anti-ILT 7 antibodies
阅读说明：本技术 抗ilt7抗体 (anti-ILT 7 antibodies ) 是由 鸭川由美子 赵民权 新井直子 石田晃司 于 2006-12-20 设计创作，主要内容包括：本发明涉及抗ILT7抗体。能够结合IPC的抗体是通过利用动物细胞来获得的,在该细胞中与ILT7结合的细胞膜蛋白是作为免疫原共表达的。本发明的抗体具有高特异性,使得该抗体能够从免疫学上将ILT7与其他ILT家族分子区分开来。本发明的抗ILT-7的抗体能够结合IPC并抑制其活性。通过本发明的抗ILT-7的抗体可以抑制IPC的活性,并且可以治疗或预防干扰素相关疾病。在IFNα存在的情况下,ILT7在IPC中的表达仍可维持。因此,在IFNα生成量增加的多种自身免疫病病人体内,可以预期抗ILT-7的抗体对于IPC活性的抑制作用。(The present invention relates to an anti-ILT 7 antibody, which is obtained by using an animal cell in which a cell membrane protein that binds to ILT7 is co-expressed as an immunogen, and which has high specificity so that the antibody can immunologically distinguish ILT7 from other ILT family molecules, an anti-ILT-7 antibody of the present invention can bind to and inhibit the activity of IPC, and an interferon-related disease can be treated or prevented by the anti-ILT-7 antibody of the present invention, and the expression of ILT7 in IPC can be maintained in the presence of IFN α, and thus, an inhibitory effect of the anti-ILT-7 antibody on the activity of IPC can be expected in various autoimmune disease patients with an increased production amount of IFN α.)
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.
2. The monoclonal antibody or a fragment thereof comprising an antigen binding region according to claim 1, wherein said monoclonal antibody is capable of binding to a human interferon producing cell.
3. A monoclonal antibody produced by the hybridoma ILT7#11 deposited under the number FERM BP-10704 or the hybridoma ILT7#17 deposited under the number FERM BP-10705, or a fragment containing an antigen binding region of the monoclonal antibody.
4. The monoclonal antibody according to claim 1, or a fragment containing an antigen-binding region thereof, wherein the monoclonal antibody contains the following amino acid sequences i) to iii) as CDR1, CDR2 and CDR3 in the heavy chain variable region and the light chain variable region:
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 light chain variable region: KASQDVGTAVA (SEQ ID NO: 61);
CDR2 of light chain variable region: WASTRHT (SEQ ID NO: 62); and
CDR3 of light chain variable region: QQYSSYPLT (SEQ ID NO: 63);
ii) CDR1 of the variable region of the heavy chain: SYWIH (SEQ ID NO: 64);
CDR2 of the heavy chain variable region: RIYPGTGSTYYNEKFKG (SEQ ID NO: 65); and
CDR3 of the heavy chain variable region: YPTYDWYFDV (SEQ ID NO: 66);
CDR1 of light chain variable region: RASQSISNYLH (SEQ ID NO: 67);
CDR2 of light chain variable region: YASQSIS (SEQ ID NO: 68);
CDR3 of light chain variable region: QQSNSWPLT (SEQ ID NO: 69);
iii) CDRL of the heavy chain variable region: SDYAWN (SEQ ID NO: 70);
CDR2 of the heavy chain variable region: YISYSGSTSYNPSLKSR (SEQ ID NO: 71);
CDR3 of the heavy chain variable region: ALPLPWFAY (SEQ ID NO: 72);
CDR1 of light chain variable region: KASQDVGTAVA (SEQ ID NO: 73);
CDR2 of light chain variable region: WASTRHT (SEQ ID NO: 74); and
CDR3 of light chain variable region: QQYSSYPYT (SEQ ID NO: 75).
5. The monoclonal antibody according to claim 1, which comprises mature sequences of amino acid sequences selected from any one of the following combinations (a) to (c) as a heavy chain variable region and a light chain variable region, or a fragment comprising an antigen binding region of the monoclonal antibody:
a) SEQ ID NO: 39 and the heavy chain variable region of SEQ ID NO: 41, the light chain variable region;
b) SEQ ID NO: 43 and the heavy chain variable region of SEQ ID NO: 45, a light chain variable region; and
c) SEQ ID NO: 47 and SEQ ID NO: 49, light chain variable region.
6. A polynucleotide encoding the monoclonal antibody of claim 4 or 5 or a fragment containing the antigen binding region of the monoclonal antibody.
7. A vector comprising a polynucleotide encoding the monoclonal antibody of claim 4 or 5 or a fragment comprising the antigen binding region of said monoclonal antibody.
8. A transformed cell carrying the vector of claim 7 in an expressible manner.
9. A method of preparing the monoclonal antibody of claim 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 claim 8, and recovering the monoclonal antibody or a fragment containing an antigen-binding region of the monoclonal antibody from the culture.
10. A hybridoma that produces the monoclonal antibody of claim 1 or 2.
11. The hybridoma ILT7#11 deposited under the accession number FERM BP-10704 or the hybridoma ILT7#17 deposited under the accession number FERM BP-10705.
12. A method of producing a monoclonal antibody, the method comprising the steps of: culturing the hybridoma of claim 11 and collecting the monoclonal antibody from the culture.
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 steps of:
(1) administering to an immunized animal a cell expressing an exogenous protein comprising the extracellular domain of human ILT7 and an exogenous molecule that binds to human ILT 7; and
(2) selecting antibody-producing cells that produce antibodies capable of binding to human ILT7 from the antibody-producing cells of the immunized animal.
14. The method according to claim 13, wherein the molecule that binds to human ILT7 is a cell membrane protein.
15. The method according to claim 14, wherein said cell membrane protein is the Fc receptor gamma chain.
16. The method according to claim 15, wherein the cell expressing human ILT7 and the molecule binding to human ILT7 is a cell carrying in an expressible manner the following (a) and (b):
(a) an exogenous polynucleotide encoding an amino acid sequence comprising the extracellular domain of human ILT 7; and
(b) an exogenous polynucleotide encoding an Fc receptor gamma chain.
17. The method according to claim 16, wherein the cell is an animal cell.
18. The method of claim 17, wherein the cell is a human cell.
19. The method of claim 18, wherein the human cell is a 293T cell.
20. The method according to claim 13, further comprising the step of cloning antibody producing cells obtained according to the method of claim 13.
21. A method of making a monoclonal antibody capable of binding the extracellular domain of human ILT7, the method comprising the steps of: culturing the antibody-producing cells obtained by the method according to claim 8, and collecting the monoclonal antibody from the culture.
22. A monoclonal antibody capable of recognizing human ILT7 or a fragment containing an antigen binding region thereof, said monoclonal antibody or fragment obtainable by the steps of:
(1) administering to an immunized animal a cell that exogenously expresses a protein comprising the extracellular domain of human ILT7 and a molecule that binds to human ILT 7;
(2) selecting antibody-producing cells that produce antibodies capable of binding to human ILT7 from the antibody-producing cells of the immunized animal; and
(3) culturing the antibody-producing cells selected in step (2), and recovering an antibody capable of recognizing human ILT7 from the culture.
23. An immunogen for use in preparing an antibody capable of binding to the extracellular domain of human ILT7, which immunogen comprises an animal cell or cell membrane fraction thereof carrying in an exogenously expressible manner (a) a polynucleotide encoding an amino acid sequence comprising the extracellular domain of human ILT7 and (b) a polynucleotide encoding a γ chain of an Fc receptor.
24. An immunogen according to claim 23, wherein said animal cells are human cells.
25. A method for detecting interferon producing cells, the method comprising the steps of:
contacting a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen binding region of the monoclonal antibody with a test cell; and
detecting the monoclonal antibody bound to the cell or a fragment containing the antigen-binding region of the monoclonal antibody.
26. A detection reagent for detecting an interferon-producing cell, the detection reagent comprising a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen-binding region of the monoclonal antibody.
27. A method of inhibiting the activity of an interferon-producing cell, the method comprising the step of contacting an interferon-producing cell with any of:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell or a fragment containing an antigen-binding region of the monoclonal antibody; and
(b) an immunoglobulin into which a complementarity determining region of the monoclonal antibody of (a) is introduced, or a fragment containing an antigen binding region of the immunoglobulin.
28. A method for inhibiting the activity of an interferon-producing cell in a living body, the method comprising the step of administering to the living body any of:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell or a fragment containing an antigen-binding region of the monoclonal antibody;
(b) an immunoglobulin into which a complementarity determining region of the monoclonal antibody of (a) is introduced, or a fragment containing an antigen binding region of the immunoglobulin; and
(c) a polynucleotide encoding the component of (a) or (b).
29. The method of claim 27 or 28, wherein the activity of the interferon producing cell is interferon producing activity, or survival of an interferon producing cell, or both.
30. An interferon-producing cell activity inhibitor comprising as an active ingredient any of the following:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell or a fragment containing an antigen-binding region of the monoclonal antibody;
(b) an immunoglobulin into which a complementarity determining region of the monoclonal antibody of (a) is introduced, or a fragment containing an antigen binding region of the immunoglobulin; and
(c) a polynucleotide encoding the component of (a) or (b).
31. The inhibitor of interferon-producing cell activity according to claim 30, wherein the activity of the interferon-producing cell is interferon-producing activity, or survival of an interferon-producing cell, or both.
Technical Field
The present invention relates to antibodies capable of binding to
Background
Interferon α (IFN α: hereinafter "interferon" is abbreviated as IFN) and interferon β (IFN β) are known as
Systemic lupus erythematosus (Shiozawa et al, Arthr. & Rheum.35, 412, 1992)
Chronic rheumatism (Hopkins et al, Clin. Exp. Immunol.73, 88, 1988)
There have been reported examples of the appearance or worsening of symptoms of autoimmune diseases after administration of
Further, there have been studies suggesting that IFN α is capable of inducing differentiation of dendritic cells (dendritic cells) which are also an antigen presenting cell, and thus, the induction of dendritic cell differentiation is considered to include an important mechanism of autoimmune diseases.A deep association between the induction of dendritic cell differentiation by IFN α and the onset of systemic lupus erythematosus has been shown (Blanco et al, Science, 16: 294, 1540-.
Cells that produce high amounts of
IPCs, on the other hand, are undifferentiated lymphoid dendritic cells that are considered precursor cells of dendritic cells. IPC may refer to Plasmacytoid dendritic cells (Plasmacytoid dendritic cells). IPCs differentiate into dendritic cells under viral stimulation and induce T cells to produce IFN γ or IL-10. IPC can also differentiate into dendritic cells under the stimulation of IL-3. Dendritic cells differentiated under IL-3 stimulation 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 stimuli.
Accordingly, IPCs are of two types: IFN-producing cells and dendritic cell precursor cells. Both cells play an important role in the immune system. In other words, IPCs are important cells that support the immune system in multiple ways.
Non-patent document 1: shiozawa et al, Arthr. & Rheum.35, 412, l992
Non-patent document 2: hopkins et al, clin. exp. immunol.73, 88, 1988
Non-patent document 3: wada et al, am.j.gastroenterol.90, 136, 1995
Non-patent document 4: parez et al, am.J.Hematol.49, 365, 1995
Non-patent document 5: bianco et al, Science, 16: 294, 1540-1543, 2001
Non-patent document 6: ju et al, gene.2004 Apr 28; 331: 159-64.
Non-patent document 7: colonna M et al, semiamines in Immunology 12: 121-127, 2000.
Non-patent document 8: nakajima h.et al, j.immunology 162: 5-8.1999
Non-patent document 9: wilson LE et al, Semin arthritis Rheum.32, 163-
Non-patent document 10: nestle FO et al, J.Exp.Med.202, 135-143, 2005
Patent document 1: WO03/12061(U.S. patent Published Application No.2003-148316)
Disclosure of Invention
[ problems to be solved by the invention ]
The object of the present invention is to provide an antibody capable of binding to Immunoglobulin-like transcript 7(Immunoglobulin-like transcript-7, ILT7), and detecting, identifying or isolating IPC. It is another object of the invention to modulate the activity of IPC.
In order to modulate the activity of a humoral factor such as IFN, it is effective to administer an antibody capable of recognizing the factor. For example, attempts to treat autoimmune diseases by antibodies against Interleukin (IL) -1 or IL-4 have been implemented (Guleret al, Arthritis rheum, 44.S307, 2001). Further, it is contemplated that neutralizing antibodies may act as therapeutic agents for autoimmune diseases like interferon (Stewart, TA. cytokine Growth Factor Rev.14; 139-154, 2003). It can be predicted that the same method as described above is equally effective for IFN produced by IPC. However, such methods are based on inhibiting the potency of humoral factor after production of the factor. If the production of the desired humoral factor can be directly controlled, a more significant therapeutic effect can be obtained.
Antibodies capable of recognizing human IPC 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). Researchers found that anti-BDCA-2 monoclonal antibodies were effective in inhibiting human IPC production of IFN (J.exp. Med.194: 1823-1834, 2001.). Furthermore, it has been reported that a monoclonal antibody capable of recognizing interferon-producing cells in mice can inhibit interferon production (Blood 2004 Jun 1; 103/11: 4201-4206.Epub 2003 Dec). Monoclonal antibodies directed against mouse plasmacytoid dendritic cells have been reported to cause a reduction in the number of dendritic cells (J.Immunol.2003, 171: 6466-6477).
Similarly, it would be useful if antibodies could be provided that recognize IPC in humans and modulate its activity. For example, the inventors of the present invention have shown that an antibody capable of recognizing Ly49Q specifically binds to IPC in mice. However, antibodies against Ly49Q did not interfere with IPC activity in mice (Blood, 1 April 2005, Vol.105, No.7, and pp.2787-2792.; WO 2004/13325). On the other hand, ILT7 is known as a molecule specifically expressed in plasmacytoid dendritic cells (JuXS et al and Gene.2004 Apr 28; 331: 159-64; WO 03/12061). However, any antibody to ILT7 has not been obtained. Therefore, the effect of antibodies on IPC is still unknown.
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 system (Colonna M et al, Seminirs in Immunology 12: 121-127, 2000.). One class of molecules with a structure similar to ILT7 is designated as the ILT family. The ILT family is also similar to the killer cell inhibitory receptor (KIR) in structure and function. ILT7 has 4C-type immunoglobulin-like domains as do other molecules of the ILT family. The researchers thought that ILT7 sends an activated signal into cells like ILT1, ILT 1-like proteins, ILT8, and LIR6a, etc. It has been confirmed that there is expression of molecules belonging to The ILT family in blood system cells (Young et al, Immunogenetics 53: 270-.
Thus, high expression of ILT7 in Plasmacytoid Dendritic Cells (PDC) and low expression in monocyte-derived dendritic cells (MDDC) was detected by subtractive hybridization. ILT2 and ILT3 were expressed not only in PDC but also in DCs obtained from MDDC or CD34 positive cells. However, since the mRNA of ILT7 was specifically expressed in PDC, it was found that the mRNA can be used as a marker for PDC. In addition, it has been found that stimulation by CpG can reduce expression of ILT7 (Ju XSet al, Gene, 2004
The inventors of the present invention confirmed by the researchers IPC that: expression of ILT7 is specifically accelerated in IPC. Thus, the inventors of the present invention tried to prepare an ILT7 antibody and elucidated its effects. For example, molecules such as ILT2 and ILT3, which constitute the ILT family, are highly conserved in amino acid sequence, particularly in the extracellular domain (fig. 9). These ILT family molecules each exhibit a characteristic expression profile in different blood cells. Therefore, it is very important to obtain an antibody capable of immunologically distinguishing 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.
Generally, a protein prepared by a gene recombination technique is used as an immunogen in order to obtain an antibody capable of recognizing a trace amount of the protein obtained from a living tissue. The present inventors have made an attempt to express human ILT7 based on the cDNA sequence of human ILT7 and the information on the amino acid sequence translated from the nucleotide sequence (GenBank Accession No. NM-012276). However, human ILT7 cannot be expressed as a recombinant under conventional conditions.
In order to obtain antibodies against proteins, it is also often attempted to use partial amino acid sequences of native proteins as immunogens. However, since the amino acid sequence homology is extremely high in the ILT family, almost no amino acid sequence is specific for
The inventors of the present invention have made clear that an antibody capable of binding IPC under such conditions can be obtained by using a specific immunogen. Further, the inventors of the present invention found that: the antibody obtained in this way is capable of specifically recognizing human IPC and further has an effect of regulating its activity, and thus the present invention has been successfully completed. That is, the present invention relates to the following anti-ILT-7 antibodies, a method for producing the same, and uses thereof.
[ Effect of the invention ]
The present invention provides an immunogen useful for the preparation of an antibody capable of recognizing human ILT7 and a method for preparing an anti-human ILT-7 antibody using the same. ILT7 is a membrane protein belonging to the ILT family. Specifically, the amino acid sequence of the extracellular region of the ILT family is highly conserved. Therefore, it is very difficult to prepare antibodies capable of distinguishing members of the ILT family by conventional immunization methods. The present inventors have shown that it is convenient to obtain an antibody capable of recognizing human ILT7 using an animal cell co-expressing 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.
In a preferred embodiment, the anti-human ILT-7 antibody provided by the present invention is capable of binding to human IPC. further, 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 producing
In a preferred embodiment, the anti-ILT-7 antibody provided by the present invention has the effect of modulating the activity of IPC in humans, therefore, the anti-ILT-7 antibody of the present invention can be used to inhibit the activity of IPC. As described above, expression of ILT7 in IPC is not reduced in the presence of IFN α. therefore, if the inhibition of the activity of IPC by the antibody of the present invention is utilized, the antibody can be expected to have a therapeutic effect on a patient suffering from an autoimmune disease in which expression of IFN α is promoted.
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, activation of the producing cells is directly inhibited. Therefore, it can be expected that a potent IFN inhibitory effect can be obtained even with a small amount of antibody, as compared with neutralizing IFN with an anti-IFN antibody. Furthermore, in the case of continuous production of IFN, 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 is expected that the inhibitory effect on the production of IFN can be effective for a long period of time.
The present invention relates to the following items.
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.
2. The monoclonal antibody according to
3. A monoclonal antibody produced by the
4. The monoclonal antibody according to
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 light chain variable region: KASQDVGTAVA (SEQ ID NO: 61);
CDR2 of light chain variable region: WASTRHT (SEQ ID NO: 62); and
light chain variable region: CDR3QQYSSYPLT (SEQ ID NO: 63);
ii) CDR1 of the variable region of the heavy chain: SYWIH (SEQ ID NO: 64);
CDR2 of the heavy chain variable region: RIYPGTGSTYYNEKFKG (SEQ ID NO: 65); and
CDR3 of the heavy chain variable region: YPTYDWYFDV (SEQ ID NO: 66);
CDR1 of light chain variable region: RASQSISNYLH (SEQ ID NO: 67);
CDR2 of light chain variable region: YASQSIS (SEQ ID NO: 68);
CDR3 of light chain variable region: QQSNSWPLT (SEQ ID NO: 69);
iii) CDRL of the heavy chain variable region: SDYAWN (SEQ ID NO: 70);
CDR2 of the heavy chain variable region: YISYSGSTSYNPSLKSR (SEQ ID NO: 71);
CDR3 of the heavy chain variable region: ALPLPWFAY (SEQ ID NO: 72);
CDR1 of light chain variable region: KASQDVGTAVA (SEQ ID NO: 73);
CDR2 of light chain variable region: WASTRHT (SEQ ID NO: 74); and
CDR3 of light chain variable region: QQYSSYPYT (SEQ ID NO: 75).
5. The monoclonal antibody according to
a) SEQ ID NO: 39 and the heavy chain variable region of SEQ ID NO: 41, the light chain variable region;
b) SEQ ID NO: 43 and the heavy chain variable region of SEQ ID NO: 45, a light chain variable region; and
c) SEQ ID NO: 47 and SEQ ID NO: 49, light chain variable region.
6. A polynucleotide encoding the monoclonal antibody of
7. A vector comprising a polynucleotide encoding the monoclonal antibody of
8. A transformed cell carrying the vector of
9. A method of preparing the monoclonal antibody of
10. A hybridoma producing the monoclonal antibody of
11. The
12. A method of producing a monoclonal antibody, the method comprising the steps of: the hybridoma of
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 steps of:
(1) administering to an immunized animal a cell expressing an exogenous protein comprising the extracellular domain of human ILT7 and an exogenous molecule that binds to
(2) selecting antibody-producing cells that produce antibodies capable of binding to human ILT7 from the antibody-producing cells of the immunized animal.
14. The method according to item 13, wherein the molecule that binds to human ILT7 is a cell membrane protein.
15. The method according to item 14, wherein said cell membrane protein is an Fc receptor gamma chain.
16. The method according to item 15, wherein the cell expressing human ILT7 and the molecule binding to human ILT7 is a cell carrying in an expressible manner the following (a) and (b):
(a) an exogenous polynucleotide encoding an amino acid sequence comprising the extracellular domain of
(b) an exogenous polynucleotide encoding an Fc receptor gamma chain.
17. The method of
18. The method of
19. The method according to item 18, wherein the human cell is a 293T cell.
20. The method according to item 13, which further comprises a step of cloning the antibody-producing cell obtained by the method according to item 13.
21. A method of making a monoclonal antibody capable of binding the extracellular domain of human ILT7, the method comprising the steps of: culturing the antibody-producing cells obtained by the method of
22. A monoclonal antibody capable of recognizing human ILT7 or a fragment containing an antigen binding region thereof, said monoclonal antibody or fragment obtainable by the steps of:
(1) administering to an immunized animal a cell that exogenously expresses a protein comprising the extracellular domain of human ILT7 and a molecule that binds to
(2) selecting antibody-producing cells that produce antibodies capable of binding to human ILT7 from the antibody-producing cells of the immunized animal; and
(3) culturing the antibody-producing cells selected in step (2), and recovering an antibody capable of recognizing human ILT7 from the culture.
23. An immunogen for use in preparing an antibody capable of binding to the extracellular domain of human ILT7, which immunogen comprises an animal cell or cell membrane fraction thereof carrying in an exogenously expressible manner (a) a polynucleotide encoding an amino acid sequence comprising the extracellular domain of human ILT7 and (b) a polynucleotide encoding a γ chain of an Fc receptor.
24. The immunogen according to item 23, wherein the animal cell is a human cell.
25. A method for detecting interferon producing cells, the method comprising the steps of:
contacting a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen binding region of the monoclonal antibody with a test cell; and
detecting the monoclonal antibody bound to the cell or a fragment containing the antigen-binding region of the monoclonal antibody.
26. A detection reagent for detecting an interferon-producing cell, the detection reagent comprising a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen-binding region of the monoclonal antibody.
27. A method of inhibiting the activity of an interferon-producing cell, the method comprising the step of contacting an interferon-producing cell with any of:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell or a fragment containing an antigen-binding region of the monoclonal antibody; and
(b) an immunoglobulin into which a complementarity determining region of the monoclonal antibody of (a) is introduced, or a fragment containing an antigen binding region of the immunoglobulin.
28. A method for inhibiting the activity of an interferon-producing cell in a living body, the method comprising the step of administering to the living body any of:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell or a fragment containing an antigen-binding region of the monoclonal antibody;
(b) an immunoglobulin into which a complementarity determining region of the monoclonal antibody of (a) is introduced, or a fragment containing an antigen binding region of the immunoglobulin; and
(c) a polynucleotide encoding the component of (a) or (b).
29. The method of
30. An interferon-producing cell activity inhibitor comprising as an active ingredient any of the following:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell or a fragment containing an antigen-binding region of the monoclonal antibody;
(b) an immunoglobulin into which a complementarity determining region of the monoclonal antibody of (a) is introduced, or a fragment containing an antigen binding region of the immunoglobulin; and
(c) a polynucleotide encoding the component of (a) or (b).
31. The inhibitor of interferon-producing cell activity according to item 30, wherein the activity of the interferon-producing cell is interferon-producing activity, or survival of an interferon-producing cell, or both.
Drawings
FIG. 1a is a photograph showing detection of the expression of mRNA of ILT7 gene by the RT-PCR method. The figure shows the results of analysis of the expression of mRNA of ILT7 gene in human immune cells.
FIG. 1b is a graph of detection and comparison of expression of mRNA of ILT7 gene in various human tissues and cells using a quantitative PCR method. The horizontal axis shows the tissues and cells examined, and the vertical axis shows the expression level of ILT7, wherein the expression level of ILT7 was normalized to that of GAPDH.
Fig. 2 is a diagram showing the structure of ILT7 protein. Wherein (a) of FIG. 2 shows the amino acid sequence of ILT7 protein, and further shows in the figure the putative secretion signal sequence and transmembrane region; FIG. 2 (b) shows a schematic diagram of the ILT7 protein encoded by the constructed expression vector.
Fig. 3 is a graph showing the results of introducing the ILT7 expression vector and the FcR γ expression vector into cells and detecting the expression of the ILT7 molecule on the cell surface using FCM. The horizontal axis represents the fluorescence intensity detected by the anti-FLAG antibody, i.e., the expression intensity of FLAG-tagged ILT7 molecule on the cell surface, while the vertical axis represents the number of cells.
FIG. 4 is a photograph showing the binding of molecules in cells into which an expression vector for ILT7 and an expression vector for FcR γ were introduced, as analyzed by immunoprecipitation and Western blotting. The left graph shows the results of imprinting molecules of ILT7 with anti-FLAG antibody after immunoprecipitation of FcR γ with anti-myc antibody (upper panel), and the results of imprinting molecules of FcR γ with anti-myc antibody (lower panel). Similarly, the right panel shows the results of blots with anti-FLAG antibodies (upper panel) and anti-myc antibodies (lower panel) after immunoprecipitation of FcR γ molecules with anti-FLAG antibodies.
Fig. 5 is a photograph showing detection of glycosylation of ILT7 molecule by introducing an ILT7 expression vector and an FcR γ expression vector into cells and treating with N-glycosidase. The left photograph shows the size of ILT7 without ILT7 treated with N-glycosidase, and the right photograph shows the size of ILT7 after ILT7 treated with N-glycosidase.
Fig. 6a is a graph of the reactivity of the anti-ILT 7 monoclonal antibody produced using FCM analysis. FIG. 6a shows the results of anti-ILT-7 antibody binding to BDCA-2 positive IPC, analyzed by human peripheral blood lymphocytes and double staining with anti-ILT-7 antibody and anti-BDCA-2 antibody. The vertical axis shows the reactivity with BDCA-2 antibody and the horizontal axis shows the reactivity with each of the various anti-ILT 7 antibodies prepared.
Fig. 6b is a graph showing the reactivity of the anti-ILT 7 monoclonal antibody prepared using FCM analysis. FIG. 6b shows the results of measurement of the binding ability of anti-ILT-7 antibody to ILT7 molecule using 293T cells into which ILT7 and FcR γ expression vectors were introduced. The vertical axis shows reactivity of the anti-FLAG antibody, i.e., the expression intensity of FLAG-tagged ILT7 molecule, and the horizontal axis shows reactivity of each anti-ILT 7 antibody.
Fig. 7 is a graph showing the reactivity of two clones of the anti-ILT 7 monoclonal antibody prepared by FCM analysis for 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 panel, each axis labeled with ILT7 indicates the reactivity of
FIG. 8 is a graph showing the results of examining the binding ability of the prepared anti-ILT 7 monoclonal
FIG. 9a is a picture showing the amino acid sequence of a family molecule with high homology to the ILT7 molecule. Alignment showing mainly the amino acid sequence of each extracellular region; FIG. 9b is a continuation of FIG. 9 a; FIG. 9c is
Fig. 9b is a continuation of the diagram.
FIG. 10 shows the results of measurement of the reactivity of the anti-ILT 7 monoclonal
FIG. 11 is a graph showing the effects of the prepared anti-ILT 7 monoclonal
Fig. 12 is a graph showing CDC activities of the prepared anti-ILT 7 monoclonal
FIG. 13 is a picture showing internalization (internalization) of the prepared anti-ILT 7 monoclonal
Detailed description of the invention
Human ILT 7(immunoglobulin-like transcript 7) has been reported to be a molecule specifically expressed in plasmacytoid dendritic cells (Gene.2004
Human ILT7 consists of the amino acid sequence set forth in SEQ ID NO: 2, and is a
Ligand signaling is believed to be conducted to the cells by binding of human ILT7 to a signaling molecule. For example, the majority of the Fc receptor gamma chain is present in the cell. In addition, the intracellular domain contains tyrosine-based activation motifs (ITAMs) of immune receptors involved in signaling. ITAMs are amino acid sequence portions that are common in adaptor molecules associated with immune receptors such as Fc receptors. A motif for a tyrosine phosphorylation target such as YxxL (SEQ ID NO: 76) is contained in ITAM and the signal is transmitted by phosphorylation. Examples of known signaling molecules containing ITAMs in the intracellular domain include CD3 ζ and DAP12 in addition to the γ chain of the Fc receptor. No ligand capable of binding human ILT7 has been found so far.
The inventors of the present invention confirmed the specific expression of ILT7 in human IPC by gene expression analysis. The inventors of the present invention considered that if an antibody capable of immunologically distinguishing human ILT7 from other immune molecules is available, the antibody can be used in studies on IPC. However, many molecules present in the intrinsic ILT family, including ILT7, have similar structures. Molecules such as ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, or LIR-8 contain highly homologous amino acid sequences, particularly in their extracellular domains. Therefore, the inventors of the present invention considered that it was difficult to obtain an antibody capable of distinguishing these molecules using an antibody containing a partial amino acid sequence constituting the extracellular domain as an immunogen. Accordingly, the present inventors have made an attempt to produce an antibody against human ILT7 using a cell expressing human ILT7 as an immunogen.
However, the use of conventional expression vectors cannot lead to the expression of the cDNA of human ILT7 in animal cells. Molecules of ILT1, which have a structure very similar to ILT7, have been reported to be associated with the gamma chain of Fc receptors. That is, when cells expressing the γ chain of Fc receptor, such as RBL (rat basophilic leukemia) cells and P815 (mouse mast cell tumor) cells, were used as host cells, expression of ILT1 on the cell surface was observed. However, if ILT1 is forced to be expressed in 293 such cells that do not otherwise express the γ chain of Fc receptors, then expression of the protein on the cell surface cannot be observed. On the other hand, studies have shown that expression of ILT1 on the cell surface can be confirmed when ILT1 is co-expressed with the gamma chain of Fc receptor (Nakajima H.et al, J.immunology 162: 5-8.1999). However, there is no information on the immunogen used for preparing the ILT7 antibody.
For example, in this report, an ILT1 antibody was prepared using RBL cells into which ILT1 gene was introduced as an immunogen. The present inventors have tried to produce an ILT7 antibody using RBL cells into which the ILT7 gene has been introduced in the same manner as described above. However, even if ILT7 was forced to be expressed in RBL cells (P815), expression of ILT7 on the cell surface was not observed, and thus the cells could not be used as antigens.
The inventors of the present invention conducted a special study to obtain an antibody capable of recognizing
In the present invention, human ILT7 is defined as: a natural molecule expressed in human IPC or a molecule immunologically equivalent to ILT7 expressed in human IPC. In the present invention, the binding of the antibody to human ILT7 can be confirmed, for example, by the following method.
-confirmation based on reactivity with human cells:
according to the findings of the present inventors, specific expression of human ILT7 was observed in human IPC. Initially, human ILT7 was isolated as the gene whose expression was observed in plasmacytoid dendritic cells (blood.2002100; 3295-3303, Gene.2004
In view of such expression profile of human ILT7, first, an important property of the antibody capable of binding to human ILT in the present invention is the binding activity with IPC or plasmacytoid dendritic cells, at least a partial subclass (サブセット). Specific cell surface markers for each cell population can be used to determine whether a cell is an IPC or a plasmacytoid dendritic cell. For example, the binding of the test antibody to the target cell can be confirmed by double staining with an antibody capable of binding to a cell surface marker and an antibody whose binding activity is to be detected. That is, the IPC in the present invention includes, for example, a
-confirmation based on reactivity with transformed cells expressing the human ILT7 gene:
the present inventors found that when the expression of the human ILT7 gene was performed under certain conditions, the immunological properties of ILT7 expressed in human IPC could be reconstructed. Therefore, the reactivity to human ILT7 can also be confirmed based on the reactivity of the antibody to cells artificially introduced with the
For example, the immunological properties of ILT7 expressed in human IPC can be maintained in cells co-transfected with two vectors, an expression vector containing DNA encoding human ILT7 and an expression vector containing DNA encoding a signal transduction molecule, respectively. Therefore, in the present invention, transformed cells co-expressing human ILT7 and a signaling molecule are preferably used as cells for confirming the affinity of the antibody of the present invention for the extracellular domain of
In the present invention, a molecule capable of inducing the expression of human ILT7 on the cell surface can be used as a co-expressed signal transduction molecule. The signal transduction molecule of the present invention can also be defined as: molecules capable of conferring the immunological properties of native human ILT7 to at least the extracellular domain of the ILT7 molecule in
In particular, the γ chain of Fc receptors or DAP12 is preferably used as a signaling molecule. In the present invention, the γ chain of Fc receptor is particularly preferred as a signal transduction molecule. The gamma chain of the Fc receptor is represented by SEQ ID NO: 16, or a pharmaceutically acceptable salt thereof. The signal transduction molecule may 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 located on the cell surface, it can be expressed in the sequence as set forth in SEQ ID NO: 16, or a mutant or an addition thereof. That is, the present invention provides a method for preparing a cell capable of preparing a monoclonal antibody capable of binding to the extracellular domain of human ILT7, the method comprising the steps of:
(1) administering to the immunized animal a cell that exogenously expresses a protein comprising the extracellular domain of human ILT7 and a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16; and
(2) antibody-producing cells producing an antibody capable of binding to human ILT7 are selected from the antibody-producing cells of the immunized animal.
Subsequently, as an antibody capable of binding to human ILT7 in the present invention, an antibody capable of cross-reacting with a cell population in which it is known that expression of other members of the ILT family other than ILT7 is not observed is preferably used. Specifically, as an antibody capable of binding to human ILT7 in the present invention, it is preferable to use an antibody capable of binding to a specified cell population known that expression of other members of the ILT family other than ILT7 is not observed and that observation is performed under the same conditions as those under which binding to IPC is confirmed. As already described, for example, ILT2 and ILT3 are expressed not only in PDC, but also in DCs obtained from MDDCs or CD 34-positive cells (Gene.2004
Expression patterns have been reported for other ILT family molecules ("The KIR Gene Cluster" Carrington, Mary and Norman, Paul. Bethesda (MD): National Library of Medicine (US), NCBI; 2003, Gene.2004
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That is, the monoclonal antibody capable of binding to the extracellular domain of human ILT7 in the present invention preferably comprises a monoclonal antibody having the following immunological properties:
a) monoclonal antibodies that bind to human IPC;
b) 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 under the condition that the monoclonal antibody binds to human IPC.
In particular, the monoclonal antibody of the present invention is preferably an antibody that is not able to bind to monocytes, macrophages, B cells, CD34 positive cells and dendritic cells derived from these cells under conditions in which human IPC binds.
Alternatively, 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:
c) a monoclonal antibody capable of binding 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, respectively;
d) binding to host cells prior to transformation may not be confirmed under the conditions described in c) for binding to cotransfected cells; or
The monoclonal antibodies of the invention include monoclonal antibodies having the following immunological properties:
e) binding to host cells expressing only the signaling molecule could not be confirmed under the conditions described in c) for the co-transfected cells.
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 of the other ILT family molecules. That is, for forced expression, cDNA encoding the amino acid sequence of each ILT family molecule is introduced into an appropriate host cell. To the obtained host cells, a monoclonal antibody against ILT7 that requires verification of cross-reactivity was added. Then, it was confirmed that if an antibody binds to a cell in which expression of ILT family molecules other than ILT7 was not observed, the antibody could immunologically distinguish ILT7 from other ILT family molecules. For example, in the following examples, the fact that the monoclonal antibody against ILT7 obtained by the method of the present invention did 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 capable of binding to ILT7, and binding of the monoclonal antibody to ILT1, ILT2 and ILT3 cannot be detected under the same conditions.
Specifically, ILT2 and ILT3 are genes that have been demonstrated to be expressed in IPC (Ju et al. Gene 331, 159-164, 2004). However, each of these molecules may show an intrinsic expression profile for each cell depending on the level of differentiation of the IPC or certain conditions, such as stimulation with viruses or other cytokines. Changes in expression of ILT7 can be specifically determined using antibodies that immunologically distinguish these ILT family molecules from
The binding of a monoclonal antibody, the binding ability of which 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 the antibody based on the principles of flow cytometry, it is convenient to pre-label the antibody with a molecule or group of atoms that can produce a detectable signal. Fluorescent or luminescent labels are typically used. The principle of flow cytometry-based techniques can be used to analyze the binding of fluorescently labeled antibodies to cells using a Fluorescence Activated Cell Sorter (FACS). The binding of various antibodies to cells can be efficiently confirmed using FACS.
In particular, for example, it has been previously found that antibody A, which enables identification of IPC, and antibody B, which requires analysis of its binding properties with IPC, react simultaneously with cell populations containing IPC. Antibody a and antibody B are pre-labeled with a fluorescent signal that can distinguish these antibodies from each other. In the case where both signals are detected in the same cell population, it can be confirmed that these antibodies bind to the same cell population. In other words, antibodies a and B have the same binding properties. In the case of antibodies binding to different cell populations, it is clear that the two antibodies have different binding properties.
Preferred monoclonal antibodies of the invention include monoclonal antibodies produced by
The specific preservation matters are as follows:
(a) depository name and address
Name: independent administrative law people national institute of integrated industrial and technology patent microorganism collection
Address:
(b) The preservation date is as follows: 10 and 21 days in 2005
(c) The preservation number is as follows: FERM BP-10704 (hybridoma ILT7#11)
(c) The preservation number is as follows: FERM BP-10705 (hybridoma ILT7#17)
The monoclonal antibody of the present invention may also be a fragment containing an antigen-binding region thereof. For example, an antibody fragment containing an antigen-binding region thereof obtained by enzymatically digesting IgG can be used as the antibody in the present invention. Specifically, it is possible to obtain, for example, Fab and F (ab') 2Such an antibody fragment. It is well known that these antibody fragments can be used as antibody molecules having affinity for antigens. Alternatively, an antibody constructed by gene recombination may be used as long as it can maintain a good antigen binding activity. Examples of antibodies constructed by gene recombination include chimeric antibodies, CDR-grafted antibodies, single-chain Fv, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments. It is well known that these antibodies can be obtained by using monoclonal antibodies or antibody-producing cells capable of producing the antibodies.
The monoclonal antibody of the present invention can be obtained using a certain transformed cell as an immunogen. That is, the present invention relates to a method for preparing a cell that produces a monoclonal antibody capable of binding to the extracellular domain of human ILT7, comprising the steps of:
(1) administering to the immunized animal a cell, wherein the cell expresses an exogenous protein comprising the extracellular domain of human ILT7 and an exogenous molecule that binds to
(2) antibody-producing cells producing an antibody capable of binding to human ILT7 are selected from the antibody-producing cells of the immunized animal.
The antibody-producing cells thus obtained or immortalized antibody-producing cells are cultured, and the desired monoclonal antibody is recovered from the culture. With respect to methods for immortalizing antibody-producing cells, many methods are known.
In the method for producing the monoclonal antibody of the present invention, examples of molecules that can be used for producing transformed cells that can be used as an immunogen and that can bind to human ILT7 include cell membrane proteins. Among them, the preferred cell membrane proteins of the present invention are signal transduction molecules that are localized on the cell membrane. "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 a stimulus for binding a ligand to the receptor into the cell. Examples of signaling molecules include Fc receptor gamma chain, DAP12, and the like. For example, a cell membrane protein preferably used in the present invention is Fc receptor gamma chain. The amino acid sequences of human DAP12 and Fc receptor gamma chain and the base sequences of cdnas encoding the amino acid sequences are widely known. The base sequence of the gamma chain of the human Fc receptor and the amino acid sequence coded by the base sequence are respectively shown as SEQ ID NO: 15 and 16.
In the present invention, the transformed cell used as the immunogen can be obtained by preparing, for example, a cell having the following (a) and (b):
(a) an exogenous polynucleotide encoding an amino acid sequence comprising the extracellular domain of
(b) an exogenous polynucleotide encoding a gamma chain of an Fc receptor.
In the present invention, an exogenous polynucleotide refers to a polynucleotide that is artificially introduced into a host cell. When human cells are used 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 exogenous polynucleotide includes ectopic expression of human ILT7 or human Fc receptor gamma chain (the other occurring promoter).
Here, "extracellular domain of
[ Signal sequence + extracellular domain + transmembrane region + intracellular region ]
Alternatively, the amino acid sequence of the extracellular domain of human ILT7 of the present invention also includes an amino acid sequence in which an intracellular region is partially deleted as described below.
[ Signal sequence + extracellular domain + transmembrane region + part of intracellular region ]
In addition, the amino acid sequence of the extracellular domain of human ILT7 in the present invention also includes a structure in which the intracellular region is deleted as described below.
[ Signal sequence + extracellular domain + transmembrane region ]
In the above structure, the region other than the extracellular domain may be a region selected from SEQ ID NO: 2, or a combination of other amino acid sequences having homology to these regions. For example, the amino acid sequence constituting the signal sequence, transmembrane region and intracellular region may be the amino acid sequence of an ILT family molecule other than
Therefore, in the present invention, any base sequence encoding an amino acid sequence having the above-mentioned structure [ signal sequence + extracellular domain + transmembrane region + intracellular region ] can be used as the polynucleotide in the exogenous polynucleotide described in (a). For example, SEQ ID NO: 2 is the amino acid sequence represented by SEQ ID NO: 1 by the nucleotide sequence of the above-mentioned nucleotide sequence.
In the present invention, in order to obtain transformed cells that can be used as an immunogen, an expressible expression vector carrying the above-mentioned polynucleotides (a) and (b) can be introduced into an appropriate host cell. The polynucleotides of (a) and (b) may be carried on one vector or on different vectors. When different vectors carry different polynucleotides, the host cells are co-transfected with both vectors.
In the present invention, preferred host cells include mammalian cells. Specific examples of host cells include cells of human, monkey, mouse or rat origin. Particularly preferred host cells are cells of human origin. For example, 293T cells are preferably used as the host cells derived from human in the present invention. 293T cells can be obtained from ATCC CRL-11268. In addition, cells derived from immunized animals may also be used as host cells. When cells derived from an immunized animal are used as an immunogen, there is little immune response to host cells. For this reason, an antibody against the exogenously expressed extracellular domain of ILT7 can be efficiently obtained. Thus, for example, when a mouse is used as the immunized animal, a cell derived from a mouse may also be used as the host cell.
The polynucleotide may be introduced into a cell by a vector which can induce expression in a host cell. Commercially available vectors that induce expression 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.
The thus-obtained transformed cells can be administered to an immunized animal together with an additional component such as an adjuvant, if necessary. Examples of adjuvants that can be used include Freund's complete adjuvant, and the like. In the case of using a mouse as an immunized animal, the number of transformed cells that can be administered is 10 4To 10 9More specifically 10 4To 10 6And (4) cells. Typically, multiple doses are administered with immunogen at intervals until the antibody titer is increased. For example, in the case of short-term immunization, the transformed cells are administered every 2 to 4 days, more particularly every 3 days. After two or three administrations, the antibody-producing cells can be recovered. Alternatively, the antibody-producing cells may be administered once a week and recovered after five or six administrations.
In the present invention, monoclonal antibodies are obtained by cloning the recovered antibody-producing cells. For cloning, the antibody-producing cells are preferably immortalized. For example, cell fusion methods typified by hybridoma methods, or methods for immortalizing antibody-producing cells by transformation with Epstein-Barr Virus (EBV) can be used
As the antibody-producing cell, one cell can produce one antibody. Thus, monoclonal antibodies can be obtained by establishing a cell population derived from one cell (i.e., cloning). The hybridoma method is a method in which antibody-producing cells are fused with an appropriate cell line, immortalized, and then cloned. Immortalized antibody-producing cells can be cloned using techniques such as limiting dilution. A number of cell lines are known for use in hybridoma methods. These cell lines perform well in the immortalization of lymphocytes and have the desired variety of genetic markers for selection of fused cells. Furthermore, when it is desired to obtain antibody-producing cells, a cell line lacking the antibody-producing ability may also be used.
For example, in the mouse or rat cell fusion method, mouse myelomas P3x63Ag8.653 (ATCCRL-1580) and P3x63Ag8U.1 (ATCCRL-1597) are widely used as cell lines. Usually, hybridomas are prepared by fusing homogeneous cells, but monoclonal antibodies can also be obtained by heterohybridomas of heterogeneous species that are relatively closely related.
Specific protocols for cell fusion are well known. That is, the antibody-producing cells of the immunized animal are mixed with an appropriate 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 fusion partners, the various cell lines described above can be used. Cell fusion can be performed using a polyethylene glycol method and an electrofusion method.
Thereafter, successfully fused cells were selected based on the selection marker of the fused cells. For example, when cell fusion is performed using a HAT sensitive cell line, cells grown in HAT medium are selected as successfully fused cells. In addition, it has been confirmed that the antibody produced by the selected cells has a desired reactivity.
Each hybridoma was screened for antibody reactivity. That is, an antibody produced by a hybridoma capable of binding to human ILT7 can be selected by the method described above. Preferably, the selected hybridomas are subcloned and the desired antibodies are finally confirmed, and the confirmed antibodies are selected as monoclonal antibodies produced by the hybridomas of the present invention.
Specifically, the desired hybridoma can be selected based on reactivity against human cells or reactivity against transformed cells expressing the human ILT7 gene. Antibodies capable of binding to cells can be detected based on the principles of immunoassays. For example, an ELISA using cells as antigens can be used to detect the desired antibody. Specifically, a culture supernatant of a hybridoma is added to a support on which an immunogen or a transformed cell is immobilized. In this case the culture supernatant contains the desired antibody, and cells immobilized on the support recruit the antibody. Then, the stationary phase is separated from the culture supernatant and, if necessary, washed. Thereafter, the antibody recruited on the stationary phase can be detected. Antibodies can be detected using antibodies that recognize the antibodies. For example, mouse antibodies can be detected by anti-mouse immunoglobulin antibodies. Such detection can be easily performed if an antibody capable of recognizing the antibody is labeled. Examples of useful labels include enzymes, fluorescent dyes, luminescent dyes, and the like.
On the other hand, microparticles and the inner wall of the microplate can be used as a support for fixing cells. Cells can be immobilized on the surface of a particle or container made of plastic by physical adsorption. Examples of supports that can be used to immobilize cells include beads made of polystyrene and reactors.
In the selection of hybridomas, it is sometimes predicted that: antibodies were raised against host cells of the transformed cells used as immunogens, but not against
Undesired antibodies can be absorbed by the antigen bound to the antibody that is supposed to be present. Specifically, for example, an antibody capable of recognizing a cell antigen of another person than human ILT7 may be taken up by a cell in which expression of human ILT7 cannot be detected. In the present invention, it is preferable to use a host cell as an immunogen as an antigen for uptake of an undesired antibody. Alternatively, a host cell that does not express the extracellular domain of human ILT7 but expresses a molecule that binds to ILT7 may be used as an antigen for antibody uptake.
As a monoclonal antibody whose activity of binding to an antigen has been confirmed, if necessary, its actual effect on IPC activity can be confirmed. Its effect on IPC can be confirmed by a method such as described below.
As the monoclonal antibody of the present invention, a hybridoma producing the monoclonal antibody is cultured and the monoclonal antibody of the present invention is recovered from the resulting culture. The hybridoma can be cultured in vivo or in vitro. In an in vitro example, the hybridoma can be cultured by a known medium such as RPMI 1640. The immunoglobulin secreted by the hybridoma accumulates in the culture supernatant. Therefore, if necessary, the monoclonal antibody of the present invention can be obtained by collecting and purifying the culture supernatant. The absence of serum in the culture medium makes purification of immunoglobulins easier. However, 10% fetal bovine serum can be added to the medium for the purpose of rapid expansion of the hybridoma and increase of antibody production.
The hybridoma can also be cultured in vivo. Specifically, intraperitoneal culture can be achieved by a method of inoculating the hybridoma into the abdominal cavity of a nude mouse. Monoclonal antibodies will accumulate in ascites fluid. Thus, if ascites fluid is obtained and purified as required, a desired monoclonal antibody can be prepared. The obtained monoclonal antibody may be appropriately modified or treated according to the intended use.
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 an appropriate expression vector. Techniques for obtaining a cDNA encoding the variable region of an antibody and expressing the cDNA in a suitable host cell are known. In addition, a method of obtaining a chimeric antibody by linking a variable region containing an antigen-binding region to a constant region is also known.
The monoclonal antibody preferred in the invention includes a monoclonal antibody produced by hybridoma #11 (accession number: FERM BP-10704), hybridoma #17 (accession number: FERM BP-10705) or
For example, mouse (variable region) -human (constant region) chimeric antibodies were prepared by linking these variable region genes to the human IgG1 heavy chain constant region and human Ig kappa light chain constant region, respectively. The amino acid sequence of the chimeric antibody and the nucleotide sequence encoding the chimeric antibody are as follows. The chimeric antibodies represented by these sequences show a preferred embodiment of the construction of the anti-ILT 7 monoclonal antibody of the present invention. In the amino acid sequences of the following chimeric antibodies, the amino acid sequence from the N-terminus to-1 corresponds to a signal sequence, and the amino acid sequence from 1 to the C-terminus corresponds to a mature protein. That is, a chimeric antibody comprising a heavy chain and a light chain, wherein the chimeric antibody comprises amino acid sequences from 1 to the C-terminal of each amino acid sequence, is preferred in the present invention.
Furthermore, the antigen-binding activity of the monoclonal antibody may be grafted to another immunoglobulin. The variable region of an immunoglobulin contains Complementarity Determining Regions (CDRs) and framework regions. Each immunoglobulin antigen binding is determined by the CDRs, and the framework maintains the structure of the antigen binding region. The amino acid sequences of the CDRs are extremely diverse, whereas the amino acid sequences of the framework portions are highly conserved. It is known that the antigen binding activity can be grafted by a method of integrating the amino acid sequences constituting the CDRs into the framework regions of other immunoglobulin molecules. A method of grafting antigen binding of different immunoglobulins to human immunoglobulins by such a process has been established. In the present invention, the "antigen binding region" may comprise CDRs grafted to a framework region. Thus, a "fragment comprising an antigen binding region" of a given monoclonal antibody includes a fragment of a human immunoglobulin comprising a variable region to which the CDRs of the monoclonal antibody are grafted. For example, the amino acid sequences of the above-mentioned variable regions respectively contain the following amino acid sequences (SEQ ID NOs) as CDRs.
Based on the information on the nucleotide sequence encoding the above amino acid sequence and the nucleotide sequence encoding the Framework (FR) of human immunoglobulin, a primer can be designed and cDNA whose nucleotide sequence is formed by joining the two nucleotide sequences can be amplified. The procedure for each framework was repeated, and variable regions formed by linking the mouse CDR1, CDR2, and CDR3 to human FR can be constructed. Furthermore, when the base sequences encoding the constant regions of human immunoglobulins are linked as desired, a humanized antibody having the constant regions can be obtained.
The antibody of the present invention preferably contains an antibody having a constant region derived from IgG or IgM as a chimeric antibody containing the above variable region or a humanized antibody grafted with a variable region composed of CDRs. The inventors of the present invention confirmed that a monoclonal antibody against ILT7 showed CDC effect against ILT 7-expressing cells. Therefore, antibodies having constant regions derived from IgG or IgM show cytotoxicity against ILT 7-expressing cells due to their CDC effects. Such antibodies can be used to inhibit the number of ILT7 expressing cells such as IPC.
The chimeric antibody or humanized antibody capable of recognizing ILT7 provided by the present invention can be prepared by using a polynucleotide encoding the chimeric antibody or humanized antibody capable of recognizing ILT7 and using genetic engineering. For example, a polynucleotide of the base sequence described in the following SEQ ID NO and a polynucleotide encoding an amino acid sequence that is a mature protein constituting each amino acid sequence can be used as the polynucleotide encoding
#11 SEQ ID NO: 50 (base sequence) SEQ ID NO: 52 (base sequence)
#17 SEQ ID NO: 54 (base sequence) SEQ ID NO: 56 (base sequence)
In the same manner as described above, for a polynucleotide encoding a humanized antibody, a polynucleotide expressing a 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 and light chains are carried by different vectors, both vectors are co-transfected into the same host cell. The heavy and light chains expressed from each vector were used to construct an immunoglobulin with two chains. Alternatively, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain may be carried on the same vector. A host cell transformed with a vector carrying two polynucleotides can express both heavy and light chains and can produce immunoglobulins with both chains.
These polynucleotides can be expressed as antibodies using host vector systems capable of expressing antibody genes. In addition, in the case where the heavy chain variable region and the light chain variable region are expressed as a single protein molecule by linking them, a signal sequence may be placed at the N-terminus of the protein molecule. Known examples of such antibody molecules include scFv molecules in which a heavy chain variable region is linked to a light chain variable region by a linker.
The monoclonal antibody of the present invention comprises each of the monoclonal antibodies thus prepared. In other words, the monoclonal antibody of the present invention includes a monoclonal antibody comprising an immunoglobulin containing an antigen-binding region encoded by a polynucleotide derived from a cDNA encoding the antigen-binding region of the monoclonal antibody.
As described above, RBL cells in which ILT1 gene is forcibly expressed can be used as an immunogen for obtaining ILT1 antibody. However, expression of ILT7 on the surface of RBL cells (P815) could not be confirmed, and thus it could not be used as an immunogen. The present inventors have 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
That is, the present invention provides an immunogen which can be used for preparing an antibody capable of binding to the extracellular domain of human ILT7, and which comprises an animal cell or a cell membrane fraction thereof in which a polynucleotide carrying for exogenous expression a polynucleotide comprising (a) a polynucleotide encoding an amino acid sequence comprising the extracellular domain of
Six years or more have passed since the discovery of the structure of human ILT7 by 1998. However, an antibody capable of specifically recognizing ILT7 has not yet been obtained. Antibodies capable of recognizing human ILT7 were provided for the first time by using the immunogens of the present invention. That is, the present invention provides an antibody capable of recognizing human ILT7, which is obtained by the steps of:
(1) administering to the immunized animal a cell capable of exogenously expressing a protein comprising the extracellular domain of human ILT7 and a molecule that binds to
(2) selecting antibody-producing cells capable of producing an antibody capable of binding to human ILT7 from the antibody-producing cells of the immunized animal; and is
(3) Culturing the antibody-producing cells selected in step (2), and recovering an antibody capable of recognizing human ILT7 from the culture.
Human ILT7 has been found to be specifically expressed in human IPC. The present inventors analyzed gene expression using SAGE and also confirmed specific expression of human ILT7 in human IPC. However, in the previous reports, the expression level of ILT7 in both examples was analyzed based on mRNA. Since an antibody capable of detecting human ILT7 cannot be provided, the expression state of the protein has not been conventionally analyzed. Analysis of the human ILT7 protein was achieved by providing antibodies of the invention that bind to the extracellular domain of
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 IPC. That is, the present invention relates to a method for detecting interferon producing cells, which comprises the steps of: adding a monoclonal antibody or a fragment containing an antigen binding region thereof that binds to the extracellular domain of human ILT7 to a test cell; and detecting the monoclonal antibody or a fragment containing an antigen-binding region thereof bound to the cell.
Whether a specific cell is IPC can be determined by the test of human ILT7 based on the present invention. That is, the present invention provides a method for identifying IPC using human ILT7 as an indicator. Alternatively, human IPC 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 IPC using human ILT7 as an indicator.
Based on the analysis by the human ILT7 antibody, it was confirmed that the expression level of ILT7 was reduced in IPC, the differentiation of which was induced by CpG. That is, IPC before its differentiation is induced can be specifically detected by using ILT7 as an indicator. In other words, the monoclonal antibody of the present invention can be particularly useful for detecting IPC before it is differentiated into dendritic cells. The term "IPC before differentiation" as used herein may be defined as a cell population having the ability to produce interferon.
In the present invention, a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen-binding region thereof may be labeled in advance. For example, antibodies can be readily detected by labeling with luminescent or fluorescent dyes. More specifically, the prepared fluorochrome-labeled antibody is added to a cell population that may contain IPC and then the antibody-bound cells of the present invention can be detected by using the fluorochrome as an indicator. Further, IPCs can be isolated by isolating cells in which fluorescent dyes are detected. A series of steps can be easily performed based on the principles of FACS.
Alternatively, the antibody of the present invention may be bound in advance to a solid support such as a magnetic particle. The antibody bound to the stationary phase support will recognize human ILT7 and IPC is captured on the stationary phase support. Therefore, IPCs can be detected and separated.
The invention can provide the antibody necessary for the IPC detection method as the IPC detection reagent. That is, the present invention provides a reagent for detecting interferon producing cells, which comprises a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen binding region thereof. This reagent for detecting IPC of the present invention can be used in combination with a positive control or a negative control, in addition to being used as an antibody. For example, transformed cells expressing the extracellular domain of human ILT7 and used as an immunogen and IPC obtained from within humans can be used as positive controls. Generally, only a few human IPCs are available from peripheral blood. Therefore, it is particularly preferable to use the transformed cells as a positive control in the reagent of the present invention. Alternatively, any cell that is incapable of expressing human ILT7 can be used as a negative control.
That is, the present invention provides a kit for detecting IPC of a human, comprising:
(a) a monoclonal antibody capable of binding to the extracellular domain of human ILT7 or a fragment containing an antigen binding region thereof; and
(b) a cell expressing a foreign protein comprising the extracellular domain of human ILT7 and a foreign molecule capable of binding to
The inventors of the present invention analyzed the effect of an antibody capable of binding to the extracellular domain of human ILT7 on IPC. Therefore, it was confirmed that an antibody capable of binding to the extracellular domain of human ILT7 inhibited IPC activity. That is, the present invention relates to a method for inhibiting the activity of an interferon-producing cell, which comprises the step of adding to the interferon-producing cell any of:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell, or a fragment containing an antigen-binding region thereof; and
(b) an immunoglobulin to which a complementarity determining region of the monoclonal antibody of (a) has been grafted, or a fragment thereof containing an antigen binding region.
Alternatively, the present invention relates to a method for inhibiting an interferon-producing cell activity in a living tissue, the method comprising the step of adding to the living tissue any of:
(a) a monoclonal antibody capable of binding to human ILT7 and inhibiting the activity of an interferon-producing cell, or a fragment thereof containing an antigen-binding region;
(b) an immunoglobulin containing a complementarity determining region into which the monoclonal antibody of (a) has been grafted, or a fragment thereof containing an antigen binding region; and
(c) a polynucleotide encoding the component of (a) or (b).
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 specifically mentioned, "IPC" includes not only precursor cells of dendritic cells but also cells having 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 IPC from other blood cells. Specifically, the cell surface marker profile of human IPC is as follows (Shortman, K.and Liu, YJ.Nature Reviews 2: 151-161, 2002). In recent years, specific reports suggest defining BDCA-2 positive cells as IPC (Dzionek, A.eta.1. J.Immunol.165: 6037-6046, 2000.).
[ human IPC cell surface antigen Spectrum ]
Positive for CD4, positive for CD123,
pedigree (CD3, CD14, CD16, CD19, CD20, CD56) negative and CD11c negative
Thus, IPCs can also be said to be cells that have expression profiles of these known markers and have the ability to produce IFN. Furthermore, even if the cell is a group of cells having expression profiles different from those of known markers, the cell is included in the IPC as long as the cell in the living tissue has the ability to produce IFN. In addition, common features of human IPCs are as follows:
[ morphological characteristics of cells ]
Similarity to plasma cells
Round cells with smooth cell surfaces
Relatively large nucleus
[ functional characteristics of cells ]
During viral infection, large amounts of
Differentiation into dendritic cells after viral infection.
"inhibiting activity of IPC" means inhibiting at least one IPC function. Examples of IPC functions include IFN production and cell survival. Cell survival can also be described as the number of cells. Therefore, in the case of inhibiting one or both of these functions, it can be said that the activity of IPC is inhibited. It has been found that
For example, the relationship between pathological states of various autoimmune diseases and IFN α has been pointed out.A large portion of IFN α is produced by IPC.therefore, the pathological state caused by IFN α can be alleviated by inhibiting the production of IFN α. Here, "inhibiting the production of IFN by IPC" means inhibiting the production of IFN by at least one IPC.
That is, the present invention relates to an inhibitor for inhibiting IFN production, which comprises, as an active ingredient, an antibody capable of binding to the extracellular domain of
The present inventors have found that, in a preferred embodiment of the present invention, a monoclonal antibody against ILT7 binds to ILT 7-expressing cells and then exerts cytotoxicity by complementation-dependent cytotoxicity (CDC). The CDC effect is an important mechanism of antibody drugs, and that, due to the CDC effect of the monoclonal antibody against ILT7 of the present invention, the antibody also has potential cytotoxicity against ILT 7-expressing cells such as IPC, that is, the inhibitory effect on the production of IFN by the cytotoxicity of IPC can be expected for a monoclonal antibody against ILT7, in addition to the mechanism of inhibition of the production of IFN in the preferred embodiment.
The antibody capable of recognizing the extracellular domain of human ILT7 used in the present invention can be obtained by the method based on the foregoing. The antibodies of the invention may be of any grade. The species from which the antibody is derived is also not particularly limited. In addition, a fragment containing an antigen-binding region of an antibody can be used as the antibody. For example, an antibody fragment containing an antigen-binding region thereof obtained by enzymatically digesting IgG can be used as the antibody in the present invention. Specifically, enzymes such as Fab and F (ab') 2Such an antibody fragment. It is well known that these antibody fragments can be used as antibody molecules having affinity for antibodies. Alternatively, an antibody constructed by genetic recombination techniques may be used as long as it can maintain good antigen binding activity. 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.
In the present invention, the antibody may be modified if necessary. According to the present invention, an antibody capable of recognizing the extracellular domain of human ILT7 has an inhibitory effect on the activity of IPC. That is, the antibody itself can be expected to have cytotoxicity against IPC. Subclasses of antibodies that exhibit potential effector activity are known. Alternatively, the inhibitory effect on IPC activity can be further enhanced by modifying the antibody with a cytotoxic agent. Examples of cytotoxic agents are described below.
Toxin: pseudomonas Endotoxin (PE), diphtheria toxin, ricin
A radioactive isotope: tc 99m、Sr 89、I 131、Y 90
Anti-tumor agents: calicheamicin, mitomycin, paclitaxel
The toxin consisting of the protein may be linked to the antibody or fragment thereof by a bifunctional agent. Alternatively, a gene encoding a toxin may be linked to a gene encoding an antibody, and a fusion protein of the two genes may be obtained. Methods of linking antibodies to isotopes are also known. For example, a method of labeling a radioisotope to an antibody with a chelating agent is known. In addition, the anti-tumor agent may be linked to the antibody using a sugar chain or a bifunctional agent.
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 after binding (internalization). Thus, the cytotoxic agent of the present invention may be transported into cells by adding an antibody linked to the cytotoxic agent to ILT 7-expressing cells. That is, the present invention provides an inhibitor of the activity of ILT 7-expressing cells, which comprises a monoclonal antibody against ILT7 linked to a cytotoxic agent as an active ingredient. Alternatively, the present invention relates to the use of a monoclonal antibody against ILT7, to which a cytotoxic agent has been linked, in the manufacture of an inhibitor of the activity of ILT7 expressing cells. In addition, the present invention provides a method for inhibiting the activity of an ILT 7-expressing cell, the method comprising the step of administering a monoclonal antibody against ILT7 linked to a cytotoxic agent.
In the present invention, an antibody whose structure has been artificially modified may be used as an active ingredient. For example, methods of modifying antibodies in order to improve their cytotoxicity and stability are known. Specifically, immunoglobulins in which the sugar chain of the heavy chain is 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 is enhanced by modifying the sugar chain. Alternatively, immunoglobulins in which the amino acid sequence of the Fc region is modified are also known. That is, ADCC activity is enhanced by artificially increasing the binding activity of immunoglobulin to Fc receptor (Shield, RL. equivalent. J. biol. chem.276; 6591-6604, 2001.).
This phenomenon has been found: IgG bound to Fc receptors will immediately integrate into the cell. IgG then binds to Fc receptors expressed within the endosome and will be released again into the blood. IgG, which has high binding activity to Fc receptors, has more chance to be released into the blood after integration into cells. Thus, the maintenance of IgG in blood is prolonged (Hinton, PR. et al. J Biol chem.279: 6213-6216.2004). In addition to this, it has been reported that modification of the amino acid sequence of the Fc region may cause alteration of the activity of complement-dependent cytotoxicity (CDC). These improved antibodies can be used as the antibody of the present invention.
The activity of IPC can be inhibited by adding an antibody capable of binding to the extracellular domain of human ILT7 to IPC. Therefore, these antibodies can be used as an inhibitor for inhibiting the activity of IPC or a method for inhibiting the activity of IPC. That is, the present invention provides an active inhibitor of IPC comprising at least one component selected from the group consisting of (a) to (c), which is used as an active ingredient. Alternatively, the present invention relates to a method for inhibiting the activity of IPC, which comprises the step of administering at least one ingredient selected from the group consisting of the following (a) to (c). Further, the present invention relates to the use of at least one ingredient selected from the group consisting of the following (a) to (c) for the preparation of an IPC activity inhibitor:
(a) a monoclonal antibody capable of binding to human ILT7, or a fragment containing an antigen binding region thereof;
(b) an immunoglobulin grafted with a complementarity determining region of the antibody described in (a), or an antigen-binding region-containing fragment thereof; and
(c) a polynucleotide encoding the component described in (a) or (b).
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 IPC activity. In the present invention, one or more monoclonal antibodies may be used. For example, one or more monoclonal antibodies capable of recognizing the extracellular domain of human ILT7 may be used in combination in the present invention.
The inhibitory effect of an antibody on the IFN production activity of an IPC can be confirmed by the following method, the IPC is stimulated with a virus to produce a large amount of IFN, the IPC is stimulated with a virus to which an antibody is added before, after or simultaneously, the IFN production ability of each IPC obtained is compared with the corresponding ability of each control to which no antibody is added, the IFN production ability is evaluated by measuring the IFN α or IFN β contained in the culture supernatant of the IPC.
In the present invention, the activity of IPC includes maintaining the number of IPCs. Therefore, the inhibition of activity of IPC in the present invention includes inhibiting the number of IPCs. When it was confirmed that the number of IPCs was suppressed in the presence of the antibody, it was found that the antibody was inhibiting the activity of IPCs. When measuring the production of IFN, inactive immunoglobulin derived from the same animal species can be used as a comparative control group for determining antibody activity. The number of IPCs can be quantitatively compared by cell counting. The number of cells can be counted by FACS or microscopy.
Further, it has been reported that IPC can differentiate into Th2 also called dendritic cell 2(DC2) due to viral infection or such stimulation. If the generation of IFN due to IPC stimulated by virus can be suppressed, it can also be suppressed from differentiating into
When an antibody capable of recognizing the extracellular domain of human ILT7 is added to a host different from the kind of tissue from which the antibody is obtained, it is necessary to process the antibody into a form that is not recognized as an exogenous component by the host. For example, the method of processing an antibody into the following molecules does not allow the immunoglobulin to be easily recognized as a foreign substance. The following techniques for immunoglobulin processing are known. Fragments containing the antigen binding region lack the constant region. (Monoclonal Antibodies: Principlysand Practice, third edition, Academic Press Limited.1995; Antibody Engineering, APracial Approach, IRL PRESS, 1996)
Chimeric antibodies comprising the antigen-binding region of a monoclonal antibody and the constant region of an immunoglobulin of a host ("Gene expression animal", Isao Ishida, Tamie Ando, eds., Kodansha, 1994)
CDR-replacing antibodies in which the CDRs of the host's immunoglobulin are replaced by Complementarity Determining Regions (CDRs) of a monoclonal antibody ("Gene Expression Experimental Manual", Isao Ishida, Tamie Ando, eds., Kodansha, 1994)
Alternatively, when a non-human animal is used, a human antibody can be obtained by a method of genetically integrating a human antibody into a non-human animal as an immunized animal. For example, human antibodies have been prepared by putting transgenic mice having human antibody genes into practical use as immunized animals (Ishidaet al, Cloning and Stem Cells, 4: 85-95, 2002). Human antibodies recognizing ILT7 can be obtained by using such animals and the above-mentioned immunogens. Preferably, human antibodies are administered to humans.
Alternatively, the variable region genes of human immunoglobulins can be obtained by phage display methods (McCaffertyJ. et al., Nature 348: 552-554, 1990; Kretzschmar T et al., Curr Opin Biotechnol.2002 Dec; 13 (6): 598-602.). In the phage display method, encoding human immunoglobulin variable region genes are integrated into the phage gene. Phage libraries can also be prepared by using multiple immunoglobulin genes as adjustments. The phage will express the variable region as a fusion protein with the proteins that make up the phage. The variable regions expressed by the phage on the phage surface retain binding activity to the antigen. In view of screening a phage library for phage expressing a variable region with the desired binding activity, phage capable of binding to an antigen or antigen-expressing cells are selected. In addition, the phage particles thus screened retain the gene encoding the variable region with the desired binding activity. That is, in the phage display method, the binding activity of the variable region can be used as an indicator to obtain a gene encoding a variable region having a desired binding activity.
In the activity inhibitor of IPC or the method of inhibiting the activity of IPC in the present invention, an antibody capable of recognizing the extracellular domain of human ILT7 or an antibody fragment containing at least an antigen-binding region of the antibody may be administered as a protein or a protein encoded by a polynucleotide. In the administration of polynucleotides, it is desirable to use a vector in which the polynucleotide encoding the desired protein is placed under the control of a suitable promoter to express the desired protein. Enhancers and terminators may also be inserted into the vector. Vectors with genes constituting the heavy and light chains of immunoglobulins and genes capable of expressing immunoglobulin molecules are known.
Administration can be carried out by introducing a vector capable of expressing immunoglobulin into cells. In administration to a living tissue, a vector that can infect cells by administration to a living tissue may be directly administered. Lymphocytes are isolated from living tissue and then the vector is introduced into lymphocytes that can be reinjected back into the living tissue (ex vivo).
In the activity inhibitor or the method for inhibiting the activity of IPC based on the present invention, the immunoglobulin is generally administered in an amount ranging from 0.5mg to 100mg per kg of body weight, for example, from 1mg to 50mg per kg of body weight, preferably from 2mg to 10mg per kg of body weight, to the living tissue. The interval between administration of the antibody to the living tissue can be suitably adjusted so as to maintain an effective concentration of immunoglobulin in the living tissue during the course of treatment. Specifically, for example, the interval between administration of the antibody may be 1 to 2 weeks. The route of administration is optional. The route of administration effective in the treatment can be appropriately selected by those skilled in the art. Specific examples thereof include oral administration or parenteral administration. The antibody may 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 the antibody is added to the cells, the immunoglobulin is added to the medium at a concentration generally in the range of 1. mu.g/ml, preferably 10. mu.g/ml, more preferably 50. mu.g/ml, still more preferably 0.5 mg/ml.
In the activity inhibitor of IPC or the method of inhibiting the activity of IPC in the present invention, the administration of the monoclonal antibody to the living tissue may be carried out by an alternative method. Typically, monoclonal antibodies are mixed with a pharmaceutically acceptable support. If desired, the monoclonal antibody can be mixed with additional agents such as thickening agents, stabilizers, preservatives, and solubilizing agents. Examples of such supports or additional agents include lactose, citric acid, stearic acid, magnesium stearate, sucrose, starch, talc, gelatin, agar, vegetable oils, and glycols. The term "pharmaceutically acceptable" means generally recognized by the pharmacopoeia of various government regulatory agencies or listed in various national pharmacopoeias or used in animals, mammals, and more particularly humans. The active inhibitors of IPCs of the present invention may also be provided in the form of single or multiple doses of lyophilized powders or tablets. In addition, the lyophilized powder or tablet can be used in combination with the use of sterile water for injection, physiological saline solution or buffer solution to dissolve the composition to the desired concentration prior to administration.
Furthermore, when administration of the monoclonal antibody is performed in the form of a vector capable of expressing immunoglobulin, plasmids having a heavy chain and a light chain are co-transfected, each plasmid being administered in the range of 0.1 to 10mg,for example, 1 to 5mg per kg body weight. For introducing the plasmid into the cells, the concentration of the vector used is 1 to 5. mu.g/10 6A cell. The present invention will be specifically described below with reference to examples.
All documents cited herein are incorporated by reference herein in their entirety.
Examples
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