U.S. patent application number 10/107907 was filed with the patent office on 2002-10-17 for cell surface molecule mediating cell adhesion and signal transmission.
This patent application is currently assigned to Japan Tobacco, Inc.. Invention is credited to Tamatani, Takuya, Tezuka, Katsunari.
Application Number | 20020151685 10/107907 |
Document ID | / |
Family ID | 27464138 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151685 |
Kind Code |
A1 |
Tamatani, Takuya ; et
al. |
October 17, 2002 |
Cell surface molecule mediating cell adhesion and signal
transmission
Abstract
Novel cell surface molecules recognized by monoclonal antibodies
against a cell surface molecule of lymphocytic cells that play an
important role in autoimmune diseases and allergic diseases have
been isolated, identified, and analyzed for their functions. The
cell surface molecules are expressed specifically in thymocytes,
lymphocytes activated by ConA-stimulation, and peripheral blood
lymphocytes, and induce cell adhesion. Antibodies against the cell
surface molecules significantly ameliorate pathological conditions
of autoimmune diseases and allergic diseases.
Inventors: |
Tamatani, Takuya; (Kanagawa,
JP) ; Tezuka, Katsunari; (Kanagawa, JP) |
Correspondence
Address: |
JANIS K. FRASER, PH.D.
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Assignee: |
Japan Tobacco, Inc.
|
Family ID: |
27464138 |
Appl. No.: |
10/107907 |
Filed: |
March 26, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10107907 |
Mar 26, 2002 |
|
|
|
09561308 |
Apr 28, 2000 |
|
|
|
09561308 |
Apr 28, 2000 |
|
|
|
09383551 |
Aug 26, 1999 |
|
|
|
09383551 |
Aug 26, 1999 |
|
|
|
PCT/JP98/00837 |
Feb 27, 1998 |
|
|
|
Current U.S.
Class: |
530/350 ;
530/388.24 |
Current CPC
Class: |
A01K 2217/05 20130101;
C07K 14/705 20130101; A01K 2217/075 20130101; A61K 38/00 20130101;
C07K 14/70521 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
530/350 ;
530/388.24 |
International
Class: |
C07K 014/705; C07K
016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 1997 |
JP |
9/62290 |
Feb 26, 1998 |
JP |
10/62217 |
Claims
What is claimed is:
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
adhesion between mitogen-stimulated lymphoblast cells; (c) an
antibody reactive to said cell surface molecule induces
proliferation of peripheral blood lymphocytes in the presence of an
antibody against CD3; (d) said cell surface molecule has a partial
amino acid sequence 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 claim 1, comprising the amino acid sequence
of 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 claim 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 claim 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 claim 1, wherein said cell surface molecule
is derived from human.
6. A gene encoding the polypeptide of claim 1.
7. The gene of claim 6, wherein said gene is a cDNA.
8. The gene of claim 7, wherein said cDNA has a nucleotide sequence
of SEQ ID NO: 1.
9. The gene of claim 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 claim 6.
11. A transformant into which the vector of claim 10 has been
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 claim 1.
14. The polypeptide fragment of claim 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 claim 13.
16. A homodimer molecule comprising two polypeptide fragments,
wherein each of the fragments comprises an extracellular region of
the polypeptide of claim 1 and said two polypeptide fragments
bridged through disulfide bonds to each other.
17. The homodimer molecule of claim 16, wherein said polypeptide is
a human-derived polypeptide having an amino acid sequence of SEQ ID
NO: 2.
18. A pharmaceutical composition comprising the polypeptide
fragment of claim 14 and a pharmaceutically acceptable carrier.
19. A fusion polypeptide comprising an extracellular region of the
polypeptide of claim 1 and a constant region of a human
immunoglobulin (Ig) heavy chain or a portion of the constant
region.
20. The fusion polypeptide of claim 19, wherein the immunoglobulin
is IgG.
21. The fusion polypeptide of claim 19, wherein the portion of the
constant region comprises a hinge region, C2 domain, and C3 domain
of IgG.
22. The fusion polypeptide of claim 19, 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 polypeptides of
claim 19, wherein the two polypeptides bridged through disulfide
bonds to each other.
24. A homodimer molecule comprising two fusion polypeptides of
claim 22, wherein the two polypeptides bridged through disulfide
bonds to each other.
25. A pharmaceutical composition comprising either of the fusion
polypeptide of claim 22 and a pharmaceutically acceptable
carrier.
26. The pharmaceutical composition of claim 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
claim 1, or the cell surface molecule comprising said
polypeptide.
28. The antibody of claim 27 or a portion thereof, 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 claim 14, or the human-derived cell surface
molecule comprising said polypeptide.
30. A monoclonal antibody or a portion thereof reactive to the
polypeptide of claim 1 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 claim 1 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 claim 29 or a portion thereof and a pharmaceutically acceptable
carrier.
33. The pharmaceutical composition of claim 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 claim 28.
35. A transgenic mouse in which a gene encoding the polypeptide of
claim 1 is integrated into its endogenous gene, wherein said gene
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.
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.
Description
TECHNICAL FIELD
[0001] The present invention relates to novel cell surface
molecules of mammals; polypeptides and their fragments constituting
the molecules; fusion polypeptides comprising the polypeptide
fragments and immunoglobulin fragments; genes encoding the
polypeptides and the fragments; vectors comprising the genes;
transformants into which the vectors are introduced; antibodies
having reactivity to the polypeptides or cell surface molecules
comprising the polypeptides; hybridomas producing the antibodies;
pharmaceutical compositions comprising the polypeptide fragments or
the fusion polypeptides; pharmaceutical compositions comprising the
antibodies; transgenic mice; and knockout mice.
BACKGROUND ART
[0002] A living body of mammals has immune response systems that
excludes pathogenic microorganisms (viruses, bacteria, parasites,
etc.) or foreign bodies (both are called "antigen" in the
following) that have invaded the living body. One of them is called
natural immune response system, another acquired immune response
system. The former is an exclusion mechanism comprising
phagocytosis by phagocytes (polymorphonuclear leukocytes,
monocytes, macrophages, etc.), attack by natural killer (NK) cells,
and non-specific recognition such as opsonization of antigen by
complements. The latter, acquired immune response system, is an
exclusion mechanism by lymphocytes (mainly, T cells and B cells)
that acquired the specificity to the antigen (namely, activated
lymphocytes). B cells that acquired antigen specificity attacks the
antigen existing outside of the cells through production of
antibodies specific to the antigen. T cells that acquired antigen
specificity (namely, activated T cells) are classified into helper
T cells and cytotoxic T cells (cytotoxic lymphocyte, CTL). The
helper T cells regulate 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)).
[0003] 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 receives 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).
[0004] However, the TcR/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.
[0005] 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 other molecules other than
TcR/CD3 complex on the T cell surface. This secondary signal avoids
cell anergy (clonal anergy) and activates the cells.
[0006] 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
antigen-presenting cells (macrophages, monocytes, dendritic cells,
and so on 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 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.
[0007] 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 antigen-presenting cells (namely,
cell adhesion through the binding between these molecules),
stabilizes mRNA of Th1-type cytokines and consequently promotes
production by T cells of a large amount of production of Th1-type
cytokines such as Il-2, IFN.gamma., and TNF.alpha.. The expression
of CTLA-4 is induced by the primary signal transmitted through
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.
[0008] 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.
[0009] 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%.
[0010] 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 higher
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 "YMNM
(Tyr-Met-Asn-Met)" and that CD28 plays an important role in
intracellular signal transmission through this "YxxM" structure.
Furthermore, it has been reported that CTLA-4 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.
[0011] 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)).
[0012] 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 (CD28, 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 have been noted.
[0013] 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 (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.
[0014] 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 fall are in the unresponsiveness state to maintain
immunological tolerance even if autoantigen-reactive T cells, which
reacts with autoantigen, 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.
[0015] From such viewpoints recently, many attempts to treat for
various 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.
[0016] It has been observed CD80, a costimulatory molecule as the
ligand of CD28 and CTLA-4, is abnormally expressed in the antigen
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 squamous lichen planus, and psoriasis. From such
observation, many attempts to treat various autoimmune diseases by
modulating the function of CD80 have been made.
[0017] 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 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 IgG1, were performed in animal model and clinical
tests (Nihon Rinsho, Vol. 55, No. 6, pp. 215-220 (1997)).
[0018] As shown in 1 to 5 below, therapeutic effects of CTLA-4-IgFc
in model animals of autoimmune diseases has been reported.
[0019] 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-IgFc 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)).
[0020] 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)).
[0021] 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. 181:1145-1155,
1995).
[0022] 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. 24:1249-1254, 1994).
[0023] 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 the administering the test drug at the
time of immunization and the production of autoantibodies (IgG1 and
IgG2) against collagen was inhibited (Eur. J. Immunol.
26:2320-2328, 1996).
[0024] 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.
DISCLOSURE OF THE INVENTION
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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
of the monoclonal antibodies was obtained has been 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
antibody" (this monoclional antibody was designated "JTT.2
antibody").
[0030] 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
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 having that mediate cell adhesion.
[0031] Cell surface molecules (designated "JTT-1 antigen" and
"JTT.2 antigen") recognized by each of these two monoclonal
antibodies were then identified, isolated, and characterized.
[0032] 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
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, activated B lymphoblast
cells, etc.) activated by stimulating thymocytes and spleen cells
with Concanavalin A (ConA), a mitogen, in particular, in the
activated 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.
[0033] Using an affinity column prepared by binding "JTT-1
antibody" 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
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
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; and when N-linked sugar chains were
digested, the molecules were identified as one molecule with about
36 kD under non-reduction condition and as one molecule with about
20 kD under reduction condition.
[0034] 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 that the adhesion was
significantly inhibited in the co-presence of "JTT.2 antibody",
indicating that "JTT-1 antigen" was the cell surface molecule
mediating cell adhesion.
[0035] Next, the present inventors cloned genes encoding "JTT-1
antigen" from rat, human, and mouse, and analyzed their
structures.
[0036] 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 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 with 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 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.
[0037] "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 of "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.
[0038] As the result that of motif search based on the amino acid
sequence 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 such as T cells, which regulates the functions of
activated lymphocytes such as activated T cells, cooperating with
the signal.
[0039] The structural similarity is as follows.
[0040] 1. 20 or more amino acid residues including cysteine
residues are highly conserved.
[0041] 2. Proline repeating sequence "Pro-Pro-Pro (PPP)" essential
as the ligand binding region, is conserved in the extracellular
region.
[0042] 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.
[0043] The locus of the gene encoding "mouse JTT-1 antigen" on
mouse 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.
[0044] Next, the effectiveness of therapy of autoimmune diseases
and allergic diseases by regulating the function of "JTT-1
antigen", 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
autoimmune diseases or allergic diseases can be treated by
regulating the functions of "JTT-1 antigen".
[0045] 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 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.
[0046] 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 "JTT-1 antigen" and/or its ligand.
[0047] 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 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.
[0048] The present inventions relate to polypeptides, genes,
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.
[0049] (1) A polypeptide constituting a cell surface molecule
having characteristics mentioned below,
[0050] (a) said cell surface molecule is expressed in at least
thymocytes and mitogen-stimulated lymphoblast cells,
[0051] (b) an antibody reactive to said cell surface molecule
induces adhesion between mitogen-stimulated lymphoblast cells,
[0052] (c) an antibody reactive to said cell surface molecule
induces proliferation of peripheral blood lymphocytes under the
coexistence within the presence of an antibody against CD3,
[0053] (d) said cell surface molecule has a partial amino acid
sequence represented by Phe-Asp-Pro-Pro-Pro-Phe in its
extracellular region, and
[0054] (e) said cell surface molecule has a partial amino acid
sequence represented by Tyr-Met-Phe-Met in its cytoplasmic
region.
[0055] (2) The polypeptide of (1) comprising the amino acid
sequence of 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.
[0056] (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.
[0057] (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.
[0058] (5) The polypeptide of any one of (1) to (4) wherein said
cell surface molecule is derived from human.
[0059] (6) A gene encoding the polypeptide of any one of (1) to
(5).
[0060] (7) The gene of (6) wherein said gene is a cDNA.
[0061] (8) The gene of (7) wherein said cDNA has a nucleotide
sequence of SEQ ID NO: 1.
[0062] (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.
[0063] (10 A vector comprising the gene of any one of (6) to
(9).
[0064] (11) A transformant into which the vector of (10) has been
introduced.
[0065] (12) A transformant distinguished identified by an
international deposit accession No. FERM BP-5725.
[0066] (13) A polypeptide fragment comprising an extracellular
region of the polypeptide of any one of (1) to
[0067] (14) The polypeptide fragment of (13) wherein said
polypeptide is a human-derived polypeptide having an amino acid
sequence of SEQ ID NO: 2.
[0068] (15) A gene encoding the polypeptide fragment of (13) or
(14).
[0069] (16) A homodimer molecule comprising two polypeptide
fragments, 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.
[0070] (17) The homodimer molecule of (16) wherein said polypeptide
is a human-derived polypeptide having an amino acid sequence of SEQ
ID NO: 2.
[0071] (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.
[0072] (19) A fusion polypeptide comprising an extracellular region
of 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.
[0073] (20) The fusion polypeptide of (19) wherein the
immunoglobulin is IgG.
[0074] (21) The fusion polypeptide of (19) wherein the portion of
the constant region comprises a hinge region, C2 domain, and C3
domain of IgG.
[0075] (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.
[0076] (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.
[0077] (24) A homodimer molecule comprising two fusion polypeptides
of (22) wherein the two polypeptides bridged through disulfide
bonds to each other.
[0078] (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.
[0079] (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.
[0080] (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.
[0081] (28) The antibody of (27) or a portion of it wherein said
antibody is a monoclonal antibody.
[0082] (29) An 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.
[0083] (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.
[0084] (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.
[0085] (32) A pharmaceutical composition comprising the monoclonal
antibody of (29) or a portion thereof and a pharmaceutically
acceptable carrier.
[0086] (33) The pharmaceutical composition of (32) wherein said
pharmaceutical composition is are utilized for treating autoimmune
diseases or allergic diseases, or for preventing said disease
symptom.
[0087] (34) A hybridoma producing the monoclonal antibody of any
one of (28) to (31).
[0088] (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 the
mouse its endogenous gene.
[0089] (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.
[0090] As described above, the cell surface molecule of the present
invention ("JTT-1 antigen") is involved in cell adhesion through
the molecule, signal transmission into lymphocytes such as T cells,
and function 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.
[0091] 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
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 cause
delayed allergy.
[0092] B cells are derived from the lymphoid stem cells
differentiated and matured in bone marrow. B cells are those
antibody-producing precursor cells since they produce antibodies
with an appropriate stimulus. B cells have immunoglobulins on their
cell surface, 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
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 Ig, abbreviation of
surface Ig, or mIg, abbreviation of membrane Ig. All Igs on the
surface of the same B cell have the same antigen binding sites.
[0093] 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
pre-stimulation with antigen, which is comparative to the case of
cytotoxic T cells. So, they are also called natural killer cells
(NK cells).
[0094] 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 they react with antigen-presenting cells. Prior
to these reactions at the cell level, the complex between of the
antigen peptides on antigen presenting cells and MHC class II
molecules binds to the corresponding 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
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
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 to
with the antigen expresses IL-2 receptor (IL-2R), are
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.gamma. or other cytokines, which are thought to have
similar actions. Thus, lymphokines secreted by T cells are
necessary for the differentiation into CTL. The lymphokines are
produced as the result that CD4-positive Th1 cells (CD4-positive T
cells secreting IL-2 or INF.gamma.) recognize the antigen peptides
derived from the same virus with class II molecules. In some cases,
without the help of 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 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, and proteoglycan, etc. Moreover, if CD8-positive cells
differentiated into CTL receive antigen stimulation, they also
secrete lymphokines such as IFNY, LT, TNF, or IL-2. Moreover, T
cells show blast transformation phenomenon, when they react with
hemagglutinin (phytohemagglutinin, PHA) or ConA.
[0095] Matured T cells not yet 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 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 or decades.
[0096] B cells not yet 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 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
antigen-presenting cells react with the antigen presented by
resting B cells. Namely, if an antigen invades, first, dendritic
cells (cells having 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 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 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 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)).
[0097] 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
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, recognition mechanism specific to the state of
cells, and, in particular, cell adhesion molecules (cell adhesion
proteins).
[0098] Cell adhesion molecules, namely,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
organic and functional contacts in a living body.
[0099] 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
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 .alpha./.beta.
heterodimer structure. Examples thereof are "VLA-1 to 6" lymphocyte
function-associated antigen-1 "LFA-1," "Mac-1," and "p150/90."
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" and "P-selectin."
Examples of cadherin family are "E-cadherin," "N-cadherin," and
"P-cadherin," and an example of CD44 family is "CD44".
[0100] 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
studies, it has been gradually revealed that adhesion molecules are
involved not only in these functions but also in various
diseases.
[0101] In particular, there are many reports on diseases and
expression abnormality of adhesion molecules. For example, as for
rheumatoid arthritis (RA), the expression of both "Mac-1" and
"p150/95" was reportedly strengthened in RA synoviocytes (Allen et
al., Arthritis Rheum., 32:947, 1989). It has also been reported
that various cells expressed "ICAM-1" strongly and ectopically on
RA synovial membrane (Hale et al., Arthritis Rheum., 32:22, 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 et al., Arthritis Rheum., 32:386, 1989).
Strong expression of "CD44" in vascular endothelial cells and
A-type synoviocytes on RA synovial membrane was reported (Heynes et
al., Arthritis Rheum., 34:1434, 1991).
[0102] There are reports on the relationship between systemic lupus
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", and "IFA-1"
to were strongly expressed (Haskard et al., Rheumatol. Int., 9:33,
1989).
[0103] In autoimmune thyroiditis diseases, it was reported that
"ICAM-1" was expressed when a thyroid follicular cells were
stimulated with interferon-.gamma., interleukin-1, and tumor
necrosis factor, and that the formation of cluster of follicular
cells and mononuclear cells was inhibited by anti-"ICAM-1" antibody
(Weetman et al., Eur. J. Immunol., 20:271, 1990).
[0104] In hepatitis, it is thought that the chances of adhesion
between hepatocytes and inflammatory cells increases since there
are two pathways of adhesion, "ICAM-1" and "LFA-3", and "LFA-1" and
"CD2", to thereby 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 in
the exclusion of viruses and "ICAM-1" promotes T cells to present
antigen and regulates inflammation reaction. In "ICAM-1"-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 that
serum "ICAM-1" 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
patients, and the concentration was high in the case of
histologically progressing active hepatitis (Mod. Phys., 15:73-76,
1995).
[0105] In a model animal of arteriosclerosis, adhesion and invasion
of monocytes and lymphocytes to vascular endothelium were observed
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-1" in
human arteriosclerosis nidus (Poston et al., Am. J. Pathol.,
140:665, 1992) and the expression of "VCAM-1" in arteriosclerosis
nidus of a hypercholesterolemia rabbit (Cybulsky et al., Science,
251:788, 1991). A recent report revealed that the expression of
"VCAM-1" was observed in human arteriosclerosis nidus, and, in
particular, strong expression in smooth muscle cells migrating to
intima and in monocytes/macrophages. In addition, since 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 12:1485-1488, 1994).
[0106] The relationship between tumor metastasis and adhesion
molecule abnormality has also been reported. For example, if
E-cadherin-decreased cancer cells showed strong invasiveness, but
the invasiveness was inhibited by introducing the cDNA of
E-cadherin into the cancer cells, the invasiveness was recovered
when E-cadherin antibodies 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 et
al., 113:173, 1991). In actual clinical cases, the relationship
between 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, suggesting that this molecule can 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,
colon large intestine cancer, lung cancer, hepatoma, or pancreatic
cancer, adhered to vascular endothelial cells through E-selectin
(Takada et al., Cancer Res., 53:354, 1993).
[0107] 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
inflammatory reaction in rat autoimmune arthritis model. It has
also been reported that the administration of anti-"ICAM-1"
antibody inhibited the onset of arthritis in adjuvant synovitis in
one of animal models of RA (Nihon et al., 14:571-577, 1991). It was
further reported that the metastasis formation of inoculated tumor
was remarkably inhibited if a large amount of peptides having REG
sequence, which is that 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-.beta.1 subunit antibody inhibited the
motion and infiltration of tumor cells (Yamada et al., Cancer Res.,
50:4485, 1990).
[0108] In the following, the present invention is described in
detail by clarifying the meanings of terms used herein the present
invention and the general production methods of polypeptides,
fusion polypeptides, genes, antibodies, transgenic mice, and
knockout mice of the present invention. However, it is needless to
say that the meanings of the terms are not to be interpreted
limitedly by the definition given herein.
[0109] "Mitogen" used herein is also called also mitogenic factor
and means a substance which induces cell division. Immunologically,
it means 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 only on B lymphocytes, and that PWM
acts on both lymphocytes.
[0110] The term "lymphoblast cell" used herein is also called also
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 duct, tonsil, etc.) and blood.
[0111] The term "activated lymphocyte" used herein, 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, and natural killer cells. T cells are classified into
CD4-positive 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 activated CD8-positive cells.
[0112] 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
CD4-positive T cells include those in such a state.
[0113] 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 their 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 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.gamma., LT, TNF, or IL-2. Activated
CD8-positive T cells include those in such a state.
[0114] T cells show blast formation phenomenon when they react with
hemagglutinin (phytohemagglutinin, PHA) or Concanavalin A (ConA).
Activated T cells comprise include those in such a state.
[0115] 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 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).
[0116] Activated natural killer cells mean those showing cytotoxic
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).
[0117] The "activated lymphoblast cell" used herein includes an
activated "lymphoblast" that is generated when the lymphoblast
mentioned above is stimulated with "mitogen" mentioned above such
as Concanavalin A.
[0118] The term "resting lymphocyte" used herein, in some case, an
non-activated lymphocyte, which has not received the stimulation to
activate cells, in contrast to an activated lymphocyte mentioned
above.
[0119] The "gene" of the present invention includes a genomic DNA
and a cDNA.
[0120] 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 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.
[0121] The "cell surface molecule" of the present invention is that
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.
[0122] Specifically, the "cell surface molecule" of the present
invention is that characterized by having, at least, properties
described below.,
[0123] (a) the cell surface molecule is expressed in, at least,
thymocytes and mitogen-stimulated lymphoblast cells;
[0124] (b) an antibody reactive to the cell surface molecule
induces adhesion between mitogen-stimulated lymphoblast cells;
[0125] (c) an antibody reactive to the cell surface molecule
induces proliferation of peripheral blood lymphocytes under the
coexistence within the presence of an antibody against CD3;
[0126] (d) the cell surface molecule has a partial amino acid
sequence represented by Phe-Asp-Pro-Pro-Pro-Phe in its
extracellular region; and
[0127] (e) the cell surface molecule has a partial amino acid
sequence represented by Tyr-Met-Phe-Met in its cytoplasmic
region.
[0128] Preferably, the "cell surface molecule" comprises the
following "polypeptide" of the present invention.
[0129] The "polypeptide" of the present invention is that which
constitutes the above-mentioned "cell surface molecule" of the
present invention. Examples thereof are as follows.
[0130] (1) A polypeptide encoded by a DNA hybridizing with a DNA
comprising a nucleotide sequence of SEQ ID NO: 1 under stringent
conditions;
[0131] (2) A polypeptide having an amino acid sequence having 60%
or more homology with an amino acid sequence of SEQ ID NO: 2;
[0132] (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);
[0133] (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);
[0134] (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);
[0135] (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);
[0136] (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
[0137] (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).
[0138] To determine the "percent homology" of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
homology between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions) .times.100). In one embodiment the two sequences are the
same length.
[0139] To determine percent homology between two sequences, the
algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-5877 is used. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul, et
al. (1990) J. Mol. Siol. 215:403-410. BLAST nucleotide searches are
performed with the NBLAST program, score=100, word length=12 to
obtain nucleotide sequences homologous to a nucleic acid molecules
of the invention. BLAST protein searches are performed with the
XBLAST program, score=50, word length=3 to obtain amino acid
sequences homologous to a VRK1 or VRK2 protein molecules. To obtain
gapped alignments for comparison purposes, Gapped BLAST is utilized
as described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
[0140] Furthermore, the present invention relates to a DNA that
specifically hybridizes under moderate or highly stringent
conditions to a DNA encoding a protein of the present invention and
comprises at least 15 nucleotide residues. The DNA can be used, for
example, as a probe to detect or isolate a DNA encoding a protein
of the present invention, or as a primer for PCR amplification. An
example is DNA consisting of at least 15 nucleotides complementary
to the nucleotide sequence of SEQ ID NO: 1, NO: 3, NO:4, NO:5 or
NO:6.
[0141] Standard hybridization conditions (e.g., moderate or highly
stringent conditions) are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6, hereby incorporated by
reference. Moderate hybridization conditions are defined as
equivalent to hybridization in 2.times.sodium chloride/sodium
citrate (SSC) at 30.degree. C., followed by one or more washes in
1.times.SSC, 0.1% SDS at 50-60.degree. C. Highly stringent
conditions are defined as equivalent to hybridization in
6.times.sodium chloride/sodium citrate (SSC) at 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C.
[0142] 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 occurs (Tm) is calculated using the following formula
and the temperature for hybridization can be determined according
to the following formula.
Tm=82.3.degree. C.+0.41.times.(G+C)%-500/n-0.61.times.(formamide)%
(n means the number of the nucleotide of probe).
[0143] Temperature=Tm -25.degree. C.
[0144] 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.
[0145] (1) Tm descends by about 1.degree. C. per 1% mismatch.
[0146] (2) Tm descends by 0.6 to 0.7.degree. C. per 1%
formamide.
[0147] Accordingly, the temperature conditions for the combination
of completely complementary strands can be set as follows.
[0148] (A) 65 to 75.degree. C. (formamide not added)
[0149] (B) 35 to 45.degree. C. (in the presence of 50%
formamide)
[0150] The temperature conditions for the combination of
incompletely complementary strands can be set as follows.
[0151] (A) 45 to 55.degree. C. (formamide not added
[0152] (B) 35 to 42.degree. C. (in the presence of 30%
formamide)
[0153] The temperature conditions when a probe with 23 or less
nucleotides is used can be 37.degree. C. or can be calculated using
the following formula.
Temperature=2.degree. C..times.(the number of A+T)+4.degree.
C..times.(the number of C+G)-5.degree. C.
[0154] Here, "having substantially the same amino acid sequence"
means 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
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.
[0155] Such substitution, deletion, or insertion of amino acids can
be performed by the usual method (Experimental Medicine:
SUPPLEMENT, "Handbook of Genetic Engineering" (1992); and so
on).
[0156] Examples thereof are synthetic oligonucleotide-directed
mutagenesis (gapped duplex method), point metagenesis by which a
point mutation is introduced at random by treatment with nitrite 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.
[0157] Synthetic oligonucleotide-directed mutagenesis (gapped
duplex 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 phage DNA mentioned above, denatured, and annealed
thereby forming "gapped duplex DNA." A synthetic oligonucleotide
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, deficient in mismatch repair activity, are
transfected with this DNA., E. coli cells without suppressor
activity are infected with the grown phages, and only phages
without amber mutation are screened.
[0158] The method by which a point mutation is introduced with
nitrite 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 "A:T", respectively, because hypoxanthine,
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).
[0159] Conservative amino acid substitutions can also be made at
one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
[0160] 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) 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.
[0161] The "polypeptide" constituting the above-mentioned "cell
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.
[0162] Here, a "transmembrane protein" means a protein that
connects 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 transmembrane protein constitutes each receptor
or cell surface molecule by existing in the form of a monomer,
homodimer, heterodimer or oligomer with another chain(s) having the
same or different amino acid sequence.
[0163] The "polypeptide fragment" of the present invention is a
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.
[0164] Here, an "extracellular region" means the whole or a portion
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 integrated incorporated into the membrane
(transmembrane region) and the region existing in the cytoplasm
following the transmembrane region in the membrane (cytoplasmic
regions).
[0165] "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 above, or a portion of them. The
immunoglobulin can be any immunoglobulin belonging to any class and
any subclass. Specifically, examples of the immunoglobulin are IgG
(IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 and IgA2), IgD, and
IgE. Preferably, the immunoglobulin is IgG (IgG1, IgG2, IgG3, or
IgG4), or IgM. Examples of particularly preferable immunoglobulin
in of the present invention are those belonging to human-derived
IgG (IgG1, IgG2, IgG3, or IgG4).
[0166] 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
bonds (S-S bonds). The light chain is composed of the light chain
variable regions (VL) and the light chain constant region (CL). The
heavy chain is composed of the heavy chain variable regions (VH)
and the heavy chain constant region (CH).
[0167] The heavy chain constant region is composed of some domains
having the amino acid sequences inherent in each class (IgG, IgM,
IgA, IgD, and IgE) and each subclass (IgG1, IgG2, IgG3, and IgG4,
IgA1, and IgA2).
[0168] The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is
composed of VH, CH1 domain, hinge region, CH2 domain, and CH3
domain in this order from N terminus.
[0169] Similarly, the heavy chain of IgG1 is composed of VH,
C.gamma..sub.11 domain, hinge region, C.gamma..sub.12 domain, and
C.gamma..sub.13 domain in this order from N terminus. The heavy
chain of IgG2 is composed of VH, C.gamma..sub.21 domain, hinge
region, C.gamma..sub.22 domain, and C.gamma..sub.23 domain in this
order from N terminus. The heavy chain of IgG3 is composed of VH,
C.gamma..sub.31 domain, hinge region, C.gamma..sub.32 domain, and
C.gamma..sub.33 domain in this order from N terminus. The heavy
chain of IgG4 is composed of VH, C.gamma..sub.41 domain, hinge
region, C.gamma..sub.42 domain, and C.gamma..sub.43 domain in this
order from N terminus.
[0170] The heavy chain of IgA is composed of VH, C.alpha.1 domain,
hinge region, C.alpha.2 domain, and C.alpha.3 domain in this order
from N terminus.
[0171] Similarly, the heavy chain of IgA1 is composed of VH,
C.alpha..sub.11 domain, hinge region, C.alpha..sub.12 domain, and
C.alpha..sub.13 domain in this order from N terminus. The heavy
chain of IgA2 is composed of VH, C.alpha..sub.21 domain, hinge
region, C.alpha..sub.22 domain, and C.alpha..sub.23 domain in this
order from N terminus.
[0172] The heavy chain of IgD is composed of VH, C.delta.1 domain,
hinge region, C.delta.2 domain, and C.delta.3 domain in this order
from N terminus.
[0173] The heavy chain of IgM is composed of VH, C.mu.1 domain,
C.mu.2 domain, C.mu.3 domain, and C.mu.4 domain in this order from
N terminus and have no hinge region as seen in IgG, IgA, and
IgD.
[0174] The heavy chain of IgE is composed of VH, C.epsilon.1
domain, C.epsilon.2 domain, C.epsilon.3 domain, and C.epsilon.4
domain in this order from N terminus and have no hinge region as
seen in IgG, IgA, and IgD.
[0175] If, for example, IgG is treated with papain, it is cleaved
at the slightly N terminal side beyond the disulfide bonds existing
in the hinge region where the disulfide bonds connect the two heavy
chains to generate two homologous Fab, in which a heavy chain
fragment composed of VH and CH1 is connected with one light chain
through a disulfide bond, and one Fc, in which two homologous heavy
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).
[0176] 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 C1 domain, or the Fc region. Specifically, examples
thereof are the region composed of hinge region, C2 domain, and C3
domain in the case from each of IgG, IgA, and IgD, and are the
region composed of C2 domain, C3 domain, and C4 domain in the case
from each of IgM and IgE. A particularly preferable example thereof
is the Fc region of human-derived IgG1.
[0177] The "fusion polypeptide" of the present invention is that
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
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
chain. Moreover, IgG1 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.
[0178] The fusion polypeptide of the present invention has the
advantage that the fusion polypeptide can be purified extremely
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.
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.
[0179] The polypeptide, polypeptide fragment, and fusion
polypeptide 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.
[0180] The "gene" of the present invention comprises a DNA encoding
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.
[0181] Examples of the gene are those encoding the polypeptide or
polypeptide fragment mentioned below.
[0182] (1) A polypeptide encoded by a DNA hybridizing with a DNA
comprising a nucleotide sequence of SEQ ID NO: 1 under stringent
conditions;
[0183] (2) A polypeptide having an amino acid sequence having 60%
or more homology with an amino acid sequence of SEQ ID NO: 2;
[0184] (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);
[0185] (4) A polypeptide having an amino acid sequence encoded by a
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);
[0186] (5) A polypeptide having an amino acid sequence encoded by a
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);
[0187] (6) A polypeptide having an amino acid sequence encoded by a
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);
[0188] (7) A polypeptide having an amino acid sequence encoded by a
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
[0189] (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 an amino acid sequence substantially the
same as said amino acid sequence (namely, a polypeptide
constituting a "human JTT-1 antigen" and its derivative).
[0190] Here, "substantially the same amino acid sequence" means as
defined above.
[0191] Specific examples of the gene of the present invention are
DNAs or their fragments mentioned below.
[0192] (1) A DNA comprising a nucleotide sequence of SEQ ID NO: 1,
and a DNA hybridizing with the DNA under stringent conditions;
[0193] (4) A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 26-625 of SEQ ID NO: 3;
[0194] (5) A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 35-637 of SEQ ID NO: 4;
[0195] (6) A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 1-603 of SEQ ID NO: 5;
[0196] (7) A DNA comprising a nucleotide sequence corresponding to
nucleotide residues 35-685 of SEQ ID NO: 6;
[0197] (8) A DNA encoding a polypeptide constituting a cell surface
molecule of the present invention, wherein the DNA is introduced
into a transformant identified by an international deposit
accession No. FERM BP-5725.
[0198] The DNA encoding a portion of a constant region of
immunoglobulin heavy chain, which is a part of a fusion polypeptide
of the present invention, can be cDNA, or genomic DNA comprised of
intons between every exon (the DNA encoding, for example, CH1
domain, hinge region, CH2 domain, CH3 domain, CH4 domain and so
on).
[0199] The DNA of the present invention includes any DNA comprised
of any codons as long as the codons encode the same amino
acids.
[0200] 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
these methods.
[0201] 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 so on.
[0202] (1) cDNA can be cloned from the mRNA encoding the
polypeptide of the present invention by, for example, the method
described below.
[0203] 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 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-thiocyanate method (Chirgwin et al., Biochemistry,
18:5294, 1979), hot phenol method, or AGPC method, and subjecting
it to affinity chromatography using oligo-dT cellulose or poly-U
Sepharose.
[0204] 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. 2:161, 1982; ibid. 3:280, 1983) or the method of Hoffman et
al. (Gene 25:263, 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.
[0205] 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, .lambda.XZAPII(lZAPII),
pUC19, .lambda.gt10, .lambda.gt11, and so on. When the vector is
applied to 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.
[0206] cDNA can be inserted into a plasmid by, for example, the
method of Maniatis et al. (Molecular Cloning, A Laboratory Manual,
second 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. 1, 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: XL1Blue MRF', DH5.alpha., HB101,
MC1061/P3, etc.).
[0207] 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, 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 example, a product from Stratagene or
Amersham).
[0208] 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.
[0209] For example, a clone comprising the desired cDNA can be
screened by a known colony hybridization method (Crunstein et al.,
Proc. Natl. Acad. Sci. USA, 72:3961. 1975) or plaque hybridization
method (Molecular Cloning, A Laboratory Manual, second edition,
Cold Spring Harbor Laboratory, p. 2.108 (1989)) using
.sup.32P-labeled chemically synthesized oligonucleotides as probes,
which are 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.
[0210] When a cDNA library prepared using a cDNA expression vector
(for example, .lambda.ZAPII 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 using PCR method is preferably used when many
clones are subjected to screening.
[0211] The nucleotide sequence of the DNA thus obtained can be
determined by Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad.
Sci. USA, 74:560, 1977) or the dideoxynucleotide synthetic chain
termination method using phage M13 (Sanger et al., Proc. Natl.
Acad. Sci. USA, 74:5463-5467, 1977). The whole or a 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.
[0212] (2) The DNA encoding the polypeptide of the present
invention can be isolated from the genomic DNA derived from the
cells expressing the polypeptide of the present invention as
mentioned above by the following methods.
[0213] 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
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
polypeptide of the present invention is obtained from the clones by
excision with restriction enzyme and so on.
[0214] 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:
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.
[0215] (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.
[0216] The present invention also relates to a recombinant vector
comprising the DNA encoding an above-mentioned cell surface
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.
[0217] The recombinant vector can easily be prepared by ligating
the 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. coli-derived plasmids such as
pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids
such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids
such as pUB110, pTP5, and pC194. Examples of phages are a
bacteriophage such as .lambda. phage, and an animal or insect virus
(pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and
nuclear polyhidrosis virus.
[0218] 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 host cells and produces this protein. Examples thereof
are pEFneo (Proc. Natl. Acad. Sci. USA 91:158-162, 1994), pEF-BOS
(Nucleic Acids Res. 18:5322, 1990), pME18S (Experimental Medicine:
SUPPLEMENT, "Handbook of Genetic Engineering" (1992)), pMAL C2, and
so on.
[0219] 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.
[0220] 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'-
and 3'-untranslated region of the gene encoding the polypeptide of
the present invention, splicing junctions, polyadenylation site,
selectable marker region, and replicon. The expression vector may
also contain, if required, a gene for gene amplification (marker)
that is usually used.
[0221] A promoter/operator region to express the polypeptide of the
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, .lambda.PL promoter, lpp
promoter, tac promoter, or the like. Examples of a promoter to
express the 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, retrovirus promoter, heat shock promoter, EF promoter,
and so on, and preferably SV40, SR.alpha., 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.
[0222] A preferable initiation codon is, for example, a methionine
codon (ATG).
[0223] The commonly used termination codon (for example, TAG, TGA,
TAA, and so on) is illustrated as a termination codon.
[0224] Usually used natural or synthetic terminators are used as a
terminator region.
[0225] 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
obtained by treating pBR322 with appropriate restriction enzymes)
for E. coli, yeast 2 .mu. 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.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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, to an appropriate replicon. 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.
[0230] Transformants of the present invention can be prepared by
introducing the expression vector mentioned above into host
cells.
[0231] 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
usually used in technical field of the present invention (for
example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces,
Pichia, etc.), animal cells, or insect cells.
[0232] E. coli or animal cells are preferably used. Specific
examples are E. coli (DH5, XL1Blue MRF', TB1, HB101, etc.),
mouse-derived 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.).
[0233] An expression vector can be introduced (transformed
(transduced)) into host cells by known method.
[0234] Transformation can be performed, for example, according to
the method of Cohen et al. (Proc. Natl. Acad. Sci. USA 69:2110,
1972), protoplast method (Mol. Gen. Genet. 168:111, 1979), or
competent method (J. Mol. Biol. 56:209, 1971) when the hosts are
bacteria (E. coli, Bacillus subtilis, etc.), the method of Hinnen
et al. (Proc. Natl. Acad. Sci. USA 75:1927, 1978), or lithium
method (J. Bacteriol. 153:163, 1983) when the host is Saccharomyces
cerevisiae, the method of Graham (Virology 52:456, 1973) when the
hosts are animal cells, and the method of Summers et al. (Mol.
Cell. Biol. 3:2156-2165, 1983) when the hosts are insect cells.
[0235] 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
mentioned above in nutrient media.
[0236] 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 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), vitamins, antibiotics (for example,
tetracycline, neomycin, ampicillin, kanamycin, etc.).
[0237] 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
of the present invention is overproduced.
[0238] Specific media and cultivation conditions used depending on
host cells are illustrated below, but are not limited thereto.
[0239] When the hosts are bacteria, actinomycetes, yeasts,
filamentous fungi, liquid media comprising the nutrient source
mentioned above are appropriate. The media with pH 5 to 8 are
preferably used.
[0240] 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.degree. C. for
about 3 to 24 hours with aeration and stirring, if necessary.
[0241] When the host is Bacillus, cultivation can be performed
usually at 30 to 40.degree. C. for about 16 to 96 hours with
aeration and stirring, if necessary.
[0242] When the host is yeast, examples of media are Burkholder
minimal media (Bostian, Proc. Natl. Acad. Sci. USA, 77:4505, 1980).
The pH of the media is preferably 5 to 8. Cultivation can be
performed usually at 20 to 35.degree. C. for about 14 to 144 hours
with aeration and stirring, if necessary.
[0243] When the host is an animal cell, examples of media are MEM
media containing about 5 to 20% fetal bovine serum (Science
122:501, 1952), DMEM media (Virology 8:396, 1959), RPMI1640 media
(J. Am. Med. Assoc. 199:519, 1967), and 199 media (Proc. Soc. Exp.
Biol. Med. 73:1, 1950). The pH of the media is preferably about 6
to 8. Cultivation can be performed usually at about 30 to
40.degree. C. for about 15 to 72 hours with aeration and stirring,
if necessary.
[0244] When the host is an insect cell, an example of media is
Grace's media containing fetal bovine serum (Proc. Natl. Acad. Sci.
USA 82:8404, 1985). The pH thereof is preferably about 5 to 8.
Cultivation can be performed usually at about 20 to 40.degree. C.
for 15 to 100 hours with aeration and stirring, if necessary.
[0245] Cultivation of transformants as mentioned above, in
particular animal cells can overexpress the polypeptide of the
present invention on the surface of the cells.
[0246] 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.
[0247] 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.
[0248] 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 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 chromatography, and a method
utilizing the difference in isoelectric point, such as isoelectric
focusing.
[0249] 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
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 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.
[0250] 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
mouse expresses the non-self polypeptide and secretes the
polypeptide into its body.
[0251] 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)).
[0252] 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 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 77:7380-7384, 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
other, homogenetic transgenic mice are obtained according to
Mendel's laws.
[0253] 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
out (inactivated). It can be prepared, for example, by
positive-negative selection method in which homologous
recombination is applied (U.S. Pat. Nos. 5,464,764; 5,487,992; and
5,627,059; Proc. Natl. Acad. Sci. USA 86:8932-8935, 1989; Nature
342:435-438, 1989; etc.).
[0254] The "antibody" of the present invention can be a polyclonal
antibody (antiserum) or a monoclonal antibody, and preferably a
monoclonal antibody.
[0255] Specifically, it is an antibody reactive to (against, which
binds to) the above-mentioned polypeptide or polypeptide fragment
of the present invention.
[0256] 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 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
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.
[0257] The monoclonal antibody includes those having any one
isotype of IgG, IgM, IgA, IgD, or IgE. IgG or IgM is
preferable.
[0258] 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
an antigen mentioned above with Freund's adjuvant, if
necessary.
[0259] 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 obtained from the animal so immunized
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.
[0260] Specifically, the monoclonal antibody can be produced as
follows. 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,
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 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 256:495-497, 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 derived from, preferably,
a mammal such as a mouse, rat, guinea pig, hamster, rabbit, or
human, or more preferably, a mouse, rat, or human.
[0261] For example, mouse-derived myeloma P3/X63-AG8.653 (653),
P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0,
Sp2), PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., or
human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,
D1R11, or CEM-T15 can be used as a myeloma used for the cell
fusion.
[0262] Hybridoma clones producing monoclonal antibodies can be
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.
[0263] 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 mammal.
[0264] 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.
[0265] 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, or RD medium. The basal media can contain, for
example, sera, hormones, cytokines, and/or various inorganic or
organic substances depending on the objective.
[0266] Monoclonal antibodies can be isolated and purified from the
culture supernatant or ascites fluid mentioned above by saturated
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.
[0267] Preferable examples of monoclonal antibodies of the present
invention are as follows.
[0268] (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;
[0269] (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 monoclonal antibody produced by a hybridoma
identified by an international deposit accession No. FERM BP-5707
on mitogen-stimulated rat lymphoblast cells; and
[0270] (3) 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 monoclonal antibody produced by a hybridoma
identified by an international deposit accession No. FERM BP-5708
on mitogen-stimulated rat lymphoblast cells.
[0271] 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.
[0272] The "chimeric monoclonal antibody" of the present invention
is 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.
[0273] The constant region derived from human immunoglobulin has
the amino acid sequence inherent in each isotype such as IgG (IgG1,
IgG2, IgG3, IgG4), 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.
[0274] 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.
[0275] A mouse/human chimeric monoclonal antibody can be prepared,
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 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 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.
[0276] Specifically, DNAs are first extracted from mouse monoclonal
antibody-producing hybridomas by the usual method, digested with
appropriate restriction enzymes (for example, EcoRI and HindIII),
electrophoresed (using, for example, 0.7% agarose gel), and
analyzed by Southern blotting. After an electrophoresed gel is
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 HCl 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 filter
is recovered and washed twice with 2.times.SSC. After the filter is
sufficiently dried, it is baked at 75.degree. C. for 3 hours. After
baking, the filter is treated with 0.1.times.SSC/0.1% SDS at
65.degree. C. for 30 minutes. Then, it is soaked in
3.times.SSC/0.1% SDS. The filter obtained is treated with
prehybridization solution in a plastic bag at 65.degree. C. for 3
to 4 hours.
[0277] Next, .sup.32P-labeled probe DNA and hybridization solution
are added to the bag and reacted at 65.degree. C. about 12 hours.
After hybridization, the filter is washed under appropriate salt
concentration, reaction temperature, and time (for example,
2.times.SSC-0.1% SDS, room temperature, 10 minutes). The filter is
put into a plastic bag with a little 2.times.SSC, and subjected to
autoradiography after the bag is sealed.
[0278] Rearranged VDJ gene and VJ gene encoding H chain and L chain
of a mouse monoclonal antibody are identified by Southern blotting
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,
.lambda.EMBL3, .lambda.EMBL4, etc.). E. coli (for example, LE392,
NM539, etc.) is transformed with the phage vector to generate a
genomic library. The genomic library is screened by plaque
hybridization such as Benton-Davis method (Science 196:180-182,
1977) using appropriate probes (H chain J gene, L chain (K) 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 comprising the desired, rearranged VH (VDJ) gene or VL (VJ)
gene are obtained.
[0279] Separately, human CH gene and human CL gene used for
chimerization are isolated. For example, when a chimeric antibody
with human IgG1 is produced, C.gamma.1 gene as a CH gene, and
C.kappa. gene as a CL gene, are isolated. These genes can be
isolated from human genomic library with mouse C.gamma.1 gene and
mouse C.kappa. gene, corresponding to human C.gamma.1 gene and
human C.kappa. gene, respectively, as probes, taking advantage of
high homology between the nucleotide sequences of mouse
immunoglobulin gene and that of human immunoglobulin gene.
[0280] Specifically, DNA fragments comprising human C.kappa. gene
and an enhancer region are isolated from human .lambda. Charon 4A
HaeIII-AluI genomic library (Cell 15:1157-1174, 1978), for example,
with a 3 kb HindIII-BamHI fragment of clone Ig146 (Proc. Natl.
Acad. Sci. USA 75:4709-4713, 1978) and a 6.8 kb EcoRI fragment of
clone MEP10 (Proc. Natl. Acad. Sci. USA 78:474-478, 1981) as
probes. In addition, for example, after human fetal hepatocyte DNA
is digested with HindIII and fractioned by agarose gel
electrophoresis, a 5.9 kb fragment is inserted into .lambda.788 and
then human C.gamma.1 gene is isolated with the probes mentioned
above.
[0281] 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 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.
[0282] 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 into the expression vector and, then,
cells producing desired chimeric monoclonal antibodies are
obtained.
[0283] Desired chimeric monoclonal antibodies are obtained from the
culture supernatant of antibody-producing cells thus screened.
[0284] The "humanized monoclonal antibody (CDR-grafted antibody)"
of 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
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.
[0285] 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
variable region means four comparatively conserved regions lying
upstream, downstream or between the three complementarity
determining regions (framework region, FR1, FR2, FR3, and FR4).
[0286] In other words, a humanized monoclonal antibody means that
in which the whole region except a portion or the whole of the
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.
[0287] The constant region derived from human immunoglobulin has
the amino acid sequence inherent in each isotype such as IgG (IgG1,
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.
[0288] 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.
[0289] For example, a recombinant humanized monoclonal antibody
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
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
above are isolated from human immunoglobulin genes.
[0290] 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 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 obtain transformants producing humanized
monoclonal antibody. By cultivating the transformants, desired
humanized monoclonal antibody is obtained from culture
supernatant.
[0291] The "human monoclonal antibody" of the present invention is
immunoglobulin in which the entire regions comprising the variable
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.
[0292] 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 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.
[0293] For example, a transgenic mouse producing human antibodies
is prepared by the methods described in Nature Genetics 7:13-21,
1994; Nature Genetics 15:146-156, 1997; JP-WA Nos. Hei 4-504365 and
Hei 7-509137; Nikkei Science 6:40-50, 1995; International patent
publication No. WO94/25585; Nature 368:856-859, 1994; and JP-WA No.
Hei 6-500233.
[0294] In addition, recently developed technique for producing a
human-derived protein from the milk of a transgenic cow or pig can
also be applied (Nikkei Science, pp. 78-84, April, 1997).
[0295] 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(ab').sub.2, Fab', Fab, Fv
(variable fragment of antibody), sFv, dsFv (disulfide stabilized
Fv), or dAb (single domain antibody) (Exp. Opin. Ther. Patents
6:441-456, 1996).
[0296] "F(ab') 2" and "Fab'" can be produced by treating
immunoglobulin (monoclonal antibody) with a protease such as pepsin
and papain, and means an antibody fragment generated by digesting
immunoglobulin near the disulfide bonds existing between the hinge
regions in each of the two F 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
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.gamma.1
(.gamma.1 region in the constant region of H chain) are connected
at their C terminal regions through a disulfide bond. Each of such
two 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(ab').sub.2.
[0297] 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
"portion of an antibody"; and a pharmaceutically acceptable
carrier.
[0298] The "pharmaceutically acceptable carrier" includes a
excipient, a diluent, an expander, a decomposition agent, a
stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a
colorant, 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 formulated
into tablets, pills, powders, granules, injections, solutions,
capsules, troches, elixirs, suspensions, emulsions, or syrups. The
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.
[0299] 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
administered to an adult in a dose of 10 .mu.g to 1000 mg (or 10
.mu.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.
[0300] 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 .mu.g 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 .mu.g to 100 mg/kg body
weight, preferably 50 .mu.g 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.
[0301] 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.
[0302] The injection can be sterilized by filtration with a
bacteria-non-penetrated filter, by mixing bactericide, 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.
[0303] 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.
[0304] The therapeutic effect of the pharmaceutical composition of
the present invention for symptom of various diseases can be tested
by the usual method by administering it to an known disease model
animal.
[0305] Examples of the model include (1) a (NZB/NZW)F1 mouse, a
model for human systemic lupus erythematosus (SLE) (Science
125:1225-1227, 1994); (2) experimental allergic encephalomyelitis
(EAE), a model for multiple sclerosis (MS) (J. Clin. Invest.
95:2783-2789, 1995); (3) an NOD (non-obese diabetes) mouse, a model
for insulin dependent diabetes mellitus (IDDM) (J. Exp. Med.
181:1145-1155, 1995); (4) rat nephritis model by renal glomerulus
basement membrane immunity, Goodpasture's nephritis model (Eur. J.
Immunol. 24:1249-1254, 1994); and (5) a DBA/1 mouse, a model for
human rheumatoid arthritis (Eur. J. Immunol. 26:2320-2328,
1996).
BRIEF DESCRIPTION OF THE DRAWINGS
[0306] FIG. 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."
[0307] 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."
[0308] FIG. 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."
[0309] 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-ICAM-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
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."
[0310] FIG. 3 shows the expression state of "JTT-1 antigen" and
"JTT-2 antigen" in various cells measured with a
flow-cytometer.
[0311] FIG. 4 shows the expression state of "JTT-1 ntigen" in
various lymphocytic cells measured with a flow-cytometer.
[0312] FIG. 5 is a photograph showing electrophoretogram of "JTT-1
antigen" analyzed by SDS-PAGE.
[0313] FIG. 6 are micrographs showing the state of adhesion of rat
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."
[0314] Subfigure (a) shows the state of adhesion of the cells to
the 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 (c)
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
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."
[0315] FIG. 7 shows the relative cell number of thymocytes adhering
to the plate coated with purified "JTT-1 antigen" measured in terms
of fluorescence intensity.
[0316] "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 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."
[0317] FIG. 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.
[0318] FIG. 9 shows the structural characteristics of amino acid
sequence of "JTT-1 antigen" revealed by hydropathy plot
analysis.
[0319] FIG. 10 shows the homology among amino acid sequences of
human, rat, and mouse "JTT-1 antigen" and "rat JTT-1 antigen"
mutant.
[0320] FIG. 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."
[0321] FIG. 12 schematically shows the protein secondary structure
of, and their similarity among "human JTT-1 antigen," "human CD28
molecule," and "human CTLA-4 molecule."
[0322] FIG. 13 schematically shows the structure of the genomic DNA
encoding "mouse JTT-1 antigen."
[0323] FIG. 14 shows the difference in amino acid sequences between
"rat JTT-1 antigen" and its alternative splicing mutant.
[0324] FIG. 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
[.sup.3H] thymidine uptake.
[0325] The ordinate shows the amount of uptake (dpm) of [.sup.3H]
thymidine into the cells.
[0326] FIG. 16 shows the therapeutic effect of the monoclonal
antibody against "JTT-1 antigen" on experimental allergic
encephalomyelitis (EAE) in a disease model rat.
[0327] The ordinate shows the scored degree of disease symptom, and
the abscissa shows the days after immunization for induction of
EAE.
[0328] FIG. 17 shows the therapeutic effect of the monoclonal
antibody against "JTT-1 antigen" on glomerulonephritis in a disease
model rat.
[0329] The ordinate shows the amount of urinary excretion of
proteins, and the abscissa shows the time course (week) after
immunization for induction of glomerulonephritis
[0330] FIG. 18 shows a column histogram in purification of fusion
polypeptide between "rat JTT-1 antigen" extracellular region and
human IgFc (rJTT-1-IgFc) with protein A Sepharose column.
[0331] FIG. 19 is a photograph showing electrophoretogram of
rJTT-1-IgFc analyzed by SDS-PAGE.
[0332] FIG. 20 shows a column histogram in purification of fusion
polypeptide between "human JTT-1 antigen" extracellular region and
human IgFc (rJTT-1-IgFc) with protein A Sepharose column.
[0333] FIG. 21 is a photograph showing electrophoretogram of
hJTT-1-IgFc analyzed by SDS-PAGE.
[0334] FIG. 22 schematically shows the structure of the gene
transfer (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
[0335] The present inventions are described in more detail with
reference to Examples below, but are not to be construed to be
limited thereto.
EXAMPLE 1
Preparation of Monoclonal Antibodies
[0336] Antibody-producing hybridomas were prepared according to the
method of Kohler et al. (Omori et al., Blood, 81:101-111, 1993),
and monoclonal antibodies were prepared according to the method of
Kannagi et al. (Handbook of Experimental Immunology,
4:117.21-117.21, 1986).
[0337] First, rat thymoma cell line FTL435 cells were administered
as an immunizing antigen to BALB/c mice into their footpad in an
amount of 10.sup.7 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 (JCR No. B0113; Stocker et al.,
Res. Disclosure, 217:155, 1982) by the usual method to obtain many
hybridomas producing monoclonal antibodies.
EXAMPLE 2
Screening of Hybridomas and Characterization of Monoclonal
Antibodies
[0338] 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.times.10.sup.6 cells/ml, 0.1 ml)
were seeded into each well of a 96-well microtiter plate and
cultivated at 37.degree. C. for an hour in the presence of culture
supernatant of each hybridoma (10 .mu.g/ml each). The results
obtained for hybridoma clones "JTT-1" and "JTT-2" are shown in FIG.
1 and FIG. 2.
[0339] It was revealed that a monoclonal antibody produced by
hybridoma clone "JTT-1" ("JTT-1 antibody") strongly agglutinated
FTL435 cells (FIG. 1(b) and FIG. 2(c)) and that addition of "JTT-2
antibody" strongly inhibited the aggregation of FTL435 cells
induced by "JTT-1 antibody" stimulation (FIG. 1(d)). The assays, in
which no hybridoma supernatant was added, were used as controls
(FIG. 1(a) and FIG. 2(a)).
[0340] In order to determine whether the aggregation of FTL435
cells induced by "JTT-1 antibody" stimulation was caused by the
cell 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.degree. C. for an hour in the presence of anti-rat
ICAM-1 antibody 1A29 (10 .mu.g/ml; IgG1) or anti-rat LFA-1 antibody
(10 .mu.g/ml; IgG2a) together with "JTT-1 antibody."
[0341] 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, FIG. 1(c) and FIG. 2(f);
anti-LFA-1 antibody, FIG. 2(d)).
[0342] 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 FIG.
2.
[0343] Similar to the effect on FTL435 cells, the aggregation of
activated lymphoblast cells was induced by "JTT-1 antibody"
stimulation (FIG. 2(i)). The aggregation of activated lymphoblast
cells by "JTT-1 antibody" stimulation was mostly inhibited by
anti-LFA-1 antibody (FIG. 2(j)) and anti-ICAM-1 antibody (FIG.
2(l)). (However, partial aggregation occurred.)
[0344] As understood from the control assay (FIG. 2(g)), in which
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) (FIG. 2(h)) or "JTT-1 antibody" (FIG. 2(i)).
Therefore, the fact that anti-LFA-1 antibody partially 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.
[0345] 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)
since Oct. 11, 1996 with an international accession Nos. FERM
BP-5707 and FERM BP-5708, respectively.
[0346] Analysis using mouse monoclonal antibody isotype
identification kit (Amersham) determined that the isotype of
monoclonal antibodies produced from each hybridoma (JTT-1 antibody
and JTT-2 antibody) were both IgG1.
EXAMPLE 3
Reactivity of "JTT-1 Antibody" and "JTT-2 Antibody" to Various
Cells
[0347] In order to analyze the expression pattern of molecules
recognized by "JTT-1 antibody" and "JTT-2 antibody" in various
cells, 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.
[0348] 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
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 .mu.g/ml
concanavalin A and 10% FCS at 37.degree. C. for 3 days to prepare
activated lymphoblasts.
[0349] FTL435 cells, thymocytes, spleen cells, and activated
lymphoblasts (5.times.10.sup.5 cells each) were reacted with "JTT-1
antibody" or "JTT-2 antibody" and then with FITC-labeled anti-mouse
IgG (Cappel). The fluorescence intensity of the stained cells was
measured with EPICS-Elite flow cytometer.
[0350] The results are shown in FIG. 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
[0351] 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.
[0352] 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 .mu.g/ml
concanavalin A (ConA) and 10% FCS at 37.degree. 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.
[0353] Each cells (5.times.10.sup.5 cells each) were reacted with
biotin-labeled anti-rat T cell antibody or biotin-labeled anti-rat
B cell antibody (10 .mu.g/ml, Seikagaku Corporation), and
subsequently with phycoerythrin-labeled streptavidin. The cells
were then reacted with 10 .mu.g/ml FITC-labeled "JTT-1 antibody."
The fluorescence intensity of the stained cells was measured with
EPICS-Elite flow cytometer.
[0354] The results are shown in FIG. 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 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
[0355] "JTT1 antigen" and "JTT-2 antigen" were characterized by
immunoprecipitation using FTL435 cells.
[0356] (1) Preparation of biotinylated soluble cell surface
molecules
[0357] FTL435 cells were washed with PBS, suspended in
physiological saline containing 100 .mu.g/ml NHS-biotin and 0.1 M
HEPES (pH 8.0) to adjust 1.times.10.sup.7 cells/ml, and incubated
at room temperature for 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.times.10.sup.7 cells/ml,
and the mixture was allowed to react at 4.degree. C. for 30 minutes
to lyse the cells. The cell lysate obtained was centrifuged, and
the supernatant comprising biotinylated soluble cell surface
molecules was stored at -80.degree. C.
[0358] (2) Immunoprecipitation and SDS-PAGE analysis
[0359] 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 adjust 2 mg/ml, and allowed to react at 4.degree. C. for
an hour to bind the antibody with the beads. After the beads were
washed, 500 .mu.l of the biotinylated FTL435 cell lysate was added
to 10 .mu.l of the beads, and the mixture was allowed to react at
4.degree. C. for 2 hours. After the beads were washed with lysis
buffer three times, 50 .mu.l 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.
[0360] An equal volume of sample buffer (Enprotech) for SDS-PAGE
was added to 5 .mu.l 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.
[0361] The results are shown in FIG. 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 FIG. 5) and about 24 kD and 28 kD under the
reduced conditions ("(+)" in FIG. 5). As the result of digestion of
N-linked sugar chains ("+N-gly" in FIG. 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." C. 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
[0362] 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.
[0363] (1) Preparation of "JTT-1 antibody"-affinity column
[0364] 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.degree. 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.
[0365] (2) Purification of "JTT-1 antigen"
[0366] 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.times.10.sup.7 cells/ml, and the mixture was allowed to
react at 4.degree. 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.degree. C.
[0367] The lysate (400 ml) was loaded onto "JTT-1
antibody"-affinity column. After the column was washed with 50 ml
of the lysis buffer 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.degree. C.
[0368] (3) Determination of N terminal amino acid sequence
[0369] 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.
[0370] (4) Adhesion experiment
[0371] 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.mu. ',7'-bis(carboxyethyl)carboxyfluorescein tetraacetoxy-methyl
ester (BCECF-AM; Molecular Probes) was added to the suspension, and
the mixture was incubated at 37.degree. 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
2.times.10.sup.7 cells/ml.
[0372] The purified "JTT-1 antigen" obtained in (2) was coated on a
96-well ELISA plate at the concentration of 10 .mu.l/well
overnight. After the plate was washed with PBS, 200 .mu.l/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.times.10.sup.7 cells/ml,
0.1 ml); (2) fluorescence-labeled thymocytes (same concentration)
and "JTT-1 antibody" Fab fragments prepared by the usual method
(5.mu.
[0373] "JTT-1 antibody" Fab fragments (same concentration), and
"JTT-2 antibody" (10.mu.
[0374] .degree. C. for an hour. In order to remove unbound cells,
each well was washed once with RPMI1640 medium containing 10% FCS.
Each well was observed with light microscope. Then, 100 .mu.l 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 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.
[0375] The results of light microscopy observation are shown in
FIG. 6.
[0376] Thymocytes significantly adhered to purified "JTT-1 antigen"
only in the presence of "JTT-1 antibody" Fab fragments (FIG. 6(c)).
The adhesion was significantly inhibited by "JTT-2 antibody" (FIG.
6(d)).
[0377] FIG. 7 shows the relative cell number of thymocytes adhered
to "JTT-1 antigen" coated on each well in terms of fluorescent
intensity.
[0378] 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"
[0379] 1. Preparation of cDNA Library
[0380] 1-(1) Extraction of Poly(A).sup.+ RNA from ConA-stimulated
Rat Lymphoblasts
[0381] ConA-stimulated lymphoblasts (ConA blast) derived from rat
spleen (about 1.times.10.sup.6 cells/ml) were centrifuged
(2,000.times.g) at 4.degree. 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.times.g at 4.degree. C. for 10 minutes to precipitate
RNA. The precipitated RNA was washed with ethanol and dissolved in
TE buffer. Poly(A).sup.+ RNA was purified from the total RNA so
obtained with "mRNA Purification Kit" (Pharmacia).
[0382] 1-(2) Preparation of cDNA
[0383] With 5 .mu.g of the poly(A).sup.+ RNA prepared above as a
template, cDNA was synthesized with "Time Saver cDNA Synthesis Kit"
(Pharmacia). "Oligo dT primer" (Pharmacia) having NotI site was
used to increase the efficiency of screening. EcoRI adapter was
added, and digestion with NotI was performed to obtain cDNA with
unidirectionality. Size fractionation was then performed with Spun
Column (Pharmacia).
[0384] 1-(3) Insertion into a Vector
[0385] The obtained cDNA having EcoRI- and NotI-ends was ligated
with pME18S (Hara et al., EMBO J., 11:1875-1884, 1992) digested
with EcoRI and NotI. "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.
[0386] 2. Screening of cDNA Library
[0387] Screening was performed according to panning method (Seed et
al., Proc. Natl. Acad. Sci. USA, 84:3365-3369, 1987).
[0388] 2-(1) Gene Transfer into COS Cells
[0389] The library so obtained was introduced into COS7 cells by
electroporation (Potter et al., Proc. Natl. Acad. Sci. USA,
85:2288-2292). 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.degree. C. for 30 minutes, the cells
were removed by pipetting. Only living cells were then collected
with "Lymphprep" (NYCOMED).
[0390] 2-(2) Concentration of Gene-expressing Cells by Panning
[0391] 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
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, J. Mol. Biol.,
26:365-369). E. coli DH10B (GIBCO BRL) were transformed with the
plasmid DNA so obtained. The plasmid DNAs were amplified and
purified with the transformants as in (1)-3 mentioned above. The
procedures described in (1) and (2) were then repeated twice.
[0392] 2-(3) Isolation of the Positive Clone
[0393] After the third panning, transformed E. coli DH10B cells
were cultivated overnight on LB plates containing ampicillin to
obtain colonies. Twenty drug-resistant colonies were cultivated,
plasmid DNAs were collected by alkaline miniprep method (Maniatis
et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.), and the insert DNA was
analyzed. Agarose gel electrophoresis revealed that the clone
having about 0.9 kb cDNA (designated "T132A7") was
concentrated.
[0394] "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 of
the stained cells was measured with EPICS-Elite flow cytometer
(Coulter). "JTT-1 antibody" and "JTT-2 antibody" strongly
recognized the "T132A7" gene product. The results are shown in FIG.
8.
[0395] 3. Determination of the Nucleotide Sequence and the Amino
Acid Sequence
[0396] 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"
(IntelliGenetics). The nucleotide sequence and the deduced amino
acid sequence were shown in SEQ ID NO: 4.
[0397] 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 6-(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."
[0398] 4. Computer Analysis
[0399] Hydropathy analysis of the primary structure of the deduced
amino acid sequence of "JTT-1 antigen" was performed according to
the method of Kite and Doolittle (Kite et al., J. Mol. Biol.,
157:105-132, 1982) (FIG. 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 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 FIG. 9 "CHO" means N-linked sugar chain
binding site; "P", phosphorylation site; "CKII", casein kinase II;
and "PKC", protein kinase C.
EXAMPLE 8
Cloning of cDNA Encoding "Human JTT-1 Antigen"
[0400] 1. Preparation of a Probe
[0401] 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 NotI, 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 .sup.32P using "Ready-To-Go DNA
labelling kit" (Pharmacia). These labeled DNA fragments were used
as probes for plaque hybridization.
[0402] 2. Preparation of cDNA Library
[0403] 2-(1) Extraction of Poly(A).sup.+ RNA
[0404] Poly(A).sup.+ RNA was extracted from ConA-stimulated
lymphoblasts (ConA blast) derived from human peripheral blood in
the same manner as in Example 7-1-(1).
[0405] 2-(2) Preparation of cDNA
[0406] With 5 .mu.g of the poly(A).sup.+ RNA so prepared as a
template, cDNAs were synthesized with "oligo dT primer" (Pharmacia)
and "Time Saver cDNA Synthesis Kit" (Pharmacia). EcoRI adapter was
then added, and size fractionation was performed with Spun Column
(Pharmacia).
[0407] 2-(3) Insertion into a Vector and Packaging
[0408] The cDNAs so obtained having EcoRI-ends were ligated with
the vector ".lambda.ZAPII" (Stratagene) digested with EcoRI. "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" (Stratagene), E. coli XL1Blue MRF' cells (Stratagene) were
transfected with the obtained phage particle to generate a cDNA
library composed of plaque comprising recombinant phage.
[0409] 3. Screening of cDNA Library
[0410] cDNA library was screened by plaque hybridization method
(Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) with "Rapid
hybridization buffer" (Amersham). First, the cDNA library so
obtained (1.times.10.sup.4) 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 .sup.32P-labeled 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 (Stratagene) and seven positive clones were collected as
plasmid DNA.
[0411] 4. Determination of the Nucleotide Sequence
[0412] The nucleotide sequences of the seven clones were determined
by dideoxy method with "Auto Read Sequencing Kit" (Pharmacia) and
"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 NO:
2, and the nucleotide sequence comprising 5' and 3' sequences is
shown in SEQ ID NO: 3 (ORF corresponds 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) deduced from the nucleotide sequence shows significant
homology with the amino acid sequence of "rat JTT-1 antigen" (FIG.
10). As shown in FIG. 10, the homology between the amino acid
sequences of human and rat "JTT-1 antigen" is 60% or more.
[0413] E. coli DH10B (GIBCO BRL) transformed with the clone
"DBSh41" 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 Oct. 25, 1996
(an international deposit accession No. FERM BP-5725).
[0414] 5. Structural Characteristics and Biological Function of
"JTT-1 Antigen"
[0415] The results of motif search for the deduced amino acid
sequence of "human JTT-1 antigen" in known human proteins revealed
that "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 (FIGS. 11 and
12). As mentioned above, "CD28" and "CTLA-4" are extremely
important molecules regulating the activation and inhibition of T
cells in immune system.
[0416] The structural similarity is as follows
[0417] 1. 20 or more amino acid residues including cysteine
residues are highly conserved.
[0418] 2. Proline repeating sequence, "Pro-Pro-Pro (PPP)", which is
essential as the ligand binding region in CD28 and CTLA-4, is
conserved.
[0419] 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.
[0420] 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"
[0421] 1. Preparation of a Probe
[0422] 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 NotI, and separated by agarose gel
electrophoresis. The DNA fragments so separated were purified with
"QUIAEX gel extraction kit" (QUIAGEN), and the DNA fragments were
labeled with .sup.32P using "Ready-To-Go DNA labelling kit"
(Pharmacia). These labeled DNA fragments were used as a probe for
plaque hybridization.
[0423] 2. Preparation of cDNA Library
[0424] 2-(1) Extraction of Poly(A).sup.+ RNA
[0425] As Example 7-1-(1), poly(A).sup.+ RNAs were extracted from
ConA-stimulated lymphoblasts derived from mouse spleen (about
1.times.10.sup.6 cells/ml).
[0426] 2-(2) Preparation of cDNA Library
[0427] With 5 mg of poly(A).sup.+ 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).
[0428] 2-(3) Insertion of cDNA into a Vector and Packaging
[0429] The cDNA so obtained having EcoRI-ends was ligated with the
vector lZAPII (Stratagene) digested with EcoRI. "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
(Stratagene), E. coli XL1Blue MRF' cells (Stratagene) were
transfected with the phage particle so obtained to generate a cDNA
library composed of plaque comprising recombinant phage.
[0430] 3. Screening of cDNA Library
[0431] Screening was performed by plaque hybridization method
(Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) using Rapid
hybridization buffer (Amersham).
[0432] The above-obtained cDNA library (1.times.10.sup.4) was
plated onto agar 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
.sup.32P-labeled probe prepared in Example 9-1. First and second
screenings were performed to obtain five positive clones. After
single plaque of each clone was isolated, in vivo excision was
performed in accordance with Instruction Manual (Stratagene) and
five positive clones were collected as plasmid DNA.
[0433] 4. Determination of the Nucleotide Sequence
[0434] The nucleotide sequences of each of the five clones were
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 NO: 5.
[0435] As understood from FIG. 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).
[0436] 5. Analysis of the Locus of "Mouse JTT-1 Antigen" Gene
[0437] The locus of the gene encoding "mouse JTT-1 antigen" was
analyzed by fluorescence in situ hybridization method.
[0438] The cDNA so obtained encoding "mouse JTT-1 antigen" was
labeled with .sup.32P to prepare hybridization probes by the usual
method. Using 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 FIG. 13.
[0439] The above-obtained genomic DNA clones were labeled with
digoxigenin dUTP by nick translation to prepare probes. The 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.times.SSC. After a slide glass for hybridization was incubated in
fluorescence-labeled anti-digoxigenin antibody, 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
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
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.
[0440] 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"
[0441] Another cDNA that is thought to encode alternative splicing
variant of "rat JTT-1 antigen" cloned in Example 7 was cloned as
follows.
[0442] 1. Preparation of a Probe
[0443] 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 NotI, 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 .sup.32P using "Ready-To-Go DNA
labeling kit" (Pharmacia). These labeled DNA fragments were used as
probes for plaque hybridization.
[0444] 2. Preparation of cDNA library
[0445] 2-(1) Extraction of Poly (A).sup.+ RNA
[0446] As in Example 7-1-(1), poly(A).sup.+ RNA was extracted from
rat thymoma cell line FTL435 (about 1.times.10.sup.6 cells/ml)
[0447] 2-(2) Preparation of cDNA Library
[0448] With 5 mg of the poly(A).sup.+ 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 adapter was added to the cDNA, size fractionation was
performed with Spun Column (Pharmacia).
[0449] 2-(3) Insertion of cDNA into a Vector and Packaging
[0450] The cDNA having EcoRI-end obtained above was ligated with
the vector nZAPII (Stratagene) digested with EcoRI. "DNA Ligation
Kit" (Takara Shuzo) was used for ligation. After in vitro packaging
of the ligated DNA was performed with GIGA PACK II GOLD
(Stratagene), E. coli XL1Blue MRF' (Stratagene) was transfected
with the obtained phage particle to generate a cDNA library
composed of plaque comprising recombinant phage.
[0451] 3. Screening of cDNA Library
[0452] Screening was performed by plaque hybridization method
(Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) with Rapid
hybridization buffer (Amersham).
[0453] The above-prepared cDNA library (1.times.10.sup.4) 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
.sup.32P-labeled probe prepared in Example 10-1. First and second
screenings were 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.
[0454] 4. Determination of the Nucleotide Sequence
[0455] The nucleotide sequences of the two clones were determined
by dideoxy method with "Auto Read Sequencing Kit" (Pharmacia) and
A.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 (FIG. 14). As shown in FIG. 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-Gln-Asp-Arg-Asn- )
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
[0456] 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 91:158-162, 1994)
treated with the same restriction enzyme EcoRI to prepare the
expression vector. CHO-K1 cells (ATCC: CCL-61) were transformed
with the vector by electroporation. By cultivating the cells in
RPMI1640 medium containing 0.8 mg/ml Geneticin (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"
[0457] The recombinant "human JTT-1 antigen"-expressing
transformants prepared in Example 11 were homogenized and
ultracentrifuged (100,000.times.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 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 (JCR No. B0113; Res. Disclosure 217:155, 1982) were mixed 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 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
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.
[0458] Each of two kinds (designated clone SA12 and SG430) among
these hybridomas (10.sup.6 to 10.sup.7 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 ascites fluid extracted by the
usual method.
EXAMPLE 13
Effect of the Monoclonal Antibodies Against "Human JTT-1 Antigen"
on Human Peripheral Blood Lymphocytes
[0459] As mentioned in Example 8, it is thought that "JTT-1
antigen" can be involved in the regulation of the activation of
lymphocytes in immune reaction like "CD28" and "CTLA-4." In order
to prove this, the effect of the monoclonal antibodies against
"human JTT-1 antigen" on human lymphocytes was analyzed in light of
cell growth as an indication.
[0460] To each well of 96-well microtiter plate were added (1)
either SA12 or SG430 (1 .mu.g/ml), the monoclonal antibody against
"human JTT-1 antigen" prepared in Example 12, or (2) a mixture of
either monoclonal antibody SA12 or SG430 (1 .mu.g/ml) with anti-CD3
monoclonal antibody OKT-3 (1 .mu.g/ml, orthodiagnostic Systems),
which is used for adding the primary signal in the activation of
lymphocytes. The plate was incubated at 37.degree. C. for 1 hour to
coat each well with the antibody. After the plate was washed with
RPMI1640 medium, normal human peripheral blood lymphocytes
(1.times.10.sup.5 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 (PMA) was added. Then,
[.sup.3H] thymidine (3.7 .mu.kBq/well) was added to each well, and
the plate was incubated at 37.degree. C. for 6 hours. The cells
were harvested, and the amount of [.sup.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 FIG. 15.
[0461] 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
monoclonal antibody SA12 or SG430 was used.
[0462] 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
costimulatory signal like "CD28" and "CTLA-4."
EXAMPLE 14
Effect of "JTT-2 Antibody" on Experimental Allergic
Encephalomyelitis (EAE)
[0463] As above mentioned in detail, recently, many attempts to
treat various autoimmune diseases (rheumatoid arthritis, multiple
sclerosis, autoimmune thyroiditis, allergic contact dermatitis,
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
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).
[0464] 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
monoclonal antibody on "JTT-1 antigen" in the model was
analyzed.
[0465] 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 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 200 mg per rat. This immunization so induces
experimental allergic encephalomyelitis (EAE).
[0466] 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.
[0467] (1) Monoclonal antibody "JTT-2 antibody" against "rat JTT-1
antigen" prepared in Example 2 (dosage: 2 mg/ml PBS, 5 mg/kg)
[0468] (2) Prednisolone, steroid agent (dosage: 4 mg/ml PBS, 10
mg/kg)
[0469] (3) Control antibody non-reactive to "rat JTT-1 antigen"
(dosage: 2 mg/ml PBS, 5 mg/kg)
[0470] Symptom was observed in the course of time after the
immunization. After the onset of EAE had been found, the degree of
the symptom was estimated by scoring the symptom based on the
following criteria.
[0471] (Score 1) Disappearance of tension of a tail
[0472] (Score 2) Dragging of hind legs, and slight paralysis
[0473] (Score 3) Dragging of hind legs, and serious paralysis
[0474] (Score 4) Paralysis of the whole body, or death
[0475] The results are shown in FIG. 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
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
prednisolone-administered group.
[0476] These results indicate that "JTT-1 antigen" is a molecule
that 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
[0477] 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.
[0478] After bovine glomerulus basement membrane (Shigei Medical
Institute) digested with collagenase was diluted with physiological
saline to 200 .mu.g/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 Wistar kyoto rats (about 200 g) under
anesthesia in an amount of about 0.2 ml per footpad (dosage: about
15 .mu.g). This immunization so induces glomerulus basement
membrane (GBM) nephritis.
[0479] 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.
[0480] (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)
[0481] (2) Prednisolone, steroid agent, as a positive control
(suspended in 0.5% carboxymethylcellulose (CMC)) (dosage: 3 mg/kg
(5 ml/kg), oral administration)
[0482] (3) 0.5% CMC as a negative control (dosage: 5 ml/kg, oral
administration)
[0483] After the administration of a test substrate, sterilized
water (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 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).
[0484] The results are shown in FIG. 17. Compared to the control
group, the urinary excretion of protein at 3 weeks after the
immunization was significantly reduced in the "JTT-2 antibody"
-administered group.
[0485] These results indicate that "JTT-1 antigen" is a molecule
that induces 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 16
Preparation of the Fusion Protein Between "JTT-1 Antigen" and
IgFc
[0486] 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-IgFc) composed of the extracellular domain of "CTLA-4" and
the Fc region of human immunoglobulin IgG1 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 various autoimmune diseases.
[0487] (1) Preparation of the fusion protein between "rat JTT-1
antigen" and human IgG1-Fc (rJTT-1-IgFc)
[0488] In order to amplify the cDNA encoding the extracellular
region of "rat JTT-1 antigen" by PCR, 5' primer having XhoI
restriction 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" 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 XhoI and BamHI restriction
sites at its both ends. The PCR products so obtained were digested
with XhoI and BamHI and separated by agarose gel electrophoresis to
isolate an 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
XhoI and BamHI. Sequence analysis with an automated fluorescence
DNA sequencer (Applied Biosystems) revealed that the cDNA fragment
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).
[0489] On the other hand, the DNA encoding the Fc of human IgG1 as
the fusion partner was cut out as an about 1.3 kb BamHI-XbaI DNA
fragment by digesting the plasmid (see Cell 61:1303-1313, 1990).
Prepared by B. Seed et al. (Massachusetts General Hospital)) with
BamHI and XbaI. This fragment comprises exons encoding human IgG1
hinge region, C.gamma..sub.12, and C.gamma..sub.13.
[0490] The XhoI-BamHI fragment encoding the extracellular region of
"rat JTT-1 antigen," and BamHI-XbaI fragment comprising exons
encoding the Fc of human IgG1 ("IgFc"), both prepared as mentioned
above, were subcloned into pBluescript II SK (+) (Stratagene)
cleaved with XhoI and XbaI.
[0491] Then, the plasmid was digested with XhoI and XbaI, and an
about 1.8 kb DNA fragment comprising the fusion DNA comprising the
extracellular region of "rat JTT-1 antigen" and human IgFc was cut
out. This fusion DNA fragment was inserted into the XhoI and XbaI
sites of the expression vector pME18S (Medical Immunology 20:27-32,
1990; Experimental Medicine: SUPPLEMENT, "Handbook of Genetic
Engineering," Yodosha, pp. 101-107, 1992) with T4 DNA ligase to
construct plasmid prJTT-1-IgFc.
[0492] HEK293 cells (ATCC CRL1573) subconfluently cultivated as
monolayer in DMEM medium containing 10% fetal calf serum and
ampicillin were transformed with prJTT-1-IgFc by electroporation to
obtain transformants.
[0493] The transformants were cultured in serum-free ASF104 medium
for 72 hours to express rJTT-1-IgFc.
[0494] Using a Protein G Sepharose affinity column (Pharmacia),
rJTT-1-IgFc was purified as follows.
[0495] The supernatant obtained by centrifuging the culture medium
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 pure rJTT-1-IgFc.
[0496] The result of affinity chromatography is shown in FIG. 18,
and the result of SDS-PAGE of the pure rJTT-1-IgFc so obtained in
FIG. 19.
[0497] (2) Preparation of the fusion protein between "human JTT-1
antigen" and human IgG1-Fc (hJTT-1-IgFc) hJTT-1-IgFc 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'-ATCCTATGGGTAACGGATCCTTCAGCTGGC-3' (SEQ ID NO: 10) were used as
primers.
[0498] The result of affinity chromatography is shown in FIG. 20,
and the result of SDS-PAGE of the pure hJTT-1-IgFc so obtained in
FIG. 21.
EXAMPLE 17
Preparation of a Transgenic Mouse in Which cDNA Encoding "Rat JTT-1
Antigen" has been Integrated
[0499] The cDNA encoding the full length of "rat JTT-1 antigen"
obtained in Example 7 was inserted into the expression vector
pCAGGS (Gene 108:193-200, 1991) having chicken .beta. actin
promoter using DNA Blunting Kit (Takara) to obtain plasmid,
prJTT-1. In order to prepare a transgenic mouse, prJTT-1 was
linearized by restriction enzyme treatment.
[0500] A female ICR mouse having a vaginal plug, obtained by mating
a white ICR mouse (Nihon LSC) with a male vasoligated white ICR
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
(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.
[0501] The "rat JTT-1 antigen" gene was introduced into the
fertilized egg under microscopy using a manipulator according to
the usual 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 microinjected into the male pronucleus of the
fertilized eggs with a DNA introduction needle at 37.degree. C.
[0502] After gene introduction, only fertilized eggs keeping normal
state were selected, and then, the fertilized egg so selected in
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).
[0503] The tail of a progeny mouse (founder mouse) born from the
foster 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
can be prepared by mating the heterozygous mice with each
other.
[0504] The microinjected construct comprising the "rat JTT-1
antigen" gene is schematically shown in FIG. 22.
EXAMPLE 18
Preparation of a Knockout Mouse Whose Endogenous Gene Encoding
"Mouse JTT-1 Antigen" has been Inactivated
[0505] (1) Construction of a Targeting Vector
[0506] 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.
[0507] The PstI-HindIII 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
HindIII was subcloned into pGEM-3 (Promega). Then, pGEM-3 was
linearized with XhoI, and neomycin resistance gene ("neo") excised
from pMC1-neo-polyA (Stratagene) by treating it with XhoI and SalI
was inserted at the upstream of the "homologous DNA" (1) and then
ligated them. The above-mentioned mouse genomic DNA clone was
digested with XhoI 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-homologous DNA (1)" has been inserted was digested with
XhoI and HindIII to cut off "neo-homologous DNA (1)." "Homologous
DNA (2)" and "neo-homologous DNA (1)" thus obtained were subcloned
into pSEAP2-CONT (Clontech) linearized with NotI and HindIII.
[0508] After the obtained plasmid, in which "homologous
(2)-neo-homologous (1)" has been inserted, was digested and
linearized at the downstream of "homologous DNA (1) with NruI,
thymidine kinase gene ("TK") obtained by digesting pMC1-TK
(Stratagene) with PvuII was inserted at the downstream of
"homologous DNA (1)" to obtain a targeting vector, in which
"homologous DNA (2)-neo-homologous (1)" was inserted.
[0509] (2) Introduction of the Targeting Vector into ES Cells
[0510] Mouse embryonic stem cells (Nature 362:255-258, 1993; Nature
326:292-295, 1987) cultured in DMEM medium containing 15% fetal
calf serum were treated with trypsin to be single cells, and the
cells were washed three times, followed by adding phosphate buffer
thereto to adjust 1.times.10.sup.7 cells/ml. The targeting vector
mentioned above (25 .mu.g 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 .mu.F). Then, 1.times.10.sup.7
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 .mu.g/ml G418 and 2 .mu.M
ganciclovir). The cells were cultivated with the medium changed
every two days. At the tenth day from the introduction of the
targeting vector, 573 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.
[0511] (3) Screening of Knockout ES Cells
[0512] 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.
[0513] For PCR, (1) primers designed and synthesized based on the
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.
[0514] Each genomic DNA was extracted from each of the
neomycin-resistant 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.degree. C. for 3 minutes, 30 cycles of
reaction at 94.degree. C. for 1 minute, at 60.degree. C. for 1
minute, and at 72.degree. C. for 3.5 minutes, and 1 cycle of
reaction at 72.degree. C. for 10 minutes, and the resulting
products were stored at 40.degree. 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.
[0515] 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.
[0516] 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.
[0517] (4) Preparation of a Knockout Mouse
[0518] 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.
[0519] 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
[0520] Each of the monoclonal antibody (50-150 .mu.g/ml), "JTT-1
antibody" and "JTT-2 antibody" against "rat JTT-1 antigen,"
prepared in Example 1, and monoclonal antibodies, "SA12" and
"SG430" against "human JTT-1 antigen," prepared in Example 12, was
added to injectable distilled water (10 ml) to prepare
injection.
[0521] Industrial Applicability
[0522] 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.
[0523] (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.
[0524] (i) 20 or more amino acid residues including cysteine
residues are highly conserved;
[0525] (ii) Proline repeating sequence, "Pro-Pro-Pro (PPP)," which
is essential as the ligand binding region, is conserved in the
extracellular region;
[0526] (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
[0527] (iv) The locus of the gene encoding "mouse JTT-1 antigen" on
mouse chromosome is "1C3", like "CD28" and "CTLA-4."
[0528] (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.
[0529] (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.
[0530] (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.
[0531] (5) The administration of the antibody against "JTT-1
antigen" significantly inhibits the symptom of experimental
allergic encephalomyelitis (EAE).
[0532] (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.
[0533] "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.
[0534] 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
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
abnormality of regulation of activated lymphocyte functions.
[0535] 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.
[0536] 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.
[0537] 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 drugs targeting the ligands.
[0538] 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
various drugs (low molecular weight compounds, antibodies,
antisense substances, polypeptides, etc.) having activity
regulating (inhibition, suppression,
Sequence CWU 1
1
26 1 600 DNA Homo sapiens CDS (1)...(597) 1 atg aag tca ggc ctc tgg
tat ttc ttt ctc ttc tgc ttg cgc att aaa 48 Met Lys Ser Gly Leu Trp
Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 gtt tta aca gga
gaa atc aat ggt tct gcc aat tat gag atg ttt ata 96 Val Leu Thr Gly
Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 ttt cac
aac gga ggt gta caa att tta tgc aaa tat cct gac att gtc 144 Phe His
Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45
cag caa ttt aaa atg cag ttg ctg aaa ggg ggg caa ata ctc tgc gat 192
Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50
55 60 ctc act aag aca aaa gga agt gga aac aca gtg tcc att aag agt
ctg 240 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser
Leu 65 70 75 80 aaa ttc tgc cat tct cag tta tcc aac aac agt gtc tct
ttt ttt cta 288 Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser
Phe Phe Leu 85 90 95 tac aac ttg gac cat tct cat gcc aac tat tac
ttc tgc aac cta tca 336 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr
Phe Cys Asn Leu Ser 100 105 110 att ttt gat cct cct cct ttt aaa gta
act ctt aca gga gga tat ttg 384 Ile Phe Asp Pro Pro Pro Phe Lys Val
Thr Leu Thr Gly Gly Tyr Leu 115 120 125 cat att tat gaa tca caa ctt
tgt tgc cag ctg aag ttc tgg tta ccc 432 His Ile Tyr Glu Ser Gln Leu
Cys Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140 ata gga tgt gca gcc
ttt gtt gta gtc tgc att ttg gga tgc ata ctt 480 Ile Gly Cys Ala Ala
Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu 145 150 155 160 att tgt
tgg ctt aca aaa aag aag tat tca tcc agt gtg cac gac cct 528 Ile Cys
Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170 175
aac ggt gaa tac atg ttc atg aga gca gtg aac aca gcc aaa aaa tct 576
Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser 180
185 190 aga ctc aca gat gtg acc cta taa 600 Arg Leu Thr Asp Val Thr
Leu 195 2 199 PRT Homo sapiens 2 Met Lys Ser Gly Leu Trp Tyr Phe
Phe Leu Phe Cys Leu Arg Ile Lys 1 5 10 15 Val Leu Thr Gly Glu Ile
Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile 20 25 30 Phe His Asn Gly
Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val 35 40 45 Gln Gln
Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp 50 55 60
Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu 65
70 75 80 Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe
Phe Leu 85 90 95 Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe
Cys Asn Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Lys Val Thr
Leu Thr Gly Gly Tyr Leu 115 120 125 His Ile Tyr Glu Ser Gln Leu Cys
Cys Gln Leu Lys Phe Trp Leu Pro 130 135 140 Ile Gly Cys Ala Ala Phe
Val Val Val Cys Ile Leu Gly Cys Ile Leu 145 150 155 160 Ile Cys Trp
Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro 165 170 175 Asn
Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser 180 185
190 Arg Leu Thr Asp Val Thr Leu 195 3 2610 DNA Homo sapiens CDS
(26)...(622) 3 ggactgttaa ctgtttctgg caaac atg aag tca ggc ctc tgg
tat ttc ttt 52 Met Lys Ser Gly Leu Trp Tyr Phe Phe 1 5 ctc ttc tgc
ttg cgc att aaa gtt tta aca gga gaa atc aat ggt tct 100 Leu Phe Cys
Leu Arg Ile Lys Val Leu Thr Gly Glu Ile Asn Gly Ser 10 15 20 25 gcc
aat tat gag atg ttt ata ttt cac aac gga ggt gta caa att tta 148 Ala
Asn Tyr Glu Met Phe Ile Phe His Asn Gly Gly Val Gln Ile Leu 30 35
40 tgc aaa tat cct gac att gtc cag caa ttt aaa atg cag ttg ctg aaa
196 Cys Lys Tyr Pro Asp Ile Val Gln Gln Phe Lys Met Gln Leu Leu Lys
45 50 55 ggg ggg caa ata ctc tgc gat ctc act aag aca aaa gga agt
gga aac 244 Gly Gly Gln Ile Leu Cys Asp Leu Thr Lys Thr Lys Gly Ser
Gly Asn 60 65 70 aca gtg tcc att aag agt ctg aaa ttc tgc cat tct
cag tta tcc aac 292 Thr Val Ser Ile Lys Ser Leu Lys Phe Cys His Ser
Gln Leu Ser Asn 75 80 85 aac agt gtc tct ttt ttt cta tac aac ttg
gac cat tct cat gcc aac 340 Asn Ser Val Ser Phe Phe Leu Tyr Asn Leu
Asp His Ser His Ala Asn 90 95 100 105 tat tac ttc tgc aac cta tca
att ttt gat cct cct cct ttt aaa gta 388 Tyr Tyr Phe Cys Asn Leu Ser
Ile Phe Asp Pro Pro Pro Phe Lys Val 110 115 120 act ctt aca gga gga
tat ttg cat att tat gaa tca caa ctt tgt tgc 436 Thr Leu Thr Gly Gly
Tyr Leu His Ile Tyr Glu Ser Gln Leu Cys Cys 125 130 135 cag ctg aag
ttc tgg tta ccc ata gga tgt gca gcc ttt gtt gta gtc 484 Gln Leu Lys
Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val 140 145 150 tgc
att ttg gga tgc ata ctt att tgt tgg ctt aca aaa aag aag tat 532 Cys
Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr 155 160
165 tca tcc agt gtg cac gac cct aac ggt gaa tac atg ttc atg aga gca
580 Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala
170 175 180 185 gtg aac aca gcc aaa aaa tct aga ctc aca gat gtg acc
cta 622 Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu 190
195 taatatggaa ctctggcacc caggcatgaa gcacgttggc cagttttcct
caacttgaag 682 tgcaagattc tcttatttcc gggaccacgg agagtctgac
ttaactacat acatcttctg 742 ctggtgtttt gttcaatctg gaagaatgac
tgtatcagtc aatggggatt ttaacagact 802 gccttggtac tgccgagtcc
tctcaaaaca aacaccctct tgcaaccagc tttggagaaa 862 gcccagctcc
tgtgtgctca ctgggagtgg aatccctgtc tccacatctg ctcctagcag 922
tgcatcagcc agtaaaacaa acacatttac aagaaaaatg ttttaaagat gccaggggta
982 ctgaatctgc aaagcaaatg agcagccaag gaccagcatc tgtccgcatt
tcactatcat 1042 actacctctt ctttctgtag ggrtgagaat tcctctttta
atcagtcaag ggagatgctt 1102 caaagctggr gctattttat ttctgagatg
ttgatgtgaa ctgtacatta gtacatactc 1162 agtactctcc ttcaattgct
gaaccccagt tgaccatttt accaagactt tagatgcttt 1222 cttgtgccct
caattttctt tttaaaaata cttctacatg actgcttgac agcccaacag 1282
ccactctcaa tagagagcta tgtcttacat tctttcctct gctgctcaat agttttatat
1342 atctatgcat acatatatac acacatatgt atataaaatt cataatgaat
atatttgcct 1402 atattctccc tacaagaata tttttgctcc agaaagacat
gttcttttct caaattcagt 1462 taaaatggtt tactttgttc aagttagtgg
taggaaacat tgcccggaat tgaaagcaaa 1522 tttawwttat tatcctattt
tctaccatta tctatgtttt catggtgcta ttaattacaa 1582 gtttagttct
ttttgtagat catattaaaa ttgcaaacaa aatcatcttt aatgggccag 1642
cattctcatg gggtagagca gaatattcat ttagcctgaa agctgcagtt actataggtt
1702 gctgtcagac tatacccatg gtgcctctgg gcttgacagg tcaaaatggt
ccccatcagc 1762 ctggagcagc cctccagacc tgggtggaat tccagggttg
agagactccc ctgagccaga 1822 ggccactagg tattcttgct cccagaggct
gaagtcaccc tgggaatcac agtggtctac 1882 ctgcattcat aattccagga
tctgtgaaga gcacatatgt gtcagggcac aattccctct 1942 cataaaaacc
acacagcctg gaaattggcc ctggcccttc aagatagcct tctttagaat 2002
atgatttggc tagaaagatt cttaaatatg tggaatatga ttattcttag ctggaatatt
2062 ttctctactt cctgtctgca tgcccaaggc ttctgaagca gccaatgtcg
atgcaacaac 2122 atttgtaact ttaggtaaac tgggattatg ttgtagttta
acattttgta actgtgtgct 2182 tatagtttac aagtgagacc cgatatgtca
ttatgcatac ttatattatc ttaagcatgt 2242 gtaatgctgg atgtgtacag
tacagtacwt aacttgtaat ttgaatctag tatggtgttc 2302 tgttttcagc
tgacttggac aacctgactg gctttgcaca ggtgttccct gagttgtttg 2362
caggtttctg tgtgtggggt ggggtatggg gaggagaacc ttcatggtgg cccacctggc
2422 ctggttgtcc aagctgtgcc tcgacacatc ctcatcccaa gcatgggaca
cctcaagatg 2482 aataataatt cacaaaattt ctgtgaaatc aaatccagtt
ttaagaggag ccacttatca 2542 aagagatttt aacagtagta agaaggcaaa
gaataaacat ttgatattca gcaactgaaa 2602 aaaaaaaa 2610 4 2072 DNA
Rattus norvegicus CDS (35)...(634) 4 ctggagggga agagtgcagc
tgttcctggc agac atg aag ccc tac ttc tcg tgc 55 Met Lys Pro Tyr Phe
Ser Cys 1 5 gtc ttt gtc ttc tgc ttc cta atc aaa ctt tta aca gga gaa
ctc aat 103 Val Phe Val Phe Cys Phe Leu Ile Lys Leu Leu Thr Gly Glu
Leu Asn 10 15 20 gac ttg gcc aat cac agg atg ttt tcg ttt cac gat
gga ggt gta cag 151 Asp Leu Ala Asn His Arg Met Phe Ser Phe His Asp
Gly Gly Val Gln 25 30 35 att tct tgt aac tac cct gag act gtc cag
cag tta aaa atg cag ttg 199 Ile Ser Cys Asn Tyr Pro Glu Thr Val Gln
Gln Leu Lys Met Gln Leu 40 45 50 55 ttc aaa gac aga gaa gtc ctc tgc
gac ctc acc aag acc aag gga agc 247 Phe Lys Asp Arg Glu Val Leu Cys
Asp Leu Thr Lys Thr Lys Gly Ser 60 65 70 gga aac acc gtg tcc atc
aag aat ccg atg tcc tgt cca tat cag ctg 295 Gly Asn Thr Val Ser Ile
Lys Asn Pro Met Ser Cys Pro Tyr Gln Leu 75 80 85 tcc aac aac agt
gtc tct ttt ttc cta gac aac gca gac agc tcc cag 343 Ser Asn Asn Ser
Val Ser Phe Phe Leu Asp Asn Ala Asp Ser Ser Gln 90 95 100 ggc agc
tac ttt tta tgc agc ctg tcg att ttc gac cca ccc cct ttt 391 Gly Ser
Tyr Phe Leu Cys Ser Leu Ser Ile Phe Asp Pro Pro Pro Phe 105 110 115
caa gaa aag aac ctt agt gga gga tat ttg ctt att tat gaa tcc cag 439
Gln Glu Lys Asn Leu Ser Gly Gly Tyr Leu Leu Ile Tyr Glu Ser Gln 120
125 130 135 ctt tgt tgc cag ctg aag ctt tgg tta ccc gta ggg tgt gca
gct ttt 487 Leu Cys Cys Gln Leu Lys Leu Trp Leu Pro Val Gly Cys Ala
Ala Phe 140 145 150 gtg gca gcg ctc ctt ttt gga tgc ata ttt atc gtc
tgg ttt gca aaa 535 Val Ala Ala Leu Leu Phe Gly Cys Ile Phe Ile Val
Trp Phe Ala Lys 155 160 165 aag aag tac aga tcc agt gtg cac gac cct
aat agc gag tac atg ttc 583 Lys Lys Tyr Arg Ser Ser Val His Asp Pro
Asn Ser Glu Tyr Met Phe 170 175 180 atg gcg gca gtc aac aca aac aaa
aag tcc aga ctt gca ggt atg acc 631 Met Ala Ala Val Asn Thr Asn Lys
Lys Ser Arg Leu Ala Gly Met Thr 185 190 195 tca taatctggaa
cacgggaacc catggaggaa ctacactgtc tagttcccct 684 Ser 200 gaaacttgaa
tggagaaagt cttctatttt ctggaccaca gggcatctga cttgattaac 744
tactgatacc tccttttggk gttttgtttg tctggatcag tgactatcag tcactcggaa
804 tttcagcaga ctgccctggg tttgctgagt ccttttaagg caaacccctt
cttatagaag 864 acccggctca tatgtattca acaaacagac ctcactggga
tacaatcccc tctttctgcg 924 cctgcttcta gctatgcacc ggccagcaag
acaaacatat ctccagcatt tttacaaaaa 984 tgccagggta tgaatctgta
aagtacacag gcagccattg accaccgtct gtcctcgttt 1044 tttcagattc
tatttttttc catagagatc agcattcctt ctagaatcag acagtagagg 1104
gagatgcttc acaacagaag ctcttatgtt tctgagatgt tgatgaattc atgctttagt
1164 accaccatgt tctctaacaa cttctatatt ccagctgatc actgcttcag
ggcttagatg 1224 cctgcttttg ccttcaagtc tccccttaaa gatactccca
caggtctact tggtggcctg 1284 cagccactct gaataggaag tttggtctac
aatttccccc ctctgctgct caaaaaaaaa 1344 aattagtaga tatgattttc
ccatattctc cctgccaaag taattttttc cagcaaagac 1404 atctaaattc
agttaatatg gtttactgtg ttgatattag tggcagtaaa catttctcag 1464
aatcaaaagc aaattaattt tgcggtggtg tttttctacc attatcttgg gtttccatgg
1524 tgctattact cacaagttta gctatttttt tatgcatcat attaaagttg
caagcaagca 1584 gagcaaccct cggttaatgg gcaaacattc tcctggggta
gaatgaattg tctatttagc 1644 ccgaaaactg cagtttctgt gggtggctgc
cagactacag ccgtgctttg ctctggcttt 1704 gacaggttga aatagycccc
atgascstgg aacagwactc cagactgtgc tggagtccca 1764 aagttaggag
ggccatggag cctgggacag gctgctgctt tggtctttag gatctaggaa 1824
raattacaga ggggccaaga cagagttccc tcccctagaa actgtgcagc ctggaagtca
1884 gccctggcac tttaagatag ccttctttag aacatgagtt agttggtagt
attctgacgt 1944 gtaaacagcc tatkgttgct cggagctgga ccattttctc
cacttccctg tctgcatgcc 2004 taagacttct agagcagcca acgtatatgc
aacattaaag aaaaaaaaaa aaaaaaaaaa 2064 aaaaaaaa 2072 5 603 DNA Mus
musculus CDS (1)...(600) 5 atg aag ccg tac ttc tgc cat gtc ttt gtc
ttc tgc ttc cta atc aga 48 Met Lys Pro Tyr Phe Cys His Val Phe Val
Phe Cys Phe Leu Ile Arg 1 5 10 15 ctt tta aca gga gaa atc aat ggc
tcg gcc gat cat agg atg ttt tca 96 Leu Leu Thr Gly Glu Ile Asn Gly
Ser Ala Asp His Arg Met Phe Ser 20 25 30 ttt cac aat gga ggt gta
cag att tct tgt aaa tac cct gag act gtc 144 Phe His Asn Gly Gly Val
Gln Ile Ser Cys Lys Tyr Pro Glu Thr Val 35 40 45 cag cag tta aaa
atg cga ttg ttc aga gag aga gaa gtc ctc tgc gaa 192 Gln Gln Leu Lys
Met Arg Leu Phe Arg Glu Arg Glu Val Leu Cys Glu 50 55 60 ctc acc
aag acc aag gga agc gga aat gcg gtg tcc atc aag aat cca 240 Leu Thr
Lys Thr Lys Gly Ser Gly Asn Ala Val Ser Ile Lys Asn Pro 65 70 75 80
atg ctc tgt cta tat cat ctg tca aac aac agc gtc tct ttt ttc cta 288
Met Leu Cys Leu Tyr His Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85
90 95 aac aac cca gac agc tcc cag gga agc tat tac ttc tgc agc ctg
tcc 336 Asn Asn Pro Asp Ser Ser Gln Gly Ser Tyr Tyr Phe Cys Ser Leu
Ser 100 105 110 att ttt gac cca cct cct ttt caa gaa agg aac ctt agt
gga gga tat 384 Ile Phe Asp Pro Pro Pro Phe Gln Glu Arg Asn Leu Ser
Gly Gly Tyr 115 120 125 ttg cat att tat gaa tcc cag ctc tgc tgc cag
ctg aag ctc tgg cta 432 Leu His Ile Tyr Glu Ser Gln Leu Cys Cys Gln
Leu Lys Leu Trp Leu 130 135 140 ccc gta ggg ttg cca gct ttc gtt gtg
gta ctc ctt ttt gga tgc ata 480 Pro Val Gly Leu Pro Ala Phe Val Val
Val Leu Leu Phe Gly Cys Ile 145 150 155 160 ctt atc atc tgg ttt tca
aaa aag aaa tac gga tcc agt gtg cat gac 528 Leu Ile Ile Trp Phe Ser
Lys Lys Lys Tyr Gly Ser Ser Val His Asp 165 170 175 cct aat agt gaa
tac atg ttc atg gcg gca gtc aac aca aac aaa aag 576 Pro Asn Ser Glu
Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 tct aga
ctt gca ggt gtg acc tca taa 603 Ser Arg Leu Ala Gly Val Thr Ser 195
200 6 836 DNA Rattus norvegicus CDS (35)...(682) 6 ctggagggga
agagtgcagc tgttcctggc agac atg aag ccc tac ttc tcg tgc 55 Met Lys
Pro Tyr Phe Ser Cys 1 5 gtc ttt gtc ttc tgc ttc cta atc aaa ctt tta
aca gga gaa ctc aat 103 Val Phe Val Phe Cys Phe Leu Ile Lys Leu Leu
Thr Gly Glu Leu Asn 10 15 20 gac ttg gcc aat cac agg atg ttt tcg
ttt cac gat gga ggt gta cag 151 Asp Leu Ala Asn His Arg Met Phe Ser
Phe His Asp Gly Gly Val Gln 25 30 35 att tct tgt aac tac cct gag
act gtc cag cag tta aaa atg cag ttg 199 Ile Ser Cys Asn Tyr Pro Glu
Thr Val Gln Gln Leu Lys Met Gln Leu 40 45 50 55 ttc aaa gac aga gaa
gtc ctc tgc gac ctc acc aag acc aag gga agc 247 Phe Lys Asp Arg Glu
Val Leu Cys Asp Leu Thr Lys Thr Lys Gly Ser 60 65 70 gga aac acc
gtg tcc atc aag aat ccg atg tcc tgt cca tat cag ctg 295 Gly Asn Thr
Val Ser Ile Lys Asn Pro Met Ser Cys Pro Tyr Gln Leu 75 80 85 tcc
aac aac agt gtc tct ttt ttc cta gac aac gca gac agc tcc cag 343 Ser
Asn Asn Ser Val Ser Phe Phe Leu Asp Asn Ala Asp Ser Ser Gln 90 95
100 ggc agc tac ttt tta tgc agc ctg tcg att ttc gac cca ccc cct ttt
391 Gly Ser Tyr Phe Leu Cys Ser Leu Ser Ile Phe Asp Pro Pro Pro Phe
105 110 115 caa gaa aag aac ctt agt gga gga tat ttg ctt att tat gaa
tcc cag 439 Gln Glu Lys Asn Leu Ser Gly Gly Tyr Leu Leu Ile Tyr Glu
Ser Gln 120 125 130 135 ctt tgt tgc cag ctg aag ctt tgg tta ccc gta
ggg tgt gca gct ttt 487 Leu Cys Cys Gln Leu Lys Leu Trp Leu Pro Val
Gly Cys Ala Ala Phe 140 145 150 gtg gca gcg ctc ctt ttt gga tgc ata
ttt atc gtc tgg ttt gca aaa 535 Val Ala Ala Leu Leu Phe Gly Cys Ile
Phe Ile Val Trp Phe Ala Lys 155 160 165 aag aag tac aga tcc agt gtg
cac gac cct aat agc gag tac atg ttc 583 Lys Lys Tyr Arg Ser Ser Val
His Asp Pro Asn Ser Glu Tyr Met Phe 170 175 180 atg gcg gca gtc aac
aca aac aaa aag tcc aga ctt gca ggt aca gca 631 Met Ala Ala
Val Asn Thr Asn Lys Lys Ser Arg Leu Ala Gly Thr Ala 185 190 195 ccc
ctt agg gct ttg ggg aga gga gaa cac tct tca tgt caa gac cgg 679 Pro
Leu Arg Ala Leu Gly Arg Gly Glu His Ser Ser Cys Gln Asp Arg 200 205
210 215 aat taatttgttt atttctattt taaaagaaag acattttttc ccctaaagat
732 Asn aatttttgta tttttatgtg aaagtctgaa tcttcatttt aactcgactt
atatactctg 792 tggtatatta aaaataatgt ttgtgaaaaa aaaaaaaaaa aaaa 836
7 27 DNA Artificial Sequence primer for PCR 7 ctgctcgaga tgaagcccta
cttctcg 27 8 32 DNA Artificial Sequence primer for PCR 8 accctacggg
taacggatcc ttcagctggc aa 32 9 30 DNA Artificial Sequence primer for
PCR 9 taactgtttc tcgagaacat gaagtcaggc 30 10 30 DNA Artificial
Sequence primer for PCR 10 atcctatggg taacggatcc ttcagctggc 30 11
35 DNA Artificial Sequence primer for PCR 11 cgtgatattg ctgaagagct
tggcggcgaa tgggc 35 12 34 DNA Artificial Sequence primer for PCR 12
cattcaagtt tcagggaact agtccatgcg tttc 34 13 200 PRT Rattus
norvegicus 13 Met Lys Pro Tyr Phe Ser Cys Val Phe Val Phe Cys Phe
Leu Ile Lys 1 5 10 15 Leu Leu Thr Gly Glu Leu Asn Asp Leu Ala Asn
His Arg Met Phe Ser 20 25 30 Phe His Asp Gly Gly Val Gln Ile Ser
Cys Asn Tyr Pro Glu Thr Val 35 40 45 Gln Gln Leu Lys Met Gln Leu
Phe Lys Asp Arg Glu Val Leu Cys Asp 50 55 60 Leu Thr Lys Thr Lys
Gly Ser Gly Asn Thr Val Ser Ile Lys Asn Pro 65 70 75 80 Met Ser Cys
Pro Tyr Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90 95 Asp
Asn Ala Asp Ser Ser Gln Gly Ser Tyr Phe Leu Cys Ser Leu Ser 100 105
110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Lys Asn Leu Ser Gly Gly Tyr
115 120 125 Leu Leu Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Leu
Trp Leu 130 135 140 Pro Val Gly Cys Ala Ala Phe Val Ala Ala Leu Leu
Phe Gly Cys Ile 145 150 155 160 Phe Ile Val Trp Phe Ala Lys Lys Lys
Tyr Arg Ser Ser Val His Asp 165 170 175 Pro Asn Ser Glu Tyr Met Phe
Met Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 Ser Arg Leu Ala Gly
Met Thr Ser 195 200 14 200 PRT Mus musculus 14 Met Lys Pro Tyr Phe
Cys His Val Phe Val Phe Cys Phe Leu Ile Arg 1 5 10 15 Leu Leu Thr
Gly Glu Ile Asn Gly Ser Ala Asp His Arg Met Phe Ser 20 25 30 Phe
His Asn Gly Gly Val Gln Ile Ser Cys Lys Tyr Pro Glu Thr Val 35 40
45 Gln Gln Leu Lys Met Arg Leu Phe Arg Glu Arg Glu Val Leu Cys Glu
50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Ala Val Ser Ile Lys
Asn Pro 65 70 75 80 Met Leu Cys Leu Tyr His Leu Ser Asn Asn Ser Val
Ser Phe Phe Leu 85 90 95 Asn Asn Pro Asp Ser Ser Gln Gly Ser Tyr
Tyr Phe Cys Ser Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe Gln
Glu Arg Asn Leu Ser Gly Gly Tyr 115 120 125 Leu His Ile Tyr Glu Ser
Gln Leu Cys Cys Gln Leu Lys Leu Trp Leu 130 135 140 Pro Val Gly Leu
Pro Ala Phe Val Val Val Leu Leu Phe Gly Cys Ile 145 150 155 160 Leu
Ile Ile Trp Phe Ser Lys Lys Lys Tyr Gly Ser Ser Val His Asp 165 170
175 Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys
180 185 190 Ser Arg Leu Ala Gly Val Thr Ser 195 200 15 216 PRT
Rattus norvegicus 15 Met Lys Pro Tyr Phe Ser Cys Val Phe Val Phe
Cys Phe Leu Ile Lys 1 5 10 15 Leu Leu Thr Gly Glu Leu Asn Asp Leu
Ala Asn His Arg Met Phe Ser 20 25 30 Phe His Asp Gly Gly Val Gln
Ile Ser Cys Asn Tyr Pro Glu Thr Val 35 40 45 Gln Gln Leu Lys Met
Gln Leu Phe Lys Asp Arg Glu Val Leu Cys Asp 50 55 60 Leu Thr Lys
Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Asn Pro 65 70 75 80 Met
Ser Cys Pro Tyr Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90
95 Asp Asn Ala Asp Ser Ser Gln Gly Ser Tyr Phe Leu Cys Ser Leu Ser
100 105 110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Lys Asn Leu Ser Gly
Gly Tyr 115 120 125 Leu Leu Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu
Lys Leu Trp Leu 130 135 140 Pro Val Gly Cys Ala Ala Phe Val Ala Ala
Leu Leu Phe Gly Cys Ile 145 150 155 160 Phe Ile Val Trp Phe Ala Lys
Lys Lys Tyr Arg Ser Ser Val His Asp 165 170 175 Pro Asn Ser Glu Tyr
Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 Ser Arg Leu
Ala Gly Thr Ala Pro Leu Arg Ala Leu Gly Arg Gly Glu 195 200 205 His
Ser Ser Cys Gln Asp Arg Asn 210 215 16 200 PRT Artificial Sequence
consensus sequence 16 Met Lys Pro Tyr Phe Xaa Xaa Val Phe Val Phe
Cys Phe Leu Ile Lys 1 5 10 15 Leu Leu Thr Gly Glu Xaa Asn Xaa Xaa
Ala Asn His Arg Met Phe Ser 20 25 30 Phe His Xaa Gly Gly Val Gln
Ile Ser Cys Xaa Tyr Pro Glu Thr Val 35 40 45 Gln Gln Leu Lys Met
Gln Leu Phe Lys Xaa Arg Glu Val Leu Cys Asp 50 55 60 Leu Thr Lys
Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Asn Pro 65 70 75 80 Met
Xaa Cys Xaa Tyr Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu 85 90
95 Xaa Asn Xaa Asp Ser Ser Gln Gly Ser Tyr Xaa Xaa Cys Ser Leu Ser
100 105 110 Ile Phe Asp Pro Pro Pro Phe Gln Glu Xaa Asn Leu Ser Gly
Gly Tyr 115 120 125 Leu Xaa Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu
Lys Leu Trp Leu 130 135 140 Pro Val Gly Cys Ala Ala Phe Val Xaa Xaa
Leu Leu Phe Gly Cys Ile 145 150 155 160 Xaa Ile Xaa Trp Phe Xaa Lys
Lys Lys Tyr Xaa Ser Ser Val His Asp 165 170 175 Pro Asn Ser Glu Tyr
Met Phe Met Ala Ala Val Asn Thr Asn Lys Lys 180 185 190 Ser Arg Leu
Ala Gly Xaa Thr Xaa 195 200 17 214 PRT Artificial Sequence
consensus sequence 17 Met Leu Xaa Leu Xaa Leu Ala Trp Xaa Leu Xaa
Leu Phe Xaa Leu Xaa 1 5 10 15 Ile Xaa Val Xaa Xaa Xaa Xaa Ile Xaa
Val Xaa Gln Xaa Xaa Xaa Xaa 20 25 30 Xaa Ala Xaa Xaa Asn Gly Xaa
Xaa Xaa Xaa Xaa Cys Lys Tyr Xaa Xaa 35 40 45 Pro Xaa Xaa Xaa Xaa
Glu Phe Arg Xaa Xaa Leu Leu Lys Gly Xaa Asp 50 55 60 Ser Xaa Val
Xaa Xaa Cys Xaa Xaa Xaa Xaa Thr Tyr Xaa Xaa Gly Asn 65 70 75 80 Xaa
Val Xaa Xaa Lys Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Leu Ser Asn 85 90
95 Asn Ser Val Xaa Phe Xaa Leu Gln Asn Leu Xaa Xaa Xaa Xaa Thr Xaa
100 105 110 Xaa Tyr Phe Cys Lys Xaa Glu Xaa Met Tyr Pro Pro Pro Tyr
Xaa Xaa 115 120 125 Xaa Xaa Xaa Asn Gly Thr Xaa Ile His Val Xaa Xaa
Xaa Xaa Leu Cys 130 135 140 Pro Xaa Xaa Xaa Phe Xaa Xaa Trp Xaa Leu
Xaa Xaa Val Xaa Xaa Xaa 145 150 155 160 Leu Xaa Xaa Tyr Ser Xaa Leu
Xaa Thr Ala Xaa Ile Xaa Xaa Xaa Xaa 165 170 175 Xaa Lys Lys Arg Ser
Xaa Leu Xaa Xaa Gly Xaa Tyr Met Xaa Met Xaa 180 185 190 Pro Xaa Xaa
Pro Xaa Xaa Xaa Xaa Lys Xaa Xaa Gln Pro Tyr Xaa Xaa 195 200 205 Asp
Phe Xaa Xaa Xaa Xaa 210 18 6 PRT Homo sapiens 18 Met Tyr Pro Pro
Pro Tyr 1 5 19 4 PRT Homo sapiens 19 Tyr Met Asn Met 1 20 4 PRT
Homo sapiens 20 Tyr Val Lys Met 1 21 6 PRT Homo sapiens 21 Phe Asp
Pro Pro Pro Phe 1 5 22 4 PRT Homo sapiens 22 Tyr Met Phe Met 1 23
216 PRT Artificial Sequence consensus sequence 23 Met Lys Pro Tyr
Phe Ser Cys Val Phe Val Phe Cys Phe Leu Ile Lys 1 5 10 15 Leu Leu
Thr Gly Glu Leu Asn Asp Leu Ala Asn His Arg Met Phe Ser 20 25 30
Phe His Asp Gly Gly Val Gln Ile Ser Cys Asn Tyr Pro Glu Thr Val 35
40 45 Gln Gln Leu Lys Met Gln Leu Phe Lys Asp Arg Glu Val Leu Cys
Asp 50 55 60 Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile
Lys Asn Pro 65 70 75 80 Met Ser Cys Pro Tyr Gln Leu Ser Asn Asn Ser
Val Ser Phe Phe Leu 85 90 95 Asp Asn Ala Asp Ser Ser Gln Gly Ser
Tyr Phe Leu Cys Ser Leu Ser 100 105 110 Ile Phe Asp Pro Pro Pro Phe
Gln Glu Lys Asn Leu Ser Gly Gly Tyr 115 120 125 Leu Leu Ile Tyr Glu
Ser Gln Leu Cys Cys Gln Leu Lys Leu Trp Leu 130 135 140 Pro Val Gly
Cys Ala Ala Phe Val Ala Ala Leu Leu Phe Gly Cys Ile 145 150 155 160
Phe Ile Val Trp Phe Ala Lys Lys Lys Tyr Arg Ser Ser Val His Asp 165
170 175 Pro Asn Ser Glu Tyr Met Phe Met Ala Ala Val Asn Thr Asn Lys
Lys 180 185 190 Ser Arg Leu Ala Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 24
16 PRT Rattus norvegicus 24 Leu Arg Ala Leu Gly Arg Gly Glu His Ser
Ser Cys Gln Asp Arg Asn 1 5 10 15 25 220 PRT Homo sapiens 25 Met
Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val 1 5 10
15 Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr
20 25 30 Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu
Phe Ser 35 40 45 Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp
Ser Ala Val Glu 50 55 60 Val Cys Val Val Tyr Gly Asn Tyr Ser Gln
Gln Leu Gln Val Tyr Ser 65 70 75 80 Lys Thr Gly Phe Asn Cys Asp Gly
Lys Leu Gly Asn Glu Ser Val Thr 85 90 95 Phe Tyr Leu Gln Asn Leu
Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys 100 105 110 Lys Ile Glu Val
Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 115 120 125 Asn Gly
Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 130 135 140
Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly 145
150 155 160 Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe
Ile Ile 165 170 175 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
Ser Asp Tyr Met 180 185 190 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
Arg Lys His Tyr Gln Pro 195 200 205 Tyr Ala Pro Pro Arg Asp Phe Ala
Ala Tyr Arg Ser 210 215 220 26 223 PRT Homo sapiens 26 Met Ala Cys
Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala 1 5 10 15 Ala
Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro 20 25
30 Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala
35 40 45 Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser
Pro Gly 50 55 60 Lys Ala Tyr Glu Val Arg Val Thr Val Leu Arg Gln
Ala Asp Ser Gln 65 70 75 80 Val Thr Glu Val Cys Ala Ala Thr Tyr Met
Thr Gly Asn Glu Leu Thr 85 90 95 Phe Leu Asp Asp Ser Ile Cys Thr
Gly Thr Ser Ser Gly Asn Gln Val 100 105 110 Asn Leu Thr Ile Gln Gly
Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile 115 120 125 Cys Lys Val Glu
Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly 130 135 140 Asn Gly
Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser 145 150 155
160 Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe
165 170 175 Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu
Lys Lys 180 185 190 Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met
Pro Pro Thr Glu 195 200 205 Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr
Phe Ile Pro Ile Asn 210 215 220
* * * * *
References