Purified non-hamster antibody, compositions comprising it and isolated cells which produce it

Purified non-hamster antibody, compositions comprising thereof and isolated cells produce it

Japan Tobacco Inc.

C. 119651 ¹²^X^-ention relates to novel 0־-״־ 11־ — of mammals; polypeptides and their fragments molecules; i־״i״״ P°lyP־PtW־s comprasung the ^lypep fragments and immunoglobulin fragments; genes J polypeptides, and the fragments; vectors comprrsrng g , transformants into which the vectors are introduced; antibodies having reactivity to the polypeptides or cell surface mol־־ ״־־ comprising the polypeptides; hybridomas producing the antrbodies, phXceulcal compositions comprising the polypeptide fragments or the fusion polypeptides; pharmaceutical compositions comprising the antibodies; transgenic mice; and knockout mice.

“³TuXTody of mammals has immune response systems that excludes pathogenic microorganisms (viruses, bacteria, paras1 es, etc.) or foreign bodies (both are called'antigen״ in the fo o g) that have invaded the living body. One of them 1־ called ״ immune response system, another acquired immune .

The former is an exclusion mechanism comprising phagocyt y phagocytes (polymorphonuclear leukocytes, monocytes macropM׳ etc.), attack by natural killer (MX) ־11־־, and no״-sp־° recognition such as opsonization of antigeu by complements The latter, acquired .immune response system, is an exclusion mechanism by lymphocytes (mainly, T cells and B cells) that specificity to the antigen (namely, activated lymphocytes) cells that acquired antigen specificity attack the antigen existing outside of the cells through production of antibodies speclfi י the antigen. I cells that acquired antigen specificity (name y, activated T cells) are classified into helper T cells and cytotox T cells (cytotoxic lymphocyte, CTL). The helper T cells regulat a differentiation of B cells and a production of antibodies, and destroy the antigen cooperating with phagocytes. The latter, CTLs attack virus-infected cells and so on by themselves (Experimental

Medicine: SUPPLEMENT, Bio Science Term Library, Immunity,

Yodosha, pp.14-17 (1995)).

This acquisition of antigen specificity by T cells (namely, activation of T cells) is initiated through recognition by T cells the antigen presented by antigen-presenting cells (APC) such as macrophage, B cells, or dendritic cells. Antigen-presenting cells process the antigens so incorporated and present these processed antigens through binding them to major histocompatibility complex (MHC). T cells receive primary signal for activation of the cells (or acquisition of specificity) by recognizing the processed antigens presented by antigen-presenting cells through a complex between T cell receptor (TcR) and CD3 antigen existing on the surface of the cell membrane (TcR/CD3 complex).

However, the T0R/CD3 complex-mediated primary signal alone cannot activate T cells sufficiently and leads to unresponsiveness or clonal anergy, so that the cells can not react with any stimulation received thereafter. The autocrine of interleukin 2 (IL-2) is necessary for T cells to be activated, to be differentiated into antigen specific T cell clones, and to be proliferated. In clonal anergy, T cells are inactivated due to no production of IL-2 and no cell division. Namely, the activation of T cells accompanied by production of cytokines such as IL-2 requires the secondary signal following the first signal through TcR/CD3 complex. This secondary signal is called costimulatory signal.

T cells receive this secondary signal and transmit it into the cells by interacting (cell adhesion) with molecules other than MHC on antigen-presenting cells through molecules other than TcR/CD3 complex on the T cell surface. This secondary signal avoids cell anergy (clonal anergy) and activates the cells.

Although some part of the mechanism of the secondary signal transmission between antigen-presenting cells and lymphocytes such as T cells have not yet been elucidated in detail, studies so far have revealed that an important factor for the secondary signal transmission is the interaction of CD28 (also named Tp44,T44, or

9.3 antigen), which is a cell surface molecule expressed mainly on T cells and .thymus cells, with CD80 (also named B7-1, B7, BB1, or B7/BB1), which is a cell surface molecule expressed on 5 antigen-presenting cells (macrophages, monocytes, dendritic cells, etc.) and with CD86 (also named B7-2 or B70), which is also a cell surface molecule on antigen-presenting cells (namely, cell adhesion through the binding between these molecules). Moreover, it has been experimentally elucidated that the interaction of 10 Cytolytic T lymphocyte associated antigen 4 (CTLA-4), whose expression is thought to be enhanced depending on the secondary signal, with the CD80 (B7-1) and CD86 (B7-2) (namely, cell adhesion through the binding between these molecules) also plays an important role in the regulation of T cell activation by the secondary signal.

In other words, the regulation of T cell activation by the transmission of the secondary signal involves at least the interaction between CD28 and CD80/CD86, the enhancement of CTLA-4 expression, which is thought to depend on the interaction, and the interaction between CTLA-4 and CD80/CD86.

CD28 is known to be a costimulator molecule transmitting the secondary signal (costimulatory signal) required for the activation of T cells and for the avoidance of anergy. The secondary signal transmitted by binding this molecule to costimulator molecules, CD80 (B7-1) and CD86 (B7-2), on 25 antigen-presenting cells (cell adhesion through the binding between these molecules), stabilizes mRNA of Thl-type cytokines and consequently promotes production by T cells of a large amount of Thl-type cytokines such as 11-2, IFN7 , and TNFa. The expression of CTLA-4 is induced by the primary signal transmitted through 30 TcR/CD3, and the expression is also enhanced by the secondary-signal transmitted by the binding between CD28 and CD80. It is being revealed that CTLA-4 receives these signals to work to inhibit T cell function, which is contrary to the activation of T cells by the secondary signal transmitted by CD28.

Human CD28 and CTLA-4 are I-type glycoproteins whose molecular weights are 44 kD and 41 to 43 kD, respectively. Both have an immunoglobulin-like domain, belong to the immunoglobulin superfamily, and have both function as a cell adhesion molecule and function as a signal transmission molecule.

Human CD28 forms a homodimer with a disulfide bond while CTLA-4 exists as a monomer. Both CD28 and CTLA-4 genes are located at 2q33 on human chromosome and 1C on mouse chromosome, and are composed of four (4) exons. Human CD28 and CTLA-4 are composed of 220 and 223 amino acids, respectively, including the leader sequences, and amino acid homology between them is 20 to 30%.

The ligands for CD28 and CTLA-4 are CD80 (B7-1) and CD86 (B7-2) in human and mice. CTLA-4 has about 20 times as high affinity to both ligands as CD28. It has been elucidated that the amino acid sequence structures MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)״ conserved through animal species is important for the binding of CD28 and CTLA-4 to CD80 (B7-1). It has also been reported that, when CD28 is stimulated, PI3 kinase (phosphoinositide 3 kinase, PI3K) associates with the phosphorylated tyrosine residue in a partial sequence YMNM (Tyr-Met-Asn-Met) of CD28 and that CD28 plays an important role in intracellular signal transmission through this YxxM structure. Furthermore, it has been reported that CTLA4 also has a sequence represented by YxxM,״ namely YVKM (Tyr-Val-Lys-Met) ״ in its cytoplasmic region and that, after being stimulated, SYP associates with this sequence.

CD28 is expressed specifically in thymocytes and peripheral blood T cells, and CTLA-4 is expressed specifically in activated T cells (Cell Engineering: SUPPLEMENT, Handbook of Adhesion Molecule, Shujunsha, pp.93-102 (1994); ibid. pp.120-136; Experimental Medicine: SUPPLEMENT, BIO SCIENCE Term Library, Immunity, Yodosha, pp.94-98 (1995); Experimental Medicine: Supplement, ״bio science Term Library, intracellular ־signal Transduction, Yodosha, pp.58-59 (1997); Nihon Rinsho,. Vol.55,, No.6, pp.215-220 (1997)).

In the regulation of T cell function (the activation and the inhibition of function of T cells), the importance of interactions among multiple molecules such as costimulator molecules (CD2 8, CD80 (B7-1), CD86 (B7-2), etc.) and CTLA-4, which cooperates with them.

(in other words, cell adhesion through the binding between these molecules) has thus been recognized, and this has been drawn attention to the relationship between these molecules and diseases, and the treatment of diseases by regulating the function of these molecules.

As described above, although a living body activates its acquired immune response system against antigens that are foreign bodies to the living body (self), it also has immunological tolerance so as to show no immune response against its own component 10 (autoantigen). If immunological tolerance breaks down by some reason, immune response to the autoantigen occurs, autoantigen-reactive T cells are induced by the same mechanism as mentioned above to fall into abnormal state of immunity, and various autoimmune diseases are caused.

In other words, since non-stimulated antigen presenting cells (APC) in normal tissues do not express costimulatory molecules when the immune system of a living body is normal, T cells are in the unresponsiveness state to maintain immunological tolerance even if autoantigen-reactive T cells, which reacts with autoantigen, 20 exist. It has been suggested that in abnormal state of immunity, more autoantigen-reactive T cells are activated due to abnormal excess and continuous expression of costimulatory molecules to thereby cause autoimmune diseases.

From such viewpoints recently, many attempts to treat various 25 autoimmune diseases by modulating the transmission of costimulatory signals, for example, the above-mentioned signal transmission between CD28/CTLA-4 and CD80/CD86, are proposed.

It has been observed CD80, a costimulatory molecule as the ligand of CD28 and CTLA-4, is abnormally expressed in the antigen 30 presenting cells at the nidus of autoimmune disease such as rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact-type dermatitis, and chronic inflammatory dermatosis such as lichen planus, and psoriasis. From such observation, many attempts to treat various autoimmune diseases 35 by modulating the function of CD80 have been made.

It has been proposed to block the function of CD80, by methods using an antibody against CD80, solubilized protein of CD28 that is a ligand of CD80, and solubilized protein of CTLA-4 that is also a ligand of CD80. Particularly, based on the fact that the binding affinity of CTLA-4 to CD80 is 20 or more times higher than that 5 of CD28, therapeutic attempts using solubilized CTLA-4,״ specifically, the fusion protein (CTLA-4-IgFc) comprising the extracellular domain of CTLA-4 and the Fc region of human immunoglobulin IgGl, were performed in animal model and clinical tests (Nihon Rinsho, Vol.55, No.6, pp.215-220 (1997)).

As shown in 1 to 5 below, therapeutic effects of CTLA-4-IgFc in model animals of autoimmune diseases has been reported.

1. In a (NZB/NZW)F1 mouse, that is a model for human systemic lupus erythematosus (SLE), the production of autoantibodies and the onset of lupus nephritis were suppressed by administration of

CTLA-4-lgFc before the onset, and the pathologic conditions were improved by administration of the drug even after the onset (Science, Vol.125, p.1225-1227 (1994)).

2. In experimental allergic encephalomyelitis (EAE), that is a model for multiple sclerosis (MS), the onset was prevented by short-term administration of CTLA-4-IgFc immediately after immunization (J. Clin. Invest., Vol.95, pp.2783-2789 (1995)).

3. In an NOD (non-obese diabetes) mouse, which is a model for insulin dependent diabetes mellitus (IDDM), the onset rate was remarkably decreased by administering CTLA-4-IgFc to the 2- or

3-week-old female mouse for two weeks (J. Exp. Med., Vol.181, pp.1145-1155 (1995)).

4. In rat nephritis by renal glomerulus basement membrane immunity. Goodpasture's nephritis model, the improvement of the symptom has been improved by the administration of CTLA-4-IgFc (Eur.

J. Immunol., Vol.24, No.6, pp.1249-1254 (1994)). ־־

5. In type II collagen-induced arthritis (CIA) using a DBA/1 mouse, that is a model for human rheumatoid arthritis, the onset of arthritis was suppressed by administering the test drug at the time of the immunization and the production of autoantibodies (IgGl and IgG2) against collagen was inhibited (Eur. J. Immunol., Vol.26, pp.2320-2328 (1996)).

6a

U.S application No. 5,484,892 discloses a series of monoclonal antibodies directed against CD22. The monoclonal antibodies (mAb) specifically block red blood cell and leukocyte adhesion (80100%־) to COS cells transfected with CD22 cDNA and also identify a region of CD22 distinct from those defined by previously described CD22 mAb.

U.S application No 5, 506, 126 discloses a rapid immunoselection cloning method for cloning cDNAs from mammalian expression libraries based on transient expression in mammalian host cells. The method is used to clone genes for cell surface antigens of human lymphocytes.

U.S application No. 5,521,288 discloses fragments and derivatives of the B7 antigen and CD28 receptor, including fusion proteins having amino acid sequences corresponding to the extracellular domains of B7 or CD28 joined to amino acid sequences encoding portions of human immunoglobulin C gamma 1.

US application No. 5,770,197 discloses the complete amino acid sequence encoding 15 human CTLA4 receptor gene and methods for expressing CTLA4 as an immunoglobulin fusion protein, for preparing hybrid CTLA4 fusion proteins, and for using the soluble fusion proteins including monoclonal antibodies reactive with B7 and CTLA4, to regulate T cell interactions and immune responses mediated by such interactions.

U.S application No. 5,914,112 discloses a method for improving clinical outcome in focal ischemic stroke in a mammal by increasing cerebral blood flow and/or reducing infarct size which involves administering an effective amount of an anti-CD18 antibody to the mammal, in the absence of removal of the arterial obstruction.

U.S application No. 6,075,181 discloses antibodies with fully human variable regions 25 against a specific antigen which can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled.

U.S application No. 2002164697A discloses Th2-specific polypeptides, proteins, nucleic acid molecules and also isolated Th2-specific fusion proteins, antigenic 30 peptides, and anti-Th2-specific antibodies.

U.S application No. 2002177191 discloses methods and compositions for the prevention and treatment of immune system disorders, including cancer, AIDS, asthmatic disorders, autoimmune diseases, organ transplant rejection and chronic viral diseases such as HCV or HBV infections comprising administering molecules that modulate the 35 activity of 8F4, thereby modulating costimulation of T cells.

U.S application No 2002182667 and German application No DE 19821060 disclose the 8F4 molecule for preventing or treating diseases which involve the immune system.

European application No. EP0984023 discloses cell surface molecules recognized by monoclonal antibodies against a cell surface molecule of lymphocytic cells that play an 40 important role in autoimmune diseases and allergic diseases have been isolated, identified, and analyzed for their functions.

International publication No. WO 98\38216 discloses a cell surface molecule recognized by a monoclonal antibody, which is expressed specifically in thymocytes, lymphocytes activated by ConA-stimulation and peripheral blood lymphocytes. An 45 antibody against this molecule significantly ameliorates conditions of autoimmune diseases and allergic diseases.

Japanese application No. JP11228442 discloses a method for administrating fructose1,6-diphosphate to a patient by phleboclysis after the organ transplantation operation.

6b

Japanese application No. JP2000154151 discloses an immunosuppressant containing an active material such as flavonoids and chromones selectively suppressing an activated T cell.

International publication No. WO 95/33770 discloses isolated ligands which bind a molecule expressed on the surface of T cells and induce antigen specific apoptosis in activated T cells. Preferably, the T cell surface molecule is CTLA4 and the ligand is a monoclonal anti-CTLA4 antibody that binds to an epitope of CTLA4 distinct from the binding sites of B7-1 and B7-2.

International publication No. WO 97/26912 discloses a method for improving clinical outcome in focal ischemic stroke in a mammal by increasing cerebral blood flow and/or reducing infarct size is described which involves administering an effective amount of an anti-CD18 antibody to the mammal, in the absence of removal of the arterial obstruction.

International publication No. WO 98/11909 discloses the use of proteins extracted with perchloric acid from animal organs, for the preparation of medicaments active against autoimmune diseases, in particular with activity against atherosclerosis, arthritis, multiple sclerosis, diabetes.

International publication No. WO 98/19706 discloses antibodies which are specific to human B7.1 antigen (CD80) and which are capable of inhibiting the binding of B7.1 to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor.

International publication No. WO 98/37415 discloses a substantially pure UC pANCA secretory vesicle antigen, including a protein immunoreactive with NANUC-1 and NANUC-2 and having the characteristic of being selectively expressed in secretory vesicle membrane.

International publication No. WO 98/45331 discloses humanized and variant antiVEGF antibodies and various uses thereoff.

International publication No. WO 00/19988 discloses Th2-specific polypeptides, proteins, and nucleic acid molecules as well as isolated Th2-specific fusion proteins, antigenic peptides, and anti-Th2-specific antibodies.

International publication No. WO 00/46240 discloses polypeptides which comprise a receptor-ligand pair involved in T-cell activation.

International publication No. WO 00/67788 discloses methods of enhancing immune responses in which soluble forms of costimulatory molecules, e.g., B7 molecules, are administered to augment immune responses to antigens, e.g., to tumor cells and infectious agents.

International publication No. WO 01/08700 discloses a method of inhibiting and diagnosing spontaneous abortion in a subject based, inter alia, on the administration of an agent that inhibits a costimulatory signal in a T cell such that spontaneous abortion in the subject is inhibited.

International publication No. WO 01/12658 discloses a soluble form of B7-3, comprising the extracellular domain shown to bind ICOS, as well as assay methods for obtaining agents for modulation of the interaction between B7-3 and ICOS.

International publications No. WO 01/15732 and European application No. EPl 125585 disclose antibodies against AILIM (also called JIT-1 antigen, JTT-2 antigen, ICOS and 8F4) exert significant therapeutic effects on joint diseases such as articular rheumatism and arthrosis deformans, graft-versus-host disease, transplantation immunological rejection, inflammation (hepatitis, inflammatory diseases, etc.) and symptoms in association with immune sensitization by a foreign antigen and the thus induced

6c hyperproduction of an antibody against the antigen.

International publications No. WO 01/18022, WO 01/32675 disclose human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins.

International publication No. WO 01/21796A discloses isolated nucleic acids molecules, designated GL50 nucleic acid molecules, which encode novel GL50 polypeptides.

International publication No. WO 01/64704 discloses hB7-H2 polypeptides useful for co-stimulating T cells.

International publication No. WO 01/87981 discloses the immunization of human antibody-producing transgenic mice, created using genetic engineering techniques, with AILIM molecule as an antigen.

International publication No. WO 02/44364 discloses polypeptides which comprise a receptor-ligand pair involved in T-cell activation.

International publication No. WO 02/070010 discloses an antibody against AILIM (also called ICOS and 8F4) and AILIM-Ig having a significant therapeutic effect of inhibiting or preventing graft rejection.

International publication No. WO 02/076504 discloses ICOS for inhibiting the onset of inflammatory bowel diseases.

Robert J. et al., (Exp Cell Res. 1997. 25;235(l):227-37) reports that antibody crosslinking of CTX in vitro inhibits the growth of tumor cells and causes morphological alterations demonstrating that multinucleation can be induced by specifically crosslinking a cell surface molecule.

Heyeck SD. et al., (Proc Natl Acad Sci USA. 1993. 15;90(2):669-73) reports on the isolation of a murine tyrosine kinase gene, called Tsk, that appears to be exclusively expressed in T lymphocytes and which mRNA levels are 5- to 10-fold higher in thymocytes than in peripheral T cells and increase in the thymus during mouse development from neonate to adult.

Marguet D. et al., (J Biol Chem. 1992. 5;267(4):2200-8) reports on the mouse homologue of the human T cell-activating ectoenzyme CD26 (dipeptidyl peptidase IV, DPP IV).

/3

The results of the experiments as mentioned above are have not yet clarified in detail the mechanism of the T cell activation by interaction between costimulatory molecules and the related molecules (in other words, cell adhesion through the binding between these molecules). Other unknown molecules may be involved in this mechanism.

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided a purified non-hamster antibody or portion thereof that binds to a polypeptide consisting of SEQ ID NO:2.

In another aspect the present invention is directed to a purified chimeric, humanized or human monoclonal antibody or a portion thereof that binds to a polypeptide consisting of SEQ ID NO:2.

In yet another aspect of the present invention there is provided a purified chimeric, humanized or human monoclonal antibody or a portion thereof that binds to a polypeptide consisting of SEQ ID NO: 13.

In a further aspect the present invention provides a purified non-hamster antibody or portion thereof that binds to a polypeptide consisting of SEQ ID NO: 14.

In yet a further aspect the present invention provides a purified chimeric, humanized or human monoclonal antibody or a portion thereof that binds to a polypeptide consisting of SEQ ID NO: 14.

DISCLOSURE OF THE INVENTION

Pharmaceuticals useful for treating or preventing various diseases such as the above-mentioned autoimmune diseases, allergic diseases, and inflammatory diseases can be developed if the mechanism of the activation of lymphocytes such as T cells by cell adhesion through the binding between molecules involved in the transmission of the secondary signal essential for the activation of lymphocytes such as T cells mentioned above and the mechanism of the regulation of lymphocyte function are clarified, and known or unknown molecules capable of mediating cell adhesion involved in the mechanism and of transmitting signals are identified and characterized.

a131584/2

An objective of the present invention is to identify novel cell surface molecules having both functions of mediating such cell adhesion and signal transmission, and to clarify its structural and biological characteristics. Another objective of the present invention is to provide pharmaceuticals useful for treating or preventing various autoimmune diseases and inflammatory diseases by using the novel molecules or antibodies against the molecules.

Passages of the description which are outside the scope of the claims do not constitute part of the claimed invention.

7b 131584/1

In order to identify such useful molecules, the present inventors focused on the fact that lymphocytes such as T cells play an important role in autoimmune diseases, and the fact that cell adhesion are essential for the signal transmission of the secondary signal (costimulatory signal) from antigen presenting cells into lymphocytes, and planned to isolate and identify cell surface molecules that are expressed specifically on lymphocytic cells and that mediate cell adhesion.

The present inventors obtained monoclonal antibodies against various cell surface molecules expressed on the surface of lymphocytic cells by immunizing animals against the lymphocytic cells, and isolated and identified desired cell surface molecules that mediate cell adhesion using the monoclonal antibodies so obtained. The methods used are described in detail below.

The present inventors first administered rat lymphocytic cell line as an antigen to mice and prepared various monoclonal antibodies. Then, the monoclonal antibodies obtained were reacted with rat lymphocytic cells used as an antigen and tested the effect of the monoclonal antibodies given to the cells. As a result, one 10 of the monoclonal antibodies was found to agglutinate the rat lymphocytic cells strongly (this monoclonal antibody was designated JTT-1 antibody״). Moreover, other one of the monoclonal antibodies was found to strongly inhibit the agglutination of rat lymphocytic cells induced by the ״JTT-1 15 antibody (this monoclonal antibody was designated JTT.2 antibody).

Since the agglutination of rat lymphocytic cells by JTT-1 antibody was not inhibited by antibodies against Intercellular adhesion molecule-1 (ICAM-1) or Lymphocyte function-associated 20 antigen-1 (LFA-1), which are the most representative known cell adhesion molecules expressed on the cells, the present inventors thought that this agglutination was caused by cell adhesion through unknown adhesion molecules that mediate cell adhesion.

Cell surface molecules (designated JTT-1 antigen and JTT.2 25 antigen) recognized by each of these two monoclonal antibodies were then identified, isolated, and characterized.

First, the analysis of the expression patterns of JTT-1 antigen and JTT.2 antigen in various cells were analyzed by flow cytometry based on fluorescent antibody technique using JTT-1 30 antibody and JTT-2 antibody. while both JTT-1 antigen and JTT.2 antigen were strongly expressed in activated lymphoblast cells (activated T lymphoblast cells, activatedB lymphoblast cells , etc.) activated by stimulating thymocytes and spleen cells with Concanavalin A (ConA), a mitogen, in particular, in the activated 35 lymphoblast cells, the expression was hardly found in spleen cells not stimulated at all (these cells are sometimes called resting lymphocytes herein). The expression patterns of molecules recognized by each of JTT-1 antibody and JTT-2 antibody were almost the same.

Using an affinity column prepared by binding JTT-1 antibody 5 to adsorbents, molecules trapped by the JTT-1 antibody, namely, JTT-1 antigens were purified from the mixture of soluble cell surface molecules prepared from the above-described rat lymphocytic cells. The molecular weights of these purified JTT-1 antigens were analyzed by immunoprecipitation using JTT-1 10 antibody and JTT-2 antibody and by SDS-PAGE. As a result, it was found that molecules immunoprecipitated by each of JTT-1 antibody and JTT-2 antibody were the same, and that each molecule was a homodimer having different sugar chains. Specifically, when N-linked sugar chains were not digested, the molecules were 15 identified as one molecule with about 47 kD under non-reduction condition, and as two molecules with about 24 kD and about 28 kD under reduction condition; when N-linked sugar chains were digested, the molecules were identified as one molecule with about 36 kD under non-reduct ion condition and as one molecule with about 20 kD under 20 reduction condition.

The adhesion of rat thymocytes to the plate coated by the purified JTT-1 antigen״ was then analyzed. As a result, thymocytes significantly adhered to the plate (namely, to JTT-1 antigen) only in the presence of JTT-1 antibody and the adhesion 25 was significantly inhibited in the co-presence of ״JTT.2 antibody, indicating that JTT-1 antigen was the cell surface molecule mediating cell adhesion.

Next, the present inventors cloned genes encoding JTT-1 antigen״ from rat, human, and mouse, and analyzed their structures. 30 First, the cDNA encoding the full length of rat JTT-1 antigen was isolated from the cDNA library made from the lymphoblasts derived from ConA-stimulated rat spleen by, expression cloning method utilizing panning method using JTT-1 antibody and a completely novel rat gene was isolated and identified by determining 35 its nucleotide sequence by dideoxy method. The cDNA encoding the full length of human JTT-1 antigen was also isolated from the cDNA library made from ConA-stimulated human peripheral blood lymphoblasts by plaque hybridization using the cDNA encoding rat JTT-1 antigen״ -so obtained as a probe and a completely novel human gene was isolated and identified by determining its nucleotide 5 sequence by dideoxy method. Similarly, the cDNA encoding the full length of mouse JTT-1 antigen״ was isolated from the cDNA library made from the lymphoblasts derived from ConA-stimulated mouse spleen and a completely novel mouse gene was isolated and identified by determining its nucleotide sequence by dideoxy method.

Furthermore, the cDNA encoding the full length of alternative splicing variant of rat JTT-1 antigen״ mentioned above was isolated similarly from the cDNA library made from the rat thymoma cell line and another completely novel rat gene was isolated and identified by determining its nucleotide sequence by dideoxy method.

JTT-1 antigen was found to be a transmembrane protein (cell surface molecule) composed of a signal sequence, an extracellular region having the glycosylation site(s), a transmembrane region, and an intracellular region by hydropathy plot analysis of the amino acid sequence encoded by the isolated cDNA of human JTT-1 antigen.

Homology search with known molecules revealed that JTT-1 antigens from rat, human, and mouse had no significant homology to any known molecules including cell adhesion molecules, indicating that they are novel cell surface molecules that mediates cell adhesion.

As the result of motif search based on the amino acid sequence 25 of human JTT-1 antigen, it was found that human JTT-1 antigen had structural similarity with the above-mentioned CD28, a cell surface molecule on lymphocytes such as T cells, which transmits costimulatory signal important for T cell activation through cell adhesion, and with CTLA-4, a cell surface molecule on lymphocytes 30 such as T cells, which regulate the functions of activated lymphocytes such as activated T cells, cooperating with the signal.

The structural similarlity is as follows.

1. 20 or more amino acid residues including cysteine residues are highly conserved.

2. Proline repeating sequence ״Pro-Pro-Pro (PPP)״ essential as the ligand binding region, is conserved in the extracellular region.

3. A sequence Tyr-Xaa-Xaa-Met (YxxM) (Xaa and x represents any amino acid) sequence essential as the signal transmitting region is conserved in the cytoplasmic region.

The locus of the gene encoding mouse JTT-1 antigen on mouse 5 chromosome was found to be 1C3, which is the same location as that of mouse CD28 and CTLA-4 using fluorescence in situ hybridization (FISH) method.

Next, the effectiveness of therapy of autoimmune diseases and allergic diseases by regulating the function of JTT-1 antigen״ 10 was examined by experiments in which JTT-2 antibody mentioned above was administered to model rats for experimental allergic encephalomyelitis (EAE) and glomerulus basement membrane (GBM) nephritis. It was found that the pathological states were significantly suppressed in both disease model animals, and that 15 autoimmune diseases or allergic diseases can be treated by regulating the functions of JTT-1 antigen.

It was also found that the monoclonal antibody against human JTT-1 antigen״ significantly proliferated human peripheral blood lymphocytes, and that the proliferation was further enhanced in 20 the co-presence of a monoclonal antibody against CD3 constituting a TcR/CD3 complex on T cells, which receives the primary signal essential for T cell activation from antigen presenting cells, indicating that JTT-1 antigen״ was a cell surface molecule involved in signal transmission into lymphocytes.

Furthermore, the present inventors succeeded in producing a fusion polypeptide comprising of the extracellular region of human JTT-1 antigen and Fc region of human immunoglobulin. The fusion polypeptide is useful as pharmaceuticals for treating autoimmune diseases, allergic diseases, and inflammatory diseases by regulating the functions of JTT-1 antigen״ and/or its ligand.

Moreover, the present inventors succeeded in preparing a transgenic mouse into which a gene encoding JTT-1 antigen of other animal species was introduced. The transgenic mouse is useful for analyzing detailed functions of JTT-1 antigen and for developing 35 pharmaceuticals for treating autoimmune—-diseases, allergic—-, diseases, and inflammatory diseases. The inventors also produced a knockout mouse in which the endogenous gene encoding mouse JTT-1 antigen״ was inactivated. This knockout mouse is also useful for the above-mentioned purpose.

The present inventions relate to polypeptides, genes, 5 antibodies, vectors, transformants, pharmaceutical compositions, transgenic mice, knockout mice and so on, which are relevant to a novel mammalian JTT-1 antigen isolated and identified as mentioned above. Specifically, the present invention are as described in (1) to (36) below.

(1) A polypeptide constituting a cell surface molecule having characteristics below, (a) said cell surface molecule is expressed in at least thymocytes and mitogen-stimulated lymphoblast cells, (b) an antibody reactive to said cell surface molecule induces 15 adhesion between mitogen-stimulated lymphoblast cells, (c) an antibody reactive to said cell surface molecule induces proliferation of peripheral blood lymphosytes in the presence of an antibody against CD3, (d) said cell surface molecule has a partial amino acid sequence 20 represented by Phe-Asp-Pro-Pro-Pro-Phe in its extracellular region, and (e) said cell surface molecule has a partial amino acid sequence represented by Tyr-Met-Phe-Met in its cytoplasmic region.

(2) The polypeptide of (1) comprising the amino acid sequence of 25 SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, or added.

(3 ) The polypeptide of (1), which is encoded by a DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions.

(4) The polypeptide of (1) comprising an amino acid sequence having 60% or more homology with an amino acid sequence of SEQ ID NO: 2.

(5) The polypeptide of any one of (1) to (4) wherein said cell surface molecule is derived from human.

(6) A gene encoding the polypeptide of any one of (1) to (5).

(7) The gene of (6) wherein said gene is a cDNA.

(8) The gene of (7) wherein said cDNA has a nucleotide sequence of SEQ ID NO: 1.

(9) The gene-of (7) wherein said cDNA comprises a nucleotide sequence corresponding to nucleotide residues 26 to 625 of SEQ ID

NO! 3, nucleotide residues 35 to 637 of SEQ ID NO: 4, nucleotide residues 1 to 603 of SEQ ID NO: 5, or nucleotide residues 35 to 685 of SEQ ID NO: 6.

(10) A vector comprising the gene of any one of (6) to (9).

(11) A transformant into which the vector of (10) has been 10 introduced.

(12) A transformant identified by an international deposit accession No. FERM BP-5725.

(13) A polypeptide fragment comprising an extracellular region of the polypeptide of any one of (1) to (5).

(14) The polypeptide fragment of (13) wherein said polypeptide is a human-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.

(15) A gene encoding the polypeptide fragment of (13) or (14).

(16) A homodimer molecule comprising two polypeptide fragments, 20 wherein each of the fragments comprises an extracellular region of the polypeptide of any one of (1) to (5) and said two polypeptide fragments bridged through disulfide bonds to each other.

(17) The homodimer molecule of (16) wherein said polypeptide is a human-derived polypeptide having an amino acid sequence of SEQ

ID NO: 2.

(18) A pharmaceutical composition comprising either of the polypeptide fragment of (14) or the homodimer molecule of (17), or both of them, and a pharmaceutically acceptable carrier.

(19) A fusion polypeptide comprising an extracellular region of 30 the polypeptide of any one of (1) to (5) and a constant region of a human immunoglobulin (Ig) heavy chain or a portion of the constant region.

(20) The fusion polypeptide of (19) wherein the immunoglobulin is IgG.

(21) The fusion polypeptide of (19) wherein the portion of the constant region comprises a hinge region, C2 domain, and C3 domain of IgG.

(22) The fusion polypeptide of any one of (19) to (21) wherein said polypeptide is a human-derived polypeptide having an amino acid sequence of SEQ ID NO: 2.

(23) A homodimer molecule comprising two fusion polypeptide of any one of (19) to (22) wherein the two polypeptides bridged through disulfide bonds to each other.

(24) A homodimer molecule comprising two fusion polypeptides of (22) wherein the two polypeptides bridged through disulfide bonds to each other.

(25) A pharmaceutical composition comprising either of the fusion polypeptide of (22) or the homodimer molecule of (24), or both of them, and a pharmaceutically acceptable carrier.

(26) The pharmaceutical composition of (25) wherein said pharmaceutical composition is utilized for treating autoimmune diseases or allergic diseases, or for preventing said disease symptom.

(27) An antibody or a portion thereof reactive to the polypeptide of any one of (1) to (5), the polypeptide fragment of (13) or (14), or the cell surface molecule comprising said polypeptide.

(28) The antibody of (27) or a portion of it wherein said antibody is a monoclonal antibody.

(29) A monoclonal antibody or a portion thereof reactive to the polypeptide having an amino acid sequence of SEQ ID NO: 2, the polypeptide fragment of (14), or the human-derived cell surface molecule comprising said polypeptide.

(30) A monoclonal antibody or a portion thereof reactive to the polypeptide of any one of (1) to (5) or the cell surface molecule comprising said polypeptide, wherein the effect of said monoclonal antibody on mitogen-stimulated lymphoblast cells is substantially the same as the effect of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-5707 on mitogen-stimulated rat lymphoblast cells.

(31) A monoclonal antibody or a portion thereof reactive to the polypeptide of any one of (1) to (5) or the cell surface molecule comprising said polypeptide, wherein the effect of said monoclonal antibody on mitogen-stimulated lymphoblast cells is substantially the same as the effect of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM

BP-5708 on mitogen-stimulated rat lymphoblast cells.

(32) A pharmaceutical composition comprising the monoclonal antibody of (29) or a portion thereof and a pharmaceutically acceptable carrier.

(33) The pharmaceutical composition of (32) wherein said pharmaceutical composition is utilized for treating autoimmune diseases or allergic diseases, or for preventing said disease symptom.

(34) A hybridoma producing the monoclonal antibody of any one of (28) to (31).

(35) A transgenic mouse in which a gene encoding the polypeptide of (1) which is a human-derived gene comprising a nucleotide sequence of SEQ ID NO: 1 or a rat-derived gene comprising a nucleotide sequence corresponding to nucleotide residues 35 to 637 of SEQ ID NO: 4, which is integrated into its endogenous gene.

(36) A knockout mouse in which its endogenous gene encoding the mouse polypeptide of claim 1 comprising the amino acid sequence encoded by the gene of SEQ ID NO: 5 is inactivated so that said mouse polypeptide is not produced.

As described above, the cell surface molecule of the present invention (JTT-1 antigen) is involved in cell adhesion, signal transmission into lymphocytes such as T cells, and regulation of function of activated lymphocytes. General knowledge of lymphocytic cells, cell adhesion molecules, and the relationship between them and diseases are described below just for general understanding of these biological events, but the following general knowledge is not for interpreting the present invention limitedly.

Lymphocytes are roughly classified into two kinds, T cells and B cells. After differentiation from multipotent stem cells in bone marrow to lymphoid stem cells, some of them flow into blood to reach thymus. Lymphocytes differentiated and matured in thymus, which are called T cells (Thymus-derived T cells) , get into blood again, and circulate through the whole body. Matured T cells have a molecule called CD3 on their surface. The existence of CD3 molecule is an marker to determine whether the cells are matured T cells or not. CD3 is a convincing T cell marker. In addition, T cells express CD4 or CD8. T cells are classified into helper T cells (Th 5 cells) assisting the antibody production by B lymphocytes, cytotoxic T cells (Tc cells, CTL) or killer T cells that are bound to target cells to destroy them directly, suppressor T cells that suppress the antibody production by B lymphocytes, and effector T cells that secrete effector substances such as lymphokines to 10 cause delayed allergy.

B cells are derived from the lymphoid stem cells differentiated and matured in bone marrow. B cells are antibody-producing precursor cells since they produce antibodies with an appropriate stimulus. B cells have immunoglobulins on their cell surface, 15 which were produced in a cell. Such immunoglobulins function as receptors for antigens. Matured B cells have both IgM and IgD on their surface. If B cells are differentiated with antigen stimulation and signals from T cells, the production of IgM increases and their C-terminal cell membrane binding regions are 20 changed to be secreted. With sufficient stimulation, not only the surface immunoglobulins change into IgG, IgE, and IgA, but also the immunoglobulins of each class are secreted. The immunoglobulin on the B cell surface is sometimes represented as sig, abbreviation of surface Ig, or mlg, abbreviation of membrane Ig. All Igs on the 25 surface of the same B cell have the same antigen binding sites.

There are lymphocytes called large granular lymphocytes (LGL) or null cells, which are neither T cells nor B cells. These cells can destroy tumor cells and virus-infected cells without prestimulation with antigen, which is comparative to the case of 30 cytotoxic T cells. So, they are also called natural killer־cells (NK cells).

Among the T cells mentioned above, CD4-positive T cells secrete various cytokines, newly express receptors for these cytokines, enlarge their own size, start cell dividing, and proliferate, when 35 they react with antigens presented by antigen-presenting cells. Prior to these reactions at the cell level, the complex of the antigen peptides on antigen presenting cells and MHC class II molecules binds to the coresponding T cell antigen receptor (TCR). This causes various biochemical changes in the cells, and the signal is transmitted into nuclei to start the transcription of 5 specific DNAs and to produce respective proteins. As a result, reactions at the cell level are raised. For example, cells infected with a virus produce virus proteins and they are degraded into peptides by proteasomes in the cytoplasm. A part of the peptides enters endoplasmic reticulum through TAP, forms stable complex with 10 MHC class I molecules just produced, and transfers to the cell surface. The peptide transferred to the cell surface is recognized specifically by CD8-positive T cells, but the T cells can not yet destroy the infected cells at this stage. These T cells reacted with the antigen expresses IL-2 receptor (IL-2R), are 15 differentiated into CTL cellular cytotoxicity upon IL-2 action, and destroy their target cells to kill them in the next time when they meet the same antigen peptide/MHC class I complex. Cytokines required for the differentiation into CTL are not only IL-2 but also IFN/ or other cytokines, which are thought to have similar 20 actions. Thus, lymphokines secreted by T cells are necessary for the differentiation into CTL. The lymphokines are produced as the result that CD4-positive Thl cells (CD4-positive T cells secreting IL-2 or IFN/ ) recognize the antigen peptides derived from the same virus with class II molecules. In some cases, without the help of 25 CD4-positive T cells, CD8-positive T cells react with antigens and produce IL-2 and other cytokines. When CD8-positive T cells are differentiated into CTL, granules increase in the cytoplasm. These granules comprise various high molecular weight proteins, represented by perforin. Perforin resembles a membrane attack 30 complex (MAC) composed of the fifth to ninth components of complement, and makes holes in the cell membrane of target cells. In addition, the granules comprise serine proteases, LT, proteoglycan, etc. Moreover, if CD8-positivecells differentiated into CTL receive antigen stimulation, they also secrete lymphokines 35 such as IFN/, LT, TNF, or IL-2. Moreover, T cells show blast transformation phenomenon, when they react with hemagglutinin (phytohemagglutinin, PHA) or ConA.

Matured T cells not stimulated at all are called resting T cells, and have smaller cell size and shorter lifetime, a few days. When they receive stimulation, the cells enlarge as already mentioned 5 above, and are apt to react with various kinds of stimulation. Such T cells are called activated T cells. A part of the activated T cells become memory T cells, which bring secondary immunoreaction if they receive the same antigen stimulation. Memory T cells are thought to be kept in circulating around the body for a few years 10 or decades.

B cells not stimulated at all are called resting B cells like in the case of T cells, and proliferating B cells stimulated with multivalent antigens or CD40L, are called activated B cells. Since resting B cells have no costimulator molecules, which stimulate 15 T cells with signals through TCR, such as B7-1 (CD80) or B7-2 (CD86), presenting antigens to resting T cells are thought only to stimulate TCR and to be unable to express CD40 ligands (CD40L) or produce lymphokines. Therefore, it is thought that activated helper T cells stimulated with antigen presented by other antigen20 presenting cells react with the antigen presented by resting B cells.

Namely, if an antigen invades, first, dendritic cells (cellshaving extremely dendritic projections) expressing B7 molecules or macrophages activated by reacting with microorganisms present the antigen and stimulate resting helper T cells to activate them so 25 as to express CD40L. The activated helper T cells then bind to resting B cells presenting the same antigen and stimulate their CD40. Once B cells are activated by stimulation with multivalent antigens or CD40L, they also express B7 molecules, activate helper T cells by stimulating CD28 on their surface with TCR, and allow 30 the helper T cells to express CD40L or produce lymphokines. B־cells that show changes such as the expansion of the cell size with Stimulation but not show antibody secretion are called activated B cells. If B cells so matured meet antigens, the IgM production increases together with the stimulation from T cells and the IgM 35 molecules so produced are secreted by turning from the membrane type into secretory type. Moreover, they produce isotypic antibodies other than igM, such as IgG upon the humoral factors from T cells. This is called isotype switching or class switching.

B cells secreting antibodies are called antibody-secreting cells.

A part of them becomes morphologically characteristic cells and is called a plasma cell (Knowledge of Immunology, Ohmsha, (1996)).

Incidentally, in various reactions of immune system, the subpopulation of white blood cells, namely, T lymphocytes, B lymphocytes, NK, neutrophils, etc. often show dynamics different from one another. Even the same lymphocytes as mentioned above show 10 dynamics different from one another depending on whether the cells are activated or resting. These facts imply the existence of recognition mechanism specific to the subpopulation of white blood cells, further, recognitionmechanismspecific to the state of cells, and, in particular, cell adhesion molecules (cell adhesion 15 proteins).

Cell adhesion molecules, or cell adhesion proteins are, in general, the molecules that adhere cells to each other in the development and differentiation of individuals or in migration of cells to local site, and are known to be essential molecules for 20 organic and functional contacts in a living body.

Cell adhesion molecules are roughly classified from their structural characteristics into five (5) families, immunoglobulin superfamily, integrin family, selectin family, cadherin family, and CD44 family. Adhesion molecules belonging to immunoglobulin 25 superfamily are characterized by the existence of repeated loop-like domains formed with disulfide bonds. Examples thereof are intercellular adhesion molecule-1 ICAM-1 and vascular cell adhesion molecule-1 VCAM-1.״ In addition, adhesion molecules belonging to integrin family are characterized by α/β heterodimer 30 structure. Examples thereof are very late antigen-1 to 6~ VLA1 to 6, lymphocyte function-associated antigen-1 LFA-1, Mac-1,״ and pl50/90. Molecules belonging to selectin family have lectin-like domain, EGF-like domain, and complement domain in this order from N terminus. Examples thereof are E-selectin״ 35 and P-selectin.״ Examples of cadherin family are ״E-cadherin,״ N-cadherin, and P-cadherin,״ and an example of CD44 family is

CD44״.

The specific function of these adhesion molecules is known to be adhesion of -white blood cells to vascular endothelial cells or of lymphocytes to antigen-presenting cells. From recent various 5 studies, it has been gradually revealed that adhesion molecules are involved not only in these functions but also in various diseases.

In particular, there are many reports on diseases and expression abnormality of adhesion molecules. For example, as for rheumatoid 10 arthritis (RA), the expression of both “Mac-1״ and pl50/95״ was reportedly strengthened in RA synoviocytes (Allen, C. et al. Arthritis Rheum., Vol. 32, p.947 (1989)). It has also been reported that various cells expressed ״ICAM-1 strongly and ectopically on RA synovial membrane (Hale, L. et al. Arthritis Rheum., Vol.32, 15 p.22 (1989)). Another report implied that ELAM-1״ was also involved in the adhesion of neutrophils to vascular endothelial cells and that the overexpression of these molecules was involved in infiltration of neutrophils (especially, into synovial fluid), which is observed in RA synovial fluid (Laffon, A. et al., Arthritis 20 Rheum., Vol.32, p.386 (1989)). Strong expression of CD44 in vascular endothelial cells and A-type synoviocytes on RA synovial membrane was reported (Heynes, B. et al., Arthritis Rheum., Vol.34, p.1434 (1991)).

There are reports on the relationship between systemic lupus 25 erythematosus (SLE) and the expression abnormality of adhesion molecules. For example, adhesion ability of T lymphocytes to cultured vascular endothelial cells was reportedly lowered in SLE patients, compared to healthy volunteers. In peripheral lymphocytes of SLE patients, adhesion molecules ICAM-1, VLA-4, 30 and IFA-1״ were strongly expressed (Haskard, D. 0. et al., Rheumatol. Int., Vol.9, p.33 (1989)).

In autoimmune thyroid deseases, it was reported that ״!CAM1 was expressed when a thyroid follicular cells were stimulated with interferon-7 , int er leukin-1, and tumor necrosis factor, and 35 that the formation of cluster of follicular cells and mononuclear cells was inhibited by anti-lCAM-l antibody (Weetman, A. P. et al., Eur. J. Immunol., Vol.20, p.271 (1990)).

In hepatitis, it is thought that the chances of adhesion between hepatocytes and inflammatory cells increases since there are two pathways of adhesion, ICAM-I״ and LFA-3, and LFA-1 and CD2, 5 to threby promote presentation of antigens and activation of inflammatory cells. In particular, in hepatitis B, LFA-3 molecules are strongly expressed in hepatocytes, in which viruses are actively proliferating, and ICAM-1״ well correlates with the degree of hepatitis. It is thus implied that LFA-3 is involved 10 in the exclusion of viruses and ICAM-1 promotes T cells to present antigen and regulates inflammation reaction. In ICAM-!-negative and HBc antigen-positive hepatocytes, chronic virus infection, a kind of immunounresponsiveness, may occur due to no interaction between lymphocytes and hepatocytes. It has also been reported 15 that serum ICAM-l in chronic liver disease may correlate with the degree of hepatocyte damage because the serum ICAM-1 concentrations in acute hepatitis patients, chronic active hepatitis patients, and liver cirrhosis patients were higher than that in healthy volunteers and chronic persisting hepatitis 20 patients, and the concentration was high in the case of histologically progressing active hepatitis (Mod. Phys., Vol.15, No.l, pp.73-76 (1995)).

In a model animal of arteriosclerosis, adhesion and invasion of monocytes and lymphocytes to vascular endothelium were observed 25 at very early stages of the onset of the disease. It is thus thought that the interaction of these hemocytes with endothelium is the first step of the onset of arteriosclerosis. Various reports show the expression of adhesion molecules in actual arteriosclerosis nidus including the expression of ICAM-I in human 30 arteriosclerosis nidus (Poston, R. N. et al. Am. J. Pathol., Vol.140, p.665 (1992)) and the expression of VCAM-1 in arteriosclerosis nidus of a hypercholesterolemia rabbit (Cybulsky, Μ. I. & Gimbrone, M. A. Jr., Science, Vol.251, p.788 (1991)). A recent report revealed that the expression of VCAM-1 was observed 35 in human arteriosclerosis nidus, and, in particular, strong expression in smooth muscle cells migrating to intima and in monocytes/macrophages. In addition, the expression of MCP-1 was enhanced in rabbit and human arteriosclerosis nidus, suggesting that MCP-1 promotes the formation of arteriosclerosis nidus through the migration of monocytes/macrophages (Current Therapy,

Vol.12, No.8 pp.1485-1488 (1994)).

The relationship between tumor metastasis and adhesion molecule abnormality has also been reported. For example, E-cadherindecreased cancer cells showed strong invasiveness, but the invasiveness was inhibited by introducing the cDNA of E-cadherin 10 into the cancer cells, and invasiveness was recovered when Ecadherin antiserum was added to the cells. This suggests the tight relationship between the decrease in the expression of E-cadherin and invasiveness of tumor cells (Frixen, U. H. et al., Vol.113, p.173 (1991)). in actual clinical cases, the relationship between 15 the decrease of the expression of E-cadherin and metastasis is pointed out in various kinds of cancer such as hepatoma, esophageal cancer, gastric cancer, and breast cancer. It has also been reported that VLA-4 molecules, a ligand for VCAM-1, were highly expressed in metastatic melanoma, gastric cancer, and breast cancer, 20 suggesting that this molecule could be utilized for the implantation to vascular endothelial cells in metastasis. In addition, based on experiments using various tumor cell lines, it has been reported that epithelial cancer, such as gastric cancer, large intestine cancer, lung cancer, hepatoma, or pancreatic cancer, adhered to 25 vascular endothelial cells through E-selectin (Takada, A. et al. Cancer Res., Vol.53, p.354 (1993)).

On the other hand, therapeutic approach to treat diseases by targeting these adhesion molecules have been made. For example, it was reported that anti-rat ICAM-1״ antibody strongly inhibited 30 inflammatory reaction in rat autoimmune arthritis model. ־It has also been reported that the administration of anti-״ICAM-l״ antibody inhibited the onset of arthritis in adjuvant synovitis in one of animal models of RA (Nihon Rinsho Meneki Gakkai Kaishi, Vol.14, No.6, pp.571-577 (1991)). It was further reported that the 35 metastasis formation of inoculated tumor was remarkably inhibited if a large amount of peptides having REG sequence, which is an amino acid sequence in an extracellular matrix protein recognized and bound by some integrins, were administered to a gallbladder cancer mouse, and that.in in vitro system RGD peptides and anti-/?l subunit antibody inhibited the motion and infiltration of tumor cells (Yamada, K. M. et al. Cancer Res., Vol.50, p.4485, (1990)).

In the following, the present invention is described in detail by clarifying the meanings of terms used herein and the general production methods of polypeptides, fusion polypeptides, genes, antibodies, transgenic mice, and knockout mice of the present 10 invention. However, it is needless to say that the meanings of the terms are not to be interpreted limitedly by the definition given herein.

Mitogen used herein is also called mitogenic factor and means a substance which induces cell division. Immunologically, it means 15 a substance inducing blastogenesis of lymphocytes polyclonally and inducing cell division. Examples thereof are lectins such as PHA and PWM, Concanavalin A (ConA), lipopolysaccharides, streptolysin S, and anti-lymphocyte antibody. It is known that Concanavalin A and PHA act only on T lymphocytes, that lipopolysaccharides act 20 only on B lymphocytes, and that PWM acts on both lymphocytes.

The term lymphoblast cell used herein is also called a large lymphocyte, lymphoblast, or immunoblast, and means a lymphocyte belonging to a large lymphocyte among lymphocytes existing in lymphoid tissues (lymph node, spleen, thymus, bone marrow, lymph 25 duct, tonsil, etc.) and blood.

The term activated lymphocyte used herein means, for example, a lymphocyte mentioned below, but is not limited thereto. For example, the term means a lymphocyte activated by some stimulation. As mentioned above, lymphocytes are classified into T cells, B cells, 30 and natural killer cells. T cells are classified into CD4-po~sitive cells and CD8-positive cells. Therefore, the activated lymphocytes of the present invention include mainly activated T cells, activated B cells, and activated natural killer cells, and activated T cells include activated CD4-positive cells and 35 activated CD8-positive cells.

Upon reacting with antigens presented by antigen-presenting cells, CD4-positive T cells secrete various cytokines, newly express receptors for these cytokines, enlarge their own size, start cell dividing,- proliferate, and are activated. Activated CD4positive T cells include those in such a state.

CD8-positive T cells express IL-2R when they react with antigens.

When IL-2 acts on IL-2R, the cells are differentiated into CTL, which has cellular cytotoxicity. CTL destroy its target cells to kill them when they meet the same antigen peptide/MHC class I complex. When CD8-positive T cells are differentiated into CTL, granules 10 increase in the cytoplasm. These granules comprise various high molecular weight proteins, represented by perforin. Perforin resembles MAC composed of the fifth to ninth components of complement, and makes holes in the cell membrane of target cells. The granules also comprise serine proteases, LT, and proteoglycan.

If CD8-positive cells receive antigen stimulation and are differentiated into CTL, they also secrete lymphokines such as IFN 7 , LT, TNF, or IL-2. Activated CD8-positive T cells include those in such a state.

T cells show blast formation phenomenon when they react with 20 hemagglutinin (phytohemagglutinin, PHA) or Concanavalin A (ConA). Activated T cells include those in such a state.

B cells express B7 molecules, activate helper T cells by stimulating CD28 on their surface with TCR, allow the helper T cells to express CD40L or produce lymphokines. When the cells receive 25 stimulation, they change to expand their cell size or proliferate. Activated B cells include those in such a state. In the present invention, activated B cells include those secreting antibodies (antibody-secreting cells and plasma cells).

Activated natural killer cells mean those showing cytotoxic 30 action on tumor cells or virus-infected cells as mentioned ־above. In the present invention, activated lymphocytes include thymus cells stimulated by Concanavalin A (ConA).

The activated lymphoblast cell used herein includes an activated .״lymphoblast that is generated when the lymphoblast 35 mentioned above is stimulated with mitogen״ mentioned above such as Concanavalin A.

The term resting lymphocyte״ used herein means, in some case, an non-activated lymphocyte, which has not received the stimulation to activate cells, in contrast to an activated lymphocyte mentioned above.

The gene of the present invention includes a genomic DNA and a cDNA.

The human-derived substance of the present invention includes natural substance isolated from a human body component (organ, tissue, cell, body fluid, etc.), and recombinant substance produced 10 by recombinant DNA technology. When the substance is protein or polypeptide, the substance includes an artificial protein and polypeptide having an amino acid sequence where one or more amino acids are substituted, deleted, or added.

The cell surface molecule of the present invention is that 15 derived from a mammal such as human, rat, mouse, guinea pig, and rabbit, preferably that derived from human, rat, or mouse, and more preferably that derived from human.

Specifically, the cell surface molecule of the present invention is that characterized by having, at least, properties 20 described below.

(a) the cell surface molecule is expressed in, at least, thymocytes and mitogen-stimulated lymphoblast cells;

(b) an antibody reactive to the cell surface molecule induces adhesion between mitogen-stimulated lymphoblast cells;

(c) an antibody reactive to the cell surface molecule induces proliferation of peripheral blood lymphocytes in the presence of an antibody against CD3;

(d) the cell surface molecule has a partial amino acid sequence represented by Phe-Asp-Pro-Pro-Pro-Phe in its extracellular region; and (e) the cell surface molecule has a partial amino acid sequence represented by Tyr-Met-Phe-Met in its cytoplasmic region.

Preferably, the cell surface molecule comprises the following polypeptide of the present invention.

The polypeptide of the present invention is that which constitutes the above-mentioned cell surface molecule of the

L present invention. Examples thereof are as follows.

(1) A polypeptide encoded by a DNA hybridizing with a DNA comprising a nucleotide sequence of SEQ ID NO: 1 under stringent conditions;

(2) A polypeptide having an amino acid sequence having 60% or more homology with an amino acid sequence of SEQ ID NO: 2;

(3) A polypeptide having an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting human JTT-1 antigen and its derivative);

(4) A polypeptide having an amino acid sequence encoded by a nucleotide sequence corresponding to nucleotide residues 26 to 625 of SEQ ID NO: 3 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting human JTT-1 antigen and its derivative);

(5) A polypeptide having an amino acid sequence encoded by a nucleotide sequence corresponding to nucleotide residues 35 to 637 of SEQ ID NO: 4 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting rat

JTT-1 antigen and its derivative);

(6) A polypeptide having an amino acid sequence encoded by a nucleotide sequence corresponding to nucleotide residues 1 to 603 of SEQ ID NO: 5 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting mouse JTT-1 antigen and its derivative);

(7) A polypeptide having an amino acid sequence encoded by a nucleotide sequence corresponding to nucleotide residues 35 to 685 of SEQ ID NO: 6 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting a mutant of rat JTT-1 antigen and its derivative); and (8) A polypeptide having an amino acid sequence encoded by a DNA encoding a polypeptide! constituting the cell surface molecule of the present invention, wherein the DNA is introduced into the transformant identified by an international deposit accession No.

FERM BP-5725 or, having amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting a human JTT-1 antigen and its derivative).

Examples of stringent conditions are as follows. When a probe with 50 or more-nucleotides is used and hybridization is performed in 0.9% NaCl, the standard of temperature where 50% dissociation 5 occurs (Tm) is calculated using the following formula and the temperature for hybridization can be determined according to the following formula.

Tm = 82.3°C + 0.41 x (G+C) % - 500/n - 0.61 x (formamide) % (n means the number of the nucleotide of probe).

Temperature « Tm -25°C.

In addition, when a probe with 100 or more nucleotides (G+C = 40 to 50%) is used, it should be considered that Tm varies as (1) and (2) mentioned below.

(1) Tm descends by about 1°C per 1% mismatch.

(2) Tm descends by 0.6 to 0.7°C per 1% formamide.

Accordingly, the temperature conditions for the combination of completely complementary strands can be set as follows.

(A) 65 to 75⁰¢ (formamide not added) (B) 35 to 45°C (in the presence of 50% formamide)

The temperature conditions for the combination of incompletely complementary strands can be set as follows.

(A) 45 to 55°C (formamide not added) (B) 35 to 42°C (in the presence of 30% formamide)

The temperature conditions when a probe with 23 or less 25 nucleotides is used can be 37°C or can be calculated using the following formula.

Temperature = 2°C x (the number of A+T) + 4°C x (the number of C+G) -5°C.

Here, having substantially the same amino acid sequence means 30 to include a polypeptide having an amino acid sequence“ where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in the amino acid sequence shown in Sequence Listing are substituted, deleted, and/or modified, and a polypeptide having an amino acid sequence where multiple amino 35 acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, are added to the amino acid sequence shown in

Sequence Listing, as long as the polypeptide has substantially the same biological properties as the polypeptide having the amino acid sequence shown-in Sequence Listing.

Such substitution, deletion, or insertion of amino acids can 5 be performed by the usual method (Experimental Medicines SUPPLEMENT, Handbook of Genetic Engineering (1992); and so on).

Examples thereof are synthetic oligonucleotide-directed mutagenesis (gapped duplex method), point mutageneseis by which a point mutation is introduced at random by treatment with nitrite 10 or sulfite, the method by which a deletion mutant is prepared with Bal31 enzyme and the like, cassette mutagenesis, linker scanning method, miss incorporation method, mismatch primer method, DNA segment synthesis method, etc.

Synthetic oligonucleotide-directed mutagenesis (gapped duplex 15 method) can be, for example, performed as follows. The region desired to be mutagenized is cloned into M13 phage vector having amber mutation to prepare the single-stranded phage DNA. After RF I DNA of M13 vector without amber mutation is linearized by restriction enzyme treatment, DNA is mixed with the single-stranded 20 phage DNA mentioned above, denatured, and annealed thereby forming gapped duplex DNA. A synthetic oligdnucleotide into which mutations are introduced is hybridized with the gapped duplex DNA and the closed-circular double-stranded DNAs are prepared by the reactions with DNA polymerase and DNA ligase. E. coli mutS cells, 25 deficient in mismatch repair activity, are transfected with this DNA. E. coll cells without suppressor activity are infected with the grown phages, and only phages without amber mutation are screened.

The method by which a point mutation is introduced with nitrite 30 utilizes, for example the principle as mentioned below. If“DNA is treated with nitrite, bases are deaminated to change adenine into hypoxanthine, cytosine into uracil, and guanine into xanthine. If deaminated DNA is introduced into cells, A:T and G:C are replaced with G:C״ and AjT, respectively, because hypoxanthine, 35 uracil, and xanthine form a base pair with cytosine, adenine, and thymine, respectively, in the DNA replication. Actually, single-stranded DNA fragments treated with nitrite are hybridized with ״gapped duplex DNA״, and thereafter mutant strains are separated by manipulating in the same way as synthetic oligonucleotide-directed mutagenesis (gapped duplex method).

Alphabetical triplet or single letter codes used to represent amino acids in the present specification or figures mean amino acids as follows. (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) 10 asparagine, (Gln/Q) glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M) methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W) tryptophane, (His/H) histidine, (Pro/P) proline.

The polypeptide״ constituting the above-mentioned cell 15 surface molecule of the present invention is a transmembrane protein, which penetrates cell membrane, and the cell surface molecule is composed of one or two of these transmembrane polypeptides.

Here, a transmembrane protein״ means a protein that connects 20 with membrane through the hydrophobic peptide region penetrating the lipid bilayer of the membrane once, or several times and whose structure is, as a whole, composed of three main regions, that is, extracellular region, transmembrane region, and cytoplasmic region, as seen in many receptors or cell surface molecules. Such a 25 transmembrane protein constitutes each receptor or cell surface molecule in the form of a monomer, homodimer, heterodimer or oligomer with another chain(s) having the same or different amino acid sequence.

The ״polypeptide fragment of the present invention is a 30 fragment from the above-defined polypeptide of the present invention, and preferably the extracellular region of the polypeptide. One to five amino acids, if desired, can be added to the N terminus and/or C terminus of this region.

Here, an ״extracellular region means the whole or a portion 35 from the partial structure (partial region) from the entire structure of the above-mentioned transmembrane protein where the partial structure exists outside of the membrane. In other words, it means the whole or a portion of the region of the transmembrane protein except the region incorporated into the membrane (transmembrane region) and the region existing in the cytoplasm following the transmembrane region (cytoplasmic region).

The constant region or a portion of the constant region of human immunoglobulin (Ig) heavy chain״ used herein means the constant region or the Fc region of human-derived immunoglobulin heavy chain (H chain) as described, or a portion of them. The immunoglobulin 10 can be any immunoglobulin belonging to any class and any subclass. Specifically, examples of the immunoglobulin are IgG (IgGl, IgG2, IgG3, and IgG4), IgM, IgA (IgAl and IgA2), IgD, and IgE. Preferably, the immunoglobulin is IgG (IgGl, IgG2, IgG3, or IgG4), or IgM. Examples of particularly preferable immunoglobulin of the present 15 invention are those belonging to human-derived IgG (IgGl, IgG2, IgG3, or IgG4).

Immunoglobulin has a Y-shaped structural unit in which four chains composed of two homologous light chains (L chains) and two homologous heavy chains (H chains) are connected through disulfide 20 bonds (S-S bonds). The light chain is composed of the light chain variable region (VL) and the light chain constant region (CL). The heavy chain is composed of the heavy chain variable region (VH) and the heavy chain constant region (CH).

The heavy chain constant region is composed of some domains 25 having the amino acid sequences inherent in each class (IgG, IgM, IgA, IgD, and IgE) and each subclass (IgGl, IgG2, lgG3, and lgG4, IgAl, and IgA2).

The heavy chain of IgG (IgGl, IgG2, IgG3, and IgG4) is composed of VH, CHI domain, hinge region, CH2 domain, and CH3 domain in this 30 order from N terminus.

Similarly, the heavy chain of IgGl is composed of VH, C/ J domain, hinge region, C/₃2 domain, and Cy₃3 domain in this order from N terminus. The heavy chain of lgG2 is composed of VH, cy₂l domain, hinge region, Cy₂2 domain, and cy₂3 domain in this order from N 35 terminus. The heavy chain of IgG3 is composed of VH, Cy₃l domain, hinge region, Cy₃2 domain, and Cy₃3 domain in this order from N terminus. The heavy chain of IgG4 is composed of VH, Cy₄l domain, hinge region, C7₄2 domain, and Cy₄3 domain in this order from N terminus.

The heavy chain of IgA is composed of VH, Cal domain, hinge region, Ca2 domain, and Ca3 domain in this order from N terminus.

Similarly, the heavy chain of IgAl is composed of VH, ca!l domain, hinge region, ca!2 domain, and Ca₂3 domain in this order from N terminus. The heavy chain of IgA2 is composed of VH, Ca₂l domain, hinge region, ca₂2 domain, and ca₂3 domain in this order from N 10 terminus.

The heavy chain of igD is composed of VH, cdl domain, hinge region, C<52 domain, and 053 domain in this order from N terminus.

The heavy chain of IgM is composed of VH, C/zl domain, C//2 domain, C/Z3 domain, and C/Z4 domain in this order from N terminus and have 15 no hinge region as seen in IgG, IgA, and IgD.

The heavy chain of IgE is composed of VH, Cel domain, Ce2 domain, C<5 3 domain, and C£4 domain in this order from N terminus and have no hinge region as seen in IgG, IgA, and IgD.

If, for example, IgG is treated with papain, it is cleaved at 20 the slightly N terminal side beyond the disulfide bonds existing in the hinge region where the disulf ide bonds connect the two heavy chains to generate two homologous Fab, in which a heavy chain fragment composed of VH and CHI is connected with one light chain through a disulfide bond, and one Fc, in which two homologous heavy 25 chain fragments composed of the hinge region, CH2 domain, and CH3 domain are connected through disulfide bonds (See ״Immunology Illustrated, original 2nd ed., Nankodo, pp.65-75 (1992); and Focus of Newest Medical Science ^,Recognition Mechanism of Immune System״׳, Nankodo, pp.4-7 (1991); and so on).

Namely, a portion of a constant region of immunoglobulin heavy chain״ of the present invention means a portion of a constant region of an immunoglobulin heavy chain having the structural characteristics as mentioned above, and preferably, is the constant region without Cl domain, or the Fc region. Specifically, examples 35 thereof are the region composed of hinge region, C2 domain, and C3 domain from each of IgG, IgA, and IgD, and are the region composed of C2 domain, C3 domain, and C4 domain from each of IgM and igE.

A particularly preferable example thereof is the Fc region of human-derived IgGl.

The fusion polypeptide of the present invention is that 5 composed of the extracellular region of the polypeptide constituting the above-described cell surface molecule of the present invention and a constant region or a portion of a constant region of human immunoglobulin (Ig) heavy chain. Preferably, it is a fusion polypeptide composed of an extracellular region of a 10 polypeptide of the present invention and a portion of a constant region of human IgG heavy chain, and particularly preferably, it is a fusion polypeptide composed of an extracellular region of a polypeptide of the present invention and the region (Fc) composed of a hinge region, CH2 domain, and CH3 domain of human IgG heavy 15 chain. Moreover, IgGl is preferable among IgG. in addition, a polypeptide derived from human, mouse, or rat (preferably, human) is preferable as the polypeptide of the present invention.

The fusion polypeptide of the present invention has the advantage that the fusion polypeptide can be purified extremely 20 easily by using affinity column chromatography using the property of protein A, which binds specifically to the immunoglobulin fragment because the fusion polypeptide of the present invention has a portion of a constant region (for example Fc) of an immunoglobulin such as IgG as mentioned above as a fusion partner. 25 Moreover, since various antibodies against the Fc of various immunoglobulin are available, an immunoassay for the fusion polypeptides can be easily performed with antibodies against the Fc.

The polypeptide, polypeptide fragment, and fusion polypeptide 30 of the present invention can be produced not only by recombinant DNA technology as mentioned below but also by a method well known in the art such as a chemical synthetic method and a cell culture method, or a modified method thereof.

The gene of the present invention comprises a DNA encoding 35 the above-mentioned polypeptide or polypeptide fragment of the present invention, and includes any gene having a nucleotide sequence encoding the polypeptide or polypeptide fragment of the present invention.

Exmaples of the gene are those encoding the polypeptide or polypeptide fragment mentioned below.

(1) A polypeptide encoded by a DNA hybridizing with a DNA comprising a nucleotide sequence of SEQ ID NO: 1 under stringent conditions;

(2) A polypeptide having an amino acid sequence having 60% or more homology with an amino acid sequence of SEQ ID NO: 2;

(3) A polypeptide having an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting ״human JTT-1 antigen״ and its derivative);

(4) A polypeptide having an amino acid sequence encoded by a 15 nucleotide sequence corresponding to nucleotide residues 26-625 of SEQ ID NO: 3 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting human JTT-1 antigen and its derivative);

(5) A polypeptide having an amino acid sequence encoded by a 20 nucleotide sequence corresponding to nucleotide residues 35-637 of SEQ ID NO: 4 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting rat JTT-1 antigen״ and its derivative);

(6) A polypeptide having an amino acid sequence encoded by a 25 nucleotide sequence corresponding to nucleotide residues 1-603 of

SEQ ID NO: 5 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting mouse JTT-1 antigen and its derivative);

(7) A polypeptide having an amino acid sequence encoded by a 30 nucleotide sequence corresponding to nucleotide residues 35-685־ of SEQ ID NO: 6 or an amino acid sequence substantially the same as the amino acid sequence (namely, a polypeptide constituting a mutant of rat JTT-1 antigen״ and its derivative); and (8) A polypeptide having an amino acid sequence encoded by a DNA 35 encoding a polypeptide constituting the cell surface molecule of the present invention, wherein the DNA is introduced into the transformant identified by an international deposit accession No.

FERM BP-5725 or, having an amino acid sequence substantially the same as said amino acid sequence (namely, a polypeptide constituting a human JTT-1 antigen and its derivative).

Here, substantially the same amino acid sequence means as defined above.

Specific examples of the gene of the present invention are DNAs or their fragments mentioned below.

(1) A DNA comprising a nucleotide sequence of SEQ ID NO: 1, and 10 a DNA hybridizing with the DNA under stringent conditions;

(4) A DNA comprising a nucleotide sequence corresponding to nucleotide residues 26-625 of SEQ ID NO: 3;

(5) A DNA comprising a nucleotide sequence corresponding to nucleotide residues 35-637 of SEQ ID NO: 4;

(6) A DNA comprising a nucleotide sequence corresponding to nucleotide residues 1-603 of SEQ ID NO: 5;

(7) A DNA comprising a nucleotide sequence corresponding to nucleotide residues 35-685 of SEQ ID NO: 6;

(8) A DNA encoding a polypeptide constituting a cell surface 20 molecule of the present invention, wherein the DNA is introduced into a transformant identified by an international deposit accession No. FERM BP-5725.

The DNA encoding a portion of a constant region of immunoglobulin heavy chain, which is a part of a fusion polypeptide of the present 25 invention, can be cDNA, or genomic DNA comprised of intons between every exon (the DNA encoding, for example, CHI domain, hinge region, CH2 domain, CH3 domain, CH4 domain and so on).

The DNA of the present invention includes any DNA comprised of any codons as long as the codons encode the same amino acids.

The DNA of the present invention can be a DNA obtained ־by any method. For example, the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining 35 these methods.

The DNA encoding the polypeptide of the present invention can be obtained by the usual method such as a method to clone cDNA from mRNA encoding the polypeptide of the present invention, a method to isolate genomic DNA and then splice them, chemical synthesis and soon.

(1) cDNA can be cloned from the mRNA encoding the polypeptide of the present invention by, for example, the method described below.

First, the mRNA encoding a cell surface molecule (polypeptide) of the present invention is prepared from tissues or cells (for example, thymus cells or spleen-derived lymphoblast cells 10 stimulated with ConA) expressing and producing a cell surface molecule (polypeptide) of the present invention. mRNA can be prepared isolating total RNA by a known method such as quanidine-thiocyanatemethod (Chirgwin, J.M. et al. Biochemistry, Vol. 18, p5294, 1979), hot phenol method, or AGPC method, and 15 subjecting it to affinity chromatography using oligo-dT cellulose or poly-U Sepharose.

Then, with the mRNA obtained as a template, cDNA is synthesized, for example, by a well-known method using reverse transcriptase such as the method of Okayama et al. (Mol. Cell. Biol. Vol.2, p.161 20 (1982); ibid. Vol.3, p.280 (1983)) or the method of Hoffman et al.

(Gene Vol.25, p.263 (1983)), and converted into double-stranded cDNA. A cDNA library is prepared by transforming E. coli with plasmid vectors, phage vectors, or cosmid vectors having this cDNA or by transfecting E, coli after in vitro packaging.

The plasmid vectors used in this invention are not limited as long as they are replicated and maintained in hosts. Any phage vectors that can be replicated in hosts can also be used. Examples of usually used cloning vectors are pME18S, λΖΑΡΙΙ(ΙΖΑΡΙΙ), pUC19, λ gtlO, λ gtll, and so on. When the vector is applied to 30 immunological screening as mentioned below, the vector having a promoter that can express a gene encoding the polypeptide of the present invention in a host is preferably used.

cDNA can be inserted into a plasmid by, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second 35 edition. Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can be inserted into a phage vector by, for example, the method of Hyunh et al. (DNA cloning, a practical approach, Vol.l, p.49 (1985)).

These methods can be simply performed by using a commercially available cloning kit (for example, a product from Takara Shuzo).

The recombinant plasmid or phage vector thus obtained is introduced into appropriate host cells such as a prokaryote (for example, E.

coli: XLlBlue MRF׳, DH5a, HB101, MC1061/P3, etc.).

Examples of a method for introducing a plasmid into a host are calcium chloride method, calcium chloride/rubidium chloride method described in Molecular Cloning, A Laboratory Manual (second edition, 10 Cold Spring Harbor Laboratory, p. 1.74 (1989)), and electroporation method. Phage vectors can be introduced into host cells by, for example, a method in which the phage DNAs are introduced into grown hosts after in vitro packaging. In vitro packaging can be easily performed with a commercially available in vitro packaging kit (for 15 example, a product from Stratagene or Amersham).

The cDNA encoding the polypeptide of the present invention can be isolated from the cDNA library so prepared according to the method mentioned above by combining general cDNA screening methods.

For example, a clone comprising the desired cDNA can be screened 20 by a known colony hybridization method (Crunstein et al. Proc. Natl.

Acad. Sci. USA, Vol.72, p.3961 (1975)) or plague hybridization method (Molecular Cloning, A Laboratory Manual, second edition. Cold Spring Harbor Laboratory, p.2.108 (1989)) using ³²P-labeled chemically synthesized oligonucleotides as probes, which are 25 corresponding to the amino acid sequence of the polypeptide of the present invention. Alternatively, a clone having a DNA fragment encoding a specific region within the polypeptide of the present invention can be screened by amplifying the region by PCR with synthetic PCR primers.

When a cDNA library prepared using a cDNA expression vector (for example, λΖΑΡΙΙ phage vector) is used, the desired clone can be screened by the antigen-antibody reaction using an antibody against the polypeptide of the present invention. A screening method us ing PCR method is preferably used when many clones are subjected to 35 screening.

The nucleotide sequence of the DNA thus obtained can be determined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci. USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic chain termination method using phage Ml3 (Sanger et al. Proc. Natl. Acad. Sci. USA, Vol.74, pp.5463-5467 (1977)). The whole or a 5 portion of the gene encoding the polypeptide of the present invention can be obtained by excising the clone obtained as mentioned above with restriction enzymes and so on.

(2) The DNA encoding the polypeptide of the present invention can be isolated from the genomic DNA derived from the cells expressing 10 the polypeptide of the present invention as mentioned above by the following methods.

Such cells are solubilized preferably by SDS or proteinase K, and the DNAs are deproteinized by repeating phenol extraction. RNAs are digested preferably with ribonuclease. The DNAs obtained 15 are partially digested with appropriate restriction enzymes, and the DNA fragments obtained are amplified with appropriate phage or cosmid to generate a library. Then, clones having the desired sequence are detected, for example, by using radioactively labeled DNA probes, and the whole or a portion of the gene encoding the 20 polypeptide of the present invention is obtained from the clones by excision with restriction enzyme and soon.

cDNA encoding a human-derived polypeptide can be obtained as follows. After a cosmid library into which human genomic DNA (chromosomal DNA) is introduced is prepared (״Laboratory Manual: 25 Human Genome Mapping״, Maruzen press), positive clones comprising the DNA of the coding region of the desired protein are obtained by screening the cosmid library. Then, the cDNA library mentioned above is screened with the coding DNA excised from the positive clone as a probe to prepare the human cDNA.

(3) The DNA of the present invention can also be chemically synthesized by the usual method, based on the nucleotide sequence of SEQ ID NO: 1, 3, 4, 5, or 6.

The present invention also relates to a recombinant vector comprising the DNA encoding an above-mentioned cell surface 35 molecule (polypeptide) of the present invention. The recombinant vector of the present invention is not limited as long as it can be replicated and maintained or can autonomously replicate in various prokaryotic and/or eukaryotic hosts. The vector of the present invention includes plasmid vectors and phage vectors.

The recombinant vector can easily be prepared by ligating the 5 DNA encoding the polypeptide of the present invention with a vector for recombination available in the art (plasmid DNA and bacteriophage DNA) by the usual method. Specific examples of the vectors for recombination used are E. coll-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids 10 such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids such as pUBllO, pTP5, and pC194. Examples of phages are a bacteriophage such as λ phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.

An expression vector is useful for expressing the DNA encoding the polypeptide of the present invention and for producing the polypeptide of the present invention. The expression vector is not limited as long as it expresses the gene encoding the polypeptide of the present invention in various prokaryotic and/or eukaryotic 20 host cells and produces this protein. Examples thereof are pEFneo (Proc. Natl. Acad. Sci. USA, Vol.91., pp.158-162 (1994)), pEF-BOS (Nucleic Acids Res. Vol.18, p.5322 (1990)), pME18S (Experimental Medicine: SUPPLEMENT, ״Handbook of Genetic Engineering1992) ״)), pMAL C2, and so on.

When bacteria, particularly E. coli are used as host cells, an expression vector is generally comprised of, at least, a promoter/operator region, an initiation codon, the DNA encoding the polypeptide of the present invention, termination codon, terminator region, and replicon.

When yeast, animal cells, or insect cells are used as ־hosts, an expression vector is preferably comprised of, at least, a promoter, an initiation codon, the DNA encoding the polypeptide of the present invention, and a termination codon. It may also comprise the DNA encoding a signal peptide, enhancer sequence, 5׳35 and !^,-untranslated region of the gene encoding the polypeptide of the present invention, splicing junctions, polyadenylation site.

selectable marker region, and repl icon. The expression vector may also contain, if required, a gene for gene amplification (marker) that is usually used.

A promoter/operator region to express the polypeptide of the 5 present invention in bacteria comprises a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia, it preferably comprises Trp promoter, lac promoter, recA promoter, λ PL promoter, Ipp promoter, tac promoter, or the like. Examples of a prompter to express the 10 polypeptide of the present invention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and so on. When the host is Bacillus, examples thereof are SL01 promoter, SP02 promoter, penP promoter and so on. When the host is a eukaryotic cell such as a mammalian cell, examples thereof are SV40-derived promoter, 15 retrovirus promoter, heat shock promoter, EF promoter, and so on, and preferably SV-40, SRa, and retrovirus-derived one. As a matter of course, the promoter is not limited to the above examples. In addition, to use an enhancer is effective for expression.

A preferable initiation codon is, for example, a methionine 20 codon (ATG).

The commonly used termination codon (for example, TAG, TGA, TAA, and so on) is illustrated as a termination codon.

Usually used natural or synthetic terminators are used as a terminator region.

A replicon means a DNA capable of replicating the whole DNA sequence in host cells, and includes a natural plasmid, an artificially modified plasmid (DNA fragment prepared from a natural plasmid), a synthetic plasmid, and so on. Examples of a preferable plasmids are pBR322 or its artificial derivatives (DNA fragment 30 obtained by treating pBR322 with appropriate restriction eiffcyiaes) for E. coli, yeast 2 Jl plasmid or yeast chromosomal DNA for yeast, and pEFneo, pME18S, pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, etc. for mammalian cells.

An enhancer sequence, polyadenylation site, and splicing junction that are usually used in the art, such as those derived from SV40 can be also used.

A selectable marker usually used can be used according to the usual method.- Examples thereof are resistance genes for antibiotics, such as tetracycline, neomycin, ampicillin, or kanamycin, and thymidine kinase gene.

Examples of a gene for gene amplification are dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene, etc.

The expression vector of the present invention can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA (gene) encoding the polypeptide of the present invention, termination codon, and terminator region, 15 to an appropriate repl icon. If desired, appropriate DNA fragments (for example, linkers, restriction sites generated with other restriction enzyme), can be used by the usual method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.

Transformants of the present invention can be prepared by 20 introducing the expression vector mentioned above into host cells.

Host cells used in the present invention are not limited as long as they are compatible with an expression vector mentioned above and can be transformed. Examples thereof are various cells such as natural cells or artificially established recombinant cells 25 usually used in technical field of the present invention (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insect cells.

E. coli or animal cells are preferably used. Specific examples are E. coli (DH5a, XLlBlue MRF׳, TB1, HB101, etc. ) , mouse-derived 30 cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, etc.), rat-derived־cells, hamster-derived cells (BHK, CHO-K1, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, etc.), and human-derived cells (HEK293, Hela, diploid fibroblast-derived cells, myeloma, Namalwa, etc.).

An expression vector can be introduced (transformed (transduced)) into host cells by known method.

Transformation can be performed, for example, according to the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69, p.2110 (1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.lll (1979)), or competent method (J. Mol. Biol., V01.56, p.209 (1971)) 5 when the hosts are bacteria (E. coli, Bacillus subtilis, etc.), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927 (1978)), or lithium method (J. Bacteriol., Vol.153, p.163 (1983)) when the host is Saccharomyces cerevisiae, the method of Graham (Virology, Vol.52, p.456 (1973) ) when the hosts are animal 10 cells, and the method of Summers et al. (Mol. Cell. Biol., Vol.3, pp.2156-2165 (1983)) when the hosts are insect cells.

The polypeptide of the present invention can be produced by cultivating transformants (in the following this term includes transductants) comprising an expression vector prepared as 15 mentioned above in nutrient media.

The nutrient media preferably comprise carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of host cells (transformants). Examples of the carbon source are glucose, dextran, soluble starch, and sucrose, and examples of the 20 inorganic or organic nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meet extract, soy bean cake, and potato extract. If desired, they may comprise other nutrients (for example, an inorganic salt (for example, calcium chloride, sodium dihydrogenphosphate, and magnesium chloride), 25 vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.).

Cultivation is performed by a method known in the art. Cultivation conditions such as temperature, pH of the media, and cultivation time are selected appropriately so that the polypeptide 30 of the present invention is overproduced.

Specific media and cultivation conditions used depending on host cells are illustrated below, but are not limited thereto.

When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi, liquid media comprising the nutrient source mentioned above 35 are appropriate. The media with pH 5 to 8 are preferably used.

When the host is E, coli, examples of preferable media are LB media, and M9 media (Miller et al. Exp. Mol. Genet., Cold Spring

Harbor Laboratory, p.431 (1972)). Using these media, cultivation can be performed usually at 14 to 43 °C for about 3 to 24 hours with aeration and stirring, if necessary.

When the host is Bacillus, cultivation can be performed usually at 30 to 40 °C for about 16 to 96 hours with aeration and stirring, if necessary.

When the host is yeast, examples of media are Burkholder minimal media (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505 (1980)). The pH of the media is preferably 5 to 8. Cultivation can be performed usually at 20 to 35°C for about 14to 144 hours with aeration and stirring, if necessary.

When the host is an animal cell, examples of media are MEM media containing about 5 to 20% fetal bovine serum (Science, Vol.122, p.501 (1952)), DMEMmedia (Virology, Vol.8, p.396 (1959)), RPMI1640 media (J. Am. Med. Assoc., Vol.199, p.519 (1967)), and 199 media (Proc. Soc. Exp. Biol. Med., Vol.73, p.l (1950)). The pH of the media is preferably about 6 to 8. Cultivation can be performed usually at about 30 to 40°C for about 15 to 72 hours with aeration and stirring, if necessary.

When the host is an insect cell, an example of media is Grace's media containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol.82, p.8404 (1985)). The pH thereof is preferably about 5to 8. Cultivation can be performed usually at about 20 to 40°C for 15 to 100 hours with aeration and stirring, if necessary.

Cultivation of transformants as mentioned above, in particular animal cells can overexpress the polypeptide of the present invention on the surface of the cells.

The polypeptide of the present invention can be produced as a soluble polypeptide fragment such as an extracellular ־region fragment by preparing the transformants as mentioned above using the DNA encoding the extracellular region or each domain and by cultivating the transformants to allow them to secrete the soluble polypeptide into the culture supernatant. In addition, a fusion polypeptide of the present invention can be prepared similarly.

Namely, a culture filtrate (supernatant) is obtained by the method such as filtration or centrifugation of the obtained culture, and the polypeptide or polypeptide fragment of the present invention is purified and isolated from the culture filtrate by the usual method commonly used in order to purify and isolate a natural or synthetic protein.

Examples of the isolation and purification method are a method utilizing solubility, such as salting out and solvent precipitation method, a method utilizing the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl 10 sulfate-polyacrylamide gel electrophoresis, a method utilizing charges, such as ion exchange chromatography and hydroxylapatite chromatography, a method utilizing specific affinity, such as affinity chromatography, a method utilizing the difference in hydrophobicity, such as reverse phase high performance liquid 15 chromatography, and a method utilizing the difference in isoelectric point, such as isoelectric focusing.

When the polypeptide or a polypeptide fragment of the present invention exists in the periplasm or cytoplasm of cultured transformants, first, the fungus bodies or cells are harvested by 20 the usual method such as filtration or centrifugation and suspended in appropriate buffer. After the cell wall and/or cell membrane of the cells and so on are disrupted by the method such as lysis with sonication, lysozyme, and freeze-thawing, the membrane fraction comprising the polypeptide of the present invention is 25 obtained by the method such as centrifugation or filtration. The membrane fraction is solubilized with a detergent such as Triton-X100 to obtain the crude extract. Finally, the polypeptide or the polypeptide fragment is isolated and purified from the crude extract by the usual method as illustrated above.

The transgenic mouse of the present invention is a transgenic mouse wherein the DNA (cDNA or genomic DNA) prepared as mentioned above encoding the polypeptide of the present invention derived from animals except mice (non-self polypeptide) have been integrated into its endogenous locus of the mouse. The transgenic 35 mouse expresses the non-self polypeptide and secretes the polypeptide into its body.

The transgenic mouse can be prepared according to the method as usually used for producing a transgenic animal (for example, see Newest Manual of Animal Cell Experiment, LIC press. Chapter

7, pp.361-408, (1990)).

Specifically, for example, embryonic stem cells (ES cells) obtained from normal mouse blastocysts are transformed with an expression vector in which the gene encoding human-derived polypeptide of the present invention (i.e. human JTT-1 antigen״) has been operably inserted. ES cells in which the gene encoding 10 the human-derived polypeptide of the present invention has been integrated into the endogenous gene are screened by the usual method. Then, the ES cells screened are microinjected into a fertilized egg obtained from another normal mouse (blastocyst) (Proc. Natl. Acad. Sci. USA, Vol.77, No.12, pp.7380-7384 (1980); U.S. Pat. No.

4,873,191). The blastocyst is transplanted into the uterus of another normal mouse as the foster mother. Then, founder mice (progeny mice) are born from the foster mother mouse. By mating the founder mice with normal mice, heterogeneic transgenic mice are obtained. By mating the heterogeneic transgenic mice with each 20 other, homogeneic transgenic mice are obtained according to Mendel’ s laws.

Knockout mouse of the present invention is a mouse wherein the endogenous gene encoding the mouse-derived polypeptide of the present invention (i.e. ״mouse JTT-1 antigen״) has been knocked 25 out (inactivated). It can be prepared, for example, by positive-negative selection method in which homologous recombination is applied (U.S. Pat. No. 5,464,764; No. 5,487,992; No. 5,627,059; Proc. Natl. Acad. Sci. USA, Vol.86, pp.8932-8935 (1989); Nature, Vol.342, pp.435-438 (1989); etc.).

The antibody״ of the present invention can be a polyclonal antibody (antiserum) or a monoclonal antibody, and preferably a monoclonal antibody.

Specifically, it is an antibody reactive to (against, which binds to) the above-mentioned polypeptide or polypeptide fragment 35 of the present invention.

The antibody of the present invention can be natural antibodies obtained by immunizing mammals such as mice, rats, hamsters, guinea pigs, and rabbits with the antigen, such as cells (natural cells, cell lines, tumor cells, etc.) expressing cell surface molecules״ of the present invention, transformants overexpressing the 5 polypeptide or cell surface molecules of the present invention on the surface thereof prepared using recombinant DNA technology on the cell surface, or polypeptide fragments or fusion polypeptides of the present invention. The antibody of the present invention also includes chimeric antibodies and humanized 10 antibodies (CDR-grafted antibodies) that can be produced by recombinant DNA technology, and human antibodies that can be produced using human antibody-producing transgenic animals.

The monoclonal antibody includes those having any one isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable.

The polyclonal antibody (antisera) or monoclonal antibody of the present invention can be produced by the known methods. Namely, a mammal, preferably, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, horse, or cattle, or more preferably, a mouse, rat, hamster, guinea pig, or rabbit is immunized, for example, with 20 an antigen mentioned above with Freund's adjuvant, if necessary.

The polyclonal antibody can be obtained from the antiserum obtained from the animal so immunized. In addition, the monoclonal antibodies are produced as follows. Hybridomas are prepared from the antibody-producing cells obtaind from the animal so immunized 25 and myeloma cells that are not capable of producing autoantibodies. The hybridomas are cloned, and clones producing the monoclonal antibodies showing the specific affinity to the antigen used for immunizing the mammal are screened.

Specifically, the monoclonal antibody can be produced as follows. 30 Immunizations are performed by injecting or implanting once or several times the antigen as mentioned above as an immunogen, if necessary, with Freund's adjuvant, subcutaneously, intramuscularly, . intravenously, through the footpad, or intraperitoneally into a non-human mammal, specifically a mouse, 35 rat, hamster, guinea pig, or rabbit, preferably a mouse, rat, or hamster (including a transgenic animal generated so as to produce antibodies derived from another animal such as the transgenic mouse producing human antibody mentioned below). Usually, immunizations are performed once to four times every one to fourteen days after the first immunization. Antibody-producing cells are obtained 5 from the mammal so immunized in about one to five days after the last immunization. The frequency and interval of immunizations can be appropriately arranged depending on property of the immunogen used. Hybridomas that secrete a monoclonal antibody can be prepared by the method of Kohler and Milstein (Nature, Vol.256, 10 pp.495-497 (1975)) and by its modified method. Namely, hybridomas are prepared by fusing antibody-producing cells contained in a spleen, lymph node, bone marrow, or tonsil obtained from the non-human mammal immunized as mentioned above, preferably a spleen, with myelomas without autoantibody-producing ability, which are 15 derived from, preferably, a mammal such as a mouse, rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse, rat, or human.

For example, mouse-derived myeloma P3/X63-AG8.653 (653), P3/NSl/l-Ag4-l (NS-l), P3/X63-Ag8.Ul (P3U1), SP2/0-Agl4 (Sp2/0, 20 Sp2), PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., or human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEMAGR, D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.

Hybridoma clones producing monoclonal antibodies can be 25 screened by cultivating hybridomas, for example, in microtiter plates and by measuring the reactivity of the culture supernatant in the well in which hybridoma growth is observed, to the immunogen used for the immunization mentioned above, for example, by enzyme immunoassay such as RIA and ELISA.

The monoclonal antibodies can be produced from hybridomas by cultivating the hybridomas in vitro or in vivo such as in the ascites fluid of a mouse, rat, guinea pig, hamster, or rabbit, preferably a mouse or rat, more preferably mouse and isolating the antibodies from the resulting the culture supernatant or ascites fluid of a 35 mammal.

Cultivating hybridomas in vitro can be performed depending on the property of cells to be cultured, on the object of a test study, and on the various conditions of a cultivating method, by using known nutrient media or any nutrient media derived from known basal media for growing, maintaining, and storing the hybridomas to produce monoclonal antibodies in culture supernatant.

Examples of basal media are low calcium concentration media such as Ham׳F12 medium, MCDB153 medium, or low calcium concentration MEM medium, and high calcium concentration media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, 10 or RD medium. The basal media can contain, for example, sera, hormones, cytokines, and/or various inorganic or organic substances depending on the objective.

Monoclonal antibodies can be isolated and purified from the culture supernatant or ascites fluid mentioned above by saturated 15 ammonium sulfate precipitation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), affinity chromatography using anti-immunoglobulin column or protein A column.

Preferable examples of monoclonal antibodies of the present 20 invention are as follows.

(1) A monoclonal antibody reactive to a polypeptide having an amino acid sequence of 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 to a polypeptide of the present invention, a polypeptide fragment derived from the polypeptide, or a cell surface molecule composed of the polypeptide, wherein the effect of the monoclonal antibody on mitogen-stimulated lymphoblast cells is substantially the same as the effect of a 30 monoclonal antibody produced by a hybridoma identif ied ־־by an international deposit accession No. FERM BP-5707 on mitogenstimulated rat lymphoblast cells; and (3) A monoclonal antibody reactive to a polypeptide of the present invention, a polypeptide fragment derived from the polypeptide, 35 or a cell surface molecule composed of the polypeptide, wherein the effect of the monoclonal antibody on mitogen-stimulated lymphoblast cells is substantially the same as the effect of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-5708 on mitogenstimulated rat lymphoblast cells.

In addition, the monoclonal antibody of the present invention includes that produced by the hybridoma identified by an international deposit accession No. FERM BP-5707 or No. FERM BP-5708.

The chimeric monoclonal antibody of the present invention is 10 a monoclonal antibody prepared by genetic engineering, and specifically means a chimeric antibody such as mouse/human chimeric monoclonal antibody whose variable regions are derived from immunoglobulin of an non-human mammal (mouse, rat, hamster, etc.) and whose constant regions are derived from human immunoglobulin.

The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG (IgGl, IgG2, IgG3, lgG4), IgM, IgA, IgD, and IgE. The constant region of the recombinant chimeric monoclonal antibody of the present invention can be that of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG.

The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.

A mouse/human chimeric monoclonal antibody can be prepared, 25 referring to Experimental Medicine: SUPPLEMENT, Vol.1.6, No. 10 (1988); and examined published Japanese patent application (JP-B) No. Hei 3-73280. Namely, it can be prepared by operably inserting CH gene (C gene encoding the constant region of H chain) obtained from the DNA encoding human immunoglobulin downstream of active 30 VH genes (rearranged VDJ gene encoding the variable region of H chain) obtained from the DNA encoding a mouse monoclonal antibody isolated from the hybridoma producing the mouse monoclonal antibody , and CL gene (C gene encoding the constant region of L chain) obtained from the DNA encoding human immunoglobulin downstream of active 35 VL genes (rearranged VJ gene encoding the variable region of L chain) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma, into the same or different vectors so as for them to be expressed, following by transforming host cells with the expression vector, and then by cultivating the transformants.

Specifically, DNAs are first extracted from mouse monoclonal antibody-producing hybridomas by the usual method, digested with appropriate restriction enzymes (for example, EcoRI andHindlll), electrophoresed (using, for example, 0.7% agarose gel), and analyzed by Southern blotting. After an electrophoresed gel is 10 stained, for example, with ethidium bromide and photographed, the gel is given with marker positions, washed twice with water, and soaked in 0.25 M HC1 for 15 minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minutes with gently stirring. The DNAs are transferred to a filter for 4 hours by the usual method. The 15 filter is recovered and washed twice with 2xSSC. After the filter is sufficiently dried, it is baked at 75°C for 3 hours. After baking, the filter is treated with 0.1 x SSC/0.1% SDS at 65°C for 30 minutes. Then, it is soaked in 3 x SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65°C for 20 3 to 4 hours.

Next, ³²P-labeled probe DNA and hybridization solution are added to the bag and reacted at 65°C about 12 hours. After hybridization, the filter is washed under appropriate salt concentration, reaction temperature, and time (for example, 2 x SSC-0.1% SDS, room 25 temperature, 10 minutes). The filter is put into a plastic bag with a little 2 x SSC, and subjected to autoradiography after the bag is sealed.

Rearranged VDJ gene and VJ gene encoding H chain and L chain of a mouse monoclonal antibody are identified by Southern blotting 30 mentioned above. The region comprising the identified DNA fragment is fractioned by sucrose density gradient centrifugation and inserted into a phage vector (for example, Charon 4A, Charon 28, AEMBL3, AEMBL4, etc.). E. coli (for example, LE392, NM539, etc.) is transformed with the phage vector to generate a genomic library. 35 The genomic library is screened by plaque hybridization such as Benton-Davis method (Science, Vol.196, pp.180-182 (1977)) using appropriate probes (H chain J gene, L chain (A?) J gene, etc.) to obtain positive clones comprising rearranged VDJ gene or VJ gene. By making the- restriction map and determining the nucleotide sequence of the clones obtained, it is confirmed that genes 5 comprising the desired, rearranged VH (VDJ) gene or VL (VJ) gene are obtained.

Separately, human CH gene and human CL gene used for chimerization are isolated. For example, when a chimeric antibody with human IgGl is produced, Cyl gene as a CH gene, and CK gene 10 as a CL gene, are isolated. These genes can be isolated from human genomic library with mouse C y 1 gene and mouse C κ gene, corresponding to human cyl gene and human CK gene, respectively, as probes, taking advantage of high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human 15 immunoglobulin gene.

Specifically, DNA fragments comprising human Ck gene and an enhancer region are isolated from human λ Charon 4A Haelll-Alul genomic library (Cell, Vol.15, pp.1157-1174 (1978)), for example, with a 3 kb Hindlll-BamHI fragment of clone Igl46 (Proc. Natl. Acad. 20 Sci. USA, Vol.75, pp.4709-4713 (1978)) and a 6.8 kb EcoRI fragment of clone MEP10 (Proc. Natl. Acad. Sci. USA, Vol.78, pp.474-478 (1981)) as probes. In addition, for example, after human fetal hepatocyte DNA is digested with Hindlll and fractioned by agarose gel electrophoresis, a 5.9 kb fragment is inserted into λ788 and 25 then human Cyl gene is isolated with the probes mentioned above.

Using mouse VH gene, mouse VL gene, human CH gene, and human CL gene so obtained, and taking promoter region and enhancer region into consideration, human CH gene is inserted downstream mouse VH gene and human CL gene is inserted downstream mouse VL gene into 30 an expression vector such as pSV2gpt or pSV2neo with appropriate restriction enzymes and DNA ligase by the usual method. In this case, chimeric genes of mouse VH gene/human CH gene and mouse VL gene/human CL gene can be respectively inserted in the same expression vector or in different expression vectors.

Chimeric gene-inserted expression vector(s) thus prepared are introduced into myelomas that do not produce antibodies, for example.

P3X63*Ag8’653 cells or SP210 cells by protoplast fusion method, DEAE-dextran method, calcium phosphate method, or electroporation method. The transformants are screened by cultivating in media containing a drug corresponding to the drug resistance gene inserted 5 into the expression vector and, then, cells producing desired chimeric monoclonal antibodies are obtained.

Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened.

The humanized monoclonal antibody (CDR-grafted antibody)״ of 10 the present invention is a monoclonal antibody prepared by genetic engineering and specifically means a humanized monoclonal antibody wherein a portion or the whole of the complementarity determining regions of the hypervariable region are derived from the complementarity determining regions of the hypervariable region 15 from a monoclonal antibody of an non-human mammal (mouse, rat, hamster, etc.), the framework regions of the variable region are derived from the framework regions of the variable region from human immunoglobulin, and the constant region is derived from human a constant region from immunoglobulin.

The complementarity determining regions of the hypervariable region exists in the hypervariable region in the variable region of an antibody and means three regions which directly and complementary binds to an antigen (complementarity-determining residues, CDR1, CDR2, and CDR3). The framework regions of the 25 variable region means four comparatively conserved regions lying upstream, downstream or between the three complementarity determining regions (framework region, FR1, FR2, FR3, and FR4).

In other words, a humanized monoclonal antibody means that in which the whole region except a portion or the whole of the 30 complementarity determining regions of the hypervariable ־region of a nonhuman mammal-derived monoclonal antibody have been replaced with their corresponding regions derived from human immunoglobulin.

The constant region derived from human immunoglobulin has the 35 amino acid sequence inherent in each isotype such as IgG (IgGl, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonal antibody in the present invention can be that from human immunoglobulin belonging to any isotype.

Preferably, it-is the constant region of human IgG. The framework regions of the constant region derived from human immunoglobulin are not particularly limited.

The humanized monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.

For example, a recombinant humanized monoclonal antibody 10 derived from mouse monoclonal antibody can be prepared by genetic engineering, referring to unexamined Japanese patent publication (JP-WA) No. Hei 4-506458 and unexamined Japanese patent publication (JP-A) No. Sho 62-296890. Namely, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the 15 mouse H chain CDR gene are isolated from hybridomas producing mouse monoclonal antibody, and human H chain gene encoding the whole regions except human H chain CDR corresponding to mouse H chain CDR mentioned above and human L chain gene encoding the whole region except human L chain CDR correspond to mouse L chain CDR mentioned 20 above are isolated from human immunoglobulin genes.

The mouse H chain CDR gene(s) and the human H chain gene(s) so isolated are operably inserted into an appropriate vector so that they can be expressed. Similarly, the mouse L chain CDR gene(s) and the human L chain gene(s) are operably inserted into another 25 appropriate vector so that they can be expressed. Alternatively, the mouse H chain CDR gene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human L chain gene(s) can be operably inserted into the same expression vector so that they can be expressed. Host cells are transformed with the expression vector thus prepared to 30 obtain trans formants producing humanized monoclonal antibody. By cultivating the transformants, desired humanized monoclonal antibody is obtained from culture supernatant.

The human monoclonal antibody of the present invention is immunoglobulin in which the entire regions comprising the variable 35 and constant region of H chain, and the variable and constant region of L chain constituting immunoglobulin are derived from the gene encoding human immunoglobulin.

The human antibody can be produced in the same way as the production method of polyclonal or monoclonal antibodies mentioned above by immunizing, with an antigen, a transgenic animal which 5 for example, at least human immunoglobulin gene(s) have been integrated into the locus of a non-human mammal such as a mouse by the usual method.

For example, a transgenic mouse producing human antibodies is prepared by the methods described in Nature Genetics, Vol.7, 10 pp. 13-21 (1994); Nature Genetics, Vol. 15, pp.146-156 (1997); JP-WA Nos. Hei 4-504365 and Hei 7-509137; Nikkei Science, No.6, pp.40-50 (1995); international patent publication No. WO94/25585; Nature, Vol.368, pp.856-859 (1994); and JP-WA No. Hei 6-500233.

In addition, recently developed technique for producing a 15 human-derived protein from the milk of a transgenic cow or pig can also be applied (Nikkei Science, pp.78-84 (April, 1997)).

The portion of an antibody used in the present invention means a partial region of the monoclonal antibody as mentioned above, and specifically, means F(ab₂(׳, Fab׳, Fab, Fv (variable fragment 20 of antbody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domain antibody) (Exp. Opin. Ther. Patents, Vol.6, No.5, pp.441-456 (1996)).

F(ab₂( ׳ and Fab״׳ can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, 25 and means an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous 30 antibody fragments in which an L chain composed of VL (L־־ chain variable region) and CL (L chain constant region) , and an H chain fragment composed of VH (H chain variable region) and CH 71 (71 region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond. Each of such two 35 homologous antibody fragments is called Fab׳. Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned

Fab׳ are connected at the hinge region. This antibody fragment is called F(ab₂(׳.

The ״pharmaceutical composition״ of the present invention comprises any one of the polypeptides of the present invention as defined above; ״homodimer molecule״, polypeptide fragment״, fusion polypeptide״ comprising the polypeptide; homodimer molecule comprising the fusion polypeptides, antibody, or 10 portion of an antibody; and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier״ includes a excipieut, a diluent, an expander, a decomposition agent, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a colorant, 15 a sweetener, a viscosity increasing agent, a flavor, a solubility increasing agent, or other additives. Using one or more of such carriers, a pharmaceutical composition can be fomulated into tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions, or syrups. The 20 pharmaceutical composition can be administered orally or parenterally. Other forms for parenteral administration include a solution for external application, suppository for rectal administration, and pessary, prescribed by the usual method, which comprises one or more active ingredient.

The dosage can vary depending on the age, sex, weight, and symptom of a patient, effect of treatment, administration route, period of treatment, or the kind of active ingredient (polypeptide or antibody mentioned above) contained in the pharmaceutical composition. Usually, the pharmaceutical composition can be 30 administered to an adult in a dose of 10 /Zg to 1000 mg (οϊ 10 ZZ g to 500 mg) per one administration. Depending on various conditions, the dosage less than that mentioned above may be sufficient in some cases, and the dosage more than that mentioned above may be necessary in other cases.

in particular, the injection can be produced by dissolving or suspending the antibody in a non-toxic, pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection with adjusting a concentration to 0.1 /zg antibody/ml carrier to 10 mg antibody/ml carrier. The injection thus produced can be administered to a human patient in need of treatment in a dose of 1 /Zg to 100 mg/kg body weight, preferably 50 /Zg to 50 mg/kg body weight once or more times a day. Examples of administration route are medically appropriate administration routes such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection, preferably intravenous injection.

The injection can also be prepared into a non-aqueous diluent (for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and alcohol such as ethanol), suspension, or emulsion.

The injection can be sterilized by filtration with a bacteria-non-penetrated filter, by mixing bacteriocide, or by irradiation. The injection can be produced in the form that is prepared upon use. Namely, it is freeze-dried to be a sterile solid composition, and can be dissolved in sterile distilled water for injection or another solvent before use.

The pharmaceutical composition of the present invention can be applied to treating or preventing various autoimmune diseases, allergic diseases, or inflammatory diseases caused by the activation of lymphocytes such as T cells and the regulation of activated lymphocyte functions. Examples of the diseases are rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, chronic inflammatory dermatosis such as lichen planus, systemic lupus erythematosus, insulin dependent diabetes mellitus, and psoriasis.

The therapeutic effect of the pharmaceutical compos it ioirof the present invention for symptom of various diseases can be tested by the usual method by administering it to an known disease model animal.

Examples of the model include (1) a (NZB/NZW)F1 mouse, a model for human systemic lupus erythematosus (SLE) (Science, Vol.125, p.1225-1227 (1994)), (2) experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis (MS) (J. Clin. Invest., Vol. 95, pp. 2783-2789 (1995)), (3) an NOD (non-obese diabetes) mouse, a model for insulin dependent diabetes mellitus (IDDM) (J. Exp. Med., Vol.181, pp.1145-1155 (1995)), (4) rat nephritis model by renal glomerulus basement membrane immunity. Goodpasture's nephritis model (Eur. J. Immunol., Vol.24, N0.6, pp.1249-1254 (1994)), and (5) a DBA/1 mouse, a model for human rheumatoid arthritis (Eur.

J. Immunol., Vol.26, pp.2320-2328 (1996)).

Brief Description of the Drawings

Figure 1 are micrographs showing the state of aggregation of FTL435 cells induced by JTT-1 antibody and the state of inhibition of the cell aggregation by JTT.2 antibody.

Subfigure (a) shows the state of the cells in the absence of any hybridoma supernatant, subfigure (b) shows the state of cell aggregation induced by JTT-1 antibody, subfigure (c) shows the state of the cell aggregation in the presence of anti-ICAM-1 antibody together with JTT-1 antibody, and subfigure (d) shows the state of the cell aggregation in the presence of ״ JTT-2 antibody together with JTT-1 antibody.

Figure 2 are micrographs showing the state of 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.

Subfigure (a) shows the state of FTL435 cells in the absence of any antibody, subfigure (b) shows the state of FTL435 cells in the presence of PMA, subfigure (c) shows the state of FTL435 cells in the presence of JTT-1 antibody, subfigure (d) shows the state of FTL435 cells in the presence of anti-LFA-1 antibody together with JTT-1 antibody, subfigure (e) shows the state of FTL435־cells in the presence of anti-CD18 antibody together with JTT-1 antibody, subfigure (f) shows the state of FTL435 cells in the presence of anti-lCAM-1 antibody together with JTT-1 antibody, subfigure (g) shows the state of activated lymphoblasts in the absence of any antibody, subfigure (h) shows the state of activated lymphoblasts in the presence of PMA, subfigure (i) shows the state of activated lymphoblasts in the presence of JTT-1 antibody, subfigure (j) shows the state of activated lymphoblasts in the presence of anti-LFA-1 antibody together with JTT-1 antibody, subfigure (k) shows the state of activated lymphoblasts in the 5 presence of anti-CD18 antibody together with JTT-1 antibody, and subfigure (1) shows the state of activated lymphoblasts in the presence of anti-ICAM-1 antibody together with JTT-1 antibody. Figure 3 shows the expression state of JTT-1 antigen and ״JTT-2 antigen in various cells measured with a flow-cytometer.

Figure 4 shows the expression state of JTT-1 antigen in various lymphocytic cells measured with a flow-cytometer.

Figure 5 is a photograph showing electrophoretogram of JTT-1 antigen analyzed by SDS-PAGE.

Figure 6 are micrographs showing the state of adhesion of rat 15 thymocytes to the microtiter plate coated with purified JTT-1 antigen, where the adhesion is induced in the presence of JTT-1 antibody, ״ and the state of inhibition of the cell adhesion by JTT-2 antibody.

Subfigure (a) shows the state of adhesion of the cells to the 20 plate which has not been coated with JTT-1 antigen, subfigure (b) shows the state of adhesion of the cells to the plate coated with JTT-1 antigen in the absence of any antibody, subfigure (cj shows the state of adhesion of the cells to the plate coated with JTT-1 antigen in the presence of the Fab fragments of JTT-1 25 antibody, and subfigure (d) shows the state of adhesion of the cells to the plate coated with JTT-1 antigen in the presence of JTT-2 antibody together with the Fab fragments of JTT-1 antibody.

Figure 7 shows the relative cell number of thymocytes adhering 30 to the plate coated with purified JTT-1 antigen measured in־ terms of fluorescence intensity.

'״Ag(-).״ shows the relative cell number in the plate which has not been coated with JTT-1 antigen, ״Ag(+)״ shows the relative cell number in the plate coated with JTT-1 antigen in the absence 35 of any antibody, Ag(+) + JTT-1 Fab shows the relative cell number in the plate coated with JTT-1 antigen in the presence of the

Fab fragments of ״JTT-1 antibody״, and ״Ag(+) + JTT-1 Fab + JTT-2״ shows the relative cell number in the plate coated with JTT-1 antigen״ in the presence of ״JTT-2 antibody״ together with the Fab fragments of JTT-1 antibody.

Figure 8 shows the expression state of rat JTT-1 antigen״ and “rat JTT-2 antigen in COS cells transformed with cDNA encoding ״rat JTT-1 antigen״ with a flow-cytometer.

Figure 9 shows the structural characteristics of amino acid sequence of JTT-1 antigen״ revealed by hydropathy plot analysis.

Fiqure 10 shows the homology among amino acid sequences of human, rat SEQ ID NO: 13 and mouse SEQ ID NO: 14 JTT-1 antigen and rat JJT-1 antigen mutant.

Figure 11 shows the homology among amino acid sequences and conservation state of motifs in ״human JTT-1 antigen,״ human CD28 molecule, and human CTLA-4 molecule.

Figure 12 schematically shows the protein secondary structure of, and their similarity among human JTT-1 antigen, ״human CD28 molecule, and ״human CTLA-4 molecule.

Figure 13 schematically shows the structure of the genomic DNA encoding mouse JTT-1 antigen.

Figure 14 shows the difference in amino acid sequences between ״rat JTT-1 antigen and its alternative splicing mutant.

Figure 15 shows the degree of the growth of human peripheral blood lymphocytes induced by the monoclonal antibody against human JTT-1 antigen, where the degree of the growth was measured by [³HJ thymidine uptake.

The ordinate shows the amount of uptake (dpm) of [³H] thymidine into the cells.

Figure 16 shows the therapeutic effect of the monoclonal antibody against JTT-1 antigen on experimental allergic encephalomyelitis (EAE) in a disease model rat.

The ordinate shows the scored degree of disease symptom, and the abscissa shows the days after immunization for induction of

EAE.

Figure 17 shows the therapeutic effect of the monoclonal antibody against ״JTT-1 antigen on glomerulonephritis in a disease model rat.

The ordinate shows the amount of urinary excretion of proteins, and the abscissa shows the time course (week) after immunization for induction of glomerulonephritis

Figure 18 shows a column histogram in purification of fusion 5 polypeptide between rat JTT-1 antigen extracellular region and human IgFc (rJTT-l-IgFc) with protein A Sepharose column.

Figure 19 is a photograph showing electrophoretogram of rJTT-l-IgFc analyzed by SDS-PAGE.

Figure 20 shows a column histogram in purification of fusion 10 polypeptide between human JTT-1 antigen extracellular region and human IgFc (hJTT-l-IgFc) with protein A Sepharose column.

Figure 21 is a photograph showing electrophoretogram of hJTT-l-IgFc analyzed by SDS-PAGE.

Figure 22 schematically shows the structure of the gene transfer 15 (targeting) vector used for preparation of a transgenic mouse into which the cDNA encoding rat JTT-1 antigen has been introduced.

Best Mode for Implementing the Invention

The present inventions are described in more detail with 20 reference to Examples below, but are not to be construed to be limited thereto.

Example 1 Preparation of monoclonal antibodies

Antibody-producing hybridomas were prepared according to the method of Kohler et al. (Omori et al., Blood, vol.81, pp.101-111 25 (1993)), and monoclonal antibodies were prepared according to the method of Kannagi et al. (Handbook of Experimental Immunology, Vol . 4, 117.21-117.21 (1986)).

First, rat thymoma cell line FTL435 cells were administered as an immunizing antigen to BALB/c mice into their footpad in an amount 30 of 10⁷ cells/mouse at intervals of 0, 7, 14, and 28 days. The mixture of the antigen with Freund's complete adjuvant was administered only in the first immunization. Two days after the last immunization, the lymph nodes of the mice were taken out and fused with mouse myeloma cells PAI (JCRNo. B0113; Stocker, J. W. et al., 35 Res. Disclosure, Vol.217, p.155 (1982)) by the usual method to obtain many hybridomas producing monoclonal antibodies.

Example 2 Screening of hybridomas and characterization of monoclonal antibodies

The hybridomas prepared in Example 1 were screened by analyzing the effect of the antibodies produced in the culture supernatant of the hybridomas on FTL435 cells, which were used as the immunogen.

FTL435 cells (5 x 10⁶ cells/ml, 0.1 ml) were seeded into each well of a 96-well microtiter plate and cultivated at 37°C for an hour in the presence of culture supernatant of each hybridoma (10 /z g/ml each). The results obtained for hybridoma clones JTT-1״ and 10 JTT-2 are shown in Figure 1 and Figure 2.

It was revealed that a monoclonal antibody produced by hybridoma clone JTT-1 (JTT-1 antibody״) strongly agglutinated FTL435 cells (Figure 1 (b) and Figure 2 (c)) and that addition of JTT-2 antibody strongly inhibited the aggregation of FTL435 cells 15 induced by JTT-1 antibody stimulation (Figure 1 (d)). Theassays, in which no hybridoma supernatant was added, were used as controls (Figure 1 (a) and Figure 2 (a)).

In order to determine whether the aggregation of FTL435 cells induced by JTT-1 antibody stimulation was caused by the cell 20 adhesion between intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1), which .is a representative known pathway of cell adhesion, FTL435 cells were cultivated at 37°C for an hour in the presence of anti-rat ICAM-1 antibody 1A29 (10 /zg/ml; IgGl) or anti-rat LFA-1 antibody (10 χζ 25 g/ml; IgG2a) together with JTT-1 antibody.

The aggregation of FTL435 cells by JTT-1 antibody״ stimulation was inhibited by neither anti-ICAM-1 antibody nor anti-LFA-1 antibody (anti-ICAM-1 antibody. Figure 1 (c) and Figure 2 (f); anti-LFA-1 antibody. Figure 2 (d)).

in order to further analyze the cell agglutination ability of JTT-1 antibody, the ability to agglutinate rat lymphoblast cells activated with concanavalin A stimulation was analyzed in the same manner as mentioned above. The results are shown in Figure 2.

Similar to the effect on FTL435 cells, the aggregation of 35 activated lymphoblast cells was induced by JTT-1 antibody stimulation (Figure 2 (i)). The aggregation of activated lymphoblast cells by JTT-1 antibody״ stimulation was mostly inhibited by anti-LFA-1 antibody (Figure 2 (j)) and anti-ICAM-1 antibody (Figure 2 (1)). (However, partial aggregation occurred.) As understood from the control assay (Figure 2 (g)), in which 5 no antibody was added, activated lymphocytes such as activated lymphoblasts showed no aggregation through cell adhesion unless they receive the stimulation with phorbol myristate acetate (PMA, which activates LFA-1) (Figure 2 (h)) or JTT-1 antibody״ (Figure 2 (I)). Therefore, the fact that anti-LFA-1 antibody partially 10 inhibited cell aggregation by JTT-1 antibody stimulation indicates that LFA-1 in activated lymphoblast cells was activated by JTT-1 antibody״ stimulation. This also indicates that molecules recognized by JTT-1 antibody״ are involved in some signal transmission.

Hybridoma clones JTT-1 and JTT-2״ have been deposited under the Budapest Treaty with international depository authority, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of international Trade and Industry, Japan (1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) 20 since October 11, 1996 with an international accession Nos. FERM BP-5707 and FERM BP-5708, respectively.

Analysis using mouse monoclonal antibody isotype identification kit (Amersham) determined that the isotype of monoclonal antibodies produced from each hybridoma (JTT-1 antibody 25 and JTT-2 antibody) were both IgGl.

Example 3 Reactivity of JTT-1 antibody״ and JTT-2 antibody to various cells

In order to analyze the expression pattern of molecules recognized by JTT-1 antibody״ and JTT-2 antibody in various cells, 30 the reactivities of the antibodies to various cells were examined.

Molecules recognized by JTT-1 antibody or JTT-2 antibody are designated JTT-1 antigen״ or JTT-2 antigen, respectively.

A five- to ten—week—old Wistar rat (150 to 250 g) was killed by anesthesia with diethyl ether. The thymus and spleen were taken 35 out of its chest and abdomen, respectively, by celiotomy, and homogenized to prepare cell suspension. The resulting spleen cells were cultivated in RPMI1640 medium containing 2 /£g/ml concanavalin

A and 10% FCS at 37°C for 3 days to prepare activated lymphoblasts.

FTL435 cells, thymocytes, spleen cells, and activated lymphoblasts (5 x 10⁵ cells each) were reacted with JTT-1 antibody״ 5 or JTT-2 antibody and then with FITC-labeled anti-mouse IgG (Cappel). The fluorescence intensity of the stained cells was measured with EPlCS-Elite flow cytometer.

The results are shown in Figure 3. In FTL435 cells, the strong expression of each ״ JTT-1 antigen and JTT-2 antigen was observed.

While the antigens were expressed in thymocytes, they were expressed only a little in spleen cells. However, in activated lymphoblasts obtained by simulating spleen cells with concanavalin A, JTT-1 antigen and JTT-2 antigen were strongly expressed. In addition, in each kind of cells, the expression pattern of JTT-1 antigen and JTT-2 antigen coincided with each other. These results indicate that JTT-1 antigen״ and JTT-2 antigen are the same molecules.

Example 4 Reactivity of JTT-1 antibody to various lymphocytic cells

In order to analyze the expression pattern of molecules (JTT-1 antigen) recognized by JTT-1 antibody in various lymphocytic cells, the reactivity of JTT-1 antibody to lymph nodes, T lymphoblasts derived from spleen, and B lymphoblasts derived from spleen of two kinds of rats (Wistar rat and F344 rat) was analyzed.

A five- to ten-week-old Wistar rat and F344 rat (150 to 250 g) were killed by anesthesia with diethyl ether. The lymph nodes and spleen were taken out of each rat by celiotomy, and homogenized to prepare cell suspension. The resulting cell suspension from spleen was cultivated in RPMI1640 medium containing 2 /1 g/ml concanavalin A (ConA) and 10% FCS at 37°C for 3 days. Activated T lymphoblasts and activated B lymphoblasts were obtained from each rat after 1-day and 3-day cultivation. In addition, spleen-derived T lymphoblasts and B lymphoblasts obtained before lymph node cells and ConA were added were used as controls.

Each cells (5 x 10⁵ cells each) were reacted with biotin-labeled anti-rat T cell antibody or biotin-labeled anti-rat B cell antibody (10 μ g/mL, Seikagaku Corporation), and subsequently with phycoerythrin-labeled streptavidin. The cells were then reacted with 10 ZZg/ml-FITC-labeled JTT-1 antibody. The fluorescence intensity of the stained cells was measured with EPICS-Elite flow cytometer.

The results are shown in Figure 4. In both activated T lymphoblasts and activated B lymphoblasts from wistar rat and F344 rat, the strong expression of JTT-1 antigen was observed from day 1 of activation with ConA stimulation. In addition, the 10 expression pattern of JTT-1 antigen in each kind of cells almost perfectly coincided with each other.

Example 5 Characterization of JTT-1 antigen״ and ״ JTT-2 antigen״ by immunoprecipitation ״JTT1 antigen and JTT-2 antigen״ were characterized by 15 immunoprecipitation using FTL435 cells.

(1) Preparation of biotinylated soluble cell surface molecules FTL435 cells were washed with PBS, suspended in physiological saline containing 100 /zg/ml NHS-biotin and 0.1 M HEPES (pH 8.0) to adjust 1 x 10⁷ cells/ml, and incubated at room temperature for 20 40 minutes. The cells were washed three times with PBS, lysis buffer (1% NP-40, 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl) was added thereto to adjust 5 x 10⁷ cells/ml, and the mixture was allowed to react at 4°C for 30 minutes to lyse the cells. The cell lysate obtained was centrifuged, and the supernatant comprising 25 biotinylated soluble cell surface molecules was stored at -80°C.

(2) immunoprecipitation and SDS-PAGE analysis

The purified sample of JTT-1 antibody purified by the usual method from the culture supernatant of the hybridoma clone JTT-1 prepared in Example 1 was mixed with protein G-Sepharose beads to 30 adjust 2 mg/ml, and allowed to react at 4 °C for an hour to bind the antibody with the beads. After the beads were washed, 500 μ 1 of the biotinylated FTL435 cell lysate was added to 10 μΐ of the beads, and the mixture was allowed to react at 4 °C for 2 hours. After the beads were washed with lysis buffer three times, 50 μ 35 1 of glycanase buffer (sodium phosphate buffer (pH 7.0) containing

0.15% SDS) was added to the beads, and the mixture was boiled to elute the bound molecules trapped by the antibody-bound beads.

1.25% NP-40 and 20 U/ml N-glycanase were added to a fraction of the sample so eluted, and the mixture was allowed to react overnight to digest N-linked sugar chains.

An equal volume of sample buffer (Enprotech) for SDS-PAGE was added to 5 /11 of the eluted sample in the presence or absence of

2-mercaptoethanol, and the mixture was boiled. After electrophoresis, the gel was transferred to a PVDF membrane. The membrane was blocked with 3% BSA-PBS and reacted with peroxidase-labeled streptavidin to detect biotinylated soluble cell surface molecules trapped by JTT-1 antibody with ECL system (Amersham) as described in the manual.

The results are shown in Figure 5. The JTT-1 antibody״recognized molecule (JTT-1 antigen) on FTL435 cells showed the molecular weight of about 47 kD under the non-reduced conditions (״(-)״ in Figure 5) and about 24 kD and 28 kD under the reduced conditions (״(+) in Figure 5). As the result of digestion of N-linked sugar chains (+N-gly in Figure 5), JTT-1 antigen was converged on a single band of about 36 kD under the non-reduced conditions and about 20 kD under the reduced conditions. These results suggest that JTT-1 antigen forms a dimer in which the same core proteins have different sugar chains. Completely the same results were obtained in the experiment performed as mentioned above using JTT-2 antibody. Considering these results together with the results of Example 3 and Example 7 below, JTT-1 antigen (molecule recognized by JTT-1 antibody) and JTT-2 antigen (molecule recognized by JTT-2 antibody) have been thought to be identical to each other.

Example 6 Adhesion experiment of rat thymocytes to purified JTT-1 antigen and N terminal amino acid analysis

The following experiments were performed to analyze whether the molecule that JTT-1 antibody recognizes (JTT-1 antigen) functions as an adhesion molecule. N-terminal amino acid analysis was also performed.

(1) Preparation of JTT-1 antibody-affinity column

The purified sample (2 mg in 2 ml) of JTT-1 antibody purified by the usual method from the culture supernatant of the hybridoma clone JTT-1 prepared in Example 1 was mixed with 1 ml of protein

G- Sepharose resin, and the mixture was allowed to react at 4°C for an hour. The resin was washed three times with 200 mM triethanolamine (pH 8.2). The resin was then incubated in triethanolamine (pH 8.2) containing 10 mM dimethyl pimelimidate (DMP) at room temperature for an hour to covalently bind JTT-1 antibody to the resin.

(2) Purification of JTT-1 antigen

FTL435 cells were cultivated in RPMI1640 medium containing 10% FCS. The cells were harvested by centrifugation to obtain a pellet and washed with PBS three times. Lysis buffer (1% NP-40, 10 mM Tris-HCl (pH 7.4), 0.15 M NaCl) was added to the washed pellet to adjust 5 x 10⁷ cells/ml, and the mixture was allowed to react at 15 4°C for 30 minutes to lyse the cells. The cell lysate obtained was centrifuged, and the supernatant containing soluble cell surface molecules was stored at -80°C.

The lysate (400 ml) was loaded onto JTT-1 antibody״-affinity column. After the column was washed with 50 ml of the lysis buffer 20 and 20 ml of PBS, JTT-1 antigen״ was eluted with 0.2 M glycine buffer (pH 2.8). 1 M Tris buffer was added to the JTT-1 antigen so eluted for neutralization. ״ JTT-1 antigen״ obtained was stored at -80°c.

(3) Determination of N terminal amino acid sequence

After the purified JTT-1 antigen״ was subjected to SDS-PAGE, the N-terminal amino acid sequence was determined by the usual method. The result revealed that JTT-1 antigen contained an amino acid sequence Glu-Leu-Asn-Asp-Leu-Ala-Asn-His-Arg.

. (4) Adhesion experiment

A five- to ten-week-old Wistar rat (150 to 250 g) was־killed by anesthesia with diethyl ether. The thymus was taken out of its chest by celiotomy and homogenized to prepare thymocyte suspension. 10 /z M 2 ׳ 7 ,׳-bis(carboxyethyl) carboxyfluorescein tetraacetoxymethyl ester (BCECF-AM; Molecular Probes) was added to the 35 suspension, and the mixture was incubated at 37°C for 30 minutes to fluorescently label the thymocytes. The cells were washed with

PBS and suspended in RPMI1640 medium containing 10% FCS to adjust x 10⁷ cells/ml.

The purified JTT-1 antigen obtained in (2) was coated on a

96-well ELISA plate at the concentration of 10 χζΐ/well overnight.

After the plate was washed with PBS, 200 /41/well of PBS containing 3% BSA was added to the plate, and blocking was performed for 2 hours. After the plate was washed with PBS, (1) only fluorescence-labeled thymocytes (2 x 10⁷ cells/ml, 0.1 ml), (2) fluorescence-labeled thymocytes (same concentration) and JTT-1 10 antibody Fab fragments prepared by the usual method (5 /Zg/ml), or (3) fluorescence-labeled thymocytes (same concentration), the JTT-1 antibody Fab fragments (same concentration), and JTT-2 antibody (10 /£g/ml), were added to each well, and cultivated at 37°C for an hour. In order to remove unbound cells, each well was 15 washed once with RPMI1640 medium containing 10% FCS. Each well was observed with light microscope. Then, 100 fil of 0.1% NP-40 was added to each well, and the cells adhered to the plate were lysed. The relative cell number of fluorescence-labeled thymocytes adhered to each well was counted by measuring the fluorescence 20 intensity at 538 nm (excited at 485 nm) with Fluoroscan II Microplate Fluorometer (Flow Laboratories). The assay in which a plate was not coated with purified JTT-1 antigen״ was used as a control.

The results of light microscopy observation are shown in Figure 6.

Thymocytes significantly adhered to purified JTT-1 antigen only in the presence of JTT-1 antibody Fab fragments (Figure 6 (c)). The adhesion was significantly inhibited by JTT-2 antibody (Figure 6 (d)).

Figure 7 shows the relative cell number of thymocytes adhered 30 to JTT-1 antigen coated on each well in terms of fluorescent intensity.

From these results, it was revealed that JTT-1 antigen functions as an adhesion molecule.

Example 7 cloning of cDNA encoding rat JTT-1 antigen

1. Preparation of cDNA library

1-(1) Extraction of poly(A)* RNA from ConA-stimulated rat lymphoblasts

ConA-stimulated lymphoblasts (ConA blast) derived from rat spleen (about lx 10⁶ cells/ml) were centrifuged (2,000 x g) at 4°C for 5 minutes. The precipitated cells were suspended with ISOGEN (Nippon Gene) and extracted with chloroform with shaking to collect the supernatant. After isopropanol was added to the obtained supernatant, the mixture was allowed to stand at room temperature for 10 minutes and centrifuged at 12,000 x g at 4°C for 10 minutes to precipitate RNA. The precipitated RNA was washed with ethanol and dissolved inTE buffer. Poly (A)⁺ RNA was purified from the total RNA so obtained with mRNA Purification Kit (Pharmacia).

1-(2) Preparation of cDNA

With 5 !4g of the poly (A)⁺ RNA prepared above as a template, cDNA was synthesized with Time Saver cDNA Synthesis Kit (Pharmacia). Oligo dT primer״ (Pharmacia) having Notl site was used to increase the efficiency of screening. EcoRI adapter was added, and digestion with Notl was performed to obtain cDNA with unidirectionality. Size fractionation was then performed with Spun Column (Pharmacia).

1- (3) Insertion into a vector

The obtained cDNA having EcoRI- and Notl-ends was ligated with pME18S (Hara, T. & Miyajima, A., EMBO J., Vol.11, pp.1875-1884 (1992)) digested with EcoRI and Notl. DNA Ligation Kit (Takara Shuzo) was used for the ligation. E, coli DH5 cells (Toyobo) were transformed with the reaction product so obtained. Transformants were cultivated until O.D. value (at 600 nm) reached 0.6 and harvested to recover plasmid DNAs with a library. QUIAGEN-Tip (QUIAGEN) was used for purification of plasmid DNAs.

2. Screening of cDNA library

Screening was performed according to panning method (Seed, B. et al. Proc. Natl. Acad. Sci. USA, Vol.84, pp.3365-3369 (1987)).

2- (1) Gene transfer into COS cells

The library so obtained was introduced into COS7 cells by electroporation (Potter, H. et al. Proc. Natl. Acad. Sci. USA, Vol.85, pp.2288-2292). The transformants were cultivated for 60 hours after introduction, the supernatant was removed, and the pellet was washed with PBS three times. After the pellet was treated with PBS (containing 0.5 mM EDTA) at 37°C for 30 minutes, the cells were .removed by pipetting. Only living cells were then collected with Lymphprep״ (NYCOMED).

2-(2) Concentration of gene-expressing cells by panning

The living cells obtained above were suspended in PBS (containing 5% FCS and 0.5 mM EDTA). The cell suspension was transferred to a Culture dish coated with JTT-1 antibody״ and incubated at room temperature for 3 hours. After cells not binding 10 to the culture dish were removed and the culture dish was washed with PBS three times, plasmid DNAs were collected from the cells binding to the culture dish by Hirt method (Hirt, B., J. Mol. Biol., Vol.26, pp.365-369). E. coli DH10B (GIBCO BRL) were transformed with the plasmid DNA so obtained. The plasmid DNAs were amplified 15 and purified with the transformants as in 1-(3) mentioned above. The procedures described in (1) and (2) were then repeated twice.

2-(3) Isolation of the positive clone

After the third panning, transformed E. coli DH10B cells were cultivated overnight on LB plates containing ampicillin to obtain 20 colonies. Twenty drug-resistant colonies were cultivated, plasmid DNAs were collected by alkaline miniprep method (Maniatis, T. et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), and the insert DNA was analyzed. Agarose gel electrophoresis revealed that the clone 25 having about 0.9 kb cDNA (designated T132A7) was concentrated.

T132A7 was transiently expressed in COS7 cells again with the method described in (1). After T132A7״-introduced cells were reacted with JTT-1 antibody or JTT-2 antibody, and then with FITC-labeled anti-mouse IgG (Cappel), the fluorescence intensity 30 of the stained cells was measured with EPlCS-Elite flow cytometer (Coulter). JTT-1 antibody״ and JTT-2 antibody strongly recognized the T132A7 gene product. The results are shown in Figure 8.

3. Determination of the nucleotide sequence and the amino acid 35 sequence

The nucleotide sequence of clone T132A7 was determined by dideoxy method with Auto Read Sequencing Kit״ (Pharmacia) and A.L.F. DNA Sequencer״ (Pharmacia). In addition, the deduced amino acid sequence of rat JTT-1 antigen encoded by the nucleotide sequence was analyzed with gene analysis software ״geneworks״ 5 (IntelliGenetics). The nucleotide sequence and the deduced amino acid sequence were shown in SEQ ID NO: 4.

The amino acid sequence (composed of 200 amino acid residues) deduced from the cloned gene comprises the same amino acid sequence as the N terminal amino acid sequence determined in Example 610 (3). Considering that clone T132A7״-introduced cells strongly react with JTT-1 antibody, it can be concluded that clone T132A7 comprises the cDNA encoding rat JTT-1 antigen.

4. Computer analysis

Hydropathy analysis of the primary structure of the deduced 15 amino acid sequence of JTT-1 antigen was performed according to the method of Kite and Doolittle (Kite, J. & Doolittle, R. F., J. Mol. Biol., Vol.157, pp.105-132 (1982)) (Figure 9). The results revealed that JTT-1 antigen is a transmembrane protein having a signal sequence at the N-terminus. In addition, the results of 20 motif analysis revealed that JTT-1 antigen has two Asn-linked sugar chain binding sites in the extracellular domain, and two casein kinase phosphorylation sites and one protein kinase C phosphorylation site in the cytoplasmic domain. In Figure 9 CHO means N-linked sugar chain binding site; P, phosphorylation site; 25 CKII, casein kinase II; and PKC, protein kinase c.

Example 8 Cloning of cDNA encoding human JTT-1 antigen

1. Preparation of a probe

The cDNA (about 0.9 kb) encoding rat JTT-1 antigen was generated by digesting the clone T132A7 obtained in Example 7 30 with restriction enzymes EcoRl and Notl, and separated by agarose gel electrophoresis. The separated DNA fragments were purified with QUIAEX gel extraction kit (QUIAGEN), and the obtained DNA fragments were labeled with ³²P using Ready-Το-Go DNA labelling kit (Pharmacia). These labeled DNA fragments were used as probes 35 for plaque hybridization.

2. Preparation of cDNA library

2-(1) Extraction of poly(A)⁺ RNA

Poly(A)⁺ RNA was extracted from ConA-stimulated lymphoblasts (ConA blast) derived from human peripheral blood in the same manner as in Example 7-1-(1).

2-(2) Preparation of cDNA

With 5 of the poly(A)⁺ RNA so prepared as a template, cDNAs were synthesized with ״oligo dT primer (Pharmacia) and Time Saver cDNA Synthesis Kit (Pharmacia). EcoRl adapter was then added, and size fractionation was performed with Spun Column (Pharmacia).

2-(3) insertion into a vector and packaging

The cDNAs so obtained having EcoRI-ends were ligated with the vector ״ λZAPH״ (Stratagene) digested with EcoRl. DNA Ligation Kit (Takara Shuzo) was used for the ligation. After in vitro packaging of the ligated DNA was performed with GIGA PACK II GOLD 15 (Stratagene), E. coli XLlBlue MRF׳ cells (Stratagene) were transfected with the obtained phage particle to generate a cDNA library composed of plaque comprising recombinant phage.

3. Screening of cDNA library cDNA library was screened by plaque hybridization method 20 (Maniatis, T. et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) with Rapid hybridization buffer (Amersham). First, the cDNA library so obtained (1 x 10⁴) was plated onto agar plates and the replica was produced with Hybond-N nylon membrane (Amersham). Plaque 25 hybridization was performed in Rapid hybridization buffer (Amersham) using the replica and the ³²p-labled probe prepared in Example 8-1. First and second screenings were performed to obtain eight positive clones. Single plaques of each clone were isolated and subjected to in vivo excision in accordance with the manual 30 (Stratagene) and seven positive clones were collected as plasmid DNA.

4. Determination of the nucleotide sequence

The nucleotide sequences of the seven clones were determined by dideoxy method with Auto Read Sequencing Kit (Pharmacia) and 35 A.L.F. DNA Sequencer (Pharmacia). The seven clones comprise the same nucleotide sequence. It was found that clone pBSh41 encodes the full length human JTT-1 antigen. The cDNA sequence corresponding to the open reading frame (ORF) of human JTT-1 antigen is shown in SEQ ID NO: 1, the full length of the deduced amino acid sequence of human JTT-1 antigen is shown in SEQ ID 5 NO: 2, and the nucleotide sequence comprising 5׳ and 3׳ sequences is shown in SEQ ID NO: 3 (ORF correspondes to the nucleotide residues 26 to 625). It is understood that the nucleotide sequence contained in the clone encodes the full length of human JTT-1 antigen because the amino acid sequence (composed of 199 amino acid residues) 10 deduced from the nucleotide sequence shows significant homology with the amino acid sequence of rat JTT-1 antigen (Figure 10). As shown in Figure 10, the homology between the amino acid sequences of human and rat JTT-1 antigen is 60% or more.

E. coli DH10B (GIBCO BRL) transformed with the clone pBSh41 15 has been deposited under the Budapest Treaty with international depository authority. National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan (1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) since October 25, 1996 (an international deposit accession No. FERM BP-5725).

5. Structural characteristics and biological function of JTT-1 antigen

The results of motif search for the deduced amino acid sequence of human JTT-1 antigen״ in known human proteins revealed that 25 human JTT-1 antigen has structural similarity to CD28 and CTLA-4, human-derived cell membrane proteins belonging to the immunoglobulin superfamily, mentioned in detail above (Figures 11 and 12). As mentioned above, CD28 and CTLA-4 are extremely important molecules regulating the activation and inhibition of 30 T cells in immune system.

The structural similarity is as follows

1. 20 or more amino acid residues including cysteine residues are highly conserved.

2. Proline repeating sequence, ״Pro-Pro-Pro (PPP), which is 35 essential as the ligand binding region in CD28 and CTLA-4, is conserved.

3. Tyr-Xaa-Xaa-Met (YxxM)״ (Xaa and x represents any amino acid) sequence essential as the signal transmitting region in CD28 and

CTLA-4 is conserved in the cytoplasmic region.

From the fact that the same structure with the specific structure of CD28 and CTLA-4״, which play an important role in regulation of activation of T cells that are main actor in immune mechanism, JTT-1 antigen of the present invention is inferred to play an important role like those molecules in regulation of activation of lymphocytes such as T cells which are main actor in immune response.

Example 9 Cloning of cDNA encoding mouse JTT-1 antigen

1. Preparation of a probe

The cDNA (about 0.9 kb) encoding rat JTT-1 antigen was obtained by digesting the clone T132A7, cloned in Example 7, with restriction enzymes EcoRI and Notl, and separeted by agarose gel electrophoresis. The DNA fragments so separated were purified with QUIAEX gel extraction kit (QUIAGEN), and the DNA fragments were labeled with ³²P using Ready-To-Go DNA labelling kit (Pharmacia). These labeled DNA fragments were used as a probe for plaque hybridization.

2. Preparation of cDNA library

2-(1) Extraction of poly(A)⁺ RNA

As Example 7-1-(1), poly(Aj RNAs were extracted from ConAstimulated lymphoblasts derived from mouse spleen (about 1 x 10⁶cells/ml).

2-(2) Preparation of cDNA library

With 5 mg of poly (A j RNAs prepared in the above as a template, cDNAs were synthesized with oligo dT primer (Pharmacia) and Time Saver cDNA Synthesis Kit (Pharmacia). After EcoRI adapter was added to the cDNA, size fractionation was performed with Spun Column (Pharmacia).

2-(3) Insertion of cDNA into a vector and packaging

The cDNA so obtained having EcoRI-ends was ligated with the vector 1ZAPII (Stratagene) digested with EcoRI. DNALigation Kit (Takara Shuzo) was used for the ligation. After in vitro packaging of the ligated DNA was per formed with GIGA PACK II GOLD (Stratagene),

E. coli XLlBlue MRF׳ cells (Stratagene) were transfected with the phage particle so obtained to generate a cDNA library composed of plaque comprising recombinant phage.

3. Screening of cDNA library

Screening was performed by plaque hybridization method (Maniatis, T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) using Rapid hybridization buffer (Amersham).

The above-obtained cDNA library (1 x 10⁴) was plated onto agar 10 plates and the replica was produced using Hybond-N nylon membrane (Amersham). Plaque hybridization was performed in Rapid hybridization buffer (Amersham) using the replica and the ³²P-labled probe prepared in Example 9-1. First and second screenings were performed to obtain five positive clones. After single plaque of 15 each clone was isolated, in vivo excision was performed in accordance with Instruction Manual (Stratagene) and five positive clones were collected as plasmid DNA.

4. Determination of the nucleotide sequence

The nucleotide sequences of each of the five clones were 20 determined by dideoxy method with Auto Read Sequencing Kit (Pharmacia) and A.L.F. DNA Sequencer (Pharmacia). The four of the five clones comprise the same nucleotide sequence. The nucleotide sequence of cDNA encoding the full length of mouse JTT-1 antigen״ and the deduced amino acid sequence are shown in SEQ ID 25 NO: 5.

As understood from Figure 10, mouse JTT-1 antigen is composed of 200 amino acid residues like rat JTT-1 antigen. The homology among the amino acid sequences of mouse, rat, and human JTT-1 antigens is significant (60% or more).

5. Analysis of the locus of mouse JTT-1 antigen gene־

The locus of the gene encoding mouse JTT-1 antigen was analyzed by fluorescence in situ hybridization method.

The cDNA so obtained encoding mouse JTT-1 antigen was labeled with ³²P to prepare hybridization probes by the usual method. Using 35 these probes, the 129 SVJ mouse genomic DNA library (Stratagene) was screened to obtain mouse genomic DNA clones comprising the exons encoding mouse JTT-1 antigen. The structure of the genomic DNA is schematically shown in Figure 13.

The above-obtained genomic DNA clones were labeled with digoxigenin dUTP by nick translation to prepare probes. The 5 labeled probes were bound to cleaved mouse DNA and hybridized with normal metaphase chromosomes derived from mouse embryonic fibroblasts in the solution containing 50% formaldehyde, 10% dextran sulfate, and 2 x SSC. After a slide glass for hybridization was incubated in fluorescence-labeled anti-digoxigenin antibody, 10 specific hybridization signal was detected by staining with DAPI.

In the first test, the part near the largest chromosome that was thought to be the chromosome 1, judging from the DNA size and emerged band, was specifically labeled. Based on this information, the above-described genomic DNA clone was co-hybridized with probes 15 specific to the centromere region of the chromosome 1. As a result, the centromere region of the chromosome 1 and the regions near them were specifically labeled. Ten samples of the chromosome 1 showing the specific hybridization were analyzed, and it was revealed that the above-mentioned genomic DNA clone was located at the position 20 of 33% of the distance from the border between heterochromatin and euchromatin to the telomere of the chromosome 1, namely, on the same band 1C3 as the loci of mouse CD28 and CTLA-4 genes.

As the result that 80 metaphase cells were analyzed, specific labeling was identified at said position for 79 cells.

These results and the results obtained in Example 8 indicating the structural similarity of JTT-1 antigen to CD28 and CTLA-4 suggest that JTT-1 antigen, like CD28 and CTLA-4, is an important molecule involved in the regulation of the transmission of costimulatory signal and/or activation of lymphocytes.

Example 10 Cloning of cDNA encoding a mutant of rat JTT-1 antigen Another cDNA that is thought to encode alternative splicing variant of rat JTT-1 antigen cloned in Example 7 was cloned as follows.

1. Preparation of a probe

The cDNA (about 0.9 kb) encoding rat JTT-1 antigen was generated by digesting the clone T132A7, obtained in Example 7, with restriction enzymes EcoRI and Notl, and separated by agarose gel electrophoresis. The separated DNA fragments were purified with QUIAEX gel extraction kit״ (QUIAGEN), and the obtained DNA fragments were labeled with ³²P using Ready-To-Go DNA labeling kit״ 5 (Pharmacia). These labeled DNA fragments were used as probes for plaque hybridization.

2. Preparation of cDNA library

2-(1) Extraction of poly(A)⁺ RNA

As in Example 7-1-(1), poly(A)⁺ RNA was extracted from rat 10 thymoma cell line FTL435 (about 1 x 10⁶ cells/ml).

2-(2) Preparation of cDNA library

With 5 mg of the poly(A)⁺ RNA prepared as mentioned above as a template, cDNAs were synthesized using oligo dT primer (Pharmacia) and Time Saver cDNA Synthesis Kit״ (Pharmacia). After EcoRI 15 adapter was added to the cDNA, size fractionation was performed with Spun Column (Pharmacia).

2-(3) Insertion of cDNA into a vector and packaging

The cDNA having EcoRl-end obtained above was ligated with the vector 1ZAPII (Stratagene) digested with EcoRI. DNALigation Kit״ 20 (Takara Shuzo) was used for ligation. After in vitro packaging of the ligated DNA was performed with GIGA PACK II GOLD (Stratagene), E. coli XLlBlue MRF׳ (Stratagene) was transfected with the obtained phage particle to generate a cDNA library composed of plaque comprising recombinant phage.

3. Screening of cDNA library

Screening was performed by plaque hybridization method (Maniatis, T. et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) with Rapid hybridization buffer (Amersham).

The above-prepared cDNA library (I x 10⁴) was plated onto agar plates and the replica was produced with Hybond-N nylon membrane (Amersham). Plaque hybridization was performed in Rapid hybridization buffer (Amersham) using the replica and the ³²P-labled probe prepared in Example 10-1. First and second screenings were 35 performed to obtain two positive clones. After single plaque of each clone was isolated, in vivo excision was performed in accordance with Instruction Manual (Stratagene), and two positive clones were collected as plasmid DNA.

4. Determination of the nucleotide sequence

The nucleotide sequences of the two clones were determined by dideoxy method with Auto Read Sequencing Kit״ (Pharmacia) andA.L.F. DNA Sequencer (Pharmacia). The two clones comprise the same nucleotide sequence. The nucleotide sequence of cDNA encoding the full length of the obtained ״rat JTT-1 antigen and the deduced amino acid sequence are shown in SEQ ID NO: 6. The amino acid sequence (SEQ ID NO: 6) deduced from the obtained cDNA sequence was compared with the amino acid sequence (SEQ ID NO: 4) deduced from the obtained cDNA sequence encoding ״rat JTT-1 antigen cloned in Example 7 (Figure 14). As shown in Figure 14, the amino acid sequence encoded by the cDNA cloned in this test was completely the same as that encoded by the cDNA encoding rat JTT-1 antigen obtained in Example 7, except that (1) C-terminal three continuous amino acid residues (Met-Thr-Ser) changes into Thr-Ala-Pro, and that (2) subsequent to the Thr-Ala-Pro, 16 continuous amino acid residues (Leu-Arg-Ala-Leu-Gly-Arg-Gly-Glu-His-Ser-Ser-Cys-Gln20 Asp-Arg-A&n) are added. This indicates that the cDNA cloned in this test encodes the alternative splicing variant of rat JTT-1 antigen obtained in Example 7.

Example 11 Preparation of recombinant human JTT-1 antigen״expressing cells

The plasmid clone pBSh41 obtained in Example 8 was digested with a restriction enzyme EcoRI, and a DNA fragment comprising the cDNA encoding the full length of human JTT-1 antigen was excised. This DNA fragment was inserted with DNA Ligation Kit (Takara Shuzo) into a plasmid pEFneo (Proc. Natl. Acad. Sci. USA, Vol.91, pp.158-162 (1994)) treated with the same restriction enzyme EcoRI to prepare the expression vector. CHO-K1 cells (ATCC: CCL-61) were transformedwiththevector by electroporation. By cultivating the cells in RPMI1640 medium containing 0.8 mg/ml Genetic in (GIBCO BRL) and 10% fetal calf serum for about two weeks, Geneticin-resistant transformants were selected. The expression of recombinant human JTT-1 antigen was confirmed by Northern blotting by the usual method.

Example 12 Preparation of monoclonal antibodies against human

JTT-1 antigen״.

The recombinant human JTT-1 antigen״-expressing transformants 5 prepared in Example 11 were homogenized and ultracentrifuged (100,000 x g). The pellet containing the cell membrane fraction was collected and suspended in PBS. The resulting suspension comprising the cell membrane fraction was injected into the footpad of a BALB/c mouse with complete Freund's adjuvant for the first 10 immunization (day 0). The cell membrane fraction antigen was further administered into its footpad at intervals of 7, 14, and 28 days. Two days after the last immunization, the lymph node cells was taken out. The lymph node cells and mouse myeloma cells PAI (JCRNo. B0113; Res. Disclosure, Vol.217, p.155 (1982)) were mixed 15 at a ratio of 5:1, and fused using polyethyleneglycol 4000 (GIBCO) as a fusing agent to prepare monoclonal antibody-producing hybridomas. The hybridomas were screened by cultivating them in HAT-containing ASF104 medium (Ajinomoto) supplemented with 10% fetal calf serum and aminopterin. The culture supernatant of each 20 hybridoma was reacted with the recombinant human jtt-1 antigen -expressing transformants prepared in Example 11, and the fluorescence intensity of cells stained by reacting them with FITC-labeled anti-mouse IgG (Cappel) was measured with EPICS-ELITE flow cytometer to confirm the reactivity of the monoclonal antibody 25 generated in each culture supernatant to human JTT-1 antigen. It has been confirmed that 10 or more kinds of hybridomas producing monoclonal antibodies reactive to human JTT-1 antigen were obtained.

Each of two kinds (designated clone SAI2 and SG430) among these 30 hybridomas (10* to 10⁷ cells/0.5 ml/mouse) was injected into a ICR nu/nu mouse (female, 7-8 weeks old) intraperitoneally. After 10 to 20 days, celiotomy of the mice was performed under anesthesia, and the two kinds of monoclonal antibodies (SA12 and SG430) reactive to human JTT-1 antigen were prepared in a large amount from the 35 ascites fluid extracted by the usual method.

Example 13 Effect of the monoclonal antibodies against human

JTT-1 antigen״ on human peripheral blood lymphocytes

As mentioned in Example 8, it is thought that JTT-1 antigen״ can be involved in the regulation of the activation of lympocytes in immune reaction like CD28 and CTLA-4. ״ In order to prove this, 5 the effect of the monoclonal antibodies against human JTT-1 antigen״ on human lymphocytes was analyzed in light of cell growth as an indication.

To each well of 96-well microtiter plate were added (1) either SA12 or SG430 (1 /Zg/ml), the monoclonal antibody against human 10 JTT-1 antigen״ prepared in Example 12, or (2) a mixture of either monoclonal antibody SA12 or SG430 (1 !1 g/ml) with anti-CD3 monoclonal antibody OKT-3 (1 /zg/ml, Orthodiagnostic Systems), which is used for adding the primary signal in the activation of lymphocytes. The plate was incubated at 37°C for 1 hour to coat 15 each well with the antibody. After the plate was washed with RPMI1640 medium, normal human peripheral blood lymphocytes (1 x 10⁵ cells/well) were added to each well and incubated in RPMI1640 medium containing 10% fetal calf serum for 3 days. If necessary, 1 ng/ml phorbol myristate acetate (PNA) was added. Then, [³H] 20 thymidine (3.7 ZZkBq/well) was added to each well, and the plate was incubated at 37°C for 6 hours. The cells were harvested, and the amount of [³H] thymidine incorporated into DNA was measured with a liquid scintillation counter (Beckman). The assay without any antibody was used as a control. The results are shown in Figure 25 15.

In the assay using the plates coated with either monoclonal antibody SA12 or SG430, the number of lymphocytes increased about 10 times compared to the control. In the co-presence of OKT3, the number of lymphocytes increased about 100 times when either 30 monoclonal antibody SA12 or SG430 was used.

These results indicate that JTT-1 antigen functions in the regulation of the lymphocyte activation. The fact that the cell growth rate was increased by using together with OKT3 indicates that JTT-1 antigen is involved in the transmission of 35 costimulatory signal like CD28 and CTLA-4.

Example 14 Effect of JTT-2 antibody on experimental allergic encephalomyelitis (EAE)

As above mentioned in detail, recently, many attempts to treat various autoimmune diseases (rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, 5 chronic inflammatory dermatosis such as lichen planus, systemic lupus erythematosus, insulin dependent diabetes mellitus, psoriasis, etc.) have been made by regulating the transmission of between CD28/CTLA-4 and CD80/CD86. The effect has been already confirmed in various model animals of autoimmune diseases ((1) a 10 model for human systemic lupus erythematosus (SLE), (2) experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis (MS), (3) a model for insulin dependent diabetes mellitus (IDDM) , (4) Goodpasture's nephritis model, and (5) human rheumatoid arthritis).

In order to determine.whether JTT-1 antigen of the present invention is a molecule involved in the activation or inhibition of lymphocytes such as CD28 and CTLA-4, model rats for experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis (MS), were produced, and the effect of the titled 20 monoclonal antibody on JTT-1 antigen in the model was analyzed.

An emulsion to be used as immunogen was prepared by mixing Hartley guinea pig cerebrospinal homogenate (800 mg/ml physiological saline) with the same amount of Freund's complete adjuvant. Immunization was performed by intradermally injecting 25 the emulsion into left and right foot pads of 15 Lewis rats (female, 6-week-old) in an amount of 0.25 ml per footpad. The administration (immunization) was adjusted so as for the dosages of the homogenate prepared to be 200mg per rat. This immunization so induces experimental allergic encephalomyelitis (EAE).

The rats so immunized were divided into three groups of five rats each, and any one of (1) to (3) below was intravenously injected into mice of each group immediately after immunization (day 0), and 3, 6, 9, and 12 days after the immunization.

(1) Monoclonal antibody JTT-2 antibody״ against rat JTT-1 35 antigen prepared in Example 2 (dosage! 2 mg/ml PBS, 5 mg/kg) (2) Prednisolone, steroid agent (dosage: 4 mg/ml PBS, 10 mg/kg) (3) Control antibody non-reactive to ״rat JTT-1 antigen״ (dosage:

mg/ml PBS, 5 mg/kg)

Symptom was observed in the course of time after the immunization. After the onset of EAE had been found, the degree of the symptom 5 was estimated by scoring the symptom based on the following criteria.

(Score 1) Disappearance of tension of a tail (Score 2) Dragging of hind legs, and slight paralysis (Score 3) Dragging of hind legs, and serious paralysis (Score 4) Paralysis of the whole body, or death

The results are shown in Figure 16. In the group to which the control antibody was administered, the symptom of EAE reached the peak (maximum score) at day 11 to 15 after the immunization, and then gradually recovered. In contrast, in the JTT-2 15 antibody״-administered group, the symptom of EAE at day 11 after the immunization was significantly inhibited. This inhibitory effect was significantly higher than that in the prednisoloneadministered group.

These results indicate that JTT-1 antigen״ is a molecule that 20 functions in the induction of immune response such as the lymphocyte activation induced by immunization by foreign antigens, and that the regulation of the function of JTT-1 antigen or its ligands can inhibit the symptom of various autoimmune diseases.

Example 15 Effect of JTT-2 antibody on glomerulonephritis 25 For the same purpose as Example 14, glomerulus basement membrane (GBM) nephritis model rats were produced, and the effect of the titled monoclonal antibody on JTT-1 antigen in the model was analyzed.

After bovine glomerulus basement membrane (Shigei Medical 30 Institute) digested with collagenase was diluted־ with physiological saline to 200 /zg/ml, the dilution was mixed with Freund's complete adjuvant to prepare an emulsion to be used as immunogen, immunization was performed by intradermally injecting the emulsion into both hind soles of 48 wister kyoto rats (about 35 200 g) under anesthesia in an amount of about 0.2 ml per footpad (dosage! about 15 /zg). This immunization so induces glomerulus basement membrane (GBM) nephritis.

The immunized rats were divided into eight groups of six rats each, and any one of (1) to (3) below was injected into rats of each group immediately after immunization (day 0), and three times a week for 5 consecutive weeks.

(1) Monoclonal antibody JTT-2 antibody against rat JTT-1 antigen prepared in Example 2 (dosage: 3 mg/kg (2 ml PBS/kg), intravenous injection) (2) Prednisolone, steroid agent, as a positive control (suspended 10 in 0.5% carboxymethylcellulose (CMC)) (dosage: 3 mg/kg (5 ml/kg), oral administration) (3) 0.5% CMC as a negative control (dosage: 5 ml/kg, oral administration)

After the administration of a test substrate, sterilized water 15 (25 ml/kg) was orally administered into each rat forcedly, and urine was collected for 5 hours from each rat which had been kept in a metabolism cage without eating and drinking. After the volume of the collected urine was measured, the urinary protein concentration was measured using Tonein TP-II (Otuka), and the urinary excretion 20 of protein per five hours was calculated (unit: mg protein/5 hours).

The above-mentioned urinary collection and urinary protein measurement were performed in the same manner at 1, 2, 3, and 4 weeks after the immunization (day 0).

The results are shown in Figure 17. Compared to the control 25 group, the urinary excretion of protein at 3 weeks after the immunization was significantly reduced in the JTT-2 antibody-administered group.

These results indicate that JTT-1 antigen is a molecule that induces immune response such as the lymphocyte activation induced 30 by immunization by foreign antigens, and that the regulation of the function of JTT-1 antigen or its ligands can inhibit the symptom of various autoimmune diseases.

Example 16 preparation of the fusion protein between JTT-1 antigen and IgFc

As mentioned in Examples 8, and 13 to 15, JTT-1 antigen of the present invention is thought to be a molecule such as CD28 and CTLA-4 involved in the transmission of costimulatory signal involved in the regulation of the activation of lymphocytes. In addition, as mentioned in Example 14, a fusion protein (CTLA-4-lgFc) composed of the extracellular domain of CTLA-4 and the Fc region 5 of human immunoglobulin IgGl reportedly has therapeutic effects on various autoimmune diseases. In this Example, a fusion protein composed of the extracellular region of ״JTT-1 antigen and human igGFc was prepared as follows in order to examine whether soluble JTT-1 antigen, like CTLA-4-IgFc, could be applied to therapy of 10 various autoimmune diseases.

(1) Preparation of the fusion protein between rat JTT-1 antigen and human igGl-Fc (rJTT-l-lgFc)

In order to amplify the cDNA encoding the extracellular region of rat JTT-1 antigen by PCR, 5׳ primer having Xhol restriction 15 site (5׳-CTGCTCGAGATGAAGCCCTACTTCTCG-3׳, SEQ ID NO: 7) and 3׳ primer having BamHI restriction site (5׳ACCCTACGGGTAACGGATCCTTCAGCTGGCAA-3 ׳, SEQ ID NO: 8) at their terminus were designed and synthesized. Using cDNA clone T132A7 obtained in Example 7 encoding the full length of rat JTT-1 antigen 20 as a template, PCR was performed with the primers to prepare the cDNA comprising the cDNA encoding the extracellular region of rat JTT-1 antigen having Xhol and BamHI restriction sites at its both ends. The PCR products so obtained were digested with Xhol and BamHI and separated by agarose gel electrophoresis to isolate an 25 about 450-bp band predicted to be the cDNA fragment encoding a desired extracellular region. The isolated cDNA fragment was subcloned into pBluescript II SK ( + ) (Stratagene) cleaved with Xhol and BamHI. Sequence analysis with an automated fluorescence DNA sequencer (Applied Biosystems) revealed that the cDNA fragment 30 comprises the region encoding amino acid sequence corresponding to the amino acid residues 1 to 141 of rat JTT-1 antigen (SEQ ID NO: 4).

On the other hand, the DNA encoding the Fc of human IgGl as the fusion partner was cut out as an about 1.3 kb BamHI-Xbal DNA fragment 35 by digesting the plasmid (see Cell, Vol.61, pp.1303-1313 (1990)j. Prepared by B. Seed et al. (Massachusetts General Hospital)) with

BamHI and Xbal. This fragment comprises exons encoding human IgGl hinge region, Cy_t2, and Cy!3.

The XhoI-BamHI fragment encoding the extracellular region of rat JTT-1 antigen, and BamHI-Xbal fragment comprising exons 5 encoding the Fc of human IgGl (IgFc), both prepared as mentioned above, were subcloned into pBluescript II SK (+) (Stratagene) cleaved with Xhol and Xbal.

Then, the plasmid was digested with Xhol and Xbal, and an about 1.8 kb DNA fragment comprising the fusion DNA comprising the 10 extracellular region of rat JTT-1 antigen and human IgFc was cut out. This fusion DNA fragment was inserted into the Xhol and Xbal sites of the expression vector pME18S (Medical Immunology, Vol.20, No.l, pp.27-32 (1990); Experimental Medicine: SUPPLEMENT,

Handbook of Genetic Engineering,״ Yodosha, pp.101-107 (1992)) 15 with T4 DNA ligase to construct plasmid prJTT-l-IgFc.

HEK293 cells (ATCC CRL1573) subconfluently cultivated as monolayer in DMEM medium containing 10% fetal calf serum and ampicillin were transformed with prJTT-l-IgFc by electroporation to obtain transformants.

The transformants were cultured in serum-free ASF104 medium for 72 hours to express rJTT-l-IgFc.

Using a Protein G Sepharose affinity column (Pharmacia), rJTT-l-lgFc was purified as follows.

The supernatant obtained by centrifuging the culture medium 25 mentioned above was loaded onto Protein G Sepharose affinity column previously equilibrated with binding buffer. After the column was washed with binding buffer, elution was performed with elution buffer. The eluate was collected and dialyzed against phosphate buffer with exchanging the external solution twice or more to obtain 30 pure rJTT-1-IgFc. ־־

The result of affinity chromatography is shown in Figure 18, and the result of SDS-PAGE of the pure rJTT-l-IgFc so obtained in Figure 19.

(2) Preparation of the fusion protein between human JTT-1 35 antigen and human IgGl-Fc (hJTT-l-IgFc) hJTT-l-lgFc was prepared as mentioned above in (1) , except for cDNA used as templates and primers for PCR. In this test, the clone pBSh41 comprising the cDNA encoding the full length human JTT-1 antigen prepared in Example 8 was used as a template, and 5׳TAACTGTTTCTCGAGAACATGAAGTCAGGC-3' (SEQ ID NO: 9) and 5׳5 ATCCTATGGGTAACGGATCCTTCAGCTGGC-3׳ (SEQ ID NO: 10) were used as primers.

The result of affinity chromatography is shown in Figure 20, and the result of SDS-PAGE of the pure hJTT-l-IgFc so obtained in Figure 21.

Example 17 Preparation of a transgenic mouse in which cDNA encoding rat JTT-1 antigen has been integrated

The cDNA encoding the full length of rat JTT-1 antigen obtained in Example 7 was inserted into the expression vector pCAGGS (Gene, Vol. 108, pp. 193-200 (1991)) having chicken β actin promoter using 15 DNA Blunting Kit (Takara) to obtain plasmid, prJTT-1. In order to prepare a transgenic mouse, prJTT-1 was linearized by restriction enzyme treatment.

A female ICR mouse having a vaginal plug, obtained by mating a white ICR mouse (Nihon LSC) with a male vasoligated white ICR 20 mouse (Nihon SLC), was used as a foster mother mouse. A mouse for obtaining fertilized eggs for introducing rat JTT-1 antigen gene thereinto was prepared by mating a female BDF-1 mouse (Nihon SLC) that had been made to superovulate by administered PEAMEX (5 units, Sankyo zoki) and Pregnil (5 units. Organon) with a male BDF-1 male 25 (Nihon SLC). After mating, the oviduct was excised from the female BDF-1 mouse, and only fertilized eggs were obtained by hyaluronidase treatment and stored in a medium.

The rat JTT-1 antigen gene was introduced into the fertilized egg under microscopy using a manipulator according to the usual 30 method. The fertilized egg was fixed with a retaining needle. A solution containing the above-mentioned linearized gene encoding rat JTT-1 antigen, which was diluted with Tris-EDTA buffer, was micorinjected into the male pronucleus of the fertilized eggs with a DNA introduction needle at 37°C.

After gene introduction, only fertilized eggs keeping normal state were selected, and then, the fertilized egg so selected in

L which the ״rat JTT-1 antigen״ genes have been introduced was inserted into the ovarian fimbria in the ovary of a foster mother mouse (white ICR mouse).

The tail of a progeny mouse (founder mouse) born from the foster 5 mother mouse was cut off and the genomic gene was collected from it. It was confirmed by PCR that the ״rat JTT-1 antigen״ gene was integrated into the mouse genome. Then, heterozygous transgenic mice highly expressing rat JTT-1 antigen״ were prepared by mating this founder mouse with a normal mouse. Homozygous transgenic mice 10 can be prepared by mating the heterozygous mice with each other.

The microinjected construct comprising the rat JTT-1 antigen״ gene is schematically shown in Figure 22.

Example 18 Preparation of a knockout mouse whose endogenous gene encoding mouse JTT-1 antigen״ has been inactivated (1) Construction of a targeting vector

A targeting vector for inactivating (knocking out) the endogenous gene encoding mouse JTT-1 antigen through homologous recombination (Nikkei Science, pp.52-62 May 1994) was prepared as follows.

The Pstl-Hindlll fragment (homologous DNA (1)״) obtained by digesting the mouse genomic DNA clone comprising the region encoding mouse JTT-1 antigen cloned in Example 9-5 with PstI and Hindlll was subcloned into pGEM-3 (Promega). Then, pGEM-3 was linerized with Xhol, and neomycin resistance gene (neo) excised from 25 pMCl-neo-polyA (Stratagene) by treating it with Xhol and Sall was inserted at the upstream of the homologous DNA1) ״) and then ligated them. The above-mentioned mouse genomic DNA clone was digested with Xhol and NotI to cut off an about 5.5 kb gene (homologous DNA (2) ״) located upstream of above-mentioned homologous DNA (1). ״

Separately, the above-mentioned pGEM-3 into which neo-homalogous DNA (1) has been inserted was digested with Xhol and Hindlll to cut off neo-homologous DNA (1). Homologous DNA (2) and neo-homologous DNA (1) thus obtained were subcloned into pSEAP2-CONT (Clontech) linerized with Notl and Hindlll.

After the obtained plasmid, in which homologous (2)-neohomologous (1) has been inserted, was digested and linerized at the downstream of homologous DNA (1) with Nrul, thymidine kinase gene (TK) obtained by digesting pMCl-TK (Stratagene) with Pvull was inserted at the downstream of homologous DNA (1) to obtain a targeting vector, in which homologous DNA (2)-neo-homologous (1) was inserted.

(2) Introduction of the targeting vector into ES cells

Mouse embryonic stem cells (Nature, Vol.362, pp.255-258 (1993); Nature, Vol.326, pp.292-295 (1987)) cultured in DMEM medium containing 15% fetal calf serum were treated with trypsin to be 10 single cells, and the cells were washed three times, followed by adding phosphate buffer thereto to adjust 1 x 10⁷ cells/ml. The targeting vector mentioned above (25 jLlg per 1 ml of the cell suspension) was added to the cell suspension, and electric pulse was delivered once under the condition of 350 V/cm (25 /ZF). Then, 15 lx 10’ of ES cells were plated on a 10-cm dish and cultivated in maintenance medium for a day, and the medium was changed to selection medium (containing 250 Ag/ml G418 and 2 /ZM ganciclovir). The cells were cultivated with the medium changed every two days. At the tenth day from the introduction of the targeting vector, 573 20 neomycin-resistant ES cell clones were obtained under microscopy with a micropipet. Each of the ES cell clones so obtained were cultivated independently on a 24-well plate coated by Feeder cells to obtain 768 neomycin-resistant ES cell replicas.

(3) Screening of knockout ES cells

It was confirmed by PCR whether the endogenous gene encoding mouse JTT-1 antigen was disrupted (knocked out) through homologous recombination in each of the neomycin-resistant ES cells.

For PCR, (1) primers designed and synthesized based on the 30 sequence of above-mentioned neomycin-resistant gene (־neo) (5 ׳ -CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC-3 ׳, SEQ ID NO: 11) and (2) primers designed and synthesized based on the sequence of above-mentioned homologous DNA (1) . (5'CATTCAAGTTTCAGGGAACTAGTCCATGCGTTTC-3 ׳, SEQ ID NO: 12) were used. 35 Each genomic DNA was extracted from each of the neomycinresistant ES cell, and PCRs were performed using the primers with the genomic DNA as a template. PCR was performed 1 cycle of reaction at 94°C for 3 minutes, 30 cycles of reaction at 94°C for 1 minute, at 60°C for 1 minute, and at 72°C for 3.5 minutes, and 1 cycle of reaction at 72°C for 10,minutes, and the resulting products were stored at 4°C. When a fragment less than about 4 kb was amplified by this PCR, it could be judged that the endogenous gene encoding mouse JTT-1 antigen״ was disrupted (knocked out) through homologous recombination in the ES cell clone.

A desired PCR product was obtained from three of 768 ES cell clones tested. Genomic southern blottings were performed for these three clones for further screening and confirmation. After genomic DNA was extracted from the three clones and digested with restriction enzyme BamHI, the digested products were subjected to agarose gel electrophoresis. The resulting DNAs were transferred to nylon membrane, and hybridization was performed with a probe prepared from the genomic DNA sequence comprising mouse JTT-1. The probe was designed based on the sequence outside the site where homologous recombination occurred, which enables to distinguish mutant type genome from normal type genome in size.

As a result, two bands corresponding to mutant type and normal type were observed in one of the three clones. This ES cell clone was used for the preparation of a knockout mouse described below.

(4) Preparation of a knockout mouse

The above-obtained ES cells (15 ES cells per blastocyst) whose endogenous gene encoding mouse JTT-1 antigen has been inactivated (knocked out) through homologous recombination were microinjected into blastocysts to, which were obtained by mating a female C57BL6 mouse (Nihon Charles River) with male one. Immediately after the microinjection, the blastocysts (about 10 blastocysts per one side of the uterus) were transplanted in the uterus of a<foster mother ICR mouse (CLEA Japan), which was 2.5 day-mouse from pseudopregnant treatment. As a result, 38 progeny mice in total were obtained, and 18 out of them were desired chimeric mice. Eleven (11) out of the chimeric mice were the chimeric-mouse in which the contribution to hair color was 80% or more.

The chimeric mice so obtained were then mated with a normal

C57BL6 mice to obtain agouti mice whose color is derived from hair color gene of the ES cells.

Example 19 Preparation of pharmaceutical composition comprising antibody

Each of the monoclonal antibody (50-150 μ g/ml), ״jtt-1 antibody and JTT-2 antibody against rat JTT-1 antigen, prepared in Example 1, and monoclonal antibodies, SAI2 and SG430 against human JTT-1 antigen, prepared in Example 12, was added to injectable distilled water (10 ml) to prepare injection.

industrial Applicability

Novel cell surface molecules (called JTT-1 antigen) of the present invention derived from mammals such as human, mouse, and rat are characterized as follows.

(1) JTT-1 antigen had the following similarity with CD28, a cell surface molecule on lymphocytes such as T cells, which transmits costimulatory signal important for T cell activation through cell adhesion, and CTLA-4, a cell surface molecule on lymphocytes such as T cells, which regulates the function of activated lymphocytes such as activated T cells, cooperating with the signal.

(1) 20 or more amino acid residues including cysteine residues are highly conserved;

(ii) Prorine repeating sequence, ״Pro-Pro-Pro (PPP),״ which is 25 essential as the ligand binding region, is conserved in the extracellular region;

(iii) ״Tyr-Xaa-Xaa-Met (YxxM)״ (Xaa and x represents any amino acid) sequence essential as the signal transmitting region is conserved in the cytoplasmic region; and (iv) The locus of the gene encoding mouse JTT-1 antigen on mouse chromosome is 1C3, like CD28״ and CTLA-4.

(2) JTT-1 antigen can mediate cell adhesion of thymocytes, lymphoblasts stimulated with mitogen such as ConA, thymomas, like CD28 and CTLA-4 that mediate cell adhesion.

(3) JTT-1 antigen is strongly expressed, at least, in thymocytes, lymphoblast cells stimulated with mitogen such as ConA (activated

T lymphoblast cells and activated B lymphoblast cells, etc.), peripheral blood lymphocytes, and thymomas.

(4) The antibody against JTT-1 antigen״ significantly proliferates human peripheral blood lymphocytes, and the proliferation is more enhanced in the presence of a monoclonal antibody against CD3 constituting TCR/CD3 complex on T cells that receive the primary signal essential for T cell activation from antigen-presenting cells.

(5) The administration of the antibody against JTT-1 antigen 10 significantly inhibits the symptom of experimental allergic encephalomyelitis (EAE).

(6) The administration of the antibody against JTT-1 antigen to a model rat for glomerulus basement membrane (GBM) nephritis significantly inhibits the symptom of this disease.

JTT-1 antigen of the present invention is, like CD28״ and CTLA-4, ״ thought to be a molecule transmitting the secondary signal (costimulatory signal) essential for the activation of lymphocytes such as T cells, and regulating the function of activated lymphocytes such as activated T cells, cooperating with the signal.

Therefore, polypeptides constituting such cell surface molecules, its polypeptide fragment, and fusion polypeptides therefrom, and antibodies thereto of the present invention can provide extremely useful pharmaceuticals for therapy or prevention of various autoimmune diseases, allergic diseases, or inflammatory 25 diseases, specifically, rheumatoid arthritis, multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis, chronic inflammatory dermatosis such as lichen planus, systemic lupus erythematosus, insulin dependent diabetes mellitus, and psoriasis, caused by the activation of lymphocytes such as T cells and the 30 abnormality of regulation of activated lymphocyte functions.

Similarly, the genes encoding polypeptides or polypeptide fragments of the present invention can be used in not only gene therapy of various diseases as mentioned above but also preparation of antisense pharmaceuticals.

Among the antibodies of the present invention, human monoclonal antibodies and their pharmaceutical compositions have dramatically increased pharmaceutical value of antibody drugs because they have no antigenicity against human, which has been a serious problem (side effect) of antibody pharmaceuticals containing nonhuman mammal-derived antibodies such as mouse-derived antibodies.

The genes (DNA), polypeptides, polypeptide fragments and antibodies of the present invention are useful not only as pharmaceuticals but also as reagents for searching molecules (ligands) interacting with the cell surface molecules of the present invention, clarifying the function of the ligand, and developing 10 drugs targeting the ligands.

Furthermore, the transgenic mouse of the present invention is extremely useful not only as a model animal for studying physiological function of JTT-1 antigen that is a cell surface molecule of the present invention but also as a tool for screening 15 various drugs (low molecular weight compounds, antibodies, antisense substances, polypeptides, etc.) having activity regulating (inhibition, suppression, activation, stimulation, etc.) the function of JTT-1 antigen. Specifically, such test . substances can be administered to the transgenic mouse to measure 20 and analyze various physiological, biological, or pharmacological parameters generated in the mouse, thereby assessing activity of the administered test substances.

In addition, the knockout mouse of the present invention can clarify the function of the cell surface molecules of the present 25 invention by analyzing the characteristics of the mouse from various viewpoints (physiological, biological, pharmacological, pathological, and genetic viewpoints).

Sequence listing (1) Name of applicant: JAPAN TABACCO INC.

(2) Title of the invention: Cell Surface Molecule Mediating Cell Adhesion and Signal Transmission (3) Reference number: J1-802PCT (4) Application number:

(5) Filing date:

(6) Country where the priority application was filed and the application number of the application:

Japan, No. Hei 9-062290

Japan, Patent Application filed on February 26, 1998 (reference number, J1-802DP1) (7) Priority date: February 27, 1997 (8) Number of sequences: 12

SEQ ID NO: 1:

SEQUENCE LENGTH: 600

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: cDNA to mRNA

ORIGINAL SOURCE:

ORGANISM: Homo sapiens

FEATURE:

NAME/KEY: CDS .

LOCATION: 1 . 600

IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 1

ATG AAG TCA GGC CTC TGG TAT TTC TTT CTC30

Met Lys Ser Gly Leu Trp Tyr Phe PheLeu

־־ 10 5 1 30

TTC TGC TTG CGC ATT AAA GTT TTA ACA GGA60

Phe Cys Leu Arg He Lys Val Leu ThrGly

20

GAA ATC AAT GGT TCT GCC AAT TAT GAG ATG90

Glu He Asn Gly Ser Ala Asn Tyr Glu Met

30

TTT ATA TTT CAC AAC GGA GGT GTA CAA ATT120

Phe He Phe His Asn Gly Gly Val GinHe

3540

TTA TGC AAA TAT CCT GAC ATT GTC CAG CAA150

Leu Cys Lys Tyr Pro Asp He Val GinGin

4550

TTT AAA ATG CAG TTG CTG AAA GGG GG6 CAA180

Phe Lys Met Gin Leu Leu Lys Gly GlyGin

5560

ATA CTC TGC GAT CTC ACT AAG ACA AAA GGA210

He Leu Cys Asp Leu Thr Lys Thr LysGly

6570

AGT GGA AAC ACA GTG TCC ATT AAG AGT CTG240

Ser Gly Asn Thr Val Ser lie Lys SerLeu

7580

AAA TTC TGC CAT TCT CAG TTA TCC AAC AAC270

Lys Phe Cys His Ser Gin Leu Ser AsnAsn

8590

AGT GTC TCT TTT TTT CTA TAC AAC TTG GAC300

Ser Val Ser Phe Phe Leu Tyr Asn LeuAsp

95100

CAT TCT CAT GCC AAC TAT TAC TTC TGC AAC330

His Ser His Ala Asn Tyr Tyr Phe CysAsn

105110

CTA TCA ATT TTT GAT CCT CCT CCT TTT AAA360

Leu Ser lie Phe Asp Pro Pro Pro PheLys

115120

GTA ACT CTT ACA GGA GGA TAT TTG CAT ATT390

Val Thr Leu Thr Gly Gly Tyr Leu HisHe

125130

TAT GAA TCA CAA CTT TGT TGC CAG CTG AAG420

Tyr Glu Ser Gin Leu Cys Cys Gin LeuLys

135140

TTC TGG TTA CCC ATA GGA TGT GCA GCC TTT450

Phe Trp Leu Pro lie Gly Cys Ala AlaPhe

145150

GTT GTA GTC TGC ATT TTG GGA TGC ATA CTT

Vai Vai Vai Cys Ile Leu Gly Cys Ile Leu

135160

ATT TGT TGG CTT ACA AAA AAG AAG TATTCA lie Cys Trp Leu Thr Lys Lys Lys TyrSer

165170

TCC AGT GTG CAC GAC CCT AAC GGT GAATAC

Ser Ser Vai His Asp Pro Asn Gly GluTyr

175180

ATG TTC ATG AGA GCA GTG AAC ACA GCC AAA Met Phe Met Arg Ala Vai Asn Thr Ala Lys 185190

AAA TCT AGA CTC ACA GAT GTG ACC CTA TAA Lys Ser Arg Leu Thr Asp Vai Thr Leu

15195

SEQUENCE ID NO: 2:

SEQUENCE LENGTH: 199

SEQUENCE TYPE: amino acid

TOPOLOGY: linear

MOLECULE TYPE: protein

ORIGINAL SOURCE:

ORGANISM: Homo sapiens

SEQUENCE DESCRIPTION: SEQ ID NO: 2

Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu 1 510

Phe Cys Leu Arg Ile Lys Vai Leu Thr Gly 1520

Glu He Asn Gly Ser Ala Asn Tyr Glu Met 30 2530

Phe Ile Phe His Asn Gly Gly Vai Gin Ile 3540

Leu Cys Lys Tyr Pro Asp lie Vai Gin Gin 4550

Phe Lys Met Gin Leu Leu Lys Gly Gly Gin 5560

He Leu Cys Asp Leu Thr Lys Thr Lys Gly

70

Ser Gly Asn Thr Val Ser lie Lys Ser Leu

80

Lys Phe Cys His Ser Gin Leu Ser Asn Asn 8590

Ser Val Ser Phe Phe Leu Tyr Asn Leu Asp 95100

His Ser His Ala Asn Tyr Tyr Phe Cys Asn

105110

Leu Ser He Phe Asp Pro Pro Pro Phe Lys 115120

Val Thr Leu Thr Gly Gly Tyr Leu His He 125130

Tyr Glu Ser Gin Leu Cys Cys Gin Leu Lys 135140

Phe Trp Leu Pro He Gly Cys Ala Ala Phe 145150

Val Val Val Cys He Leu Gly Cys He Leu 20 155160

He Cys Trp Leu Thr Lys Lys Lys Tyr Ser 165170

Ser Ser Val His Asp Pro Asn Gly Glu Tyr 175180

Met Phe Met Arg Ala Val Asn Thr Ala Lys 185190

Lys Ser Arg Leu Thr Asp Val Thr Leu 195

SEQUENCE ID NO: 3:

SEQUENCE LENGTH: 2610

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acids (cDNA containing 3'- and 5׳-untranslated 35 sequences)

ORIGINAL SOURCE:

ORGANISM: Homo sapiens

FEATURE:

NAME/KEY: CDS

LOCATION: 26 . 625

IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 3

GGACTGTTAA CTGTTTCTGG CAAAC25

ATG AAG TCA GGC CTC TGG TAT TTC TTT CTC55

Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu

510

TTC TGC TTG CGC ATT AAA GTT TTA ACA GGA85

Phe Cys Leu Arg lie Lys Val Leu Thr Gly

1520

GAA ATC AAT GGT TCT GCC AAT TAT GAG ATG115

Glu He Asn Gly Ser Ala Asn Tyr Glu Met

2530

TTT ATA TTT CAC AAC GGA GGT GTA CAA ATT145

Phe lie Phe His Asn Gly Gly Val Gin He

3540

TTA TGC AAA TAT CCT GAC ATT GTC CAG CAA175

Leu Cys Lys Tyr Pro Asp lie Val Gin Gin

4550

TTT AAA ATG CAG TTG CTG AAA GGG GGG CAA205

Phe Lys Met Gin Leu Leu Lys Gly Gly Gin

5560

ATA CTC TGC GAT CTC ACT AAG ACA AAA GGA235

He Leu Cys Asp Leu Thr Lys Thr Lys Gly

6570

AGT GGA AAC ACA GTG TCC ATT AAG AGT CTG265

Ser Gly Asn Thr Val Ser He Lys Ser Leu

7580

AAA TTC TGC CAT TCT CAG TTA TCC AAC AAC295

Lys Phe Cys His Ser Gin Leu Ser Asn Asn

8590

AGT GTC TCT TTT TTT CTA TAC AAC TTG GAC325

Ser Val Ser Phe Phe Leu Tyr Asn Leu Asp

95100

CAT TCT CAT GCC AAC TAT TAG TTC TGC AAC355

His Ser His Ala Asn Tyr Tyr Phe CysAsn

105110

CTA TCA ATT TTT GAT CCT CCT CCT TTT AAA385

Leu Ser lie Phe Asp Pro Pro Pro PheLys

115120

GTA ACT CTT ACA GGA GGA TAT TTG CAT ATT415

Val Thr Leu Thr Gly Gly Tyr Leu HisHe

125130

TAT GAA TCA CAA CTT TGT TGC CAG CTG AAG445

Tyr Glu Ser Gin Leu Cys Cys Gin LeuLys

135140

TTC TGG TTA CCC ATA GGA TGT GCA GCC TTT475

Phe Trp Leu Pro He Gly Cys Ala AlaPhe

145150

GTT GTA GTC TGC ATT TTG GGA TGC ATA CTT505

Val Val Val Cys lie Leu Gly Cys lieLeu

155160

ATT TGT TGG CTT ACA AAA AAG AAG TAT TCA535

He Cys Trp Leu Thr Lys Lys Lys TyrSer

165170

TCC AGT GTG CAC GAC CCT AAC GGT GAA TAG565

Ser Ser Val His Asp Pro Asn Gly GluTyr

175180

ATG TTC ATG AGA GCA GTG AAC ACA GCC AAA595

Met Phe Met Arg Ala Val Asn Thr AlaLys

185190

AAA TCT AGA CTC ACA GAT GTG ACC CTA TAA625

Lys Ser Arg Leu Thr Asp Val Thr Leu

TATGGAACTC TGGCACCCAG GCATGAAGCA CGTTGGCCAG TTTTCCTCAA 675

CTTGAAGTGC AAGATTCTCT TATTTCCGGG ACCACGGAGA GTCTGACTTA 725

ACTACATACA TCTTCTGCTG GTGTTTTGTT CAATCTGGAA GAATGACTGT 775

ATCAGTCAAT GGG6ATTTTA ACAGACTGCC TTG6TACTGC CGAGTCCTCT 825

CAAAACAAAC ACCCTCTTGC AACCAGCTTT GGAGAAAGCC CAGCTCCTGT 875

6TGCTCACTG GGAGTG6AAT CCCTGTCTCC ACATCTGCTC CTAGCAGTGC 925 ATCAGCCAGT AAAACAAACA CATTTACAAG AAAAAT6TTT TAAAGATGCC 975 A6GGGTACT6 AATCTGGAAA GCAAATGAGC AGCCAAGGAC CAGCATCTGT 1025 CCGCATTTCA CTATCATACT ACCTCTTCTT TCTGTA6G6R TGAGAATTCC 1075 5 TCTTTTAATC AGTCAA6GGA GATGCTTCAA AGCTGGRGCT ATTTTATTTC 1125

TGAGAT6TTG AT6T6AACTG TACATTA6TA CATACTCA6T ACTCTCCTTC 1175 AATTGCTGAA CCCCAGTTGA CCATTTTACC AAGACTTTAG ATGCTTTCTT 1225 GTGCCCTCAA TTTTCTTTTT AAAAATACTT CTACAT6ACT GCTTGACAGC 1275 CCAACAGCCA CTCTCAATAG AGAGCTATGT CTTACATTCT TTCCTCTGCT 1325 10 GCTCAATA6T TTTATATATC TAT6CATACA TATATACACA CATATGTATA 1375

TAAAATTCAT AATGAATATA TTTGCCTATA TTCTCCCTAC AAGAATATTT 1425 TTGCTCCAGA AAGACATGTT CTTTTCTCAA ATTCAGTTAA AATGGTTTAC 1475 TTT6TTCAA6 TTAGTGGTAG GAAACATTGC CCGGAATTGA AAGCAAATTT 1525 AWTTATTAT CCTATTTTCT ACCATTATCT ATGTTTTCAT GGTGCTATTA 1575 15 ATTACAAGTT TAGTTCTTTT T6TAGATCAT ATTAAAATTG CAAACAAAAT 1625

CATCTTTAAT GGGCCAGCAT TCTCATGGGG TAGAGCAGAA TATTCATTTA 1675 GCCTGAAAGC TGCAGTTACT ATAGGTTGCT GTCAGACTAT ACCCATGGTG 1725 CCTCTGGGCT TGACAGGTCA AAATG6TCCC CATCAGCCT6 GAGCAGCCCT 1775 CCAGACCTGG GTGGAATTCC AGGGTTGAGA GACTCCCCTG AGCCAGAGGC 1825 20 CACTAGGTAT TCTT6CTCCC AGAGGCTGAA GTCACCCTGG GAATCACAGT 1875

G6TCTACCTG CATTCATAAT TCCAGGATCT 6T6AAGAGCA CATATGTGTC 1925 AGGGCACAAT TCCCTCTCAT AAAAACCACA CAGCCTGGAA ATTGGCCCTG 1975 6CCCTTCAAG ATAGCCTTCT TTA6AATAT6 ATTTGGCTAG AAAGATTCTT 2025 AAATATGTGG AATATGATTA TTCTTAGCTG GAATATTTTC TCTACTTCCT 2075 25 GTCTGCATGC CCAAGGCTTC TGAAGCAGCC AATGTCGATG CAACAACATT 2125

TGTAACTTTA G6TAAACTGG GATTATGTTG TAGTTTAACA TTTTGTAACT 2175 6TGTGCTTAT AGTTTACAAG TGAGACCCGA TATGTCATTA TGCATACTTA 2225 TATTATCTTA AGCATGTGTA ATGCTGGATG TGTACAGTAC AGTACWTAAC 2275 TTGTAATTTG AATCTAGTAT GGTGTTGTGT TTTCAGCTGA CTTGGACAAC 2325 30 CTGACTGGCT TTGCACAGGT GTTCCCTGAG TTGTTTGCAG GTTTCTGTGT 2375

GTGGGGTGGG GTATGGGGAG GAGAACCTTC ATGGTGGCCC ACCTGGCCTG 2425 GTT6TCCAAG CTGT6CCTCG ACACATCCTC ATCCCAAGCA TGGGACACCT 2475 CAAGATGAAT AATAATTCAC AAAATTTCTG TGAAATCAAA TCCA6TTTTA 2525 AGAGGAGCCA CTTATCAAAG AGATTTTAAC AGTAGTAAGA AGGCAAAGAA 2575 35 TAAACATTT6 ATATTCAGCA ACTGAAAAAA AAAAA 2610

SEQ ID NO: 4:

SEQUENCE LENGTH: 2072

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acids (cDNA containing 3׳- and 5׳-untranslated sequences)

ORIGINAL SOURCE:

ORGANISM: Rattus

FEATURE:

NAME/KEY: CDS

LOCATION: 35 . 637

IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 4

CTGGAGGGGA AGAGTGCAGC TGTTCCTGGC AGAC34

ATG AAG CCC TAC TTC TCG TGC GTC TTT GTC64

Met Lys Pro Tyr Phe Ser Cys Vai PheVai

510

TTC TGC TTC CTA ATC AAA CTT TTA ACA GGA94

Phe Cys Phe Leu lie Lys Leu Leu ThrGly

1520

GAA CTC AAT GAC TTG GCC AAT CAC AGG ATG124

Glu Leu Asn Asp Leu Ala Asn His ArgMet

2530

TTT TCG TTT CAC GAT GGA GGT GTA CAG ATT154

Phe Ser Phe His Asp Gly Gly Vai Ginlie

3540

TCT TGT AAC TAC CCT GAG ACT GTC CAG CAG 184;

Ser Cys Asn Tyr Pro Glu Thr Vai GinGin

4550

TTA AAA ATG CAG TTG TTC AAA GAC AGA GAA 214־־

Leu Lys Met Gin Leu Phe Lys Asp ArgGlu

5560

GTC CTC TGC GAC CTC ACC AAG ACC AAG GGA244

Vai Leu Cys Asp Leu Thr Lys Thr LysGly

6570

AGC GGA AAC ACC GTG TCC ATC AAG AAT CCG274

Ser Gly Asn Thr Val Ser He Lys Asn Pro

80

ATG TGC TGT CCA TAT CAG CTG TCC AAC AAC 304

Met Ser Cys Pro Tyr Gin Leu Ser Asn Asn

85 90

AGT GTC TCT TTT TTC CTA GAC AAC GCA GAC334

Ser Val Ser Phe Phe Leu Asp Asn AlaAsp

95100

AGC TCC CAG GGC AGC TAC TTT TTA TGC AGC364

Ser Ser Gin Gly Ser Tyr Phe Leu CysSer

105110

CTG TC6 ATT TTC GAC CCA CCC CCT TTT CAA394

Leu Ser He Phe Asp Pro Pro Pro PheGin

115120

GAA AAG AAC CTT AGT GGA GGA TAT TTG CTT424

Glu Lys Asn Leu Ser Gly Gly Tyr LeuLeu

125130

ATT TAT GAA TCC CAG CTT TGT TGC CAG CTG454 lie Tyr Glu Ser Gin Leu Cys Cys GinLeu

135140

AAG CTT TGG TTA CCC GTA GGG TGT GCA GCT484

Lys Leu Trp Leu Pro Val Gly Cys AlaAla

145150

TTT GTG GCA GCG CTC CTT TTT GGA TGC ATA514

Phe Val Ala Ala Leu Leu Phe Gly CysHe

155160

TTT ATC GTC TGG TTT GCA AAA AAG AAG TAC544

Phe He Val Trp Phe Ala Lys Lys LysTyr

165170

AGA TCC AGT GTG CAC GAC CCT AAT AGC GAG574

Arg Ser Ser Val His Asp Pro Asn SerGlu

175180

TAC ATG TTC ATG GCG GCA GTC AAC ACA AAC604

Tyr Met Phe Met Ala Ala Val Asn ThrAsn

185. 190

AAA AAG TCC AGA CTT GCA GGT ATG ACC TCA634

Lys Lys Ser Arg Leu Ala Gly Met Thr Ser

195 200

TAA 637

TCTGGAACAC GGGAACCCAT GGAGGAACTA CACTGTCTAG TTCCCCTGAA 687 5 ACTT6AATGG A6AAAGTCTT CTATTTTCTG GACCACAGGG CATCTGACTT 737 GATTAACTAC TGATACCTCC TTTTGGKGTT TTGTTTGTCT GGATCAGTGA 787 CTATCAGTCA CTC6GAATTT CAGCAGACTG CCCTGGGTTT GCTGAGTCCT 837 TTTAAGGCAA ACCCCTTCTT ATAGAAGACC CGGCTCATAT GTATTCAACA 887 AACAGACCTC ACT6GGATAC AATCCCCTCT TTCTGCGCCT GCTTCTAGCT 937 10 ATGCACCGGC CAGCAAGACA AACATATCTC CAGCATTTTT ACAAAAAT6C 987

CAGGGTATGA ATCTGTAAAG TACACAGGCA GCCATTGACC ACCGTCTGTC 1037 י CTCGTTTTTT CA6ATTCTAT TTTTTTCCAT AGAGATCAGC ATTCCTTCTA 1087

GAATCAGACA GTAGAGGGAG ATGCTTCACA ACAGAAGCTC TTATGTTTCT 1137 GA6ATGTT6A TGAATTCATG CTTTAGTACC ACCATGTTCT CTAACAACTT 1187 15 CTATATTCCA GCTGATCACT GCTTCAGGGC TTAGATGCCT 6CTTTTGCCT 1237

TCAAGTCTCC CCTTAAAGAT ACTCCCACAG GTCTACTTG6 TGGCCTGCAG 1287 CCACTCTGAA TAG6AAGTTT GGTCTACAAT TTCCCCCCTC TGCTGCTCAA 1337 AAAAAAAAAT TAGTAGATAT GATTTTCCCA TATTCTCCCT GCCAAAGTAA 1387 TTTTTTCCAG CAAAGACATC TAAATTCAGT TAATATGGTT TACTGTGTTG 1437 20 ATATTAGTGG CAGTAAACAT TTCTCAGAAT CAAAAGCAAA TTAATTTTGC 1487

GGTGGTGTTT TTCTACCATT ATCTTGGGTT TCCATGGTGC TATTACTCAC 1537 AAGTTTAGCT ATTTTTTTAT GCATCATATT AAAGTTGCAA GCAAGCAGAG 1587 CAACCCTCGG TTAATGGGCA AACATTCTCC TGGGGTAGAA TGAATT6TCT 1637 ATTTAGCCCG AAAACTGCAG TTTCTGTGGG TGGCTGCCAG ACTACAGCCG 1687 25 TGCTTTGCTC TGGCTTTGAC AGGTTGAAAT AGYCCCCAT6 ASCSTGGAAC 1737

AGWACTCCAG ACTGTGCTGG AGTCCCAAAG TTAGGAGGGC CATGGAGCCT 1787 GGGACAGGCT GCTGCTTTGG TCTTTAGGAT CTAGGAARAA TTACAGAGGG 1837 GCCAAGACAG AGTTCCCTCC CCTAGAAACT GTGCAGCCT6 6AAGTCAGCC 1887 CTGGCACTTT AAGATAGCCT TCTTTAGAAC ATGAGTTAGT TG6TAGTATT 1937 30 CTGACGTGTA AACAGCCTAT KGTTGCTCGG AGCTGGACCA TTTTCTCCAC 1987

TTCCCTGTCT GCATGCCTAA GACTTCTAGA GCAGCCAACG TATATGCAAC 2037 ATTAAAGAAA AAAAAAAAAA AAAAAAAAAA AAAAA 2072

SEQ ID NO: 5:

SEQUENCE LENGTH: 603

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: cDNA to mRNA

ORIGINAL SOURCE: ORGANISM: Mus 5 FEATURE:

NAME/KEY: CDS LOCATION: 1 .. 603 IDENTIFICATION METHOD: E SEQUENCE DESCRIPTION: SEQ ID NO: 5 10 ATG AAG CC6 TAC TTC TGC CAT GTC TTT GTC

Met Lys Pro Tyr Phe Cys His Vai PheVai

510

TTC TGC TTC CTA ATC AGA CTT TTA ACAGGA

Phe Cys Phe Leu lie Arg Leu Leu ThrGly

1520

GAA ATC AAT GGC TCG GCC GAT CAT AGGATG

Glu lie Asn Gly Ser Ala Asp His Arg Met 2530

TTT TCA TTT CAC AAT GGA GGT GTA CAGATT

Phe Ser Phe His Asn Gly Gly Vai GinHe

3540

TCT TGT AAA TAC CCT GAG ACT GTC CAGCAG

Ser Cys Lys Tyr Pro Glu Thr Vai GinGin

4550

TTA AAA ATG CGA TTG TTC AGA GAG AGAGAA

Leu Lys Met Arg Leu Phe Arg Glu Arg Glu 5560

GTC CTC TGC GAA CTC ACC AAG ACC AAGGGA

Vai Leu Cys Glu Leu Thr Lys Thr LysGly

6570

AGC GGA AAT GCG GTG TCC ATC AAG AAT CCA

Ser Gly Asn Ala Vai Ser He Lys Asn Pro

7580

ATG CTC TGT CTA TAT CAT CTG TCA AAC AAC

Met Leu Cys Leu Tyr His Leu Ser Asn Asn

8590

AGC GTC TCT TTT TTC CTA AAC AAC CCA GAC 300

Ser Val Ser Phe Phe Leu Asn Asn Pro Asp •95 100

AGC TCC CAG GGA AGC TAT TAC TTC TGC AGC 330

Ser Ser Gin Gly Ser Tyr Tyr Phe Cys Ser

105110

CTG TCC ATT TTT GAC CCA CCT CCT TTT CAA360

Leu Ser He Phe Asp Pro Pro Pro PheGin

115120

GAA AGG AAC CTT AGT GGA GGA TAT TTG CAT390

Glu Arg Asn Leu Ser Gly Gly Tyr LeuHis

125130

ATT TAT GAA TCC CAG CTC TGC TGC CAG CTG420

He Tyr Glu Ser Gin Leu Cys Cys GinLeu

135140

AAG CTC TGG CTA CCC GTA GGG TTG CCA GCT450

Lys Leu Trp Leu Pro Val Gly Leu ProAla

145150

TTC GTT GTG GTA CTC CTT TTT GGA TGC ATA480

Phe Val Val Val Leu Leu Phe Gly CysHe

155160

CTT ATC ATC TGG TTT TCA AAA AAG AAA TAC510

Leu He He Trp Phe Ser Lys Lys LysTyr

165170

GGA TCC AGT GTG CAT GAC CCT AAT AGT GAA540

Gly Ser Ser Val His Asp Pro Asn SerGlu

175180

TAC ATG TTC ATG GCG GCA GTC AAC ACA AAC570

Tyr Met Phe Met Ala Ala V&1 Asn ThrAsn

185190

AAA AAG TCT AGA CTT GCA GGT GTG ACC TCA600

Lys Lys Ser Arg Leu Ala Gly Val ThrSer

195200

TAA603

SEQ ID NO: 6:

SEQUENCE LENGTH: 836

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear .

MOLECULE TYPE: other nucleic acids (cDNA containing 3׳- and 5׳-untranslated sequences)

ORIGINAL SOURCE:

ORGANISM: Rattus

FEATURE:

NAME/KEY: CDS

LOCATION: 35 . 685 IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 6

CT6GAGGG6A AGAGTGCAGC TGTTCCTGGC AGAC34

ATG AAG CCC TAC TTC TCG TGC GTC TTT GTC64

Met Lys Pro Tyr Phe Ser Cys Val PheVal

510

TTC TGC TTC CTA ATC AAA CTT TTA ACA GGA

Phe Cys Phe Leu Ile Lys Leu Leu TheGly

1520

GAA CTC AAT GAC TTG GCC AAT CAC AGG ATG124

Glu Leu Asn Asp Leu Ala Asn His ArgMet

2530

TTT TCG TTT CAC GAT GGA GGT 6TA CAG ATT154

Phe Ser Phe His Asp Gly Gly Val GinHe

3540

TCT TGT AAC TAC CCT GAG ACT GTC CAG CAG184

Ser Cys Asn Tyr Pro Glu Thr Val GinGin

4550

TTA AAA ATG CAG TTG TTC AAA GAC AGA GAA214

Leu Lys Met Gin Leu Phe Lys Asp ArgGlu

5560

GTC CTC TGC GAC CTC ACC AAG ACC AAG GGA244

Val Leu Cys Asp Leu Thr Lys Thr LysGly

6570

AGC GGA AAC ACC GTG TCC ATC AAG AAT CCG274

Ser Gly Asn Thr Val Ser Ile Lys Asn Pro

80

ATG TCC TGT CCA TAT CAG CTG TCC AAC AAC 304

Met Ser Cys Pro Tyr Gin Leu Ser Asn Asn

90

AGT GTC TCT TTT TTC CTA GAC AAC GCA GAC 334

Ser Val Ser Phe Phe Leu Asp Asn Ala Asp

95100

AGC TCC CAG GGC AGC TAC TTT TTA TGC AGC364

Ser Ser Gin Gly Ser Tyr Phe Leu CysSer

105110

CTG TCG ATT TTC GAC CCA CCC CCT TTT CAA394

Leu Ser He Phe Asp Pro Pro Pro PheGin

115120

GAA AAG AAC CTT AGT GGA GGA TAT TTG CTT424

Glu Lys Asn Leu Ser Gly Gly Tyr LeuLeu

125130

ATT TAT GAA TCC CAG CTT TGT TGC CAG CTG454 lie Tyr Glu Ser Gin Leu Cys Cys GinLeu

135140

AAG CTT TGG TTA CCC GTA GGG TGT GCA GCT484

Lys Leu Trp Leu Pro Val Gly Cys AlaAla

145150

TTT GTG GCA GCG CTC CTT TTT GGA TGC ATA514

Phe Val Ala Ala Leu Leu Phe Gly CysHe

155160

TTT ATC GTC TGG TTT GCA AAA AAG AAG TAC544

Phe He Val Trp Phe Ala Lys Lys LysTyr

165170

AGA TCC AGT GTG CAC GAC CCT AAT AGC GAG574

Arg Ser Ser Val His Asp Pro Asn SerGlu

175180

TAC ATG TTC ATG GCG GCA GTC AAC ACA AAC604

Tyr Met Phe Met Ala Ala Val Asn ThrAsn.

185190

AAA AAG TCC AGA CTT GCA GGT ACA GCA CCC634

Lys Lys Ser Arg Leu Ala Gly Thr Ala Pro

195 200

CTT AGG GCT TTG G66 AGA GGA GAA CAC TCT 664

Leu Arg Ala Leu Gly Arg Gly Glu His Ser

205 210

TCA TGT CAA GAC CGG AAT TAA 685

Ser Cys Gin Asp Arg Asn

TTTGTTTATT TCTATTTTAA AAGAAAGACA TTTTTTCCCC TAAAGATAAT 735

TTTTGTATTT TTATGTGAAA GTCTGAATCT TCATTTTAAC TCGACTTATA 785

TACTCTGTGG TATATTAAAA ATAATGTTTG TGAAAAAAAA AAAAAAAAAA 835 A 836

SEQ ID NO: 7:

SEQUENCE LENGTH: 27

SEQUENCE TYPE: nucleic acid TOPOLOGY: linear

MOLECULE TYPE: other nucleic acids (synthetic DNA) FEATURE:

NAME/KEY: primer bind

LOCATION: 1 . 27

IDENTIFICATION METHOD: E SEQUENCE DESCRIPTION: SEQ ID NO: 7 CTGCTCGAGA TGAAGCCCTA CTTCTCG 27

SEQ ID NO: 8:

SEQUENCE LENGTH: 32

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acids (synthetic DNA)

FEATURE:

NAME/KEY: primer bind

LOCATION: 1 . 32

IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 8

ACCCTACGGG TAACGGATCC TTCAGCTGGC AA 32

SEQ ID NO: 9:

SEQUENCE LENGTH: 30

SEQUENCE TYPE! nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acid (synthetic DNA) FEATURE:

NAME/KEY: primer bind

LOCATION: 1. 30

IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 9

TAACTGTTTC TC6AGAACAT GAAGTCAGGC 30

SEQ ID NO: 10:

SEQUENCE LENGTH: 30

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acid (synthetic DNA)

FEATURE:

NEME/KEY: primer bind

LOCATION: 1 . 30

IDENTIFICATION METHOD: E SEQUENCE DESCRIPTION: SEQ ID NO: 10 ATCCTATGGG TAACGGATCC TTCAGCTGGC 30

SEQ ID NO: 11:

SEQUENCE LENGTH: 35

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acid (synthetic DNA)

FEATURE:

NEME/KEY: primer bind

LOCATION: 1. 35

IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 11

CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGC 35

SEQ ID NO: 12:

SEQUENCE LENGTH: 34

SEQUENCE TYPE: nucleic acid

TOPOLOGY: linear

MOLECULE TYPE: other nucleic acid (synthetic DNA) FEATURE:

NAME/KEY: primer bind

LOCATION: 1 . 34 IDENTIFICATION METHOD: E

SEQUENCE DESCRIPTION: SEQ ID NO: 12 CATTCAAGTT TCAGGGAACT AGTCCATGCG TTTC

107a

SEQ ID NO: 13

SEQUENCE LENGTH : 34 SEQUENCE TYPE: PRT ORGANISM: Rattus norvegicus

SEQUENCE: 13

Met Lys Pro Tyr Phe Ser Cys Vai Phe Vai Phe Cys Phe Leu lie Lys 15 1015

Leu Leu Thr Gly Glu Leu Asn Asp Leu Ala Asn His Arg Met Phe Ser 20 2530

Phe His Asp Gly Gly Vai Gin He Ser Cys Asn Tyr Pro Glu Thr Vai 35 4045

Gin Gin Leu Lys Met Gin Leu Phe Lys Asp Arg Glu Vai Leu Cys Asp 50 5560

Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Vai Ser He Lys Asn Pro 65 70 7580

Met Ser Cys Pro Tyr Gin Leu Ser Asn Asn Ser Vai Ser Phe Phe Leu 85 9095

Asp Asn Ala Asp Ser Ser Gin Gly Ser Tyr Phe Leu Cys Ser Leu Ser 100 105110

He Phe Asp Pro Pro Phe Gin Glu Lys Asn Leu Ser Gly Gly Tyr

115 120125

Leu Leu lie Tyr Glu Ser Gin Leu Cys Cys Gin Leu Lys Leu TrpLeu

130 135140

Pro Vai Gly Cys Ala Ala Phe Vai Ala Ala Leu Leu Phe Gly CysHe

145 150 155160

Phe He Vai Trp Phe Ala Lys Lys Lys Tyr Arg Ser Ser Vai His Asp 165 170175

Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Vai Asn Thr Asn Lys Lys 180 185190

Ser Arg Leu Ala Gly Met Thr Ser

195200

107b

SEQUENCE ID NO: 14

SEQUENCE LENGTH: 200

SEQUENCE TYPE: PRT

ORGANISM: Mus musculus

SEQUENCE: 14

Met Lys Pro Tyr Phe Cys His Val Phe Val Phe Cys Phe Leu lie Ary 1510

Leu Leu Thr Gly Glu He Asn Gly Ser Ala Asp His Arg Met Phe Ser 20 254U

Phe His Asn Gly Gly Val Gin lie Ser Cys Lys Tyr Pro Glu Thr Val 3540

Gin Gin Leu Lys Met Arg Leu Phe Arg Glu Arg Glu Val Leu Cys Glu cn 5500

Leu Thr Lys Thr Lys Gly Ser Gly Asn Ala Val Ser He Lys Asn Pro, ης 70 ⁷⁵°

Met Leu Cys Leu Tyr His Leu Ser Asn Asn Ser Val Ser Phe Phe Leu

. כל 9085

Asn Asn Pro Asp Ser Ser Gin Gly Ser Tyr Tyr Phe Cys Ser Leu Ser 100 105 lie Phe Asp Pro Pro Pro Phe Gin Glu Arg Asn Leu Ser Gly Gly Tyr 115 120125

Leu His lie Tyr Glu Ser Gin Leu Cys Cys Gin Leu Lys Leu Trp Leu 130 125140

Pro Val Gly Leu Pro Ala Phe Val Val Val Leu Leu Phe Gly Cys He 145 150 155_

Leu He lie Trp Phe Ser Lys Lys Lys Tyr Gly Ser Ser Val His Asp

165 I^701/b

Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys 180 I⁸⁵¹⁹⁰

Ser Arg Leu Ala Gly Val Thr Ser 195200

Passages of the description, which are not within the scope of the claims, do not consist part of the claimed invention.