JP2004147665A - Cell surface molecule mediating intercellular adhesion and signaling - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicine useful for treating or preventing several kinds of autoimmune diseases and inflammatory diseases by identifying a new cell surface molecule simultaneously having both functions of mediating intercellular adhesion between lymphoid cells and of signaling of control of the cell activation and/or cell function, revealing its structural and biological properties and using the molecule or an antibody against the molecule. <P>SOLUTION: The monoclonal antibody differentially manifested in thymocyte, ConA-stimulation activated lymphocyte and peripheral blood lymphocyte and introducing intercellular adhesion of the cells is found out in monoclonal antibody to the cell surface molecule significantly working in autoimmune diseases and allergic diseases. The cell surface molecule recognized by the monoclonal antibody is separated and identified and functions of the molecule is analyzed. The antibody to the molecule significantly suppresses disease conditions of autoimmune diseases and allergic diseases. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to a novel cell surface molecule of a mammal, a polypeptide constituting the molecule and a fragment thereof, a fusion polypeptide comprising the polypeptide fragment and an immunoglobulin fragment, a gene encoding the polypeptide and the fragment, A vector containing a gene, a transformant into which the vector has been introduced, an antibody having reactivity with the polypeptide or a cell surface molecule constituted by the polypeptide, a hybridoma producing the antibody, the polypeptide fragment or the fusion polypeptide The present invention relates to a pharmaceutical composition comprising a peptide, a pharmaceutical composition comprising the antibody, a transgenic mouse, and a knockout mouse.

  BACKGROUND ART Living organisms of mammals have an immune response system for eliminating pathogenic microorganisms (viruses, bacteria, parasites, etc.) and foreign foreign substances (hereinafter collectively referred to as “antigens”) that have entered the body. One is called the innate immune response system and the other is called the acquired immune response system. The former are elimination mechanisms by nonspecific recognition, such as phagocytosis by phagocytes (polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack by natural killer (NK) cells, and opsonization of antigen by complement. is there. The latter acquired immune response system is a mechanism of elimination by lymphocytes (mainly T cells and B cells) that have acquired (activated) specificity for the antigen. B cells that have acquired antigen specificity attack the extracellular antigen by producing antibodies specific to the antigen. T cells that have acquired (activated) antigen specificity are classified into helper T cells and cytotoxic T cells (cytotoxic T cells; CTLs), the former regulating B cell differentiation and antibody production. Together with the phagocytes to destroy the antigen. The latter attacks cells infected with viruses and the like (Experimental Medicine (separate volume), "Bio Science Terminology Library [Immunity]", Yodosha, p.14-17, 1995).

  The acquisition (activation) of antigen specificity by T cells is achieved by T cells recognizing antigens presented by antigen-presenting cells (APCs) such as macrophages, B cells or dendritic cells. Be started. The antigen presenting cell processes (processes) the taken-in antigen, binds the processed antigen to the major histocompatibility complex (MHC), and presents the antigen. T cells recognize the processed antigen presented by the antigen presenting cell through a complex of a T cell receptor (TcR) and a CD3 antigen (TcR / CD3 complex) on the surface of the cell membrane, and thereby recognize the cell. Receive the first signal for activation (acquisition of specificity).

  However, only the first signal through the TcR / CD3 complex not only does not result in sufficient activation of the T cells, but also becomes unresponsive or unresponsive to any subsequent stimuli. It falls into a condition called clonal anergy. Autocrine interleukin-2 (IL-2) is required for T cell activation to differentiate and proliferate into antigen-specific T cell clones. In this case, IL-2 is not produced and cell division does not occur, and T cells are inactivated. That is, activation of T cells with production of cytokines such as IL-2 requires a second signal following the first signal via the TcR / CD3 complex. This second signal is called the costimulatory signal (costimulatory signal).

  The T cell interacts with another molecule, distinct from the MHC on the antigen presenting cell (cell-cell adhesion), through a molecule different from the TcR / CD3 complex on the surface of the T cell, thereby forming this second signal. Receiving and transmitting it into cells. The second signal avoids cell anergy (clonal paralysis) and activates the cell.

  Although the mechanism of transmission of the second signal between antigen presenting cells and lymphocytes such as T cells has not yet been elucidated in detail, according to previous studies, this second signal transmission involves: CD28 (also known as Tp44, T44 or 9.3 antigen), which is a cell surface molecule mainly expressed on T cells and thymocytes, and cell surface molecule expressed on antigen presenting cells (macrophages, monocytes, dendritic cells, etc.) Interactions between CD80 (also known as B7-1, B7, BB1, or B7 / BB1) and CD86 (also known as B7-2 or B70), which is also a cell surface molecule of an antigen presenting cell-like, Cell-cell adhesion through intermolecular bonds) has been shown to be extremely important. Further, in controlling the activation of T cells by the second signal, CTLA-4 (Cytolytic T lymphocyte associated antigen 4), which is considered to be enhanced in expression depending on the second signal, and the T cell It is experimentally revealed that the interaction between CD80 (B7-1) and CD86 (B7-2) (ie, cell-cell adhesion through the binding between their molecules) also plays an important role. Have been. That is, the control of T cell activation by the transmission of the second signal includes at least the interaction between CD28 and CD80 / CD86, the enhancement of CTLA-4 expression which is considered to depend on the interaction, It has also been shown that the interaction between CTLA-4 and CD80 / CD86 is involved.

  CD28 has been shown to be a costimulator molecule that transmits a second signal (costimulatory signal) required for T cell activation and avoidance of anergy. The binding of this molecule to the costimulator molecules CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (in other words, cell-cell adhesion via binding between those molecules) Signal stabilizes the mRNA of Th1-type cytokines, thereby encouraging the production of large quantities of Th1-type cytokines such as IL-2, IFNγ and TNFα from T cells. On the other hand, it is known that the expression of CTLA-4 is induced by the first signal entering through TcR / CD3 and the expression is also enhanced by the second signal entering by binding of CD28 and CD80. . CTLA-4 has been shown to act on T cell function in response to these signals, as opposed to T cell activation by a second signal coming from CD28.

  Human CD28 and CTLA-4 are type I glycoproteins with molecular weights of 44 kD and 41-43 kD, respectively. Both have one immunoglobulin-like domain, belong to the immunoglobulin superfamily, and have both functions as an intercellular adhesion molecule and a function as a signal transduction molecule into cells.

  It has been shown that human CD28 forms a homodimer via disulfide bonds, while CTLA-4 exists as a monomer. The positions of the CD28 and CTLA-4 genes on the chromosome are both "2q33" in humans and "1C" in mice, and each consist of four exons. Human CD28 and CTLA-4 are composed of 220 and 223 amino acids, respectively, including the leader sequence, and their amino acid homology is about 20 to 30%.

  The ligands for CD28 and CTLA-4 have been elucidated in humans and mice as CD80 (B7-1) and CD86 (B7-2). CTLA-4 has a higher affinity for both ligands than CD28, with a difference of about 20-fold. For binding of CD28 and CTLA-4 to CD80 (B7-1), "MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)", an amino acid sequence conserved across animal species, is important It has been revealed that In addition, when CD28 is stimulated, phosphoinositide 3 kinase (PI3K) associates with a phosphorylated tyrosine residue in the intracellular partial sequence “YMNM (Tyr-Met-Asn-Met)”. It has been shown that CD28 plays an important role in intracellular signal transmission through this “YxxM” structure. In addition, the intracellular region of CTLA4 also has a sequence represented by “YxxM”, that is, “YVKM (Tyr-Val-Lys-Met)”. After stimulation, SYP associates with this sequence. It is shown.

  It has been shown that CD28 is expressed exclusively on thymocytes and peripheral blood T cells, while CTLA-4 is specifically expressed on activated T cells. Junsha Publishing, pp. 93-102, 1994; The Journal, pp. 120-136; Experimental Medicine, Separate Volume, "BIO SCIENCE Term Library / Immunity", Yodosha Publishing, pp. 94-98, 1995; Experiment Medicine, separate volume, "BIO SCIENCE Terminology Library, Intracellular Signaling," published by Youtosha, pp. 58-59, 1997; Japan Clinical Society, Vol. 55, No. 6, pp. 215-220, 1997) .

  Thus, costimulator molecules (such as CD28, CD80 (B7-1) and CD86 (B7-2)) and cooperating CTLA-4, etc. in controlling T cell functions (T cell activation and function suppression) The importance of the interaction between multiple molecules (in other words, cell-cell adhesion through bonding between those molecules) has been proposed, and the elucidation of the relationship between those molecules and diseases, and Attempts to treat diseases by controlling the function of these molecules have received attention.

  As described above, a living body activates an adaptive immune response system against an antigen that is foreign to the living body (self), but has an immune tolerance that does not show an immune response against its own biological component (self antigen). Have. However, when immune tolerance is disrupted for any reason, an immune response to self-antigen occurs, and self-antigen-reactive T cells are induced by the same mechanism as described above, resulting in an immune abnormality and various autoimmune diseases. .

  That is, when the immune system of a living body is in a normal state, unstimulated antigen presenting cells (APCs) of normal tissues do not express costimulatory molecules, so that, for example, autoantigen reactivity which reacts with self antigens Even if T cells are present, self tolerance is maintained because the T cells are in an unresponsive state, but in an immune abnormal state, self-tolerance is caused by excess or continuous expression of costimulatory molecules. It has been suggested that antigen-reactive T cells can be activated to cause autoimmune diseases.

  From this viewpoint, in recent years, attempts have been made to treat various autoimmune diseases by regulating the transmission of costimulatory signals, for example, the signal transmission between CD28 / CTLA-4-CD80 / CD86. Many have been made.

  In autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, and chronic inflammatory skin diseases lichen planus and psoriasis, antigen-presenting cells in the focus of the disease Abnormal expression of CD80, a costimulatory molecule as a ligand for CD28 and CTLA-4, has been confirmed, and in particular, attempts to treat various autoimmune diseases by suppressing the function of CD80 Many have been made.

  As a method for blocking the function of CD80, a method using an antibody against CD80, a solubilized protein of CD28 which is a ligand of CD80, or a solubilized protein of CTLA-4 which is also a ligand of CD80 has been studied. Among them, “solubilized CTLA-4” based on the fact that the binding affinity of CTLA-4 to CD80 is at least 20 times that of CD28, specifically the extracellular domain of “CTLA-4” and human immunoglobulin IgG1 Therapeutic trials using a fusion protein (CTLA-4-IgFc) consisting of the Fc region of Escherichia coli have been conducted in animal models and clinical trials (Japanese clinical studies, Vol. 55, No. 6, pp. 215-220, 1997).

The following reports (i) to (v) have been made on the therapeutic effects of CTLA-4-IgFc in autoimmune disease model animals.
(i) In (NZB / NZW) F1 mice, a model of human systemic lupus erythematosus (SLE), suppression of autoantibody production and suppression of lupus nephritis by pre-symptomatic administration An improvement was observed (Science, Vol. 125, p. 1225-1227, 1994).
(ii) In experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis (MS), the disease could be prevented by short-term administration immediately after immunization (J. Clin. Invest., Vol. 95, p.2783-2789, 1995).
(iii) The incidence of NOD (non-obese diabetes) mice, which is an insulin-dependent diabetes mellitus (IDDM) model, was significantly reduced by administration to females of 2 to 3 weeks of age for 2 weeks (J. Exp. Med., Vol. 181, p. 1145-1155, 1995).
(iv) In rats with nephritis induced by glomerular basement membrane immunity, which is a model of nephritis in Goodpasture (Goodpasture), the symptoms were improved by administration (Eur. J. Immunol., Vol. 24, No. 6, p. .1249-1254, 1994).
(v) In type II collagen-induced arthritis (CIA) using DBA / 1 mouse, a human rheumatoid arthritis model, the onset of arthritis is suppressed by immunization, and autoantibodies against collagen (IgG1 and IgG2) Production was suppressed (Eur. J. Immunol., Vol. 26, p. 2320-2328, 1996).

  However, while treatment attempts have been made as described above, the interaction between costimulator molecules and related molecules (in other words, cell-cell adhesion through binding between those molecules) has caused T. The mechanism of cell activation has not been elucidated in detail, and it remains possible that other unidentified molecules may be involved in this mechanism.

  Activation of lymphocytes such as T cells and function of lymphocytes by cell-cell adhesion through binding between molecules involved in transmission of a second signal essential for activation of lymphocytes such as T cells as described above. The elucidation of the mechanism of the regulation of, and the identification and characterization of known or unknown molecules that have both the ability to mediate cell-cell adhesion and the ability to transmit signals involved in the mechanism, and their characterization, have been described in various contexts. It is possible to develop and provide a drug useful in treating or preventing various diseases such as an autoimmune disease, an allergic disease, and an inflammatory disease.

  That is, an object of the present invention is to identify a novel cell surface molecule having both functions of mediating cell-cell adhesion and signaling, and to clarify its structural and biological properties. I do. Another object of the present invention is to provide a medicament useful for treating or preventing various autoimmune diseases and inflammatory diseases by using the novel molecule or an antibody against the molecule.

  In order to identify such useful molecules, the present inventors have determined that lymphocytes such as T cells play an important role in autoimmune diseases and allergic diseases, etc. Focusing on the fact that cell-cell adhesion is essential for signal transduction of signal 2 (costimetry signal) into lymphocyte cells, it is specifically expressed in lymphoid cells and cell-cell adhesion It was considered that a cell surface molecule having a function of mediating DNA was isolated and identified.

  As a method, first, monoclonal antibodies against various cell surface molecules expressed on the surface of the lymphoid cells are obtained by immunizing the lymphoid cells themselves, and the obtained monoclonal antibodies are used to mediate cell-cell adhesion. A technique for isolating and identifying a target cell surface molecule having the function of Hereinafter, the method will be specifically described.

  The present inventors first administered a mouse lymphocyte cell line as an immunogen to mice to prepare various monoclonal antibodies. Next, the obtained monoclonal antibody was allowed to act on rat lymphoid cells used as an immunogen, and the effect of the monoclonal antibody on the cells was examined. As a result, it was found that one of the obtained monoclonal antibodies had a property of strongly agglutinating the rat lymphoid cells (this monoclonal antibody was named "JTT.1 antibody"). Furthermore, in the same manner, among the prepared monoclonal antibodies, a monoclonal antibody that strongly inhibits the aggregation of rat lymphoid cells induced by the “JTT.1 antibody” was found (this monoclonal antibody was referred to as “JTT. 2 antibody).)

  Aggregation of rat lymphoid cells by the "JTT.1 antibody" is caused by the interplay of ICAM-1 (Intercellular adhesion molecule-1) and LFA-1 (ICAM-1), the most typical known cell-cell adhesion molecules expressed in the cells. Since the aggregation was not inhibited by an antibody against Lymphocyte function-associated antigen-1), this aggregation was considered to be aggregation due to intercellular adhesion via an unknown adhesion molecule having an intercellular adhesion-mediated action.

  Next, identification, isolation, and characterization of cell surface molecules (each named “JTT.1 antigen” and “JTT.2 antigen”) respectively recognized by the two monoclonal antibodies were performed.

  First, the expression patterns of “JTT.1 antigen” and “JTT.2 antigen” in various cells were determined by flow cytometry based on the fluorescent antibody method using “JTT-1 antibody” and “JTT-2 antibody”. Was analyzed. As a result, activated lymphoblasts that were activated by stimulating thymocytes and spleen cells with mitogen Concanavalin A (ConA) in both “JTT.1 antigen” and “JTT.2 antigen” Spherical cells (activated T lymphoblasts, activated B lymphoblasts, etc.), especially spleen cells which have been strongly expressed in the activated lymphoblasts but have not been stimulated at all, Such cells are sometimes referred to as “quiescent lymphocytes” in the present invention). In addition, they found that the expression patterns of the molecules confirmed by each of the “JTT-1 antibody” and the “JTT-2 antibody” were almost the same.

  Then, using an affinity column prepared by adsorbing the `` JTT-1 antibody '' on the adsorbent, the mixture of soluble cell surface molecules prepared from the rat lymphoid cells was trapped by the `` JTT-1 antibody '' from the mixture. Molecule, ie, the “JTT-1 antigen” was purified. The molecular weight of this purified “JTT-1 antigen” was analyzed by immunoprecipitation using “JTT-1 antibody” and “JTT-2 antibody” and SDS-PAGE. As a result, it was found that the molecules immunoprecipitated by each of the “JTT-1 antibody” and “JTT-2 antibody” were the same molecule, and that the molecules were homodimers having different sugar chain modifications. . Specifically, when the N-glycan is not digested, it is identified as one molecule of about 47 kD in non-reduction and as two molecules of about 24 kD and about 28 kD in reduction, When digestion was carried out, it was identified as one molecule of about 36 kD in non-reduction and as one molecule of about 20 kD in reduction.

  Next, the adhesion of rat thymic cells to the plate coated with the purified “JTT.1 antigen” was analyzed. As a result, thymocytes significantly adhered to the plate only in the presence of “JTT.1 antibody” (that is, adhered to “JTT.1 antigen”), and the adhesion was significantly significant in the presence of “JTT.2 antibody”. It was confirmed that "JTT.1 antigen" was actually a cell surface molecule that mediates cell-cell adhesion.

  Next, the present inventors cloned the gene encoding the "JTT.1 antigen" of rat, human and mouse, and analyzed the structure thereof.

  Specifically, first, from the cDNA library of rat spleen derived rat spleen stimulated with ConA, "rat JTT.1 antigen" was expressed by an expression cloning method using a panning method using "JTT.1 antibody". We succeeded in isolating the cDNA encoding the full length of "", and isolated and identified a completely novel rat gene by determining its nucleotide sequence by the dideoxy method. Further, by using plaque hybridization using a cDNA encoding the obtained "rat JTT.1 antigen" as a probe, "human JTT.1" was extracted from the cDNA library of human peripheral blood lymphoblasts stimulated with ConA. We succeeded in isolating the cDNA encoding the entire length of the "antigen" and isolated and identified a completely novel human gene by determining its base sequence by the dideoxy method. Similarly, a cDNA encoding the full length of "mouse JTT.1 antigen" was successfully isolated from a cDNA library of mouse spleen-derived lymphoblasts stimulated with ConA, and its nucleotide sequence was determined by the dideoxy method. As a result, a completely novel mouse gene was isolated and identified. Furthermore, in the same manner, from the cDNA library of the rat thymoma cell line, the cDNA encoding the full-length alternative splicing variant of the "rat JTT.1 antigen" was successfully isolated, By determining the nucleotide sequence by the dideoxy method, another completely novel rat gene was isolated and identified.

  From the hydropathic blot analysis of the amino acid sequence encoded by the isolated cDNA of the human JTT-1 antigen, the JTT.1 antigen showed a signal sequence, an extracellular region having a glycosylation site, a transmembrane region, And a cell transmembrane protein composed of an intracellular region (that is, a cell surface molecule). Furthermore, as a result of homology search with known molecules, none of the rat, human, and mouse "JTT.1 antigens" have significant homology with any known molecules including cell-indirected molecules, and It has been found that it is a completely novel cell surface molecule having a function of mediating adhesion between cells.

  Furthermore, as a result of conducting a motif search based on the amino acid sequence of “human JTT-1 antigen”, “human JTT-1 antigen” was found to activate T cells through cell-cell adhesion as described in detail above. "CD28", a cell surface molecule of lymphocytes such as T cells that transmit important costimulatory signals, and T cells that control the function of activated lymphocytes such as activated T cells in conjunction with the signals Has the following structural similarity to “CTLA-4”, a cell surface molecule of lymphocytes.

  That is, (i) 20 or more amino acid residues including cysteine residues are well conserved. (ii) A continuous sequence of proline residues “Pro-Pro-Pro (PPP)” essential as a ligand binding region is conserved in the extracellular region. In addition, (iii) a sequence “Tyr-Xaa-Xaa-Met (YxxM) (Xaa and x mean any amino acid) essential for a signal transduction region is conserved in the intracellular region.

  Next, the position of the gene encoding “mouse JTT-1 antigen” on the mouse chromosome was analyzed using fluorescence in situ hybridization (FISH). "1C3", which is the same as the position of "CTLA-4".

  Next, in order to analyze the efficacy of treatment of autoimmune diseases and allergic diseases by controlling the function of "JTT-1 antigen", the above-mentioned "JTT-2 antibody" was used in experimental allergic encephalomyelitis. Experiments were performed in which rats (EAE) and glomerular basement membrane (GBM) nephritis were administered to model rats. As a result, we found that any disease model animal showed significant suppression of the disease state, and it was possible to treat autoimmune diseases and allergic diseases by controlling the function of “JTT-1 antigen” I found that.

  Furthermore, the monoclonal antibody against "human JTT-1 antigen" significantly expands human peripheral blood lymphocytes, and the expansion receives a first signal from an antigen presenting cell essential for T cell activation. In the presence of a monoclonal antibody against CD3, which constitutes the TcR / CD3 complex on T cells, we discovered that even higher proliferation was observed, and that "JTT-1 antigen" was involved in signal transduction to lymphocytes. Was found to be a molecule.

  Furthermore, cells of "human JTT-1 antigen" useful as pharmaceuticals for treating autoimmune diseases, allergic diseases or inflammatory diseases by controlling the function of "JTT-1 antigen" and / or its ligand A fusion polypeptide consisting of the outer region and the Fc region of human immunoglobulin was successfully produced.

  Furthermore, it is useful to analyze the detailed function of "JTT-1 antigen" and to develop pharmaceuticals for the treatment of autoimmune diseases, allergic diseases and inflammatory diseases. We succeeded in producing transgenic mice into which the gene encoding the "antigen" was introduced, and knockout mice in which the endogenous gene encoding the "mouse JTT-1 antigen" was inactivated.

That is, the present invention provides polypeptides, genes, antibodies, vectors, transformants, pharmaceutical compositions, transgenics related to the novel mammalian "JTT-1 antigen" isolated and identified as described above. The present invention relates to a mouse, a knockout mouse, and the like. Specifically, the present invention relates to the inventions described in the following (1) to (36).
(1) A polypeptide constituting a cell surface molecule having the following characteristics:
(A) expressed at least in thymocytes and mitogen-stimulated lymphoblasts;
(B) antibodies reactive with the cell surface molecules induce adhesion between mitogen-stimulated lymphoblast cells;
(C) an antibody reactive with the cell surface molecule induces proliferation of peripheral blood lymphocytes in the presence of an antibody reactive with CD3;
(D) having a partial amino acid sequence represented by Phe-Asp-Pro-Pro-Pro-Phe in the extracellular region; and (e) having a partial amino acid sequence represented by Tyr-Met-Phe-Met in the intracellular region. .
(2) the polypeptide has the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been substituted, deleted, or added; The polypeptide according to the above (1), which is characterized in that:
(3) The polypeptide according to (1), wherein the polypeptide is encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1.
(4) The polypeptide according to (1), wherein the polypeptide has an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 2.
(5) The polypeptide according to any one of (1) to (4), wherein the cell surface molecule is a human-derived cell surface molecule.
(6) A gene encoding the polypeptide according to any one of (1) to (5).
(7) The gene according to (6), wherein the gene is cDNA.
(8) The gene according to (7), wherein the cDNA has the nucleotide sequence of SEQ ID NO: 1.
(9) the cDNA comprises the nucleotide sequence of nucleotides 26 to 625 of SEQ ID NO: 3, the nucleotide sequence of nucleotides 35 to 637 of SEQ ID NO: 4, the nucleotide sequence of nucleotides 1 to 603 of SEQ ID NO: 5 The gene according to the above (7), which comprises a sequence or any one of the nucleotide sequences of base numbers 35 to 685 of SEQ ID NO: 6.
(10) A vector containing the gene according to any one of (6) to (9).
(11) A transformant into which the vector according to (10) has been introduced.
(12) A transformant identified by International Accession Number FERM BP-5725.
(13) A polypeptide fragment comprising the extracellular region of the polypeptide according to any of (1) to (5).
(14) The polypeptide fragment of (13), wherein the polypeptide is a human-derived polypeptide having the amino acid sequence of SEQ ID NO: 2.
(15) A gene encoding the polypeptide fragment according to (13) or (14).
(16) A homodimer molecule formed by binding a polypeptide fragment comprising the extracellular region of the polypeptide according to any one of (1) to (5) by a disulfide bond.
(17) The homodimer molecule according to (16), wherein the polypeptide is a human-derived polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(18) A pharmaceutical composition comprising one or both of the polypeptide fragment of (14) or the homodimer molecule of (17) and a pharmaceutically acceptable carrier.
(19) A fusion polypeptide comprising the extracellular region of the polypeptide according to any of (1) to (5) and a constant region or a part of a constant region of a heavy chain of human immunoglobulin (Ig). .
(20) The fusion polypeptide according to the above (19), wherein the immunoglobulin is an IgG.
(21) The fusion polypeptide according to (19), wherein a part of the constant region comprises an IgG hinge region, a C2 domain, and a C3 domain.
(22) The fusion polypeptide according to any of (19) to (21), wherein the polypeptide is a human-derived polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(23) A homodimer molecule formed by bonding the fusion polypeptide according to any of (19) to (22) by a disulfide bond.
(24) A homodimer molecule formed by binding the fusion polypeptide according to (22) by a disulfide bond.
(25) A pharmaceutical composition comprising one or both of the fusion polypeptide according to (22) or the homodimer molecule according to (24) and a pharmaceutically acceptable carrier.
(26) The pharmaceutical composition according to the above (25), wherein the pharmaceutical composition is used for treating an autoimmune disease or an allergic disease or suppressing the symptoms of the disease.
(27) Reactivity with the polypeptide according to any of (1) to (5), the polypeptide fragment according to (13) or (14), or a cell surface molecule constituted by the polypeptide Or a portion thereof.
(28) The antibody or a part thereof according to (27), wherein the antibody is a monoclonal antibody.
(29) a polypeptide having the amino acid sequence of SEQ ID NO: 2, a polypeptide fragment of (14) or a monoclonal antibody reactive with a human-derived cell surface molecule composed of the polypeptide, or a monoclonal antibody thereof; part.
(30) A monoclonal antibody reactive to the polypeptide according to any of (1) to (5) or a cell surface molecule constituted by the polypeptide, and stimulated with the mitogen of the monoclonal antibody A monoclonal antibody whose effect on lymphoblast cells is substantially the same as that exhibited by mitogen-stimulated rat lymphoblast cells, wherein the monoclonal antibody produced by the hybridoma identified by International Accession No.FERM BP-5707 part.
(31) A monoclonal antibody reactive to the polypeptide according to any of the above (1) to (5) or a cell surface molecule constituted by the polypeptide, and stimulated with the mitogen of the monoclonal antibody A monoclonal antibody or an antibody which has substantially the same effect on lymphoblast cells as the monoclonal antibody produced by the hybridoma identified by International Accession No.FERM BP-5708 exhibits on mitogen-stimulated rat lymphoblast cells. part.
(32) A pharmaceutical composition comprising the monoclonal antibody according to (29) or a part thereof and a pharmaceutically acceptable carrier.
(33) The pharmaceutical composition according to (32), wherein the pharmaceutical composition is used for treating an autoimmune disease or an allergic disease or for suppressing the symptoms of the disease.
(34) A hybridoma that produces the monoclonal antibody according to any of (28) to (31).
(35) A gene encoding the polypeptide according to (1), which is a human-derived gene comprising the nucleotide sequence of SEQ ID NO: 1 or the nucleotides of nucleotides 35 to 637 of SEQ ID NO: 4 A transgenic mouse, wherein a rat-derived gene containing the sequence is integrated into an endogenous mouse gene.
(36) A mouse encoding the polypeptide according to (1), wherein the polypeptide is derived from a mouse having the amino acid sequence encoded by the gene set forth in SEQ ID NO: 5, so that the mouse-derived polypeptide is not produced. A knockout mouse, wherein an endogenous gene has been inactivated.

The novel cell surface molecule (referred to as "JTT-1 antigen") derived from mammals such as human, mouse and rat provided by the present invention has the following characteristics.
(1) “CD28”, a cell surface molecule of lymphocytes such as T cells, which transmits costimulatory signals important for T cell activation through cell-cell adhesion, and activation in conjunction with the signals It has the following similarities to “CTLA-4” which is a cell surface molecule of lymphocytes such as T cells that controls the function of activated lymphocytes such as T cells.
(i) 20 or more amino acid residues including cysteine residues are well conserved.
(ii) A continuous sequence of proline residues essential for a ligand binding region, “Pro-Pro-Pro (PPP)”, is conserved in the extracellular region.
(iii) The essential sequence “Tyr-Xaa-Xaa-Met (YxxM) (Xaa and x mean any amino acid)” as a signal transduction region is conserved in the intracellular region.
(iv) The position of the gene encoding “mouse JTT-1 antigen” on the mouse chromosome is “1C3”, as is the position of “CD28” and “CTLA-4” in mice.
(2) Like "CD28" and "CTLA-4", which have a function of mediating cell-cell adhesion, "JTT-1 antigen" is used for thymocytes, lymphoblasts and thymoma cells stimulated with mitogens such as ConA. Has the ability to mediate cell-cell adhesion.
(3) It is strongly expressed in at least thymocytes, lymphoblasts (activated T lymphoblasts and activated B lymphoblasts) stimulated with a mitogen such as ConA, peripheral blood lymphocytes and thymoma cells.
(4) Antibodies directed against “JTT-1 antigen” significantly proliferate human peripheral blood lymphocytes, and the proliferation receives a first signal from an antigen presenting cell essential for T cell activation. Higher proliferation is induced by the coexistence of a monoclonal antibody against CD3 constituting the TcR / CD3 complex on T cells.
(5) By administering an antibody directed to the "JTT-1 antigen" to experimental allergic encephalomyelitis (EAE), the disease state is significantly suppressed.
(6) By administering an antibody directed to the "JTT-1 antigen" to a model rat of glomerular basement membrane (GBM) nephritis, the disease state is significantly suppressed.

  Due to such characteristics, the "JTT-1 antigen" of the present invention, like the above-mentioned "CD28" and "CTLA-4", comprises a second signal (costiy) essential for activation of lymphocytes such as T cells. It is considered to be a molecule that controls the transmission of a stimulating lymphocyte, such as an activated T cell, in conjunction with the transmission of such signals.

  Therefore, the polypeptide, polypeptide fragment, fusion polypeptide and antibody of the present invention constituting such a cell surface molecule are caused by abnormal activation of lymphocytes such as T cells and abnormal control of the functions of activated lymphocytes. Various autoimmune diseases, allergic diseases or inflammatory diseases, specifically rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, flat inflammatory skin diseases It is possible to provide a medicament which is extremely useful for treating or preventing lichen, systemic lupus erythematosus, insulin-dependent diabetes mellitus and psoriasis.

  Similarly, the gene encoding the polypeptide or polypeptide fragment of the present invention not only enables gene therapy for the various diseases, but also can provide an antisense drug.

  Among the antibodies of the present invention, a human monoclonal antibody and a pharmaceutical composition thereof are an antigen for humans, which has been a major problem (side effect) in the therapeutic treatment of an antibody pharmaceutical comprising an antibody derived from a non-human mammal such as a mouse-derived antibody. Since it has no properties, it dramatically increases the value as a pharmaceutical.

  In addition, the gene (DNA), polypeptide, polypeptide fragment and antibody of the present invention are not only useful as pharmaceuticals, but also search for molecules (ligands) that interact with the cell surface molecule of the present invention, and functions of the ligand. It is useful as a reagent for elucidating and for developing a therapeutic drug targeting the ligand.

  Further, the transgenic mouse of the present invention is not only useful as a model animal for elucidating the physiological function of the cell surface molecule of the present invention, `` JTT-1 antigen, '' but also a `` JTT-1 antigen '' It is extremely useful as a tool for screening various drugs (low molecular weight compounds, antibodies, antisense, polypeptides, etc.) having an activity of controlling (inhibiting, suppressing, activating, stimulating, etc.) the function. That is, such a test substance is administered to the transgenic mouse, and various physiological, biological or pharmacological parameters occurring in the living body of the mouse are measured and analyzed to determine the activity of the administered test substance. It is possible to evaluate.

  Further, the knockout mouse of the present invention is characterized by analyzing the characteristics of the mouse from various aspects (physiological, biological, pharmacological, pathological, genetic, etc.) to obtain the cell surface of the present invention. It is possible to elucidate the functions of molecules.

  As described above, the cell surface molecule of the present invention (that is, the “JTT-1 antigen”) is used for cell-cell adhesion through the molecule, transmission of signals to lymphocytes such as T cells, and function of activated lymphocytes. It has aspects that play a role in control. Thus, general knowledge about lymphoid cells, intercellular adhesion molecules, and their relationship to disease is described below for the sole purpose of providing a general understanding of such biological events. Therefore, the general findings described below are not intended to limit the present invention.

  Lymphocytes are roughly divided into two types, T cells and B cells. When lymphoid stem cells differentiate from pluripotent stem cells in the bone marrow, some of them enter the bloodstream to reach the thymus. Lymphocytes differentiated and matured in the thymus are called T cells (Thymus-derived cells: T cells) and enter the blood again and go around the whole body. Mature T cells have a molecule called CD3 on their surface, and having this molecule is a marker to distinguish whether the cell is a mature T cell. CD3 is a potent T cell marker. In addition, T cells express CD4 or CD8. T cells are also helper T cells (Th cells: helper T cells) that support B lymphocyte antibody production, and cytotoxic T cells (Tc cells: cytotoxic T cells: CTL) that attach to target cells and destroy them directly ) Or killer T cells, suppressor T cells that suppress B-lymphocyte antibody production, and effector T cells that secrete effect substances such as lymphokines and cause delayed allergy.

  Among lymphoid stem cells, B cells are directly differentiated and matured in bone marrow. B cells are cells that will produce antibodies when appropriately stimulated and are antibody-producing precursor cells. On the surface is an immunoglobulin synthesized in B cells, which functions as an antigen receptor. Mature B cells have both IgM and IgD on the surface, and when stimulated by antigen and differentiated by signals from T cells, IgM production increases, and the C-terminal cell membrane-binding portion is altered and secreted. With sufficient stimulation, the immunoglobulin on the surface also changes to IgG, IgE, and IgA, and secretes each class of immunoglobulin. Immunoglobulins on the surface of B cells are sometimes referred to as sIg for surface Ig or mIg for cell membrane Ig. Igs on the surface of one B cell all have the same antigen binding site.

  There are lymphocytes that are neither T cells nor B cells and are called LGL (laege granular lymphocute) or null cells. These cells have the ability to damage tumor cells and virus-infected cells, but unlike cytotoxic T cells, they do not require prior antigen stimulation. Therefore, it is also called natural killer cell (NK cell).

  Among the above T cells, CD4 positive T cells secrete various cytokines in response to the antigen presented by the antigen-providing cells, and receptors for those cytokines are newly expressed, and the cells themselves become larger, Initiate divisions and multiply. Prior to such a cell-level reaction, a complex of an antigen peptide on an antigen-donor cell and an MHC class II molecule binds to an antigen receptor (T cell antigen receptor: TCR) specific to each T cell. This causes various biochemical changes in the cell, transmitting signals into the nucleus, starting transcription of specific DNA, and synthesizing each protein. As a result, a response at the cellular level can be seen. Regarding CD8-positive T cells, for example, considering a cell infected with a certain virus, the infected cell synthesizes a viral protein, and the protein is degraded by a proteasome in the cytoplasm, and some of the peptide reacts with TAP. After that, it enters the endoplasmic reticulum, forms a stable complex with the just-synthesized MHC class I molecule, and emerges on the cell surface. This is recognized by specific CD8 + T cells but has not yet destroyed them at this stage. These T cells that responded to the antigen express the IL-2 receptor (IL-2R), and when IL-2 acts on the T cells, they differentiate into CTLs that have cytotoxic activity, and then form the same antigen peptide / MHC class I complex. When they meet, they destroy and kill their target cells. It is said that not only IL-2 but also cytokines necessary for differentiating into CTLs have a similar effect on IFNγ and other cytokines. Thus, differentiation of CTL requires lymphokines secreted by T cells, and these lymphokines are derived from the same virus as CD4 + Th1 cells (CD4 + T cells secreting IL-2, IFNγ, etc.) And recognizes the antigen peptide together with the class II molecule. Even without the help of CD4-positive T cells, CD8-positive T cells may produce IL-2 and the like in response to antigens. When CD8-positive T cells differentiate into CTL, granules increase in the cytoplasm. Various high molecular proteins are contained in the granules, and perforin is a typical example. Perforin is very similar to the membrane attack complex (MAC), composed of components 5-9 of complement, and has the effect of puncturing the cell membrane of target cells. In addition, serine protease, LT, proteoglycan and the like are also included. When differentiated into CTLs and stimulated with antigen, they also secrete lymphokines such as IFNγ, LT, TNF and IL-2. In addition, T cells respond to pan-haemagglutinin (plant agglutinin, PHA) or concanavalin A (Con A) to exhibit a blast formation phenomenon.

  Mature T cells that have not been stimulated at all are called resting T cells, and have a small cell size and a short life span of several days. When stimulated, the cells grow larger and are more responsive to various stimuli, as already mentioned. Such T cells are called activated T cells. Some activated T cells become memory T cells, which, when subjected to the same antigenic stimulus, will result in a secondary immune response. It is said that memory T cells continue to circulate in the body for years or decades.

  B cells that have not been stimulated yet are called resting B cells, just like T cells, and proliferated when stimulated with a multivalent antigen or CD40L. Such B cells are called activated B cells. Resting phase B cells have no costimulator molecules (molecules that stimulate T cells together with TCR-mediated signals) such as B7-1 (CD80) and B7-2 (CD86). It is said that even if the antigen is provided, it is not possible to express CD40 ligand (CD40L) or produce lymphokine by merely stimulating the TCR. Therefore, it is considered that the helper T cells activated and stimulated by other antigen-providing cells respond to antigen presentation of quiescent B cells. That is, when an antigen has entered, first, dendritic cells expressing B7 molecules (cells having remarkable dendrites) or macrophages activated in response to microorganisms present the antigen. It is thought that it stimulates resting helper T cells and activates them to express CD40L, and then binds to quiescent B cells presenting the same antigen to stimulate CD40. Once stimulated with a polyvalent antigen or CD40L and activated once, B cells also express B7 molecules and stimulate CD28 on the surface with TCR to activate their helper T cells to express CD40L, Or produce lymphokines. In addition, B cells that have undergone a change such as an increase in cells upon stimulation, but have hardly seen antibody secretion are called activated B cells. When matured B cells meet the antigen, the production of IgM increases due to stimulation from T cells, and the produced IgM changes from a membrane type to a secretory type and becomes secreted. In addition, humoral factors from T cells cause IgM to produce antibodies of other isotypes, such as IgG. This is called an isotype switch or a class switch. B cells that begin to secrete antibodies come to be called antibody-secreting cells, some of which become morphologically distinctive cells, and are called plasma cells (plasma cells). Knowledge of Science, Ohmsha (1996)).

  By the way, in various reactions of the immune system, the subpopulations of leukocytes, ie, T lymphocytes, B lymphocytes, NK, neutrophils, etc., often show different dynamics. Further, as described above, even the same lymphocyte shows different dynamics depending on the state of the cell, that is, whether the cell is activated or in the stationary phase. These facts suggest a recognition mechanism specific to subpopulations of leukocytes, and also a recognition mechanism specific to the state of cells, particularly the presence of cell adhesion molecules (cell adhesion proteins).

  A cell adhesion molecule, that is, a cell adhesion protein is generally a molecule having a function of adhering cells to each other at the time of development and differentiation of an individual, or at the time of migration of a cell to a local area, and is an organic substance of a living organism. It is known to be an essential molecule for functional communication.

  Cell adhesion molecules are broadly classified into five immunoglobulin superfamily, integrin family, secretin family, cadherin family, and CD44 family based on their structural characteristics. Adhesion molecules belonging to the immunoglobulin superfamily are characterized by repeatedly having a loop-like domain formed by disulfide bonds, and are characterized by "ICAM-1" (intercellular adhesion molecule-1) and "VCAM-1" (vascular cell adhesion molecule). Molecules such as molecule-1) are known. Adhesion molecules belonging to the integrin family are characterized by an α / β heterodimer structure, and are characterized by “VLA-1-6” (very late antigen-1-6) and “LFA-1” (lymphocyte function-associated antigen-1). ), “Mac-1” and “p150 / 90” are known. Molecules belonging to the secretin family have, in order from the N-terminus, a lectin-like domain, an EGF-like domain, and a complement domain, and "E-selectin", "P-selectin" and the like are known. Further, “E-cadherin”, “N-cadherin”, and “P-cadherin” are known as cadherin families, and “CD44” is known from the CD44 family.

  Specific functions of these adhesion molecules are known, such as adhesion of leukocytes to vascular endothelial cells and adhesion of lymphocytes to antigen-presenting cells. It has gradually become clear that it is also related to various diseases.

  In particular, many reports have been made on diseases and abnormal expression of adhesion molecules. For example, in rheumatoid arthritis (RA), it has been reported that the expression of both “Mac-1” and “p150 / 95” is enhanced in RA synovial cells (Allen, C et al. Arthritis Rheum., 32, 947 (1989)). Further, it has been reported that various cells in the RA synovium strongly and ectopically express "ICAM-1" (Hale, L. et al. Arthritis Rheum., 32, 22 ( 1989)). In addition, ELAM-1 is also involved in the adhesion of neutrophils to vascular endothelial cells, and the overexpression of these molecules is due to the infiltration of neutrophils found in RA synovial fluid (particularly into synovial fluid). (Laffon, A., et al. Arthritis Rheum., 32, 386 (1989)). Furthermore, it has been reported that “CD44” is also strongly expressed on vascular endothelial cells and type A synovial cells in RA synovium (Heynes, B. et al. Arthritis Rheum., 34, 1434 (1991)). ).

  There are also examples of the relationship between systemic lupus erythematosus (SLE) and abnormal expression of adhesion molecules. For example, the ability of T lymphocytes to adhere to cultured vascular endothelial cells in SLE patients is lower than that in healthy individuals Have been reported. In addition, in studies of the expression of adhesion molecules in peripheral lymphocytes of SLE patients, there is a tendency to enhance “ICAM-1,” “VLA-4,” and “IFA-1” (Haskard, DO et al, Rheumatol. Int, 9, 33 (1989)).

  Regarding autoimmune thyroid disease, stimulation of thyroid follicle cells with interferon-γ, interleukin-1, and tumor necrosis factor causes the expression of “ICAM-1” and the formation of clusters between follicular cells and mononuclear cells. It is reported that it is suppressed by the "ICAM-1" antibody (Weetman, AP, et al. Eur. J. Immunol., 20, 271 (1990)).

  In hepatitis, the adhesion between hepatocytes and inflammatory cells is increased by two pathways, ICAM-1 and LFA-3, and LFA-1 and CD2. It is thought that presentation and activation of inflammatory cells are promoted. In particular, in studies on hepatitis B, "LFA-3" was strongly expressed in hepatocytes where virus proliferation was active, and "ICAM-1" correlated well with the degree of hepatitis. It has been suggested that ICAM-1 is involved in virus elimination and that it promotes antigen presentation to T cells and regulates the inflammatory response. In the case of "ICAM-1" negative and HBc antigen-positive hepatocytes, it is thought that there is no interaction between cells with lymphocytes and a kind of immune unresponsiveness such as chronic infection of virus occurs. Serum "ICAM-1" concentration in patients with acute hepatitis, chronic active hepatitis and cirrhosis is higher than in healthy and chronic persistent hepatitis patients, and high values are found in histologically advanced patients among active hepatitis patients Thus, it has been reported that serum "ICAM-1" in chronic liver disease correlates with the degree of hepatocyte damage (Mod. Phys. 15, (1), 73-76 (1995)).

  In arteriosclerosis model animals, monocytes and lymphocytes adhere to and invade the vascular endothelium very early in the onset of the lesion, and the interaction between these blood cells and the endothelium is considered to be the first step in the onset of arteriosclerosis. ing. Various reports have been made on the expression of adhesion molecules in actual arteriosclerotic lesions. For example, expression of “ICAM-1” in human atherosclerotic lesions (Poston RN, et al. Am J Patol, 140, 665 (1992)) and expression of “VCAM-1” in hypercholesterolemic rabbit atherosclerotic lesions (Cybulsky) MI, Gimbrone MA Jr. Science, 251, 788 (1991)). More recently, the expression of "VCAM-1" has been observed in human atherosclerotic lesions, and its expression was particularly observed in smooth muscle cells and monocytes / macrophages that migrated to the intima. Have been reported. Increased expression of “MCP-1” has been observed in rabbit and human atherosclerotic lesions, indicating that “MCP-1” promotes the formation of atherosclerotic lesions through monocyte / macrophage migration. There is some suggestion (current therapy, 12, (8), 1485-1488 (1994)).

  In addition, there is a report on the relationship between cancer metastasis and abnormal adhesion molecules. For example, cancer cells with reduced E-cadherin show strong invasiveness, but the introduction of E-cadherin cDNA suppresses the invasiveness, and the invasiveness is restored when E-cadherin antiserum is added. It has been suggested that reduced expression of E-cadherin is closely related to invasiveness of cancer cells (Frixen, UH, et al. 113, 173 (1991)). Even in actual clinical cases, it has been pointed out that various cancers such as liver cancer, esophagus cancer, stomach cancer and breast cancer have a relationship between reduced E-cadherin expression and metastasis. In addition, it has been reported that VLA-4, a ligand of VCAM-1, is highly expressed in metastatic melanoma, gastric cancer, breast cancer, etc., and these are used for implantation into vascular endothelial cells during metastasis. Has been suggested. In addition, experiments using various cancer cell lines have reported that epithelial cancers such as gastric cancer, colon cancer, lung cancer, liver cancer, and pancreatic cancer adhere to vascular endothelial cells via E-selectin (Takada, A., et al. Cancer Res., 53, 354 (1993)).

  On the other hand, attempts have been made to treat diseases targeting these adhesion molecules. For example, it has been reported that an anti-rat “ICAM-1” antibody strongly suppresses the inflammatory response in a rat autoimmune arthritis model. It has also been reported that the administration of anti-ICAM-1 antibody suppresses the development of arthritis in adjuvant synovitis, which is one of the animal models of RA (Japanese Journal of Clinical Immunology) , 14, (6), 571-577 (1991)). Furthermore, when a large amount of a peptide of the REG sequence, which is an amino acid sequence on the extracellular matrix protein that recognizes and binds to some integrins, is administered to bile cancer mice, the metastasis of inoculated tumors is significantly suppressed, and in vitro In the system, it has been reported that the RGD peptide and the anti-β1 subunit antibody suppress the movement and invasion of cancer cells (Yamada, KM, et al. Cancer Res, 50, 4485, (1990)).

  Hereinafter, the present invention will be described in detail by clarifying the meanings of the terms used in the present invention and general methods for producing the polypeptide, fusion polypeptide, gene, antibody, transgenic mouse, knockout mouse, and the like of the present invention. However, it is needless to say that the meaning of the term is not to be construed as being limited by such definition.

  The “mitogen” in the present invention is also called a mitogen and refers to a substance that induces cell division. Immunologically, it refers to an antigen nonspecific (polyclonal) lymphocyte blast that induces division. Examples include lectins such as PHA and PWM, Concanavalin A (ConA), lipopolysaccharide, streptolysin S, and anti-lymphocyte antibody. It is known that concanavalin A and PHA act only on T lymphocytes, lipopolysaccharide acts only on B lymphocytes, and PWM acts on both lymphocytes.

  As used herein, the term “lymphoblasts” is also called large lymphocytes, lymphoblasts or immunoblasts, and refers to lymphoid tissues (lymph nodes, spleen, thymus, bone marrow, lymph vessels, tonsils) Gland) and lymphocytes that belong to large lymphocytes among lymphocytes that are present in the blood.

  The term "activated lymphocytes" as used herein means, for example, but not limited to, the following lymphocytes. For example, it refers to lymphocytes activated by some stimulus. As described above, lymphocytes are classified into T cells, B cells, and natural killer cells, and T cells can be further classified into CD4 positive cells and CD8 positive cells. Therefore, the “activated lymphocytes” of the present invention mainly include activated T cells, activated B cells, and activated natural killer cells, and activated T cells further include activated CD4 positive cells and activated CD4 cells. CD8-positive cells.

  CD4 positive T cells secrete various cytokines in response to the antigen presented by the antigen-providing cells, and the receptors for those cytokines are newly expressed, the cells themselves become larger, start dividing, proliferate and proliferate. Be activated. Activated CD4-positive T cells refer to CD4-positive T cells in such a state.

  CD8-positive T cells express IL-2R in response to antigens, differentiate into cytotoxic CTLs when acted on by IL-2, and then meet the same antigen peptide / MHC class I complex The target cells are destroyed and killed. As CD8-positive T cells differentiate into CTLs, granules increase in the cytoplasm. Various high-molecular proteins are contained in the granules, and perforin is a typical example. Perforin closely resembles the MAC composed of components 5-9 of complement and has the effect of puncturing the cell membrane of target cells. In addition, serine protease, LT, proteoglycan and the like are also included. When differentiated into CTL and stimulated with antigen, they also secrete lymphokines such as IFNγ, LT, TNF or IL-2. Activated CD8-positive T cells refer to CD8-positive T cells in such a state.

  T cells respond to pan-hemagglutinin (plant agglutinin, PHA) or concanavalin A (Con A) to exhibit a blast formation phenomenon, and T cells in such a state are also included in activated T cells.

  B cells express the B7 molecule, stimulate CD28 on the surface together with the TCR to activate their helper T cells, express CD40L, produce lymphokines, and become stimulated to grow cells, Changes such as proliferation are observed. Activated B cells refer to B cells in such a state, and in the present invention, B cells that are now secreting antibodies (antibody-secreting cells and plasma cells) Are also included in activated B cells.

  Activated natural killer cells refer to natural killer cells that exhibit a damaging effect on tumor cells and virus-infected cells as described above. In the present invention, thymocytes stimulated with concanavalin A (Con A) are also included in the activated lymphocytes.

  The “activated lymphoblast cells” used in the present invention include lymphoblasts obtained by stimulating the aforementioned “lymphoblasts” with the “mitogen” such as concanavalin A and stimulating them. It is.

  As used herein, the term "quiescent lymphocyte", as the case may be, refers to a non-activated lymphocyte that has not been stimulated for cell activation, in contrast to the aforementioned activated lymphocytes. .

  The “gene” in the present invention includes genomic DNA and cDNA.

  In the present invention, “derived from human” includes natural substances isolated from human biological components (organs, tissues, cells, body fluids, and the like), and recombinant substances produced using genetic recombination techniques. When the substance is a protein or polypeptide, artificial proteins and polypeptides having an amino acid sequence in which one or several amino acid sequences in the amino acid sequence have been substituted, deleted or added are also included.

  The `` cell surface molecule '' of the present invention is a cell surface molecule derived from mammals such as human, rat, mouse, guinea pig and rabbit, and preferably a cell surface molecule derived from human, rat or mouse. Preferably, it is a cell surface molecule of human origin.

The “cell surface molecule” of the present invention is specifically a cell surface molecule having at least the following properties.
(A) expressed on at least thymocytes and mitogen-stimulated lymphoblasts;
(B) antibodies reactive with the cell surface molecules induce adhesion between mitogen-stimulated lymphoblast cells;
(C) an antibody reactive with the cell surface molecule induces proliferation of peripheral blood lymphocytes in the presence of an antibody reactive with CD3;
(D) having a partial amino acid sequence represented by Phe-Asp-Pro-Pro-Pro-Phe in the extracellular region; and (e) having a partial amino acid sequence represented by Tyr-Met-Phe-Met in the intracellular region. .

  A preferred embodiment is a cell surface molecule composed of the “polypeptide” of the present invention described below.

The “polypeptide” of the present invention is a polypeptide constituting the above “cell surface molecule” of the present invention, and specific examples thereof include the following.
(1) A polypeptide encoded by a DNA that hybridizes to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions.
(2) A polypeptide having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 2.
(3) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence (that is, a polypeptide constituting “human JTT-1 antigen” and derivatives thereof).
(4) an amino acid sequence encoded by the nucleotide sequence of nucleotides 26 to 625 of SEQ ID NO: 3 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (that is, a “human JTT-1 antigen” Constituent polypeptides and derivatives thereof).
(5) a polypeptide having the amino acid sequence encoded by the nucleotide sequence of nucleotides 35 to 637 of SEQ ID NO: 4 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (that is, “rat JTT-1 antigen” Constituent polypeptides and derivatives thereof)
.
(6) an amino acid sequence encoded by the nucleotide sequence of any of nucleotide numbers 1 to 603 of SEQ ID NO: 5 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (that is, a “mouse JTT-1 antigen” Constituent polypeptides and derivatives thereof).
(7) an amino acid sequence encoded by the nucleotide sequence of nucleotides 35 to 685 of SEQ ID NO: 6 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (ie, “mutation of rat JTT-1 antigen” Polypeptides and their derivatives).
(8) the amino acid sequence encoded by the DNA encoding the polypeptide constituting the cell surface molecule of the present invention introduced into the transformant identified by International Accession No. FERM BP-5725, or substantially the same as the amino acid sequence (I.e., a polypeptide constituting the "human JTT-1 antigen" and a derivative thereof).

Here, examples of the “stringent conditions” include the following conditions. For example, in the case of performing hybridization under 0.9% NaCl using a probe having 50 bases or more, an estimate of the temperature (Tm) at which 50% dissociation occurs is obtained from the following formula, and the hybridization temperature is determined as follows. It can be set like a calculation formula.
Tm = 82.3 ° C. + 0.41 × (G + C)% − 500 / n−0.61 × (formamide)%
(N indicates the number of bases of the probe.)
Temperature = Tm-25 ° C
When a probe having 100 bases or more (when G + C = 40 to 50%) is used, it is intended that Tm changes as shown in the following (1) and (2).
(1) Tm is reduced by about 1 ° C. for each 1% mismatch.
(2) For every 1% of formamide, Tm is lowered by 0.6 to 0.7 ° C.

Therefore, the temperature conditions in the case of a combination of perfectly complementary strands can be as follows.
(A) 65-75 ° C (without formamide)
(B) 35-45 ° C (in the presence of 50% formamide)

The temperature conditions for the combination of incomplete complementary chains can be as follows.
(A) 45-55 ° C (without formamide)
(B) 35-42 ° C (in the presence of 30% formamide)

In addition, when a probe having 23 bases or less is used, the temperature condition can be set to 37 ° C., and the following formula can be used as a guide.
Temperature = 2 ° C x (number of A + T) + 4 ° C x (number of C + G)-5 ° C
Here, "having substantially the same amino acid sequence" refers to a plurality of amino acid sequences in the amino acid sequence as long as they have substantially the same biological properties as the polypeptide having the amino acid sequence described in the sequence listing. A polypeptide having an amino acid sequence in which amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids are substituted, deleted and / or modified, and a plurality of amino acids in the amino acid sequence; It is meant to include a polypeptide having an amino acid sequence to which 1 to 10 amino acids are added, particularly preferably 1 to 5 amino acids.

  Such amino acid substitution, deletion, or insertion can be performed according to a conventional method (Experimental Medicine Supplement, “Genetic Engineering Handbook” (1992), etc.).

  For example, a synthetic oligonucleotide-directed mutagenesis method (gapped duplex method), a method of randomly introducing point mutations by nitrite or sulfite treatment, a method of producing deletion mutants using Ba131 enzyme, etc., a cassette mutation method, a linker Examples of the method include a scanning method, a mismatch incorporation method, a mismatch primer method, and a DNA segment synthesis method.

  The synthetic oligonucleotide-directed mutagenesis (gapped duplex) method can be performed, for example, as follows. A region to be mutagenized is cloned into an M13 phage vector having an amber mutation, and a single-stranded phage DNA is prepared. The R13 DNA of the M13 vector having no amber mutation is linearized by treatment with a restriction enzyme, mixed with the above single-stranded phage DNA, denatured, and annealed to form “gapped duplex DNA”. A synthetic oligonucleotide having a mutation introduced therein is hybridized, and a closed circular double-stranded DNA is obtained by a reaction between DNA polymerase and DNA ligase. This DNA is transfected into Escherichia coli mutS strain deficient in mismatch modification ability, and the grown phage is infected into Escherichia coli having no suppressor function, and only phages having no amber mutation are selected.

  In addition, a method for introducing a point mutation with nitrite utilizes, for example, the following principle. When DNA is treated with nitrite, the base is deaminated, adenine becomes hypoxanthine, cytosine becomes uracil, and guanine becomes xanthine. When the deaminated DNA is introduced into cells, hypoxanthine and cytosine form hypoxanthine and uracil and adenine and xanthine form base pairs with thymine during DNA replication, so `` A: T '' becomes `` G: C '', `` "G: C" replaces "A: T". Actually, a single-stranded DNA fragment treated with nitrite is hybridized to "gapped duplex DNA", and the mutant strain may be isolated by the same procedure as in the synthetic oligonucleotide-directed mutagenesis (gapped duplex) method. .

In the specification and the drawings of the present application, three letters or one letter of an alphabet used to represent an amino acid mean the following amino acids, respectively.
(Gly / G) glycine, (Ala / A) alanine, (Val / V) valine, (Leu / L) leucine, (Ile / I) isoleucine, (Ser / S) serine, (Thr / T) threonine, Asp / D) aspartic acid, (Glu / E) glutamic acid, (Asn / N) asparagine, (Glu / Q) glutamine, (Lys / K) lysine, (Arg / R) arginine, (Cys / C) cysteine, ( (Met / M) methionine, (Phe / F) phenylalanine, (Tyr / Y) tyrosine, (Trp / W) tryptophan, (His / H) histidine, (Pro / P) proline.

  The “polypeptide” constituting the “cell surface molecule” of the present invention described above is a cell transmembrane protein that penetrates a cell membrane, and the “cell surface molecule” is constituted by one or two of the cell membrane penetrating polypeptide.

  As used herein, the term “transmembrane protein” refers to a cell that is linked to a membrane by a hydrophobic peptide region that penetrates the lipid bilayer once or several times, as seen in many receptors or cell membrane surface molecules. It refers to a protein having a structure composed of three main regions: an extracellular region, a transmembrane region, and a cytoplasmic region. Furthermore, such transmembrane proteins can be homodimers, heterodimers, as monomers or with another chain having the same amino acid sequence or a chain having a different amino acid sequence, respectively. Alternatively, each receptor or cell surface molecule is constituted by the presence of an oligomer.

  The “polypeptide fragment” of the present invention is a polypeptide fragment of the “polypeptide” of the present invention as defined above, and is preferably an extracellular region of the polypeptide. The region may have 1 to 5 amino acids added to its N-terminal and / or C-terminal as desired.

  Here, the “extracellular region” refers to all or a part of the partial structure (partial region) existing on the outer side of the membrane to which the membrane protein is connected, of the entire structure of the cell membrane transmembrane protein as described above. In other words, all or part of the region other than the region taken up in the membrane (transmembrane region) and the region present in the cytoplasm following the region in the membrane (intracellular region) means.

  The “constant region or part of the constant region of the heavy chain of human immunoglobulin (Ig)” in the present invention refers to the constant region of the heavy chain (Heavy Chain, H chain) of human-derived immunoglobulin as described above. Constant region), Fc region or a part thereof. The immunoglobulin may be an immunoglobulin belonging to any class and subclass, specifically, IgG (IgG1, IgG2, IgG3 and IgG4), IgM, IgA (IgA1 and IgA2), IgD and IgE. Can be mentioned. Preferably, it is IgG (IgG1, IgG2, IgG3 or IgG4) or IgM. Particularly preferred examples in the present invention are immunoglobulins belonging to human-derived IgG (IgG1, IgG2, IgG3 or IgG4).

  Immunoglobulin is a Y-shape in which four homologous light chains (Light Chains, L chains) and two homologous heavy chains (Heavy Chains, H chains) are linked by disulfide bonds (SS bonds). Having the following structural unit. The light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH).

  The heavy chain constant region is comprised of several domains each having a unique amino acid sequence for each class (IgG, IgM, IgA, IgD and IgE) and subclass (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). .

  The heavy chain of IgG (IgG1, IgG2, IgG3 and IgG4) is composed of VH, CH1 domain, hinge region, CH2 domain and CH3 domain in order from the N-terminus.

  Similarly, the heavy chain of IgG1 is composed of VH, Cγ11 domain, hinge region, Cγ12 domain, and Cγ13 domain in order from the N-terminus. The heavy chain of IgG2 is composed of, in order from the N-terminus, VH, Cγ21 domain, hinge region, Cγ22 domain, and Cγ23 domain. The heavy chain of IgG3 is composed of VH, Cγ31 domain, hinge region, Cγ32 domain and Cγ33 domain in order from the N-terminus. The heavy chain of IgG4 is composed of VH, Cγ41 domain, hinge region, Cγ42 domain and Cγ43 domain in order from the N-terminus.

  The IgA heavy chain is composed of VH, Cα1 domain, hinge region, Cα2 domain, and Cα3 domain in order from the N-terminus.

  Similarly, the heavy chain of IgA1 is composed of VH, Cα11 domain, hinge region, Cα12 domain, and Cα13 domain in order from the N-terminus. The heavy chain of IgA2 is composed of, in order from the N-terminus, VH, Cα21 domain, hinge region, Cα22 domain, and Cα23 domain.

   The heavy chain of IgD is composed of VH, Cδ1 domain, hinge region, Cδ2 domain and Cδ3 domain in order from the N-terminus.

  The IgM heavy chain is composed of, in order from the N-terminus, VH, Cμ1, Cμ2, Cμ3, and Cμ4 domains, and does not have a hinge region as found in IgG, IgA, and IgD.

  The heavy chain of IgE is composed of VH, Ce1 domain, Ce2 domain, Ce3 domain and Ce4 domain in order from the N-terminus, and does not have a hinge region as found in IgG, IgA and IgD.

  Furthermore, taking IgG as an example, when IgG is treated with papain, it is cleaved slightly at the N-terminal side of the disulfide bond present in the hinge region connecting the two heavy chains to form a heavy chain consisting of VH and CH1. This results in two homologous Fabs in which the chain fragment and one light chain are linked by a disulfide bond, and one Fc in which two homologous heavy chain fragments consisting of the hinge region, CH2 domain and CH3 domain are linked by a disulfide bond (see above). , "Immunology Illustrated," Original Edition, 2nd ed., Pp. 65-75, 1992, published by Nankodo, and "Focus on the Latest Medical Science" Immune System Recognition Mechanism "," pp. 4-7, 1991. , Published by Nankodo).

  That is, “a part of the constant region of the immunoglobulin heavy chain” in the present invention means a part of the constant region of the immunoglobulin heavy chain having the structural characteristics as described above. The constant region or the Fc region that is missing. Specifically, in the case of IgG, IgA or IgD, a region consisting of each hinge region, C2 domain and C3 domain is mentioned, and in the case of IgM or IgE, respective C2 domain, C3 domain and C4 domain Region. A particularly preferred example is the Fc region of human IgG1.

  The “fusion polypeptide” of the present invention refers to the extracellular region of the “polypeptide” constituting the “cell surface molecule” of the present invention as defined above and the “heavy chain of human immunoglobulin (Ig)”. A constant region or a portion of a constant region. " Preferably, it is a fusion polypeptide of the extracellular region of the polypeptide of the present invention and a part of the constant region of the heavy chain of human IgG. Particularly preferably, the extracellular region of the polypeptide of the present invention and the heavy chain of human IgG are It is a fusion polypeptide with a region (Fc) consisting of a hinge region, a CH2 domain and a CH3 domain. In addition, IgG1 is preferable as IgG. Further, as the polypeptide of the present invention, a polypeptide derived from human, mouse or rat (preferably human) is preferable.

  Since the fusion polypeptide of the present invention has a part of the constant region (for example, Fc) of immunoglobulin such as IgG as described above as a fusion partner, the fusion polypeptide of protein A that specifically binds to the immunoglobulin fragment is used. The use of affinity column chromatography based on properties has the advantage that the fusion polypeptide can be very easily purified. Further, since various antibodies against Fc of various immunoglobulins are provided, immunoassay of the fusion polypeptide can be easily performed using antibodies against the Fc.

  The polypeptides, polypeptide fragments and fusion polypeptides of the present invention may be, as well as genetic recombination techniques as described below, chemical synthesis methods, known methods known in the art such as cell culture methods or the like. It can be produced by appropriately using a modification method.

  The “gene” of the present invention is a gene consisting of DNA encoding the above-described polypeptide or polypeptide fragment of the present invention, and a gene having any base sequence capable of encoding the polypeptide or polypeptide fragment of the present invention. Also included.

A specific embodiment is a gene encoding the following polypeptide or a polypeptide fragment thereof.
(1) A polypeptide encoded by a DNA that hybridizes to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions.
(2) A polypeptide having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 2.
(3) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence (that is, a polypeptide constituting “human JTT-1 antigen” and derivatives thereof).
(4) an amino acid sequence encoded by the nucleotide sequence of nucleotides 26 to 625 of SEQ ID NO: 3 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (that is, a “human JTT-1 antigen” Constituent polypeptides and derivatives thereof).
(5) a polypeptide having the amino acid sequence encoded by the nucleotide sequence of nucleotides 35 to 637 of SEQ ID NO: 4 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (that is, “rat JTT-1 antigen” Constituent polypeptides and derivatives thereof).
(6) an amino acid sequence encoded by the nucleotide sequence of any of nucleotide numbers 1 to 603 of SEQ ID NO: 5 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (that is, a “mouse JTT-1 antigen” Constituent polypeptides and derivatives thereof).
(7) an amino acid sequence encoded by the nucleotide sequence of nucleotides 35 to 685 of SEQ ID NO: 6 or a polypeptide having an amino acid sequence substantially identical to the amino acid sequence (ie, “mutation of rat JTT-1 antigen” Polypeptides and their derivatives).
(8) the amino acid sequence encoded by the DNA encoding the polypeptide constituting the cell surface molecule of the present invention introduced into the transformant identified by International Accession No. FERM BP-5725, or substantially the same as the amino acid sequence (I.e., a polypeptide constituting the "human JTT-1 antigen" and a derivative thereof).

  Here, “having substantially the same amino acid sequence” has the meaning as defined above.

More specific embodiments include the following DNAs or fragments thereof.
(1) A DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and a DNA that hybridizes to the DNA under stringent conditions.
(4) a DNA comprising the nucleotide sequence of nucleotides 26 to 625 of SEQ ID NO: 3;
(5) a DNA comprising the nucleotide sequence of base numbers 35 to 637 of SEQ ID NO: 4;
(6) a DNA comprising the nucleotide sequence of any of nucleotides 1 to 603 of SEQ ID NO: 5;
(7) a DNA comprising the nucleotide sequence of nucleotides 35 to 685 of SEQ ID NO: 6;
(8) DNA encoding a polypeptide constituting the cell surface molecule of the present invention, which has been introduced into a transformant identified by International Accession No. FERM BP-5725.

  The DNA encoding a part of the constant region of the heavy chain of immunoglobulin, which is a part of the fusion polypeptide in the present invention, may be cDNA or each exon (for example, CH1 domain, hinge region). , A genomic DNA containing an intron between DNA, CH2 domain, CH3 domain, CH4 domain, etc.).

  In the present invention, any codon that encodes the same amino acid is included in the DNA.

  The DNA of the present invention may be obtained by any method. For example, complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA obtained by chemical synthesis, DNA obtained by amplifying by PCR using RNA or DNA as a template, and appropriately combining these methods It also encompasses all DNAs constructed by the above method.

  The DNA encoding the polypeptide of the present invention can be obtained by a method of cloning cDNA from mRNA of the polypeptide of the present invention, a method of isolating and splicing genomic DNA, a method of chemically synthesizing, etc. according to a conventional method. it can.

  (1) For example, the following method is exemplified as a method for cloning cDNA from mRNA of the polypeptide of the present invention.

  First, mRNA encoding the polypeptide of the present invention is prepared from a tissue or a cell that expresses / produces the cell surface molecule (polypeptide) of the present invention (eg, thymocytes or ConA-stimulated spleen-derived lymphoblast cells). . For preparation of mRNA, total RNA prepared by a known method such as the guanidine thiocyanate method (Chirgwin JM et al, Biochemistry, Vol. 18, p. 5294, 1979), the hot phenol method or the AGPC method is oligo- (dTd). ) Affinity chromatography using cellulose or poly-U-sepharose can be carried out.

  Then, using the obtained mRNA as a template, a known method such as, for example, using reverse transcriptase, for example, the method of Okayama et al. (Mol. Cell. Biol., Vol. 2, pp. 161, 1982) And the same journal, Vol. 3, p. 280, 1983) and the method of Hoffman et al., Gene, Vol. 25, p. 263, 1983, etc. Conversion to strand cDNA is performed. This cDNA is incorporated into a plasmid vector, a phage vector or a cosmid vector, and transformed into Escherichia coli, or after in vitro packaging, transfected into Escherichia coli to prepare a cDNA library.

  The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host, and any phage vector can be used as long as it can be propagated in the host. Examples of commonly used cloning vectors include pME18S, λZAPII (lZAPII), pUC19, λgt10, λgt11 and the like. However, when subjected to the immunological screening described below, a vector having a promoter capable of expressing a gene encoding the polypeptide of the present invention in a host is preferred.

  Examples of the method of incorporating cDNA into a plasmid include the methods of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition), Cold Spring Harbor Laboratory, 1.53, 1989). Examples of the method for incorporating cDNA into a phage vector include the method of Hyunh et al. (DNA cloning, a practical approach, Vol. 1, p. 49, 1985). . For convenience, a commercially available cloning kit (for example, Takara Shuzo) can also be used. The thus obtained recombinant plasmid or phage vector is introduced into a suitable host such as a prokaryotic cell (for example, E. coli: XL1Blue MRF ', DH5α, HB101 or MC1061 / P3).

  A method for introducing a plasmid into a host is described in (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, page 1.74, 1989). The described calcium chloride method, the calcium chloride / rubidium chloride method, the electroporation method and the like can be mentioned. Examples of a method for introducing a phage vector into a host include a method in which phage DNA is packaged in vitro and then introduced into a grown host. In vitro packaging can be easily performed by using a commercially available in vitro packaging kit (for example, Stratagene, Amersham, etc.).

  The method for isolating the cDNA encoding the polypeptide of the present invention from the cDNA library prepared by the above method can be performed by combining general cDNA screening methods.

  For example, after separately synthesizing an oligonucleotide which is considered to correspond to the amino acid sequence of the polypeptide of the present invention, labeling it with 32 P to form a probe, a known colony hybridization method (Crunstein et al., Proc. Natl. Acid. Sci. USA, Vol. 72, pp. 3961, 1975) or plaque hybridization (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory). Pp. 2.108 (1989)), a method for screening a clone containing the desired cDNA, a PCR primer was prepared, a specific region of the polypeptide of the present invention was amplified by the PCR method, and a DNA fragment encoding the region was amplified. Methods for selecting clones having .

  When a cDNA library prepared using a vector capable of expressing cDNA (for example, λZAPII phage vector) is used, an antigen-antibody reaction using an antibody reactive with the polypeptide of the present invention may be used. The desired clone can be selected. When a large number of clones are processed, it is preferable to use a screening method using a PCR method.

  The nucleotide sequence of the DNA thus obtained is determined by the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA. 74: 560, 1977) or phage M13. The method of terminating the dideoxynucleotide synthesis chain (Sanger et al., Proc. Natl. Acad. Sci. USA., 74, 5463-5467, 1977). The gene encoding the polypeptide of the present invention can be obtained by cutting out all or a part of the clone obtained as described above using a restriction enzyme or the like.

  (2) As a preparation method by isolating DNA encoding the polypeptide of the present invention from genomic DNA derived from cells expressing the polypeptide of the present invention as described above, for example, the following method is exemplified. Is done.

  The cells are preferably lysed using SDS or proteinase K and the like, and DNA extraction is performed by repeating extraction with phenol. The RNA is digested, preferably by ribonuclease. The obtained DNA is partially digested with an appropriate restriction enzyme, and the obtained DNA fragment is amplified with an appropriate phage or cosmid to prepare a library. Then, a clone having the desired sequence is detected by, for example, a method using a radiolabeled DNA probe, and the whole or a part of the gene encoding the polypeptide of the present invention is cut out from the clone with a restriction enzyme or the like and obtained.

  When obtaining cDNA encoding a human-derived polypeptide, a cosmid dry slurry into which human genomic DNA (chromosomal DNA) has been introduced is further prepared (“Lab Manual Human Genome Mapping”, Maruzen Publishing) and the cosmid library is prepared. By screening, a positive clone containing the DNA of the coding region of the target protein can be obtained, and the cDNA library can be prepared by screening the aforementioned cDNA library using the coding DNA cut out from the positive clone as a probe.

  (3) The production of the DNA of the present invention by chemical synthesis can be carried out according to a conventional method based on the nucleotide sequence represented by SEQ ID NO: 1, 3, 4, 5, or 6.

  Further, the present invention relates to a recombinant vector containing a DNA encoding the above-mentioned cell surface molecule (polypeptide) of the present invention. The recombinant vector of the present invention is not particularly limited as long as it can replicate and maintain or self-proliferate in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and phage vectors.

  The recombinant vector can be simply prepared by ligating a DNA encoding the polypeptide of the present invention to a vector for recombination (plasmid DNA and bacterial phage DNA) available in the art in a conventional manner. . Specific examples of the recombination vector used include, for example, pBR322, pBR325, pUC12, pUC13, and pUC19 as plasmids derived from Escherichia coli, pSH19 and pSH15 as plasmids derived from yeast, and pUB110, pTP5, and pC194 as plasmids derived from Bacillus subtilis. And the like. Examples of the phage include bacteriophage such as λ phage, and animal and insect viruses (pVL1393, manufactured by Invitrogen) such as retrovirus, vaccinia virus, and nucleopolyhedrovirus.

  For the purpose of expressing the DNA encoding the polypeptide of the present invention to produce the polypeptide of the present invention, an expression vector is useful. The expression vector is not particularly limited as long as it has a function of expressing the gene encoding the polypeptide of the present invention in various prokaryotic and / or eukaryotic host cells and producing these proteins. For example, pEFneo (Proc. Natl. Acad. Sci. USA, 91, 158-162, 1994), pEF-BOS (Nucleic Acid Research, 18, 5322, 1990), pME18S (Experimental Medicine Supplement “Genetic Engineering Handbook”, 1992 Year etc.) or pMAL C2.

  When a bacterium, particularly Escherichia coli, is used as a host cell, the expression vector generally comprises at least a promoter / operator region, an initiation codon, DNA encoding the polypeptide of the present invention, a stop codon, a terminator region, and a replicable unit.

  When yeast, animal cells or insect cells are used as a host, the expression vector preferably contains at least a promoter, an initiation codon, a DNA encoding the polypeptide of the present invention, and a stop codon. DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5 ′ and 3 ′ sides of the gene encoding the polypeptide of the present invention, splicing junctions, polyadenylation sites, selectable marker regions or replicable units, etc. May be included. Further, it may contain a gene amplification gene (marker) usually used depending on the purpose.

  Promoter / operator regions for expressing the polypeptides of the present invention in bacteria include promoters, operators and Shine-Dalgarno (SD) sequences (eg, AAGG, etc.). For example, when the host is a bacterium belonging to the genus Escherichia, those containing a Trp promoter, a lac promoter, a recA promoter, a λPL promoter, an lpp promoter, a tac promoter and the like are preferably exemplified. Examples of a promoter for expressing the polypeptide of the present invention in yeast include a PH05 promoter, a PGK promoter, a GAP promoter, and an ADH promoter. When the host is a bacterium belonging to the genus Bacillus, the SL01 promoter, the SP02 promoter, the penP promoter, and the like. Is mentioned. When the host is a eukaryotic cell such as a mammalian cell, examples thereof include an SV40-derived promoter, a retrovirus promoter, a heat shock promoter, and an EF promoter. Preferably, they are SV-40, SRα, and retrovirus. However, it is not particularly limited to these. Use of an enhancer is also an effective method for expression.

  A suitable initiation codon is, for example, a methionine codon (ATG).

  Examples of the stop codon include common stop codons (eg, TAG, TGA, TAA, etc.).

  As the terminator region, a commonly used natural or synthetic terminator can be used.

  A replicable unit refers to DNA capable of replicating its entire DNA sequence in a host cell, including native plasmids, artificially modified plasmids (DNA fragments prepared from native plasmids) and Includes synthetic plasmids and the like. Suitable plasmids include plasmid pBR322 in E. coli or an artificially modified product thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme), yeast in the case of yeast 2μ plasmid, or yeast chromosomal DNA; Examples of mammalian cells include plasmids pEFneo, pME18S, pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmid pdBPV-MMTneo ATCC 37224, and plasmid pSV2neo ATCC 37149.

  As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, such as those derived from SV40, for example, can be used.

  As the selection marker, a commonly used marker can be used by a conventional method. For example, an antibiotic resistance gene such as tetracycline, neomycin, ampicillin, or kanamycin or a thymidine kinase gene is exemplified.

  Examples of gene amplification genes include dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, and aspartate transcarbamylase gene. And the like.

  The expression vector of the present invention is obtained by linking at least the above-mentioned promoter, initiation codon, DNA (gene) encoding the polypeptide of the present invention, termination codon and terminator region to an appropriate replicable unit in a continuous and circular manner. Can be prepared. At this time, if desired, an appropriate DNA fragment (for example, a linker, another restriction site, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

  The transformed cell of the present invention can be prepared by introducing the above-described expression vector into a host cell.

  The host cell used in the present invention is not particularly limited as long as it is compatible with the above-mentioned expression vector and can be transformed, and natural cells or artificially established cells usually used in the technical field of the present invention are used. Various cells such as recombinant cells (for example, bacteria (genus Escherichia, genus Bacillus), yeasts (genus Saccharomyces, genus Pichia), animal cells or insect cells) are exemplified.

  Preferred are Escherichia coli or animal cells, specifically Escherichia coli (DH5α, XL1Blue MRF ′, TB1, HB101, etc.), mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1, or NIH3 T3, etc.), Rat-derived cells, hamster-derived cells (such as BHK, CHO-K1 and CHO), monkey-derived cells (such as COS1, COS3, COS7, CV1 and Velo) and human-derived cells (HEK293, Hela, diploid fibroblasts) , Myeloma cells, Namalwa, etc.).

  Introduction (transformation (transfection)) of an expression vector into a host cell can be performed using a conventionally known method.

  For example, in the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA., Vol. 69, pp. 2110, 1972), the protoplast method (Mol. Gen. Genet., 168, 111, 1979) or the competent method (Journal of Molecular Biology (J. Mol. Biol.), 56, 209, 1971). In the case of cerevisiae, for example, the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA., Vol. 75, p. 1927, 1978) and the lithium method (J. Bacteriol., Vol. 153, Vol. 163, 1983), in the case of animal cells, for example, the method of Graham (Virology, Vol. 52, p. 456, 1973), and in the case of insect cells, for example, Summers. (Mol. Cell. Biol., No. 3) Vol., 2156 to 2165, 1983).

  The polypeptide of the present invention can be produced by culturing a transformed cell containing the expression vector prepared as described above (hereinafter referred to as including a transfectant) in a nutrient medium.

  The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, and examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, and so on. Examples include soybean meal and potato extract. If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be contained. .

  The culturing is performed by a method known in the art. Culture conditions such as temperature, medium pH and culture time are appropriately selected so that the polypeptide of the present invention is produced in large quantities.

  Specific examples of the culture medium and culture conditions used according to the host cell are shown below, but the invention is not limited thereto.

  When the host is a bacterium, an actinomycete, a yeast or a filamentous fungus, for example, a liquid medium containing the above-mentioned nutrients is suitable. Preferably, the medium has a pH of 5 to 8.

  When the host is E. coli, preferred mediums include LB medium and M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, page 431, 1972). In such a case, the cultivation can be carried out usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and stirring as necessary.

  When the host is a bacterium belonging to the genus Bacillus, the reaction can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary.

  When the host is yeast, the medium includes, for example, Burkholder minimum culture (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980), and the pH is 5-8. It is desirable that The cultivation is usually performed at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and stirring can be performed.

  When the host is an animal cell, for example, a MEM medium containing about 5 to 20% of fetal bovine serum (Science, Vol. 122, p. 501, 1952), a DMEM medium (Virology, 8, 396, 1959), RPMI 1640 medium (J. Am. Med. Assoc., 199, 519, 1967), 199 medium (proc. Soc. Exp. Biol. Med. 73, page 1, 1950) and the like. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.

  When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 82, 8404, 1985) and the like can be mentioned. Preferably it is. The cultivation is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring may be performed.

  The polypeptide of the present invention enables high expression of a target molecule on the cell surface by culturing the above-mentioned transformed cells, particularly animal cells.

  On the other hand, when the polypeptide of the present invention is produced as a soluble polypeptide fragment such as an extracellular region fragment, a transformant is prepared as described above using the DNA encoding the extracellular region or each domain. Then, it can be produced by culturing the exogenous transformant to secrete it into the culture supernatant. Further, the fusion polypeptide of the present invention can be produced in the same manner.

  That is, the culture obtained is filtered or centrifuged to obtain a culture filtrate (supernatant), and the present invention is subjected to a conventional method generally used for purifying and isolating a natural or synthetic protein from the culture filtrate. Is purified and isolated.

  Isolation and purification methods include, for example, methods using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, and methods using molecular weight differences such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods using charge such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using difference in hydrophobicity such as reverse phase high performance liquid chromatography, isoelectric A method utilizing the difference between isoelectric points, such as point electrophoresis, may be used.

  On the other hand, when the polypeptide or polypeptide fragment of the present invention is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to a conventional method such as filtration or centrifugation to collect cells or cells. After suspending in an appropriate buffer, disrupting the cell walls and / or cell membranes of cells and the like by, for example, sonication, lysozyme, and freeze-thawing, the polypeptide of the present invention is contained by a method such as centrifugation or filtration. Obtain a membrane fraction. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. Then, the crude solution can be isolated and purified by using a conventional method as exemplified above.

  The “transgenic mouse” in the present invention is a DNA (cDNA or genomic DNA) encoding a polypeptide (non-self polypeptide) of an animal species other than the mouse of the present invention, which can be prepared according to the method described above. A transgenic mouse integrated into a sex locus, wherein the transgenic mouse expresses and secretes the non-self polypeptide in the body.

  The transgenic mouse can be prepared by a conventional method (for example, the latest manual for animal cell experiments, published by LCI, Chapter 7, pp. 361 to 408, 1990) as usually used in the production of transgenic animals. Reference).

  Specifically, for example, embryonic stem cells (Embryonic Stem Cells, ES Cells) obtained from normal mouse blastcysts can be used, for example, with the polypeptide of the present invention derived from human (ie, “human JTT-1 antigen”). ") Is transformed with an expression vector into which a gene encoding the gene has been inserted. ES cells in which the gene encoding the human polypeptide of the present invention has been integrated on an endogenous gene are selected by a conventional method. Next, the selected ES cells are microinjected into fertilized eggs (pulmonary blastocysts) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7384). U.S. Pat. No. 4,873,191). The blastocyst is implanted into the uterus of another normal mouse as a foster mother. Then, a founder mouse (child mouse) is born from the foster parent mouse. A heterotransgenic mouse is obtained by mating the founder mouse with a normal mouse. By crossing the heterogeneic transgenic mice, a homogenic transgenic mouse is obtained according to Mendel's law.

  The “knockout mouse” of the present invention is a mouse in which an endogenous gene encoding a polypeptide of the present invention derived from a mouse (ie, “mouse JTT-1 antigen”) has been knocked out (inactivated). It can be prepared using the applied positive negative selection method (US Pat. Nos. 5,464,764, 5,487,992, 5,627,059, Proc. Natl. Acad. Sci. USA, Vol. 86, 8932-8935, 1989, Nature, Vol. 342, 435-438, 1989).

  The "antibody" in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, and is preferably a monoclonal antibody.

  Specifically, it is an antibody reactive with the aforementioned polypeptide or polypeptide fragment of the present invention.

  The “antibody” of the present invention may be a cell (natural cell, cell line, tumor cell, etc.) expressing the “cell surface molecule” of the present invention, the polypeptide of the present invention produced using a gene recombination technique, or the like. Using a transformant in which a cell surface molecule is highly expressed on the cell surface, or a "polypeptide fragment" or "fusion polypeptide" of the present invention as an antigen, the antigen is used as a mammal such as a mouse, rat, hamster, guinea pig or rabbit. Natural antibodies obtained by immunizing animals, chimeric antibodies and human antibodies (CDR-grafted antibodies) that can be produced using gene recombination techniques, and transgenic animals that produce human antibodies can be used. Also encompasses human antibodies.

  In the case of a monoclonal antibody, a monoclonal antibody having any isotype such as IgG, IgM, IgA, IgD, or IgE is also included. Preferably, it is IgG or IgM.

  The polyclonal antibody (antiserum) or monoclonal antibody referred to in the present invention can be produced by an existing general production method. That is, for example, a mammal, preferably a mouse, a rat, a hamster, a guinea pig, a rabbit, a cat, a dog, a pig, a goat, a horse, or a cow, together with an antigen as described above, if necessary, together with Freund's Adjuvant. And more preferably immunize a mouse, rat, hamster, guinea pig or rabbit.

  Polyclonal antibodies can be obtained from serum obtained from the immunized animal. In addition, a monoclonal antibody is prepared by preparing a hybridoma from the antibody-producing cells obtained from the immunized animal and a myeloma cell line (myeloma cell) having no autoantibody-producing ability, cloning the hybridoma, and immunizing a mammal. It is produced by selecting a clone producing a monoclonal antibody showing specific affinity for the antigen.

  A monoclonal antibody can be specifically produced as follows. That is, a non-human mammal, specifically, a mouse, rat, hamster, guinea pig or rabbit is used as an immunogen with the antigen as described above, together with Freund's adjuvant, if necessary. Preferably subcutaneously, intramuscularly, intravenously, in mice, rats or hamsters (including transgenic animals created to produce antibodies from other animals such as human antibody-producing transgenic mice described below); Immunization is performed by one or several injections or transplantation into the footpad or intraperitoneal cavity. Usually, immunization is performed 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization. The number of immunizations and the time interval can be appropriately changed depending on the nature of the immunogen used. Hybridomas secreting monoclonal antibodies can be prepared according to the method of Koehler and Milstein et al. (Nature, 256, 495-497, 1975) and modifications according thereto. That is, the antibody-producing cells contained in the spleen, lymph node, bone marrow, tonsil, etc., and preferably in the spleen obtained from the non-human mammal immunized as described above, and preferably mouse, rat, guinea pig, hamster, rabbit Alternatively, it is prepared by cell fusion with a myeloma cell having no autoantibody-producing ability derived from a mammal such as a human, more preferably a mouse, a rat or a human.

  Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1 ), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used.

  Screening of a hybridoma clone producing a monoclonal antibody is performed by culturing the hybridoma, for example, in a microtiter plate, and measuring the reactivity of the culture supernatant of the well in which proliferation was observed with the immunizing antigen used in the above-described immunization. The measurement can be performed by an enzyme immunoassay such as RIA or ELISA.

  Production of the monoclonal antibody from the hybridoma is carried out in vitro on the hybridoma or in vivo in a mouse, rat, guinea pig, hamster or rabbit or the like, preferably in a mouse or rat, more preferably in ascites of a mouse, and the obtained culture supernatant. Alternatively, it can be performed by isolating from ascites of a mammal.

  When culturing in vitro, the hybridoma is grown, maintained, and preserved in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culture method, to produce a monoclonal antibody in the culture supernatant. It can be carried out using a known nutrient medium or any nutrient medium derived and prepared from a known basal medium.

  As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium, etc. The basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

  For isolation and purification of the monoclonal antibody, the above-mentioned culture supernatant or ascites is obtained by using a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or It can be performed by subjecting it to affinity column chromatography such as a protein A column.

The monoclonal antibody of the present invention preferably includes the following monoclonal antibodies.
(1) A monoclonal antibody having reactivity with a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, a polypeptide fragment derived from the polypeptide, or a human-derived cell surface molecule composed of the polypeptide.
(2) A monoclonal antibody reactive with the polypeptide of the present invention, a polypeptide fragment derived from the polypeptide or a cell surface molecule constituted by the polypeptide, and stimulated with the mitogen of the monoclonal antibody The monoclonal antibody produced by the hybridoma identified by International Accession No.FERM BP-5707 has substantially the same effect on mitogen-stimulated rat lymphoblast cells. .
(3) A monoclonal antibody reactive with the polypeptide of the present invention, a polypeptide fragment derived from the polypeptide or a cell surface molecule constituted by the polypeptide, and stimulated with the mitogen of the monoclonal antibody The monoclonal antibody produced by the hybridoma identified by International Accession No.FERM BP-5708 has substantially the same effect on mitogen-stimulated rat lymphoblast cells. .

  Further, the monoclonal antibody of the present invention also includes a monoclonal antibody produced by a hybridoma identified by International Accession No. FERM BP-5707 and FERM BP-5708.

  The “chimeric monoclonal antibody” in the present invention is a monoclonal antibody produced by genetic engineering, and specifically, the variable region thereof is derived from a non-human mammal (mouse, rat, hamster, etc.) immunoglobulin. A chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody, which is a variable region and whose constant region is derived from human immunoglobulin.

  The constant region derived from human immunoglobulin has an amino acid sequence unique to each of isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE. The region may be a human immunoglobulin constant region belonging to any isotype. Preferably, it is a human IgG constant region.

  The chimeric monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that the present invention is not limited to such a manufacturing method.

  For example, a mouse / human chimeric monoclonal antibody can be prepared with reference to Experimental Medicine (Extra Issue), Vol. 1.6, No. 10, 1988, and Japanese Patent Publication No. 3-73280. That is, a human immunogen is located downstream of an active VH gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from DNA encoding a mouse monoclonal antibody isolated from a hybridoma producing the mouse monoclonal antibody. The CH gene (C gene encoding the H chain constant region) obtained from the DNA encoding glomlulin and the active VL gene (L chain variable region) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma. Downstream of the rearranged VJ gene that encodes the C gene (the C gene that encodes the constant region of the L chain) obtained from the DNA that encodes human immunoglobulin, Inserted into separate expression vectors, and transformed host cells with the expression vectors. It can be produced by culturing the conversion cells.

  Specifically, first, after extracting DNA from a mouse monoclonal antibody-producing hybridoma by a conventional method, the DNA is digested with an appropriate restriction enzyme (for example, EcoRI, HindIII, etc.) and subjected to electrophoresis (for example, Use 7% agarose gel) Perform Southern blotting. The electrophoresed gel is stained with, for example, ethidium bromide or the like, and after photographing, a marker is attached, the gel is washed twice with water, and immersed in a 0.25 M HCl solution for 15 minutes. It is then immersed in a 0.4N NaOH solution for 10 minutes while gently shaking. After being transferred to a filter according to a conventional method, the filter is collected after 4 hours and washed twice with 2 × SSC. After sufficiently drying the filter, baking (75 ° C., 3 hours) is performed. After baking, the filter is placed in a 0.1 × SSC / 0.1% SDS solution and treated at 65 ° C. for 30 minutes. Then, immerse in 3 × SSC / 0.1% SDS solution. The obtained filter is put in a plastic bag together with the prehybridization solution, and treated at 65 ° C. for 3 to 4 hours.

  Next, the 32 P-labeled probe DNA and the hybridization solution are put therein, and reacted at 65 ° C. for about 12 hours. After hybridization, the filter is washed under an appropriate salt concentration, reaction temperature and time (for example, 2 × SSC-0.1% SDS solution, room temperature, 10 minutes). The filter is placed in a plastic bag, a small amount of 2 × SSC is added, sealed, and autoradiography is performed.

  Rearranged VDJ and VJ genes encoding the H and L chains of the mouse monoclonal antibody, respectively, are identified by the Southern blot method described above. The region containing the identified DNA fragment is fractionated by sucrose density gradient centrifugation, incorporated into a phage vector (eg, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.), and Escherichia coli (eg, LE392, NM539, etc.) ) To create a genomic library. Using the genomic library with an appropriate probe (H chain J gene, L chain (κ) J gene, etc.), for example, the Benton Davis method (Science, vol. 196, pp. 180-182, 1977) ), Plaque hybridization is performed to obtain rearranged VDJ genes or positive clones each containing the VJ gene. A restriction enzyme map of the obtained clone is prepared, its nucleotide sequence is determined, and it is confirmed that the desired rearranged gene containing the VH (VDJ) gene or the VL (VJ) gene has been obtained.

  On the other hand, a human CH gene and a human CL gene used for chimerization are separately isolated. For example, when producing a chimeric antibody with human IgG1, the Cγ1 gene as the CH gene and the Cκ gene as the CL gene are isolated. These genes use the mouse Cγ1 gene and mouse Cκ gene corresponding to the human Cγ1 gene and human Cκ gene as probes by utilizing the high homology of the nucleotide sequences of the mouse immunoglobulin gene and the human immunoglobulin gene. It can be obtained by isolation.

  Specifically, for example, a 3 kb HindIII-BamHI fragment from clone Ig146 (Procedures National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 75, 4709-4713, 1978). Using a 6.8 kb EcoRI fragment from clone MEP10 (Procedures National Academy of Sciences (Proc. Natl. Acad. Sci. USA), Vol. 78, 474-478, 1981) as a probe, From the HaeIII-AluI genomic library of Lambda Charon 4A (Cell, vol. 15, pp. 1157-1174, 1978) containing a human Cκ gene and retaining an enhancer region. Isolate. The human Cγ1 gene is isolated, for example, by cutting human fetal hepatocyte DNA with HindIII, fractionating by agarose gel electrophoresis, inserting a 5.9 kb band into λ788, and using the aforementioned probe.

  Using the mouse VH gene and mouse VL gene, and the human CH gene and human CL gene thus isolated, the human CH gene is placed downstream of the mouse VH gene, taking into account the promoter region and the enhancer region. The human CL gene is inserted downstream of the mouse VL gene into an expression vector such as pSV2gpt or pSV2neo using an appropriate restriction enzyme and DNA ligase according to a conventional method. At this time, the chimeric gene of the mouse VH gene / human CH gene and the mouse VL gene / human CL gene may be simultaneously arranged in one expression vector, or may be arranged in separate expression vectors.

  The thus prepared chimeric gene insertion / expression vector is used for protoplast fusion, DEAE-dextran, calcium phosphate or electroporation in myeloma cells which do not produce antibodies themselves, for example, P3X63 / Ag8 / 653 cells or SP210 cells. Introduced by the perforation method. The transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain a desired chimeric monoclonal antibody-producing cell.

  The desired chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.

  The “human-type monoclonal antibody (CDR-grafted antibody)” in the present invention is a monoclonal antibody produced by genetic engineering, and specifically, a part or all of the complementarity determining region of the hypervariable region. A non-human mammal (mouse, rat, hamster, etc.) is a complementarity determining region of a hypervariable region derived from a monoclonal antibody, the framework region of the variable region is a human immunoglobulin-derived variable region framework region, and A humanized monoclonal antibody, wherein the constant region is a human immunoglobulin-derived constant region.

  Here, the complementarity-determining regions of the hypervariable region are three regions (Complementarity-determining residue; CDR1 and CDR2) which are present in the hypervariable region in the variable region of the antibody and which directly bind complementarily to the antigen. , CDR3), and the framework region of the variable region refers to four relatively conserved regions (Framework; FR1, FR2, FR3, FR4) intervening before and after the three complementarity determining regions.

  In other words, it means a monoclonal antibody in which all but a part or all of the complementarity determining region of the hypervariable region of a non-human mammal-derived monoclonal antibody has replaced the corresponding region of human immunoglobulin.

  The constant region derived from human immunoglobulin has an amino acid sequence unique to each isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. May be a human immunoglobulin constant region belonging to any isotype. Preferably, it is a human IgG constant region. Further, the framework region of the variable region derived from human immunoglobulin is not limited.

  The humanized monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that the present invention is not limited to such a manufacturing method.

  For example, a recombinant humanized monoclonal antibody derived from a mouse monoclonal antibody can be prepared by genetic engineering with reference to Japanese Patent Application Laid-Open No. 4-506458 and Japanese Patent Application Laid-Open No. 62-296890. That is, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated from a hybridoma producing a mouse monoclonal antibody, and the mouse H chain CDR gene is isolated from a human immunoglobulin gene. A human H chain gene encoding the entire region other than the human H chain CDR corresponding to the chain CDR and a human L chain gene encoding the entire region other than the human L chain CDR corresponding to the pre-mouse L chain CDR are isolated.

  The isolated mouse H chain CDR gene and the human H chain gene are introduced into an appropriate expression vector so that they can be expressed, and similarly, the mouse H chain CDR gene and the human L chain gene can be appropriately expressed so that they can be expressed. Into another expression vector. Alternatively, the mouse H chain CDR gene / human H chain gene and the mouse L chain CDR gene / human L chain gene can be introduced so that they can be expressed in the same expression vector. By transforming a host cell with the expression vector thus prepared, a human monoclonal antibody-producing transformed cell is obtained, and by culturing the transformed cell, a target human monoclonal antibody is obtained from the culture supernatant. obtain.

  The “human monoclonal antibody” in the present invention means that all regions including the variable region of the H chain and the constant region of the H chain, and the variable region of the L chain and the constant region of the L chain that constitute immunoglobulin encode human immunoglobulin. Immunoglobulin derived from the gene expressed.

  Human antibodies can be prepared by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into a locus of a non-human mammal such as a mouse, using an antigen, according to a conventional method. The polyclonal antibody or the monoclonal antibody can be produced in the same manner as described above.

  For example, transgenic mice producing human antibodies are described in Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; JP-T-Hei 7-509137; Nikkei Science, June, pp. 40-50, 1995; International Application Publication WO 94/25585; Nature, Vol. 368, p. 856-859, 1994; And a method described in JP-A-6-500233 or the like.

  It is also possible to apply a method for producing a human-derived protein from transgenic bovine or porcine milk, a technique recently developed (Nikkei Science, April 1997, pp. 78-84). .

  The “part of the antibody” in the present invention means a partial region of the monoclonal antibody as described above, and specifically, F (ab ′) 2, Fab ′, Fab, Fv (variable fragment of antibody), Means sFv, dsFv (disulphide stabilized Fv) or dAb (single domain antibody) (Expert Opin. Ther. Patents, Vol. 6, No. 5, No. 441-456, 1996).

  Here, "F (ab ') 2" and "Fab'" are produced by treating an immunoglobulin (monoclonal antibody) with pepsin or papain, which is a protease, and two fragments in the hinge region. Means an antibody fragment produced by digestion before and after a disulfide bond existing between H chains of the above. For example, when IgG is treated with papain, an L chain consisting of VL (light chain variable region) and CL (light chain constant region) is cleaved upstream of a disulfide bond existing between two heavy chains in the hinge region; And two homologous antibody fragments in which an H chain fragment consisting of VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region) are linked by a disulfide bond at a C-terminal region can be produced. Each of these two homologous antibody fragments is called Fab '. When IgG is treated with pepsin, it is cleaved downstream of the disulfide bond existing between the two heavy chains in the hinge region to produce an antibody fragment slightly larger than the two Fab's connected by the hinge region. Can be. This antibody fragment is called F (ab ') 2.

  The "pharmaceutical composition" of the present invention includes the "polypeptide" of the present invention as defined above, a "homodimer molecule" composed of the polypeptide, a "polypeptide fragment" or a "fusion polypeptide", and the fusion A pharmaceutical composition comprising any of a “homodimer molecule”, an “antibody” or a “part of an antibody” composed of a polypeptide, and a pharmaceutically acceptable carrier.

  Here, "pharmaceutically acceptable carrier" refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, coloring agents, sweeteners, thickeners. , A flavoring agent, a solubilizer or other additives. By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, liquids, capsules, troches, elixirs, suspensions, emulsions, syrups, etc. A composition can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions containing one or more active substances and formulated in a conventional manner, suppositories and pessaries for enteral administration.

  The dose varies depending on the age, sex, weight and condition of the patient, therapeutic effect, administration method, treatment time, or the type of active ingredient (such as the polypeptide or antibody) contained in the pharmaceutical composition. It can be administered in a dose of 10 μg to 1000 mg (or 10 μg to 500 mg) at a time per adult. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.

  In particular, in the case of an injection, the concentration is 0.1 μg antibody / ml carrier to 10 mg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. Can be produced by dissolving or suspending as described above. The injection thus produced is applied to a human patient in need of treatment at a rate of 1 μg to 100 mg per kg body weight, preferably 50 μg to 50 mg per day, in a single administration. It can be administered once to several times. Examples of the administration form include medically appropriate administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection. Preferably it is an intravenous injection.

  Injectables can also be prepared as non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions and emulsions. .

  Such injections can be sterilized by filtration sterilization through a bacteria retaining filter, blending of a bactericide or irradiation. Injectables can be manufactured in the form of preparation for use. That is, it can be used as a sterile solid composition by freeze-drying or the like, dissolved in sterile distilled water for injection or another solvent before use.

  INDUSTRIAL APPLICABILITY The pharmaceutical composition of the present invention is applied to the treatment and prevention of various autoimmune diseases, allergic diseases or inflammatory diseases caused by activation of lymphocytes such as T cells and abnormal control of the functions of activated lymphocytes. Is possible. Examples of the disease include rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, lichen planus, which is a chronic inflammatory skin disease, systemic lupus erythematosus, insulin-dependent diabetes mellitus, psoriasis and the like. Can be

  In addition, the therapeutic effect of the pharmaceutical composition of the present invention for various disease symptoms can be tested and examined by administering to a known disease model animal according to a conventional method.

  For example, (i) (NZB / NZW) F1 mouse (Science, Vol. 125, p. 1225-1227, 1994) which is a model of human systemic lupus erythematosus (SLE), and (ii) multiple sclerosis (MS) Experimental allergic encephalomyelitis (EAE) model (J. Clin. Invest., Vol. 95, p. 2783-2789, 1995), (iii) NOD (non-insulin dependent diabetes mellitus (IDDM) model) -obese diabetes) mice (J. Exp. Med., Vol. 181, p. 1145-1155, 1995), (iv) rat nephritis due to renal glomerular basement membrane immunity, a model of nephritis of Goodpasture Model (Eur. J. Immunol., Vol. 24, No. 6, p. 1249-1254, 1994), (v) DBA / 1 mouse, a human rheumatoid arthritis model (Eur. J. Immunol., Vol. 26, p. 2320-2328, 1996).

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of Monoclonal Antibody The preparation of the antibody-producing hybridoma described below was performed with reference to the method of Kohler et al. (Blood, Vol. 81, pp. 101-111, 1993, Omori et al.). The preparation of the monoclonal antibody was carried out with reference to the method of Kanagi et al. (Handbook of Experimental Immunology, Vol. 4, pages 117.21-117.21, 1986).

  First, rat thymoma cell line FTL435 cells were used as immunizing antigens, and the antigens were administered to BALB / c mice at intervals and in amounts of day 0 (107 cells / animal), days 7, 14, and 28. The foot pad was administered. Only the first immunization was performed by mixing the antigen with Freund's complete adjuvant. Two days after the last immunization, lymph nodes of the mouse were collected, and mouse myeloma cell PAI (JCR No. B0113; Res. Disclosure, Vol. 217, p. 155, 1982, Stocker, JWet) was obtained by a conventional method. al.), a number of monoclonal antibody-producing hybridomas were obtained.

[Example 2] Screening of hybridomas and analysis of properties of monoclonal antibodies By analyzing the effects of the monoclonal antibodies produced in the culture supernatants of the various hybridomas prepared in Example 1 on FTL435 cells, which are immunogens, Hybridomas were screened.

  FTL435 cells (0.1 ml of 5 × 10 6 cells / ml) were inoculated into each well of a 96-well microtiter plate, and the culture supernatant of each hybridoma (10 μg / ml each) was added, followed by culturing at 37 ° C. for 1 hour. . The results for the hybridoma clone “JTT-1” and the clone “JTT-2” are shown in FIG. 1 and FIG.

  It was confirmed that the monoclonal antibody ("JTT-1 antibody") produced by the hybridoma clone "JTT-1" had an action of strongly aggregating FTL435 cells (FIGS. 1 (b) and 2 (c)). . On the other hand, it was confirmed that the addition of the “JTT-2 antibody” together with the “JTT-1 antibody” strongly suppressed the aggregation of FTL435 cells due to the “JTT.1 antibody” stimulation (FIG. 1 (d)). In addition, a system to which neither hybridoma supernatant was added was used as a control (FIGS. 1 (a) and 2 (a)).

  Aggregation of FTL435 cells by this "JTT-1 antibody" stimulation is caused by a typical known adhesion molecule pathway between ICAM-1 (Intercellular adhesion molecule-1) and LFA-1 (Lymphocyte function-associated antigen-1). In order to confirm whether or not this was due to cell adhesion, anti-rat ICAM-1 antibody 1A29 (10 μg / ml; IgG1) or anti-rat LFA-1 antibody (10 μg / ml; IgG2a) was used together with “JTT-1 antibody”. In addition, the cells were cultured at 37 ° C. for 1 hour.

  Aggregation of FTL435 cells stimulated by “JTT-1 antibody” was not suppressed by either the anti-ICAM-1 antibody or the anti-LFA-1 antibody ([anti-ICAM-1 antibody]: FIG. 1 (c) and FIG. 2). (f) and [Anti-LFA-1 antibody]: FIG. 2 (d)).

  In order to further analyze the cell aggregation ability of the "JTT-1 antibody", the aggregation ability of rat activated lymphoblast cells activated by stimulation with concanavalin A was analyzed in the same manner as described above. FIG. 2 shows the results.

  Similar to the action on FTL435 cells, stimulation of the "JTT-1 antibody" induced aggregation of activated lymphoblast cells (FIG. 2 (i)). However, for activated lymphoblast cells, most of the cell aggregation induced by the "JTT-1 antibody" stimulation was caused by the anti-LFA-1 antibody (FIG. 2 (j)) and the anti-ICAM-1 antibody (FIG. 2 (l )) (Although partial aggregation remained).

  Activated lymphocytes such as activated lymphoblasts have a function to activate PMA (Phorbol myristate acetate: LFA-1), as is clear from the control system without any antibody (Fig. 2 (g)). (Fig. 2 (h)) and no aggregation by cell adhesion occurs unless stimulated by the "JTT-1 antibody" (Fig. 2 (i)). Therefore, the fact that the cell aggregation caused by the stimulation of “JTT-1 antibody” was partially suppressed by the anti-LFA-1 antibody is due to the fact that LFA-1 was stimulated by the stimulation of “JTT-1 antibody” in activated lymphoblast cells. It indicates that it is activated. This indicates that the molecule recognized by the “JTT-1 antibody” has a function related to transmission of some signal.

  The hybridoma clones “JTT-1” and “JTT-2” were registered on October 11, 1996 by the Ministry of International Trade and Industry of Japan, International deposit ([JTT-1]: International deposit number FERM BP-5707, [JTT-2]: International deposit number FERM BP-5708).

  Analysis using a mouse monoclonal antibody isotype identification kit (Amersham) determined that the isotypes of the monoclonal antibodies (JTT-1 antibody and JTT-2 antibody) produced from each hybridoma were both IgG1.

[Example 3] Reactivity of "JTT.1 antibody" and "JTT.2 antibody" against various cells Expression patterns of molecules recognized by "JTT.1 antibody" and "JTT.2 antibody" in various cells were determined. For the purpose of analysis, the reactivity of the antibody to various cells was confirmed. The molecule recognized by "JTT-1 antibody" is named "JTT-1 antigen", and the molecule recognized by "JTT-2 antibody" is named "JTT-2 antigen".

  Five to ten week old Wistar rats (150 to 250 g) were anesthetized and killed with diethyl ether. The abdomen was opened by surgery, and the thymus and spleen were respectively excised from the chest and abdomen, and ground to prepare a cell suspension. Further, activated lymphoblasts were prepared by culturing the spleen cells in RPMI1640 medium containing Concanavalin A (2 μg / ml) and 10% FCS at 37 ° C. for 3 days.

  FTL435 cells, thymocytes, spleen cells and activated lymphoblasts (5 × 105 each) were reacted with “JTT.1 antibody” or “JTT.2 antibody”, and then FITC-labeled anti-mouse IgG (Cappel) After the reaction, the fluorescence intensity of the stained cells was measured using an EPICS-Elite flow cytometer.

  The results are shown in FIG. In FTL435 cells, strong expression of "JTT.1 antigen" and "JTT.2 antigen" was observed. Thymocytes also expressed the same molecule, but spleen cells only slightly expressed it. However, activated lymphoblasts obtained by stimulating spleen cells with concanavalin A showed strong expression of “JTT.1 antigen” and “JTT.2 antigen”. In each cell type, the expression patterns of "JTT.1 antigen" and "JTT.2 antigen" were consistent. This result suggests that "JTT-1 antigen" and "JTT-2 antigen" are the same molecule.

[Example 4] Reactivity of "JTT.1 antibody" against various lymphoid cells Analysis of expression pattern of molecule ("JTT-1 antigen") recognized by "JTT.1 antibody" in various lymphoid cells For the purpose of analysis, the reactivity of "JTT-1 antibody" to lymph nodes, spleen-derived T lymphoblasts, and spleen-derived B lymphoblasts of two kinds of rats (Wistar rats and F344 rats) was analyzed.

  Wistar rats and F344 rats (150 to 250 g) aged 5 to 10 weeks were anesthetized and killed with diethyl ether. Each rat was laparotomized by surgery to remove lymph nodes and spleen, and ground to prepare a cell suspension. In addition, the spleen cell preparation was cultured at 37 ° C. for 3 days in RPMI1640 medium containing Concanavalin A (ConA; 2 μg / ml) and 10% FCS. Activated T lymphoblasts and activated B lymphoblasts were obtained for each rat after culturing for 1 day and 3 days. As controls, spleen-derived T lymphoblasts and B lymphoblasts obtained before adding lymph node cells and ConA (day 0) were used.

  Each of the cells (5 × 10 5 cells) was reacted with a biotin-labeled anti-rat T cell antibody or a biotin-labeled anti-rat B cell antibody (10 μg / ml, manufactured by Seikagaku Corporation), and then reacted with picoerythrin-labeled streptavidin. Was. Next, the cells were reacted with FITC-labeled “JTT-1 antibody” (10 μg / ml), and the fluorescence intensity of the stained cells was measured using an EPICS-Elite flow cytometer.

  FIG. 4 shows the results. In both Wistar rats and F344 rats, strong expression of “JTT.1 antigen” was observed in activated T lymphoblasts and activated B lymphoblasts from day 1 of activation by ConA stimulation. The expression pattern of "JTT.1 antigen" in each cell type was almost the same.

[Example 5] Analysis of properties of "JTT.1 antigen" and "JTT.2 antigen" by immunoprecipitation experiment To analyze the properties of "JTT.1 antigen" and "JTT.2 antigen", FTL435 cells were used. To perform immunoprecipitation experiments.
(1) Preparation of biotinylated soluble cell surface molecule
After washing the FTL435 cells with PBS, the cells were suspended in 0.1 M HEPES-containing physiological saline (pH 8.0) containing 100 μg / ml NHS-biotin at 1 × 10 7 cells / ml and reacted at room temperature for 40 minutes. . After washing the cells three times with PBS, a solubilization buffer (1% NP-40, 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl) was added to 5 × 10 7 cells / ml, and the cells were added at 4 ° C., 30 ° C. The cells were lysed by reacting for minutes. The obtained cell lysate was centrifuged, and the centrifuged supernatant containing the biotinylated soluble cell surface molecule was stored at -80 ° C.

(2) Immunoprecipitation and SDS-PAGE analysis The purified JTT.1 antibody purified from the culture supernatant of the hybridoma clone "JTT-1" prepared in Example 1 by a conventional method is 2 mg / ml. Was mixed with protein G-Sepharose beads as described above, and reacted at 4 ° C. for 1 hour to bind the antibody to the beads. The beads were washed, and 500 μl of a biotinylated FTL435 cell lysate was added to 10 μl of the beads, and reacted at 4 ° C. for 2 hours. The beads were washed three times with a solubilization buffer, and 50 μl of glycanase buffer (sodium phosphate buffer containing 0.15% SDS (pH 7.0)) was added thereto. The molecules were eluted. 1.25% NP-40 and N-glycanase (20 U / ml) were added to a part of the eluted sample and reacted overnight to digest the N-type sugar chain.

  Equivalent volume of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (manufactured by Enprotech) is added to 5 μl of the eluted sample in the presence or absence of 2-mercaptoethanol, and the mixture is boiled. Was transferred to a PVDF membrane. After blocking the transfer membrane with 3% BSA-PBS, and then reacting with peroxidase-labeled streptavidin, trap with "JTT-1 antibody" using the ECL system (manufactured by Amersham) according to the instruction manual. The detected biotinylated soluble cell membrane surface molecules were detected.

  FIG. 5 shows the results. The molecule on FTL435 cells ("JTT-1 antigen") recognized by "JTT.1 antibody" has a molecular weight of about 47 kD under non-reducing conditions (indicated by "(-)" in FIG. 5). Under reduction (indicated by "(+)" in FIG. 5), it had a molecular weight of about 24 kD and about 28 kD. In addition, by digesting the N-type sugar chain (indicated by "+ N-gly" in FIG. 5), "JTT.1 antigen" is about 36 kD under non-reducing and about 20 kD under reducing. It converged on a book band. From the above results, it was presumed that "JTT.1 antigen" had a different sugar chain modification, and a core protein formed a dimer consisting of the same molecule. In addition, in the test performed in the same manner as described above using the “JTT.2 antibody”, completely the same results were obtained. When this result is compared with the results of Example 3 and Example 7 described later, “JTT.1 antigen” (a molecule recognized by “JTT-1 antibody”) and “JTT.2 antigen” (“JTT-2 Molecules recognized by the "antibody") were considered to be identical molecules.

[Example 6] Adhesion experiment of rat thymocytes to purified "JTT.1 antigen" and N-terminal amino acid analysis A molecule recognized by "JTT.1 antibody"("JTT-1antigen") has a function as an adhesion molecule The following experiment was performed in order to analyze whether or not it was performed. In addition, N-terminal amino acid analysis was performed.

(1) Preparation of “JTT.1 antibody” affinity column 2 mg (2 ml) of “JTT.1 antibody” purified from the culture supernatant of hybridoma clone “JTT-1” prepared in Example 1 by an ordinary method Was mixed with 1 ml of protein G-sepharose resin and reacted at 4 ° C. for 1 hour. The resin was washed three times with 200 mM triethanolamine (pH 8.2). Further, the “JTT.1 antibody” was covalently bound to the resin by incubating for 1 hour at room temperature in triethanolamine (pH 8.2) containing 10 mM dimethylpimelimidate (DMP).

(2) Purification of "JTT.1 antigen"
FTL435 cells were cultured using RPMI1640 medium containing 10% FCS. The cells were collected by centrifugation, and the pellet was washed three times with PBS. To the pellet after washing, a solubilization buffer (1% NP-40, 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl) is added to a concentration of 5 × 107 cells / ml, and the mixture is reacted at 4 ° C. for 30 minutes to allow cells to react. Dissolved. The obtained cell lysate was centrifuged, and the centrifuged supernatant containing soluble cell surface molecules was stored at -80 ° C.
400 ml of the lysate was added to the “JTT.1 antibody” affinity column. After washing the column with 50 ml of solubilization buffer and 20 ml of PBS, "JTT-1 antigen" was eluted with 0.2 M glycine buffer (pH 2.8). The eluted "JTT-1 antigen" was neutralized by adding 1M Tris buffer. The obtained “JTT.1 antigen” was stored at −80 ° C.

(3) Determination of N-terminal amino acid sequence After developing the purified “JTT-1 antigen” by SDS-PAGE, the N-terminal amino acid sequence was determined by a conventional method, and Glu-Leu-Asn-Asp-Leu was determined. It was revealed that the protein contained the amino acid sequence of -Ala-Asn-His-Arg.

(4) Adhesion experiment
Five to ten week old Wistar rats (150 to 250 g) were anesthetized and killed with diethyl ether. The thymus was excised by laparotomy of the chest by surgical operation, and the thymus was ground to prepare a thymocyte suspension. 10 μM of 2 ′, 7′-bis (carboxyethyl) carboxyfluorine tetracetoxymethyl ester (BCECF-AM; manufactured by Molecular Probes) is added to the cell suspension and cultured at 37 ° C. for 30 minutes to obtain fluorescence. Labeling was performed. After washing the cells with PBS, they were resuspended in RPMI1640 medium containing 10% FCS to a concentration of 2 × 107 cells / ml.

The purified “JTT.1 antigen” obtained in (2) was coated on a 96-well ELISA plate at 10 μl / well overnight. After the plate was washed with PBS, 200 μl / well of PBS containing 3% BSA was added, and blocking was performed for 2 hours. After the plate was washed with PBS, each well was prepared using only (i) fluorescence-labeled thymocytes (0.1 ml of 2 × 10 7 cells / ml), (ii) fluorescence-labeled thymocytes (same concentration) and a conventional method. Fab fragment of the JTT.1 antibody (5 μg / ml), and (iii) fluorescence-labeled thymocytes (same concentration), Fab fragment of the “JTT.1 antibody” (same concentration) and “JTT.2 antibody” ( 10 μg / ml) and cultured at 37 ° C. for 1 hour. Each well was washed once with RPMI 1640 medium containing 10% FCS to remove unbound cells. Each well was observed with an optical microscope. Next, 100 μl of a 0.1% NP-40 solution was added to each well to lyse the cells bound to the plate. By measuring the fluorescence intensity at a wavelength of 538 nm (excitation at 485 nm) using a Fluoroscan II microplate fluorometer (manufactured by Flow Laboratories), the fluorescence-labeled thymocytes bound to each well were measured. The relative cell number was counted. A system not coated with purified “JTT-1 antigen” was used as a control.
FIG. 6 shows the results of optical microscope observation.
Thymocytes significantly adhered to the purified "JTT.1 antigen" only in the presence of the Fab fragment of "JTT.1 antibody" (FIG. 6 (c)). The adhesion was significantly inhibited by the “JTT.2 antibody” (FIG. 6 (d)).
FIG. 7 shows the results obtained by measuring the relative cell number of thymocytes adhered to “JTT.1 antigen” coated on each well based on the fluorescence intensity.
From the above results, it was confirmed that “JTT.1 antigen” has a function as an adhesion molecule.

[Example 7] Cloning of cDNA encoding rat "JTT.1 antigen" Preparation of cDNA library 1- (1) Extraction of poly (A) + RNA from ConA-stimulated rat lymphoblasts
The rat spleen-derived lymphoblasts (Con A blast) (about 1 × 10 6 cells / ml) stimulated with ConA were centrifuged at 4 ° C. for 5 minutes (2,000 × g), and the precipitated cells were subjected to ISOGEN (Nippon Gene). The resultant was suspended in chloroform and immersed in chloroform for extraction, and the supernatant was recovered. After isopropanol was added to the obtained supernatant and allowed to stand at room temperature for 10 minutes, the mixture was centrifuged at 12,000 × g at 4 ° C. for 10 minutes to precipitate RNA. The precipitated RNA was washed with ethanol and then dissolved in TE buffer. From the obtained total RNA, poly (A) + RNA was purified using “mRNA Purification Kit” (Pharmacia).
1- (2) Preparation of cDNA Using 5 μg of the prepared poly (A) + RNA as a template, cDNA was synthesized using “Time Saver cDNA Synthesis Kit” (Pharmacia). To increase the screening efficiency, "oligo dT primer" (Pharmacia) having a NotI cleavage site was used. Next, EcoRI was added to the adapter, and NotI digestion was performed to obtain a unidirectional cDNA. Furthermore, size fractionation was performed using a span column (Pharmacia).
1- (3) Incorporation into Vector The obtained cDNA having EcoRI and NotI ends was treated with EcoRI and NotI, and vector pME18S (Hara, T. and Miyajima, A. EMBOJ., 11, 1875-1884, 1992) ). A "DNA ligation Kit" (Takara Shuzo) was used for the ligation reaction. E. coli DH5 (manufactured by Toyobo) was transformed using the obtained reaction product. The transformant was cultured until the OD value (600 nm) reached 0.6, and then collected, and the plasmid DNA containing the library was recovered. For purification of plasmid DNA, QUIAGEN-Tip (manufactured by QUIAGEN) was used.

2. Screening of cDNA Library Screening was performed according to the panning method (Seed, B. et al. Proc. Natl. Acad. Sci USA, 84, 3365-3369, 1987).
2- (1) Gene transfer into COS cells The obtained library was transferred into COS7 cells by electroporation (Potter, H. et al. Proc. Natl. Acad. Sci. USA, 85, 2288-2292). Introduced. The cells were cultured for 60 hours after the introduction, the supernatant was removed, and the cells were washed three times with PBS. Then, the cells were treated with PBS (0.5 mM EDTA) (37 ° C., 30 minutes), and then the cells were detached by pipetting. Further, only live cells were recovered using "Lymphprep" (manufactured by NYCOMED).
2- (2) Enrichment of Gene-Expressing Cells by Panning The obtained living cells were suspended in PBS (5% FCS, 0.5 mM EDTA). The cell suspension was transferred to a culture dish coated with “JTT.1 antibody” and allowed to act at room temperature for 3 hours. After removing unbound cells from the culture dish and washing the culture dish three times with PBS, plasmid DNA was recovered from the cells bound to the culture dish by the Hirt method (Hirt, BJMol. Biol., 26.365-369). E. coli DH10B (GIEBCO BRL) was transformed with the obtained plasmid DNA. Using the transformant, plasmid DNA was amplified and purified in the same manner as in 1- (3) above. Using the obtained DNA, the operations described in the above (1) and this (2) were further repeated twice.
2- (3) Isolation of positive clone
After the third panning, the transformed E. coli DH10B was cultured overnight on an LB plate containing ampicillin to obtain colonies. Twenty drug-resistant colonies were cultured and plasmid DNA was recovered and inserted by alkaline miniprep method (Maniatis, T. et al. Molecular Cloning; A Larolatory Mnual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). DNA (insert DNA) was analyzed. As a result of agarose gel electrophoresis, it was revealed that a clone having a cDNA of about 0.9 kb (hereinafter, this clone is referred to as “T132A7”) was concentrated.
Using the method described in the above (1), "T132A7" was once again transiently expressed in COS7 cells. The cells transfected with "T132A7" were reacted with "JTT.1 antibody" or "JTT.2 antibody", and then reacted with FITC-labeled anti-mouse IgG (Cappel), and the fluorescence intensity of the stained cells was determined by EPICS. The measurement was performed using -Elite flow cytometer (manufactured by Coulter). “JTT.1 antibody” and “JTT.2 antibody” strongly recognized the “T132A7” gene product. FIG. 8 shows the results.

3. Determination of base sequence and amino acid sequence The base sequence of clone "T132A7" was determined by the dideoxy method using "Auto Read Sequencing Kit" (Pharmacia) and "ALF DNA sequencer" (Pharmacia). In addition, the deduced amino acid sequence of “rat JTT.1 antigen” encoded by the nucleotide sequence was analyzed using gene analysis software “GENEWORKS” (manufactured by IntelliGenetics). The nucleotide sequence and the deduced amino acid sequence are set forth in SEQ ID NO: 4.
The amino acid sequence (consisting of 200 amino acid residues) deduced from the cloned gene contained the same amino acid sequence as the N-terminal amino acid sequence determined in Example 6- (3). Considering that clone "T132A7" transfected cells strongly react with "JTT-1 antibody", it is concluded that clone "T132A7" contains cDNA encoding "rat JTT.1 antigen". it can.

4. Computer analysis The primary structure of the deduced amino acid sequence of the "JTT.1 antigen" was determined to be hydropathy according to the method of Kite and Doolittle (Kite, J. & Doolittle, RFJ Mol. Biol. 157, 105-132, 1982). -Blot analysis was performed (Fig. 9). As a result, it was revealed that "JTT.1 antigen" is a cell transmembrane protein having a signal sequence at the N-terminus. In addition, as a result of motif analysis, the extracellular domain of “JTT.1 antigen” has two asparagine-linked sugar chain binding sites, and the intracellular domain has two casein kinase phosphorylation sites and one It was found to have a protein kinase C phosphorylation site. In FIG. 9, “CHO” indicates an N-type sugar chain binding site, “P” indicates a phosphorylation site, “CKII” indicates casein kinase II, and “PKC” indicates protein kinase C.

[Example 8] Cloning of "human JTT.1 antigen" cDNA Preparation of probe The clone "T132A7" obtained in Example 7 was digested with restriction enzymes EcoRI and NotI, and about 0.9 kb of cDNA encoding "rat JTT.1 antigen" was cut out, followed by agarose gel electrophoresis. Separated. The separated DNA fragment was purified using a “QUIAEX gel extraction kit” (manufactured by QUIATEN), and the obtained DNA fragment was labeled with 32 P using a “Ready-To-Go DNA labeling kit” (manufactured by Pharmacia). . This labeled DNA fragment was used as a probe for plaque hybridization.

2. Preparation of cDNA library 2- (1) Extraction of poly (A) + RNA In the same manner as in Example 7- (1), from lymphocytes derived from human peripheral blood stimulated with ConA (Con A blast) poly (A) + RNA was extracted.
2- (2) Preparation of cDNA cDNA was synthesized using “oligo dT Primer” (Pharmacia) and “Time Saver cDNA Synthesis Kit” (Pharmacia) using 5 μg of the prepared poly (A) + RNA as a template. did. Next, after adding an EcoRI adapter, size fractionation was performed using a span column (Pharamacia).
2- (3) Incorporation into vector and packaging The obtained cDNA having an EcoRI end was ligated to a vector "λZAPII" (Stratagene) treated with EcoRI. A "DNA ligation Kit" (Takara Shuzo) was used for the ligation reaction. After in vitro packaging using "GIGA PACK II GOLD" (manufactured by Strategene), E. coli XL1Blue MRF '(manufactured by Strategene) is used using the obtained phage particles. A cDNA library consisting of a plaque containing the recombinant phage was prepared as a host.

3. Screening of cDNA library Screening was performed by a plaque hybridization method (Maniatis, T. et al. Molecular Cloning: A Labolatory Manual, Cold Spring Harbor Laboratory, using "Rapid hybridization buffer" (Amersham)). Cold Spring Harbor, New York). The obtained cDNA library (1 × 104) was spread on an agar plate, and a replica was prepared using “Hybond-N nylon menbrane” (manufactured by Amersham). Using the replica and the 32P-labeled probe prepared in Example 8-1, plaque hybridization was carried out in "Rapid hybridization buffer" (Amersham). Primary screening and secondary screening were performed to obtain eight positive clones. After each clone was isolated by a single plaque, it was subjected to in vivo excision according to the manual (Strategene), and seven positive clones were recovered as plasmid DNA.

4. Nucleotide sequence determination The nucleotide sequences of the seven clones were determined by the dideoxy method using an "Auto Read Sequencing Kit" (Pharmacia) and an "ALFDNA sequencer" (Pharmacia). All seven clones contained the same nucleotide sequence. Among them, it was confirmed that clone "pBSh41" encoded the full length of "human JTT-1 antigen". The nucleotide sequence of the cDNA corresponding to the open reading frame (ORF) of "human JTT-1 antigen" is shown in SEQ ID NO: 1, and the estimated full-length amino acid sequence of "human JTT.1 antigen" is shown in SEQ ID NOs: 2 and 5. The nucleotide sequence containing the 'and 3' sequences is shown in SEQ ID NO: 3 (ORF is nucleotides 26 to 625). The fact that the nucleotide sequence contained in the clone encodes the entire length of “human JTT-1 antigen” means that the amino acid sequence (consisting of 199 amino acid residues) deduced from the nucleotide sequence is “ This is evident from the fact that it shows significant homology with the amino acid sequence of "rat JTT.1 antigen" (FIG. 10). Further, as shown in FIG. 10, the amino acid sequence homology of human and rat “JTT.1 antigen” is 60% or more.
E.coli.DH10B (GIBCO BRL) transformed with the clone “pBSh41” was used as a depositary institution certified by the Ministry of International Trade and Industry, Ministry of International Trade and Industry, Japan under the Budapest Treaty on October 25, 1996. Deposited with the Institute of Engineering Technology (Deposit No .: FERM BP-5725).

5. Structural characteristics and biological function of "JTT-1 antigen" As a result of performing a motif search with a known human protein for the deduced amino acid sequence of "human JTT-1 antigen", "human JTT-1 antigen" It has been confirmed that it has structural similarity with human-derived cell membrane molecules “CD28” and “CTLA-4” belonging to the immunoglobulin superfamily described in detail above (FIG. 11 and FIG. 12). As described above, “CD28” and “CTLA-4” are extremely important molecules in the immune system that control T cell activation and suppression.
That is, (i) 20 or more amino acid residues including cysteine residues are well conserved. (ii): A continuous sequence of proline residues “Pro-Pro-Pro (PPP)” essential as a ligand binding region in CD28 and CTLA-4 is preserved. (Iii): In the intracellular domain, a sequence “Tyr-Xaa-Xaa-Met (YxxM) (Xaa and x mean any amino acid)” essential as a signal transduction region in CD28 and CTLA-4 is conserved. Have been.
The “JTT-1 antigen” of the present invention has the same structure as that of “CD28” and “CTLA-4”, which play an important role in controlling the activation of T cells, which are the main players in such an immune response. Because of its structure, the "JTT-1 antigen" of the present invention is a molecule that also plays an important role in controlling the activation of lymphocytes such as T cells, which are the main components of the immune response, as well as those molecules. It is speculated that there is.

Example 9 Cloning of cDNA Encoding “Mouse JTT-1 Antigen” Preparation of probe The clone "T132A7" obtained in Example 7 was digested with restriction enzymes EcoRI and NotI, and about 0.9 kb of cDNA encoding "rat JTT.1 antigen" was cut out and separated by agarose gel electrophoresis. did. The separated DNA fragment was purified using a “QUIAEX gel extraction kit” (manufactured by QUIATEN), and the obtained DNA fragment was labeled with 32 P using a “Ready-To-Go DNA labeling kit” (manufactured by Pharmacia). . This labeled DNA fragment was used as a probe for plaque hybridization.

2. Preparation of cDNA library 2- (1) Extraction of poly (A) + RNA In the same manner as in Example 7- (1), mouse spleen-derived lymphoblasts stimulated with ConA (about 1 × 10 6 cells / ml) Poly (A) + RNA from was extracted.
2- (2) Preparation of cDNA Library Using oligo (dT) primer (Pharmacia) and "Time Saver cDNA Synthesis Kit" (Pharmacia) as a template, 5 mg of poly (A) + RNA prepared above was used as a template. Was synthesized. After adding an EcoRI adapter to the cDNA, size fractionation was performed using a span column (Pharmacia).
2- (3) Incorporation into vector and packaging The cDNA having an EcoRI end obtained above was ligated to EcoRI-treated vector lZAPII (Stratagene). A "DNA ligation Kit" (Takara Shuzo) was used for the ligation reaction. After in vitro packaging using GIGA PACK II GOLD (manufactured by Stratagene), the resulting phage particles are used to construct a plaque containing recombinant phage using E coli XL1Blue MRF '(manufactured by Stratagene) as a host. A cDNA library was prepared.

3. Screening of cDNA library Screening was performed according to the plaque hybridization method (Maniatis, T. et al. Molecular Cloning: A Labolatory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) using Rapid hybridization buffer (manufactured by Amersham). went.
The cDNA library (1 × 104) obtained above was seeded on an agar plate, and a replica was prepared using Hybond-N nylon menbrane (Amersham). Plaque hybridization was performed in a Rapid hybridization buffer (Amersham) using the replica and the 32P-labeled probe prepared in 9-1. The primary screening and the secondary screening were performed, and five positive clones were obtained. After each clone was isolated with a single plaque, it was subjected to in vivo excision according to the Instruction Manual of Stratagene, and 5 clones were recovered as plasmid DNA.

4. Nucleotide Sequence Determination The nucleotide sequence of each of the five clones was determined by the dideoxy method using the “Auto Read Sequencing Kit” (Pharmacia) and the “ALFDNA sequencer” (Pharmacia). Four of the five clones all contained the same nucleotide sequence. The nucleotide sequence and deduced amino acid sequence of cDNA encoding the full length of “mouse JTT-1 antigen” are described in SEQ ID NO: 5.
As is clear from FIG. 10, “mouse JTT-1 antigen” is composed of 200 amino acid residues similarly to “rat JTT-1 antigen”, and mouse, rat and human “JTT-1 antigen” Each had significant amino acid sequence homology (60% or more).

5. Analysis of Chromosome Position of "Mouse JTT-1 Antigen" Gene The chromosome position of the gene encoding "mouse JTT-1 antigen" was analyzed by a fluorescent in situ hybridization method.

  The cDNA encoding the obtained “mouse JTT-1 antigen” was labeled with 32 P to prepare a hybridization probe according to a conventional method. Using this probe, a 129 SVJ mouse genomic DNA library (STRATAGENE) was screened to obtain a mouse genomic DNA clone containing an exon encoding “mouse JTT-1 antigen”. FIG. 13 schematically shows the structure of the genomic DNA.

  The genomic DNA clone obtained above was labeled with digoxigenin dUTP by nick translation to obtain a probe. After binding of the labeled probe-separated mouse DNA, normal metaphase chromosome derived from mouse embryonic fibroblasts was cultured in a solution containing 50% formaldehyde, 10% dextran sulfate and 2 × SSC. Was hybridized. The specific hybridization signal was detected by incubating the hybridization slide in a fluorescently labeled anti-digoxigenin antibody, followed by staining with DAPI. In the first test, specific labeling was performed on the vicinity of the largest chromosome, probably chromosome 1, based on the size of the DNA and the appearance of the band. Based on this information, the genomic DNA clones were co-hybridized with a probe specific to the centromere region of chromosome 1. As a result, the centromere region of chromosome 1 and its neighboring region were specifically labeled. As a result of analyzing chromosome 1 (10 samples) in which specific hybridization was observed, the genomic DNA clone was located at a position 33% of the distance from the boundary between heterochromatin and euchromatin to the telomere of chromosome 1, ie, It was revealed that the genes of mouse "CD28" and "CTLA-4" were present in the same band "1C3" as the position on the chromosome. As a result of analyzing a total of 80 metaphase cells, specific labeling was confirmed at this position in 79 cells.

  Considering this result and the result obtained in Example 8, ie, the structural similarity between “JTT-1 antigen” and “CD28” and “CTLA-4”, “JTT-1 antigen” was changed to “CD28”. It is presumed that it is an important molecule involved in the control of costimulatory signal transmission and / or lymphocyte activation similar to that of "CTLA-4".

Example 10 Cloning of cDNA Encoding a Mutant of “Rat JTT-1 Antigen” A cDNA encoding an alternative splicing variant of “rat JTT-1 antigen” cloned in Example 7. One other possible cDNA was cloned as described below.

1. Preparation of probe The clone "T132A7" obtained in Example 7 was digested with restriction enzymes EcoRI and NotI, and about 0.9 kb of cDNA encoding "rat JTT.1 antigen" was cut out and separated by agarose gel electrophoresis. did. The separated DNA fragment was purified using a “QUIAEX gel extraction kit” (manufactured by QUIATEN), and the obtained DNA fragment was labeled with 32 P using a “Ready-To-Go DNA labeling kit” (manufactured by Pharmacia). . This labeled DNA fragment was used as a probe for plaque hybridization.

2. Preparation of cDNA Library 2- (1) Extraction of poly (A) + RNA Poly (A) + RNA was extracted from rat thymoma cell line FTL435 (about 1 × 10 6 cells / ml) in the same manner as in Example 7- (1). A) + RNA was extracted.
2- (2) Preparation of cDNA library Using oligo (dT) primer (Pharmacia) and "Time Saver cDNA Synthesis Kit" (Pharmacia) with 5 mg of poly (A) + RNA prepared as described above as a template. To synthesize cDNA. After adding an EcoRI adapter to the cDNA, size fractionation was performed using a span column (Pharmacia).
2- (3) Incorporation into Vector and Packaging The cDNA having an EcoRI end obtained as described above was ligated to EcoRI-treated vector lZAPII (Stratagene). A "DNA ligation Kit" (Takara Shuzo) was used for the ligation reaction. After in vitro packaging using GIGA PACK II GOLD (manufactured by Stratagene), the resulting phage particles are used to construct a plaque containing recombinant phage using E coli XL1Blue MRF '(manufactured by Stratagene) as a host. A cDNA library was prepared.

3. Screening of cDNA library Screening was performed according to a plaque hybridization method (Maniatis, T. et al. Molecular Cloning: A Labolatory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) using Rapid hybridization buffer (manufactured by Amersham). went.
The prepared cDNA library (1 × 104) was spread on an agar plate, and a replica was prepared using Hybond-N nylon menbrane (Amersham). Plaque hybridization was performed in a Rapid hybridization buffer (Amersham) using the replica and the 32P-labeled probe prepared in 10-1. The primary screening and the secondary screening were performed to obtain two positive clones. After each clone was isolated with a single plaque, it was subjected to in vivo excision according to the Instruction Manual of Stratagene, and two clones were recovered as plasmid DNA.

4. Nucleotide sequence determination The nucleotide sequence of each of the two clones was determined by the dideoxy method using the "Auto Read Sequencing Kit" (Pharmacia) and the ALF DNA sequencer (Pharmacia). All two clones contained the same nucleotide sequence. SEQ ID NO: 6 shows the nucleotide sequence and deduced amino acid sequence of the obtained cDNA encoding the full length of "rat JTT-1 antigen". The amino acid sequence deduced from the obtained cDNA sequence (SEQ ID NO: 6) was compared with the amino acid sequence deduced from the cDNA sequence encoding “rat JTT-1 antigen” cloned in Example 7 (SEQ ID NO: 4). (FIG. 14). As a result, as apparent from FIG. 14, the amino acid sequence encoded by the cDNA cloned in this test was the same as the amino acid sequence encoded by the cDNA encoding “rat JTT.1 antigen” obtained in Example 7. In comparison with (i) that three consecutive amino acid sequences at the C-terminus (Met-Thr-Ser) are changed to Thr-Ala-Pro, and (ii) an additional 16 amino acids following Thr-Ala-Pro. Except that two consecutive amino acid residues (Leu-Arg-Ala-Leu-Gly-Arg-Gly-Glu-His-Ser-Ser-Cys-Gln-Asp-Arg-Asn) are extended Has exactly the same amino acid sequence. From this, it is considered that the cDNA cloned in this test encodes an alternative splicing variant of “rat JTT.1 antigen” obtained in Example 7.

[Example 11] Preparation of cells expressing recombinant "human JTT-1 antigen" The plasmid clone pBSh41 obtained in Example 8 was digested with a restriction enzyme EcoRI to encode the full-length "human JTT-1 antigen". A DNA fragment containing the cDNA to be cloned was cut out. This DNA fragment was inserted into a plasmid pEFneo (Proc. Natl. Acad. Sci. USA, 91, 158-162, 1994), similarly treated with the restriction enzyme EcoRI, using a DNA ligation kit (manufactured by Takara Shuzo). Was prepared. CHO-K1 cells (ATCC: CCL-61) were transformed with the vector by electroporation. Geneticin-resistant transformed cells were selected by culturing the cells for about 2 weeks in RPMI1640 medium containing Geneticin (0.8 mg / ml; manufactured by GIBCO BRL) and 10% fetal calf serum. Expression of the recombinant "human JTT-1 antigen" was confirmed by Northern blotting according to a conventional method.

Example 12 Preparation of Monoclonal Antibody Against “Human JTT.1 Antigen” The transformed cells expressing the recombinant “human JTT-1 antigen” prepared in Example 11 were homogenized and ultracentrifuged (100,000 × g). ), And the centrifugation residue containing the cell membrane fraction was collected and suspended in PBS. The obtained cell membrane fraction was initially immunized (day 0) by injecting it into the footpad of a BALB / c mouse together with complete Freund's adjuvant. Further, the cell membrane fraction antigen was administered into the foot pad at intervals of 7, 14, and 28 days. Two days after the last immunization, lymph node cells were collected. The lymph node cells and mouse myeloma cells PAI (JCR No. B0113; Res. Disclosure, Vol. 217, p. 155, 1982) were mixed at a ratio of 5: 1, and polyethylene glycol 4000 (manufactured by GIBCO) was used as a fusion agent. Thus, a monoclonal antibody-producing hybridoma was prepared by cell fusion. Hybridomas were selected by culturing in HAT-containing ASF104 medium (manufactured by Ajinomoto) containing 10% fetal bovine serum and aminopterin. The reactivity of the monoclonal antibody produced in the culture supernatant of each hybridoma with respect to “human JTT-1 antigen” was determined using the recombinant “human JTT-1 antigen” prepared in Example 11 for each culture supernatant. After reacting with the expressed transformed cells, the cells were reacted with FITC-labeled anti-mouse IgG (manufactured by Cappel), and the fluorescence intensity of the cells stained was measured using an EPICS-ELITE flow cytometer. As a result, it was confirmed that 10 or more hybridomas producing monoclonal antibodies reactive with “human JTT-1 antigen” were obtained.

  Two of these hybridomas (named clones SA12 and SG430 respectively) (106 to 107 cells / 0.5 ml / mouse) were injected intraperitoneally into ICR nu / nu mice (female, 7 to 8 weeks old) did. After 10 to 20 days, the mice were laparotomized under anesthesia, and two types of monoclonal antibodies (SA12 and SG430) reactive with "human JTT-1 antigen" were prepared from ascites collected according to a conventional method.

Example 13 Effect of Monoclonal Antibody Against “Human JTT-1 Antigen” on Human Peripheral Blood Lymphocytes As described in Example 8, “JTT-1 antigen” is similar to “CD28” or “CTLA-4”. It is likely that it has a role in controlling the activation of lymphocyte cells in the immune response. To demonstrate this possibility, the effect of monoclonal antibodies against "human JTT-1 antigen" on human lymphocytes was analyzed using cell proliferation as an index.

  In each well of the 96-well macrotiter plate, (i) either one of the monoclonal antibodies SA12 or SG430 (1 μg / ml) against “human JTT-1 antigen” prepared in Example 12 alone, or (ii) the monoclonal antibody SA12 or Mixed solution of one of SG430 (1 μg / ml) and anti-CD3 monoclonal antibody OKT-3 (1 μg / ml, manufactured by Ortho Dianostic Systems) for giving a first signal in lymphocyte activation Was added and cultured at 37 ° C. for 1 hour, and each well was coated with the antibody. After washing the plate with RPMI1640 medium, normal human peripheral blood lymphocytes (1 × 105 cells / well) were added to each well, and the plate was cultured in RPMI1640 medium containing 10% fetal bovine serum for 3 days. PMA (phorbol myristate acetate: 1 ng / ml) was added as needed. Next, [3H] thymidine (3.7 μkBq / well) was added to each well, and the cells were cultured at 37 ° C. for 6 hours. The cells were collected (harvested), and the amount of [3H] thymidine incorporated in the DNA was measured using a liquid scintillation counter (Beckman). The culture system without any antibody was used as a control. The results are shown in FIG.

  When treated with either monoclonal antibody SA12 or SG430 alone, lymphocytes grew approximately 10-fold when using either antibody as compared to the control. Furthermore, when used in combination with OKT3, lymphocytes grew approximately 100-fold when either monoclonal antibody SA12 or SG430 was used.

  This result indicates that “JTT-1 antigen” functions in controlling lymphocyte activation. In addition, the cell proliferation rate was amplified in combination with OKT3, indicating that "JTT-1 antigen" is responsible for the same costimulatory signal transmission as "CD28" and "CTLA-4" Things.

[Example 14] Effect of "JTT-2 antibody" on experimental allergic encephalomyelitis (EAE) As described in detail above, recently, signaling between CD28 / CTLA-4-CD80 / CD86 was detected. Various autoimmune diseases (rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, lichen planus, a chronic inflammatory skin disease, systemic lupus erythematosus, insulin dependence Many attempts have been made to treat (eg, diabetes mellitus and psoriasis). Already, various autoimmune disease model animals ((i) a model of human systemic lupus erythematosus (SLE), (ii) a model of multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE), (iii) ) Its effects have been confirmed in the insulin-dependent diabetes mellitus (IDDM) model, (iv) the nephritis model of Goodpasture, and (v) the rheumatoid arthritis model in humans.

  To confirm whether the "JTT-1 antigen" of the present invention is a molecule involved in the activation or suppression of lymphocytes such as "CD28" and "CTLA-4", multiple sclerosis (MS An experimental allergic encephalomyelitis (EAE) model rat was prepared as a model in (1), and the effect of a monoclonal antibody against “JTT-1 antigen” in this model was analyzed.

  Hartley guinea pig cerebral spinal cord homogenate (800 mg / ml saline) was mixed with an equal amount of Freund's complete adjuvant to prepare an emulsion as an immunizing antigen. The emulsion was immunized intradermally by injecting 0.25 ml each of the left and right toes of Lewis rats (female, 6 weeks old, 15 rats). The administration was performed such that the dose of the homogenate was 200 mg per rat. This immunization can induce allergic encephalomyelitis (EAE).

The immunized rats were divided into three groups of 5 rats, and for each group, one of the following (i) to (iii) was administered immediately after the immunization (day 0) and for 3 days after the immunization. Intravenous administration on days 6, 6, 9 and 12.
(i) Monoclonal antibody “JTT-2 antibody” against “rat JTT-1 antigen” prepared in Example 2 (dose: 2 mg / ml PBS, 5 mg / kg);
(ii) steroid prednisolone (dose: 4 mg / ml PBS, 10 mg / kg); and
(iii) Control antibody that does not react with “rat JTT-1 antigen” (dose: 2 mg / ml PBS, 5 mg / kg
).

Symptoms were observed over time after the immunization, and the degree of symptoms was evaluated by scoring the symptoms according to the following criteria from the time when EAE development was observed.
(Score 1) loss of tail tension;
(Score 2) hind limb drag and mild paralysis;
(Score 3) hind limb drag and severe paralysis; and (score 4) general paralysis or death.

FIG. 16 shows the results. In the group to which the control antibody was administered, EAE symptoms peaked (maximum score) on days 11 to 15 after immunization, and gradually recovered thereafter. On the other hand, in the group administered with the “JTT-2 antibody”, the onset of EAE symptoms on day 11 after sensitization was significantly suppressed. This inhibitory effect was significantly higher than that of the prednisolone administration group.
This result indicates that “JTT-1 antigen” is a molecule that functions in the induction of immune responses including activation of lymphocytes induced by immunization with a foreign antigen, and that “JTT-1 antigen” or its It shows that the symptoms of various autoimmune diseases can be suppressed by controlling the function of the ligand.

[Example 15] Effect of "JTT-2 antibody" on glomerulonephritis For the same purpose as in Example 14, a glomerular basement membrane (GBM) nephritis model rat was prepared, and "JTT-1 antigen" in this model was prepared. Was analyzed for the effect of the monoclonal antibody.

  Bovine glomerular basement membrane (manufactured by Shigei Medical Research Institute) digested with collagenase was diluted with physiological saline to a concentration of 200 μg / ml, and then mixed with Freund's complete adjuvant to prepare an emulsion as an immunizing antigen. Under ether anesthesia, immunization was carried out by injecting about 0.2 ml (antigen amount: about 15 μg) into each plantar skin of both hind limbs of Wistar kyoto rats (about 200 g, 48 rats). This immunization induces glomerular basement membrane (GBM) nephritis.

The immunized rats were divided into 8 groups of 6 rats, and for each group, one of the following (i) to (iii) was administered immediately after the immunization (day 0) and thereafter 3 times a week. It was administered for 5 weeks.
(i) Monoclonal antibody "JTT-2 antibody" against "rat JTT-1 antigen" prepared in Example 2 (dose: 3 mg / kg (2 ml PBS / kg), intravenous administration);
(ii) a steroid drug prednisolone (suspended in 0.5% CMC (carboxymethylcellulose)) as a positive control (dose: 3 mg / kg (5 ml / kg), oral administration); and
(iii) 0.5% CMC as a negative control (dose: 5 ml / kg, oral administration).

  After the administration of the test substance, sterilized water (25 ml / kg) was orally administered to each rat, and each rat was placed in a metabolic cage, and urine was collected for 5 hours under fasted water. After measuring the amount of the collected urine, the urine protein concentration was measured using Tonein TP-II (manufactured by Otsuka Pharmaceutical), and the urinary protein excretion per 5 hours (unit: mg protein / 5 hours) was calculated. . The urine collection and the measurement of the amount of protein in the urine were performed in the same manner at each of the first week, the second week, the third week, and the fourth week after the immunization (day 0).

The results are shown in FIG. Compared with the control group, the “JTT-2 antibody” -administered group significantly reduced urinary protein excretion three weeks after immunization.
This result indicates that “JTT-1 antigen” is a molecule that functions in the induction of immune responses including activation of lymphocytes induced by immunization with a foreign antigen, and that “JTT-1 antigen” or its It shows that the symptoms of various autoimmune diseases can be suppressed by controlling the function of the ligand.

[Example 16] Preparation of fusion protein of "JTT-1 antigen" and IgFc As described in Examples 8, 13 to 15, "JTT-1 antigen" of the present invention includes "CD28" and "CTLA- It is thought to be a molecule involved in costimulation signaling involved in controlling lymphocyte activation, such as 4 ". Further, as described in Example 14, the fusion protein comprising the extracellular domain of “CTLA-4” and the Fc region of human immunoglobulin IgG1 (CTLA-4-IgFc) has a therapeutic effect on various autoimmune diseases. It has been reported. In this example, in order to confirm the applicability of the same solubilized JTT-1 antigen as CTLA-4-IgFc to the treatment of various autoimmune diseases, the extracellular region of the `` JTT-1 antigen '' was replaced with human IgGFc. Was prepared as follows.

(1) Preparation of fusion protein (rJTT-1-IgFc) of "rat JTT-1 antigen" and human IgG1-Fc To amplify the cDNA encoding the extracellular region of "rat JTT-1 antigen" by PCR And a 5 'primer having an XhoI cleavage site at the end (5'-CTGCTCGAGATGAAGCCCTACTTCTCG-3', SEQ ID NO: 7) and a 3 'primer having a BamHI cleavage site (5'-ACCCTACGGGTAACGGATCCTTCAGCTGGCAA-3', SEQ ID NO: 8) are designed and synthesized. did. The cDNA encoding the full length of the "rat JTT-1 antigen" obtained in Example 7 was subjected to PCR using the clone "T132A7" as a template and the primers, and having XhoI and BamHI cleavage sites at both ends. A cDNA containing a cDNA encoding the extracellular region of “rat JTT-1 antigen” was prepared. The obtained PCR product was digested with XhoI and BamHI, and subjected to agarose gel electrophoresis to isolate a band of about 450 bp which was predicted to be a cDNA fragment encoding the extracellular region of interest. The isolated cDNA fragment was subcloned into a plasmid pBluescript II SK (+) (Stratagene) digested with XhoI and BamHI. Sequence analysis using an automatic fluorescent DNA sequencer (manufactured by Applied Biosystems) confirmed that the cDNA fragment contained a region encoding the region from amino acid No. 1 to 141 of "rat JTT-1 antigen" (SEQ ID NO: 4). .

  On the other hand, DNA encoding the Fc of human IgG1 as a fusion partner has been prepared by a plasmid (see Cell, Vol. 61, p. 1303-1313, 1990. Made by Dr. B. Seed of Massachusetts General Hospital). ) Was cut out as a BamHI-XbaI DNA fragment (about 1.3 kb) by digestion with BamHI and XbaI. This fragment contains exons encoding the hinge region, Cγ12, and Cγ13, respectively, of human IgG1.

  The XhoI-BamHI fragment encoding the extracellular region of “rat JTT-1 antigen” and the BamHI-XbaI fragment containing the exon encoding Fc of human IgG1 (abbreviated as “IgFc”) prepared as described above were , XhoI and XbaI and subcloned into plasmid pBluescript II SK (+) (Stratagene).

  Next, the plasmid was digested with XhoI and XbaI to cut out a DNA fragment of about 1.8 kb containing a fusion DNA consisting of the extracellular region of “rat JTT-1 antigen” and human IgFc. Using T4 DNA ligase, this fusion DNA fragment was expressed in the expression vector pME18S (Medical Immunology, Vol. 20, No. 1, p. 27-32, 1990, and Experimental Medicine (separate volume): “Genetic Engineering Handbook”, The plasmid was inserted into the XhoI and XbaI sites of Yodosha, pp. 101-107, 1992) to construct a plasmid prJTT-1-IgFc.

  By electroporation, HEK293 cells (ATCC CRL1573) cultured in subconfluent monolayer in DMEM medium containing 10% fetal bovine serum and ampicillin are transformed with plasmid prJTT-1-IgFc to obtain transformed cells. did.

  The transformed cells were cultured in serum-free ASF104 medium for 72 hours to express rJTT-1-IgFc.

  rJTT-1-IgFc was purified using a Protein G Sepharose affinity column (Pharmacia) as follows.

  The centrifuged supernatant obtained by centrifuging the culture supernatant was added to a Protein G Sepharose affinity column that had been equilibrated with a binding buffer in advance. Next, the column was washed with a binding buffer and then eluted with an elution buffer. The eluate was recovered and dialyzed by exchanging the external solution with a phosphate buffer at least twice to obtain purified rJTT-1-IgFc.

  FIG. 18 shows the result of affinity column chromatography, and FIG. 19 shows the result of SDS-PAGE of the purified rJTT-1-IgFc obtained.

(2) Preparation of fusion protein (hJTT-1-IgFc) of "human JTT-1 antigen" and human IgG1-Fc In the same manner as in the above (1), except for cDNA and primers as templates for PCR. Prepared. In this test, a clone “pBSh41” containing a cDNA encoding the full length of “human JTT-1 antigen” prepared in Example 8 was used as a template, and 5′-TAACTGTTTCTCGAGAACATGAAGTCAGGC-3 ′ ( SEQ ID NO: 9) was used as a 3 'primer, and 5'-ATCCTATGGGTAACGGATCCTTCAGCTGGC-3' (SEQ ID NO: 10) was used.
The results of affinity column chromatography are shown in FIG. 20, and the results of SDS-PAGE of the purified hJTT-1-IgFc obtained are shown in FIG.

[Example 17] Production of cDNA-transgenic transgenic mouse encoding "rat JTT-1 antigen" The cDNA encoding the full-length "rat JTT-1 antigen" obtained in Example 7 was transformed with a chicken β actin promoter. It was inserted into an expression vector pCAGGS (Gene, Vol. 108, p. 193-200, 1991) using a DNA end blunting kit (manufactured by Takara) to obtain a plasmid prJTT-1. For the production of transgenic mice, prJTT-1 was treated with a restriction enzyme to make it linear.

  The foster mother mouse has a plug (or vaginal plug) obtained by mating a white ICR mouse (female, manufactured by Japan SLC) with a white ICR mouse (male, manufactured by Japan SLC) ligated in vas deferens. Female ICR mice were used. In addition, the mouse for egg collection to obtain a fertilized egg for introducing the “rat JTT-1 antigen” gene should be administered with PEAMEX (5 units, manufactured by Sankyo Zoki) and pregnyl (5 units, manufactured by Organon). BDF-1 mice (female, manufactured by Nippon SLC Co., Ltd.) that had been superovulated by BDF-1 were crossed with BDF-1 mice (male, manufactured by Nippon SLC Co., Ltd.). After mating, the oviduct was excised from the BDF-1 mouse (female), and only a fertilized egg was obtained by treatment with hyaluronidase and stored in a medium.

  The introduction of the “rat JTT-1 antigen” gene into the fertilized egg was performed by a conventional method using a manipulator under a microscope. The fertilized egg was fixed with a retaining needle, and the solution containing the linear gene encoding “rat JTT-1 antigen” diluted with Tris EDTA buffer at 37 ° C. was transferred to the fertilized egg using a DNA introduction needle. Was injected (microinjected) into the male pronucleus.

  After gene transfer, only fertilized eggs that maintained a normal state were selected, and the “rat JTT-1 antigen” gene-transferred fertilized eggs were inserted into the fallopian tubes in the ovaries of a foster parent mouse (white ICR mouse).

The tail of a mouse (founder) born to the foster mother was cut off, the genomic gene was recovered, and PCR confirmed that the “rat JTT-1 antigen” gene had been introduced into the mouse genome. Next, the founder was crossed with a normal mouse to prepare a heterotransgenic mouse that highly expresses “rat JTT-1 antigen”. A homo mouse can be produced by crossing the hetero mice.
A schematic diagram of a microinjected construct containing the "rat JTT-1 antigen" gene is shown in FIG.

[Example 18] Preparation of knockout mouse in which endogenous gene encoding "mouse JTT-1 antigen" is inactivated (1) Construction of targeting vector Homologous set of endogenous gene encoding "mouse JTT-1 antigen" A targeting vector for inactivation (knockout) by replacement (Nikkei Science, May 1994, pp. 52-62) was constructed as follows.

  A PstI-HindIII fragment ("homologous DNA (1)") obtained by digesting a mouse genomic DNA clone containing a region encoding "mouse JTT-1 antigen" cloned in Example 9-5 with PstI and HindIII was used. And subcloned into plasmid pGEM-3 (promega). Next, pGEM-3 was opened with XhoI, and the plasmid pMC1-neo-polyA (manufactured by Stratagene) was treated with XhoI and SalI. The neomycin resistance gene (“neo”) cut out was homologous DNA (1). Was inserted upstream of and ligated.

  The mouse genomic DNA clone was digested with XhoI and NotI to cut out a gene of about 5.5 kb (“homologous DNA (2)”) located upstream from the above “homologous DNA (1)”. On the other hand, pGEM-3 into which the aforementioned "neo-homologous DNA (1)" was inserted was digested with XhoI and HindIII to cut out "neo-homologous DNA (1)". The obtained "homologous DNA (2)" and "neo-homologous DNA (1)" were subcloned into plasmid pSEAP2-CONT (Clontech) opened with NotI and HindIII.

  The resulting plasmid ("homologous DNA (2) -neo-homologous DNA (1)" is inserted) is digested with NruI downstream of "homologous DNA (1)", and the plasmid is opened. The thymidine kinase gene ("TK") obtained by digesting pMC1-TK (manufactured by Stratagene) with PvuII is inserted and ligated downstream of "homologous DNA (1)", and the targeting vector ("homologous DNA (2)- neo-homologous DNA (1) -TK ").

(2) Introduction of targeting vector into ES cells
Mouse embryonic stem cells (ES cells; embryonic stem cells) cultured in DMEM medium containing 15% fetal bovine serum (Nature, Vol. 362, p. 255-258, 1993 and Nature, Vol. 326, p. 292) -295, 1987) was treated with trypsin to form single cells, and then washed three times with a phosphate buffer to adjust the cell concentration to 1 × 10 7 cells / ml. 25 μg of the targeting vector was added per 1 ml of the cell suspension, and an electric pulse was applied once under the condition of 350 V / cm (25 μF). Next, 1 × 10 7 ES cells were seeded on a Petri dish (10 cm), cultured in a maintenance medium for 1 day, and then replaced with a selection medium (containing G418 (250 μg / ml) and 2 μM ganciclovir). did. Thereafter, the medium was exchanged every two days and cultured. Ten days after the introduction of the targeting vector, 573 neomycin-resistant ES cell clones were obtained under a microscope using a micropipette. The obtained ES cell clones were separately cultured in a 24-well plate on which Feeder cells were spread, thereby obtaining 768 neomycin-resistant ES cell replicas.

(3) Screening of knockout ES cells For each of the obtained neomycin-resistant ES cells, it was determined by PCR whether or not disruption (knockout) of an endogenous gene encoding “mouse JTT-1 antigen” by homologous recombination had occurred. confirmed.

  In PCR, (1) primers (5′-CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC-3 ′, SEQ ID NO: 11) designed and synthesized based on the sequence of the neomycin resistance gene (“neo”)) and (2) the “homologous DNA (1 )), A primer designed and synthesized based on the sequence (5′-CATTCAAGTTTCAGGGAACTAGTCCATGCGTTTC-3 ′, SEQ ID NO: 12) was used.

  Genomic DNA was extracted from each neomycin-resistant ES cell, and PCR was performed using the extracted genomic DNA as a template and the primers. PCR consisted of one cycle of a reaction at 94 ° C. for 3 minutes, 30 cycles of a reaction consisting of three reactions of 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 3 minutes and 30 seconds, and 10 minutes at 72 ° C. After 1 cycle of the reaction, the mixture was stored at 4 ° C. When a fragment of about 4 kb is amplified by this PCR, it can be determined that the endogenous gene encoding “mouse JTT-1 antigen” has been disrupted (knocked out) by homologous recombination in the ES cell clone. .

As a result, out of the 768 ES cell clones tested, the desired PCR product was obtained in 3 clones. Genomic southern blotting was performed on these three clones, and further selection and confirmation were performed. The genomic DNA of the three clones was extracted, digested with the restriction enzyme BamHI, and electrophoresed on an agarose gel. This was transferred to a nylon membrane, and hybridization was carried out using a probe prepared with the genomic DNA sequence of “mouse JTT-1”. The probe was designed based on the sequence outside the site where the homologous recombination occurs, and at the site where the mutant genome and the normal genome can be distinguished in size.
As a result, two bands of a mutant type and a normal type were confirmed in one of the three clones. This ES cell clone was used for producing a knockout mouse described below.

(4) Preparation of knockout mouse ES cells in which the endogenous gene encoding the “mouse JTT-1 antigen” obtained above was inactivated (knockout) by homologous recombination were transferred to C57BL6 mouse (Charles River Japan). The blastocysts obtained by mating males and females were injected (microinjection) into 15 blastocysts per embryo. Immediately after the injection, about 10 blastocysts per one side of the uterus were implanted into the uterus of a foster parent ICR mouse (manufactured by CLEA Japan) 2.5 days after the pseudopregnancy treatment. As a result, a total of 38 babies were obtained, 18 of which were the target chimeric mice. Eleven chimeric mice contributed 80% or more to coat color.

  Next, the obtained chimeric mouse was bred to a normal C57BL6 mouse to obtain an agouti-colored mouse with a hair-color gene derived from ES cells.

[Example 19] Preparation of antibody pharmaceutical composition Each of monoclonal antibodies "JTT-1 antibody" and "JTT-2 antibody" against "rat JTT-1 antigen" prepared in Example 1 and prepared in Example 12 Each of the monoclonal antibodies “SA12” and “SG430” (50-150 μg / ml) against “human JTT-1 antigen” was added to distilled water for injection (10 ml) to prepare an injection.

5 is a micrograph showing the state of cell aggregation of FTL435 cells induced by “JTT.1 antibody” and the state of inhibition of the cell aggregation by “JTT.2 antibody”. The diagram (a) shows the state of the cells when no hybridoma supernatant was added, the diagram (b) shows the state of cell aggregation by the "JTT-1 antibody", and the diagram (c) shows the "JTT Figure 1 shows the state of cell aggregation when "anti-ICAM-1 antibody" was added together with "antibody-1", and figure (d) shows the cells when "JTT-2 antibody" was added together with "JTT-1 antibody". This shows the state of aggregation. FIG. 2 is micrographs showing the state of cell aggregation of FTL435 cells and rat activated lymphoblasts induced by “JTT.1 antibody” and the state of inhibition of the cell aggregation by “JTT.2 antibody”. (A) shows the state of FTL435 cells when no antibody was added, (b) shows the state of FTL435 cells when PMA was added, and (c) shows the state of cells. The cell state of FTL435 cells when "JTT-1 antibody" was added is shown. The division diagram (d) shows the cell state of FTL435 cells when anti-LFA-1 antibody was added together with "JTT-1 antibody". (E) shows the state of FTL435 cells when the anti-CD18 antibody was added together with the "JTT-1 antibody", and (f) shows the anti-ICAM-1 antibody together with the "JTT-1 antibody". (G) shows the state of activated lymphoblasts without any antibody, and (h) shows the state of PML. (I) shows the state of the activated lymphoblast cells when the “JTT-1 antibody” was added, and (j) shows the state of the activated lymphoblast cells when the “JTT-1 antibody” was added. Shows the state of activated lymphoblasts when anti-LFA-1 antibody is added together with the "JTT-1 antibody". Figure (k) shows the activity when the anti-CD18 antibody is added together with the "JTT-1 antibody". The state of activated lymphoblast cells is shown, and the diagram (l) shows the state of activated lymphoblast cells when an anti-ICAM-1 antibody was added together with the "JTT-1 antibody". It is a figure which shows the state of expression of "JTT.1 antigen" and "JTT.2 antigen" in various cells measured using the flow cytometer. FIG. 3 is a view showing the state of expression of “JTT.1 antigen” in various lymphoid cells measured using a flow cytometer. It is a photograph showing an electrophoresis image of “JTT.1 antigen” analyzed by SDS-PAGE electrophoresis. The state of adhesion of rat thymocytes to a microplate coated with purified "JTT.1 antigen" induced in the presence of "JTT.1 antibody", and the state of inhibition of said cell adhesion by "JTT.2 antibody" FIG. Panel (a) shows the state of cell attachment to a plate not coated with “JTT-1 antigen”, and panel (b) shows the state of “JTT-1 antigen” when neither antibody was added. The state of adhesion of cells to the coated plate is shown. The diagram (c) shows the state of adhesion of cells to the plate coated with "JTT-1 antigen" when the Fab fragment of "JTT-1 antibody" was added. And (d) shows the state of cell adhesion to a plate coated with “JTT-1 antigen” when “JTT-2 antibody” was added together with a Fab fragment of “JTT-1 antibody”. . FIG. 4 is a view showing relative cell numbers based on the fluorescence intensity of thymocytes adhered to a plate coated with purified “JTT.1 antigen”. "Ag (-)" indicates the relative cell number in the plate not coated with "JTT-1 antigen", and "Ag (+)" was coated with "JTT-1 antigen" when no antibody was added. Shows the relative cell number on the plate, "Ag (+) + JTT.1 Fab" indicates the relative cell number on the plate coated with "JTT-1 antigen" when the Fab fragment of "JTT-1 antibody" is added `` Ag (+) + JTT.1 Fab + JTT.2 '' is the `` JTT-1 antigen '' on the plate coated with `` JTT-1 antigen '' when `` JTT-2 antibody '' is added together with the Fab fragment of `` JTT-1 antibody '' The relative cell number is shown. FIG. 5 shows the expression states of “rat JTT-1 antigen” and “rat JTT-2 antigen” in COS cells transformed with cDNA encoding “rat JTT-1 antigen” measured using a flow cytometer. FIG. FIG. 4 is a view showing a structural characteristic of an amino acid sequence of “JTT.1 antigen” by hydropathy blot analysis. FIG. 3 is a diagram showing the homology of the amino acid sequences of human, rat, and mouse “JTT.1 antigen” and mutants of “rat JTT-1 antigen”. FIG. 3 is a view showing the amino acid sequence homology in the amino acid sequences of “human JTT-1 antigen”, “human CD28 molecule” and “human CTLA-4 molecule” and the state of conservation of the motif. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram schematically showing the protein secondary structures of “human JTT-1 antigen”, “human CD28 molecule” and “human CTLA-4 molecule” and their similarities. FIG. 2 is a diagram schematically showing the structure of genomic DNA encoding “mouse JTT-1 antigen”. It is a figure which shows the difference of each amino acid sequence of "rat JTT-1 antigen" and its alternative splicing variant. FIG. 2 is a graph showing the degree of proliferation of human peripheral blood lymphocytes induced by a monoclonal antibody against “human JTT-1 antigen” measured by a [3 H] thymidine incorporation test. The vertical axis indicates the uptake (dpm) of [3H] thymidine into cells. It is a figure which shows the therapeutic effect by the monoclonal antibody with respect to "JTT-1 antigen" of experimental allergic encephalomyelitis (EAE) in a disease model rat. The vertical axis shows the score of the disease symptoms, and the horizontal axis shows the number of days after immunization for EAE induction. It is a figure which shows the therapeutic effect by the monoclonal antibody with respect to "JTT-1 antigen" of glomerulonephritis in a disease model rat. The vertical axis indicates urinary protein excretion, and the horizontal axis indicates elapsed time (weeks) after immunization for glomerulonephritis induction. It is a figure which shows the column histogram in the refinement | purification of the fusion polypeptide (rJTT-1-IgFc) of the extracellular region of "rat JTT-1 antigen" and human IgFc by a protein A Sepharose column. 1 is a photograph showing an electrophoretic image of rJTT-1-IgFc analyzed by electrophoresis by SDS-PAGE. FIG. 3 is a view showing a column histogram in purification of a fusion polypeptide of an extracellular region of “human JTT-1 antigen” and human IgFc (hJTT-1-IgFc) using a protein A Sepharose column. 4 is a photograph showing an electrophoretic image of hJTT-1-IgFc analyzed by electrophoresis by SDS-PAGE. FIG. 1 is a diagram schematically showing the structure of a gene transfer vector for producing a transgenic mouse into which cDNA encoding “rat JTT-1 antigen” has been introduced.

Claims (36)

A polypeptide comprising a cell surface molecule having the following characteristics: (a) expressed at least in thymocytes and mitogen-stimulated lymphoblast cells;
(B) antibodies reactive with the cell surface molecules induce adhesion between mitogen-stimulated lymphoblast cells;
(C) an antibody reactive with the cell surface molecule induces proliferation of peripheral blood lymphocytes in the presence of an antibody reactive with CD3;
(D) having a partial amino acid sequence represented by Phe-Asp-Pro-Pro-Pro-Phe in the extracellular region; and (e) having a partial amino acid sequence represented by Tyr-Met-Phe-Met in the intracellular region. . The polypeptide has the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been substituted, deleted, or added. 2. The polypeptide of claim 1, wherein 2. The polypeptide according to claim 1, wherein the polypeptide is encoded by a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1. 2. The polypeptide according to claim 1, wherein the polypeptide has an amino acid sequence having 60% or more homology with the amino acid sequence set forth in SEQ ID NO: 2. The polypeptide according to any one of claims 1 to 4, wherein the cell surface molecule is a human-derived cell surface molecule. A gene encoding the polypeptide according to any one of claims 1 to 5. The gene according to claim 6, wherein the gene is cDNA. The gene according to claim 7, wherein the cDNA has the nucleotide sequence of SEQ ID NO: 1. The cDNA comprises the nucleotide sequence of nucleotides 26 to 625 of SEQ ID NO: 3, the nucleotide sequence of nucleotides 35 to 637 of SEQ ID NO: 4, the nucleotide sequence of nucleotides 1 to 603 of SEQ ID NO: 5, or The gene according to claim 7, comprising any one of the nucleotide sequences of base numbers 35 to 685 of SEQ ID NO: 6. A vector containing the gene according to any one of claims 6 to 9. A transformant into which the vector according to claim 10 has been introduced. A transformant identified by International Accession No. FERM BP-5725. A polypeptide fragment comprising the extracellular region of the polypeptide according to any one of claims 1 to 5. 14. The polypeptide fragment according to claim 13, wherein the polypeptide is a human-derived polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. A gene encoding the polypeptide fragment according to claim 13 or 14. A homodimer molecule formed by binding a polypeptide fragment comprising the extracellular region of the polypeptide according to any one of claims 1 to 5 by a disulfide bond. 17. The homodimer molecule according to claim 16, wherein the polypeptide is a human-derived polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. A pharmaceutical composition comprising one or both of the polypeptide fragment according to claim 14 and the homodimer molecule according to claim 17, and a pharmaceutically acceptable carrier. A fusion polypeptide comprising the extracellular region of the polypeptide according to any one of claims 1 to 5 and a constant region or a part of a constant region of a heavy chain of human immunoglobulin (Ig). 20. The fusion polypeptide according to claim 19, wherein the immunoglobulin is IgG. 20. The fusion polypeptide of claim 19, wherein a portion of the constant region comprises an IgG hinge region, a C2 domain, and a C3 domain. The fusion polypeptide according to any one of claims 19 to 21, wherein the polypeptide is a human-derived polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. A homodimer molecule formed by binding the fusion polypeptide according to any one of claims 19 to 22 by a disulfide bond. A homodimer molecule formed by linking the fusion polypeptide of claim 22 by a disulfide bond. A pharmaceutical composition comprising one or both of the fusion polypeptide of claim 22 or the homodimer molecule of claim 24 and a pharmaceutically acceptable carrier. 26. The pharmaceutical composition according to claim 25, wherein the pharmaceutical composition is used for treating an autoimmune disease or an allergic disease or for suppressing the symptoms of the disease. An antibody reactive with the polypeptide according to any one of claims 1 to 5, the polypeptide fragment according to claim 13 or claim 14, or a cell surface molecule constituted by the polypeptide, or a part thereof. . 28. The antibody or part thereof according to claim 27, wherein said antibody is a monoclonal antibody. 15. A polypeptide having the amino acid sequence of SEQ ID NO: 2, a polypeptide fragment according to claim 14, or a monoclonal antibody or a part thereof, which is reactive with a human-derived cell surface molecule composed of the polypeptide. A monoclonal antibody reactive to the polypeptide according to any one of claims 1 to 5 or a cell surface molecule constituted by the polypeptide, and the lymphocyte is stimulated with the mitogen of the monoclonal antibody. Wherein the monoclonal antibody produced by the hybridoma identified by International Accession No. FERM BP-5707 has substantially the same effect as that exhibited by mitogen-stimulated rat lymphoblast cells. A monoclonal antibody reactive to the polypeptide according to any one of claims 1 to 5 or a cell surface molecule constituted by the polypeptide, and the lymphocyte is stimulated with the mitogen of the monoclonal antibody. Or a portion thereof, wherein the action of the monoclonal antibody produced by the hybridoma identified by International Accession No. FERM BP-5708 is substantially the same as that exhibited by mitogen-stimulated rat lymphoblast cells. A pharmaceutical composition comprising the monoclonal antibody according to claim 29 or a part thereof and a pharmaceutically acceptable carrier. 33. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is used for treating an autoimmune disease or an allergic disease or suppressing the symptoms of the disease. A hybridoma that produces the monoclonal antibody according to any one of claims 28 to 31. A gene encoding the polypeptide according to claim 1, which is a human-derived gene comprising the nucleotide sequence of SEQ ID NO: 1 or a rat comprising the nucleotide sequence of nucleotides 35 to 637 of SEQ ID NO: 4 A transgenic mouse, wherein the derived gene is integrated into a mouse endogenous gene. An endogenous mouse gene encoding the polypeptide of claim 1, such that the mouse-derived polypeptide having the amino acid sequence encoded by the gene set forth in SEQ ID NO: 5 is not produced. A knockout mouse characterized by being inactivated.

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