JP2003052374A - New dendritic cell membrane molecule and dna encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new a human dendritic cell membrane molecule expressed by maturation of the human dendritic cell. SOLUTION: Disclosed are the new the human dendritic cell membrane molecule, a mutant thereof, a DNA encoding the membrane molecule or the mutant, a antibody specifically and immunologically binding to the membrane molecule, the mutant thereof or their fragments, and a method for separating or detecting the dendritic cell using the antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to human dendritic cells (Den
dritic cell; hereinafter also referred to as DC), particularly a membrane molecule expressed in mature DC, a DNA encoding the membrane molecule, an antibody against the membrane molecule, and a DC separation method and a DC detection method using the antibody.

[0002]

2. Description of the Related Art The development of autoimmune diseases has a genetic background,
Exposure to self-antigens that were not originally exposed to the immune system due to tissue inflammatory reaction, and local environment derived from inflammatory reaction of viruses and bacteria, that is, homology of activated T cell recognition peptide Changes in the local environment, such as the activation of so-called cross-reactive T cells, and further costimulatory molecules on the cell membrane.
It is assumed that factors such as the presence of autoreactive T cells induced by failure of tolerance due to abnormal expression of ule) are involved.

[0003] Antigen-presenting cells (APC) that have not been stimulated by normal tissues are regulated by the expression of costimulatory molecules even if they present self-antigens, so that T cells are in an anergy and activated. However, self-tolerance is maintained. However, it has been suggested that autoimmune diseases may occur as a result of activation of autoreactive T cells due to excessive or continuous abnormal expression of costimulatory molecules in an abnormal immune state. In particular, the molecules belonging to the CD28 family expressed on the T cell side, CD28 / CD152 (CTLA-4) and the molecules belonging to the B7 family expressed on the APC side, CD80 (B7-1) / CD86 (B7-
The signal generated between 2) plays an important and unique role in controlling T cell activation, and immunotherapy by regulating this signal has been tried in a mouse experimental model, and in fact clinical trials have begun in humans. .

It has been clarified that the costimulation between CD40 and CD154 (CD40L) is indispensable, in addition to the signal generated between CD28 / CD152 and CD80 / CD86. CD40, which belongs to the tumor necrosis factor receptor (TNFR) superfamily, is constitutively expressed in APC. T cells stimulated through TCR from APC express CD154 (belonging to TNF family) in an activation-dependent manner. It is known that APC stimulated by CD40 is activated and matured, and expression of CD80 / CD86 is increased.

[0005] Dendritic cells (DC) are differentiated and matured in vivo by using CD34-positive cells existing in bone marrow as progenitor cells to produce APCs.
Is known to play an important role in the induction, maintenance, expansion and regulation of immune responses. In the 1990s, it became possible to differentiate and induce DCs from progenitor cells with cytokines, and it became possible to handle large amounts of DCs.
The importance of the role of DCs in the immune response at the cellular and biological levels has become clear, and it has become a focus of attention as a target for immune regulation.

[0006] From the results of biological research so far, human monocytes (also called Mo) are also called DC (monocyte-derived DC; mo-DC) by culturing in the presence of GM-CSF and IL-4. ) Has been demonstrated [J. Leucocyte Biol., 59, 208-218 (1996).
Year)]. Although this in vitro differentiation induction system has an artificial part, it has become clear that it actually functions as a DC. The important function of DC is to take up the antigen into the cell (phagocytosis) and transmit information on the antigen to the T cell to stimulate and activate the T cell. In addition, DC may be immature DC or mature DC (Mat) depending on the stage of differentiation.
ure DC). Antigens taken up in immature DCs are processed intracellularly and presented as antigen-derived peptides on DC surface MHC class II molecules. CD4-positive antigen-specific helper T cells recognize the complex of the peptide derived from the antigen and MHC class II molecule at the antigen receptor, and are stimulated and activated by the stimulation from the costimulatory molecule. At this step, CD40 stimulation is particularly important for activation of antigen presenting cells. T with TCR stimulation
Cells express CD154 (CD40 ligand) and CD154-mediated DC
CD40-mediated stimulation of above and promotion of DC maturation, IL-1
Production of cytokines including 2 is seen.

[0007] CD is also mediated by CD via MHC class I molecules.
8-positive cytotoxic T cells (CTL) are also stimulated and activated. Phagocytosis is strong in immature mo-DC and weak in mature mo-DC. The ability to present antigens to T cells is related to CD40, C
Consistent with the degree of expression of D80, CD86, MHC class I molecule, and MHC class II molecule, it is weak in immature mo-DC and strong in mature mo-DC.

By the way, it is not difficult to imagine that a change in the membrane molecule presented on the cell surface occurs with a change in the function from antigen uptake ability to antigen presentation ability, but this maturation (matu
When considering what kind of event is occurring in the process of (ration), there is a fact that the expression of the molecule newly expressed on the cell membrane surface does not always occur from the expression of mRNA. For example, it is known that HLA-DR, which is expressed from the immature stage, translocates from the intracellular surface onto the cell membrane surface with little change in the expression levels of mRNA and protein with maturation. Has been. There is also a case where there is almost no correlation when the actual mRNA expression level is compared with the protein expression level, or the mRNA is expressed but not translated as a protein [Biochem. Biophys. Res. Commun., 231
Vol. 1-6 (1997)]. Up 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 various methods can be devised. It is also true that if changes in the existing proteins can be tracked, then such changes better reflect intracellular events and are closer to reality.

[0009] Regarding DC, in addition to the above findings, it has been clarified that there are several subsets in DC. It is known that such subsets differ in function between immature and mature. There is. However, the answer to what caused such functional differences between subsets is not available. At least, the elucidation of the membrane molecules that are considered to be involved in intracellular signaling, especially the membrane molecules that are expressed as DCs mature.

[0010] As described above, the proliferation (proliferation) and survival (s
It is known that various membrane molecules are involved in urvival) and differentiation. Among them, the superfamily of Tumor Necrosis Factor (TNF) and its receptor (TNFR) is famous as a group of molecules involved in inflammation, apoptosis, autoimmunity, and organ formation. Molecule belonging to TNF superfamily (TNFSFP)
And molecules belonging to the TNFR superfamily (TNFRSF
P) are all known to form symmetrical trimers [Microscopy Res. Tech., 50, 184-195 (2000).
Year)]. TNF SFP is a type II protein and has a compact jellyroll beta strand structure, and molecules belonging to SF have 25-30% homology in amino acid identity. On the other hand, TNFRSFP is a type I protein whose structure is maintained by disulfide bonds, and is a cysteine-rich domain (cyst
eine-rich domains (CRDs). TNFRSF usually has 1 to 5 CRDs, and the CRD at the most amino-terminal side is CRD1a, CRD1b, C from the position of the cysteine residue.
Broadly divided into RD1c and CRD1d. Regarding the membrane protein of TNFRSF, the signal transduced into cells is TRAFs (TNF-recep
tor associated factors) Through the molecule [Exp. Cel
l Res., 254, 14-24 (2000)] and death domain (DD) molecule [Cell., 103
Vol., Pp. 273-282 (2000)]. Of the TNFRSF, those that pass through DD (Fas, TNFR1, DR, etc.) are intracellular 60
It has six conserved alpha helices over amino acids and is unique to individual TNFR-specific death receptors (Death Receptor).
Molecules called ceptors (DRs) (Fas → FADD (Fas-associ
ated DD protein), TNFRI → TRADD (TNFR-associated DD
signal) via). These DD
The protein activates a cysteine protease called caspase and induces cell death [Curr. Op.
in. Immunol., 11: 277-285 (1999)]. About TRAF molecule, TRAF1 to TRAF6 are known, and each TN
It is known that TRAFs that can be bound by FRSFPs are different, and this binding is determined depending on the sequence of the intracellular domain of each TNFRSFP. For example, CD40 → TRAF2,3,5,6, OX40
(CD134) → TRAF1,2,3,5,4-1BB (CD137) → TRAF1,2,3, RAN
K → TRAF 1,2,3,5,6. TRAF2,5,6 are transcription factors NF-kapp
aB [J. Biol. Chem., 103, 2042-2045 (1997)]
And c-Jun N-terminal kinase (JNK) [Cell., 87, 565-
576 (1996)] is known to be activated.
In addition, TRAF6 is an extracellular signal-regulated kinase (extracell
It is known that ular signal-regulated kinase (ERK) is also activated [J. Exp. Med., 187, 237-244 (1998)].

In the immune system, adaptive immunity (adaptive i
mmunity) and antigen-direct immunity
It is known that TNF / TNFR plays a central role in. For example, LT (lymphotoxin) β or LTβ
Analysis of receptor knockout mice revealed lymph nodes
(LN), Peyer's patches and other secondary lymphoid tissues are not developed, and humoral immunity is not established [Ann
u. Rev. Immunol., 17, 399-433 (1999)]. In addition, RANK (receptor activator of nuclear factor kap
paB) or RANK ligand (RANKL), the structure of Peyer's patches and spleen is not affected, but peripheral and mesenteric LN are completely eliminated [J. Exp. Med., 19
Volume 2, 1467-1478 (2000)]. Thus LT α1 β2
The / LT β receptor and RANK / RANKL are molecules that appear to have similar functions but are not complementary to each other.
In the former, the expression is found in both hematopoietic cells and stromal cells, whereas in the latter, the expression is mainly localized in CD4-positive progenitor cells. This means that when CD4-positive progenitor cells migrate to LN, RANK / RANKL is used as a survival / differentiation signal in an autocrine manner, whereas LT α1 β2 / LT β receptors undergo maturation of LN. Have been suggested to be involved [J. Exp. Med., 192
Volume, 1467-1478 (2000)].

On the other hand, acute immune response
It is known that TNF / TNFRSFP also plays an important role in e). Of particular importance is the CD40 / CD154 interaction. T cell-dependent antigen response and germinal center
Center: GC) formation requires CD40-mediated stimulation of B cells and APCs. In mice lacking CD40 or CD154, CD4 + helper T cell priming, follicular DC differentiation, GC formation, and class switching (high IgM due to poor class switching to IgG)
Syndrome), etc. [Annu. Rev. Immuno
l., 16, 111-135 (1998)]. Also, LT α, LT
Mice deficient in β, TNF, and TNFR1 show severe abnormalities in follicle and GC formation [Annu. Rev. Immunol., 17, 399]
-Page 433 (1999)]. In addition, BlyS expressed in activated DC
(B lymphocyte stimulator) is the TACI (transmemb
rane activator and CAML interactor), BCMA (B cell m
aturation molecule) and promotes its survival. BlyS transgenic mice have an increased number of B cells and exhibit autoimmune disease symptoms. Blocking TACI suppresses antibody production in normal mice, suppresses GC formation, and causes autoimmunity (autoimmune-prone mice)
Of autoantibody formation and lethal immune complex-induced nephritis in Escherichia coli [Science, 285, 260-263 (1999)
Year)].

T cell activation is also regulated by TNF / TNFRSFP. RANKL is an early thymocyte differentiation (thymocyte
Other TNF / TNFRSFPs are important for development, but thymocyte differentiation, CD4 / CD8 lineage commitment (lineage co
mmitment), but is not involved in Th1 / Th2 differentiation. But drai
TN is required for the original activation of T cells by DC in ning LN.
F / TNFR SFP plays an important role. For example, TNFR1
And Fas are known to be involved in co-stimulation of T cell activation under physiological conditions [Nat. Immunol., 1
Vol. 469-474 (2000)] and expressed on T cells.
TNFSF such as 4 (OX40), CD27, and CD137 (4-1BB) also expresses their ligands (OX40L, CD7) on DC or B cells.
0, 4-1BBL) is used to control the survival or proliferation of CD4 + or CD8 + T cells. These TN
Most F / TNFRSFPs are expressed in activated T cells, but it is awaited to elucidate the difference in the role of each molecule in the immune function.

There is also a group of membrane proteins extracellularly having an EGF (epidermal growth factor) -like domain. This structure is involved in cell interaction, receptor-ligand interaction, blood coagulation and the like. The EGF-like domain is composed of approximately 50 amino acids and has 6 cysteine residues that form 3 disulfide bonds. The EGF-like domain is a Ca ²⁺ -binding consensus sequence ((D / N) X (D / N) (E / Q) Xm (D / N *) Xn (Y / F): m, n
Has a natural number and * has a possibility of b-hydroxylation). In relation to pathological conditions, natural mutations in fibrin-1, which has an EGF-like domain, LDL receptor, factor IX, and protein S cause Marfan syndrome [Hum.
l. Genet., 4, 1799-1809 (1995)], familial hypercholesterolemia [Hum. Mutat., 1, 445-466 (1
992)], hemophilia B (hemophilia B) [Nucleic. Aci
ds. Res., 24, 103-118 (1996)], protein
It is known to fall into S deficiency [Blood, 85, 130-138 (1995)].

On the other hand, there are several subsets of the immunoglobulin superfamily (IgSF), which occupies most of the membrane molecules. Of which, CD2 family (CD2F), B
It has been clarified that the 7 family (B7F) plays an important role in interaction between DC and other immunocompetent cells, activation, differentiation, cytokine production and the like. As described above, it is known that DC, which is a strong APC existing in the body, expresses CD80 / CD86 belonging to B7F at a high level when it matures upon activation stimulus. Regarding the relationship with autoimmune diseases, for example, DCs present in synovial fluid of rheumatoid arthritis and lesion tissues of psoriasis, and skin tissues of allergic contact dermatitis may have CD80 / CD86 aberrant expression. Are known.

CD28 has the characteristic of potently enhancing the activation of naive T cells. The presence of the signal from CD28 enhances the production of IL-2 and the expression of IL-2 receptor, enhances the proliferative response, and consequently enhances various effector functions of T cells. CD28 signal is IL
-2 as well as IL-4, IL-5, IL-13, IFN-g, TNF-a, GM-CS
It also enhances the production of various cytokines such as F. Also CD40
It is also involved in the expression of T cell activation antigens such as ligands and chemokines such as IL-8 and RANTES. CD80 or CD86
There are many reports showing that the binding of CD4 to CD28 is involved in the induction of differentiation of CD4-positive T cells into Th1 or Th2. However, CD80 is not unilateral like Th1 and CD86 is Th2, and the determination of the differentiation direction of naive CD4-positive T cells is not limited to CD28 and CD152 signals, and antigen or APC
It is considered to be affected by various factors that are defined by itself. However, the fact that the CD28 signal plays an important role in the Th2 differentiation and activation of naive T cells, which is not compensated by the function of other molecules, is a Th2 cytokine-dependent immunoglobulin IgG1, IgG2b in CD28 knockout mice. Selective production disorder [Science, 261, 609-612 (1993)]
DO11-10 TCR transgenic mouse with CD4 positive T cells
Selective inhibition of Th2 differentiation by antigen stimulation by APC derived from CD80 / CD86 double knockout mouse [J. Immunol., 161
Vol., Pages 2762-271 (1998)]. Another important role of the CD28 signal is to enhance the expression of survival factors such as bcl-xL and suppress T cell apoptosis after antigen stimulation.

Although CD28 is constitutively expressed on T cells, the expression of CD80 / CD86 is regulated in tissue cells. The mechanism is such that no immune response occurs. If the tolerance induction system fails, various diseases such as autoimmune diseases and allergic diseases may be caused. CD to islet β cells
Inflammatory lymphocyte infiltration into the pancreas was observed only by forced expression of 80 or CD86, but it did not lead to the development of insulin-dependent diabetes mellitus (IDDM), which is autoimmune diabetes, and the major histocompatibility complex ( MHC) class II molecule, TNF-α, or a viral protein as a self-antigen can be expressed together with CD80 to develop IDDM accompanied by β cell disruption. This suggests that autoimmune diseases may develop only when placed in an environment that promotes enhancement of T cell receptor (TCR) signals in addition to CD28-CD80 / CD86 signals.

Expression of CD80 / CD86 has been reported in professional APCs such as DC and macrophages in several human organ-specific autoimmune diseases. Thus, in normal tissue APC, C is under control.
A large number of cases where D80 / CD86 is abnormally expressed in the local area of the disease have been found, and it has been suggested that it is associated with autoimmune diseases.

At present, the main treatment for autoimmune diseases is nonspecific immunosuppressive drugs which have many side effects. Costimula
inhibition has an antigen-specific suppression and a sustained effect after the end of administration, and it can be expected to reduce side effects by maintaining the administration period and maintain an immune response to prevent infection. It is expected that more effective treatment will be performed as the interactions and functions of immune system cells become clear.

Further, CD28 is expressed in activated T cells
ICOS (inducible T cell co-st) as a family molecule
imulator) and PD-1 are being found. Recent findings on ICOS include a report on the analysis results of ICOS KO mice [Nature, 409, 97-109 (2001)]. I
COS plays an important role in Ig isotype class switching, and the number and size of GC formation in ICOS ^-/- were significantly reduced, indicating that B cell activation in the T cell region was mostly caused. However, it has become clear that the transition to the FDC network to form the GC has not occurred. The greatest feature of ICOS stimulation is that it induces Th2 differentiation.

Regarding PD-1, its ligand, PD-L1
[J. Exp. Med., 192, 1027-1034 (2000)], PD
-L2 [Nat. Immunol., Vol. 2, pp. 261-268 (2001)] have been reported one after another. PD-L1 is expressed in the heart and placenta, whereas PD-L2 is expressed in the heart, placenta, pancreas, lung and liver. In the blood cell system, Mo IFN-γ stimulation, B
Expression of PD-L1 and PD-L2 is increased by anti-Ig stimulation of cells. PD
-The interaction between L1 / L2 and PD-1 inhibits TCR-mediated reactions, and PD-L1 / L2 and PD-1 interact with T7 even in the presence of B7 / CD28 signals.
It has been clarified that both cell proliferation and cytokine production can be inhibited.

Next, CD2F is known as a molecule involved in lymphocyte activation and / or adhesion. The molecules belonging to CD2F include CD2 (LFA-2), CD48, CD58 (LFA-3), CD84, and C.
D150 (SLAM), CD229 (Ly-9), CD244 (2B4) and the like are known. The extracellular domain of molecules belonging to CD2F is composed of two Ig domains of IgV-IgC2 except CD229 (CD22
9 is IgV-IgC2-IgV-IgC2). CD84, CD150, CD229, CD244
Is a tyrosine-based motif
otif) (TxYxxV / I / A) and binds to this sequence and transmits a signal SAP / SH2D1A (SLAM-associated protein / SH2
When regulation by domain containing gene 1A) fails, X-linked
ymphoproliferative syndrome) -like phenotype [Na
ture, 395, pp. 462-469 (1998)]. The previously defined ligands of molecules belonging to CD2F are also known to belong to CD2F. In humans CD2 is CD58, CD48
CD244 and CD150 are CD150 (CD84 and CD229 ligands are orphans).

CD2 is expressed on T cells and natural killer (NK) cells, and in T cells, it is an adhesion molecule important for transient interaction with APC in the early stage [Nature,
339, 312-314 (1989)]. When activated through the TCR by the complex of MHC and antigen, the SH3 domain-containing adapter molecule CD2AP induces the intracellular end of CD2 (cytoplasmic t
ail) and immunological synaps (immunological synaps)
e) [Cell, 94, 667-677 (1998)].
On the other hand, when CD2BP1 having the same binding domain binds, CD2
Is known to be down-regulated in adhesion through EMBO [EMBO J., 17: 7320-7336 (1998
Year)]. CD2, a ligand of CD2, is widely expressed in both hematopoietic and non-hemocyte systems.

Signals mediated by CD244 and CD150 are competitively transmitted by SAP or SHP2 (SH2-domain containing protein tyrosine phosphatase 2) binding to a tyrosine-based motif. CD244 is NK cell, CD8 positive T cell,
Expression is seen in γδ positive T cells. When CD244 on NK cells binds to the target CD48-positive leukocytes, the target cells undergo cytolysis. CT in CD8-positive cells even if CD244 binds
L is not induced, but CD8 + CD244 + cells have MHC non-restricted cytotoxicity [Immunology, 96, 491-497.
Page (1999)]. CD150 is expressed on activated T cells and B cells. CD150 binding on T cells leads to costimulation, and IFN-γ
Production is seen. B cells show proliferation and Ig production.

Although CD84 and CD229 have two tyrosine-based motifs in the cell, they are orphans and their functions are not clear as described above. CD84 is B cell, thymocyte,
In memory T cells, monocytes and platelets, CD229 is expressed on thymocytes, T cells, B cells and lymphoid cells of bone marrow.

CD48 and CD58 have GPI anchors and no intracellular domain. In addition, there are splicing variants in CD84, CD150, and CD244, and intracellular tyrosine-based motifs are 2 or 0, 3 or 1, respectively.
4 or 1 There are also secretory forms of CD150.

[0027]

DISCLOSURE OF THE INVENTION The present inventors focused on DC maturation, which is important for the functional expression of DC, and focused on the identification of membrane molecules whose expression level increases due to DC maturation. Have been carried out based on the comprehensive identification of membrane molecules whose expression is regulated by DC maturation by proteomics. Proteomics is a term that corresponds to the genomics of genes (genomics) for proteins, and conducts large-scale research on proteins. Investigate properties such as protein expression levels, post-translational modifications, and interactions to determine what is happening at the level of expressed proteins in normal and cancer cells, the overall biological network of intracellular networks and processes, etc. Get information. If membrane molecules that are specifically present in DC are identified,
It will be possible to prepare an antibody against the membrane molecule and use the antibody in the medical field.

The purpose of the present invention is to develop a mature ADC that is a powerful APC.
This is to control autoimmune diseases by controlling the function of DC by targeting this molecule expressed above. In addition, since the molecule is expected to be expressed in APCs such as mature DC and is involved in the activation and regulation of other immunocompetent cells, an antibody that specifically recognizes the molecule should be selected. It is to provide a method for separating and detecting APCs such as mature DCs.

[0029]

[Means for Solving the Problems] As a result of exploratory research on membrane molecules whose expression is regulated by maturation of DC, the present inventors have found that mature DC is specifically expressed between immature DC and mature DC. We identified a membrane molecule in which the phenotype is present and found that its expression at the protein level increased markedly as DC matured. One is considered to belong to TNFRSF (A), one is considered to have an EGF-like domain (B), and three are considered to belong to IgSF (C,
D, E). Of the three molecules considered to belong to IgSF, it is considered that two molecules belong to B7F (C, D) and one molecule belongs to CD2F (E).

From recent research results, 1) DC is a heterogeneous cell population with several subsets [Stem
Cells, Vol. 15, 409-419 (1997)], 2) DC subsets have different functions in T cell activation (apoptosis induction, induction of T cell differentiation into Th1 and Th2, etc.) [Science, 283]. Volume, 1183-1186 (1999)], 3) DC
It is possible to control the immune response via
Therapy, DC-specific drugs, antibodies, cytokines, etc.),
Has become clear.

Human DC subsets are roughly classified into myeloid DCs and lymphocyte DCs. It has been shown that myeloid DCs have two differentiation pathways [J. Ex.
p. Med., 184, 695-706 (1996)], CD14-positive in a system in which CD34-positive hematopoietic stem cells are cultured with GM-CSF and TNF-α.
CD1a negative and CD14 negative CD1a positive progenitor cells appear,
It is known that the former differentiates into dermal DC and the latter differentiates into epidermal Langerhans cells. The mo-DC used by the present inventors is similar to the DC derived from the former in vivo and is considered to belong to myeloid DC. on the other hand,
In human lymphoid DC, plasmacytoid CD4 + T cells are IL-3
It is known that it differentiates into immature DC (CD11c-negative, CD14-negative) in the presence and functionally matures as DC upon stimulation with IL-3 and CD40 ligand [J. Exp. Med., Vol. 185. , 1
101-1111 (1997)]. In addition, it is reported that mo-DC differentiates into so-called DC1 which has a function of differentiating and inducing Th1 into Th1 by stimulation with CD40 ligand or endotoxin [Science, 283, 1183-1186 (1
999)]. As described above, it is understood that the mo-DC used by the present inventors is a group that can differentiate into at least DC1. In addition, there are at least two DC subsets in peripheral blood, which are lineage marker (CD3, CD19, CD56, CD14) negative, HLA-DR positive, CD11c positive and lineage marker (CD3, CD1).
9, CD56, CD14) negative, HLA-DR positive, CD11c negative, CD123
It shows a strongly positive phenotype. After the DCs mature, the former DC
1. The latter is known as DC2, which has the function of differentiating and inducing naive T cells into Th2 [Blood, 95
Vol., Pp. 2484-2490 (2000)].

As described above, the fact that the expression of this membrane molecule is remarkably increased with the maturation of mo-DC is
It is considered to be a molecule related to cell activation, and is also expected as a target molecule for controlling the immune response. On the other hand, a pilot study of a cancer vaccine using autologous peripheral blood DC pulsed with an antigen is also being conducted [Nature Me
d., 2, 52-58 (1996)], using the specificity of the expression of this membrane molecule in mature DCs to confirm the maturation of DCs after pulse using specific antibodies. You can It is also useful for separating mature DC and immature DC and detecting mature DC.

The present invention is summarized as follows based on the above findings. (1) Human dendritic cell membrane molecule having the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9, or deletion, substitution, insertion and / or deletion of one or more amino acid residues in the amino acid sequence A variant thereof, which is capable of controlling an immune response, comprising an addition and having 80% or more homology with the amino acid sequence. (2) The homology is 95% or more,
The human dendritic cell membrane molecule or a mutant thereof according to (1) above.

(3) A DNA encoding the human dendritic cell membrane molecule described in (1) or (2) above or a mutant thereof. (4) The DN according to (3) above, which has the nucleotide sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10 or has 95% or more homology with the nucleotide sequence.
A. (5) An antibody that specifically immunologically binds to the human dendritic cell membrane molecule described in (1) or (2) above, a mutant thereof, or a fragment thereof.

(6) The antibody according to (5) above, which is characterized in that it does not immunologically recognize immature dendritic cells but recognizes mature dendritic cells. (7) The antibody according to (5) above, which recognizes an extracellular region of the human dendritic cell membrane molecule. (8) The antibody according to any one of (5) to (7) above, which is a polyclonal antibody or a monoclonal antibody.

(9) A method for separating dendritic cells derived from humans or other animals using the antibody described in any of (5) to (8) above. (10) The method according to (9) above, wherein mature dendritic cells are separated. (11) A method for detecting dendritic cells using the antibody according to any one of (5) to (8) above. (12) The method according to (11) above, which comprises detecting mature dendritic cells.

The term "specifically immunologically binds" with respect to the antibody of the present invention as used herein means that the antibody of the present invention immunologically cross-reacts with an epitope possessed only by the membrane molecule. To do. Such an epitope is obtained by aligning and comparing the amino acid sequence of the membrane molecule of the present invention and the amino acid sequence of another protein, for example. Preferably at least 15 amino acids).

As used herein, the term "homology" refers to sequence identity or similarity between two or more amino acid sequences or base sequences, which sequences include, for example, the diagonal pattern method and the frequency distribution. Comparisons can be made by conventional methods including methods. The term "capable of regulating an immune response" as used herein may have the same or substantially the same ability to activate T cells as the native membrane molecule of the invention, or It means that it may have the ability to suppress or inhibit the activation of T cells.

[0039]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below. Novel Membrane Molecule and DNA Encoding It As described above, the present invention is based on the finding that this membrane molecule is expressed specifically in mature DC in association with maturation in DC.

The human dendritic cell membrane molecule of the present invention stimulates mo-DC isolated from human monocytes with lipopolysaccharide,
After obtaining mature DC based on the possibility, prepare the cell membrane,
Fractionation of the soluble protein using membrane methods such as concanavalin A sepharose chromatography, wheat embryo agglutinin sepharose chromatography, SDS-polyacrylamide gel electrophoresis,
It was identified by microanalysis by LC / MS (particularly LCQ manufactured by Thermoquest) (see Examples 1 to 5 described later). LC / M
By synthesizing a primer based on a plurality of partial amino acid sequences identified by the S method, using the mature DC-derived cDNA library as a template to carry out polymerase chain reaction (PCR) to amplify and obtain the gene fragment of the membrane molecule of the present invention, A clone containing the gene of the membrane molecule of the present invention was selected by colony hybridization using this gene fragment as a probe, and its nucleotide sequence (SEQ ID NO: 2, 4, 6, 8, 10) and amino acid sequence (SEQ ID NO: 1, 3, 5, 7, 9) were determined (see Example 6 below).

The membrane molecules (A) to (E) of the present invention were analyzed by hydropathic plot analysis [J. Exp. Med., Volume 157, 105-132].
Page, 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 430 amino acid residues. From the results of hydropathic plot analysis (Fig. 1), a signal sequence of 27 amino acid residues, an extracellular region of 136 amino acid residues, 23 amino acids It is considered to consist of a transmembrane region of residues and an intracellular region of 244 amino acid residues. Furthermore, from the results of homology and motif searches, the extracellular region is rich in cysteine (Cys) and is predicted to have three CRDs, and there is one potential asparagine-linked glycosylation site (149th position). To do. The extracellular region has an amino acid identity of 29% with OX40, but CRD1 is CRD1c from the position of its cysteine residue. 3 potential protein kinase C phosphorylation sites in the intracellular domain
There are two sites (232, 257, 299), two potential casein kinase II phosphorylation sites (257, 299), and one potential tyrosine kinase phosphorylation site (413). Positions 356 to 360 are sequences (PXQXT / S) that have the ability to bind to TRAF1 / TRAF2 / TRAF3.

Membrane molecule (B) Initiation Methionine could not be cloned, but within the range identified so far, it consists of partial length 619 amino acid residues, extracellular region of 195 amino acid residues, transmembrane region of 19 residues. It is thought to be composed of an intracellular region of 405 residues (Fig. 2). Furthermore, from the results of homology search and motif search, the extracellular region is rich in cysteine residues, and CXCX ₅ GX ₂ C and CXCX ₂ (G /
P) (F / Y / W) (X ₄ / X ₈ ) C has a sequence that applies to it, so it is expected to have at least 5 EGF-like domains, and there are 3 potential asparagine-linked glycosylation sites. (63rd, 118th, 156th) exist. In the intracellular domain, there are three potential cAMP- and cGMP-dependent protein kinase phosphorylation sites (450, 591, and 601) and nine potential protein kinase C phosphorylation sites (226
Rank, 249th, 295th, 361st, 448th, 455th, 466th, 473
Positions, 589), 10 potential casein kinase II phosphorylation sites (226, 249, 285, 301, 305, 348)
Position, 359th position, 366th position, 453th position, 515th position) and one ATP / GTP-binding site motif (524th position). This molecule is predicted to have an EGF-like domain also because it has an amino acid identity with the acetyl LDL receptor and has a homology of 39% with respect to the identified extracellular domain (partial length).

Membrane molecule (C) This membrane molecule consists of 468 amino acid residues. From the result of hydropathic plot analysis (Fig. 3), a signal sequence of 21 amino acid residues, an extracellular region of 384 amino acid residues, 21 amino acids It is considered to be composed of a transmembrane region of residues and an intracellular region of 43 amino acid residues. Furthermore, from the results of homology search and motif search, there is no potential asparagine-linked glycosylation site in the extracellular region, but it is called IgV-IgC2-IgC2 from the position of the six cysteine residues involved in disulfide bond formation. It is predicted to consist of three Ig domains, and the sequences characteristic of potential Ig and MHC (major histocompatibility complex proteins signat
ure) (380th), it is expected to belong to IgSF. One potential protein kinase C phosphorylation site (position 461) in the cell, potential casein kinase
II There is one phosphorylation site (position 447). CD155 (poliovirus receptor) throughout the extracellular region
Has the highest homology (27% in amino acid identity), but B7
B7-H3 and 29 for the IgV domain reported to be important for binding to the CD28 family in family members
It has a% homology.

Membrane molecule (D) This membrane molecule consists of 282 amino acid residues. From the result of hydropathic plot analysis (Fig. 4), a signal sequence of 22 amino acid residues, an extracellular region of 237 amino acid residues, 17 amino acids It is considered to consist of a transmembrane region of residues and an intracellular region of 6 amino acid residues. From the results of homology search and motif search, it is expected to consist of two Ig domains, IgV-IgC2, from the position of four cysteine residues, and the extracellular domain IgV-IgC2 has the highest homology. Is B7-H3 (B7-homologue3)
The amino acid identity was 31%. There are 7 potential asparagine-linked glycosylation sites (112, 160, 190, 19
6th, 205th, 216th, 220th) exist.

Membrane molecule (E) This membrane molecule consists of 331 amino acid residues. From the result of hydropathic plot analysis (Fig. 5), a signal sequence of 21 amino acid residues, an extracellular region of 205 amino acid residues, 22 amino acids It is considered to consist of a transmembrane region of residues and an intracellular region of 83 amino acid residues. Furthermore, from the results of homology search and motif search, there were 7 potential asparagine-linked glycosylation sites in the extracellular domain (58, 8
7th, 137th, 144th, 161, 178th, 203th) exist.
The intracellular region has one potential cAMP- and cGMP-dependent protein kinase phosphorylation site (251) and four potential protein kinase C phosphorylation sites (259).
Position, 267 position, 290 position, 298 position), there are three potential casein kinase II phosphorylation sites (290 position, 298 position, 325 position). This molecule has amino acid identity with CD84 belonging to CD2F,
Since it has 29% homology with respect to the identified extracellular domain and the position of the cysteine residue necessary for Ig domain formation is conserved, it is expected to consist of two Ig domains, IgV-IgC2, It is considered to be a molecule belonging to CD2F.

The membrane molecules of the present invention can be synthesized in large quantities by utilizing gene cloning and DNA recombination techniques. General techniques therefor include, for example, Sambrook et al., Molecular Cloning: A Laboratory Manu.
al, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY (1989) and Ausubel et al., Curren
tProtocols in Molecular Biology, Greene Publishing
Standard techniques such as those described in Associates 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. SEQ ID NOs: 11-15
The desired cDNA can be obtained by screening the library using a specific probe synthesized based on the sequence described in (1) above. Alternatively, based on the sequences of SEQ ID NOs: 11 to 15, synthesize sense and antisense primers for amplifying a sequence containing the mature sequence of the target molecule, perform PCR using the cDNA library as a template, and target cDNA. Can be amplified. PCR is an automatic thermal cycler (automate
d thermal cycler), and the reaction is performed in the presence of a thermostable polymerase (Taq, etc.), template DNA and primers to denature the DNA (eg, 94 ° C, 15-30 ° C).
Second), primer annealing (eg 55 ° C, 30 seconds ~
1 minute), and an extension reaction in the coexistence of four types of substrates (dNTP) (eg 72 ° C, 30 seconds to 10 minutes) is about 25 cycles per cycle.
It can be carried out by carrying out ~ 40 cycles and further heating at 70 to 75 ° C for 5 to 15 minutes. The size of the primer is usually at least 15 nucleotides.

The cDNA encoding the membrane molecule of the present invention is incorporated into an expression vector (eg, plasmid, phage, cosmid, virus, etc.) containing an appropriate transcription / translation control sequence, and then introduced into an appropriate host cell (eukaryotic or prokaryotic). Cells) or transfection or transduction.

Transcription / translation control sequences can include promoters and enhancers selected depending on the host / vector system used. Examples of promoters include P _L , P _R , Ptrp, Plac in bacterial systems, PHO5, GAP, ADH, AOX1 promoters in yeast systems, and SV40 early promoter, retrovirus promoter, heat shock promoter in animal cells. You can

Host cells include prokaryotic cells such as Escherichia, Bacillus and Pseudomonas, yeasts such as Saccharomyces and Pichia, human embryonic kidney cells, human leukemia cells, African green monkey kidney cells, Chinese hamster ovary cells (CHO ) And other animal cells. In addition, insect cells and plant cells can also be used.

Various expression vectors are commercially available or deposited depending on the host, or they are described in publications such as literatures, and they can be used. For example, pQE (Qiagen), pBluescript II SK + (Stratagene), pET (Novagen), etc. for bacteria. Examples of the method for transforming or transfecting a host cell with a vector include a method using calcium ions, an electroporation method, a protoplast method, a microinjection method and the like.

The transformed or transfected host cell is cultured in an appropriate culture medium to express the gene of interest, and the produced membrane molecule of the present invention is recovered from the medium or from the host cell. When 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, etc. to obtain a cell-free extract. Isolation and purification of membrane molecules can be carried out by general methods used for protein purification, such as gel filtration, ion exchange chromatography,
Chromatography such as affinity chromatography and hydrophobic chromatography, HPLC, electrophoresis,
The desalting method, the organic solvent precipitation method and the like can be performed in any combination.

Variants The present invention also includes, in addition to the human dendritic cell membrane molecule having the amino acid sequence shown in SEQ ID NOs: 1, 3, 5, 7 and 9, one or more (preferably one) in the amino acid sequence. Or several) amino acid residue deletions, substitutions, insertions and / or additions, and
Also provided are variants thereof capable of controlling the activation of T cells, which have a homology of 0% or more, preferably 90% or more, more preferably 95% or more, most preferably 98% or more.

The variant may have all or part of the function of the native membrane molecule. In vivo, an antibody is produced against a certain exogenous antigen that has invaded, but at this time, antigen-presenting cells, T cells, and B cells are involved. The antigen-presenting cell takes up the antigen into the cell and reduces it to read the property of the antigen. If the antigen is a component other than self,
Signals to T cells and activates T cells for antibody production. The variants of the present 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 inserted into an appropriate expression vector (for example, an adenovirus vector), and then the gene is introduced into the genome of the target DC by homologous recombination, It is conceivable to express the gene to obtain DC having the membrane molecule mutant in the DC membrane.

Alternatively, on the contrary, this mutant can be used for inhibiting or suppressing T cell activation.
In this case, the variant should be able to antagonize a membrane molecule on the native DC membrane, for example such variants include those that have a defect in a domain essential for T cell activation. To be

Any variant is within the scope of the invention as long as it has a high homology (> 80%) with the native membrane molecule and has the ability to control T cell activation. Such a mutant can be produced, for example, by introducing a desired modification (deletion, substitution, insertion and / or addition) into the native membrane molecule using site-directed mutagenesis, PCR, etc. (See Sambrook et al., And Ausubel et al., Supra). Such modifications include, for example, conservative amino acid substitutions,
For example acidic amino acids (aspartic acid and glutamic acid)
And basic amino acids (lysine and arginine) and hydrophobic amino acids (leucine, isoleucine, valine, etc.).

When the mutant of the present invention is obtained from a natural source or the like, a probe is prepared based on the nucleotide sequence, hybridized under moderate or high stringent conditions, and then subjected to high stringent conditions. The gene encoding the mutant of interest is extracted by washing, and the mutant of interest can be obtained by incorporating the gene into an appropriate vector and expressing it. Examples of high stringent conditions are 0.5M NaHPO ₄ , 7% SDS, 1 mM EDTA, hybridization at 65 ° C, followed by 0.1 x SSC / 0.1% SD.
S, 68 ° C wash (see Ausubel et al., Supra). Hybridization conditions can be determined by appropriately selecting temperature and ionic strength, but usually, the higher the temperature and the lower the ionic strength, the higher the stringency. Therefore, one skilled in the art can select suitable hybridization conditions. In the present invention, the nucleotide sequence and 80
% Or more, preferably 90% or more, more preferably 95% or more,
DNAs encoding the above variants, most preferably having 98% homology, are also included.

The expression of the membrane molecules belonging to the antibodies TNFRSF, B7F and CD2F, and the membrane molecules having the EGF-like domain is increased mainly with activation, and as described above, it is inferred from the properties of the family that mature DC, activation It is expected that the expression of this membrane molecule is also found in macrophages, activated monocytes, activated B cells and the like. By utilizing this finding, it is possible to obtain an antibody against this membrane molecule that specifically recognizes activated antigen-presenting cells (APC).

Although any antibody having the above characteristics is included in the present invention, the antigenic epitope for obtaining the desired antibody has a highly antigenic region or superficial in the amino acid sequence of the present membrane molecule group. It may be selected from a region, a region which may not have a secondary structure, a region having no or low homology with other proteins (in particular, other proteins of the family to which each membrane molecule belongs). Here, the highly antigenic region is
Parker et al. [Biochemistry, 25, 5425-5432 (1
986)]. The superficial region can be estimated, for example, by calculating and plotting the hydropathy index. Areas that may not have a secondary structure are described in, for example, the method of Chou and Fasman [Adv Enzymol Relat Areas Mol Biol., Vol.
Page (1978)]. Furthermore, the region having no or low homology with other proteins to which each membrane molecule belongs can be estimated by homology comparison between the amino acid sequence of each membrane molecule and the amino acid sequence of another protein.

Based on the partial amino acid sequence of the present membrane molecule deduced by the above method, a peptide consisting of the amino acid sequence can be synthesized by utilizing the peptide synthesis method. The peptide of interest is, for example, RB Merrifield
[Science, 232, 341-347 (1986)], which was synthesized by using a commercially available peptide synthesizer based on solid-phase peptide synthesis, and after removing the protecting group, ion exchange chromatography, Purification is performed by gel filtration chromatography, reverse phase chromatography, etc., alone or in combination. The obtained purified peptide can be used as an immunogen by binding with a carrier protein such as keyhole limpet hemocyanin (KLH) and albumin.

Furthermore, a polyclonal antibody or a monoclonal antibody against the present membrane molecule can be prepared by a known method using the gene recombinant main membrane molecule as an immunogen. In this case, the term "recombinant" as used in reference to the 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 (eg bacteria such as Escherichia coli) and eukaryotic organisms (eg 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.
An "antibody" is subcutaneous, intraperitoneal, for the purpose of producing lymphocytes that produce or will produce antibodies that will specifically bind to proteins used to immunize mice or other suitable host animals. It can be obtained by immunizing with antigen or antigen-expressing cells by the intramuscular or intramuscular route. Furthermore, as a host animal, an antigen or antigen-expressing cells may be administered to a transgenic animal having a repertoire of human antibody genes to obtain a desired human antibody [Proc. Natl. Acad. Sci. USA, vol. 97, 722]. -Page 727 (2000), International Publication WO96 / 33735, WO97 / 07671, WO97 / 1
3852, see WO98 / 37757]. Instead, in lymphocytes
It may be immunized in vitro. A polyclonal antibody can be obtained by collecting and purifying a fraction that binds to the antigen from the serum obtained from the host animal. Alternatively, a monoclonal antibody can be prepared by fusing lymphocytes with myeloma cells using a suitable fusion reagent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal A
ntibodies: Principals and Practice, pages 59-103, Acad
emic press, 1986). For example, the monoclonal antibody of the present invention is obtained by the hybridoma method [Nature, 256, 495 (19
1975)] or by the recombinant DNA method (Cabilly et al., US Pat. No. 4,816,567).

The antigen protein can be prepared by expressing DNA encoding all or a partial sequence of the protein of the present membrane molecule in Escherichia coli, yeast, insect cells, animal cells and the like. The gene-recombinant membrane molecule is purified by affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, etc., alone or in combination,
This purified preparation is used as an immunogen. Further, the antibody of the present invention may be an intact antibody, or (Fab ′) ₂
It may be an antibody fragment such as Fab or Fab.

A chimeric antibody in which the constant region is replaced with a human constant region (eg, mouse-human chimeric antibody; Cabil
Ly et al., U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. A.
cad. Sci. USA, 89, 6851 (1984), constant region and hypervariable region (or Complementary-determinin).
humanized antibody (Carter et al., Proc. Natl. Acad.) in which all variable regions except g region (CDR) are replaced with human sequences.
Sci. USA, 89, 4285, 1992 and Carter et al., BioTec.
hnology, 10: 163, 1992) are also included in the antibody 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.

The various antibodies against the present membrane molecule thus obtained can be used in various applications in which their characteristics can be utilized. Utilizing the fact that this membrane molecule is specifically expressed in mature DC, this antibody is labeled with a fluorescent substance (rhodamine, fluoresamine, etc.), and the target DC is detected using well-known FACS.・ Separation or i of mo-DC
In vitro differentiation can be confirmed. Furthermore, since this membrane molecule is a protein whose expression level remarkably increases with the maturation of DCs, immature DCs and mature DCs can be separated by FACS using this antibody. The detection of this membrane molecule is not limited to the detection by FACS. For example, it is expected that the antibody can be detected by allowing the antibody to act as a primary antibody in Western blotting, and the expression can be confirmed at the protein level. In addition, this antibody is bound to a solid phase (polystyrene beads, microtiter well surface, latex beads, etc.) to carry out immunological reactions in a heterogeneous system or in a homogeneous system to detect and quantify homologous membrane molecules (fluorescence). Antibody method, ELISA, radioimmunoassay, etc.) can be used. In this case, the immunological reaction may be a competitive reaction or a non-competitive reaction. A reaction by a sandwich method using two or more antibodies (monoclones or polyclones) can also be used. For the detection and quantification in the above, any immunological method known in the art can be used.

In addition, it can be used for the purpose of evaluating the function of the present membrane molecule. Mature DCs are potent APCs that stimulate CD4-positive T cells via MHC class II molecules and activate MHC
It is known to stimulate and activate CD8-positive cytotoxic T cells via class I molecules. To confirm whether these functions can be controlled, allogeneic MLR (MixedLy
mphocyte reaction) function confirmation, whether antigen-specific CTL-induced CTL function inhibition can be confirmed, and whether it is a molecule involved in DC antigen presentation, etc. It can also be used for in vitro assays.

The antibodies of the present invention can also be used to control the immune response in vivo. As described above, this group of membrane molecules is expected to be molecules involved in costimulation, and is also expected to be membrane molecules 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 the ligand on T cells, it is expected to suppress T cell activation. Thus, it is expected that the action of the antibody capable of controlling the function of the membrane molecule group can control the immune response by controlling the DC function and the T cell function. Furthermore, solubilized molecules of the present membrane molecule and ligands of the present membrane molecule (that is, molecules corresponding to the extracellular region) and antibodies to these molecules are also expected to have an activity of controlling the immune response.

It is also possible to obtain a ligand for the present membrane molecule using the antibodies of the present invention or a solubilized molecule of the present membrane molecule. The solubilized membrane-membrane molecule ligand can directly act on the membrane-membrane molecule and control the signal mediated by the membrane-membrane molecule on DC.
In addition, low molecular weight substances that can modulate the interaction between the membrane molecule ligand and this membrane molecule, and low molecular weight substances that modulate intracellular signal pathways related to this membrane molecule ligand and this membrane molecule are also useful for signal control. Is.

When the antibodies of the present invention or the solubilized molecule of the present membrane molecule, the solubilized molecule of the ligand of the present membrane molecule, or the above small molecule is used for treatment, for example, cancer, autoimmune disease, organ transplantation, It is applicable to the treatment of diseases such as infectious diseases and allergies. The administration method and dosage form are not particularly limited, but may be intravenous, intraarterial administration, intramuscular administration, oral administration, suppository administration, etc., for oral use in combination with a pharmaceutically acceptable excipient or diluent. Or it can be prescribed parenterally,
Parenteral administration is preferred. Administration may be carried out once or in several divided doses per day depending on the severity of the patient, age,
It is determined according to conditions such as sex and weight, and is a range that does not cause side effects.

[0071]

EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples. Example 1 Preparation of Cell Membrane of DC In the case of a cell membrane protein, its original expression level is low and its handling is difficult. As one of the means to solve 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 with a higher specific gravity can be obtained by density gradient centrifugation.
Cell membranes were prepared [J. Biol. Chem., 258, 10062-100.
72 pages (1983)].

Due to the low expression level of the target membrane molecule and the difficulty of handling, in order to collect a large amount of human DC, monocytes were separated from the apheresis product (mononuclear cell fraction) to prepare large amounts of mo-DC. did. LPS (lipopolysaccharide) stimulation was performed with and without the same number of cells, and the same number (5
X10 ⁸ cells) were obtained as immature DCs and mature DCs.

Example 2 Sensitive detection of protein and in gel
Proteins were detected by silver staining according to the method of digestion Mann et al. [Anal. Chem., 68, 850-858 (1996)]. In gel digestion using trypsin for enzymatic digestion
Method [Anal. Chem., 224, pp. 451-455 (1995)] and used as the analytical sample used below.

Example 3 Fractionation of Membrane Protein After solubilizing the cell membrane protein from the cell membrane obtained in Example 1 using a surfactant, concanavalin A (ConA)
It was applied to Sepharose and washed with a buffer containing a surfactant. These flow-through 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 (WGA) and washed with a surfactant-containing buffer. These flow-through fractions were called WGA FT. The adsorbed fraction was eluted with a buffer containing N-acetylglucosamine and a surfactant (WGA E
L). 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 Microanalysis by LC / MS The sample obtained in Example 3 was analyzed using LC / MS (LCQ manufactured by Thermoquest). 95% solution A (0.1% formic acid) and 5%
PepMap reverse-phase column (inner diameter 0.075 mm x length 150 mm) equilibrated with solution B (0.08% formic acid, 80% acetonitrile)
(Manufactured by LC Packing Co.) and washed at a flow rate of 180 nanoliter / min for 5 minutes, and then the ratio of solution B was linearly increased to 50% over 67.5 minutes for sequential elution and introduction into MS.
For the sample introduced into MS, data was acquired in the next repeated cycle to obtain sequence information. 1. Ful
l MS Scan: Observation of molecular weight of 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: Zoom MS Scan
Observation of daughter ions generated when He gas is applied to the molecule measured at. It was identified by the identification method (SEQUEST algorithm) that scores and ranks how many and how many matches with the theoretical b, y series [J. Am. Soc. Mass Spectrom., Volume 5] , Pages 976-989 (19
1994)].

Example 5 Identification of Membrane Membrane Molecules Among the molecules identified by the method of Example 4, the membrane molecule group was identified only from mature DC. The fragments to be identified are m / z = 751.9 (divalent ion), m / z = 1295.7 (monovalent ion), m / z = 1176.1 (divalent ion), m / z = 1487.2 (divalent ion). , M / z = 1121.6 (monovalent ion), and the mass of the parent ion and the MS / MS pattern are the partial amino acid sequence RGVEVAAGASSGGETR (16 residues from molecule (A)) of this membrane molecule group, respectively. (SEQ ID NO: 11), GPCPAGFYGLGCR
(13 residues derived from molecule (B)) (SEQ ID NO: 12), TAAWFMANV
QVSGGGPSISLVMK (23 residues from molecule (C)) (SEQ ID NO: 1)
3), HSITVTTVASAGNIGEDGILSCTFEPDIK (29 residues from molecule (D)) (SEQ ID NO: 14), SPEIHVTNPK (10 residues from molecule (E)) (SEQ ID NO: 15) were assigned very well.
As a result of a homology search of these partial sequences against a non-redundant DNA database, it was revealed that there is no other base sequence having these partial sequences.

Example 6 Gene Cloning of Membrane Membrane A partial amino acid sequence of each molecule assigned in Example 5 was searched against a database (Ensembl Genscan) that predicted ORF (open reading frame) from the human genome database. However, (A) AP000763.00001 and (B) AC respectively
005500.00001, (C) AC006064.00001, (D) AL080312.0000
1, it was found to be a gene listed in (E) AC012471.00021. Although Ensembl Genscan is excellent as a database, there are many examples in which the 5'side and 3'side have large redundancy and erroneous sequences. Therefore, Example 5
We designed a primer corresponding to the vicinity of the partial amino acid sequence that was revealed in 1. and attempted to obtain the partial length by nested PCR with the vector primer. Those containing the target nucleotide sequence were selected from the obtained gene fragments. Using this gene fragment as a probe, clones containing the gene of this membrane molecule group were selected by colony hybridization to determine the nucleotide sequence. The putative ORF structures are shown in SEQ ID NO: 2, 4, 6,
8 and 10, the deduced amino acid sequences are shown in SEQ ID NOs: 1, 3,
5, 7, and 9 are shown.

Regarding the primary structure of the deduced amino acid sequence of this membrane molecule group, the method of Kyte and Doolittle (J. Exp. Med., 157).
Vol., Pp. 105-132, 1982), and hydropathic plot analysis was performed (Figs. 1 to 5). As a result, it was revealed that the present membrane molecule groups (A) to (E) are type I transmembrane proteins having a signal sequence at the N-terminus.

(A) The present membrane molecule consists of 430 amino acid residues, and the result of hydropathy plot analysis (FIG. 1) shows that
It is considered to be composed of a signal sequence of 7 amino acid residues, an extracellular region of 136 amino acid residues, a transmembrane region of 23 amino acid residues, and an intracellular region of 244 amino acid residues. Furthermore, from the results of homology and motif searches, the extracellular region is rich in cysteine residues and is predicted to have three CRDs, and there is one potential asparagine-linked glycosylation site (149th position). . About the extracellular region
It has 29% homology in amino acid identity with OX40 (CD134), but CRD1 has CRD1 at the position of its cysteine residue.
c. The intracellular region is longer than any other TNFRSF molecule, but it has no DD and three potential protein kinase C phosphorylation sites (232, 257, 299),
There are two potential casein kinase II phosphorylation sites (257
Position, position 299), and one potential tyrosine kinase phosphorylation site (position 413). TRAF1 / TR in the 356th to 360th place
This is a sequence (PXQXT / S) capable of binding to AF2 / TRAF3.

(B) The initiation methionine could not be cloned, but within the range identified so far, the partial length was 61
It consists of 9 amino acid residues, and is considered to be composed of an extracellular region of 195 amino acid residues, a transmembrane region of 19 residues, and an intracellular region of 405 residues (Fig. 2). Furthermore, from the results of homology search and motif search, the extracellular region is rich in cysteine residues, which is characteristic of EGF-like domain.
CX ₅ GX ₂ C and CX CX ₂ (G / P) (F / Y / W) (X ₄ / X ₈ ) C have at least 5 EGF-like domains, and are potentially There are three asparagine-linked glycosylation sites (63, 118, 156). The intracellular domain has three potential cAMP- and cGMP-dependent protein kinase phosphorylation sites (450, 591).
Positions, 601, and 9 potential protein kinase C phosphorylation sites (226, 249, 295, 361, 448, 455)
Positions, 466th, 473th, 589th), 10 potential casein kinase II phosphorylation sites (226th, 249th, 285th, 301st)
No., 305, 348, 359, 366, 453, 515), AT
There is one P / GTP-binding site motif (position 524). This molecule has an amino acid identity with the acetyl LDL receptor and has a homology of 39% in the identified extracellular domain (partial length), which suggests that it has an EGF-like domain.

(C) This membrane molecule consisted of 468 amino acid residues, and was found to be 2 from the results of hydropathic plot analysis (FIG. 3).
It is considered to be composed of a signal sequence of 1 amino acid residue, an extracellular region of 384 amino acid residues, a transmembrane region of 21 amino acid residues, and an intracellular region of 43 amino acid residues. Furthermore, from the results of homology search and motif search, there is no potential asparagine-linked glycosylation site in the extracellular region, but it is called IgV-IgC2-IgC2 from the position of the six cysteine residues involved in disulfide bond formation. Expected to consist of three Ig domains, potential Ig and MHC
Characteristic sequence (major histocompatibility complex p
roteins signature) (380th place)
Expected to belong to. There is one potential protein kinase C phosphorylation site (position 461) and one potential casein kinase II phosphorylation site (position 447) in the cell. It has the highest homology to CD155 (poliovirus receptor) throughout the extracellular domain (27% in amino acid identity), but is reported to be important for binding to the CD28 family in B7 family members. It has 29% homology with H3.

(D) This membrane molecule consists of 282 amino acid residues, and from the result of hydropathic plot analysis (FIG. 4),
It is considered to be composed of a signal sequence of 22 amino acid residues, an extracellular region of 237 amino acid residues, a transmembrane region of 17 amino acid residues, and an intracellular region of 6 amino acid residues. Furthermore, from the results of homology search and motif search, it was predicted that it would consist of two Ig domains, IgV-IgC2, from the position of the four cysteine residues, and this extracellular domain
The highest homology molecule in the unit of IgV-IgC2 is B7-H
3 (B7-homologue3) had an amino acid identity of 31%. There are 7 potential asparagine-linked glycosylation sites (112
Rank, 160 rank, 190 rank, 196 rank, 205 rank, 216 rank, 220 rank) exist.

(E) This membrane molecule consists of 331 amino acid residues, and from the result of hydropathic plot analysis (FIG. 5),
It is considered to be composed of a signal sequence of 21 amino acid residues, an extracellular region of 205 amino acid residues, a transmembrane region of 22 amino acid residues, and an intracellular region of 83 amino acid residues. Furthermore, from the results of homology search and motif search, there were 7 potential asparagine-linked glycosylation sites in the extracellular domain (58th, 87th, 137th, 144th, 16th, 178th).
Position, 203)) exists. Potential cAMP in the intracellular region
-And 1 cGMP-dependent protein kinase phosphorylation site (251), 4 potential protein kinase C phosphorylation sites (259, 267, 290, 298), potential casein kinase II 3 phosphorylation sites (290
No., 298, 325) exist. This molecule is a CD belonging to CD2F
It has amino acid identity with 84, has 29% homology in the identified extracellular domain, and the position of the cysteine residue necessary for Ig domain formation is conserved. , And is considered to be a molecule belonging to CD2F.

Example 7 Peptide antibody against the present membrane molecule
And preparation of peptide antibody In order to prepare the above-mentioned specific peptide antibody against the present membrane molecule, design is carried out from the deduced amino acid sequence of the present membrane molecule.
First, select a highly antigenic portion according to Parker's law (above), and then select a superficial portion based on the method of Chou and Fasman (above), or a portion that may not have a secondary structure. In addition, a single peptide is synthesized with respect to a portion where sugar chain addition is not expected and a portion which does not contain a cysteine residue. A single peptide with a cysteine residue added to the C-terminus of this sequence is synthesized. This single peptide is purified and conjugated to about 1 mg / ml keyhole limpet hemocyanin (KLH) at a concentration of about 0.2 mg / ml via a cysteine residue. Rabbits are repeatedly immunized several times with this KLH-binding peptide (about 100 μg) as an immunogen to prepare peptide antibodies.

[0085]

INDUSTRIAL APPLICABILITY According to the present invention, not only can DC be selectively separated from other blood cells with high purity,
It also allows the separation of mature and immature DCs, allowing the supply of the cells for DC therapy. The separated DC is expected to be used as a cancer vaccine by pulsing the antigen and then returning it to the patient. It is also expected that the immune response can be regulated by inhibiting the interaction between DCs and other immunocompetent cells using an antibody against the membrane molecule or a solubilized molecule of the membrane molecule.

[0086]

[Sequence list]                          SEQUENCE LISTING <110> Kirin Beer Kabushiki Kaisha <120> New dendritic cell membrane molecules and DNA encoding       the same <130> P01-0582 <140> <141> <160> 15 <170> PatentIn Ver. 2.0 <210> 1 <211> 429 <212> PRT <213> Homo sapiens <400> 1 Met Lys Pro Ser Leu Leu Cys Arg Pro Leu Ser Cys Phe Leu Met Leu   1 5 10 15 Leu Pro Trp Pro Leu Ala Thr Leu Thr Ser Thr Thr Leu Trp Gln Cys              20 25 30 Pro Pro Gly Glu Glu Pro Asp Leu Asp Pro Gly Gln Gly Thr Leu Cys          35 40 45 Arg Pro Cys Pro Pro Gly Thr Phe Ser Ala Ala Trp Gly Ser Ser Pro      50 55 60 Cys Gln Pro His Ala Arg Cys Ser Leu Trp Arg Arg Leu Glu Ala Gln  65 70 75 80 Val Gly Met Ala Thr Arg Asp Thr Leu Cys Gly Asp Cys Trp Pro Gly                  85 90 95 Trp Phe Gly Pro Trp Gly Val Pro Arg Val Pro Cys Gln Pro Cys Ser             100 105 110 Trp Ala Pro Leu Gly Thr His Gly Cys Asp Glu Trp Gly Arg Arg Ala         115 120 125 Arg Arg Gly Val Glu Val Ala Ala Gly Ala Ser Ser Gly Gly Glu Thr     130 135 140 Arg Gln Pro Gly Asn Gly Thr Arg Ala Gly Gly Pro Glu Glu Thr Ala 145 150 155 160 Ala Gln Tyr Ala Val Ile Ala Ile Val Pro Val Phe Cys Leu Met Gly                 165 170 175 Leu Leu Gly Ile Leu Val Cys Asn Leu Leu Glu Arg Lys Gly Tyr His             180 185 190 Cys Thr Ala His Lys Glu Val Gly Pro Gly Pro Gly Gly Gly Gly Ser         195 200 205 Gly Ile Asn Pro Ala Tyr Arg Thr Glu Asp Ala Asn Glu Asp Thr Ile     210 215 220 Gly Val Leu Val Arg Leu Ile Thr Glu Lys Lys Glu Asn Ala Ala Ala 225 230 235 240 Leu Glu Glu Leu Leu Lys Glu Tyr His Ser Lys Gln Leu Val Gln Thr                 245 250 255 Ser His Arg Pro Val Ser Lys Leu Pro Pro Ala Pro Pro Asn Val Pro             260 265 270 Arg Ile Cys Pro His Arg His His Leu His Thr Val Gln Gly Leu Ala         275 280 285 Ser Leu Ser Gly Pro Cys Cys Ser Arg Cys Ser Gln Lys Lys Trp Pro     290 295 300 Glu Val Leu Leu Ser Pro Glu Ala Val Ala Ala Thr Thr Pro Val Pro 305 310 315 320 Ser Leu Leu Pro Asn Pro Thr Arg Val Pro Lys Ala Gly Ala Lys Ala                 325 330 335 Gly Arg Gln Gly Glu Ile Thr Ile Leu Ser Val Gly Arg Phe Arg Val             340 345 350 Ala Arg Ile Pro Glu Gln Arg Thr Ser Ser Met Val Ser Glu Val Lys         355 360 365 Thr Ile Thr Glu Ala Gly Pro Ser Trp Gly Asp Leu Pro Asp Ser Pro     370 375 380 Gln Pro Gly Leu Pro Pro Glu Gln Gln Ala Leu Leu Gly Ser Gly Gly 385 390 395 400 Ser Arg Thr Lys Trp Leu Lys Pro Pro Ala Glu Asn Lys Ala Glu Glu                 405 410 415 Asn Arg Tyr Val Val Arg Leu Ser Glu Ser Asn Leu Val             420 425 <210> 2 <211> 1293 <212> DNA <213> Homo sapiens <400> 2 atgaagccaa gtctgctgtg ccggcccctg tcctgcttcc ttatgctgct gccctggcct 60 ctcgccaccc tgacatcaac aaccctttgg cagtgcccac ctggggagga gcccgacctg 120 gacccagggc agggcacatt atgcaggccc tgccccccag gcaccttctc agctgcatgg 180 ggctccagcc catgccagcc ccatgcccgt tgcagccttt ggaggaggct ggaggcccag 240 gtgggcatgg caactcgaga tacactctgt ggagactgct ggcctgggtg gtttgggcct 300 tggggggttc cccgcgttcc atgtcaacca tgttcctggg cacctctggg tactcatggc 360 tgtgatgagt gggggcggcg ggcccgacgt ggcgtggagg tggcagcagg ggccagcagc 420 ggtggtgaga cacggcagcc tgggaacggc acccgggcag gtggcccaga ggagacagcc 480 gcccagtacg cggtcatcgc catcgtccct gtcttctgcc tcatggggct gttgggcatc 540 ctggtgtgca acctcctcga gcggaagggc taccactgca cggcgcacaa ggaggtcggg 600 cccggccctg gaggtggagg cagtggaatc aaccctgcct accggactga ggatgccaat 660 gaggacacca ttggggtcct ggtgcgcttg atcacagaga agaaagagaa tgctgcggcc 720 ctggaggagc tgctgaaaga gtaccacagc aaacagctgg tgcagacgag ccacaggcct 780 gtgtccaagc tgccgccagc gcccccgaac gtgccacgca tctgcccgca ccgccaccat 840 ctccacaccg tgcagggcct ggcctcgctc tctggcccct gctgctcccg ctgtagccag 900 aagaagtggc ccgaggtgct gctgtcccct gaggctgtag ccgccactac tcctgttccc 960 agccttctgc ctaacccgac cagggttccc aaggccgggg ccaaggcagg gcgtcagggc 1020 gagatcacca tcttgtctgt gggcaggttc cgcgtggctc gaattcctga gcagcggaca 1080 agttcaatgg tgtctgaggt gaagaccatc acggaggctg ggccctcgtg gggtgatctc 1140 cctgactccc cacagcctgg cctcccccct gagcagcagg ccctgctagg aagtggcgga 1200 agccgtacaa agtggctgaa gcccccagca gagaacaagg ccgaggagaa ccgctatgtg 1260 gtccggctaa gtgagagcaa cctggtcatc tga 1293 <210> 3 <211> 619 <212> PRT <213> Homo sapiens <400> 3 Cys His Pro Val Asp Gly Thr Cys Ala Cys Glu Pro Gly Tyr Arg Gly   1 5 10 15 Lys Tyr Cys Arg Glu Ala Arg Gly Pro Cys Pro Ala Gly Phe Tyr Gly              20 25 30 Leu Gly Cys Arg Arg Arg Cys Gly Gln Cys Lys Gly Gln Gln Pro Cys          35 40 45 Thr Val Ala Glu Gly Arg Cys Leu Thr Cys Glu Pro Gly Trp Asn Gly      50 55 60 Thr Lys Cys Asp Gln Pro Cys Ala Thr Gly Phe Tyr Gly Glu Gly Cys  65 70 75 80 Ser His Arg Cys Pro Pro Cys Arg Asp Gly His Ala Cys Asn His Val                  85 90 95 Thr Gly Lys Cys Thr Arg Cys Asn Ala Gly Trp Ile Gly Asp Arg Cys             100 105 110 Glu Thr Lys Cys Ser Asn Gly Thr Tyr Gly Glu Asp Cys Ala Phe Val         115 120 125 Cys Ala Asp Cys Gly Ser Gly His Cys Asp Phe Gln Ser Gly Arg Cys     130 135 140 Leu Cys Ser Pro Gly Val His Gly Pro His Cys Asn Val Thr Cys Pro 145 150 155 160 Pro Gly Leu His Gly Ala Asp Cys Ala Gln Ala Cys Ser Cys His Glu                 165 170 175 Asp Thr Cys Asp Pro Val Thr Gly Ala Cys His Leu Glu Thr Asn Gln             180 185 190 Arg Lys Gly Val Met Gly Ala Gly Ala Leu Leu Val Leu Leu Val Cys         195 200 205 Leu Leu Leu Ser Leu Leu Gly Cys Cys Cys Ala Cys Arg Gly Lys Asp     210 215 220 Pro Thr Arg Arg Glu Leu Ser Leu Gly Arg Lys Lys Ala Pro His Arg 225 230 235 240 Leu Cys Gly Arg Phe Ser Arg Ile Ser Met Lys Leu Pro Arg Ile Pro                 245 250 255 Leu Arg Arg Gln Lys Leu Pro Lys Val Val Val Ala His His Asp Leu             260 265 270 Asp Asn Thr Leu Asn Cys Ser Phe Leu Glu Pro Pro Ser Gly Leu Glu         275 280 285 Gln Pro Ser Pro Ser Trp Ser Ser Arg Ala Ser Phe Ser Ser Phe Asp     290 295 300 Thr Thr Asp Glu Gly Pro Val Tyr Cys Val Pro His Glu Glu Ala Pro 305 310 315 320 Ala Glu Ser Arg Asp Pro Glu Val Pro Thr Val Pro Ala Glu Ala Pro                 325 330 335 Ala Pro Ser Pro Val Pro Leu Thr Thr Pro Ala Ser Ala Glu Glu Ala             340 345 350 Ile Pro Leu Pro Ala Ser Ser Asp Ser Glu Arg Ser Ala Ser Ser Val         355 360 365 Glu Gly Pro Gly Gly Ala Leu Tyr Ala Arg Val Ala Arg Arg Glu Ala     370 375 380 Arg Pro Ala Arg Ala Arg Gly Glu Ile Gly Gly Leu Ser Leu Ser Pro 385 390 395 400 Ser Pro Glu Arg Arg Lys Pro Pro Pro Pro Asp Pro Ala Thr Lys Pro                 405 410 415 Lys Val Ser Trp Ile His Gly Lys His Ser Ala Ala Ala Ala Gly Arg             420 425 430 Ala Pro Ser Pro Pro Pro Pro Gly Ser Glu Ala Ala Pro Ser Pro Ser         435 440 445 Lys Arg Lys Arg Thr Pro Ser Asp Lys Ser Ala His Thr Val Glu His     450 455 460 Gly Ser Pro Arg Thr Arg Asp Pro Thr Pro Arg Pro Pro Gly Leu Pro 465 470 475 480 Glu Glu Ala Thr Ala Leu Ala Ala Pro Ser Pro Pro Arg Ala Arg Ala                 485 490 495 Arg Gly Arg Gly Pro Gly Leu Leu Glu Pro Thr Asp Ala Gly Gly Pro             500 505 510 Pro Arg Ser Ala Pro Glu Ala Ala Ser Met Leu Ala Ala Glu Leu Arg         515 520 525 Gly Lys Thr Arg Ser Leu Gly Arg Ala Glu Val Ala Leu Gly Ala Gln     530 535 540 Gly Pro Arg Glu Lys Pro Ala Pro Pro Gln Lys Ala Lys Arg Ser Val 545 550 555 560 Pro Pro Ala Ser Pro Ala Arg Ala Pro Pro Ala Thr Glu Thr Pro Gly                 565 570 575 Pro Glu Lys Ala Ala Thr Asp Leu Pro Ala Pro Glu Thr Pro Arg Lys             580 585 590 Lys Thr Pro Ile Gln Lys Pro Pro Arg Lys Lys Ser Arg Glu Ala Ala         595 600 605 Gly Glu Leu Gly Arg Ala Gly Ala Pro Thr Leu     610 615 <210> 4 <211> 1860 <212> DNA <213> Homo sapiens <400> 4 tgccaccctg tggacggcac gtgtgcctgc gagccgggct accgcggcaa gtactgtcgc 60 gaggcacgag ggccgtgccc cgccggcttc tacggcttgg gctgtcgccg ccggtgtggc 120 cagtgcaagg gccagcagcc gtgcacggtg gccgagggcc gctgcttgac gtgcgagccc 180 ggctggaacg gaaccaagtg cgaccagcct tgcgccaccg gtttctatgg cgagggctgc 240 agccaccgct gtccgccatg ccgcgacggg catgcctgta accatgtcac cggcaagtgt 300 acgcgctgca acgcgggctg gatcggcgac cggtgcgaga ccaagtgtag caatggcact 360 tacggcgagg actgcgcctt cgtgtgcgcc gactgcggca gcggacactg cgacttccag 420 tcggggcgct gcctgtgcag ccctggcgtc cacgggcccc actgtaacgt gacgtgcccg 480 cccggactcc acggcgcgga ctgtgctcag gcctgcagct gccacgagga cacgtgcgac 540 ccggtcactg gtgcctgcca cctagaaacc aaccagcgca agggcgtgat gggcgcgggc 600 gcgctgctcg tcctgctcgt ctgcctgctg ctctcgctgc tcggctgctg ctgcgcttgc 660 cgcggcaagg accctacgcg ccgggagctt tcgcttggga ggaagaaggc gccgcaccga 720 ctatgcgggc gcttcagtcg catcagcatg aagctgcccc ggatcccgct ccggaggcag 780 aaactaccca aagtcgtagt ggcccaccac gacctggata acacactcaa ctgcagcttc 840 ctggagccac cctcagggct ggagcagccc tcaccatcct ggtcctctcg ggcctccttc 900 tcctcgtttg acaccactga tgaaggccct gtgtactgtg taccccatga ggaggcacca 960 gcggagagcc gggaccccga agtccccact gtccctgccg aggcgccggc gccgtcccct 1020 gtgcccttga ccacgccagc ctccgccgag gaggcgatac ccctccccgc gtcctccgac 1080 agcgagcggt cggcgtccag cgtggagggg cccggagggg ctctgtacgc gcgcgtggcc 1140 cgacgcgagg cccggccggc ccgggcccgg ggcgagattg ggggcctgtc gctgtcgcca 1200 tcgcccgagc gcaggaaacc gccgccacct gaccccgcca ccaagcctaa ggtgtcctgg 1260 atccacggca agcacagcgc cgctgcagct ggccgtgcgc cctcaccacc gccgccaggc 1320 tccgaggccg cgcccagccc cagcaagagg aaacggacgc ccagcgacaa atcggcgcat 1380 acggtcgaac acggcagccc ccggacccgc gacccaacgc cgcggccccc cgggctgccc 1440 gaggaggcga cagccctcgc tgcgccctcg ccgcccaggg cccgagcgcg gggccgcggc 1500 cccggcctct tggagcccac ggacgccggc ggtcccccgc gaagcgcgcc cgaggctgcc 1560 tccatgttgg ccgctgagct gcgcggcaag actcgcagcc tgggccgcgc cgaggtggcc 1620 ctgggcgcgc agggccccag ggaaaagccg gcgcccccac agaaagccaa gcgctccgtg 1680 ccgccagcct cgcccgcccg cgcgccccca gcgaccgaaa ccccggggcc tgagaaggcg 1740 gcgaccgact tgcccgcgcc tgagaccccc cggaagaaga cccccatcca gaagccgccg 1800 cgcaagaaga gccgggaggc ggcgggcgag ctgggcaggg cgggcgcacc caccctgtag 1860 <210> 5 <211> 468 <212> PRT <213> Homo sapiens <400> 5 Met Gly Thr Gln Glu Gly Trp Cys Leu Leu Leu Cys Leu Ala Leu Ser   1 5 10 15 Gly Ala Ala Glu Thr Lys Pro His Pro Ala Glu Gly Gln Trp Arg Ala              20 25 30 Val Asp Val Val Leu Asp Cys Phe Leu Val Lys Asp Gly Ala His Arg          35 40 45 Gly Ala Leu Ala Ser Ser Glu Asp Arg Ala Arg Ala Ser Leu Val Leu      50 55 60 Lys Gln Val Pro Val Leu Asp Asp Gly Ser Leu Glu Asp Phe Thr Asp  65 70 75 80 Phe Gln Gly Gly Thr Leu Ala Gln Asp Asp Pro Pro Ile Ile Phe Glu                  85 90 95 Ala Ser Val Asp Leu Val Gln Ile Pro Gln Ala Glu Ala Leu Leu His             100 105 110 Ala Asp Cys Ser Gly Lys Glu Val Thr Cys Glu Ile Ser Arg Tyr Phe         115 120 125 Leu Gln Met Thr Glu Thr Thr Val Lys Thr Ala Ala Trp Phe Met Ala     130 135 140 Asn Val Gln Val Ser Gly Gly Gly Pro Ser Ile Ser Leu Val Met Lys 145 150 155 160 Thr Pro Arg Val Ala Lys Asn Glu Val Leu Trp His Pro Thr Leu Asn                 165 170 175 Leu Pro Leu Ser Pro Gln Gly Thr Val Arg Thr Ala Val Glu Phe Gln             180 185 190 Val Met Thr Gln Thr Gln Ser Leu Ser Phe Leu Leu Gly Ser Ser Ala         195 200 205 Ser Leu Asp Cys Gly Phe Ser Met Ala Pro Gly Leu Asp Leu Ile Ser     210 215 220 Val Glu Trp Arg Leu Gln His Lys Gly Arg Gly Gln Leu Val Tyr Ser 225 230 235 240 Trp Thr Ala Gly Gln Gly Gln Ala Val Arg Lys Gly Ala Thr Leu Glu                 245 250 255 Pro Ala Gln Leu Gly Met Ala Arg Asp Ala Ser Leu Thr Leu Pro Gly             260 265 270 Leu Thr Ile Gln Asp Glu Gly Thr Tyr Ile Cys Gln Ile Thr Thr Ser         275 280 285 Leu Tyr Arg Ala Gln Gln Ile Ile Gln Leu Asn Ile Gln Ala Ser Pro     290 295 300 Lys Val Arg Leu Ser Leu Ala Asn Glu Ala Leu Leu Pro Thr Leu Ile 305 310 315 320 Cys Asp Ile Ala Gly Tyr Tyr Pro Leu Asp Val Val Val Thr Trp Thr                 325 330 335 Arg Glu Glu Leu Gly Gly Ser Pro Ala Gln Val Ser Gly Ala Ser Phe             340 345 350 Ser Ser Leu Arg Gln Ser Val Ala Gly Thr Tyr Ser Ile Ser Ser Ser         355 360 365 Leu Thr Ala Glu Pro Gly Ser Ala Gly Ala Thr Tyr Thr Cys Gln Val     370 375 380 Thr His Ile Ser Leu Glu Glu Pro Leu Gly Ala Ser Thr Gln Val Val 385 390 395 400 Pro Pro Glu Arg Arg Thr Ala Leu Gly Val Ile Phe Ala Ser Ser Leu                 405 410 415 Phe Leu Leu Ala Leu Met Phe Leu Gly Leu Gln Arg Arg Gln Ala Pro             420 425 430 Thr Gly Leu Gly Leu Leu Gln Ala Glu Arg Trp Glu Thr Thr Ser Cys         435 440 445 Ala Asp Thr Gln Ser Ser His Leu His Glu Asp Arg Thr Ala Arg Val     450 455 460 Ser Gln Pro Ser 465 <210> 6 <211> 1407 <212> DNA <213> Homo sapiens <400> 6 atgggcacac aggagggctg gtgcctgctg ctctgcctgg ctctatctgg agcagcagaa 60 accaagcccc acccagcaga ggggcagtgg cgggcagtgg acgtggtcct agactgtttc 120 ctggtgaagg acggtgcgca ccgtggagct ctcgccagca gtgaggacag ggcaagggcc 180 tcccttgtgc tgaagcaggt gccagtgctg gacgatggct ccctggagga cttcaccgat 240 ttccaagggg gcacactggc ccaagatgac ccacctatta tctttgaggc ctcagtggac 300 ctggtccaga ttccccaggc cgaggccttg ctccatgctg actgcagtgg gaaggaggtg 360 acctgtgaga tctcccgcta ctttctccag atgacagaga ccactgttaa gacagcagct 420 tggttcatgg ccaacgtgca ggtctctgga gggggaccta gcatctcctt ggtgatgaag 480 actcccaggg tcgccaagaa tgaggtgctc tggcacccaa cgctgaactt gccactgagc 540 ccccagggga ctgtgcgaac tgcagtggag ttccaggtga tgacacagac ccaatccctg 600 agcttcctgc tggggtcctc agcctccttg gactgtggct tctccatggc accgggcttg 660 gacctcatca gtgtggagtg gcgactgcag cacaagggca ggggtcagtt ggtgtacagc 720 tggaccgcag ggcaggggca ggctgtgcgg aagggcgcta ccctggagcc tgcacaactg 780 ggcatggcca gggatgcctc cctcaccctg cccggcctca ctatacagga cgaggggacc 840 tacatttgcc agatcaccac ctctctgtac cgagctcagc agatcatcca gctcaacatc 900 caagcttccc ctaaagtacg actgagcttg gcaaacgaag ctctgctgcc caccctcatc 960 tgcgacattg ctggctatta ccctctggat gtggtggtga cgtggacccg agaggagctg 1020 ggtggatccc cagcccaagt ctctggtgcc tccttctcca gcctcaggca aagcgtggca 1080 ggcacctaca gcatctcctc ctctctcacc gcagaacctg gctctgcagg tgccacttac 1140 acctgccagg tcacacacat ctctctggag gagccccttg gggccagcac ccaggttgtc 1200 ccaccagagc ggagaacagc cttgggagtc atctttgcca gcagtctctt ccttcttgca 1260 ctgatgttcc tggggcttca gagacggcaa gcacctacag gacttgggct gcttcaggct 1320 gaacgctggg agaccacttc ctgtgctgac acacagagct cccatctcca tgaagaccgc 1380 acagcgcgtg taagccagcc cagctga 1407 <210> 7 <211> 282 <212> PRT <213> Homo sapiens <400> 7 Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile   1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser              20 25 30 Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile          35 40 45 Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu      50 55 60 Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val  65 70 75 80 His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met                  85 90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn             100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr         115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu     130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln                 165 170 175 Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser             180 185 190 Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met         195 200 205 Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser     210 215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val 225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser                 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp Ala Leu             260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys         275 280 <210> 8 <211> 849 <212> DNA <213> Homo sapiens <400> 8 atggcttccc tggggcagat cctcttctgg agcataatta gcatcatcat tattctggct 60 ggagcaattg cactcatcat tggctttggt atttcaggga gacactccat cacagtcact 120 actgtcgcct cagctgggaa cattggggag gatggaatcc tgagctgcac ttttgaacct 180 gacatcaaac tttctgatat cgtgatacaa tggctgaagg aaggtgtttt aggcttggtc 240 catgagttca aagaaggcaa agatgagctg tcggagcagg atgaaatgtt cagaggccgg 300 acagcagtgt ttgctgatca agtgatagtt ggcaatgcct ctttgcggct gaaaaacgtg 360 caactcacag atgctggcac ctacaaatgt tatatcatca cttctaaagg caaggggaat 420 gctaaccttg agtataaaac tggagccttc agcatgccgg aagtgaatgt ggactataat 480 gccagctcag agaccttgcg gtgtgaggct ccccgatggt tcccccagcc cacagtggtc 540 tgggcatccc aagttgacca gggagccaac ttctcggaag tctccaatac cagctttgag 600 ctgaactctg agaatgtgac catgaaggtt gtgtctgtgc tctacaatgt tacgatcaac 660 aacacatact cctgtatgat tgaaaatgac attgccaaag caacagggga tatcaaagtg 720 acagaatcgg agatcaaaag gcggagtcac ctacagctgc taaactcaaa ggcttctctg 780 tgtgtctctt ctttctttgc catcagctgg gcacttctgc ctctcagccc ttacctgatg 840 ctaaaataa 849 <210> 9 <211> 331 <212> PRT <213> Homo sapiens <400> 9 Met Leu Trp Leu Phe Gln Ser Leu Leu Phe Val Phe Cys Phe Gly Pro   1 5 10 15 Gly Asn Val Val Ser Gln Ser Ser Leu Thr Pro Leu Met Val Asn Gly              20 25 30 Ile Leu Gly Glu Ser Val Thr Leu Pro Leu Glu Phe Pro Ala Gly Glu          35 40 45 Lys Val Asn Phe Ile Thr Trp Leu Phe Asn Glu Thr Ser Leu Ala Phe      50 55 60 Ile Val Pro His Glu Thr Lys Ser Pro Glu Ile His Val Thr Asn Pro  65 70 75 80 Lys Gln Gly Lys Arg Leu Asn Phe Thr Gln Ser Tyr Ser Leu Gln Leu                  85 90 95 Ser Asn Leu Lys Met Glu Asp Thr Gly Ser Tyr Arg Ala Gln Ile Ser             100 105 110 Thr Lys Thr Ser Ala Lys Leu Ser Ser Tyr Thr Leu Arg Ile Leu Arg         115 120 125 Gln Leu Arg Asn Ile Gln Val Thr Asn His Ser Gln Leu Phe Gln Asn     130 135 140 Met Thr Cys Glu Leu His Leu Thr Cys Ser Val Glu Asp Ala Asp Asp 145 150 155 160 Asn Val Ser Phe Arg Trp Glu Ala Leu Gly Asn Thr Leu Ser Ser Gln                 165 170 175 Pro Asn Leu Thr Val Ser Trp Asp Pro Arg Ile Ser Ser Glu Gln Asp             180 185 190 Tyr Thr Cys Ile Ala Glu Asn Ala Val Ser Asn Leu Ser Phe Ser Val         195 200 205 Ser Ala Gln Lys Leu Cys Glu Asp Val Lys Ile Gln Tyr Thr Asp Thr     210 215 220 Lys Met Ile Leu Phe Met Val Ser Gly Ile Cys Ile Val Phe Gly Phe 225 230 235 240 Ile Ile Leu Leu Leu Leu Val Leu Arg Lys Arg Arg Asp Ser Leu Ser                 245 250 255 Leu Ser Thr Gln Arg Thr Gln Gly Pro Glu Ser Ala Arg Asn Leu Glu             260 265 270 Tyr Val Ser Val Ser Pro Thr Asn Asn Thr Val Tyr Ala Ser Val Thr         275 280 285 His Ser Asn Arg Glu Thr Glu Ile Trp Thr Pro Arg Glu Asn Asp Thr     290 295 300 Ile Thr Ile Tyr Ser Thr Ile Asn His Ser Lys Glu Ser Lys Pro Thr 305 310 315 320 Phe Ser Arg Ala Thr Ala Leu Asp Asn Val Val                 325 330 <210> 10 <211> 996 <212> DNA <213> Homo sapiens <400> 10 atgttgtggc tgttccaatc gctcctgttt gtcttctgct ttggcccagg gaatgtagtt 60 tcacaaagca gcttaacccc attgatggtg aacgggattc tgggggagtc agtaactctt 120 cccctggagt ttcctgcagg agagaaggtc aacttcatca cttggctttt caatgaaaca 180 tctcttgcct tcatagtacc ccatgaaacc aaaagtccag aaatccacgt gactaatccg 240 aaacagggaa agcgactgaa cttcacccag tcctactccc tgcaactcag caacctgaag 300 atggaagaca caggctctta cagagcccag atatccacaa agacctctgc aaagctgtcc 360 agttacactc tgaggatatt aagacaactg aggaacatac aagttaccaa tcacagtcag 420 ctatttcaga atatgacctg tgagctccat ctgacttgct ctgtggagga tgcagatgac 480 aatgtctcat tcagatggga ggccttggga aacacacttt caagtcagcc aaacctcact 540 gtctcctggg accccaggat ttccagtgaa caggactaca cctgcatagc agagaatgct 600 gtcagtaatt tatccttctc tgtctctgcc cagaagcttt gcgaagatgt taaaattcaa 660 tatacagata ccaaaatgat tctgtttatg gtttctggga tatgcatagt cttcggtttc 720 atcatactgc tgttacttgt tttgaggaaa agaagagatt ccctatcttt gtctactcag 780 cgaacacagg gccccgagtc cgcaaggaac ctagagtatg tttcagtgtc tccaacgaac 840 aacactgtgt atgcttcagt cactcattca aacagggaaa cagaaatctg gacacctaga 900 gaaaatgata ctatcacaat ttactccaca attaatcatt ccaaagagag taaacccact 960 ttttccaggg caactgccct tgacaatgtc gtgtaa 996 <210> 11 <211> 16 <212> PRT <213> Homo sapiens <400> 11 Arg Gly Val Glu Val Ala Ala Gly Ala Ser Ser Gly Gly Glu Thr Arg   1 5 10 15 <210> 12 <211> 13 <212> PRT <213> Homo sapiens <400> 12 Gly Pro Cys Pro Ala Gly Phe Tyr Gly Leu Gly Cys Arg   1 5 10 <210> 13 <211> 23 <212> PRT <213> Homo sapiens <400> 13 Thr Ala Ala Trp Phe Met Ala Asn Val Gln Val Ser Gly Gly Gly Pro   1 5 10 15 Ser Ile Ser Leu Val Met Lys              20 <210> 14 <211> 29 <212> PRT <213> Homo sapiens <400> 14 His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile Gly Glu   1 5 10 15 Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys              20 25 <210> 15 <211> 10 <212> PRT <213> Homo sapiens <400> 15 Ser Pro Glu Ile His Val Thr Asn Pro Lys   1 5 10

[Brief description of drawings]

Fig. 1 Primary structure of deduced amino acid sequence of this membrane molecule (A) Kyte and Doolittle method (J. Exp. Med., 157: 1)
05-132, 1982) shows a hydropathic plot.

FIG. 2: Primary structure of deduced amino acid sequence of the present membrane molecule (B). Kyte and Doolittle method (J. Exp. Med., 157: 1).
05-132, 1982) shows a hydropathic plot.

FIG. 3: Primary structure of deduced amino acid sequence of the present membrane molecule (C). Kyte and Doolittle method (J. Exp. Med., 157: 1).
05-132, 1982) shows a hydropathic plot.

Fig. 4 Primary structure of deduced amino acid sequence of the present membrane molecule (D). Kyte and Doolittle method (J. Exp. Med., 157: 1).
05-132, 1982) shows a hydropathic plot.

FIG. 5: Kyte and Doolittle's method (J. Exp. Med., 157: 1) on the primary structure of the deduced amino acid sequence of this membrane molecule (E).
05-132, 1982) shows a hydropathic plot.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/15 G01N 33/53 D 4H045 33/50 M 33/53 33/566 C12P 21/08 33/566 C12N 15/00 ZNAA // C12P 21/08 5/00 EF Term (reference) 2G045 AA24 AA40 BA11 BB50 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA31 BA80 CA03 HA15 4B063 QA01 QQ08 QR48 QR55 QS33 4B064 CC01 CA24 CA19 4B065 AA94X BA25 BA30 CA44 CA46 4H045 AA10 AA11 AA30 BA10 CA40 DA75 DA76 DA86 EA20 EA50 FA71 FA72 FA74

Claims (11)

[Claims] 1. A human dendritic cell membrane molecule having the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9, or a deletion, substitution, insertion and deletion of one or more amino acid residues in the amino acid sequence. A variant thereof which is capable of controlling an immune response, which contains / or addition and has 80% or more homology with the amino acid sequence. 2. A DNA encoding the human dendritic cell membrane molecule according to claim 1 or a variant thereof. 3. The DNA according to claim 2, which has the nucleotide sequence shown in SEQ ID NO: 2, 4, 6, 8 or 10. 4. An antibody that specifically immunologically binds to the human dendritic cell membrane molecule according to claim 1 or a variant thereof or a fragment thereof. 5. An immunologically unrecognized immature dendritic cell,
The antibody according to claim 4, which recognizes mature dendritic cells. 6. The antibody according to claim 4, which recognizes an extracellular region of the human dendritic cell membrane molecule. 7. The antibody according to any one of claims 4 to 6, which is a polyclonal antibody or a monoclonal antibody. 8. A method for separating human or other animal-derived dendritic cells using the antibody according to any one of claims 4 to 7. 9. The method according to claim 8, characterized in that mature dendritic cells are isolated. 10. A method for detecting dendritic cells using the antibody according to claim 4. 11. The method according to claim 10, characterized in that mature dendritic cells are detected.