibody, a polynucleotide expressing the humanized antibody can be prepared by using a base sequence encoding a protein having a signal sequence added to the N-terminus of the protein. When the heavy chain and the light chain are carried by different vectors, the two vectors are co-transfected into the same host cell. The heavy chain and the light chain expressed by each vector are used to construct an immunoglobulin having two chains. Alternatively, a polynucleotide encoding the heavy chain and a polynucleotide encoding the light chain can also be carried on the same vector. The host cell transformed with the vector carrying the two polynucleotides can express the heavy chain and the light chain and can prepare an immunoglobulin having two chains.

[0219] Using a host vector system capable of expressing antibody genes, these polynucleotides can be expressed as antibodies. In addition, in an example where it is expressed as a single protein molecule by linking the heavy chain variable region and the light chain variable region, a signal sequence can be placed at the N-terminus of the protein molecule. Known examples of such antibody molecules include scFv molecules, in which the heavy chain variable region is linked to the light chain variable region by a linker.

[0220] The monoclonal antibodies of the present invention include each monoclonal antibody thus prepared. In other words, the monoclonal antibodies in the present invention include monoclonal antibodies composed of immunoglobulins containing an antigen-binding region encoded by a polynucleotide derived from the cDNA encoding the antigen-binding region of the above-mentioned monoclonal antibody.

[0221] As described above, RBL cells with forced expression of the ILT1 gene can be used as an immunogen for obtaining ILT1 antibodies. However, the expression of ILT7 on the surface of RBL cells (P815) cannot be confirmed, so it cannot be used as an immunogen. The inventors of the present invention found that the expression of human ILT7 on the cell surface can be induced by co-expressing human ILT7 with other cell membrane proteins that bind to human ILT7. Thus, the inventors of the present invention found that an antibody capable of binding human IPC can be obtained by using transformed cells as an immunogen, and completed the present invention, wherein the expression of the transformed cells is induced by the above method.

[0222] That is to say, the present invention provides an immunogen that can be used to prepare an antibody that can bind to the extracellular domain of human ILT7, and includes animal cells or their cell membrane components, in which the following polynucleotides are carried for exogenous expression, and the polynucleotides include (a) a polynucleotide encoding an amino acid sequence containing the extracellular domain of human ILT7; and (b) a polynucleotide encoding the γ chain of the Fc receptor.

[0223] Since the structure of human ILT7 was discovered in 1998, six years or more have passed. However, an antibody that can specifically recognize ILT7 has still not been obtained. An antibody that can recognize human ILT7 is provided for the first time by using the immunogen of the present invention. That is to say, the present invention provides an antibody that can recognize human ILT7, and the antibody is obtained through the following steps:

[0224] (1) Administering cells to an immunized animal, the cells being capable of exogenous expression of a protein containing the extracellular domain of human ILT7 and a molecule that binds to human ILT7;

[0225] (2) Selecting antibody-producing cells from the antibody-producing cells of the immunized animal that are capable of producing antibodies that can bind to human ILT7; and

[0226] (3) Culture the antibody-producing cells selected in step (2), and recover from the culture an antibody capable of recognizing human ILT7.

[0227] Human ILT7 has been found to be specifically expressed in human IPCs. The inventors of the present invention analyzed gene expression using SAGE and also confirmed the specific expression of human ILT7 in human IPCs. However, in previous reports, the expression levels of ILT7 in two cases were analyzed based on mRNA. Since antibodies capable of detecting human ILT7 were not available, the expression state of the protein was not analyzed as a matter of course. Analysis of the human ILT7 protein was achieved by providing the antibody of the present invention that binds to the extracellular domain of human ILT7.

[0228] The inventors of the present invention actually confirmed that a monoclonal antibody capable of binding to the extracellular domain of human ILT7 according to the present invention can specifically detect human IPCs. That is, the present invention relates to a method for detecting interferon-producing cells, the method comprising the steps of: adding to a test cell a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing its antigen-binding region; and detecting the monoclonal antibody or the fragment containing its antigen-binding region bound to the cell.

[0229] By detecting human ILT7 according to the present invention, it can be determined whether a specific cell is an IPC. That is, the present invention provides a method for identifying IPCs using human ILT7 as an indicator. Alternatively, human IPCs can be isolated by isolating cells in which human ILT7 is detected, wherein the method for detecting human ILT7 is based on the present invention. That is, the present invention provides a method for isolating IPCs using human ILT7 as an indicator.

[0230] Based on the analysis using the human ILT7 antibody, it was confirmed that the expression level of ILT7 in IPCs was reduced, wherein the differentiation of the IPCs was induced by CpG. That is, by using ILT7 as an indicator, IPCs before their differentiation is induced can be specifically detected. In other words, the monoclonal antibody of the present invention can be particularly used to detect a cell before it differentiates into a dendritic cell. The term "IPC before differentiation" used herein can be defined as a cell population having the ability to produce interferon.

[0231] In the present invention, monoclonal antibodies capable of binding to the extracellular domain of human ILT7 or fragments containing its antigen-binding region can be pre-labeled. For example, antibodies can be easily detected by labeling with luminescent dyes or fluorescent dyes. More specifically, the prepared antibody labeled with a fluorescent dye is added to a cell population that may contain IPC, and then the cells bound by the antibody of the present invention can be detected by using the fluorescent dye as an indicator. Further, IPC can be isolated by separating the cells that detect the fluorescent dye. A series of steps can be easily carried out based on the principle of FACS.

[0232] Alternatively, the antibodies of the present invention can be pre-bound to a solid-phase support such as magnetic particles. The antibodies bound to the solid-phase support will recognize human ILT7 and then IPC will be captured on the solid-phase support. Thus, IPC can be detected and isolated.

[0233] Based on the present invention, antibodies necessary for the method of detecting IPC can be provided as reagents for detecting IPC. That is to say, the present invention provides a reagent for detecting interferon-producing cells, which contains a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing its antigen-binding region. In addition to using the reagent for detecting IPC of the present invention as an antibody, it can also be used in combination with a positive control or a negative control. For example, transformed cells expressing the extracellular domain of human ILT7 and used as an immunogen, as well as IPC obtained from the human body, can be used as positive controls. Generally, only a small amount of human IPC can be obtained from peripheral blood. Therefore, it is particularly preferred to use transformed cells as positive controls in the reagent of the present invention. On the other hand, any cells that do not express human ILT7 can be used as negative controls.

[0234] That is to say, the present invention provides a kit for detecting human IPC, which comprises:

[0235] (a) A monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing its antigen-binding region; and

[0236] (b) Cells expressing an exogenous protein and an exogenous molecule, the exogenous protein containing the extracellular domain of human ILT7, and the exogenous molecule being capable of binding to human ILT7.

[0237] The inventors of the present invention analyzed the effect of antibodies capable of binding to the extracellular domain of human ILT7 on IPC. Therefore, it can be confirmed that antibodies capable of binding to the extracellular domain of human ILT7 inhibit the activity of IPC. That is to say, the present invention relates to a method for inhibiting the activity of interferon-producing cells, which comprises the step of adding any of the following components to interferon-producing cells:

[0238] (a) A monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of interferon-producing cells, or a fragment containing its antigen-binding region; and

[0239] (b) An immunoglobulin into which the complementarity-determining regions of the monoclonal antibody described in (a) have been transplanted, or a fragment thereof containing an antigen-binding region.

[0240] Alternatively, the present invention relates to a method for inhibiting the activity of interferon-producing cells in a living tissue, the method comprising the step of adding any of the following components to the living tissue:

[0241] (a) A monoclonal antibody that can bind to human ILT7 and inhibit the activity of interferon-producing cells, or a fragment thereof containing an antigen-binding region;

[0242] (b) An immunoglobulin containing the complementarity-determining regions of the monoclonal antibody described in (a) that have been transplanted, or a fragment thereof containing an antigen-binding region; and

[0243] (c) A polynucleotide encoding the component described in (a) or (b).

[0244] Here, "Interferon Producing cell (IPC)" refers to a cell that has the ability to produce IFN and expresses ILT7 on the cell surface. Hereinafter, unless otherwise specified, "IPC" includes not only precursor cells of dendritic cells but also cells that have the ability to produce IFN and express ILT7 on the cell surface. Methods for identifying such IPCs are well known. Some cell surface markers can be used as indicators to distinguish IPCs from other blood cells. Specifically, the cell surface marker profile of human IPCs is as follows (Shortman, K. and Liu, YJ. Nature Reviews 2: 151-161, 2002). In recent years, specific reports have proposed defining BDCA-2-positive cells as IPCs (Dzionek, A. et al. J. Immunol. 165: 6037-6046, 2000.).

[0245] [Cell surface antigen profile of human IPCs]

[0246] CD4 positive, CD123 positive,

[0247] Lineage (CD3, CD14, CD16, CD19, CD20, CD56) negative and CD11c negative

[0248] Therefore, it can also be said that IPCs are cells that have the expression profile of these known markers and have the ability to produce IFN. In addition, even if the cells are a group of cells with different expression profiles of these known markers, as long as the cells in the living tissue have the ability to produce IFN, the cells are also included in IPCs. In addition, the common characteristics of human IPCs are as follows:

[0249] [Morphological characteristics of cells]

[0250] - Similar to plasma cells

[0251] - Round cells with a smooth cell surface

[0252] - Relatively large nucleus

[0253] [Functional characteristics of cells]

[0254] - During the process of virus infection, a large amount of type I interferon is produced in a short time.

[0255] - Differentiate into dendritic cells after virus infection.

[0256] "Inhibiting the activity of IPC" refers to the inhibition of at least one IPC function. Examples of IPC functions include IFN production and cell survival. Cell survival can also be referred to as the number of cells. Therefore, in examples where one or both of these two functions are inhibited, it can be said that the activity of IPC is inhibited. It has been found that type I IFN produced by IPC can cause various diseases. Therefore, the inhibition of the number of IPCs and IFN production can be used in medical treatment methods for these diseases.

[0257] For example, the relationship between the pathological states of various autoimmune diseases and IFNα has been pointed out. Most IFNα is produced by IPCs. Therefore, the pathological state caused by IFNα can be alleviated by inhibiting the production of IFNα. Here, "inhibiting IFN produced by IPC" refers to inhibiting the production of at least one type of IFN produced by IPC. The preferred IFN in the present invention is type I IFN. Among them, IFNα is important.

[0258] That is to say, the present invention relates to an inhibitor for inhibiting IFN production, which inhibitor comprises an antibody capable of binding to the extracellular domain of ILT7 as an active ingredient. Alternatively, the present invention provides a method for inhibiting the production of IFN, which method comprises the step of administering an antibody capable of binding to the extracellular domain of ILT7. In addition, the present invention relates to the use of an antibody capable of binding to the extracellular domain of ILT7 in the production of a medicinal ingredient for inhibiting IFN production.

[0259] Among the IPCs are cells that produce large amounts of IFN from a small number of cells. For example, by stimulating precursor cells of dendritic cells with a virus or conditions such as these, the cells produce most of the IFN produced in vivo. Inhibition of the number of IPCs capable of producing a lot of IFN can inhibit the production of IFN. Therefore, by inhibiting the number of IPCs, the pathological state caused by IFNα can be alleviated. It has been confirmed that in a preferred embodiment of the present invention, a monoclonal antibody against ILT7 binds to ILT7-expressing cells and then exerts a cytotoxic effect through complement-dependent cytotoxicity (CDC). The CDC effect is an important mechanism of antibody drugs. Due to the CDC effect of the monoclonal antibody against ILT7 of the present invention, the antibody also has potential cytotoxicity against ILT7-expressing cells such as IPCs. That is to say, for the monoclonal antibody against ILT7, in addition to the mechanism of inhibiting IFN production in the preferred embodiment, an inhibitory effect on IFN production can be expected through cytotoxicity against IPCs.

[0260] The antibodies used in the present invention that can recognize the extracellular domain of human ILT7 can be obtained by the methods described above. The antibodies in the present invention can be of any class. There are also no special restrictions on the species from which the antibodies are derived. In addition, fragments containing the antigen-binding region of the antibody can be used as antibodies. For example, antibody fragments containing their antigen-binding regions obtained by enzymatic digestion of IgG can be used as the antibodies in the present invention. Specifically, enzymatic digestion can be carried out using papain or pepsin to obtain antibody fragments such as Fab and F(ab’)2. It is well known that these antibody fragments can be used as antibody molecules with antibody affinity. Alternatively, as long as good antigen-binding activity can be maintained, antibodies constructed by genetic recombination techniques can also be used. Examples of antibodies constructed by genetic recombination include chimeric antibodies, CDR-grafted antibodies, single-chain Fv, diabodies, linear antibodies, and multispecific antibodies composed of antibody fragments. It is known that these antibodies can be obtained by using monoclonal antibodies.

[0261] In the present invention, the antibody can be modified if necessary. According to the present invention, an antibody that can recognize the extracellular domain of human ILT7 has an inhibitory effect on the activity of IPCs. That is to say, it can be expected that the antibody itself has cytotoxicity against IPCs. Subclasses of antibodies that exhibit potential effector activity are known. Alternatively, the inhibitory effect of the antibody on IPC activity can be further enhanced by modifying the antibody with a cytotoxic agent. Examples of cytotoxic agents are described below.

[0262] Toxins: Pseudomonas endotoxin (PE), diphtheria toxin, ricin

[0263] Radioisotopes: Tc 99m , Sr89 、I 131 、Y 90

[0264] Antitumor agents: calicheamicin, mitomycin, paclitaxel

[0265] A toxin composed of a protein can be linked to an antibody or a fragment thereof by a bifunctional reagent. Alternatively, a gene encoding a toxin can be linked to a gene encoding an antibody to obtain a fusion protein of the two genes. Methods for linking an antibody to an isotope are also known. For example, a method of labeling a radioactive isotope to an antibody using a chelating reagent is known. In addition, an antitumor agent can be linked to an antibody using a sugar chain or a bifunctional reagent.

[0266] The inventors of the present invention have confirmed a phenomenon that a monoclonal antibody bound to ILT7 expressed on the cell surface is integrated into the cell (internalization) after binding. Therefore, the cytotoxic reagent of the present invention can be transported into the cell by adding an antibody linked to the cytotoxic reagent to ILT7-expressing cells. That is, the present invention provides an inhibitor of the activity of ILT7-expressing cells, which comprises a monoclonal antibody against ILT7 linked to a cytotoxic reagent as an active ingredient. Alternatively, the present invention relates to the use of a monoclonal antibody against ILT7 in the production of an inhibitor of the activity of ILT7-expressing cells, the antibody being linked to a cytotoxic reagent. In addition, the present invention provides a method for inhibiting the activity of ILT7-expressing cells, which comprises the step of administering a monoclonal antibody against ILT7 linked to a cytotoxic reagent.

[0267] In the present invention, an antibody with a structurally modified form can also be used as an active ingredient. For example, methods for modifying an antibody to improve its cytotoxicity and stability are known. Specifically, immunoglobulins in which the sugar chain of the heavy chain has been modified are known (Shinkawa, T. et al. J. Biol. Chem. 278: 3466-3473, 2003). The antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immunoglobulin was enhanced by modifying the sugar chain. Alternatively, immunoglobulins in which the amino acid sequence of the Fc region has been modified are also known. That is, the ADCC activity was enhanced by artificially increasing the binding activity of the immunoglobulin to the Fc receptor (Shield, R. L. et al. J. Biol. Chem. 276; 6591-6604, 2001).

[0268] This phenomenon has been discovered: IgG bound to Fc receptors is immediately incorporated into cells. Then, the IgG binds to Fc receptors expressed on endosomes and is released back into the bloodstream. IgG with high binding activity to Fc receptors has more opportunities to be released into the bloodstream after being incorporated into cells. Therefore, the retention time of IgG in the bloodstream is prolonged (Hinton, P.R. et al. J Biol Chem. 279: 6213 - 6216. 2004). In addition, it has been reported that modification of the amino acid sequence in the Fc region can cause changes in the activity of complement-dependent cytotoxicity (CDC). These modified antibodies can be used as the antibodies in the present invention.

[0269] Adding an antibody capable of binding to the extracellular domain of human ILT7 to IPC can inhibit the activity of IPC. Therefore, these antibodies can be used as inhibitors for inhibiting the activity of IPC or in a method for inhibiting the activity of IPC. That is to say, the present invention provides an inhibitor of the activity of IPC, which inhibitor comprises at least one component selected from the group consisting of (a) to (c), and this component is used as an active ingredient. Alternatively, the present invention relates to a method for inhibiting the activity of IPC, which method comprises the step of administering at least one component selected from the group consisting of the following (a) to (c). In addition, the present invention relates to the use of at least one component selected from the group consisting of the following (a) to (c) for preparing an inhibitor of IPC activity:

[0270] (a) A monoclonal antibody capable of binding to human ILT7, or a fragment containing its antigen-binding region;

[0271] (b) An immunoglobulin grafted with the complementarity-determining regions of the antibody described in (a), or a fragment thereof containing an antigen-binding region; and

[0272] (c) A polynucleotide encoding the component described in (a) or (b).

[0273] In the present invention, a monoclonal antibody capable of recognizing the extracellular domain of human ILT7 can be used as a monoclonal antibody capable of inhibiting the activity of IPC. In the present invention, one or more monoclonal antibodies can be used. For example, one or more monoclonal antibodies capable of recognizing the extracellular domain of human ILT7 can be used in combination in the present invention.

[0274] The inhibitory effect of an antibody on the IFN-producing activity of IPC can be confirmed by the following method. Stimulating IPC with a virus produces a large amount of IFN. Before, after, or simultaneously with stimulating IPC with the virus, an antibody is added to the IPC. The ability of each resulting IPC to produce IFN is compared with the ability of each corresponding control without the added antibody. The ability to generate IFN is evaluated by measuring IFNα or IFNβ contained in the culture supernatant of the IPC. As a result of the comparison, when the amount of IFN in the supernatant of the group to which the antibody has been added is significantly reduced, it can be confirmed that the tested antibody has the effect of inhibiting the ability to generate IFN. Methods for measuring IFN are known. In vivo, IPC produces most of the IFN. Therefore, the production state of IFN in vivo can be regulated by inhibiting the ability of IPC to generate IFN.

[0275] In the present invention, the activity of IPC includes maintaining the number of IPC. Therefore, the inhibition of the activity of IPC in the present invention includes inhibiting the number of IPC. When it is confirmed that the number of IPC is inhibited in the presence of an antibody, it can be found that the antibody inhibits the activity of IPC. When measuring the production of IFN, inactive immunoglobulin derived from the same animal species can be used as a comparative control group for measuring the antibody activity. The number of IPC can be quantitatively compared by cell counting. The number of cells can be counted by FACS or microscopy.

[0276] In addition, it has been reported that due to viral infection or such stimulation, IPC can differentiate into Th2, also known as dendritic cell 2 (DC2). If the IFN produced by stimulating IPC with a virus can be inhibited, then its differentiation into Th2 can also be inhibited. Therefore, it can be expected that the monoclonal antibody of the present invention capable of inhibiting the production of IFN may also have a therapeutic effect on various allergic diseases.

[0277] When an antibody capable of recognizing the extracellular domain of human ILT7 is added to a host different from the tissue type from which the antibody was obtained, the antibody needs to be processed into a form that will not be recognized as a foreign component by the host. For example, the immunoglobulin is made not to be easily recognized as a foreign substance by processing the antibody into the following molecule. The following techniques for processing immunoglobulins are known. The fragment containing the antigen-binding region lacks the constant region. (Monoclonal Antibodies: Principles and Practice, third edition, Academic Press Limited. 1995; Antibody Engineering, A Practical Approach, IRL PRESS, 1996)

[0278] - Chimeric antibodies containing the antigen-binding region of a monoclonal antibody and the constant region of the host's immunoglobulin ("Gene Expression Experiment Manual", Isao Ishida, Tamie Ando, eds., Kodansha, 1994)

[0279] - CDR replacement antibodies, in which the complementarity-determining regions (CDRs) of a monoclonal antibody are substituted for the CDRs of the host's immunoglobulin ("Gene Expression Experiment Manual", Isao Ishida, Tamie Ando, eds., Kodansha, 1994)

[0280] Alternatively, when using non-human animals, human antibodies can be obtained by integrating human antibody genes into the non-human animals used as immunized animals. For example, transgenic mice having human antibody genes have been put into practical use as immunized animals to prepare human antibodies (Ishida et al., Cloning and Stem Cells, 4: 85-95, 2002). Human antibodies capable of recognizing ILT7 can be obtained by using such animals and the above-mentioned immunogens. Preferably, human antibodies are administered to humans.

[0281] Alternatively, genes for the variable regions of human immunoglobulins can be obtained by phage display methods (McCafferty J. et al., Nature 348: 552-554, 1990; Kretzschmar T et.al., Curr Opin Biotechnol. 2002 Dec; 13(6): 598-602.). In phage display methods, genes encoding the variable regions of human immunoglobulins are integrated into phage genes. Phage libraries can also be prepared by using various immunoglobulin genes as templates. The phage expresses the variable regions as fusion proteins of the proteins constituting the phage. The variable regions expressed on the phage surface by the phage retain the binding activity to antigens. Considering screening phages expressing variable regions having the desired binding activity from the phage library, phages capable of binding to antigens or cells expressing antigens are selected. In addition, the phage particles thus screened retain the genes encoding variable regions having the desired binding activity. That is to say, in phage display methods, the binding activity of the variable regions can be used as an indicator to obtain genes encoding variable regions having the desired binding activity.

[0282] In the IPC activity inhibitors of the present invention or in the methods for inhibiting the activity of IPC, an antibody capable of recognizing the extracellular domain of human ILT7 or an antibody fragment containing at least the antigen-binding region of the antibody can be administered as a protein or a protein encoded by a polynucleotide. In the administration of the polynucleotide, a vector is required to express the desired protein, and in this vector, the polynucleotide encoding the desired protein is placed under the control of a suitable promoter. Enhancers and terminators can also be inserted into the vector. Vectors carrying genes encoding the heavy and light chains of immunoglobulins and genes capable of expressing immunoglobulin molecules are known.

[0283] It can be administered by introducing a vector capable of expressing an immunoglobulin into cells. In the administration to living tissues, for vectors that can infect cells by administration to living tissues, they can be directly administered. First, lymphocytes are isolated from the living tissue, and then the vector is introduced into the lymphocytes that can be reinjected back into the living tissue (ex vivo method).

[0284] In the IPC activity inhibitors of the present invention or in the methods for inhibiting the activity of IPC, for the amount of monoclonal antibody administered to living tissues, the range of immunoglobulin administered is usually 0.5 mg to 100 mg per kilogram of body weight, for example, 1 mg to 50 mg per kilogram of body weight, preferably 2 mg to 10 mg per kilogram of body weight. The interval for administering the antibody to living tissues can be appropriately adjusted so as to maintain an effective concentration of immunoglobulin in the living tissue during the treatment. Specifically, for example, the interval for administering the antibody can be 1 to 2 weeks. The administration route is optional. Those skilled in the art can appropriately select an administration route that is effective in the treatment. Specific examples thereof include oral administration or parenteral administration. The antibody can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or such methods. Suitable dosage forms for parenteral administration in the present invention include injection solutions, suppositories and sprays. When adding an antibody to cells, an immunoglobulin is added to the culture medium, and the range of the added concentration is usually 1 μg / ml, preferably 10 μg / ml, more preferably 50 μg / ml, and even more preferably 0.5 mg / ml.

[0285] In the IPC activity inhibitors of the present invention or in the methods for inhibiting the activity of IPC, monoclonal antibodies can be administered to living tissues by an optional method. Generally, the monoclonal antibody is mixed with a pharmaceutically acceptable carrier. If necessary, the monoclonal antibody can be mixed with additive reagents such as thickening agents, stabilizers, preservatives, and solubilizers. Examples of such carriers or additive reagents include lactose, citric acid, stearic acid, magnesium stearate, sucrose, starch, talc, gels, agar, vegetable oils, and ethylene glycol. The term "pharmaceutically acceptable" means recognized by various government regulatory agencies or listed in the pharmacopoeias of various countries or commonly recognized by the pharmacopoeias used for animals, mammals, and more specifically humans. The IPC activity inhibitors of the present invention can also be provided in the form of single-dose or multi-dose lyophilized powders or tablets. In addition, the lyophilized powder or tablet can be used in combination with sterile water for injection, physiological saline solution, or buffer solution to dissolve the composition to the desired concentration before administration.

[0286] In addition, when the monoclonal antibody is administered in the form of a vector capable of expressing immunoglobulins, plasmids having heavy and light chains are co-transfected, and the dosage range of each plasmid is 0.1 to 10 mg, for example, 1 to 5 mg per kilogram of body weight. To introduce the plasmid into cells, the concentration of the vector used is 1 to 5 μg / 10 6 cells. The present invention will be specifically described below in conjunction with examples.

[0287] All the documents cited herein are hereby incorporated by reference in their entirety. Examples

[0288] Example 1

[0289] A. Analysis of ILT7 Expression

[0290] A-1) Analysis using the SAGE library

[0291] By SAGE TM (Serial Analysis of Gene Expression) method, the expression of genes in human monocytes, IPC, and HSV-treated IPC was compared and analyzed. The analysis method is as follows. Monocytes as CD14-positive cells were isolated from human peripheral blood using a cell sorter, and IPC as BDCA-4-positive cells was isolated. In addition, IPC was cultured for 12 hours in the presence of herpes simplex virus (HSV) to prepare differentiated IPC. Using I-SAGE TMRNA was obtained from various cells using a kit (manufactured by Invitrogen), and then a SAGE library was prepared. The base sequence data of approximately 100,000 tags obtained was analyzed using SAGE analysis software (manufactured by Invitrogen). As a result, the known gene ILT7 (Gen Bank Acc#NM_012276) was discovered, and for this gene, the scores for monocytes / IPC / IPC+HSV were 0 / 16 / 0, that is, this gene showed IPC-specific expression. ILT7 is a membrane protein with an immunoglobulin-like domain encoded by the base sequence shown in SEQ ID NO: 1( Figure 2 as shown in (a)). The expression of ILT7 mRNA in IPC has been reported (Blood 100, 3295 - 3303 (2002)).

[0292] A-2) RT-PCR

[0293] The expression of ILT7 in blood cells was examined in more detail. Each type of cell was isolated from human peripheral blood using a cell sorter. RNA was isolated from each isolated cell population, and cDNA was synthesized using this RNA as a template. Quantitative PCR was performed using the obtained cDNA in the usual manner, and the expression level of ILT7 mRNA was analyzed. The PCR conditions and the base sequences of the primers used are as follows:

[0294] Forward primer: 5’CTC CAA CCC CTA CCT GCT GTC 3’ (SEQ ID NO: 3)

[0295] Reverse primer: 5’TTC CCA AGG CTC CAC CAC TCT 3’ (SEQ ID NO: 4)

[0296] 94°C for 3 minutes, 1 cycle

[0297] [94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute], 25 cycles

[0298] 72°C for 6 minutes, 1 cycle

[0299] When monocytes, IPC, IPC stimulated with HSV, CD19-positive cells (i.e., B cells), CD3-positive cells (i.e., T cells), T cells stimulated with PMA, and CD56-positive cells (i.e., NK cells) were examined, specific expression of ILT7 in IPC was found( Figure 1a ).

[0300] A-3) Quantitative RT-PCR

[0301] In addition, quantitative PCR was performed using ABI PRISM 7000 (manufactured by Applied Biosystem) to detect the expression in other tissues and organs. As cDNA templates (cDNA panels), BD TM MTC multiple tissue cDNA panels (MTC multiple tissue cDNA panel; Human I; Cat.No.636742, Human immune; Cat.No.636748, Human blood fractions; Cat.No.636750; all manufactured by Becton Dickinson) and the same blood cell-derived cDNA as in 2) above were used.

[0302] The base sequences of the primers used are as follows:

[0303] Forward primer for ILT7: 5’C CT CAA TCC AGC ACA AAA GAA GT 3’ (SEQ ID NO: 5)

[0304] Reverse primer for ILT7: 5’CGG ATG AGA TTC TCC ACT GTG TAA 3’ (SEQ ID NO: 6)

[0305] Forward primer for GAPDH: 5’CCA CCC ATG GCA AAT TCC 3’ (SEQ ID NO: 7)

[0306] Reverse primer for GAPDH: 5’TGG GAT TTC CAT TGA TGA CAA G3’ (SEQ ID NO: 8)

[0307] PCR was performed using ABI PRISM 7000 (manufactured by Applied Biosystem) and SYBR green PCR premix kit (manufactured by the same company). Analysis was performed using Sequence Detection System Software (manufactured by the same company).

[0308] The reaction conditions were as follows:

[0309] Step 1: 50°C for 2 minutes, 1 cycle

[0310] Step 2: 95°C for 10 minutes, 1 cycle

[0311] Step 3: (95°C for 15 seconds, 60°C for 1 minute), 40 cycles

[0312] The expression of the ILT7 gene in each tissue was compared by normalizing the ILT7 gene against the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, the expression of which is known to be stable. As a result, it was observed that ILT7 was not expressed in organs other than lymphoid tissues and was specifically expressed in IPCs.

[0313] B. Preparation of ILT7 and FcRγ expression vectors

[0314] Thereafter, cloning of the gene and preparation of the expression vector were carried out to express the ILT7 protein.

[0315] B-1) Cloning of the ILT7 gene

[0316] Poly(A) + RNA was extracted from IPCs, which were isolated from human peripheral blood, and cDNA was synthesized using an oligo dT primer and the Super Script Choice System for cDNA Synthesis kit. An EcoRI adapter was ligated to the synthesized cDNA, and then the cDNA was ligated to the pME18S vector digested with EcoRI. As a result, a human IPC cDNA library was prepared.

[0317] Using the prepared cDNA library as a template, the ILT7 gene was amplified by PCR using primers with the following base sequences. 1 unit of KOD Plus DNA polymerase (manufactured by TOYOBO CO., LTD) was used in the PCR reaction. The reaction conditions were: 94°C for 2 minutes, 1 cycle; then, [94°C for 15 seconds, 55°C for 30 seconds, 68°C for 2 minutes], 25 cycles.

[0318] Forward primer: 5’CAG GGC CAG GAG GAG GAG ATG 3’ (SEQ ID NO: 9)

[0319] Reverse primer: 5’TCA GCA GAC ACT TCC CCA ACT 3’ (SEQ ID NO: 10)

[0320] The amplified approximately 2 kb ILT7 cDNA fragment was separated and recovered by electrophoresis using 1% agarose gel, and then cloned into the pCR4 Blunt-TOPO plasmid vector (manufactured by Invitrogen) using the Zero Blunt TOPO PCR Cloning Kit (manufactured by Invitrogen). The nucleotide sequence of the obtained gene was analyzed, and it was confirmed that the target ILT7 gene shown in SEQ ID NO: 1 was obtained.

[0321] B-2) Preparation of ILT7 expression vector with FLAG tag

[0322] The following expression plasmids were constructed, which expressed proteins formed by fusing FLAG tags at the N-terminus or C-terminus of ILT7. ILT7 was fused with the tag, and the expression of ILT7 protein could be confirmed by detecting the tag. Using the ILT7 gene obtained in 1) as a template and primers with the following nucleotide sequences, the target sequence was amplified by PCR. 1 unit of KOD Plus DNA polymerase (manufactured by TOYOBO CO., LTD) was used in the PCR reaction. The reaction conditions were: 94°C for 2 minutes, 1 cycle; then, [94°C for 15 seconds, 55°C for 30 seconds, 68°C for 2 minutes], 25 cycles.

[0323] For N-FLAG ILT7

[0324] Forward primer (SEQ ID NO: 11): 5’CCG ctc gag ATG ACC CTC ATT CTC ACA AGC CTGCTC TTC TTT GGG CTG AGC CTG GGC [GAT TAC AAG GAT GAC GAC GAT AAG] CCC AGG ACCCGG GTG CAG GCA GAA 3’

[0325] Reverse primer (SEQ ID NO: 12): 5’C TAG act agt TCA GAT CTG TTC CCA AGG CTC 3’

[0326] For C-FLAGILT7

[0327] Forward primer (SEQ ID NO: 13): 5’CCG ctc gag ATG ACC CTC ATT CTC ACA AGC 3’

[0328] Reverse primer (SEQ ID NO: 14): 5’C TAG act agt TCA [CTT ATC GTC GTC ATC CTT GTA ATC]GAT CTG TTC CCA AGG CTC 3’

[0329] In the above base sequence, the underlined part within the parentheses shows the base sequence encoding the FLAG tag, and each set of lowercase letters shows the cleavage site of the restriction enzyme XhoI or SpeI. The DNA fragment amplified by PCR was digested with XhoI and SpeI, and then the fragment was separated by gel electrophoresis. A DNA fragment of about 2 kb was recovered and then ligated into the pME18X vector digested with XhoI and SpeI in the same manner as above. Thus, two plasmids capable of expressing the target protein, namely pME18X-N-FLAG ILT7 and pME18X-C-FLAG ILT7, were constructed respectively.

[0330] B-3) Cloning of the FcRγ gene

[0331] The FcRγ protein is considered to be a protein that can bind to the ILT7 protein. This molecule is a gene (Genbank Acc#NM_004106, J. Biol. Chem. 265, 6448-6452 (1990)) having the base sequence and amino acid sequence shown in SEQ ID NOs: 15 and 16. This molecule is a molecule (γ chain) that constitutes FcεRI (high-affinity IgE receptor). Although it is also named FcεRIγ, here this molecule is called FcRγ. In this regard, it is known that this molecule is also a component molecule of FcγR or FcαR. This gene was cloned by the PCR method shown below to prepare an expression vector.

[0332] Using the human IPC cDNA library prepared in 1) as a template and primers having the following base sequences, the FcRγ gene was amplified by the PCR method. 1 unit of KOD Plus DNA polymerase (manufactured by TOYOBO CO., LTD) was used in the PCR reaction. The reaction conditions were: 94°C for 2 minutes, 1 cycle; then, [94°C for 15 seconds, 55°C for 30 seconds, 68°C for 1 minute], 25 cycles.

[0333] Forward primer: 5’CCC AAG ATG ATT CCA GCA GTG 3’ (SEQ ID NO: 17)

[0334] Reverse primer: 5’GGA AGA ACC AGA AGC CAA AGA 3’ (SEQ ID NO: 18)

[0335] The amplified approximately 0.3 kb FcRγ cDNA fragment was separated and recovered using 2% agarose gel, and then cloned into the pCR4Blunt-TOPO plasmid vector (produced by Invitrogen) using the Zero Blunt TOPO PCR Cloning Kit (produced by Invitrogen). The base sequence of the obtained gene was analyzed, and it was confirmed that the target FcRγ gene shown in SEQ ID NO: 15 had been cloned.

[0336] B-4) Preparation of FcRγ expression vector with Myc tag

[0337] An expression plasmid capable of expressing a protein with a Myc tag linked to the C-terminus was constructed to confirm the expression of the FcRγ protein. Using the FcRγ gene prepared in 3) above as a template and primers with the following base sequences for PCR, the target sequence was amplified. 1 unit of KOD Plus DNA polymerase (manufactured by TOYOBO CO., LTD) was used in the PCR reaction.. The reaction conditions were: 94°C for 2 minutes, 1 cycle; [94°C for 15 seconds, 55°C for 30 seconds, 68°C for 1 minute], 25 cycles.

[0338] Forward primer (SEQ ID NO: 19): 5’CCG ctc gag ATG ATT CCA GCA GTG GTC TTG 3’

[0339] Reverse primer (SEQ ID NO: 20): 5’CTA Gac tag tCT A [CA GAT CCT CTT CAG AGA TGA GTT TCT GCT C] CT GTG GTG GTT TCT CAT G 3’

[0340] In the above base sequence, the underlined part in parentheses shows the base sequence encoding the Myc tag, and each group of lowercase letters shows the cleavage sites of the restriction enzymes XhoI or SpeI. The DNA fragment amplified by PCR was digested with XhoI and SpeI, and then the fragment was separated by gel electrophoresis. The approximately 0.3 kb DNA fragment was recovered and then ligated into the pME18X vector digested with XhoI and SpeI by the same method as above. Thus, a plasmid capable of expressing the desired protein, namely pME18X-Myc-FcRγ, was constructed.

[0341] C. Expression of ILT7 in animal cells

[0342] The expression of ILT7 in animal cells was detected using the expression vector prepared above.

[0343] C-1) Expression in 293T cells

[0344] The following five combinations of DNA were introduced into 293T cells (7 x 10 5 cells) using the Effectene Transfection Kit (manufactured by Qiagen). Two days after the introduction, flow cytometry analysis (FCM analysis) was performed.

[0345] (1) pME18X-N-FLAG ILT7 2 μg

[0346] (2) pME18X-C-FLAG ILT7 2 μg

[0347] (3) pME18X-N-FLAG ILT7 1 μg + pME18X-Myc-FcRγ 1 μg

[0348] (4) pME18X-C-FLAG ILT7 1 μg + pME18X-Myc-FcRγ 1 μg

[0349] (5) pME18X-Myc-FcRγ 2 μg

[0350] FCM analysis was performed according to the same method as described in A-4 of Example 2 below. The reaction was carried out using an anti-FLAG antibody conjugated with Cy3 (manufactured by Sigma), and the analysis was performed using a FACScan (manufactured by Becton Dickinson). As a result, it was clarified that: when reacted alone, only a small amount of ILT7 was expressed on the cell surface; however, when coexisting with FcRγ, ILT7 was strongly expressed extracellularly ( Figure 3 ). Moreover, it is known that murine FcRγ has a high degree of homology with human FcRγ, but when using p815 cells (murine mastocytoma) that endogenously express murine FcRγ as the host, no expression of ILT7 was observed.

[0351] C-2) Analysis by immunoprecipitation reaction and Western blotting

[0352] It was confirmed that ILT7 was co-expressed with FcRγ on the cell surface according to the following method. 293T cells co-expressing two genes in the form of the above combinations (1) to (5) were analyzed using various antibodies after immunoprecipitation reaction.

[0353] As in 1) above, DNA was introduced into 293T cells (7 x 10 5Cells) were harvested after two days. The cell components were lysed with lysis buffer (0.5% Triton, 150 mM NaCl), and the solution was then incubated on ice for 20 minutes. Subsequently, pipetting was performed several times using a needle (27G), and then centrifugation was carried out at 15K rpm for 20 minutes. Anti-myc antibody (2 μg, produced by Santa cruz biotechnology) or anti-FLAG antibody (2 μg, produced by Sigma) was added to 200 μg of the obtained lysate, and then rotation stirring was performed at 4°C for 4 hours. Then, Protein A / G Sepharose 4 Fast Flow mix (produced by Amersham bioscience) was added thereto, and rotation stirring was performed at 4°C for 1 hour. Then, the obtained precipitated component was washed 3 times with lysis buffer having the following components.

[0354] Lysis buffer:

[0355] 0.5% Triton X-100,

[0356] 50 mM HEPES (pH 7.6),

[0357] 150 mM NaCl,

[0358] 1 mM EDTA,

[0359] 10% glycerol,

[0360] 1 mM DTT,

[0361] 2 mM PMSF,

[0362] 1 μg / ml aprotinin,

[0363] 1 μg / ml leupeptin,

[0364] 1 μg / ml pepstatin A,

[0365] 0.1 μg / ml chymostatin,

[0366] 1 mM Na3VO4,

[0367] 0.1 mM β-glycerophosphate

[0368] SDS-PAGE sample buffer was added to the washed precipitate, boiled for 5 minutes and centrifuged, and then electrophoresis was carried out using a 10% SDS gel. After electrophoresis, the sample was transferred from the gel to a PVDF membrane (Immobilon-p-transfer membrane: produced by Millipore) according to the conventional method. Blotting experiments were carried out using anti-FLAG antibody and anti-myc antibody. Since its presence was observed in each immunoprecipitation fraction, it was confirmed that both ILT7 and FcRγ were bound and present in 293T cells ( Figure 4 ).

[0369] C-3) Analysis of sugar chains

[0370] Since several bands of ILT7 were observed in Western analysis, the possibility that ILT7 was glycosylated was investigated. Immunoprecipitation was carried out on 200 μg of 293T cell lysates expressing N-FLAG ILT7 and Myc-FcRγ using anti-FLAG antibody according to the method described in 1) and 2). Thereafter, the precipitate was resuspended in 60 μL of N-glycosidase buffer having the following components, and 30 μL was dispensed into each of two tubes.

[0371] N-glycosidase buffer:

[0372] 10 mM EDTA,

[0373] 0.2% SDS,

[0374] 0.5% Triton X100,

[0375] PBS (phosphate buffer) containing 1% 2-mercaptoethanol

[0376] Then, 3 μL of 3 activity units of N-glycosidase (#1365177, produced by Roche) was added to one tube, and the reaction was carried out at 37 °C for 15 hours. And, 7 μL of loading buffer was added thereto, heated at 100 °C for 5 minutes, and then electrophoresis was carried out using a 10% SDS gel. After electrophoresis, the gel was transferred to a PVDF membrane, 1 μg of the anti-ILT7 polyclonal antibody described in 4) was added, and the reaction was carried out overnight at 4 °C. Washed with TBS-T buffer and reacted with a 100,000-fold diluted HRP-labeled anti-rabbit antibody (manufactured by Jackson) at room temperature. Then, color development was carried out using an ECL Western Blotting Detection System (produced by Amersham bioscience). As a result, the apparent molecular weight decreased after N-glycosidase treatment. Therefore, it was considered that ILT7 carried sugar chains ( Figure 5 ).

[0377] C-4) Preparation of anti-ILT7 polyclonal antibody

[0378] The anti-ILT7 polyclonal antibody used in 3) was prepared as follows. A peptide corresponding to 23 amino acids at the C-terminus of ILT7 (CSQEANSRKDNAPFRVVEPWEQI; SEQ ID NO: 21) was chemically synthesized, and the peptide was conjugated with KLH protein as a carrier as an immunogen. A rabbit was immunized intradermally with the immunogen mixed with Freund's complete adjuvant. After a total of six immunizations (once a week), an increase in antibody titer in the serum was confirmed and whole blood was collected. Then, a part of the serum was affinity-purified using a peptide affinity column with the same sequence as the anti-ILT7 polyclonal antibody.

[0379] Example 2

[0380] A. Preparation of anti-ILT7 monoclonal antibody

[0381] A-1) Preparation of immunogen

[0382] Genes were introduced into 293T cells by the method described below, and an immunogen was prepared by this method. To 3 mL of opti-MEM (GIBCO) covering the entire bottom of a 100 mm / Collagen Coated Dish (culture dish) (IWAKI), 46.4 μg of transgenic genes (23.2 μg of pME18X-C-FLAGILT7 and 23.2 μg of pME18X-Myc-FcRγ) were added and mixed. Thereafter, the transgenic gene solution was left aside, and 58 μL of Lipofectamine (trade name) 2000 (Invitrogen) was diluted with 3 mL of opti-MEM and left at room temperature for 5 minutes to prepare a Lipofectamine solution by such steps. Thereafter, the Lipofectamine solution was added to the culture dish containing the transgenic gene solution and mixed. After leaving at room temperature for 20 minutes, 293T-cells were diluted with DMEM medium (SIGMA) containing 10% FBS (fetal bovine serum) into a cell solution of 1 x 10 6 cells / mL, and then 10 mL of this cell solution was gently added to the culture dish. The cells were statically cultured in a CO2 incubator at 37 °C for 48 hours, and then the cells were recovered by pipette as an immunogen with a transfectant.

[0383] A-2) Preparation of hybridoma

[0384] One day before cell immunization, 50 μL of an emulsion was injected into the soles of the feet of four 4-week-old female Balb / c mice for immunization. The emulsion was prepared by mixing 200 μL of PBS with 200 μL of adjuvant (complete Freund's adjuvant) (RM606-1, produced by Mitsubishi Kagaku Iatron, Inc.). One day later, 2 x 10 7 cells were suspended in 400 μL of PBS, and 50 μL of each was used for immunization. Thereafter, the second and third immunizations were performed every three days. Three days after the third immunization, cell fusion was performed as follows.

[0385] Cells were collected from the popliteal lymph nodes of the immunized mice. These were mixed with mouse myeloma cells P3-X63-Ag8-U1 cultured in RPMI 1640 medium (SIGMA) containing 10% FBS such that the ratio of cells obtained from the lymph nodes and myeloma was 2:1 to 10:1, and then the cells were recovered by centrifugation. G4000 (MERCK) diluted with an equal volume of RPMI 1640 medium was added to the resulting cell fraction for cell fusion. After washing the cells, they were resuspended in 160 mL of 15% FBS-HAT medium with supplements and then the mixture was inoculated into 16 96-well plates at 200 μL / well. The medium was changed three days later. After one to two weeks of observing clone formation, primary screening was performed.

[0386] A-3) Screening of hybridomas by Cell ELISA

[0387] Hybridomas producing the target antibody were screened by the following Cell ELISA. At 1 x 10 7The concentration of cells per 96-well plate was prepared using the cells prepared in 1), resuspended with 0.5% BSA / 2 mM EDTA / PBS solution, and then aliquoted into a culture plate for Cell ELISA (NUNC 249570 96V NW PS) at a volume of 100 μL / well. Centrifugation was performed at 2,000 rpm for 2 minutes at 4°C, and then the supernatant was discarded. The sampled supernatant was added at a volume of 50 μL / well and reacted at room temperature for 30 minutes. Two washing operations were performed, and the washing operation was as follows: 0.5% BSA / 2 mM-EDTA / PBS was added to each well, centrifuged at 2,000 rpm for 2 minutes at 4°C, and then the supernatant was discarded. After washing, 50 μL of a 10,000-fold diluted peroxidase-labeled goat anti-mouse IgG antibody (IM0819; Beckman coulter) was added to each well and reacted for 30 minutes. Three washing operations were performed using 0.5% BSA / 2 mM-EDTA / PBS, and then a chromogenic solution was added to measure OD450nm - 620nm, and the wells showing a positive reaction were selected.

[0388] A-4) Investigation of antibody reactivity using flow cytometry (FCM)

[0389] The hybridoma culture supernatant was analyzed by the method of flow cytometry (FCM). The cells prepared in 1) above were resuspended with 0.5% BSA / 2 mM EDTA / PBS, and then collected into a centrifuge tube at a volume of 1 x 10 5 per sample. Thereafter, 40 μL of each culture supernatant was added and reacted at room temperature for 30 minutes. Two washing operations were performed, and the washing operation was as follows: 1 ml of 0.5% BSA / 2 mM-EDTA / PBS was added to each tube, centrifuged at 1,200 rpm for 3 minutes at 4°C, and then the supernatant was discarded. After washing, 40 μL of a 100-fold diluted FITC-labeled goat anti-mouse IgG antibody (IM0819; Beckman coulter) was added to each well and reacted at room temperature for 30 minutes. Two washing operations using 0.5% BSA / 2 mM-EDTA / PBS were performed, and then analysis was performed using a flow cytometer FC500 (Beckman coulter). Hybridomas producing antibodies were selected, and the antibodies did not react with host cells but specifically reacted with cells transfected with the gene. The selected hybridomas were cloned by the limiting dilution method, and hybridomas #11 and #17 producing monoclonal antibodies were obtained.

[0390] B. Investigation of antibody reactivity against ILT-7

[0391] In 293T cells, ILT7 and FcRγ molecules with a FLAG tag linked to the N-terminus were co-expressed by the same method as described in C-1) of Example 1. Then, the reactivity of the antibody obtained in Example 2 was confirmed by FCM analysis using a FACScan (Becton Dickinson). As a result, it was confirmed that the antibodies produced by hybridomas #11 and #17 obtained in A were both reactive to cells into which the ILT7 gene had been introduced and that expressed ILT7 ( Figure 6b ).

[0392] In addition, lymphocytes were isolated from human peripheral blood using Ficoll, and then double-stained with the prepared anti-ILT-7 antibody and a PE-labeled anti-BDCA-2 antibody (Miltenyi). Then, the reactivity of this antibody to lymphocytes was examined. As a result, binding of the monoclonal antibodies produced by hybridomas #11 and #17 to BDCA-2-positive cells was detected. That is, it was confirmed that both antibodies recognize the ILT7 molecule expressed on IPCs ( Figure 6a ). These monoclonal antibodies were named anti-ILT-7 antibody #11 and anti-ILT-7 antibody #17, respectively. More detailed analysis was also performed.

[0393] Human peripheral blood lymphocytes were analyzed by multi-staining using the prepared anti-ILT-7 antibody, anti-lineage 1 antibody (anti-CD3, CD14, CD16, CD19, CD56 antibody; Becton Dickinson), anti-CD123 antibody (Becton Dickinson), and anti-BDCA-2 antibody (Miltenyi). For the ILT7 antibody-positive fraction, the lineage marker was negative, CD123 was positive, and BDCA-2 was positive. From these results, it was confirmed that ILT7#11 and ILT7#17 stained only IPCs ( Figure 7 ).

[0394] In addition, when peripheral blood lymphocytes were stimulated with CpG or IFNα for 24 hours, the expression of various molecules was detected by FCM analysis. CpG ODN 2216 was used as CpG A that induces IPC to produce IFN, and CpG ODN 2006 was used as CpG B that promotes dendritic cell maturation (Moseman et al. J. Immunology. 173, 4433 - 4442, 2004). Standards were set for the lineage marker-negative fraction. When analyzing the reactivity of anti-BDCA-2 antibody and anti-ILT-7 antibody against the CD123-positive cell population, after 24 hours of CpG stimulation, the ILT7-positive fraction basically disappeared. On the other hand, for BDCA-2, some cells still showed positive after 24 hours of CpG stimulation( Figure 8 ). It is considered that IPC differentiates into different cells immediately after CpG stimulation, which indicates that the anti-ILT-7 antibody of the present invention can be used as a stage-specific antibody for IPC. In addition, it was confirmed that in the presence of IFNα, IPC in peripheral blood lymphocytes does not differentiate and has a high survival rate, but at this time the expression of ILT7 is maintained on IPC, and in autoimmune diseases where IFN in serum may be at a high level, ILT7 on IPC stably exists.

[0395] C. Investigation of the specificity of the anti-ILT-7 antibody

[0396] ILT7 belongs to the ILT / LIR family, and many molecules in this family have high homology, especially in the extracellular region( Figures 9a - 9c ). The expression of mRNAs such as ILT2 and ILT3 has been reported in IPC (Ju et al. Gene 331, 159 - 164, 2004). Therefore, the reactivity of such molecules was confirmed using transgenic cells.

[0397] C-1) Cloning of the ILT1 molecule and preparation of the expression vector

[0398] Using an oligo dT primer and a SuperScript Choice System cDNA synthesis kit, cDNA was synthesized using RNA obtained from human tonsils as a template. A NotI linker was ligated to the synthesized cDNA, and then it was ligated to the pME18S vector digested with NotI enzyme, and as a result, a human tonsil cDNA library was prepared.

[0399] Using the obtained cDNA library as a template and primers with the following base sequences, the ILT1 gene with a FLAG tag linked to the C-terminus was amplified by PCR. 1 unit of KOD Plus DNA polymerase (manufactured by TOYOBO CO., LTD) was used in the PCR reaction. The reaction conditions were: 94°C for 2 minutes, 1 cycle; then, [94°C for 15 seconds, 55°C for 30 seconds, 68°C for 2 minutes], 25 cycles.

[0400] Forward primer (SEQ ID NO: 22): 5’CCG ctc gag ATG ACC CCC ATC CTC ACG GTC C 3’

[0401] Reverse primer (SEQ ID NO: 23): 5’CTA Gac tag tTC A CT TAT CGT CGT CAT CCT TGT AAT C CC TCC CGG CTG CAT CTT G 3’

[0402] In the above primer sequences, the underlined part in parentheses shows the base sequence encoding the FLAG tag, and each set of lowercase letters shows the cleavage sites of the restriction enzymes XhoI or SpeI. The DNA fragment amplified by PCR was digested with XhoI and SpeI, and then the fragment was separated by gel electrophoresis. A DNA fragment of about 2 kb was recovered and then ligated into the pME18X vector digested with XhoI and SpeI in the same method as above. Thus, a plasmid capable of expressing the target fusion protein, namely pME18X-C-FLAGILT1, was constructed. Its base sequence and amino acid sequence are shown in SEQ ID NO: 24 and 25.

[0403] C-2) Preparation of expressing cells and investigation of antibody reactivity

[0404] For ILT2 (SEQ ID NO: 26) and ILT3 (SEQ ID NO: 28), expression vectors prepared by cloning the two genes into the XbaI and XhoI sites of pcDNA4.1 (produced by Invitrogen) were used. The following DNA combinations were introduced into 293T cells (7 x 10 5 cells) using the same method as described in C-1). Two days after the introduction, flow cytometry analysis (FCM analysis) was performed to analyze the anti-ILT7 antibody.

[0405] (1) pME18X-N-FLAG ILT7 1 μg + pME18X-Myc-FcRγ 1 μg

[0406] (2) 0.5 μg of pME18X-C-FLAG ILT1 + 0.5 μg of pME18X-Myc-FcRγ + 0.5 μg of pcDNA4.1-ILT2 + 0.5 μg of pcDNA4.1-ILT3

[0407] As a result, no antibody reacted with the cells expressing ILT1 or the like. For this reason, it was demonstrated that the anti-ILT7 antibody specifically recognizes the ILT7 molecule on IPCs ( Figure 10 ).

[0408] Example 3

[0409] Effect of anti-ILT-7 antibody on the ability to produce human IFN

[0410] Human peripheral blood lymphocytes were seeded into 96-well plates at a density of 2 x 10 5 cells / well and then reacted with various antibodies at 37°C, with the amount of each antibody being 5 μg / mL. After culturing for 1 hour, influenza virus PR8 was added thereto. After culturing for 24 hours, IFNα in the culture supernatant was measured using an ELISA kit (Bender Med System). As a result, the production of IFN was inhibited by the addition of the anti-ILT-7 antibody ( Figure 11 ). That is, it was clarified that the anti-ILT-7 antibody of the present invention affects the IFN-producing activity of IPCs.

[0411] Example 4

[0412] CDC activity of anti-ILT-7 antibody

[0413] A. Preparation of monoclonal antibody against ILT7

[0414] Clones producing monoclonal antibodies were obtained by the same method described in A-1) to A-4) of Example 2. Reactivity was measured by the same method described in B of Example 2, and specificity was measured by the same method described in C of Example 2. As a result, hybridomas #37, #28, and #33 producing anti-ILT7 monoclonal antibodies with good reactivity and specificity were obtained. The CDC activity was measured using the anti-ILT7 monoclonal antibodies produced by these three hybridomas according to the following method.

[0415] B. Determination of CDC activity

[0416] B-1) On the previous day, the following DNA was introduced into CHO-k1 cells using Effectene Transfection Reagent (manufactured by QLAGEN). The CHO-k1 cells were seeded on 6 cm φ dishes at a density of 6 x 10 5 cells per dish, and then resistant strains were selected using 800 μg / ml Zeocin (manufactured by Invitrogen).

[0417] Introduced DNA: 1 μg of pcDNA3.1-C-FLAG ILT7 + 2 μg of pME18X-Myc FcRγ

[0418] Thereafter, a cell line that highly expressed ILT7 was obtained using a cell sorter (BD FACSAria, manufactured by Becton Dickinson). The selected cell line was confirmed to highly express ILT7 by FCM analysis. FCM analysis was performed in the same manner as described in A-4) of Example 2, except that BD FACSCaliber (manufactured by BD) was used for FCM. The following antibodies were used as the first antibody and the second antibody, respectively.

[0419] First antibody: 5 μg / ml mouse anti-ILT-7 antibody (#37),

[0420] Second antibody: R-phycoerythrin (R-PE)-conjugated goat anti-mouse immunoglobulin-specific polyclonal antibody (BD)

[0421] B-2) Reaction of target cells with anti-ILT7 antibody

[0422] The target cells (ILT7-CHO cells) obtained in B-1) were recovered using a 5 mM EDTA / PBS solution, and then the cells were resuspended in CDC medium containing the following components to obtain a concentration of 4 x 10 5 cells / ml. The resuspended solution was dispensed into V-bottom 96-well plates at a concentration of 50 μl / well.

[0423] CDC medium:

[0424] RPMI1640

[0425] 0.1% BSA

[0426] 100 units of activity / ml penicillin

[0427] 100 μg / ml streptomycin

[0428] 10 mM Hepes (pH 7.6)

[0429] 2 mM L - Glutamine

[0430] 50 μl of anti - ILT - 7 antibody solution prepared with CDC medium was added to each well and mixed to a final antibody concentration of 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, and 5 μg / ml. In addition, 50 μl of complement - containing CDC medium with the following components was added thereto and mixed to a final complement concentration of 6%, and then cultured at 37 °C for 2 hours.

[0431] Complement - containing CDC medium:

[0432] 1 ml of complement from young rabbits (Catalog No.: CL3441, produced by CEDARLANE)

[0433] CDC medium (same as above)

[0434] Then, the suspension was centrifuged (centrifugation conditions: 250 G for 4 minutes) and the supernatant was recovered, taking care not to introduce cell contamination during recovery. The LDH in the supernatant was measured by a conventional method, and this result was determined as "the amount of LDH leaked from the target cells due to complement activity (experimental sample)".

[0435] The following parameters were also measured to determine the activity of CDC.

[0436] - Target Cell Spontaneous LDH Release: Only the target cells of the same volume as the sample were cultured and prepared.

[0437] - Target Cell Maximum LDH Release: Only the target cells of the same volume as the sample were cultured, and then 60 minutes before recovering the supernatant, TritonX - 100 solution included in the kit was added thereto to a final concentration of 0.8% for preparation.

[0438] - Volume Correction Control: The same amount of TritonX - 100 as that added in preparing the target cell maximum LDH release was added to the medium of the same volume as the sample for preparation.

[0439] - Culture Medium Background: A culture medium having the same volume as the sample was prepared, and a solution was made by adding complement-containing CDC medium to the culture medium to have the same volume as the sample.

[0440] The following corrections were made: The absorption of the culture medium having the same volume as the sample was subtracted from the target maximum and target spontaneous absorptions, and the absorption of the solution made by adding complement-containing CDC medium to the culture medium to have the same volume as the sample was subtracted from the absorption of the experimental sample.

[0441] The CDC activity was calculated by the following equation. The results are shown in Table 1 and Figure 12 and are presented below. In examples using anti-ILT7 monoclonal antibodies obtained from any hybridoma, when the antibody concentration was 0.5 μg / ml or higher, 80% or more CDC activity was exhibited.

[0442] CDC activity = [(experimental sample - target spontaneous) / (target maximum - volume control - target spontaneous)] × 100

[0443] [Table 1]

[0444]

[0445] Comparative Example 1

[0446] Except for replacing the anti-ILT7 antibody with mouse IgG2a, all other procedures were carried out in exactly the same manner as described in B and C of Example 4. The results are shown together with those of Example 4 in Table 5 and Figure 12 and are presented below. No CDC activity against the target cells was observed in antibodies other than the anti-ILT7 monoclonal antibody.

[0447] Example 5

[0448] Internalization of Anti-ILT-7 Antibody on Target Cells

[0449] A. Anti-ILT7 Monoclonal Antibodies

[0450] The following anti-ILT7 monoclonal antibodies were used. Anti-ILT7 monoclonal antibodies: #17, #26, #37, #28, and #33

[0451] B. Observation of Internalization

[0452] B-1) Preparation of Target Cell Line (ILT7-CHO Cell Line)

[0453] The target cell line (ILT7-CHO cell line) was prepared by the same method as described in B-1 of Example 4.

[0454] B-2) Reaction of the target cells with anti-ILT-7 antibody

[0455] The recovered ILT7-CHO cells were resuspended in ice-cold (T(-)+10% FBS) buffer containing the following components at a concentration of 1 x 10 6 cells / ml, and a 5 mM EDTA / PBS solution was used when recovering the cells.

[0456] T(-) medium:

[0457] RPMI1640

[0458] 100 units of penicillin / ml

[0459] 100 μg / ml streptomycin

[0460] 10 mM Hepes (pH 7.6)

[0461] 2 mM L-glutamine (Glutamin)

[0462] 1 mM sodium pyruvate

[0463] 50 μM 2-mercaptoethanol

[0464] 10% heat-inactivated fetal bovine serum

[0465] 1 ml of the above suspension was placed in a 15 ml centrifuge tube and centrifuged (centrifugation conditions: centrifuged at 1200 rpm for 5 minutes at 4°C), and then the supernatant was discarded. 200 μL of anti-ILT7 monoclonal antibody suspension (10 μg / ml) was added to the cell pellet, then it was mixed and incubated at 4°C for 30 minutes, and then washed twice with ice-cold T(-) medium (amount of medium used: 10 ml each time, centrifugation conditions: centrifuged at 1200 rpm for 5 minutes at 4°C).

[0466] B-3) Modification of the ILT7-anti-ILT-7 antibody immune complex present on the surface of the target cells

[0467] Subsequently, the ILT7 - anti - ILT - 7 antibody immune complex present on the cell surface was modified with a secondary antibody, enabling detection by fluorescence. The specific method is described below. To the cell pellet obtained in B - 2), ice - pre - cooled T(-) medium containing APC - labeled goat anti - mouse IgG polyclonal antibody (Catalog No.: 550826BD, produced by Biosciences) was added, and then incubated at 4°C in the dark for 20 minutes. Thereafter, it was washed twice with ice - pre - cooled T(-) medium (amount of medium used: 10 ml for each wash, centrifugation conditions: centrifuged at 1200 rpm at 4°C for 5 minutes). Then, ice - pre - cooled T(-) medium was added thereto as a suspension at a concentration of 1 x 10 6 cells / ml.

[0468] B - 4) Inducing internalization by incubation at 37°C

[0469] The suspension obtained in B - 3) was equally divided into two tubes (i.e., tube (a) and (b)). Tube (a) and (b) were incubated at 37°C and 4°C in the dark for 60 minutes, respectively. After incubation, 1% FBS / PBS (ice - pre - cooled) was added thereto to terminate internalization. It was centrifuged (centrifugation conditions: centrifuged at 1200 rpm at 4°C for 5 minutes), then the supernatant was discarded, and then washed twice with 1% FBS / PBS (ice - pre - cooled) (amount of solution: 10 ml for each wash, centrifugation conditions: centrifuged at 1200 rpm at 4°C for 5 minutes).

[0470] B - 5) Modification of the ILT7 - anti - ILT - 7 antibody immune complex remaining on the surface of target cells after incubation

[0471] The ILT7 - anti - ILT - 7 antibody immune complex remaining on the cell surface after incubation was modified with a tertiary antibody, enabling detection by fluorescence. The specific method is described below. To the cell pellet obtained in B - 4), 20 μL of a suspension containing the tertiary antibody (FITC - labeled donkey anti - goat IgG antibody (Catalog No.: sc - 2024, produced by Santa cruz Biotechnology)) was added, and then incubated at 4°C in the dark for 15 minutes. Then, the resulting solution was washed with 1% FBS / PBS (amount of solution: 10 ml for each wash, centrifugation conditions: centrifuged at 1200 rpm at 4°C for 5 minutes). B - 6) Analysis of the anti - ILT - 7 antibody present in target cells

[0472] Subsequently, 150 μL of 1% FBS / PBS was added to the cell pellet obtained in B-5), and then it was resuspended and collected into a 1.2 mL microtitration tube, and FCM analysis was performed on it. In the analysis, the mean fluorescence intensity (MPI) of each cell was analyzed for FITC and APC. Further, the fluorescence intensity ratio (%) was calculated according to the following equation.

[0473] Fluorescence intensity ratio (%) = (Mean fluorescence intensity of cells incubated at 37°C for 60 minutes / Mean fluorescence intensity of cells incubated at 4°C for 60 minutes) × 100

[0474] This result is shown in Table 2, Table 3 and Figure 13 in.

[0475] [Table 2]

[0476]

[0477] [Table 3]

[0478]

[0479] The fluorescence intensity of FITC is an indicator of the amount of the ILT7-anti-ILT-7 antibody immune complex remaining on the cell surface after incubation. Compared with the cells incubated at 4°C, the mean fluorescence intensity of FITC in the cells incubated at 37°C for 60 minutes decreased to approximately 50%.

[0480] On the other hand, the fluorescence intensity of APC is an indicator of the amount of the ILT7-anti-ILT-7 antibody immune complex present on the cell surface before incubation. After incubation, this complex can be detected whether it is present on the cell surface or integrated into the cell. In Example 5, the APC fluorescence intensity after incubation was equal in the example incubated at 37°C compared with the example incubated at 4°C. This result indicates that the ILT7-anti-ILT-7 antibody immune complex is present in any part of the target cells regardless of incubation at either of the two temperatures. As described above, it can be seen that incubation at 37°C caused the internalization of ILT7 by the anti-ILT7 monoclonal antibody.

[0481] Comparative Example 2

[0482] Except that the anti-ILT7 antibody was replaced with mouse IgG2a, otherwise the same method as described in Example 5 was performed exactly. Its results are shown together with Example 5 in Table 2, Table 3 and Figure 13 in. In the example using mouse IgG2a, no change in the fluorescence intensity of FITC was observed, so it can be seen that mouse IgG2a did not cause the internalization of ILT7.

[0483] Example 6

[0484] Structure of mouse anti-human ILT7 monoclonal antibody

[0485] [Sequence of variable region]

[0486] A. Cloning of cDNA encoding variable region of mouse anti-ILT-7 antibody

[0487] A-1) Hybridoma producing mouse anti-ILT7 antibody

[0488] The following hybridomas were used as hybridomas producing mouse anti-ILT7 antibody.

[0489] - Hybridoma #11 (Accession No.: FERM BP-10704)

[0490] - Hybridoma #17 (Accession No.: FERM BP-10705)

[0491] A-2) Isolation of total RNA

[0492] Total RNA was extracted from the hybridomas described in A-1) using a commercially available kit “RNeasy Mini Kit” (Catalog No.: 74106, manufactured by Qiagen) according to the instructions attached to the kit. Approximately 200 μg of total RNA was obtained from 1 x 10 7 hybridoma cells.

[0493] A-3) Amplification and fragmentation of cDNA encoding mouse heavy chain variable region

[0494] Using 5 μg of the total RNA isolated in A-2) as a template, cDNA encoding the mouse heavy chain variable region was amplified by the 5’RACE method. For the amplification, a commercially available kit “5’RACE System for Rapid Amplification of cDNA Ends, Version 2.0 Kit” (Catalog No.: 18374-058, manufactured by Invitrogen) was used. The specific description is as follows. First, the first strand of cDNA was synthesized using reverse transcriptase with the total RNA isolated in A-2) as a template. At this time, the base sequence of the antisense primer (GSP1) is shown in Table 4.

[0495] [Table 4]

[0496] Primers for amplifying mouse heavy chain variable region encoding gene

[0497]

[0498] Thereafter, total RNA was degraded using RNaseH, and the first strand of cDNA remaining in single-stranded form was purified using the low melting point agarose method (1.5%). In addition, dC (i.e., nucleotide homopolymer) was ligated to the 3'-end of the first strand of cDNA using terminal deoxynucleotidyl transferase (TdT). The cDNA was amplified by PCR using an anchor primer (SEQ ID NO: 34) and a reverse primer (GSP2) as shown in Table 4, wherein the anchor primer had a nucleotide homopolymer complementary to dC (anchor sequence) at the 3'-end. In addition, the obtained PCR product was used as a template, and the cDNA was amplified by nested PCR using the AUAP primer (SEQ ID NO: 35) and the reverse primer as shown in Table 4. In addition, the PCR product was purified by the low melting point agarose method (1.5%).

[0499] Anchor primer for 5' RACE (SEQ ID NO: 34)

[0500] 5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3' (36 oligonucleotides)

[0501] AUAP primer for 5' RACE (SEQ ID NO: 35)

[0502] 5'-GGC CAC GCG TCG ACT AGT AC-3' (20 oligonucleotides)

[0503] A-4) Amplification and fragmentation of cDNA encoding the variable region of the mouse light chain

[0504] Using the total RNA isolated in A-2) as a template, the cDNA encoding the variable region of the mouse light chain was amplified using the same method as described in A-3). At this time, the base sequences of the primers used are shown in Table 5. The obtained PCR product was purified using the low melting point agarose method (1.5%).

[0505] [Table 5]

[0506] Primers for amplifying the gene encoding the variable region of the mouse light chain

[0507]

[0508] A-5) Confirmation of the cDNA base sequence and determination of the CDR region

[0509] Using the commercially available kit "Zero Blunt TOPO PCR Cloning Kit" (Catalog No.: 1325137, manufactured by Invitrogen), the cDNA fragments of the heavy chain variable region obtained in A-3) and the light chain variable region obtained in A-4) were cloned into the pCR4Blunt-TOPO vector according to the instructions attached to the kit. Then, the resulting vector was introduced into Escherichia coli competent cells to obtain Escherichia coli transformants. The above plasmid was obtained from the above transformants, and then the cDNA base sequence in the plasmid was confirmed using an automated DNA sequencer "PCR-based ABI PRISM 3100 Genetic Analyzer" (manufactured by Applied Biosystems). The correct sequence was obtained by excluding transcripts derived from inactive RNA, which was caused by frameshift and nonsense mutations around the complementarity-determining region (hereinafter referred to as the "CDR region"). In addition, the homology of the cDNA base sequence contained in the plasmid was compared with the Kabat database, and the sequences of the CDR region and the variable region in each variable region were determined.

[0510] Similarly, for the hybridoma #37 prepared in Example 4, the sequences of the CDR region and the variable region in the variable region were determined according to the procedures described in A-1) to A-5) of Example 6 using hybridoma #17. Shown in the following SEQ ID NO are the base sequences of the cDNA of the heavy chain variable region and the light chain variable region of the anti-ILT7 monoclonal antibody produced by each hybridoma and the amino acid sequences encoded by the sequences.

[0511]

[0512]

[0513] [Confirmation of constant region isotype]

[0514] For the hybridoma culture supernatant, the isotype of the constant region of the prepared monoclonal antibody was confirmed using a commercially available mouse monoclonal antibody typing kit (Catalog No.: MMT1, manufactured by Serotec Product). The heavy chain constant region of mouse anti-human ILT-7 antibody #11 is Igγ3, and the light chain constant region is Igκ. In addition, the heavy chain constant regions of mouse anti-human ILT-7 antibody #17 and mouse anti-human ILT-7 antibody #37 are both Igγ2a, and their light chain constant regions are both Igκ.

[0515] Example 7

[0516] Preparation of chimeric antibody

[0517] A. Cloning of cDNA Encoding Human IgG Constant Region

[0518] The heavy chain constant region of human IgG1 and the light chain constant region of human Igκ were selected from a cDNA library of human IPC. Then, using the commercially available kit "Zero Blunt TOPO PCR Cloning Kit" (Catalog No.: 1325137, produced by Invitrogen), the selected regions were cloned into the pCR4 Blunt-TOPO vector according to the instructions attached to the kit. Then, the resulting vector was introduced into competent Escherichia coli cells to obtain Escherichia coli transformants. The above plasmid was obtained from the above transformants, and then the base sequence of the cDNA in the plasmid was confirmed using an automated DNA sequencer "PCR-based ABI PRISM 3100 Genetic Analyzer" (produced by Applied Biosystems).

[0519] B. Ligation and Cloning of Variable Region and Constant Region

[0520] The cDNA encoding the heavy chain constant region obtained in A and the cDNA encoding the heavy chain variable region obtained in A-5 of Example 6 were used respectively. The two DNAs have a region with overlapping DNA base sequences. Therefore, double-stranded DNA was obtained using the overlap extension method in this region. The detailed method is as follows.

[0521] C-1) Preparation of cDNA Encoding the Heavy Chain of Chimeric ILT7 Antibody

[0522] The "plasmid having cDNA encoding the heavy chain variable regions of #11 and #17" obtained in A-5) was digested with restriction enzymes NotI and XbaI, and then purified by the agarose gel method (1.5%). The resulting product was dissolved in TE buffer having the following composition to prepare a solution of cDNA fragment encoding the heavy chain variable region at a concentration of 100 pmol / μL.

[0523] TE Buffer:

[0524] 10 mM Tris-HCl

[0525] 1 mM EDTA

[0526] pH 7.5 - 8.0

[0527] In addition, the "plasmid with cDNA encoding the heavy chain constant region" obtained in B was processed by the same method as described above to prepare a solution with a concentration of 100 pmol / μL. Subsequently, the two solutions were mixed, and then the overlapping regions of the two were hybridized by initially incubating the mixture at 70°C for 10 minutes and then at 37°C for 5 minutes. Thereafter, the cDNA was amplified by PCR, and the resulting cDNA was digested with restriction enzymes NotI and XbaI, and then purified using the low melting point agarose gel method (1.5%).

[0528] C-2) Preparation of cDNA encoding the light chain of the chimeric ILT7 antibody

[0529] The cDNA encoding the light chain constant region obtained in A and the cDNA encoding the light chain variable region obtained in A-5 of Example 6 were used respectively. Using these cDNAs, the cDNA encoding the light chain of the chimeric ILT7 antibody was obtained by the same method as described in C-1).

[0530] C-3) Cloning

[0531] The cDNA obtained in C-1) was cloned into the plasmid vector pcDNA3.1-Zeocin (produced by Invitrogen) using NotI and XbaI as cloning sites to prepare a chimeric ILT7 heavy chain expression vector. In addition, the cDNA obtained in C-2) was cloned into the plasmid vector pcDNA3.1-hygromycin (produced by Invitrogen) using NotI and XbaI as cloning sites to prepare a chimeric ILT7 light chain expression vector.

[0532] The names of each vector are shown in Table 6.

[0533] [Table 6]

[0534] Name of plasmid vector

[0535] For chimeric ILT7 heavy chain expression For chimeric ILT7 light chain expression #11 pcDNA - #11VH pcDNA - #11VL #17 pcDNA - #17VH pcDNA - #17VL

[0536] D. Expression of chimeric ILT7 antibody

[0537] D-1) Transient transfection

[0538] Using an Effectine transfection kit (Catalog No.: 301427, manufactured by Qiagen), 1 μg of the chimeric ILT7 antibody heavy chain expression vector and 1 μg of the chimeric ILT7 antibody light chain expression vector were co-transfected into 293T cells, and the two vectors were prepared in C-3). Thereafter, culture was carried out at 37 °C using DMEM medium supplemented with 2% Low IgG FBS.

[0539] DMEM medium supplemented with 2% Low IgG FBS:

[0540] DMEM medium (Catalog No.: D5796, produced by Sigma)

[0541] 2% Low IgG FBS (Catalog No.: SH30151.03, produced by HyClone)

[0542] 2 mM L-glutamine

[0543] 100 U / ml penicillin

[0544] 100 μg / ml streptomycin

[0545] pH 7.2 - pH 7.4

[0546] After the vectors were introduced, the cells were cultured for 96 hours, and the culture supernatant was collected. Then, cell debris was removed by centrifugation to obtain a crude antibody solution.

[0547] D-2) Stable transfection

[0548] Using an Effectine transfection kit (Catalog No.: 301427, manufactured by Qiagen), 1 μg of the chimeric ILT7 antibody heavy chain expression vector and 1 μg of the chimeric ILT7 antibody light chain expression vector were co-transfected into YB2 / 0 cells (the cells are from rat myeloma, ATCC#CRL-1622), and the two vectors were prepared in C-3). Among the plasmid vectors used, the heavy chain expression vector was marked with Zeocin resistance, and the light chain expression vector was marked with hygromycin resistance. Therefore, the cells transfected with the two vectors can grow in a medium supplemented with both Zeocin and hygromycin. Then, the cells were cultured using RPMI medium supplemented with Zeocin and hygromycin, and resistant strains were selected.

[0549] RPMI medium supplemented with Zeocin - hygromycin:

[0550] RPMI1640 medium (Catalog No.: R8758, produced by Sigma)

[0551] 10% FBS

[0552] 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid sodium salt)

[0553] 1 mM sodium pyruvate

[0554] 2 mM L-glutamine

[0555] 100 U / mL penicillin

[0556] 100 μg / mL streptomycin

[0557] 55 μM 2-mercaptoethanol

[0558] 0.5 mg / mL Zeocin

[0559] 0.5 mg / mL hygromycin

[0560] pH 7.2 - pH 7.4

[0561] Three days after this operation, the antibody production in the culture supernatant was determined by ELISA method. ILT7 chimeric antibody-producing cell lines with high antibody expression levels and significantly increased cells were selected. In addition, monoclonalization was performed by cell sorting, and the following cell lines were obtained.

[0562] #11 ILT7 chimeric antibody-producing cell lines: #11-5 cell line and #11-16 cell line

[0563] #17 ILT7 chimeric antibody-producing cell line: #17-24 cell line

[0564] The above cell lines (#11-5 cell line, #11-16 cell line, and #17-24 cell line) were cultured separately in RPMI medium supplemented with 5% FBS and having the following components. The incubation temperature and incubation time were set at 37 °C and 96 hours, respectively.

[0565] RPMI medium supplemented with 5% FBS:

[0566] RPMI1640 medium (Catalog No.: R8758, manufactured by Sigma)

[0567] 5% FBS

[0568] 0.01 M HEPES

[0569] 1 mM sodium pyruvate

[0570] 2 mM L-glutamine

[0571] 100 U / mL penicillin

[0572] 100 μg / mL streptomycin

[0573] 55 μM 2-mercaptoethanol

[0574] pH 7.2 - pH 7.4

[0575] The culture supernatant was collected and cell debris was removed by centrifugation to obtain a crude antibody solution.

[0576] E. Purification of Antibody

[0577] Each crude antibody solution obtained in D-1 and D-2 was purified by a Protein A affinity column (rProtein A Sepharose FF, catalog number: 17-1279-01, manufactured by Amersham Pharmacia). The purification conditions were as follows. PBS(-) buffer with the following composition was used as the adsorption buffer, and 0.1 M sodium citrate buffer (pH 3) was used as the elution buffer. Affinity purification was performed according to the attached instructions. 1 M Tris-HCl (pH 8.0) was added to the eluted fraction to adjust the pH to approximately 7.2. For the antibody solution with adjusted pH, it was exchanged into PBS(-) using a dialysis membrane to obtain the purified anti-ILT7 chimeric antibody. Regarding the concentration of the purified antibody, the absorbance value at 280 nm was measured and converted at 1.38 OD as 1 mg / mL. The relationship among the obtained chimeric ILT7 antibody, the hybridoma from which the variable region gene was derived, and the host cell is summarized in Table 7.

[0578] PBS(-) buffer:

[0579] 0.2 g / L potassium dihydrogen phosphate

[0580] 0.2 g / L potassium chloride

[0581] 8 g / L sodium chloride

[0582] 1.15 g / L disodium hydrogen phosphate anhydrous

[0583] [Table 7]

[0584] Prepared chimeric antibody

[0585]

[0586] The amino acid sequences of the heavy and light chains of the prepared chimeric antibody and the cDNA base sequences are shown below. In each amino acid sequence, from the amino acid sequence N-terminus to -1 is the amino acid sequence of the signal sequence, and from position 1 to the C-terminus of the amino acid sequence is the amino acid sequence of the mature protein. That is to say, the heavy and light chains that make up these chimeric antibodies are composed of the amino acid sequences from position 1 to the C-terminus of each of the following amino acid sequences.

[0587]

[0588] Industrial Applicability

[0589] The present invention provides an immunogen that can be used to produce an antibody specifically recognizing human ILT7, and a method for producing an anti-ILT-7 antibody using the immunogen. The antibody specifically recognizing human ILT7 of the present invention can specifically recognize ILT7 in the presence of the ILT family. Therefore, the antibody of the present invention can be used to assay and isolate human ILT7. For example, the antibody of the present invention can also be used to analyze the localization of ILT7. It has been recognized that ILT7 is a molecule closely related to the differentiation and function of IPCs or dendritic cells. Therefore, an antibody that can recognize ILT7 and has high specificity can be used to analyze the function of IPCs or dendritic cells. IPC-like (characterized by expressing BDCA-2) cancer cells are known (Chaper of L et al., Eur. J. Immunol. 34; 418-426, 2004, Maeda T et al., Int. J. Hematol. 81; 148-154, 2005). Confirmation of the expression of ILT7 in these cells may lead to the success of cancer diagnosis and treatment.

[0590] In the case of autoimmune diseases, for example, it has been pointed out that there is a deep relationship between IFNα produced by IPCs and the development of psoriasis, which is a skin disease (Nestle FO et al., J. Exp. Med. 202, 135-143, 2005). Therefore, the degree of psoriasis can be determined by identifying IPCs in the skin tissue of psoriasis patients, that is, using an anti-ILT-7 antibody in biopsy specimens.

[0591] It is known that the development of AIDS in HIV-infected patients is related to the number of IPCs. That is, a large number of IPCs have been observed in patients who have not shown symptoms, and a decrease in IPCs has been observed at the time of onset (Soumells V. et al., Blood 98; 906-912, 2001). Therefore, the prognosis of the infection of viruses such as HIV can be effectively predicted.

[0592] For example, ILT7 is a molecule specifically expressed in human IPC. Therefore, the anti-ILT-7 antibody of the present invention can be used to detect, identify or isolate IPC. IPC is the cell that produces most type I interferons. Therefore, the detection, identification or isolation of this molecule is an important indicator in the diagnosis and study of diseases related to type I interferons. Diseases such as various autoimmune diseases and infections that may have been shown to involve interferons in the process of pathological state formation.

[0593] In addition, the anti-ILT-7 antibody of the present invention has an inhibitory effect on the activity of IPC. Therefore, the anti-ILT-7 antibody of the present invention can be used to inhibit the activity of IPC. In addition, diseases involving type I interferons can be treated by inhibiting the activity of IPC. Specifically, the anti-ILT-7 antibody of the present invention can be used for diseases such as various autoimmune diseases and infections that involve interferons in the process of pathological state formation. Particularly, due to the high specificity of the anti-ILT-7 antibody, the antibody can effectively remove IPC.

Claims

1. Use of an anti-human immunoglobulin-like transcript 7 (ILT7) monoclonal antibody comprising the extracellular domain of human ILT7 of SEQ ID NO: 2, or a polynucleotide encoding said monoclonal antibody, in the preparation of a reagent for detecting interferon-producing cells, wherein the antibody comprises, as CDR1, CDR2 and CDR3 in the heavy chain variable region and the light chain variable region, an amino acid sequence according to any one of the following i) to iii): i) CDR1 of the heavy chain variable region: SDYAWN (SEQ ID NO: 58); CDR2 of the heavy chain variable region: YISYSGSTSYNPSLKSR (SEQ ID NO: 59); and CDR3 of the heavy chain variable region: SPPYYAMDY (SEQ ID NO: 60); CDR1 of the light chain variable region: KASQDVGTAVA (SEQ ID NO: 61); CDR2 of the light chain variable region: WASTRIAT (SEQ ID NO: 62); and CDR3 of the light chain variable region: QQYSSYPLT (SEQ ID NO: 63); ii) CDR1 of the heavy chain variable region: SYWIH (SEQ ID NO: 64); CDR2 of the heavy chain variable region: RIYPGTGSTYNNEKFKG (SEQ ID NO: 65); and CDR3 of the heavy chain variable region: YPTYDWYFDV (SEQ ID NO: 66); CDR1 of the light chain variable region: RASQSISNYLH (SEQ ID NO: 67); CDR2 of the light chain variable region: YASQSIS (SEQ ID NO: 68); and CDR3 of the light chain variable region: QQSNSWPLT (SEQ ID NO: 69); iii) CDR1 of the heavy chain variable region: SDYAWN (SEQ ID NO: 70); CDR2 of the heavy chain variable region: YISYSGSTSYNPSLKSR (SEQ ID NO: 71); and CDR3 of the heavy chain variable region: ALPLPWFAY (SEQ ID NO: 72); CDR1 of the light chain variable region: KASQDVGTAVA (SEQ ID NO: 73); CDR2 of the light chain variable region: WASTRHT (SEQ ID NO: 74); and CDR3 of the light chain variable region: QQYSSYPYT (SEQ ID NO: 75).

2. Use according to claim 1, wherein, The activity of the interferon-producing cells is interferon-producing activity, or the survival of interferon-producing cells, or both.

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