JP2006204243A - New dendritic cell(dc) membrane molecule and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separating and detecting mature DC or the like with an antibody for specifically recognizing a molecule expressed on the mature DC of strong APC by controlling infectious diseases, autoimmune diseases or the like by the function control of DC with the molecule as a target. <P>SOLUTION: A protein comprising one of the following amino acid sequences. (a) One of two specific amino acid sequences of membrane molecules; (b) an amino acid sequence which is mentioned in (a), in which one or more amino acid residues are lacked, replaced, inserted and/or added, and which can control immune responses; or (c) an amino acid sequence which has a homology of ≥80% to the amino acid sequence mentioned in (a) and can control immune responses. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a membrane molecule of mouse or human dendritic cell (hereinafter also referred to as DC), DNA encoding the membrane molecule, an antibody against the membrane molecule, a mature DC separation method using the antibody, and mature DC The detection method.

  The onset of autoimmune diseases is derived from (1) genetic background, (2) exposure of self-antigens that were not exposed to the immune system, which were released due to tissue inflammatory reactions, and inflammatory reactions such as viruses and bacteria Changes in the local environment resulting from the activation of so-called cross-reactive T cells due to the homology of the recognition peptides of activated T cells, and (3) costimulatory molecules on the cell membrane It is assumed that factors such as the presence of self-reactive T cells induced by the failure of tolerance due to abnormal expression) are involved.

  Antigen-presenting cells that have not been stimulated by normal tissues are controlled by the expression of costimulatory molecules, even if they present autoantigens. Has been. However, it has been suggested that autoimmune diseases may occur as a result of activation of autoreactive T cells due to excessive or continued abnormal expression of costimulatory molecules in abnormal immune states. In particular, the signal generated between CD28 / CD152 and CD80 / CD86 plays an important and unique role in the control of T cell activation, and immunotherapy by regulating this signal has been tried in a mouse experimental model, and is actually directed against humans. Clinical trials are also being conducted.

  While the mechanism of so-called acquired immunity based on the mechanism for recognizing such antigenic peptides has been considerably elucidated, the so-called innate immunity based on the mechanism for recognizing foreign glycolipids, proteins, DNA, and RNA has been elucidated. Is currently being revealed at the molecular level. There are also many unclear points about the molecular mechanism that links innate immunity to acquired immunity.

  Current understanding of innate immunity in mammals begins with infection defense mechanisms analyzed in the development and genetics of flies and plants. In other words, the mammalian homolog Toll-like Receptor (TLR) of the Drosophila pathogen recognition molecule Toll was discovered, and it became clear that it controls pathogen recognition in innate immunity (Non-patent Document 1). This fact means that the concept that innate immunity is a common pathogen recognition mechanism from flies to humans is established. Acquired immunity is a system that is completed at the individual level (specificity of antibodies and T cell receptors is acquired by rearranging genes individually and is not transmitted to offspring), whereas innate immunity is shared by the genome. It is a record of evolution. In other words, it is natural immunity that directs which specificity to respond to among the various specificities of acquired immunity, and TLR is considered to be a group of the most important molecules. The role of TLRs in innate immunity is the expression of costimulatory molecules and cytokine production by recognition and signaling of adjuvants. In the acquired immunity, the target protein itself has low immunogenicity, whereas pathogen-derived glycolipids (such as lipopolysaccharide (LPS)) strongly activate the immune system. For example, it is a good example that an adjuvant is required in addition to an antigen when preparing an antibody. In other words, it is no exaggeration to say that the control of acquired immunity triggers recognition by TLR.

  Dendritic cells (DCs) play a key role in determining and controlling acquired immunity. Th1 / Th2 skewing, cytotoxic T cells (CTLs) and activated macrophage induction (cellular immunity), antibody production (humoral immunity) ). This induced immune response varies depending on the type of pathogen. Cellular immunity predominates in the case of bacterial infection, but humoral immunity predominates in the case of certain parasites and viruses. In the case of Adjuvant, antibody production is dominant like aluminum hydroxide (Alum). This may be because the qualitative or quantitative changes in recognition by TLR and acquired immunity by DC are controlled.

  TLR is a type I membrane protein having a leucine-rich repeat related to protein interaction called LRR outside the cell. In the cell, TLR domain (Toll / IL-1R homology domain) has a domain related to signal transduction. (Non-Patent Document 2) So far, 10 types of TLR molecules have been reported in humans (TLR1-TLR10), and at the same time, identification of ligands is also progressing. For example, TLR2 is bacterial peptidoglycan (PG) and lipoprotein (Non-patent Documents 3 and 4), TLR4 is Grams-negative bacterial LPS and Gram-positive bacterial lipoteichoic acid (LTA) (Non-patent Document 5) TLR5 is bacterial flagella Constituent proteins (Non-patent document 6), TLR2 / 6 is mycoplasma-derived lipopeptide and yeast-derived zymosan (Non-patent document 7), TLR7 and TLR8 are bacterial-derived single-stranded RNA (Non-patent document 8), and TLR9 is It has been reported that bacteria-derived CpG DNA is recognized (Non-patent Document 9).

  Signal transduction from TLR has been analyzed mainly in TLR2 (MALP-2 stimulation, etc.), TLR4 (LPS stimulation, etc.), TLR9 (CpG DNA stimulation, etc.) in macrophages or dendritic cells. First, it was revealed that MyD88, an adapter molecule essential for signal transduction in the IL-1R family as a molecule associated with the TIR domain, is also an essential molecule for TLR4 signal transduction, and cytokine production from macrophages induced by LPS , B cell blasting, induction of shock symptoms, etc. were completely absent (Non-patent Document 10). However, activation of NF-kappaB in response to LPS stimulation was observed in macrophages derived from MyD88-deficient mice, suggesting the presence of the NFkappaB pathway independent of MyD88. As an explanation for this pathway, the analysis result of DC in MyD88-deficient mice is one answer. That is, IL-12 production by LPS stimulation in CD11c-positive DC is completely absent in MyD88-deficient mice, but is not affected by the increased expression of costimulatory molecules such as CD40, CD80, and CD86 (Non-patent Document 11). TIRAP / MAL reported as a novel TLR4 adapter molecule inhibits DC maturation by TLR4, suggesting that it may be involved in pathways necessary for the expression of costimulatory molecules in DC (Non-Patent Documents 12 and 13). ). In addition to this report, it was revealed that IRF3 required for expression of IFN-inducible genes such as IP-10 and ISRE is activated in MyD88-deficient mouse-derived macrophages and DC (Non-patent Document 14).

  On the other hand, regarding the signal transduction of TLR2 and TLR9, MyD88 was essential for cytokine production and NF-kappaB activation in the analysis results in MyD88-deficient mice (Non-patent Document 15). Apart from this, there is a report that a DNA-PKcs-deficient mouse belonging to the PI3K family and also known as a causative gene of SCID mice is unresponsive to CpG DNA (Non-patent Document 16). This suggests that DNA-PKcs exists downstream of TLR9, or that both TLR9 and DNA-PKcs are required for cell activation. Regarding signal transduction, analysis using TLR-deficient mice and MyD88-deficient mice is the main. Regarding humans, there are reports that different cytokines and chemokines are induced by stimulation with TLR2 and TLR4 in MoDC (Non-patent Document 17), and considering the specificity of TLR expression in human DC, we are interested in the analysis results in humans. .

As mentioned above, I do not wait to say that the signal from TLR is important for innate immunity activation, but there is a presence of molecules necessary for acquired immunity activation in response to innate immunity activation apart from TLR innate immunity signal. Expected to be. TREM (T riggering R eceptor E xpressed by M yeloid Cells) -1 is known as a molecule to enhance the adaptive immune reactions undergo innate immune response (Non-Patent Document 18). TREM-1 is a type I cell membrane protein that has one immunoglobulin domain outside of cells expressed on CD14 strongly positive monocytes and neutrophils, and is known to associate with the adapter molecule DAP12 and transmit signals. Expression of TREM-1 is enhanced in CD14 strongly positive monocytes and neutrophils stimulated by bacteria, fungi or their constituents. It is known that when anti-TREM-1 agonistic antibody is allowed to act on neutrophils, large amounts of IL-8 and myeloperoxidase are produced, and monocytes produce IL-1beta, TNF-alpha or MCP-1. ing. At the same time, the expression of adhesion molecules such as ICAM-1, CD11b, CD49e, and CD29 and costimulatory molecules such as CD40 and CD86 are also enhanced. In addition, the expression of these inflammatory cytokines and chemokines is enhanced in coordination with LPS stimulation, causing septic shock. It has been clarified that TREM-1 plays an important function in a model of septic shock (Non-patent Document 19).

  Dendritic cells (DC) differentiate and mature in bone marrow as CD34-positive cells in vivo as progenitor cells, and as antigen-presenting cells (APC), they play an important role in the induction, maintenance, expansion, and regulation of immune responses. It is known to be responsible. DC, a professional APC, is activated by stimulation by an innate immune response and not only triggers an antigen-specific acquired immune response, but also plays an important role in the innate immune response during infection by producing various cytokines. Play a role. In DC, the expression of each TLR is clearly different between MoDC, myeloid DC (MDC) and plasmacytoid DC (PDC) in peripheral blood, and the expression of cytokines and chemokine receptors produced when a ligand is applied is also different. (Non-Patent Documents 20 and 21). In addition, there is a report that a large amount of IL-12p70 is produced by CpG + CD40L in PDC (Non-patent Document 22), and in vivo, which DC subset is subjected to what innate immune response to autocrine / paracrine It is interesting to see what type of phenotype matures under the influence of the acting cytokine.

  In the 1990s, it became possible to differentiate and induce DC from progenitor cells with cytokines, and it became possible to handle a large amount of DC, and the importance of the role of DC in the immune response at the molecular, cellular, and biological levels became clear At the same time, it has attracted attention as a target for immune control.

  From previous biological research results, human monocytes (also referred to as Mo) are cultured in the presence of GM-CSF and IL-4 to induce DC (Monocyte-derived DC; also referred to as MoDC). (Non-Patent Document 23). Although this in vitro differentiation induction system has an artificial part, it has been clarified that it actually has a function as a DC. An important function of DC is to receive an innate immune reaction, to take up an antigen into the cell (phagocytosis) and to transmit information on the antigen to the T cell to stimulate and activate the T cell. DC can be classified into two types, immature DC and mature DC, depending on the stage of differentiation. Antigens taken up into immature DCs are processed intracellularly and presented as antigen-derived peptides on MHC class II molecules on the DC surface. CD4-positive antigen-specific helper T cells recognize antigen-derived peptides and MHC class II molecule complexes as antigen receptors, and are sensitized and activated by stimulation from costimulatory molecules. DCs also stimulate and activate CD8-positive cytotoxic T cells (CTLs) via MHC class I molecules. Phagocytosis is strong in immature MoDC and weak in mature MoDC. The ability to present antigens to T cells is weak in immature MoDCs and strong in mature MoDCs, consistent with the level of expression of CD40, CD80, CD86, MHC class I molecules, and MHC class II molecules involved. Regarding mice, myeloid dendritic cells are cultured by culturing bone marrow cells in the presence of GM-CSF (Non-patent Document 27), and myeloid dendritic cells and plasmacytoid trees are cultured by culturing in the presence of Flt-3 ligand. It has become clear that it is possible to induce dendritic cells (Non-patent Document 28). It is known that myeloid dendritic cells have almost the same function as human MoDC.

  It is no exaggeration to say that the functional change from the antigen uptake ability to the antigen presentation ability in this DC plays a part of invoking acquired immunity in response to the above-mentioned innate immunity reaction. It is not difficult to imagine that the change in the membrane molecules presented on the cell surface will occur with this functional change, but when considering what events are occurring during this maturation process, There is the fact that not all expression of newly expressed molecules on the cell membrane surface arises from mRNA expression. For example, it is known that HLA-DR, which is expressed from the immature stage, translocates from the cell to the cell membrane surface with little change in the expression level of mRNA and protein with maturation. It has been. Further, there are cases where there is almost no correlation when the actual mRNA expression level is compared with the protein expression level, or there are cases where mRNA is expressed but not translated as a protein (Non-patent Document 24). Until now, the gene has been vigorously analyzed because it can be amplified, it can be analyzed in large quantities, it is easy to handle, and many methods can be devised. It is also true that if you can follow changes in the proteins that are present, such changes better reflect intracellular events and can be considered realistic.

  Regarding DC, in addition to the above-mentioned findings, it has also been clarified that several subsets exist in DC, but such subsets are known to be mature and immature and have different functions. However, no answer has been obtained as to why such functional differences between subsets are caused. Elucidation of at least membrane molecules thought to be involved in intracellular signaling, particularly membrane molecules that are expressed as DC matures, is awaited.

  As described above, it is known that DC, which is a powerful APC present in the living body, highly expresses CD80 / CD86 belonging to the B7 family when it matures upon activation stimulation. Regarding the relationship with autoimmune diseases, CD80 / CD86 is abnormally expressed in DCs present in the joint fluid of rheumatoid arthritis, the lesion tissue of psoriasis, and the skin tissue of allergic contact dermatitis, for example. Are known.

  CD28 has the characteristic of potently enhancing the activation of naive T cells. The presence of the signal by CD28 enhances IL-2 production and IL-2 receptor expression, thereby enhancing the proliferative response and consequently enhancing various effector functions of T cells. CD28 signal enhances not only IL-2 but also production of various cytokines such as IL-4, IL-5, IL-13, IFN-gamma, TNF-alpha, and GM-CSF. It is also involved in the expression of T cell activation antigens such as CD40 ligand and chemokines such as IL-8 and RANTES. There are many reports showing that the binding of CD80 or CD86 to CD28 is involved in inducing differentiation of CD4 positive T cells into Th1 or Th2. However, CD80 is not one-sided like Th1 and CD86 is Th2, and the direction of differentiation of naive CD4-positive T cells is determined by various factors specified by antigen or APC itself in addition to CD28 and CD152 signals. It is thought that it is influenced by. However, the CD28 signal plays an important role that is not compensated by the function of other molecules, particularly in the differentiation and activation of naive T cells. (1) Th2 cytokine-dependent immunoglobulin in CD28 knockout mice Selective inhibition of IgG1 and IgG2b production (Non-patent document 25) and (2) Selective inhibition of Th2 differentiation by antigen stimulation with APC from DO11-10TCR transgenic mouse CD4 positive T cells and CD80 / CD86 double knockout mice (Non-patented) This is supported by the results of Document 26). Another important role of the CD28 signal is to enhance the expression of survival factors such as bcl-xL and suppress apoptosis of T cells after antigen stimulation.

  Despite the constant expression of CD28 in T cells, CD80 / CD86 expression is regulated in tissue cells, so excessive immune response even in the presence of T cells that respond to autoantigens It has become a mechanism that does not occur. When this tolerance induction system fails, there is a possibility that various diseases such as autoimmune diseases and allergic diseases are caused. Inflammatory lymphocyte infiltration into the pancreas is observed only by forcibly expressing CD80 or CD86 in islet beta cells, but it does not lead to the development of insulin-dependent diabetes mellitus (IDDM), an autoimmune diabetes, and is compatible with major tissues It is possible to develop IDDM with beta cell disruption only when antigen (MHC) class II molecule, TNF-alpha, or viral protein as a self-antigen is expressed together with CD80. This suggests that in addition to CD28-CD80 / CD86 signals, autoimmune diseases may develop only when placed in an environment that promotes the enhancement of T cell receptor (TCR) signals.

  In some human organ-specific autoimmune diseases, the expression of CD80 / CD86 in DC, which is a professional APC, has been reported. In this way, CD80 / CD86 under control in APC of normal tissues has been found to be abnormally expressed locally in the disease, suggesting an association with autoimmune diseases.

  At present, the treatment of autoimmune diseases is mainly non-specific immunosuppressive agents with many side effects. Inhibition of costimulation has antigen-specific suppression and a sustained effect after the end of administration, and can be expected to reduce side effects and maintain an immune response against infection by shortening the administration period. It is expected that more effective treatment will be performed as the interaction and function of immune system cells become clear. ICOS (inducible T cell co-stimulator) and PD-1 have been found as molecules of the CD28 family that are expressed in activated T cells. It is clear that B7-H2 and B7-H1 belonging to the B7 family are the ligands, respectively, and the elucidation of the difference in the effect of T cell costimulation by APC through multiple B7 family and CD28 family is awaited. .

  In addition, from the viewpoint of biological defense against infectious diseases and the like, the elucidation of the molecular mechanism that links innate immunity and acquired immunity is awaited. In other words, LPS and CpG induce DC maturation with mass production of IL-12 and enhanced expression of CD40 or CD80 / CD86, but no detailed molecular mechanism has been clarified.

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  The present inventors focused on the maturation of DC, which is important for the functional expression of DC, and carried out exploratory research centered on the identification of membrane molecules whose expression level increases with the maturation of DC, based on the proteomics (DC) It has been carried out based on the comprehensive identification of membrane molecules whose expression is regulated as they mature. Proteomics is a term corresponding to gene genomics for proteins, and is used to conduct large-scale research on proteins. Investigate properties such as protein expression levels, post-translational modifications and interactions, for example, what is happening at the expressed protein level in normal and cancer cells, the overall biological such as intracellular networks and processes Get information. If a membrane molecule specifically present in DC is identified, it will be possible to produce an antibody against the membrane molecule and use the antibody in the medical field.

  The object of the present invention is to control infectious diseases, control of autoimmune diseases, etc. by controlling the function of DC targeting the present molecule expressed on mature DC, which is a powerful APC. In addition, since the molecule is expected to be a molecule involved in T cell activation expressed in APC such as mature DC, a method for separating APC such as mature DC using an antibody that specifically recognizes the molecule And providing a method of detecting.

  As a result of searching for a membrane molecule whose expression is regulated by DC maturation, the present inventors have identified a novel membrane molecule that is expressed between immature DC and mature DC. This time, it was found that the expression at the protein level markedly increases with the maturation of. It was revealed that this membrane molecule has homology to TREM-1, which is known to be expressed in macrophages and neutrophils known in the past. This membrane molecule was presumed to have one immunoglobulin (Ig) domain characteristic of the TREM family, and the position of the Cys residue involved in the S—S bond essential for maintaining the three-dimensional structure was preserved. Based on these facts, this membrane molecule group is considered to be a novel membrane molecule belonging to the TREM family, which is expressed in DC, which plays a leading role in connecting innate immunity and acquired immunity.

  From recent research results (1) DC is a heterogeneous cell population and there are several subsets [Stem Cells, Vol. 15, pp. 409-419 (1997)], (2) DC subsets are T cells Have different functions in activation (induction of apoptosis, induction of differentiation of T cells into Th1, Th2, etc.) [Science, 283, 1183-1186 (1999)], (3) Immune response via DC It has become clear that it can be controlled (DC therapy, DC-specific drugs, antibodies, cytokines, etc.).

  The human DC subsets are broadly classified into myeloid DCs and lymphoid DCs. Two differentiation pathways have been shown to exist in myeloid DCs [J. Exp. Med., 184, 695-706 (1996)], and CD34 positive hematopoietic stem cells were identified as GM-CSF and TNF. It is known that CD14-positive CD1a-negative and CD14-negative CD1a-positive progenitor cells appear in the system cultured with -alpha, and differentiate into dermal DC from the former and epidermal Langerhans cells from the latter. MoDC used by the present inventors is similar to DC derived from the former in vivo and is considered to belong to myeloid DC. On the other hand, human lymphoid DCs differentiate into immature DCs (CD11c negative, CD14 negative) in the presence of IL-3 in the presence of plasmacytoid CD4-positive T cells and function as DCs by stimulation with IL-3 and CD40 ligand [J. Exp. Med., 185, 1101-1111 (1997)]. In addition, there is a report that MoDC produces a large amount of IL-12 by stimulation with CD40 ligand or endotoxin and differentiates into DC having a function of differentiating and inducing naive T cells into Th1 [Science, 283, 1183-1186. Page (1999)]. Thus, it can be seen that the MoDC used by the present inventors is a group capable of differentiating at least naive T cells into Th1 type. Peripheral blood has at least two DC subsets, negative for lineage markers (CD3, CD19, CD56, CD14), HLA-DR positive, CD11c positive and lineage markers (CD3, CD19, CD56, CD14) negative , HLA-DR positive, CD11c negative, CD123 strong phenotype. After maturation, the former is MDC producing a large amount of IL-12, the naive T cell is Th1, and the latter is a PDC / NIPC (natural type I interferon producing cell) producing a large amount of type I IFN. Known [Science, 284, 1835-1837 (1999)].

  Thus, this membrane molecule is also expected as a target molecule for controlling the immune response, based on the knowledge that the expression is remarkably increased with the maturation of MoDC.

  On the other hand, a pilot study of a cancer vaccine using autologous peripheral blood DC pulsed with an antigen has also been carried out [Nature Med., 2, 52-58 (1996)]. Utilizing the specificity of expression, DC maturation after pulsing can be confirmed using specific antibodies. In addition, it is useful for separation of mature DC and immature DC and detection of mature DC.

The present invention has been completed based on the above findings. That is, according to the present invention, a protein comprising any one of the following amino acid sequences is provided.
(A) the amino acid sequence set forth in SEQ ID NO: 2 or 4;
(B) an amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2 or 4, and capable of controlling an immune response; or (c ) Amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or 4 and capable of controlling an immune response:

According to another aspect of the present invention, DNA encoding the protein of the present invention is provided.
According to still another aspect of the present invention, DNA comprising any of the following base sequences is provided.
(A) the nucleotide sequence set forth in SEQ ID NO: 1 or 3;
(B) a base sequence encoding an amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more bases in the base sequence set forth in SEQ ID NO: 1 or 3, and capable of controlling an immune response; Or (c) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary sequence thereof under stringent conditions and encodes an amino acid sequence capable of controlling an immune response:

According to still another aspect of the present invention, a recombinant vector comprising the DNA of the present invention is provided.
According to still another aspect of the present invention, a transformant having the DNA or recombinant vector of the present invention is provided.

According to still another aspect of the present invention, there is provided an antibody that specifically immunologically binds to the protein of the present invention or a fragment thereof.
Preferably, the antibodies of the invention recognize dendritic cells immunologically.
Preferably, the antibody of the present invention recognizes the extracellular region of the protein of the present invention.
Preferably, the antibody of the present invention is a polyclonal antibody or a monoclonal antibody.

According to yet another aspect of the present invention, a method for isolating human or other animal-derived immunocompetent cells using the antibody of the present invention is provided.
Preferably, dendritic cells are isolated.

According to still another aspect of the present invention, a method for detecting immunocompetent cells using the antibody of the present invention is provided.
Preferably, dendritic cells are detected.

  According to the present invention, not only can DC be selectively separated with high purity from other blood cells, but also separation of mature DC and immature DC is possible. Enable supply. The separated DC is expected to be used as a cancer vaccine by pulsing the antigen and returning it to the patient again. It is also expected that the immune response can be controlled by inhibiting the interaction between DC and T cells using an antibody against this membrane molecule or a solubilized molecule of this membrane molecule.

The present invention is described in further detail below.
(1) Novel membrane molecule of the present invention and DNA encoding the same
As described above, the present invention is based on the finding that expression of the present membrane molecule increases with maturation in DC.

  The human and mouse dendritic cell membrane molecule of the present invention stimulates mouse bone marrow-derived immature dendritic cells (DCs) induced by culturing mouse bone marrow cells in the presence of GM-CSF with lipopolysaccharide. After obtaining mature DC, the cell membrane is prepared, and the soluble protein is fractionated using techniques such as concanavalin A sepharose chromatography, wheat germ agglutinin sepharose chromatography, and SDS-polyacrylamide gel electrophoresis. , LC / MS (especially QTRAP manufactured by Applied Biosystems) was identified through microanalysis (see Examples 1 to 5 below). Furthermore, primers based on multiple partial amino acid sequences identified by the LC / MS method are synthesized, and a polymerase chain reaction (PCR) is performed using a mouse mature DC-derived cDNA library as a template to amplify the gene fragment of the membrane molecule of the present invention. A clone containing the gene of the membrane molecule (A) of the present invention was selected by colony hybridization using this gene fragment as a probe, and its base sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) were determined. (See Example 6 below).

  In addition, for the molecule (B) that is considered to be a human ortholog, a database search using the BLAST method was performed based on the base sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the molecule (A) identified in the mouse. To identify candidate orthologs and induce human monocytes by immature DC derived from human monocytes cultured in the presence of GM-CSF and IL-4 or by stimulating them with lipopolysaccharide A cDNA library is prepared from the matured human monocyte-derived mature dendritic cells, and a clone containing the gene of the membrane molecule of the present invention is selected by the same colony hybridization as described above, and the nucleotide sequence (SEQ ID NO: 3) and amino acid The sequence (SEQ ID NO: 4) was determined.

  Membrane molecules (A) and (B) of the present invention are obtained by hydropathy plot analysis [J. Exp. Med., 157, 105-132, 1982], signal sequence prediction analysis [Protein Eng., 10, 1-6, 1997], it was suggested that all are type I membrane proteins having a signal sequence.

Membrane molecule (A)
This membrane molecule consists of 209 amino acid residues. From the results of hydropathy plot analysis (Fig. 1) and signal sequence prediction analysis, 17 amino acid residue signal sequence, 140 amino acid residue extracellular region, 21 amino acid residue It is thought to be composed of a transmembrane region and an intracellular region of 31 amino acid residues. Furthermore, from the results of homology search and motif search, it is predicted to have one Ig domain (IgV set) from the position of cysteine (Cys) residue necessary for disulfide bond formation, and potential asparagine-linked glycosylation sites There are two places (asparagine residues at positions 84 and 97). In addition, since a lysine (Lys) residue exists in the transmembrane region, it is expected to associate with some adapter molecule by ionic bond.

Membrane molecule (B)
This membrane molecule consists of 201 amino acid residues. From the results of hydropathy plot analysis (Fig. 1) and signal sequence prediction analysis, 17 amino acid residue signal sequence, 134 amino acid residue extracellular region, 21 amino acid residue It is thought to be composed of a transmembrane region and an intracellular region of 29 amino acid residues. Furthermore, from the results of homology search and motif search, it is predicted to have one Ig domain (IgV set) from the position of cysteine (Cys) residue necessary for disulfide bond formation, and the potential asparagine-linked glycosylation site is It is not present in the extracellular domain.

  The membrane molecules (A) and (B) are considered to be in an ortholog relationship, and as a result of CLUSTAL-W analysis, it was revealed that the entire extracellular domain has 55% homology with amino acid identity. (FIG. 2).

  The membrane molecules of the present invention can be synthesized in large quantities by utilizing gene cloning and DNA recombination techniques. Common techniques for this include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates. Standard techniques as described in and Wiley Interscience, NY (1989) can be used.

  MRNA is extracted from human mature DC by the above standard technique to prepare a cDNA library. The target cDNA can be obtained by screening the library using a specific probe synthesized based on the sequence shown in SEQ ID NO: 1 or 3. Alternatively, a sense and antisense primer for amplifying a sequence containing the mature sequence of the target molecule is synthesized based on the sequence described in SEQ ID NO: 1 or 3, and PCR is performed using the cDNA library as a template. CDNA can be amplified. PCR is preferably performed using an automated thermal cycler, and the reaction is performed in the presence of a thermostable polymerase (such as Taq), template DNA and primers (eg 94 ° C, 15-30 ° C). Seconds), primer annealing (for example, 55 ° C., 30 seconds to 1 minute), and extension reaction in the presence of four kinds of substrates (dNTP) (for example, 72 ° C., 30 seconds to 10 minutes) It can be carried out by carrying out ˜40 cycles and further heating at 70 to 75 ° C. for 5 to 15 minutes. Primer size is usually at least 15 nucleotides.

  By the above-described technique, the DNA encoding the membrane molecules (A) and (B) of the present invention can be cloned. Specific examples of the DNA encoding the membrane molecules (A) and (B) of the present invention include DNA comprising the base sequence described in SEQ ID NO: 1 or 3.

Variant In addition to the mouse or human dendritic cell membrane molecule having the amino acid sequence described in SEQ ID NO: 2 or 4, the present invention further includes one or more amino acids in the amino acid sequence described in SEQ ID NO: 2 or 4. 80% or more, preferably 90% or more, more preferably an amino acid sequence comprising a residue deletion, substitution, insertion and / or addition and capable of controlling an immune response, and the amino acid sequence set forth in SEQ ID NO: 2 or 4 Provides a protein (hereinafter also referred to as a mutant protein) having an amino acid sequence having a homology of 95% or more, most preferably 98% or more and capable of controlling an immune response.

  As used herein, “one or more” amino acid residues are preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 8, particularly preferably 1 to 5, Most preferably, it means 1-3 amino acid residues.

  As used herein, the term “homology” refers to sequence identity or similarity between two or more amino acid sequences or base sequences, such as diagonal graphic method or frequency distribution method. Comparison can be made by conventional methods including.

  As used herein, the term “can control immune response” has the same or substantially the same ability to activate T cells as the native membrane molecules (A) and (B) of the present invention. It may mean that it may have the ability to suppress or inhibit the activation of T cells.

  DNA encoding the mutant protein as described above is also within the scope of the present invention. Such DNA includes, for example, an amino acid sequence that includes one or more base deletions, substitutions, insertions and / or additions in the base sequence set forth in SEQ ID NO: 1 or 3, and can control an immune response. A nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 1 or 3 or a complementary sequence thereof, and a nucleotide that encodes an amino acid sequence capable of controlling an immune response Examples include DNA consisting of a sequence.

  The “one or more” bases referred to in this specification are preferably 1 to 20, more preferably 1 to 10, further preferably 1 to 8, particularly preferably 1 to 5, and most preferably. Means 1-3 bases.

As used herein, the term “base sequence that hybridizes under stringent conditions” means, for example, hybridization at 0.5 M NaHPO ₄ , 7% SDS, 1 mM EDTA, 65 ° C., followed by 0.1 × SSC / 0.1 % SDS, base sequence detected when washed at 68 ° C.

  The mutant protein of the present invention can have all or part of the function of a natural membrane molecule. In vivo, antibodies are produced against certain exogenous antigens that have invaded. At this time, antigen-presenting cells, T cells, and B cells are involved. Antigen-presenting cells take the antigen into the cell and reduce it to read the nature of the antigen. When the antigen is a component other than self, a signal is transmitted to the T cell to activate the T cell for antibody production. The variants of the invention can be used to activate T cells during antibody production. As an actual application example of such a mutant, a gene encoding the mutant is incorporated into an appropriate expression vector (for example, an adenovirus vector), and then introduced into the genome of the target DC by homologous recombination. It is conceivable to obtain DC having the membrane molecule mutant in the DC membrane by expressing the gene.

  Alternatively, the mutant can be used to inhibit or suppress the activation of T cells. In this case, the mutant should be able to antagonize membrane molecules on the natural DC membrane, such as those having a defect in a domain essential for T cell activation. It is done.

  Any mutant is within the scope of the present invention as long as it has high homology (over 80%) with a native membrane molecule and has the ability to control T cell activation. Such variants can be produced, for example, by introducing desired modifications (deletions, substitutions, insertions and / or additions) into natural membrane molecules using site-directed mutagenesis, PCR, etc. (See Molecular Cloning and Current Protocols in Molecular Biology above). Such modifications include, for example, conservative amino acid substitutions such as substitution between acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine and arginine), and hydrophobic amino acids (leucine, isoleucine, valine, etc.). Can be mentioned.

When obtaining the mutant of the present invention from a natural source or the like, prepare a probe based on the nucleotide sequence, and wash under high stringency conditions after hybridization under moderate or high stringency conditions. By taking out a gene encoding the target mutant, incorporating it into an appropriate vector and expressing it, the target mutant can be obtained. An example of high stringency conditions is 0.5 M NaHPO ₄ , 7% SDS, 1 mM EDTA, hybridization at 65 ° C., followed by washing at 0.1 × SSC / 0.1% SDS, 68 ° C. (Current Protocols in See Molecular Biology). Although hybridization conditions can be determined by appropriately selecting temperature and ionic strength, stringency usually increases with higher temperature and lower ionic strength. Accordingly, those skilled in the art can select suitable hybridization conditions. The present invention also includes DNAs encoding the above mutants having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98%, with the base sequence.

(2) Recombinant vector and transformant of the present invention The DNA of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes.

  Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is operably linked to an appropriate transcription / translation regulatory sequence (for example, a promoter). Examples of expression vectors include plasmids, phages, cosmids, viruses, and the like. A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected according to the type of host.

Transcription / translation regulatory sequences can include promoters and enhancers that are selected for the host / vector system used. Examples of promoters include bacterial systems such as P _L , P _R , Ptrp, and Plac; yeast systems such as the PHO5, GAP, ADH, and AOX1 promoters; animal cells include the SV40 early promoter, retroviral promoter, heat shock promoter Can do. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa, calihornica, polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, and baculovirus 39K delayed early gene promoter. There is.

  Moreover, the DNA of the present invention may be functionally bound to an appropriate terminator as necessary. The recombinant vector of the present invention may further have elements such as a polyadenylation signal (for example, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (for example, SV40 enhancer) and the like.

  The recombinant vector of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). .

  The recombinant vector of the present invention may further contain a selection marker. Selectable markers include, for example, genes that lack their complement in host cells, such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin. Methods for ligating the gene, promoter, and, if desired, terminator and / or secretory signal sequence of the present invention and inserting them into an appropriate vector are well known to those skilled in the art.

  Various expression vectors are commercially available or deposited depending on the host, or are described in publications such as literature, and these can be used. For example, pQE (Qiagen), pBluescript II SK + (Stratagene), and pET (Novagen) are used for bacteria.

A transformant can be prepared by introducing the DNA or recombinant vector of the present invention into an appropriate host.
As the host cell into which the DNA or recombinant vector of the present invention is introduced, any cell can be used as long as it can express the protein of the present invention. As host cells, prokaryotic cells, yeasts, animal cells, fungal cells, insect cells or plant cells can be used.

  Examples of prokaryotic cells include, for example, Escherichia, Bacillus, Pseudomonas and the like. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method.

  Examples of yeast cells include cells of the genus Saccharomyces, Schizosaccharomyces or Pichia, such as Saccharomyces cerevis 1ae or Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

  Examples of animal cells include human fetal kidney cells, human leukemia cells, African green monkey kidney cells, Chinese hamster ovary cells (CHO) and the like. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

  Examples of fungal cells are those belonging to filamentous fungi such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

  When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manua1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).

  As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.

  Insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company), New York, which is an ovarian cell of Spodoptera frugiperda (1992)], HiFive (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.

  Examples of the method for co-introducing a recombinant gene introduction vector into insect cells and the baculovirus for preparing a recombinant virus include the calcium phosphate method and the lipofection method.

  The transformed or transfected host cell is cultured in an appropriate culture medium to express the target gene, and the produced membrane molecule of the present invention is recovered from the medium or from the host cell. In the case of recovering from cells, after completion of the culture, the cells are separated by centrifugation or the like, suspended in an aqueous buffer solution, and disrupted by sonication, French press, dynomill or the like to obtain a cell-free extract. Isolation and purification of membrane molecules can be performed by general methods used for protein purification, such as gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, etc., HPLC, electrophoresis, desalting, An organic solvent precipitation method or the like can be arbitrarily combined.

(3) Antibody of the present invention The antibody of the present invention is an antibody that specifically immunologically binds to the protein of the present invention or fragment thereof described in (1) above.

  As used herein, the term “specifically immunocombines” with respect to the antibody of the present invention means that the antibody of the present invention immunologically cross-reacts with an epitope possessed only by the membrane molecule, and other such as the TREM family. Means that it does not cross-react with other proteins. Such epitopes can be obtained by, for example, aligning and comparing amino acid sequences of the membrane molecules of the present invention with amino acid sequences of other proteins (subsequently at least 5 amino acids, preferably at least 8 amino acids, Preferably at least 15 amino acids).

  Membrane molecules belonging to the TREM family increase mainly with activation. As described above, this is also applied to mature DCs, activated macrophages, activated monocytes, activated B cells, etc. It is expected that membrane molecules will be expressed. By utilizing this finding, an antibody against this membrane molecule that specifically recognizes activated antigen-presenting cells (APC) can be obtained.

  Any antibody having the above-mentioned characteristics is encompassed by the present invention. Antigen epitopes for obtaining the target antibody include regions having high antigenicity, superficial regions, two regions in the amino acid sequence of this membrane molecule group. It may be selected from regions that may not have the following structure, regions that have no or low homology with other proteins (especially other members of the family to which each membrane molecule belongs). Here, the region having high antigenicity can be estimated by the method of Parker et al. [Biochemistry, 25, 5425-5432 (1986)]. The superficial region can be estimated by calculating and plotting a hydropathic index, for example. A region that may not have a secondary structure can be estimated by, for example, the method of Chou and Fasman [Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978)]. Furthermore, in particular, a region having no or low homology with other proteins to which each membrane molecule belongs can be estimated by comparing the homology between the amino acid sequence of each membrane molecule and the amino acid sequence of the other protein.

  Based on the partial amino acid sequence of the present membrane molecule estimated by the above method, a peptide comprising the amino acid sequence can be synthesized by using a peptide synthesis method. The peptide of interest can be synthesized, for example, using a commercially available peptide synthesizer based on solid phase peptide synthesis developed by RB Merrifield [Science, 232, 341-347 (1986)] After desorption, the product is purified by ion exchange chromatography, gel filtration chromatography, reverse phase chromatography or the like alone or in combination. The resulting purified peptide can be used as an immunogen by binding to a carrier protein such as keyhole limpet hemocyanin (KLH) or albumin.

  Furthermore, a polyclonal antibody or a monoclonal antibody against the membrane molecule can be prepared by a known technique using the recombinant membrane membrane as an immunogen. In this case, the term “recombinant” as used with respect to the present membrane molecule, monoclonal antibody, polyclonal antibody, or other protein means that these proteins are produced by recombinant DNA in the host cell. . As a host cell, both prokaryotic organisms (for example, bacteria such as E. coli) and eukaryotic organisms (for example, yeast, CHO cells, insect cells, etc.) can be used.

  The “antibody” of the present invention may be a peptide antibody, a polyclonal antibody, or a monoclonal antibody. “Antibodies” are produced subcutaneously, intraperitoneally, in order to produce antibodies that will or will produce antibodies that will specifically bind to the protein used to immunize mice or other suitable host animals. It is obtained by immunization with an antigen or antigen-expressing cells by an intramuscular or intramuscular route. Further, as a host animal, a desired human antibody may be obtained by administering an antigen or an antigen-expressing cell to a transgenic animal having a repertoire of human antibody genes [Proc. Natl. Acad. Sci. USA, 97, 722]. -727 (2000), international publications WO96 / 33735, WO97 / 07671, WO97 / 13852, WO98 / 37757]. Alternatively, lymphocytes may be immunized in vitro. Polyclonal antibodies can be obtained by collecting and purifying fractions that bind to the antigen from serum obtained from the host animal. In addition, to form hybridoma cells, monoclonal antibodies can be prepared by fusing lymphocytes with myeloma cells using an appropriate fusion reagent such as polyethylene glycol (Goding, Monoclonal Antibodies: Principals and Practice, pages 59-103, Academic press, 1986). For example, the monoclonal antibody of the present invention can be prepared using either the hybridoma method [Nature, 256, 495 (1975)] or the recombinant DNA method (Cabilly et al., US Pat. No. 4,816,567). it can.

  Antigen proteins can be prepared by expressing DNA encoding all or a partial sequence of the protein of this membrane molecule in E. coli, yeast, insect cells, animal cells, and the like. The recombinant membrane molecule is purified by a method such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography or the like alone or in combination, and this purified sample is used as an immunogen.

The antibody of the present invention may be an intact antibody or an antibody fragment such as (Fab ′) ₂ or Fab.

  Also, a chimeric antibody in which the constant region is replaced with a human constant region (eg, mouse-human chimeric antibody; Cabilly et al., US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 89, 6851 (1984) ), Humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, vol. 89) in which all variable regions except the constant region and the hypervariable region (or CDR) are replaced with human sequences. 4285, 1992 and Carter et al., BioTechnology, 10, 163, 1992) are also included in the antibodies of the present invention.

  Further, an antibody against the antibody of the present invention thus obtained, that is, an anti-idiotype antibody is also included in the present invention.

  Various antibodies against the present membrane molecule thus obtained can be used in various applications that can make use of the characteristics. Labeling this antibody with a fluorescent substance (rhodamine, fluoresamine, etc.) using this membrane molecule that is expressed specifically in mature DC, and detecting the target DC using well-known FACS -Isolation and differentiation of mo-DC in vitro can be confirmed. Furthermore, since this membrane molecule is a protein whose expression level is markedly increased with DC maturation, immature DC and mature DC can be separated by FACS using this antibody. The detection of this membrane molecule is not limited to detection by FACS. For example, it is expected that this antibody can be detected as a primary antibody in Western blotting, and the expression can be confirmed at the protein level. In addition, this antibody can be bound to a solid phase (polystyrene beads, microtiter well surfaces, latex beads, etc.) to detect and quantify (fluorescence) homogenous membrane molecules by heterogeneous or homogeneous immunological reactions. Antibody method, ELISA, radioimmunoassay, etc.). In this case, the immunological reaction may be a competitive reaction or a non-competitive reaction. A reaction by sandwich method using two or more antibodies (monoclone or polyclone) can also be used. For the detection and quantification described above, any immunological technique known in the art can be used.

  In addition, it can be used for applications for evaluating the function of the present membrane molecule. Mature DC is a potent APC and is known to stimulate and activate CD4 positive T cells via MHC class II molecules and CD8 positive cytotoxic T cells via MHC class I molecules Yes. In order to confirm whether these functions can be controlled, it is confirmed whether the functions of allogenic MLR (Mixed Lymphocyte Reaction) can be suppressed. When antigen-specific CTL is induced, the functions are suppressed. It can also be used for in vitro assays such as confirmation of whether or not it is possible, and confirmation of whether or not it is a molecule involved in DC antigen presentation.

  The antibodies of the present invention can also be used to control immune responses in vivo. As described above, this membrane molecule group is expected to be a molecule involved in costimulation, and is expected to be a membrane molecule involved in T cell activation. Therefore, if the antibody of the present invention is an antibody that inhibits the binding between the membrane molecule group and a ligand on T cells, it is expected to suppress T cell activation. Thus, it is expected that the immune response by controlling the function of DC and further the function of T cell can be controlled by acting the antibody capable of controlling the function of this membrane molecule group.

  Furthermore, the membrane molecule, a solubilized molecule of the ligand of the membrane molecule (that is, a molecule corresponding to the extracellular region) and an antibody against these molecules are expected to have an activity of controlling the immune response.

  It is also possible to obtain a ligand of the present membrane molecule using the antibodies of the present invention or a solubilized molecule of the present membrane molecule. The solubilized main membrane molecule ligand directly acts on the main membrane molecule, and can control a signal through the main membrane molecule on DC. In addition, low molecular weight substances that can modulate the interaction between the membrane molecule ligand and the membrane molecule, and low molecular weight substances that modulate the intracellular signal pathways associated with the ligand and the membrane molecule are also useful for signal control. It is.

  When the antibodies of the present invention or a solubilized molecule of the present membrane molecule, a solubilized molecule of the present membrane molecule, or the above small molecule is used for treatment, for example, cancer, autoimmune disease, organ transplantation, infectious disease, It can be applied to the treatment of diseases such as allergies.

Administration method and dosage form are not particularly limited, but are intravenous, intraarterial administration, intramuscular administration, oral administration, suppository administration, etc. Orally combined with pharmaceutically acceptable excipients and diluents Or it can be formulated for parenteral use, but parenteral administration is preferred. Administration is carried out once or divided into several times per day, and the dosage is determined according to the patient's severity, age, sex, weight, and other conditions and does not cause side effects.
Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1 Preparation of DC cell membrane In the case of a cell membrane protein, the expression level is originally low and handling is difficult. As one means for solving this, it is necessary to establish a method for obtaining a cell membrane with higher purity. By coating the cell membrane surface and crushing it uniformly, a cell membrane having a high specific gravity was obtained by density gradient centrifugation to prepare a cell membrane with high purity [J. Biol. Chem., 258, 10062- 10072 (1983)].

Because of the low expression level of the target membrane molecules and the difficulty of handling, bone marrow cell-derived immature DCs were prepared in large quantities by culturing bone marrow cells in the presence of GM-CSF to collect large amounts of mouse DCs. With and without LPS (lipopolysaccharide) stimulation, the same number of cells was used to obtain the same number (5 × 10 ⁸ cells) of immature DC and mature DC.

Example 2 Sensitive detection of protein and in gel digestion
Proteins were detected by silver staining according to the method of Mann et al. [Anal. Chem., 68, 850-858 (1996)]. The enzyme digestion method was performed by the in gel digestion method using trypsin [Anal. Chem., 224, 451-455 (1995)], and used as an analysis sample to be used below.

Example 3 Fractionation of membrane protein Cell membrane protein was solubilized from the cell membrane obtained in Example 1 using a surfactant, applied to concanavalin A sepharose, and washed with a surfactant-containing buffer. These passing fractions were called ConA FT. The adsorbed fraction was eluted with a buffer containing methyl-alpha-D-glucopyranoside and a surfactant (ConA EL). ConA FT was reapplied to wheat germ agglutinin sepharose and washed with detergent-containing buffer. These passing fractions were referred to as WGA FT. The adsorbed fraction was eluted with a buffer containing N-acetylglucosamine and a surfactant (WGA EL). ConA EL, WGA EL, and WGA FT were used as molecular identification samples. After developing these fractions by SDS-PAGE, the protein was detected by the method of Example 2, and the gel cut into strips was subjected to in gel digestion to obtain an analysis sample.

Example 4 Trace analysis by LC / MS
The sample obtained in Example 3 was analyzed using LC / MS (Applied Biosystems QTRAP). Apply to a PepMap reverse phase column (inner diameter 0.075 mm x length 150 mm) (LC Packings) equilibrated with 95% solution A (0.1% formic acid) and 5% solution B (0.08% formic acid, 80% acetonitrile). After washing for 5 minutes at a flow rate of nanoliter / minute, the ratio of solution B was linearly increased to 50% over 67.5 minutes, and then eluted and introduced into MS. Samples introduced into MS were subjected to data acquisition in the next repetitive cycle to obtain sequence information. 1. Full MS Scan: Observation of the molecular weight of the parent ion in the range of m / z = 0 to 2000. 2. Zoom MS Scan: Observation of the valence of the parent ion identified by Full MS Scan. 3. MS / MS Scan: Observation of daughter ions generated when He gas is applied to molecules measured by Zoom MS Scan. Identified by an identification method (SEQUEST algorithm) that scores and ranks how many lines match the theoretical b, y series and how much intensity [American Society for Mass Spectrometry, 5 Volume 976-989 (1994)].

Example 5 Identification of Main Membrane Molecules Among the molecules identified by the method of Example 4, a novel membrane molecule was identified. The fragment to be identified is a divalent ion of m / z = 1470.67, and its MS / MS pattern is very well assigned to the partial amino acid sequence of this membrane molecule (27 residues) DTMTSSNQLPWPTVDGSTDMVSSDLQK (SEQ ID NO: 5). It was. As a result of homology search of these partial sequences with respect to the non-redundant DNA database, it was revealed that there are no other base sequences having these partial sequences.

Example 6 Gene Cloning of the Membrane Molecule When the partial amino acid sequence (SEQ ID NO: 5) identified in Example 5 was searched against the mouse gene database, it was the only gene having a partial sequence completely matching NM_199221. It turned out to be. Therefore, primers were synthesized based on the gene sequence corresponding to the partial amino acid sequence revealed in Example 5, and RT-PCR was performed using a mouse mature DC cDNA library as a template. The primers were 30-mer and 40-mer, respectively, and sense primer 5′-ATGAGTAAGACACTCAGAGGCAACCTGGAC-3 ′ (SEQ ID NO: 6) and antisense primer 5′-CTAAGGAGAAATGTCTTTAGCTAGAAGTTCAGAGTCCAAA-3 ′ (SEQ ID NO: 7) were used. As a result, a gene fragment of the membrane molecule of about 800 bp was obtained. A clone containing the gene of this membrane molecule was selected by colony hybridization using this gene fragment as a probe, and the base sequence was determined. The predicted open reading frame structure is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2. Although the obtained gene sequence is the same as NM_199221 reported as a known gene, it is clear that the reading frame is shifted and the amino acid sequence is different from the middle because it is missing one base from the report It became. In addition, human orthologs were obtained in the same manner using a cDNA library of human monocyte-derived dendritic cells as a template. The primers were 28 mer and 30 mer, respectively, and sense primer 5′-ATGTGCAGAAGGTGCAAGCCAGAGCTCA-3 ′ (SEQ ID NO: 8) and antisense primer 5′-CTAAGTGGCCATGTCTTTAGTCAGAGGTTC-3 ′ (SEQ ID NO: 9) were used. The predicted open reading frame structure is shown in SEQ ID NO: 3, and the predicted amino acid sequence is shown in SEQ ID NO: 4. The resulting gene corresponds to NM_174892.

  Hydropathy plot analysis was performed on the primary structure of the deduced amino acid sequence of this membrane molecule according to the method of Kyte and Doolittle (J. Exp. Med., 157, 105-132, 1982) (FIG. 1). As a result, it was revealed that the present membrane molecule is a type I transmembrane protein having a signal sequence at the N-terminus. The membrane molecules (A) and (B) are each composed of (A) 209 and (B) 201 amino acid residues, and from the hydropathy plot analysis results, a signal sequence of 17 residues, (A) 140 residues, (B) It is expected to have an extracellular region of 134 residues, a transmembrane region of 21 residues, (A) 31 residues, and (B) an intracellular region of 29 residues. Furthermore, from the results of homology search and motif search, the extracellular region is (A) 2 sites, (B) 0 asparagine-linked glycosylation sites, 2 cysteine residues necessary for immunoglobulin (Ig) domain formation. It had one immunoglobulin-like structure. It was also inferred that this extracellular Ig domain has a V set structure. Furthermore, although (A) and (B) do not have a motif capable of transmitting a signal to the intracellular region, both (A) and (B) have a lysine residue in the transmembrane region, and thus associate with the adapter molecule. And was expected to transduce signals through this associated adapter molecule. In addition, this membrane molecule is structurally homologous to or similar to TREM-1 due to having one IgV set outside the cell and having a positively charged lysine residue in the transmembrane region. Is done. The mouse membrane molecule has 26% amino acid identity to mouse TREM-1, and the human membrane molecule has 26% amino acid identity to human TREM-1, and the cysteine residues necessary for IgV set formation are conserved. .

Example 7 Production of Solubilized Recombinant Recombinant Membrane Molecules Based on the results of hydropathy plot analysis (FIG. 2), a recombinant in which the region considered to be an extracellular domain and the Fc region of human IgG1 were combined was prepared . The Fc region of human IgG1 was substituted with three amino acids in order to prevent binding to Fc gamma receptor present on various cell surfaces. Specifically, up to amino acid position 157 of SEQ ID NO: 1 in (A) was selected as the extracellular region. The amino acid sequence of this solubilized recombinant is shown in SEQ ID NO: 10. The sites where amino acid substitution was performed in the Fc region correspond to positions 177 (Ala → Leu), 178 (Glu → Leu), and 344 (Asp → Val) shown in SEQ ID NO: 10. This gene was transiently expressed in 293T cells under the control of the CMV promoter, and the expression product was purified from the obtained culture supernatant by affinity chromatography using a resin bound with protein A having binding properties to the Fc region ( FIG. 3).

Example 8 Search for Endogenous Ligands for the Main Membrane Molecules Using the recombinant described in Example 7 to search for the presence of molecules that bind to the main membrane molecules on mouse spleen cells using a flow cytometer did. Specifically, the recombinant is recognized using a phycoerythrin-labeled F (ab ′) ₂ anti-human IgG Fc region antibody (manufactured by Beckman Coulter) as a secondary antibody. As a result, it was confirmed that the present membrane molecule was bound to all confirmed cells such as CD3-positive T cells, CD19-positive B cells, NK1.1-positive NK cells, and CD11c-positive dendritic cells. (Fig. 4)

FIG. 1 shows the results of hydropathy plot analysis of the membrane molecules using the Kyte and Doolittle method. FIG. 2 shows the results of alignment analysis between the mouse and human of this membrane molecule by CLUSTAL W. FIG. 3 shows the expression and purification of the recombinant. FIG. 4 shows the search for endogenous ligands using recombinants.

SEQUENCE LISTING
<110> RIKEN
<120> New Dendritic Cell Membrane Molecule and Its Uses
<130> A42008A
<160> 10
<210> 1
<211> 768
<212> DNA
<213> mouse
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atgagtaaga cactcagagg caacctggac aaacagaacg agtcagctga gatctccagg 60
gctttgggta tcccgctgag atttggattt gctgctggct gttcatcaag gagtgcagca 120
aagtgggaag gcaggaccat gtggctgtcc ccagctttgc ttcttctcag ttttccaggc 180
tgcctctcca tccaaggccc agcattggtg aggggtccag agcaggggtc agtgactgtg 240
caatgtcgct atagctcaag atggcaaacc aacaagaagt ggtggtgccg gggagcaagc 300
tggagcactt gcagggtcct catccgatcc actgggtcag agaaagaaac gaagagcggc 360
cggctgtcca tcagggacaa tcagaaaaat cactcattcc aggttaccat ggagatgctc 420
aggcaaaatg acacggacac ttactggtgt ggtattgaaa agttcggaac tgaccgtggg 480
accagagtta aagtgaacgt ctactcggtg ggtaaagata ccatgtcgac ttctaatcaa 540
cttccctggc ccactgtgga cggcagtaca gacatggtgt cttctgactt gcagaagagg 600
acctattaca tgctcctggt atttgtgaag gtgcctgcct tgctcatctt ggttggtgct 660
gtcctctggc tgaagaggtc aactcagaag gtccctgagg aacagtggag acacactctc 720
tgtagcgatt tggactctga acttctagct aaagacattt ctccttag 768
<210> 2
<211> 209
<212> PRT
<213> mouse
<400> 2
Met Trp Leu Ser Pro Ala Leu Leu Leu Leu Ser Phe Pro Gly Cys Leu
1 5 10 15
Ser Ile Gln Gly Pro Ala Leu Val Arg Gly Pro Glu Gln Gly Ser Val
20 25 30
Thr Val Gln Cys Arg Tyr Ser Ser Arg Trp Gln Thr Asn Lys Lys Trp
35 40 45
Trp Cys Arg Gly Ala Ser Trp Ser Thr Cys Arg Val Leu Ile Arg Ser
50 55 60
Thr Gly Ser Glu Lys Glu Thr Lys Ser Gly Arg Leu Ser Ile Arg Asp
65 70 75 80
Asn Gln Lys Asn His Ser Phe Gln Val Thr Met Glu Met Leu Arg Gln
85 90 95
Asn Asp Thr Asp Thr Tyr Trp Cys Gly Ile Glu Lys Phe Gly Thr Asp
100 105 110
Arg Gly Thr Arg Val Lys Val Asn Val Tyr Ser Val Gly Lys Asp Thr
115 120 125
Met Ser Thr Ser Asn Gln Leu Pro Trp Pro Thr Val Asp Gly Ser Thr
130 135 140
Asp Met Val Ser Ser Asp Leu Gln Lys Arg Thr Tyr Tyr Met Leu Leu
145 150 155 160
Val Phe Val Lys Val Pro Ala Leu Leu Ile Leu Val Gly Ala Val Leu
165 170 175
Trp Leu Lys Arg Ser Thr Gln Lys Val Pro Glu Glu Gln Trp Arg His
180 185 190
Thr Leu Cys Ser Asp Leu Asp Ser Glu Leu Leu Ala Lys Asp Ile Ser
195 200 205
Pro

<210> 3
<211> 722
<212> DNA
<213> human
<400> 3
tctagatgtg cagaaggtgc aagccagagc tcaggcagaa cttccagagt gcatctggga 60
tctgcatttg ccactggttg cagatcaggc ggacgaggag ccgggaaggc agagccatgt 120
ggctgccccc tgctctgctc cttctcagcc tctcaggctg tttctccatc caaggcccag 180
agtctgtgag agccccagag caggggtccc tgacggttca atgccactat aagcaaggat 240
gggagaccta cattaagtgg tggtgccgag gggtgcgctg ggatacatgc aagatcctca 300
ttgaaaccag agggtcggag caaggagaga agagtgaccg tgtgtccatc aaggacaatc 360
agaaagaccg cacgttcact gtgaccatgg aggggctcag gcgagatgac gcagatgttt 420
actggtgtgg gattgaaaga agaggacctg accttgggac tcaagtgaaa gtgatcgttg 480
acccagaggg agcggcttcc acaacagcaa gctcacctac caacagcaat atggcagtgt 540
tcatcggctc ccacaagagg aaccactaca tgctcctggt atttgtgaag gtgcccatct 600
tgctcatctt ggtcactgcc atcctctggt tgaaggggtc tcagagggtc cctgaggagc 660
caggggaaca gcctatctac atgaacttct ccgaacctct gactaaagac atggccactt 720
ag 722
<210> 4
<211> 201
<212> PRT
<213> human
<400> 4
Met Trp Leu Pro Pro Ala Leu Leu Leu Leu Ser Leu Ser Gly Cys Phe
1 5 10 15
Ser Ile Gln Gly Pro Glu Ser Val Arg Ala Pro Glu Gln Gly Ser Leu
20 25 30
Thr Val Gln Cys His Tyr Lys Gln Gly Trp Glu Thr Tyr Ile Lys Trp
35 40 45
Trp Cys Arg Gly Val Arg Trp Asp Thr Cys Lys Ile Leu Ile Glu Thr
50 55 60
Arg Gly Ser Glu Gln Gly Glu Lys Ser Asp Arg Val Ser Ile Lys Asp
65 70 75 80
Asn Gln Lys Asp Arg Thr Phe Thr Val Thr Met Glu Gly Leu Arg Arg
85 90 95
Asp Asp Ala Asp Val Tyr Trp Cys Gly Ile Glu Arg Arg Gly Pro Asp
100 105 110
Leu Gly Thr Gln Val Lys Val Ile Val Asp Pro Glu Gly Ala Ala Ser
115 120 125
Thr Thr Ala Ser Ser Pro Thr Asn Ser Asn Met Ala Val Phe Ile Gly
130 135 140
Ser His Lys Arg Asn His Tyr Met Leu Leu Val Phe Val Lys Val Pro
145 150 155 160
Ile Leu Leu Ile Leu Val Thr Ala Ile Leu Trp Leu Lys Gly Ser Gln
165 170 175
Arg Val Pro Glu Glu Pro Gly Glu Gln Pro Ile Tyr Met Asn Phe Ser
180 185 190
Glu Pro Leu Thr Lys Asp Met Ala Thr
195 200
<210> 5
<211> 27
<212> PRT
<213> mouse
<400> 5
Asp Thr Met Ser Thr Ser Asn Gln Leu Pro Trp Pro Thr Val Asp Gly
1 5 10 15
Ser Thr Asp Met Val Ser Ser Asp Leu Gln Lys
20 25
<210> 6
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: a primer
<400> 6
atgagtaaga cactcagagg caacctggac 30
<210> 7
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: a primer
<400> 7
ctaaggagaa atgtctttag ctagaagttc agagtccaaa 40
<210> 8
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: a primer
<400> 8
atgtgcagaa ggtgcaagcc agagctca 28
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: a primer
<400> 9
ctaagtggcc atgtctttag tcagaggttc 30
<210> 10
<211> 390
<212> PRT
<213> mouse / human
<400> 10
Met Trp Leu Ser Pro Ala Leu Leu Leu Leu Ser Phe Pro Gly Cys Leu
1 5 10 15
Ser Ile Gln Gly Pro Ala Leu Val Arg Gly Pro Glu Gln Gly Ser Val
20 25 30
Thr Val Gln Cys Arg Tyr Ser Ser Arg Trp Gln Thr Asn Lys Lys Trp
35 40 45
Trp Cys Arg Gly Ala Ser Trp Ser Thr Cys Arg Val Leu Ile Arg Ser
50 55 60
Thr Gly Ser Glu Lys Glu Thr Lys Ser Gly Arg Leu Ser Ile Arg Asp
65 70 75 80
Asn Gln Lys Asn His Ser Phe Gln Val Thr Met Glu Met Leu Arg Gln
85 90 95
Asn Asp Thr Asp Thr Tyr Trp Cys Gly Ile Glu Lys Phe Gly Thr Asp
100 105 110
Arg Gly Thr Arg Val Lys Val Asn Val Tyr Ser Val Gly Lys Asp Thr
115 120 125
Met Ser Thr Ser Asn Gln Leu Pro Trp Pro Thr Val Asp Gly Ser Thr
130 135 140
Asp Met Val Ser Ser Asp Leu Gln Lys Arg Thr Tyr Tyr Leu Glu Pro
145 150 155 160
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
165 170 175
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
180 185 190
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
195 200 205
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
210 215 220
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
225 230 235 240
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
245 250 255
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
260 265 270
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
275 280 285
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
290 295 300
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
305 310 315 320
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
325 330 335
Thr Pro Pro Val Leu Asp Ser Val Gly Ser Phe Phe Leu Tyr Ser Lys
340 345 350
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
355 360 365
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
370 375 380
Ser Leu Ser Pro Gly Lys
385 390

Claims (13)

A protein comprising any of the following amino acid sequences.
(A) the amino acid sequence set forth in SEQ ID NO: 2 or 4;
(B) an amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 2 or 4, and capable of controlling an immune response; or (c ) Amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2 or 4 and capable of controlling an immune response: DNA encoding the protein according to claim 1. DNA consisting of any of the following base sequences:
(A) the nucleotide sequence set forth in SEQ ID NO: 1 or 3;
(B) a base sequence encoding an amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more bases in the base sequence set forth in SEQ ID NO: 1 or 3, and capable of controlling an immune response; Or (c) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or 3 or a complementary sequence thereof under stringent conditions and encodes an amino acid sequence capable of controlling an immune response: A recombinant vector comprising the DNA according to claim 2 or 3. A transformant comprising the DNA according to claim 2 or 3 or the recombinant vector according to claim 4. An antibody that specifically immunologically binds to the protein of claim 1 or a fragment thereof. The antibody of claim 6, which immunologically recognizes dendritic cells. The antibody according to claim 6, which recognizes an extracellular region of the protein according to claim 1. The antibody according to any one of claims 6 to 8, which is a polyclonal antibody or a monoclonal antibody. A method for separating immunocompetent cells derived from humans or other animals using the antibody according to claim 6. The method of claim 10, wherein the dendritic cells are isolated. A method for detecting immunocompetent cells using the antibody according to claim 6. The method of claim 12, wherein dendritic cells are detected.