Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
T~f I~iNTGAL FTET,D
The present invention relates to human proteins having
transmembrane domains and cDNAs coding for these proteins as well
IO as eucaryotic cells expressing said cDNAs. The proteins of the
present invention can be employed as pharmaceuticals or as
antigensfor preparing antibodiesagainstsaid proteins. Thehuman
cDNAs of the present invention can be utilized as probes for the
gene diagnosis and gene sources for the gene therapy. Furthermore,
the cDNAs can be utilized as gene sources for large-scale
production of the proteins encoded by said cDNAs . Cells, wherein
these membrane protein genes are introduced and membrane proteins
are expressed in large amounts, can be utilized for detection of
the corresponding ligands, screening of novel low-molecular
pharmaceuticals, and so on.
BACKGROUND RT
Membraneproteinsplayimportant roles, assignal receptors,
ion channels, transporters, etc. in the material transportation
and the information transmission which are mediated by the cell
membrane. Examples thereof include receptors for a variety of
cytokines, ion channels for the sodium ion, the potassium ion,
the chloride ion, etc., transporters for saccharides and amino
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acids, and so on, where the genes of many of them have been cloned
already.
It has been clarified that abnormalities of these membrane
proteins are associated with a number of hitherto-cryptogenic
diseases. For instance, a gene of a membrane protein having twelve
transmembrane domains was identified as the gene responsible for
cystic fibrosis [Rommens, J. M. et al., Science 245: 1059-1065
(1989) ] . In addition, it has been clarified that several membrane
proteins act as receptors when a virus infects the cells. For
instance, HIV-1 is revealed to infect into the cells through
mediation of a membrane protein fusin having a membrane protein
on the T-cell membrane, a CD-4 antigen, and 7 transmembrane domains
[Feng, Y. et al., Science 272: 872-877 (1996)]. Therefore,
discovery of a new membrane protein is anticipated to lead to
elucidation of the causes of many diseases, so that isolation of
a new gene coding for the membrane protein has been desired.
Heretofore, owing to difficulty in the purification, many
membrane proteins have been isolated by an approach from the gene
side. A general method is the so-called expression cloning which
comprises transfection of a cDNA library in eucaryotie cells to
express cDNAs and then detection of the cells expressing the target
membrane protein on the membrane by an immunological technique
using an antibody or a physiological technique on the change in
the membrane permeability. However, this method is applicable only
to cloning of a gene of a membrane protein with a known function.
In general, membrane proteins possess hydrophobic
transmembrane domains inside the proteins, wherein, after
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synthesis thereof in the ribosome, these domains remain in the
phospholipid membrane to be trapped in the membrane. Accordingly,
the evidence of the cDNA for encoding the membrane protein is
provided by determination of the whole base sequence of a
lull-length cDNA followed by detection of highly hydrophobic
transmembrane domains in the amino acid sequence of the protein
encoded by said cDNA.
DTS .OS 1R . OF TN~J .NTTC)N
The object of the present invention is to provide novel
human proteins having transmembrane domains and DNAs coding for
said proteins as well as transformation eucaryotie cells that are
capable of expressing said cDNAs.
As the result of intensive studies, the present inventors
have been successful in cloning of cDNAs coding for proteins having
transmembrane domains from the human full-length cDNA bank,
thereby completing the present invention. In other words, the
present invention provides human proteins having transmembrane
domains, namely proteins containing any of the amino acid
sequences represented by Sequence Nos. 1 to 6. Moreover, the
present invention provides DNAs coding for the above-mentioned
proteins, exemplified by cDNAs containing any of the base
sequences represented by Sequence Nos. 7 to 12, as well as
transformation eucaryotie cells that are capable of expressing
said cDNAs.
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BRT_EF D . RT_ TT(~N CAF ]] AWT~j
Figure l: A figure depicting the
hydrophobicity/hydrophilicity profile of the protein encoded by
clone HP00956.
Figure 2: A figure depicting the
hydrophobicity/hydrophilicity profile of the protein encoded by
clone HP01535.
Figure 3: A figure depicting the
hydrophobicity/hydrophilicity profile of the protein encoded by
clone HP10089.
Figure 4: A figure depicting the
hydrophobicity/hydrophilicity profile of the protein encoded by
clone HP10216.
Figure 5: A figure depicting the
hydrophobicity/hydrophilicity profile of the protein encoded by
clone HP10420.
Figure 6: A figure depicting the
hydrophobicity/hydrophilicity profile of the protein encoded by
clone HP10441.
BEST MODE FOR RRvTN, O T H TNVFNTTnN
The proteins of the present invention can be obtained, for
example, by a method for isolation from human organs, cell lines,
etc., a method for preparation of peptides by the chemical
synthesis, or a method for production with the recombinant DNA
technology using the DNAs coding for the transmembrane domains
of the present invention, wherein the method for obtainment by
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the recombinant DNA technology is employed preferably. For
instance, in vitro expression of the proteins can be achieved by
preparation of an RNA by in vitro transcription from a vector having
one of cDNAs of the present invention, followed by in vitro
5 translation using this RNA as a template. Also, recombination of
the translation region into a suitable expression vector by the
method known in the art leads to production of a large amount of
the encoded protein by using prokaryotic cells such as Escherichia
coli, Bacillus subtilis, etc., and eucaryotic cells such as yeasts,
insect cells, mammalian cells, etc.
In the case in which a protein of the present invention
is produced by a microorganism such as Escherichia coli etc., a
recombinant expression vector bearing the translation region in
the cDNA of the present invention is constructed in an expression
vector having an origin, a promoter, a ribosome-binding site, a
cDNA-cloning site, a terminator etc., which can be replicated in
the microorganism, and, after transformation of the host cells
with said expression vector, the thus-obtained transformant is
incubated, whereby the protein encoded by said cDNA can be produced
on a large scale in the microorganism. In this case, a protein
fragment containing an optional region can be obtained by carrying
out the expression with inserting an initiation codon and a
termination codon in front of and behind an optional translation.
region. Alternatively, a fusion protein with another protein can
be expressed. Only a protein portion coding for said cDNA can be
obtained by cleavage of said fusion protein with a suitable
protease.
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In the case in which one of the proteins of the present
invention is produced in eucaryotie cells, the protein of the
present invention can be produced as a transmembrane protein on
the cell-membrane surface, when the translation region of said
cDNA is subjected to recombination to an expression vector for
eucaryotie cells that has a promoter, a splicing region, a poly (Ay
insertion site, etc., followed byintroductioninto theeucaryotic
cells. The expression vector is exemplified by pKAl, pED6dp2,
pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2,
and so on. Examples of eucaryotie cells to be used in general
include mammalian culture cells such as simian kidney cells COS7,
Chinese hamster ovary cells CHO, etc., budding yeasts, fission
yeasts, silkworm cells, Xenopus laevis egg cells, and so on, but
any eucaryotie cells may be used, provided that they are capable
of expressing the present proteins on the membrane surface. The
expression vector can be introduced in the eucaryotie cells by
methods known in the art such as the electroporation method, the
potassium phosphate method, the liposome method, the DEAF-dextran
method, and so on.
After one of the proteins of the present invention is
expressed in prokaryotic cells or eucaryotie cells, the objective
protein can be isolated from the culture and purified by a
combination of separation procedures known in the art. Such
examples include treatment with a denaturing agent such as urea
or a surface-active agent, sonication, enzymatic digestion,
salting-out or solvent precipitation, dialysis, centrifugation,
ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing,
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ion-exchange chromatography, hydrophobic chromatography,
affinity chromatography, reverse phase chromatography, and so on.
The proteins of the present invention include peptide
fragments (more than S amino acid residues) containing any partial
amino acid sequence in the amino acid sequences represented by
Sequence Nos. 1. to 6. These peptide fragments can be utilized
as antigens for preparation of antibodies. Hereupon, among the
proteins of the present invention, those having the signal
sequence are secreted in the form of maturation proteins on the
surface of the cells, after the signal sequences are removed.
Therefore, these maturation proteins shall come within the scope
of the present invention. The N-terminal amino acid sequences of
the maturation proteins can be easily identified by using the
method for the cleavage-site determination in a signal sequence
Japanese Patent Kokai Publication No. 1996-187100]. Furthermore,
some membrane proteins undergo the processing on the cell surface
~o be converted to the secretory forms . Such proteins or peptides
in the secretory forms shall come within the scope of the present
invention. When sugar chain-binding sites are present in the amino
acid sequences, expression in appropriate eucaryotic cells
affords proteins wherein sugar chains are added. Accordingly, such
proteins or peptides wherein sugar chains are added shall come
within the scope of the present invention.
The DNAs of the present invention include all DNAs coding
for the above-mentioned proteins. Said DNAs can be obtained by
using a method by chemical synthesis, a method by cDNA cloning,
and so on.
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The cDNAs of the present invention can be cloned, for
example, from cDNA libraries of the human cell origin. These cDNA
are synthesized by using as templates poly(A)' RNAs extracted from
human cells. The human cells may be cells delivered from the human
body, for example, by the operation or may be the culture cells .
The cDNAs can be synthesized by using any method selected from
the Okayama-Berg method [Okayama, H, and Berg, P. , Mol . Cell . Biol .
2: 161-170 (1982)], the Gubler-Hoffman method [Gubler, U. and
Hoffman, J. Gene 25: 263-269 (1983) ], and so on, but it is preferred
to use the capping method [Kato, S. et al., Gene 150: 243-250
(1994)], as exemplified in Examples, in order to obtain a
full-length clone in an effective manner.
The primary selection of one of the cDNAs coding for the
human proteins having transmembrane domains is carried out by
sequencing of a partial base sequence of a cDNA clone selected
at random from cDNA libraries, sequencing of the amino acid
sequence encoded by the base sequence, and recognition of the
presence or absence of a hydrophobic site in the resulting N-
terminal amino acidsequenceregion.Next, thesecondaryselection
is carried out by determination of the whole sequence by the
sequencing and the protein expression by in vitro translation.
Ascertainment of cDNAs of the present invention for encoding the
proteins having secretory signal sequences is carried out by using
the signal sequence detection method [Yokoyama-Kobayashi, M. et
al . , Gene 163 : 193-196 ( 1995 ) ] . In other words, the ascertainment
for a coding portion of an inserted cDNA fragment to function as
a signal sequence is provided by fusing a cDNA fragment coding
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for the N-terminus of the target protein with a cDNA coding for
the protease domain of urokinase and then expressing the resulting
cDNA in COS7 cells to detect the urokinase activity in the cell
culture medium. On the other hand, in the case in which the
urokinase activity is not detectable in the cell medium, the
N-terminal region is judged to remain in the membrane.
The cDNAs of the present invention are characterized by
containing either of the base sequences represented by Sequence
Nos. 7 to 12 or the base sequences represented by Sequence Nos.
13, 15, 17, 19, 21 and 23. Table 1 summarizes the clone number
(HP number) , the cells affording the cDNA, the total base number
of the cDNA, and the number of the amino acid residues of the encoded
protein, for each of the cDNAs.
Table 1
Sequene No. HP No. - Cell Number Number of
of
bases amino acids
1, 7, 1 3 HP00956 l--2 OS 867 168
2, 8, 1 4 H P 0 1 5 Stomach cancer7 2 0 1 6 4
3 5
3, 9, 1 5 HP10089 Liver 566 141
4, 1 0, 1 6 H P 1 0 2 HT-1080 1 0 7 1 4 2
1 6 8
5, 1 1 1 7 H P 1 0 4 Stomach cancer1 3 1 3 4 6
, 2 0 0
6 , 1 2 1 8 H P 1 0 4 Stomach cancer7 8 1 6 6
, 4 1
Hereupon, the same clones as the cDNAs of the presen~
invention can be easily obtained by screening of the cDNA libraries
constructed from the human cell lines and human tissues utilized
in the present invention by the use of an oligonucleotide probe
synthesized on the basis of the cDNA base sequence described in
any of Sequence Nos. 7, to 12, 13, 15, 17, 19, 21 and 23.
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In general, the polymorphism due to the individual
difference is frequently observed in human genes. Accordingly,
any cDNA that is subj ected to insertion or deletion of one or plural
nucleotides and/or substitution with other nucleotides in
5 Sequence Nos . 7 to 12, 13, 15, 17, 19, 21 and 23 shall come within
the scope of the present invention.
In a similar manner, any protein that is formed by these
modifications comprising insertion or deletion of one or plural
amino acids and/or substitution with other amino acids shall come
10 within the scope of the present invention, as far as the protein
possesses the activity of any protein having the amino acid
sequences represented by Sequence Nos. 1 to 6.
The cDNAs of the present invention include cDNA fragments
(more than 10 bp) containing any partial base sequence in the base
sequences represented by Sequence Nos. 7 to 12 or in the base
sequences represented by Sequence Nos . 13, 15, 17, 19, 21 and 23.
Also, DNA fragments consisting of a sense chain and an anti-sense
chain shall come within this scope. These DNA fragments can be
utilized as the probes for the gene diagnosis.
In addition to the activities and uses described above;
the polynucleotides and proteins of the present invention may
exhibit one or more of the uses or biological activities (including
those associated with assayscited herein) identified below. Uses
or activities described for proteins of the present invention may
be provided by administration or use of such proteins or by
administration or use of polynucleotides encoding such proteins
(such as, for example, in gene therapies or vectors suitable for
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introduction of DNA).
Research Uses and 1 i 1 i i a
The polynucleotides provided by the present invention can
be used by the research community for various purposes. The
polynucleotides can be used to express recombinant protein for
analysis, characterization or therapeutic use; as markers for
tissues in which the corresponding protein is preferentially
expressed (either constitutively or at a particular stage of
tissue differentiation or development or in disease states); as
molecular weight markers on Southern gels; as chromosome markers
or tags (when labeled) to identify chromosomes or to map related
gene positions; to compare with endogenous DNA sequences in
patients to identify potential genetic disorders; as probes to
hybridize and thus discover novel, related DNA sequences; as a
source of information to derive PCR primers for genetic
fingerprinting; as a probe to "subtract-out" known sequences in
the process of discovering other novel polynucleotides; for
selecting and making oligomers for attachment to a "gene chip"
or other support, including for examination of expression
patterns; to raise anti-protein antibodiesusing DNA immunization
techniques; and as an antigen to raise anti-DNA antibodies or
elicit another immune response. Where the polynucleotide encodes
a protein which binds or potentially binds to another protein (such
as, ror example, in a receptor-ligand interaction), the
polynucleotide can also be used in interaction trap assays ( such
as, for example, that described in Gyuris et al., Cell 75:791-803
(1993)) to identify polynucleotides encoding the other protein
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with which binding occurs or to identify inhibitors of the binding
interaction.
The proteins provided by the present invention can
similarly be used in assay to determine biological activity,
including in a panel of multiple proteins for high-throughput
screening; to raise antibodies or to elicit another immune
response; as a reagent ( including the labeled reagent ) in assays
designed to quantitatively determine levels of the protein (or
its receptor) in biological fluids; as markers for tissues in which
the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state) ; and, of course, to isolate
correlative receptors or ligands. Where the protein binds or
potentially binds to another protein (such as, for example, in
a receptor-ligand interaction), the protein can be used to
identify the other protein with which binding occurs or to identify
inhibitors of the bindinginteraction. Proteins involved inthese
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization
as research products.
Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include without limitation "Molecular Cloning: A Laboratory
Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in
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Enzymology: Guide to Molecular Cloning Techniques", Academic
Press, Berger, S.L. and A.R. Kimmel eds., 1987.
jQu r i i ona 1 1~
Polynucleotides and proteins of the present invention can
also be used as nutritional sources or supplements. Such uses
include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and
use as a source of carbohydrate. In such cases the protein or
polynucleotide of the invention can be added to the feed of a
particular organism or can be administered as a separate solid
or liquid preparation, such as in the form of powder, pills,
solutions, suspensions or capsules. In the case of microorganisms,
the protein or polynucleotide of the invention can be added to
the medium in or on which the microorganism is cultured.
CytOki ne arid 1 1 rol i farad nn/T1i ffPrani-i ati nn Anti ~r; tw
A protein of the present invention may exhibit cytokine,
cell proliferation (either inducing or inhibiting) or cell
differentiation (either inducing or inhibiting) activity or may
induce production of other cytokines in certain cell populations.
Many protein factors discovered to date, including all known
cytokines, have exhibited activity in one or more factor dependent
cell proliferation assays, and hence the assays serve as a
convenient confirmation of cytokine activity. The activity of
a protein of the present invention is evidenced by any one of a
number of routine factor dependent cell proliferation assays for
cell lines including, without limitation, 32D, DA2, DA1G, T10,
B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RBS, DAl, 123, T1165,
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HT2, CTLL2, TF-l, Mo7e and CMK.
The activity of a protein of the invention may, among other
means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include
without limitation those described in: Current Protocols in
Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse
Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in
Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al . , J. Immunol . 145: 1706-1712, 1990; Bertagnolli
et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et
al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol.
152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of
spleen cells, lymph node cells or thymocytes include, without
limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.19, John
Wiley and Sons, Toronto. 1994; and Measurement of mouse and human
Interferon y, Schreiber, R. D. In Current Protocols in Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-5.8.8, John Wiley and Sons,
Toronto. 1994.
Assays for proliferation and differentiation of
hematopoietic andlymphopoietic cells include, without limitation,
those described in: Measurement of Human and Murine Interleukin
2 and Interleukin 9, Bottomly, K., Davis, L.S, and Lipsky, P.E.
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In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1
pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et
al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
5 L'.S.A. 80:2931-2938, 1983; Measurement of mouse and human
interleukin 6-Nordan, R. In Current Protocols in Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A.
83: 1857-1861, 1986; Measurement of human Interleukin 11 - Bennett,
10 ~., Giannotti, J., Clark, S.C. and Turner, K. J. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1
John Wiley and Sons, Toronto. 1991; Measurement of mouse and human
Interleukin 9 - Ciarletta, A., Giannotti, J., Clark,S.C. and
Turner, K. J. In Current Protocols in Immunology. J.E.e.a. Coligan
15 eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will
identify, among others, proteins that affect APC-T cell
interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include, without
limitation, those described in: Current Protocols in Immunology,
Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach,
W Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse
Lymphocyte Function; Chapter 6, Cytokines and their cellular
receptors; Chapter 7, Immunologic studies in Humans) ; Weinberger
et al . , Proc. Natl . Acad. Sci . USA 77 : 6091-6095, 1980; Weinberger
et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol.
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137 : 3494-3500, 1986; Takai et al . , J. Immunol . 140: 508-512, 1988 .
Immune ' mn l a i g~~ or SLnx~rP~ ~ i nd" Activi tutu
A protein of the present invention may also exhibit immune
stimulating or immune suppressing activity, including without
limitation the activities for which assays are described herein.
A protein may be useful in the treatment of various immune
deficiencies and disorders (including severe combined
immunodeficiency (SCID) ) , e. g. , in regulating (up or down) growth
and proliferation of T and/or B lymphocytes, as well as effecting
the cytolytic activity of NK cells and other cell populations.
These immune deficiencies may be genetic or be caused by viral
(e. g. , HIV) as well as bacterial orfungal infections, or may result
from autoimmune disorders. More specifically, infectious
diseases causes by viral, bacterial, fungal or other infection
may be treatable using a protein of the present invention,
including infections by HIV, hepatitis viruses, herpesviruses,
mycobacteria, Leishmania spp., malaria spp. and various fungal
infections such as candidiasis. Of course, in this regard, a
protein of the present invention may also be useful where a boost
to the immune system generally may be desirable, i.e., in the
treatment of cancer.
Autoimmune disorders which may be treated using a protein
of the present invention include, for example, connective tissue
disease, multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin
dependent diabetes mellitis, myasthenia gravis, graft-versus-
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host disease and autoimmune inflammatory eye disease. Such a
protein of the present invention may also to be useful in the
treatment of allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems.
Other conditions, in which immune suppression is desired
(including, for example, organ transplantation), may also be
treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible
to immune responses, in a number of ways. Down regulation may
be in the form of inhibiting or blocking an immune response already
in progress or may involve preventing the induction of an immune
response. The functions of activated T cells may be inhibited
by suppressing T cell responses or by inducing specific tolerance
in T cells, or both. Immunosuppression of T cell responses is
generally an active, non-antigen-specific, process which requires
continuous exposure of the T cells to the suppressive agent.
Tolerance, which involves inducing non-responsiveness or anergy
in T cells, is distinguishable from immunosuppression in that it
is generally antigen-specific and persists after exposure to the
tolerizing agent has ceased. Operationally, tolerance can be
demonstrated by the lack of a T cell response upon reexposure to
specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen functions
(such as , for example, B7)), e.g., preventing high level
lymphokine synthesis by activated T cells, will be useful in
situations of tissue, skin and organ transplantation and in
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graft-versus-host disease (GVHD) . For example, blockage of T cell
function should result in reduced tissue destruction in tissue
transplantation. Typically, in tissue transplants, rejection of
the transplant is initiated through its recognition as foreign
by T cells, followed by an immune reaction that destroys the
transplant. The administration of a molecule which inhibits or
blocks interaction of a B7 lymphocyte antigen with its natural
ligand(s) on immune cells (such as a soluble, monomeric form of
a peptide having B7-2 activity alone or in conjunction with a
monomeric form of a peptide having an activity of another B
lymphocyte antigen (e. g., B7-l, B7-3) or blocking antibody), prior
to transplantation can lead to the binding of the molecule to the
natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte
antigen function in this matter prevents cytokine synthesis by
immune cells, such as T cells, and thus acts as an immunosuppressant.
Moreover, the lack of costimulation may also be sufficient to
anergize the T cells, thereby inducing tolerance in a subject.
Induction of long-term tolerance by B lymphocyte antigen-blocking
reagents may avoid the necessity of repeated administration of
these blocking reagents. To achieve sufficient
immunosuppression or tolerance in a subject, it may also be
necessary to block the function of a combination of B lymphocyte
antigens.
The efficacy of particular blocking reagents in preventing
organ transplant rejection or GVHD can be assessed using animal
models that are predictive of efficacy in humans. Examples of
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appropriate systems which can be used include aliogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice,
both of which have been used to examine the immunosuppressive
effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992) . In addition, murine models
of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New
York, 1989, pp. 846-847) can be used to determine the effect of
blocking B lymphocyte antigen function in vivo on the development
of that disease.
Blocking antigen function may also be therapeutically
useful for treating autoimmune diseases. Many autoimmune
disorders are the result of inappropriate activation of T cells
that are reactive against self tissue and which promote the
production of cytokines and autoantibodies involved in the
pathology of the diseases. Preventing the activation cf
autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells
by disrupting receptor:ligand interactions of B lymphocyte
antigens can be used to inhibit T cell activation and prevent
production of autoantibodies or T cell-derived cytokines which
may be involved in the disease process. Additionally, blocking
reagents may induce antigen-specific tolerance of autoreactive
T cells which could lead to long-term relief from the disease.
The efficacy of blocking reagents in preventing or alleviating
autoimmune disorders can be determined using a number of
well-characterized animal models of human autoimmune diseases.
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2~ 0
Examples include murine experimental autoimmune encephalitis,
systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD
mice and BB rats, and murine experimental myasthenia gravis (see
Paul ed., Fundamental Immunology, Raven Press, New York, 1989,
pp. 840-856).
Upregulation of an antigen function (preferably a B
lymphocyte antigen function) , as a means of up regulating immune
responses, may also be useful in therapy. Upregulation of immune
responses may be in the form of enhancing an existing immune
response or eliciting an initial immune response. For example,
enhancing an immune response through stimulating B lymphocyte
antigen function may be useful in cases of viral infection. In
addition, systemic viral diseases such as influenza, the
commoncold, and encephalitis might be alleviated by the
administration of stimulatory forms of B lymphocyte
antigens systemically.
Alternatively, anti-viral immune responses may be enhanced
in an infected patient by removing T cells from the patient,
costimulating the T cells in vitro with viral antigen-pulsed APCs
either expressing a peptide of the present invention or together
with a stimulatory form of a soluble peptide of the present
invention and reintroducing the in vitro activated T cells into
the patient. Another method of enhancing anti-viral immune
responses would be to isolate infected cells from a patient,
transfect them with a nucleic acid encoding a protein of the present
invention as described herein such that the cells express all or
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a portion of the protein on their surface, and reintroduce the
transfected cells into the patient. The infected cells would now
be capable of delivering a costimulatory signal to, and thereby
activate, T cells in vivo.
In another application, up regulation or enhancement of
antigen function (preferably B lymphocyte antigen function) may
be useful in the induction of tumor immunity. Tumor cells (e.g.,
sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma)
transfected with a nucleic acid encoding at least one peptide of
the present invention can be administered to a subj ect to overcome
tumor-specific tolerance in the subject. If desired, the tumor
cell can be transfected to express a combination of peptides. For
example, tumor cells obtained from a patient can be transfected
ex vivo with an expression vector directing the expression of a
peptide having B7-2-like activity alone, or in conjunction with
a peptide having B7-1-like activity and/or B7-3-like activity.
The transfected tumor cells are returned to the patient to result
in expression of the peptides on the surface of the transfected
cell. Alternatively, gene therapy techniques can be used to
target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having
the activity of a B lymphocyte antigens) on the surface of the
tumor cell provides the necessary costimulation signal to T cells
to induce a T cell mediated immune response against the transfected
tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II molecules, or which fail to reexpress sufficient
amounts of MHC class I or MHC class II molecules, can be transfected
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with nucleic acid encoding all or a portion of (e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain
protein and (3z microglobulin protein or an MHC class IIa chain
protein and an MHC class II(3 chain protein to thereby express MHC
class I or MHC class II proteins on the cell surface. Expression
of the appropriate class I or class II MHC in conjunction with
a peptide having the activity of a B lymphocyte antigen (e. g.,
B7-l, B7-2, B7-3) induces a T cell mediated immune response against
the transfected tumor cell. Optionally, a gene encoding an
antisense construct which blocks expression of an MHC class II
associated protein, such as the invariant chain, can also be
cotransfected with a DNA encoding a peptide having the activity
of a B lymphocyte antigen to promote presentation of tumor
associated antigens and induce tumor specific immunity. Thus,
the induction of a T cell mediated immune response in a human
subject may be sufficient to overcome tumor-specific tolerance
in the subject.
The activity of a protein of the invention may, among other
means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity
include, without limitation, those described in: Current
Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic
studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
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1982; Handa et al. , J. Immunol . 135: 1564-1572, 1985; Takai et al . ,
J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology
61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al . , Cellular Immunology 133: 327-341, 1991; Brown
et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and
isotype switching (which will identify, among others, proteins
that modulate T-cell dependent antibody responses and that affect
Thl/Th2 profiles) include, without limitation, those described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for
B cell function: In vitro antibody production, Mond, J.J. and
Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan
eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify,
among others, proteins that generate predominantly Thl and CTL
responses) include, without limitation, those described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene
Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro
assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,
Immunologic studies in Humans); Takai et al., J. Immunol.
137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
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Dendritic cell-dependent assays (which will identify,
among others, proteins expressed by dendritic cells that activate
naive T-cells) include, without limitation, those described in:
Guery et al . , J. Immunol . 134 : 536-544, 1995; Inaba et al . , Journal
of Experimental Medicine 173:549-559, 1991; Macatonia et al.,
Journal of Immunology 154:5071-5079, 1995; Porgador et al.,
Journal of Experimental Medicine 182:255-260, 1995; Nair et al.,
Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965, 1994; Macatonia et al., Journal of Experimental
Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-B07, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will
identify, among others, proteins that prevent apoptosis after
superantigen induction and proteins that regulate lymphocyte
homeostasis) include, without limitation, those described in:
Darzynkiewiczet al., Cytometryl3:795-808, 1992; Gorczyca etal.,
Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research
53:1995-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of
Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA
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88:7548-7551, 1991.
H -m ot~o,'_ i R _,c,~ul ati nq Acti vi t«
A protein of the present invention may be useful in
regulation of hematopoiesis and, consequently, in the treatment
5 of myeloid or lymphoid cell deficiencies. Even marginal
biological activity in support of colony forming cells or of
factor-dependent cell lines indicates involvement in regulating
hematopoiesis, e.g. in supporting the growth and proliferation
of erythroid progenitor cells alone or in combination with other
10 cytokines, thereby indicating utility, for example, in treating
various anemias or for use in conjunction with
irradiation/chemotherapy to stimulate the production of erythroid
precursors and/or erythroid cells; in supporting the growth and
proliferation of myeloid cells such as granulocytes and
15 monocytes/macrophages (i.e., traditional CSF activity) useful,
for example, in conjunction with chemotherapy to prevent or treat
consequent myelo-suppression; in supporting the growth and
proliferation of megakaryocytes and consequently of platelets
thereby allowing prevention or treatment of various platelet
20 disorders such as thrombocytopenia, and generally for use in place
of orcomplimentary to platelet transfusions; and/orinsupporting
the growth and proliferation of hematopoietic stem cells which
are capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
25 various stem cell disorders (such as those usually treated with
transplantation, including, without limitation, aplastic anemia
and paroxysmal nocturnal hemoglobinuria), as well as in
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repopulating the stem cell compartment post
irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in
conjunction with bone marrow transplantation or with peripheral
progenitor cell transplantation (homologous or heterologous)) as
normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other
means, be measured by the following methods:
Suitable assays for proliferation and differentiation of
various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will
identify, among others, proteins that influence embryonic
differentiation hematopoiesis) include, without limitation,
those described in: Johansson et al. Cellular Biology 15:141-
151, 1995; Keller et al., Molecular and Cellular Biology
13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which
will identify, among others, proteins that regulate lympho
hematopoiesis) include, without limitation, those described in:
Methylcellulose colony forming assays, Freshney, M.G. In Culture
of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 265-268,
Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl.
Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony
forming cells with high proliferative potential, McNiece, I.K.
and Briddell, R.A. In Culture of Hematopoietic Cells. R.I.
Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York,
NY. 1994; Neben et al . , Experimental Hematology 22 : 353-359, 1994;
Cobblestone area forming cell assay, Ploemacher, R.E. In Culture
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of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21,
Wiley-Liss, Inc., New York, NY. 1994; Long term bone marrow
cultures in the presence of stromal cells, Spooncer, E., Dexter,
M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney,
et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994;
Long term culture initiating cell assay, Sutherland, H.J. In
Culture of Hematopoietic Cells. R.I. Freshney, et al, eds. Vol
pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
A protein of the present invention also may have utility
in compositions used for bone, cartilage, tendon, ligament and/or
nerve tissue growth or regeneration, as well as for wound healing
and tissue repair and replacement, and in the treatment of burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage
and/or bone growth in circumstances where bone is not normally
formed, has application in the healing of bone fractures and
cartilage damage or defects in humans and other animals. Such
a preparation employing a protein of the invention may have
prophylactic use in closed as well as open fracture reduction and
also in the improved fixation of artificial joints. De novo bone
formation induced by an osteogenic agent contributes to the repair
of congenital, trauma induced, or oncologic resection induced
craniofacial defects, and also is useful in cosmetic plastic
surgery.
A protein of this invention may also be used in the treatment
of periodontal disease, and in other tooth repair processes . Such
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agents may provide an environment to attract bone-forming cells,
stimulate growth of bone-forming cells or induce differentiation
of progenitors of bone-forming cells . A protein of the invention
may also be useful in the treatment of osteoporosis or
osteoarthritis, such as through stimulation of bone and/or
cartilage repair or by blocking inflammation or processes of
tissue destruction (collagenase activity, osteoclast activity,
etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may
be attributable to the protein of the present invention is
tendon/ligament formation. A protein of the present invention,
which induces tendon/ligament-like tissue or other tissue
formation in circumstances where such tissue is not normally
formed, has application in the healing of tendon or ligament tears,
deformities and other tendon or ligament defects in humans and
other animals. Such a preparation employing a
tendon/ligament-like tissue inducing protein may have
prophylactic use in preventing damage to tendon or ligament tissue,
as well as use in the improved fixation of tendon or ligament to
bone or other tissues, and in repairing defects to tendon or
ligament tissue. De novo tendon/ligament-like tissue formation
induced by a composition of the present invention contributes to
the repair of congenital, trauma induced, or other tendon or
ligament defects of other origin, and is also useful in cosmetic
plastic surgery for attachment or repair of tendons or ligaments.
The compositions of the present invention may provide an
environment to attract tendon or ligament-forming cells,
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stimulate growth of tendon- or ligament-forming cells, induce
differentiation of progenitors of tendon- or ligament-forming
cells, or induce growth of tendon/ligament cells or progenitors
ex vivo for return in vivo to effect tissue repair. The
compositions of the invention may also be useful in the treatment
of tendinitis, carpal tunnel syndrome and other tendon or ligament
defects. The compositions may also include an appropriate matrix
and/or sequestering agent as a carrier as is well known in the
art.
The protein of the present invention may also be useful
for proliferation of neural cells and for regeneration of nerve
and brain tissue, i . a . for the treatment of central and peripheral
nervous system diseases and neuropathies, as well as mechanical
and traumatic disorders, which involve degeneration, death or
trauma to neural cells or nerve tissue. More specifically, a
protein may be used in the treatment of diseases of the peripheral
nervous system, such as peripheral nerve injuries, peripheral
neuropathy and localized neuropathies, and central nervous system
diseases, such as Alzheimer's, Parkinson's disease, Huntington's
disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
Further conditions which may be treated in accordance with the
present invention include mechanical and traumatic disorders,
such as spinal cord disorders, head trauma and cerebrovascular
diseases such as stroke. Peripheral neuropathies resulting from
chemotherapy or other medical therapies may also be treatable
using a protein of the invention.
Proteins of the invention may also be useful to promote
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better or faster closure of non-healing wounds, including without
limitation pressure ulcers, ulcers associated with vascular
insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may
5 also exhibit activity for generation or regeneration of other
tissues, such as organs (including, for example, pancreas, liver,
intestine, kidney, skin, endothelium), muscle (smooth, skeletal
or cardiac) and vascular (including vascular endothelium) tissue,
or for promoting the growth of cells comprising such tissues . Part
10 of the desired effects may be by inhibition or modulation of
fibrotic scarring to allow normal tissue to regenerate. Aprotein
of the invention may also exhibit angiogenic activity.
A protein of the present invention may also be useful for
gut protection or regeneration and treatment of lung or liver
15 fibrosis, reperfusion injury in various tissues, and conditions
resulting from systemic cytokine damage.
A protein of the present invention may also be useful for
promoting or inhibiting differentiation of tissues described
above from precursor tissues or cells; or for inhibiting the growth
20 of tissues described above.
The activity of a protein of the invention may, among other
means, be measured by the following methods:
Assays for tissue generation activity include, without
limitation, those described in: International Patent Publication
25 No. W095/16035 (bone, cartilage, tendon); International Patent
Publication No. W095/05846 (nerve, neuronal); International
Patent Publication No. W091/07491 (skin, endothelium ).
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Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical
Publishers, Inc., Chicago, as modified by Eaglstein and Mertz,
J. Invest. Dermatol 71:382-84 (1978).
Activin/T_nhi_bin A ivit-v
A protein of the present invention may also exhibit activin-
or inhibin-related activities. Inhibins are characterized by
their ability to inhibit the release of follicle stimulating
hormone (FSH), while activins and are characterized by their
ability to stimulate the release of follicle stimulating hormone
(FSH). Thus, a protein of the present invention, alone or in
heterodimers with a member of the inhibin oc family, may be useful
as a contraceptive based on the ability of inhibins to decrease
fertility in female mammals and decrease spermatogenesis in male
mammals. Administration of sufficient amounts of other inhibins
can induce infertility in these mammals. Alternatively, the
protein of the invention, as a homodimer or as a heterodimer with
other protein subunits of the inhibin-(3 group, may be useful as
a fertility inducing therapeutic, based upon the ability of
activin molecules in stimulating FSH release from cells of the
anterior pituitary. See, for example, United States Patent
4,798,885. A protein of the invention may also be useful for
advancement of the onset of fertility in sexually immature mammals,
so as to increase the lifetime reproductive performance of
domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other
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32
means, be measured by the following methods:
Assays for activin/inhibin activity include, without
limitation, those described in: Vale et al., Endocrinology
91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663,
1985; Forage et al. , Proc. Natl . Acad. Sci . USA 83: 3091-3095, 1986.
Chemo-a i / h mokin i A ivitv
A protein of the present invention may have chemotactic
or chemokinetic activity (e.g., act as a chemokine) for mammalian
cells, including, for example, monocytes, fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells. Chemotactic and chemokinetic proteins can be
used to mobilize or attract a desired cell population to a desired
site of action. Chemotactic or chemokinetic proteins provide
particular advantages in treatment of wounds and other trauma to
tissues, as well as in treatment of localized infections. For
example, attraction of lymphocytes, monocytes or neutrophils to
tumors or sites of infection may result in improved immune
responses against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a
particular cell population if it can stimulate, directly or
indirectly, the directed orientation or movement of such cell
population. Preferably, the protein or peptide has the ability
to directly stimulate directed movement of cells. Whether a
particular protein has chemotactic activity for a population of
cells can be readily determined by employing such protein or
peptide in any known assay for cell chemotaxis.
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The activity of a protein of the invention may, among other
means, be measured by the following methods:
Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce
the adhesion of one cell population to another cell population.
Suitable assays for movement and adhesion include, without
limitation, those described in: Current Protocols in Immunology,
Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach,
W.Strober, Pub. Greene Publishing Associates and Wiley-
Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller
et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol.
152: 5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
Hemo a i and Thrnmboj~rti r A~t; ~T; r«
A protein of the invention may also exhibit hemostatic or
thrombolytic activity. As a result, such a protein is expected
to be useful in treatment of various coagulation disorders
(includinghereditary disorders, such as hemophilias) or to
enhancecoagulation and otherhemostaticeventsin treating wounds
resulting from trauma, surgery or other causes. A protein of the
invention may also be useful for dissolving or inhibiting
formation of thromboses and for treatment and prevention of
conditions resulting therefrom (such as, for example, infarction
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34
of cardiac and central nervous system vessels (e. g., stroke).
The activity of a protein of the invention may, among other
means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include,
without limitation, those described in: Linet et al., J. Clin.
Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.
45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991);
Schaub, Prostaglandins 35:467-474, 1988.
Recen o /,igand A ivi v
A protein of the present invention may also demonstrate
activity as receptors, receptor ligands or inhibitors or agonists
of receptor/ligand interactions. Examples of such receptors and
ligands include, without limitation, cytokine receptors and their
ligands, receptor kinases and their ligands, receptor
phosphatases and their ligands, receptors involved in cell-cell
interactions and their ligands (including without limitation,
cellular adhesion molecules (such as selectins, integrins and
their ligands) and receptor/ligand pairs involved in antigen
presentation, antigen recognition and development of cellular and
humoral immune responses) . Receptors and ligands are also usef-ul
for screening of potential peptide or small molecule inhibitors
of the relevant receptor/ligand interaction. A protein of the
present invention (including, without limitation, fragments of
receptors and ligands) may themselves be useful as inhibitors of
receptor/ligand interactions.
The activity of a protein of the invention may, among other
means, be measured by the following methods:
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Suitable assays for receptor-ligand activity include
without limitation those described in:Current Protocols in
Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
5 Wiley-Interscience (Chapter 7.28, Measurement of Cellular
Adhesion under static conditions 7.28.1-7.28.22), Takai et al.,
Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al.,
J. Exp. Med. 168: 1145-1156, 1988; Rosenstein et al., J. Exp. Med.
169:149-160 1989; Stoltenborg et al., J. Immunol. Methods
10 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Proteins of the present invention may also exhibit
anti-inflammatory activity. The anti-inflammatory activity may
be achieved by providing a stimulus to cells involved in the
15 inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for example, cell adhesion) , by inhibiting
or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by
stimulating or suppressing production of other factors which more
20 directly inhibit or promote an inflammatory response. Proteins
exhibiting such activities can be used to treat inflammatory
conditions including chronic or acute conditions), including
without limitation inflammation associated with infection (such
as septic shock, sepsis or systemic inflammatory response syndrome
25 (SIRS)), ischemia-reperfusion injury, endotoxin lethality,
arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or chemokine-induced lung injury, inflammatory bowel
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36
disease, Crohn's disease or resulting from over production of
ytokines such as TNF or IL-1. Proteins of the invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic
substance or material.
Timor Inh,'_bi t-; ~n A i vi v
In addition to the activities described above for
immunological treatment or prevention of tumors, a protein of the
invention may exhibit other anti-tumor activities. A protein may
inhibit tumor growth directly or indirectly (such as, for example,
via ADCC). A protein may exhibit its tumor inhibitory activity
by acting on tumor tissue or tumor precursor tissue, by inhibiting
formation of tissues necessary to support tumor growth (such as,
for example, by inhibiting angiogenesis), by causing production
of other factors, agents or cell types which inhibit tumor growth,
or by suppressing, eliminating or inhibiting factors, agents or
cell types which promote tumor growth
A protein of the invention may also exhibit one or more of
the following additional activities or effects: inhibiting the
growth, infection or function of, or killing, infectious agents,
including, without limitation, bacteria, viruses, fungi and other
parasites; effecting (suppressing or enhancing) bodily
characteristics, including, without limitation, height, weight,
hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ or body part size or shape (such as, for
example, breast augmentation or diminution, change in bone form
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37
or shape); effecting biorhythms or caricadic cycles or rhythms;
effecting the fertility of male or female subjects; effecting the
metabolism, catabolism, anabolism, processing, utilization,
storage or elimination of dietary fat, lipid, protein, carbohydrate,
vitamins, minerals, cofactors or other nutritional factors or
component(s); effecting behavioral characteristics, including,
without limitation, appetite, libido, stress, cognition (including
cognitive disorders), depression (including depressive disorders)
and violent behaviors; providing analgesic effects or other pain
reducing effects; promoting differentiation and growth of
embryonic stem cells in lineages other than hematopoietic lineages;
hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies of the enzyme and treating deficiency-related
diseases; treatment of hyperproliferative disorders (such as, for
example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the
ability to act as an antigen in a vaccine composition to raise an
immune response against such protein or another material or entity
which is cross-reactive with such protein.
Examples
The present invention is embodied in more detail by the
following examples, but this embodiment is not intended to
restrict the present invention. The basic operations and the
enzyme reactions with regard to the DNA recombination are carried
out according to the literature ["Molecular Cloning. A Laboratory
Manual", Cold Spring Harbor Laboratory, 1989]. Unless otherwise
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38
stated, restrictive enzymes and a variety of modification enzymes
to be used were those available from TAKARA SHUZO. The
manufacturer's instructions were usedfor the buffercompositions
as well as for the reaction conditions, in each of the enzyme
reactions. The cDNA synthesis was carried out according to the
literature [Kato, S. et al., Gene 150: 243-250 (1994)].
(1) Preparation of Poly(A)' RNA
The fibrosarcoma cell line HT-1080 (ATCC CCL 121), the
osteosarcoma cell line U-2 OS (ATCC HTB 96), tissues of stomach
cancer delivered by the operation, and the liver were used for
human cells to extract mRNAs. The cell lines were incubated by
a conventional procedure.
After about 1 g of the human cells was homogenized in 20
ml of a 5.5 M guanidinium thiocyanate solution, a total mRNA was
prepared according to the literature [Okayama, H. et al., "Method
in Enzymology", Vol. 164, Academic Press, 1987]. This was
subjected to chromatography on oligo(dT)-cellulose column washed
with a 20 mM Tris-hydrochloride buffer solution (pH 7.6), 0.5 M
NaCl, and 1 mM EDTA to obtain a poly(A)' RNA according to the
above-described literature.
(2) Construction of cDNA Library
Ten micrograms of the above-mentioned poly(A)' RNA were
dissolved in a 100 mM Tris-hydrochloride buffer solution (pH 8) ,
one unit of an RNase-free, bacterial alkaline phosphatase was
added, and the reaction was run at 37°C for one hour. After the
reaction solution was subjected to phenol extraction, followed
by ethanol precipitation, the resulting pellet was dissolved in
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a solution containing 50 mM sodium acetate (pH 6) , 1 mM EDTA, 0. 1 0
2-mercaptoethanol, and 0.010 Triton X-100. Thereto was added one
unit of a tobacco-origin acid pyrophosphatase (Epicentre
Technologies) and a total 100 ~.1 volume of the resulting mixture
was reacted at 37'C for one hour. After the reaction solution was
subjected to phenol extraction, followed by ethanolprecipitation,
the resulting pellet was dissolved in water to obtain a solution
of a decapped poly(A)' RNA.
The decapped poly(A)' RNA and 3 nmol of a chimeric DNA-
RNA oligonucleotide (5'-dG-dG-dG-dG-dA-dA-dT-dT-dC-dG-dA-G-G-
A-3') were dissolved in a solution containing 50 mM Tris-
hydrochloride buffer solution (pH 7.5), 0.5 mM ATP, 5 mM MgCl~,
10 mM 2-mercaptoethanol, and 25o polyethylene glycol, whereto was
added 50 units of T4RNA ligase and a total 30 ~1 volume of the
resulting mixture was reacted at 20°C for 12 hours. After the
reaction solution was subjected to phenol extraction, followed
by ethanol precipitation, the resulting pellet was dissolved in
water to obtain a chimeric-oligo-capped poly(A)' RNA.
After digestion of vector pKAl (Japanese Patent Kokai
Publication No. 1992-117292) developed by the present inventors
with KpnI, about 60 dT tails were added using a terminal transferase.
A vector primer to be used below was prepared by digestion of this
product with EcoRV to remove a dT tail at one side.
After 6 ~tg of the previously-prepared chimeric-oligo-
capped poly(A)' RNA was annealed with 1.2 ~g of the vector primer,
the resulting product was dissolved in a solution containing 50
mM Tris-hydrochloride buffer solution (pH 8.3), 75 mM KC1, 3 mM
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MgCl2, 10 mM dithiothreitol, and 1.25 mM dNTP (dATP + dCTP + dGTP
t dTTP), 200 units of a reverse transcriptase (GIBCO-BRL) were
added, and the reaction in a total 20 ~tl volume was run at 42 C
for one hour. After the reaction solution was subjected to phenol
5 extraction, followed by ethanol precipitation, the resulting
pellet was dissolved in a solution containing 50 mM Tris-
hydrochloride buffer solution (pH 7.5) , 100 mM NaCl, 10 mM MgCl,,
and 1 mM dithiothreitol. Thereto were added 100 units of EcoRI
and a total 20 ~1 volume of the resulting mixture was reacted at
10 37°C for one hour. After the reaction solution was subjected to
phenol extraction, followed by ethanol precipitation, the
resulting pellet was dissolved in a solution containing 20 mM
Tris-hydrochloride buffer solution (pH 7. 5) , 100 mM KC1, 4 mMMgCl,,
10 mM (NHq) ~SOa, and 50u g/ml of the bovine serum albumin. Thereto
15 were added 60 units of an Escherichia coli DNA ligase and the
resulting mixture was reacted at 16°C for 16 hours . To the reaction
solution were added 2 ~tl of 2 mM dNTP, 4 units of Escherichia coli
DNA polymerase I, and 0.1 unit of Escherichia coli RNase H and
the resulting mixture was reacted at 12°C for one hour and then
20 at 22°C for one hour.
Next, the cDNA-synthesis reaction solution was used for
transformation of Escherichia coli DH12S (GIBCO-BRL). The
transformation was carried out by the electroporation method. A
portion of the transformant was sprayed on the 2xYT agar culture
25 medium containing 100 ~g/ml ampicillin and the mixture was
incubated at 37°C overnight. A colony formed on the agar medium
was picked up at random and inoculated on 2 ml of the 2xYT culture
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medium containing 100 ~tg/ml ampicillin. After incubation at 37°C
overnight, the culture mixture was centrifuged to separate the
mycelia, from which a plasmid DNA was prepared by the alkaline
lysis method. The plasmid DNA was subjected to double digestion
with EcoRI and NotI, followed by 0. 8 ~ agarose gel electrophoresis,
to determine the size of the cDNA insert. Furthermore, using the
thus-obtained plasmid as a template, the sequence reaction was
carried out by using an M13 universal primer labeled with a
fluorescent dye and a Taq polymerase (a kit of Applied Biosystems)
and then the product was examined with a fluorescent DNA sequencer
(Applied Biosystems) to determine an about 400-by base sequence
at the 5'-terminus of the cDNA. The sequence data were filed as
the homo/protein cDNA bank database.
(3) Selection of cDNAs Encoding Proteins Having Transmembrane
Domains
A base sequence registered in the homo/protein cDNA bank
was converted to three frames of amino acid sequences and the
presence or absence of an open reading frame (ORF) beginning from
the initiation codon was examined. Then, the selection was made
for the presence of a signal sequence that is characteristic to
a secretory protein at the N-terminus of the portion encoded by
the ORF. These clones were sequenced from the both 5' and 3'
directions by the use of the deletion method using exonuclease
III to determine the whole base sequence. The
hydrophobicity/hydrophilicity profiles were obtained for
proteins encoded by the ORF by the Kyte-Doolittle method [Kyte,
J. & Doolittle, R. F. , J. Mol . Biol . 157 : 105-132 ( 1982 ) ) to examine
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the presence or absence of a hydrophobic region. In the case in
which there is a hydrophobic region of a putative transmembrane
domain in the amino acid sequence of an encoded protein, this
protein was judged as a membrane protein.
(4) Functional Verification of Secretory Signal Sequence or
Transmembrane Domains
It was verified by the method described in the literature
[Yokoyama-Kobayashi, M. et al., Gene 163: 193-196 (1995)] that
the N-terminal hydrophobic region in the secretory protein clone
candidate obtained in the above-mentioned steps functions as a
secretory signal sequence. First, the plasmid containing the
target cDNA was cleaved at an appropriate restriction enzyme site
existing at the downstream of the portion expected for encoding
the secretory signal sequence. In the case in which this
restriction site was a protruding terminus, the site was
blunt-ended by the Klenow treatment or treatment with the
mung-bean nuclease. Digestion with HindIII was further carried
out and a DNA fragment containing the SV40 promoter and a cDNA
encoding the secretory signal sequence at the downstream of the
promoter was separated by agarose gel electrophoresis. The
resulting fragment was inserted between HindIII in pSSD3
(DDBJ/EMBL/GenBank Registration No. AB007632) and a restriction
enzyme site selected so as to match with the urokinase-coding frame,
thereby constructing a vector expressing a fusion protein of the
secretory signal sequence of the target cDNA and the urokinase
protease domain.
After Escherichia coli (host: JM109) bearing the
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fusion-protein expression vector was incubated at 37~ for 2 hours
in 2 ml of the 2xYT culture medium containing 100 ~g/ml of
ampicillin, the helper phage M13K07 (SOU 1) was added and the
incubation was continued at 37°C overnight. A supernatant
separated by centrifugation underwent precipitation with
polyethylene glycol to obtain single-stranded phage particles.
These particles were suspended in 100 ~tl of 1 mM Tris-0.1 mM EDTA,
pH 8 (TE). Also, there were used as controls suspensions of
single-stranded phage particles prepared in the same manner from
pSSD3 and from the vector pKAl-UPA containing a full-length cDNA
of urokinase [Yokoyama-Kobayashi, M. et al., Gene 163: 193-196
( 1995 ) ] .
The culture cells originating from the simian kidney, COS7,
were incubated at 37°C in the presence of 5 o CO, in the Dulbecco' s
modified Eagle's culture medium (DMEM) containing loo fetal calf
albumin. Into a 6-well plate (Nunc Inc., 3 cm in the well diameter)
were inoculated 1 x 10' COS7 cells and incubation was care i ed out
at 37°C for 22 hours in the presence of 5 o CO., . After the culture
medium was removed, the cell surface was washed with a phosphate
buffer solution and then washed again with DMEM containing 50 mM
Tris-hydrochloric acid (pH 7.5) (TDMEM). To the resultina cells
was added a suspension of 1 ~tl of the single-stranded phage
suspension, 0.6 ml of the DMEM culture medium, and 3 ~tl of
TRANSFECTAMT'' (IBF Inc.) and the resulting mixture was incubated
at 37~C for 3 hours in the presence of 5o CO~. After the sample
solution was removed, the cell surface was washed with TDMEM, 2
ml per well of DMEM containing 10 o fetal calf albumin was added,
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and the incubation was carried out at 37°C for 2 days in the presence
of 5o CO~.
To 10 ml of 50 mM phosphate buffer solution (pH 7.4)
containing 2% bovine fibrinogen (Miles Inc.), 0.5o agarose, and
1 mM calcium chloride were added 10 units of human thrombin (Mochida
Pharmaceutical Co., Ltd.) and the resulting mixture was solidified
in a plate of 9 cm in diameter to prepare a fibrin plate. Ten
microliters of the culture supernatant of the tansfected COS7
cells were spotted on the fibrin plate, which was incubated at
37 C for 15 hours. In the case in which a clear circle appears on
the fibrin plate, it is judged that the cDNA fragment codes for
the amino acid sequence functioning as a secretory signal sequence.
On the other hand, in case in which a clear circle is not formed,
the cells were washed well, then the fibrin sheet was placed on
the cells, and incubation was carried out at 37°C for 15 hours.
In case in which a clear portion is formed on the fibrin sheet,
it indicates that the urokinase activity was expressed on the cell
surface. In other words, the cDNA fragment is judged to code for
the transmembrane domains.
(5) Protein Synthesis by In Vitro Translation
The plasmid vector bearing the cDNA of the present invention
was used for in vitro transcription/translation with a T~,T rabbit
reticulocyte lysate kit (Promega). In this case, [-SS]methionine
was added to label the expression product with a radioisotope.
Each of the reactions was carried out according to the protocols
attached to the kit. Two micrograms of the plasmid was reacted
at 30°C for 90 minutes in a total 25 ~1 volume of the reaction
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solution containing 12 . 5 a 1 of Tr,T rabbit reticulocyte lysate, 0. 5
~1 of a buffer solution (attached to kit) , 2 ~tl of an amino acid
mixture (methionine-free), 2 ~tl of [3-'S]methionine (Amersham)
(0.37 MBq/~11), 0.5 ~.1 of T7RNA polymerase, and 20 U of RNasin.
5 To 3 ~tl of the resulting reaction solution was added 2 ~.1 of the
SDS sampling buffer ( 125 mM Tris-hydrochloric acid buffer, pH 6. 8,
120 mM 2-mercaptoethanol, 2 o SDS solution, 0 . 025 bromophenol blue,
and 20x glycerol) and the resulting mixture was heated at 95~
for 3 minutes and then subjected to SDS-polyacrylamide gel
10 electrophoresis. The molecular weight of the translation product
was determined by carrying out the autoradiograph.
(6) Expression by COS7
Escherichia coli bearing the expression vector of the
protein of the present invention was infected with helper phage
15 M13K07 and single-stranded phage particles were obtained by the
above-mentioned procedure. The thus-obtained phage was used for
introducing each expression vector in the culture cells
originating from the simian kidney, COS7. After incubation at 37°C
for 2 days in the presence of 5 ~ CO~, the incubation was continued
20 for one hour in the culture medium containing [ '-S ] cystine or
[3'S]methionine. Collection and dissolution of the cells, followed
by subjecting to SDS-PAGE, allowed to observe the presence of a
band corresponding to the expression product of each protein,
which did not exist in the COS7 cells. For instance, HP01535,
25 HP10216 and HP10420 produced respectively bands of 18 kDa, 20 kDa
and 76 kDa in the supernatant of the culture medium. HP10441 also
produced a band of 7 kDa in the membrane fraction.
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(7) Clone Examples
<HP00956> (Sequence Nos. 1, 7, and 13)
Determination of the whole base sequence of the cDNA insert
of clone HP00956 obtained from cDNA libraries of the human
osteosarcoma cell line revealed the structure consisting of a
211-by 5'-nontranslation region, a 507-by ORF, and a 149-by
3' -nontranslation region. The ORF codes for a protein consisting
of 168 amino acid residues and there existed one transmembrane
domain at the C-terminal. Figure 1 depicts the
hydrophobicity/hydrophilicity profile, obtained by the Kyte-
Doolittle method, of the present protein. In vitro translation
resulted in formation of a translation product of 20 kDa that was
almost consistent with the molecular weight of 18,750 predicted
from the ORF.
The search of the protein data base by using the amino acid
sequence of the present protein revealed that the protein was
analogous to the human H-rev107 protein homologue (SWISS-PROT
Accession No. P53816) . Table 2 shows the comparison of the amino
acid sequence between the human protein of the present invention
(HP) and the human H-rev107 protein homologue (HR). Therein, the
marks of -, *, and , represent a gap, an amino acid residue identical
with the protein of the present invention, and an amino acid residue
analogous to the protein of the present invention, respectively.
The both proteins possessed a homology of 46.So.
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Table 2
HS MAFNDCFSLNYPGNPCPGDLIEVFRPGYQHWALYLGDGYVINIAPVDGIP-ASFTSAKSV
. * ******. *** *. ***. *. *****. . . ** . . . . *. . *. . *.
HR . MRAPIPEPKPGDLIEIFRPFYRHWAIYVGDGYVVHLAPPSEVAGAGAASVMSA
HS FSSKALVKMQLLKDVVG\DTYRINNKYDETYPPLPVEEIIKRSEFVIGQEVAYNLLVVNC
. . . **, **. . ** **. *, *, *, . ***, *. . *. *** . . **. *, * . . **** *. *
, **
HR LTDKAIVKKELLYDVAGSDKYQVNNKHDDKYSPLPCTKIIQftAEELVGQEVLYKLTSENC
HS EHFVTLLRYGEGVSEQANRAISTVEFVTAAVGVFSFLG-LFPKGQRAKYY
****. **** , *. *. . . * . . . . . . . . . . *. . * . *. . . . *.
HR EHFVNELRYGVARSDQVRDVIIAASVAGMGLAAMSLIGVMFSRNKRQKQ
Furthermore, the search of the GenBank using the base
sequences of the present cDNA has revealed the presence of
sequences that possessed a homology of 90~ or more ( for example,
Accession No. AA478132) in EST, but, since they are partial
sequences, it can not be judged whether or not any of these
sequences codes for the same protein as the protein of the present
invention.
The human H-rev107 protein is one of proteins which are
specifically expressed in H-ras resistant fibroblast cells
[Hajnal, A. et al., Oncogene 9: 479-490 (1994) ] . Accordingly, the
present protein has been considered to be associated with
phenotypic expression of cancer cells.
<HP01535> (Sequence Nos. 2, 8, and 15)
Determination of the whole base sequence of the cDNA insert
of clone HP01535 obtained from cDNA libraries of human stomach
cancer revealed the structure consisting of a 24-by 5'-
nontranslation region, a 495-by ORF, and a 201-by 3'-
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nontranslation region. The ORF codes for a protein consisting of
164 amino acid residues and there existed one transmembrane domain
at the C-terminal. Figure 2 depicts the
hydrophobicity/hydrophilicity profile, obtained by the Kyte-
Doolittle method, of the present protein. In vitro translation
resulted in formation of a translation product of 20 kDa that was
almost consistent with the molecular weight of 18,179 predicted
from the ORF.
The search of the protein data base by using the amino acid
sequence of the present protein revealed that the protein was
analogous to the human H-rev107 protein homologue (SWISS-PROT
Accession No. P53816) . Table 3 shows the comparison of the amino
acid sequence between the human protein of the present invention
(HP) and the human H-rev107 protein homologue (HR). Therein, the
marks of -, *, and . represent a gap, an amino acid residue identical
with the protein of the present invention, and an amino acid residue
analogous to the protein of the present invention, respectively.
Both proteins possessed a homology of 51.2=.in the entire region.
Table 3
HS MASPHQEPKPGDLIEIFRLGYEHWALYIGDGYVIHLAPPSEYPGAGSSSVFSVLSNSAEV
* . * . ************ * ***, *. *****. ******* . ***, . **, *, *. . .
HR MRAPIPEPKPGDLIEIFRPFYRHWAIYVGDGYVVHLAPPSEVAGAGAASVMSALTDKAIV
HS KRERLEDVVGGCCYRVNNSLDHEYQPRPVEVIISSAKEMVGQKMKYSIVSRNCEHFVTQL
*. * * **. *. *. ***. *. . * * * . ** . *. *. ***. . *. . . * ******. .
HR KKELLYDVAGSDKYQVNNKHDDKYSPLPCTKIIQRAEELVGQEVLYKLTSENCEHFVNEL
HS RYGKSRCKQVEKAKVEVGVAT-ALGILVVAGCSFAIRRYQKKATA
*** . *. . ** . . . . . . **. . *. . . * *. . . **.
HR RYGVARSDQVRDVIIAASVAGMGLAAMSLIGVMFSR~\KRQKQ
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Furthermore, the search of the GenBank using the base
sequences of the present cDNA has revealed the presence of
sequences that possessed a homology of 900 or more (for example,
Accession No. 855646) in EST, but, since they are partial sequences,
it can not be judged whether or not any of these sequences codes
for the same protein as the protein of the present invention.
The human H-rev107 protein is one of proteins which are
specifically expressed in H-ras resistant fibroblast cells
[Hajnal, A. et al., Oncogene 9: 479-490 (1994) ] . Accordingly, the
present protein has been considered to be associated with
phenotypic expression of cancer cells.
<HP10089> (Sequence Nos. 3, 9, and 17)
Determination of the whole base sequence of the cDNA insert
of clone HP10089 obtained from cDNA libraries of human liver
revealed the structure consisting of a 73-by 5'-nontranslation
region, a 426-by ORF, and a 67-by 3'-nontranslation region. The
ORF codes for a protein consisting of 141 amino acid residues and
there existed four transmembrane domains. Figure 3 depicts the
hydrophobicity/hydrophilicity profile, obtained by the Kyte-
Doolittle method, of the present protein. In vitro translation
resulted in formation of a translation product of 14 kDa that was
almost consistent with the molecular weight of 14,852 predicted
from the ORF.
The search of the protein data base using the amino acid
sequence of the present protein has not revealed the presence of
any known protein having an analogy. Also, the search of the GenBank
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using the base sequences of the present cDNA has revealed the
presence of sequences that possessed a homology of 90~ or more
and contained an initiation codon (for example, Accession No.
N56722) in EST, but, since they are partial sequences, it can not
5 be judged whether or not any of these sequences codes for the same
protein as the protein of the present invention.
<HP10216> (Sequence Nos. 4, 10, and 19)
Determination of the whole base sequence of the cDNA insert
of clone HP10216 obtained from cDNA libraries of the human
10 fibrosarcoma cell line HT-1080 revealed the structure consisting
of a 4-by 5'-nontranslation region, a 429-by ORF, and a 645-bt~
3'-nontranslation region. The ORF codes for a protein consisting
of 142 amino acid residues and there existed one transmembrane
domain. Figure 4 depicts the hydrophobicity/hydrophilicity
15 profile, obtained by the Kyte-Doolittle method, of the present
protein. In vitro translation resulted in formation of a
translation product of 22 kDa that was larger than the molecular
weight of 15,521 predicted from the ORF.
The search of the protein data base using the amino acid
20 sequence of the present protein has not revealed the presence of
any known protein having an analogy. Also, the search of the GenBank
using the base sequences of the present cDNA has revealed the
presence of sequences that possessed a homology of 90% or more
and contained an initiation codon (for example, Accession No.
25 AA316462) in EST, but any of the sequences was shorter than the
present cDNAs and was not found to contain the initiation codon.
<HP10420> (Sequence Nos. 5, 11, and 21)
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Determination of the whole base sequence of the cDNA insert
of clone HP10420 obtained from cDNA libraries of human stomach
cancer revealed the structure consisting of an 81-by 5'-
nontranslation region, a 1041-by ORF, and a 188-by 3'-
nontranslation region. The ORF codes for a protein consisting of
346 amino acid residues and there existed a signal-like sequence
at the N-terminus and one transmembrane domain at the C-terminus .
Figure 5 depicts the hydrophobicity/hydrophilicity profile,
obtained by the Kyte-Doolittle method, of the present protein.
Introduction of an expression vector, wherein the HindIII-PvuII
fragment containing a cDNA portion coding for the N-terminal 39
amino acid residues of the present protein was inserted into the
HindIII-SmaI site of pSSD3, into the COS7 cells revealed the
urokinase activity in the culture medium to indicate that the
present protein is a type-I membrane protein. In vitro translation
resulted in formation of a translation product of 39 kDa that was
almost consistent with the molecular weight of 35,970 predicted
from the ORF. Application of the (-3,-1) rule, a method for
predicting the cleavage site in the secretory signal sequence,
allows to expect that the maturation protein starts from leucine
at position 31.
The search of the protein data base using the amino acid
sequence of the present protein has not revealed the presence of
any known protein having an analogy. Also, the search of the GenBank
using the base sequences of the present cDNA has revealed the
presence of sequences that possessed a homology of 900 or more
and contained an initiation codon (for example, Accession No.
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T70513) in EST, but, since they are partial sequences, it can not
be judged whether or not any of these sequences codes for the same
protein as the protein of the present invention.
<HP10441> (Sequence Nos. 6, 12, and 23)
Determination of the whole base sequence of the cDNA insert
of clone HP10441 obtained from cDNA libraries of the human stomach
cancer revealed the structure consisting of a 341-by 5'-
nontranslation region, a 201-by ORF, and a 239-by 3'-
nontranslation region. The ORF codes for a protein consisting of
66 amino acid residues and there existed one transmembrane domain
at the C-terminus. Figure 6 depicts the
hydrophobicity/hydrophilicity profile, obtained by the Kyte-
Doolittle method, of the present protein. In vitro translation
resulted in formation of a translation product of 10 kDa that was
almost consistent with the molecular weight of 7,374 predicted
from the ORF.
The search of the protein data base using the amino acid
sequence of the present protein has revealed the presence of
sequences that were analogous to the nematode putative protein
F59F4.2 (GenBank Accession No. 281095). Table 4 shows the
comparison of the amino acid sequence between the human protein
of the present invention (HP) and the nematode putative protein
F59F4.2 (CE). Therein, the marks of -, *, and , represent a gap,
an amino acid residue identical with the protein of the present
invention, and an amino acid residue analogous to the protein of
the present invention, respectively. The both proteins possessed
a homology of 52.3- in the entire region.
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Table 4
HS MVAKQRIRMANEKHSK\ITQRGNVAKTSRNAPEEKASVGPWLLALFIFVVCGSAIFQIIQ
*. . ***. . **. . ***. . . ******. . . . *, * . . . ***. . **. *******. *. **.
CE MAPKQIL~ITLANKQFSK\VV'NRGNVAKSLK-PAEDKYPAAPWLIGLFVFVVCGSAVFEIIR
HS SIRMGM
. . **
CE YVKMG
Furthermore, the search of the GenBank using the base
sequences of the present cDNA has revealed the presence of
sequences that possessed a homology of 900 or more and contained
the initiation codon. (for example, Accession No. AA232459) in
EST, but many sequences were not distinct and the same ORF as that
in the present cDNA was not found.
INDU T TAT. APPT,TCA3_TT TTY
The present invention provides human proteins having
~ransmembrane domains and cDNAs coding for these proteins as well
as eucaryotic cells expressing said cDNAs. All of the proteins
of the present invention exist in the cell membrane, so that they
are considered to be proteins controlling the proliferation and
the differentiation of the cells. Accordingly, the proteins of
the present invention can be employed as pharmaceuticals such as
carclnostatic agents relating to the control of the proliferation
and the differentiation of the cells or as antigens for preparing
antibodies against said proteins. The cDNAs of the present
invention can be utilized as probes for the gene diagnosis and
gene sources for the gene therapy. Furthermore, the cDNAs can be
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54
utilized for large-scale expression of said proteins. Cells,
wherein these membrane protein genes are introduced and membrane
proteins are expressed in large amounts, can be utilized for
detection of the corresponding ligands, screening of novel
low-molecular pharmaceuticals, and so on.
The present invention also provides genes corresponding
to the polynucleotide sequences disclosed herein.
"Corresponding genes" are the regions of the genome that are
transcribed to produce the mRNAs from which cDNA polynucleotide
sequences are derived and may include contiguous regions of the
genome necessary for the regulated expression of such genes.
Corresponding genes may therefore include but are not limited to
coding sequences, 5' and 3' untranslated regions, alternatively
spliced exons, introns, promoters, enhancers, and silencer or
suppressor elements . The corresponding genes can be isolated in
accordance with known methods using the sequence information
disclosed herein. Such methods include the preparation of probes
or primers from the disclosed sequence information for
identification and/or amplification of genes in appropriate
genomic libraries or other sources of genomic materials. An
"isolated gene" is a gene that has been separated from the adjacent
coding sequences, if any, present in the genome of the organism
from which the gene was isolated.
Organisms that have enhanced, reduced, or modified
expression of the genes) corresponding to the polynucleotide
sequences disclosed herein are provided. The desired change in
gene expression can be achieved through the use of antisense
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polynucleotides or ribozymes that bind and/or cleave the mRNA
transcribed from the gene (Albert and Morris, 1994, Trends
Pharmacol. Sci. 15(7) : 250-254; Lavarosky et al., 1997, Biochem.
Mol . Med. 62 ( 1 ) : 11-22; and Hampel, 1998, Prog. Nucleic Acid Res .
5 Mol. Biol. 58: 1-39; all of which are incorporated by reference
herein). Transgenic animals that have multiple copies of the
genes) corresponding to the polynucleotide sequences disclosed
herein, preferably produced by transformation of cells with
genetic constructs that are stably maintained within the
10 ;ransformed cells and their progeny, are provided. Transgenic
animals that have modified genetic control regions that increase
or reduce gene expression levels, or that change temporal or
spatial patterns of gene expression, are also provided (see
European Patent No. 0 649 464 B1, incorporatedby reference herein) .
15 In addition, organisms are provided in which the genes)
corresponding to the polynucleotide sequences disclosed herein
have been partially or completely inactivated, through insertion
of extraneous sequences into the corresponding gene (s) or through
deletion of all or part of the corresponding gene(s). Partial
20 or complete gene inactivation can be accomplished through
insertion, preferably followed by imprecise excision, of
transposable elements (Plasterk, 1992, Bioessays 14 (9) : 629-633;
Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90 (16) : 7431-7435;
Clark et al., 1994, Proc. Natl. Acad. 5ci. USA 91(2): 719-722;
25 all of which are incorporated by reference herein), or through
homologous recombination, preferably detected by
positive/negative genetic selection strategies (Mansour et al.,
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1988, Nature 336: 348-352; U. S. Patent Nos. 5, 464, 764; 5, 487, 992;
5, 627, 059; 5, 631, 153; 5, 614, 396; 5, 616, 491; and 5, 679, 523; all
of which are incorporated by reference herein). These organisms
with altered gene expression are preferably eukaryotes and more
preferably are mammals. Such organisms are useful for the
development of non-human models for the study of disorders
involving the corresponding gene(s), and for the development of
assay systems for the identification of molecules that interact
with the protein products) of the corresponding gene(s).
Where the protein of the present invention is
membrane-bound (e. g., is a receptor), the present invention also
provides for soluble forms of such protein. In such forms part
or all of the intracellular and transmembrane domains of the
protein are deleted such that the protein is fully secreted from
the cell in which it is expressed. The intracellular and
transmembrane domains of proteins of the invention can be
identified in accordance with known techniques for determination
of such domains from sequence information.
Proteins and protein fragments of the present invention
include proteins with amino acid sequence lengths that are at least
25o(more preferably at least 50°s, and most preferably at least
75~) of the length of a disclosed protein and have at least 60=
sequence identity (more preferably, at least 75o identity; most
preferably at least 900 or 95o identity) with that disclosed
protein, where sequence identity is determined by comparing the
amino acid sequences of the proteins when aligned so as to maximize
overlap and identity while minimizing sequence gaps. Also
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included in the present invention are proteins and protein
fragments that contain a segment preferably comprising 8 or more
(more preferably 20 or more, most preferably 30 or more) contiguous
amino acids that shares at least 75o sequence identity (more
preferably, at least 85~ identity; most preferably at least 95
identity) with any such segment of any of the disclosed proteins.
Species homologs of the disclosed polynucleotides and
proteins are also provided by the present invention. As used
herein, a "species homologue" is a protein or polynucleotide with
a different species of origin from that of a given protein or
polynucleotide, but with significant sequence similarity to the
given protein or polynucleotide, as determined by those of skill
in the art. Species homologs may be isolated and identified by
making suitable probes or primers from the sequences provided
herein and screening a suitable nucleic acid source from the
desired species.
The invention also encompasses allelic variants of the
disclosed polynucleotides or proteins; that is, naturally-
occurring alternative forms of the isolated polynucleotide which
also encode proteins which are identical, homologous, or related
to that encoded by the polynucleotides.
The invention also includes polynucleotides withsequences
complementary to those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides
capable of hybridizing under reduced stringency conditions, more
preferably stringent conditions, and most preferably highly
stringent conditions, to polynucleotides described herein.
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Examples of stringency conditions are shown in the table below:
highly stringent conditions are those that are at least as
stringent as, for example, conditions A-F; stringent conditions
are at least as stringent as, for example, conditions G-L; and
reduced stringency conditions are at least as stringent as, for
example, conditions M-R.
Table 5
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Buffers Temperature
(b and Buffers
$
A DNA : DNA >_50 65C; IxSSC -or- 65C; 0.3xSSC
42C; lxSSC.50% formamide
B DNA : DNA <50 TB*; IxSSC TB*; lxSSC
C DNA : RNA >_50 67C; lxSSC -or- 67C; 0.3xSSC
45C: IxSSC.50% formamide
D DNA : RNA <50 Tp*; IxSSC TD*; lxSSC
E RNA : RNA >_50 70'C; IxSSC -or- 70C; 0.3xSSC
50C; IxSSC.50% formamide
RNA : RNA <50 TF*: lxSSC TF*; IxSSC
G DNA : DNA >50 65C; 4xSSC -or- 85C; IxSSC
42'C: 4xSSC.50% formamide
H DNA : DNA <50 TH*: 4xSSC TH*; 4xSSC
I DNA : RNA >50 67'C; 4xSSC -or- 67C; IxSSC
45C: 4xSSC.50% formamide
J DNA : RNA <50 T~*; 4xSSC T,,*; 4xSSC
RNA : RNA >50 70C; 4xSSC -or- 67C; IxSSC
50C; 4xSSC.50% formamide
L RNA : RNA <50 TL*; 2xSSC TL*; 2xSSC
M DNA : DNA >_50 50C; 4xSSC -or- 50C: 2xSSC
40'C: 6xSSC.50% formamide
N DNA : DNA <50 T~;*; 6xSSC TN*; 6xSSC
0 DNA : RNA >_50 55'C: 4xSSC -or- 55C: 2xSSC
42C; 6xSSC.50% formamide
P DNA : RNA <50 TP*; 6xSSC Tp*; 6xSSC
Q RNA : RNA >_50 60'C; 4xSSC -or- 60C; 2xSSC
45C: 6xSSC.50% formamide
R RNA : RNA <50 TR*; 4xSSC TR*; 4xSSC
$ : The hybrid length is that anticipated for the hybridized regions) of the
hybridizing
polynucleotides. When hybridizing a polynucleotide to a target polynucleotide
of unknown
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sequence. the hybrid length is assumed to be that of the hybridizing
polynucleotide. When
polynucleotides of known sequence are hybridized. the hybrid length can be
determined by
aligning the sequences of the polynucleotides and identifying the region or
regions of
optimal sequence complementarity.
-~ : SSPE (IxSSPE is 0.15M NaCI, lOmM NaH2P04, and 1.25mM EDTA, pH7.4) can be
substituted for SSC (IxSSC is 0.15M NaCI and l5mM sodium citrate) in the
hybridization
and wash buffers: washes are performed for 15 minutes after hybridization is
complete.
*TH - TR : The hybridization temperature for hybrids anticipated to be less
than 50 base
pairs in length should be 5-10°C less than the melting temperature (Tm)
of the hybrid,
where Tm is determined according to the following equations. For hybrids less
than 18
base pairs in length, Tm('C)=2(#of A + T bases) + 4(# of G + C bases). For
hybrids between
18 and 49 base pairs in length, Tm(°C)=81.5 + 16.6(log,o[Na+]) + 0.41
(%G+C) - (600/N),
where N is the number of bases in the hybrid, and [Na'] is the concentration
of sodium ions
in the hybridization buffer ([Na+] for IxSSC=0.165M).
Additional examples of stringency conditions for
polynucleotide hybridization are provided in Sambrook, J., E.F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY, chapters 9 and 11, and Current Protocols in Molecular Biology,
1995, F.M. Ausubel et al., eds., John Wiley & Sons, Inc.,
sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a
length that is at least 25 0 (more preferably at least 50g, and most
preferably at least 75~) of the length of the polynucleotide of
the present invention to which it hybridizes, and has at least
60°s sequence identity (more preferably, at least 75~ identity;
most preferably at least 900 or 95o identity) with the
polynucleotide of the present invention to which it hybridizes,
where sequence identity is determined by comparing the sequences
of the hybridizing polynucleotides when aligned so as to maximize
overlap and identity while minimizing sequence gaps.
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Sequence listing
<110> Sagami Chemical Research Center
<120> Human Proteins Having Transmembrane Domains and DNAs Encoding these
Proteins
<130> 660854
<140>
<141>
<150> Japan 9-276269
<151> 1997-10-08
I5 <160> 24
<170> Windows 95 (Word 98)
<210> 1
<211> 168
<212> PRT
<213> Homo Sapiens
<400> 1
Net Ala Phe Asn Asp Cys Phe Ser Leu Asn Tyr Pro Gly Asn Pro Cys
1 5 10 15
Pro Gly Asp Leu Ile Glu Val Phe Arg Pro Gly Tyr Gln His Trp Ala
20 25 30
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Leu Tyr Leu Gly Asp Gly Tyr Val Ile Asn ile Ala Pro Val Asp Gly
35 40 45
Ile Pro Ala Ser Phe Thr Ser Ala Lys Ser Val Phe Ser Ser Lys Ala
50 55 60
Leu Val Lys Met Gln Leu l.eu Lys Asp Val Val Gly Asn Asp Thr Tyr
65 70 75 80
Arg Ile Asn Asn Lys Tyr Asp Glu Thr Tyr Pro Pro Leu Pro Val Glu
85 90 95
Glu Ile Ile Lvs Arg Ser Glu Phe Val Ile Gly Gln Glu Val Ala Tyr
100 I05 110
Asn Leu Leu Val Asn Asn Cys Glu His Ph a Val Thr Leu Leu Arg Tyr
I15 120 125
Gly Glu Gly Val Ser Glu Gln rlla Asn Arg Ala Ile Ser Thr Val Glu
I30 135 140
Phe Val Thr Ala Ala Val Gly Val Phe S.er Phe Leu Gly Leu Phe Pro
145 150 155 160
Lys Gly Gln Arg Ala Lcs Tyr Tyr
165
<210> 2
<211> 164
<212> PRT
<213> Homo sapiens
<400> 2
Met Ala Ser Pro His Gln Glu Pro Lys Pro Gly Asp Leu Ile Glu Ile
I 5 10 15
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Phe Arg Leu Gly Tyr Glu His Trp Ala Leu Tyr Ile Gly Asp Gly Tyr
20 25 30
Val Ile His Leu Ala Pro Pro Ser Glu Tyr Pro Gly Ala Gly Ser Ser
35 40 45
Ser Val Phe Ser Val Leu Ser Asn Ser Ala Glu Val Lys Arg Glu Arg
50 55 60
Leu Glu Asp Val Val Gly Gly Cys Cys Tyr Arg Val Asn Asn Ser Leu
65 70 75 80
.asp His Glu Tyr Gln Pro tlrg Pro Val Glu Val Ile Ile Ser Ser Ala
85 90 95
Lys Glu Met Val Gly Gln Lys Met. L.vs Tyr Ser Ile Val Ser tlrg Asn
100 105 110
Cys Glu His Phe Val Thr Gln Leu Arg Tyr Gly Lys Ser Arg Cys Lys
115 120 125
Gln V'al Glu Lys Ala Lys Val Glu Val Gly Val Ala Thr Ala Leu Glv
130 135 140
Ile Leu V'al Val Ala Gly Cys Ser Phe Ala Ile .arg Arg Tyr Gln Lys
145 150 155 160
Lvs Ala Thr Ala
<210> 3
<211> 141
<212> PRT
<213> Homo sapiens
<400> 3
Met Ala Pro Lys Val Phe Arg Gln Tvr Trp Asp Ile Pro Asp Gly Thr
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1 5 10 15
:asp Cys His Arg Lys Ala Tyr Ser Thr Thr Ser Ile .ala Ser Val Ala
20 25 30
Gly Leu Thr Ala Ala Ala Tyr Arg 4'al Thr Leu Asn Pro Pro Gly Thr
35 40 45
Phe Leu Glu Gly Val Ala Lys Val Gly Gln Tyr Thr Phe Thr Ala Ala
50 55 60
Ala Val Gly Ala Val Phe Gly Leu Thr Thr Cys Ile Ser Ala His Val
65 70 75 80
:\rg Glu Lys Pro Asp :lsp Pro I_eu Asn Tyr Phe Leu Gly Gly Cys Ala
85 90 95
Gly Gly Leu Thr Leu Gly Ala Arg Thr His Asn Tyr Gly Ile Gly Ala
100 105 110
Ala Ala Cys Val Tyr Phe Gly Ile Ala Ala Ser Leu Val Lys Met Gly
115 120 125
Arg Leu Glu Gly Trp Glu Val Phe Ala Lys Pro Lys Val
13U 135 140
<210> 4
<211> 142
<212> PRT
<213> Homo sapiens
<400> 4
Net .ala Ala Ala Val Ala Ala Ala Gly tlla Gly Glu Pro Gln Ser Pro
1 5 10 15
Asp Glu Leu Leu Pro Lys Gly Asp Ala Glu Lys Pro Glu Glu Glu Leu
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20 25 30
Glu Glu Asp Asp Asp Glu Glu Leu Asp Glu Thr Leu Ser Glu Arg Leu
35 40 45
Trp Gly Leu Thr Glu 5let Phe Pro Glu Arg Val Arg Ser Ala Ala Gly
50 55 60
Ala Thr Phe Asp Leu Ser Leu Phe Val Ala Gln Lys Met Tyr Arg Phe
65 70 75 80
Ser Arg Ala Ala Leu Trp Ile Gly Thr Th r Ser Phe Met Ile Leu Val
85 90 95
Leu Pro Val Val Phe Glu Thr Glu Lys Leu Gln Met Glu Gln Gln Gln
100 105 110
Gln Leu Gln Gln Arg Gln Ile Leu Leu Gly Pro Asn Thr Gly Leu Ser
115 120 125
Gly Gly Met Pro Gly rlla Leu Pro Ser Leu Pro Gly Lys Ile
130 I35 140
<210> 5
<211> 346
<212> PRT
<2I3> Homo sapiens
<400>
Met Asp Pro Ala Arg Lys Ala Gly Ala Gln Ala Met Ile Trp Thr Ala
1 5 10 15
Gly Trp Leu Leu Leu Leu Leu Leu Arg Gly Gly Ala Gln Ala Leu GIu
20 25 30
Cys Tyr Ser Cys Val Gln Lys Ala Asp Asp Gly Cys Ser Pro Asn Lys
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35 40 45
VletLysThrValLysCysAlaProGlyValAspValCysThrGluAla
50 55 60
Val GlyAlaValGluThrIleHisGlyGlnPheSerLeuAlaValArg
65 70 75 80
Gly CysGlySerGlyLeuProGlyLysAsnAspArgGlyLeuAspLeu
85 90 95
His GlyLeuLeuAlaPheIleGlnLeuGlnGlnCysAlaGlnAspArg
100 105 110
Cys AsnAlaLysLeuAsnLeu'fhrSer,argAlaLeuAspProAlaGly
115 120 125
Asn GluSerAlaTyrProProAsnGlyValGluCysTyrSerCysVal
130 135 140
Gly LeuSerArgGluAlaCysGlnGlyThrSerProProValValSer
145 150 155 160
Cys TyrAsnAlaSer.aspHisValTyrLysGlyCysPheAspGlyAsn
165 170 175
Val ThrLeu'fhrAlaAlaAsnValThrValSerLeuProVal:lrgGlv
180 185 190
Cys V'alGlnAspGluPheCysThrArgAspGlyValThrGlyProGly
195 200 205
Phe ThrLeuSerGlySerCysCysGlnGlySerArgCysAsnSerAsp
210 215 220
l.euArgAsnLysThrTyrPheSerProArgIleProProLeuValArg
225 230 235 240
Leu ProProProGluProThrThrVal,AlaSerThrThrSerValThr
245 250 255
Thr SerThrSerAlaProValArgProThrSerThrThrLysProMet
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260 265 270
Pro Ala Pro Thr Ser Gln Thr Pro Arg Gln Gly Val Glu His Glu Ala
275 280 285
Ser Arg Asp Glu Glu Pro Arg Leu Thr Gly Gly Ala Ala Gly His Gln
290 295 300
Asp Arg Ser Asn Ser Gly Gln Tyr Pro Ala Lys Gly Gly Pro Gln Gln
305 310 315 320
Pro His Asn Lys Gly Cys Val Ala Pro Thr Ala Glv Leu Ala Ala Leu
325 330 335
Leu Leu Ala Val Ala A1a Gly Val Leu Leu
340 345
<210> 6
<211> 66
<212> PRT
<213> Homo sapiens
<400> 6
Met Val tlla Lys Gln Arg Ile Arg Met. Ala Asn Glu Lys His Ser Lys
1 5 10 15
Asn Ile Thr Gln Arg Gly Asn Val Ala Lys Thr Ser ,arg :\sn Ala Pro
20 25 30
Glu Glu Lys Ala Ser Val Gly Pro Trp Leu Leu Ala Leu Phe Ile Phe
35 40 45
Val Val Cys Gly Ser Ala Ile Phe Gln Ile Ile Gln Ser Ile Arg Met.
50 55 60
Glv Met
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<210> 7
5 <211> X04
<212> DNA
<213> Homo Sapiens
<400> 7
10 atggcgtt.taatgattgcttcagttt.gaactaccctggcaacccctgcccaggggacttg60
atcgaagt.gt tccgtcctggctatcagcactgggccctgtacttgggtgatggttacgtt.120
atcaacatag cacctgtagatggcattcctgcgtcctttacaagcgccaagtctgtattc180
agcagtaagg ccctggtgaaaatgcagctcttgaaggatgtt.gtgggaaatgacacatac240
agaataaaca ataaatacgat.gaaacgtacccccctctccctgtggaagaaatcataaag300
15 cggtcagagtttgtaattggacaggaggtggcctataactt.acttgtcaacaactgtgaa360
cattt.tgtga cattgcttcgctatggagaaggagt.ttcagagcaggccaaccgagcgata-120
agtaccgttg agttt.gtgacagctgctgttggtgtcttctcattcctgggctt.gtttcca-180
aaaggacaaa gagcaaaatactat X04
<210>8
<211>492
<212>DNA
<213>Homo Sapiens
<400> 8
atggcttcgc cacaccaaga gcccaaacct, ggagacctga ttgagattt.t ccgcctt.ggc 60
tatgagcact gggccctgta tataggagat ggctacgtga tccatctggc tcctccaagt. 120
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gagtaccccg gggctggctcctccagtgtcttctcagtcctgagcaacagtgcagaggtg180
aaacgggagc gcctggaagatgtggtgggaggctgttgctatcgggtcaacaacagcttg240
gaccatgagt accaaccacggcccgtggaggtgatcatcagttctgcgaaggagatggtt300
ggtcagaaga tgaagtacagtattgtgagcaggaactgtgagcactttgtcacccagctg360
agatatggcaagtcccgctgtaaacaggtggaaaaggccaaggttgaagtcggtgtggcc120
acggcgcttg gaatcctggttgttgctggatgctcttttgcgattaggagataccaaaaa=180
aaagcgacag cc 492
<210> 9
<211> 423
<212> DNA
<213> Homo sapiens
<400> 9
atggcgccga aggtttttcgtcagtactgggatatccccgatggcaccgattgccaccgc60
aaagcctaca gcaccaccagtattgccagcgt.cgctggcctgaccgccgctgcctacaga120
gtcacactca at.cct.ccgggcacct.tccttgaaggagtggctaaggttggacaatacacg180
ttcactgcag ctgctgtcggggccgtgtttggcctcaccacctgcatcagcgcccatgtc240
cgcgagaagcccgacgaccccctgaactactt.cctcggtggctgcgccggaggcctgact.300
ctgggagcac gcacgcacaact.acgggattggcgccgccgcctgcgtgtacttt.ggcata360
gcggcctccc t.ggtcaagatgggccggctggagggctgggaggtgtttgcaaaacccaag120
gtg 423
<210> to
<2I1> 426
<212> DNA
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<213> Homo sapiens
<400> 10
atggctgccg ccgtcgctgc t.gccggtgca ggggaacccc agtccccgga cgaatt.gctc 60
ccgaaaggcgacgcggagaagcctgaggaggagctggaggaggacgacgatgaggagcta120
gatgagaccc tgtcggagagactatggggcct.gacggagatgtttccggagagggtccgg180
tccgcggccg gagccacttttgatctttccctctttgtggctcagaaaatgtacaggttt240
tccagggcag ccttgtggatt.gggaccacttcctt.tatgatcctggttcttcccgttgtc300
tttgagacgg agaagttgcaaatggagcaacagcagcaactgcagcagcggcagatact:t.360
ctaggacct.aacacagggctctcaggaggaatgccaggggct.ctaccctcacttcctgga420
aagatc =126
<210> 11
<211>1038
<212> DNA
<213> Homo sapiens
<400> 11
atggaccccgccaggaaagcaggtgcccaggccatgatctggactgcaggctggctgctg60
ctgctgctgc ttcgcggaggagcgcaggccctggagtgctacagctgcgtgcagaaagca120
gatgacggat gctccccgaacaagatgaagacagtgaagt.gcgcgccgggcgtggacgtc180
tgcaccgagg ccgtgggggcggtggagaccat.ccacggacaattctcgctggcagtgcgg240
ggttgcggtt cgggact.ccccggcaagaatgaccgcggcctggatct.t.cacgggcttct.g300
gcgttcatccagctgcagcaatgcgctcaggatcgctgcaacgccaagctcaacctcacc360
tcgcgggcgc tcgacccggcaggtaatgagagtgcatacccgcccaacggcgtggagtgc=120
tacagctgtg tgggcctgagccgggaggcgtgccagggtacatcgccgccggtcgtgagc480
tgctacaacg ccagcgatcat.gtctacaagggctgcttcgacggcaacgtcaccttgacg540
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gcagctaatg tgactgtgtccttgcctgtccggggctgtgtccaggatgaattctgcact600
cgggatggag taacaggcccagggttcacgctcagtggctcctgttgccaggggtcccgc660
tgtaactctg acctccgcaacaagacctacttctcccctcgaatcccaccccttgtccgg720
ctgccccctc cagagcccacgactgtggcctcaaccacatctgtcaccacttctacctcg780
gccccagtgagacccacatccaccaccaaacccatgccagcgccaaccagtcagactccg840
agacagggag tagaacacgaggcctcccgggatgaggagcccaggttgactggaggcgcc900
gctggccacc aggaccgcagcaattcagggcagtatcctgcaaaaggggggccccagcag960
ccccataata aaggctgtgtggctcccacagctggattggcagcccttctgttggccgtg1020
gctgctggtg tcctactg 1038
lU
<210> 12
<211> 198
<212> DNA
<213> Homo sapiens
<400> 12
atggtcgcca agcaaaggat. ccgtatggcc aacgagaagc acagcaagaa catcacccag 60
cgcggcaacg tcgccaagac ct.cgagaaat gcccccgaag agaaggcgtc t.gtaggaccc 120
2U tggttattgg ctctcttcat tt.ttgttgtc tgtggttctg caattttcca gattattcaa I80
agtatcagga tgggcatg lgg
<210> 13
<211> 867
<212> DNA
<213> Homo sapiens
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<400> 13
cgttt.cagcg tggcggcgctggtgctggcgttggccctggaggacggccccgagtgatgg60
ctggcgcctg cctcccgggtgtctcccgggtacagatggagtcgtcccgcggccgccggc120
ggcaaggtcg gcagct.gcgaggccaagagagaccccaggacacacacagctgcctcccgg180
tgcgagaagaagaccccggct.tgagagtgag atg ttt aat tgc ttc 232
gcg gat
Met Ala Phe Asn Cys Phe
tlsp
I 5
agt tt.g tac cct. tgc cca gac tt.g gaa gtg 280
aac ggc ggg atc
aac
ccc
Ser Leu Asn Tyr Pro y :lsn Asp Leu Glu Val
Gl Pro Ile
Cys
Pro
Gly
10 15 20
ttc cgt. cct ggc t.at cag cac tgg gcc ctg tac ttg ggt gat ggt tac 328
Phe Arg Pro Gly Tyr Gln llis Trp Ala Leu Tyr Leu Gly ;lsp Gly Tyr
25 30 35
gtt atc aac ata gca cct gta gat. ggc att cct gcg tcc t.tt aca agc 376
Val Ile Asn Ile Ala Pro Val Asp Gly Ile Pro Ala Ser Phe 'fhr Ser
40 45 ~0 55
gcc aag tct gta t.tc agc agt aag gcc ctg gtg aaa atg cag ctc ttg =124
:11a Lys Ser Val Phe Ser Ser l.ys rlla Leu Val Lr,~s filet. Gln Leu Leu
60 65 70
aag gat gtt gtg gga aat gac aca tac aga ata aac aat aaa tac gat 472
Lys Asp Val Val Gly Asn Asp Thr Tyr Arg Ile Asn Asn Lys Tyr Asp
75 80 85
gaa acg tac ccc cct ctc cct gtg gaa gaa atc ata aag cgg tca gag 520
Glu Thr Tyr Pro Pro Leu Pro Val Glu Glu Ile Ile Lys Arg Ser Glu
90 95 100
tt.t gta at.t gga cag gag gtg gcc tat aac tta ctt gtc aac aac tgt 568
Phe Val Ile Gly Gln Glu Val Ala Tyr Asn Leu Leu Val Asn Asn Cvs
105 110 115
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gaa cat ttt gtg aca ttg ctt cgc tat gga gaa gga gtt tca gag cag 616
Glu His Phe Val Thr Leu Leu Arg Tyr Gly Glu Gly Val Ser Glu Gln
120 125 130 135
gce aac cga gcg ata agt acc gtt gag ttt gtg aca get get gtt ggt 661
Ala Asn Arg Ala Ile Ser Thr Val Glu Phe Val Thr Ala Ala Val Glv
140 1 ~l5 150
gtc ttc tca ttc ctg ggc ttg ttt cca aaa gga caa aga gca aaa tac 712
Val Phe Ser Phe Leu Gly Leu Phe Pro Lys Gly Gln Arg Ala Lys Tyr
I~~ 160 165
tat taaca at.Ltaccaaa gagatattga t.attgaagga atttgggagg aggaaaagaa 7i0
Tyr
acctggggtg aatacttatt ttcagt.gcat cattactgtt ccagatt.cct atgatggatg 830
gcagactctt taataaattg ct.tactgata ttatctt 867
<210> 19
<2il> 168
<212> PRT
<213> Homo sapiens
<400> l~l
Net Ala Phe Asn Asp Cys Phe
1 5
Ser I_eu Asn Tyr Pro Gly Asn Pro Cys Pro Gly Asp Leu Ile Glu Val
10 15 20
Phe Arg Pro Gly Tyr Gln His Trp Ala Leu Tyr Leu Gly Asp Gly Tyr
25 30 35
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Val Ile Asn Ile Ala Pro Val Asp Gly Ile Pro Ala Ser Phe Thr Ser
40 45 50 55
Ala Lys Ser Val Phe Ser Ser Lys Ala Leu 4'al Lys Met Gln Leu Leu
60 65 70
Lys Asp Val Val Gly Asn Asp Thr Tyr ,4rg Ile Asn Asn Lys Tyr Asp
75 80 85
Glu Thr Tyr Pro Pro Leu Pro Val Glu Glu Ile Ile Lys Arg Ser Glu
90 95 100
Phe bal Ile Gly Gln Glu Val Ala Tyr .asn Leu Leu Val Asn Asn Cys
105 110 115
Glu His Phe Val Thr Leu Leu rlrg Tyr Gly Glu Gly Val Ser Glu Gln
120 125 130 135
Ala Asn Arg rlla Ile Ser Ttir Val Glu Phe Val Thr Ala Ala Val Gly
140 145 150
Val Phe Ser Phe Leu Gly Leu Phe Pro Lys Gly Gln Arg Ala Lys Tyr
i55 160 165
Tvr
<210> I5
<211> 720
<212> DNA
<213> Homo sapiens
<400> 15
aceagacctc ctettggett cgag at.g get teg cca cac caa gag ccc aaa 51
Met Ala Ser Pro His Gln Glu Pro Lys
1 5
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
15/28
cct gga gac ctg att gag att ttc cgc ctt ggc tat gag cac tgg gcc 99
Pro Gly Asp Leu Ile Glu Ile Phe Arg Leu Gly Tyr Glu His Trp Ala
15 20 25
ctg tat ata gga gat ggc tac gtg at.c cat ctg get cct. cca agt gag 147
5 Leu Tyr Ile Gly Asp Gly Tyr Val Ile His Leu Ala Pro Pro Ser Glu
30 35 40
tac ccc ggg get gge tcc t.cc agt gtc ttc tca gtc ctg agc aac agt 195
Tyr Pro Gly Ala Gly Ser Ser Ser Val Phe Ser V'al Leu Ser Asn Ser
45 50 55
10 gca gag gt.g aaa cgg gag cgc ctg gaa gat gtg gt.g gga ggc t.gt t.gc 243
Ala Glu Val Lys Arg Glu Arg Leu Glu Asp Val Val Gly Gly Cys Cys
60 60 70
tat cgg gtc aac aac agc ttg gac cat gag tac caa cca cgg ccc gtg 291
Tyr Arg Val Asn Asn Ser Leu Asp His Glu Tyr Gln Pro Arg Pro Val
75 80 85
gag gt.g atc atc agt tct gcg aag gag atg gtt ggt cag aag at.g aag 339
Glu Val Ile Ile Ser Ser Ala Lys Glu Met Val Gly Gln Lys Met Lys
90 95 100 105
tac agt att gtg agc agg aac tgt gag cac ttt gtc acc cag ctg aga 387
Tyr Ser Ile Val Ser Arg Asn Cys Glu His Phe Val Thr Gln Leu Arg
110 115 120
tat ggc aag tcc cgc tgt aaa cag gt.g gaa aag gcc aag gt,t gaa gtc 435
Tyr Gly Lys Ser Arg Cys Lys Gln Val Glu Lys Ala Lys Val Glu Val
125 130 135
2 5 ggt gtg gcc acg geg ctt gga atc ctg gtt gtt get gga tgc tct ttt 483
Gly Val Ala Thr Ala Leu Gly Ile Leu Val Val Ala Gly Cys Ser Phe
140 145 150
gcg att agg aga tac caa aaa aaa gcg aca gcc tgaa gcagccacaa 530
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
16/28
Ala Ile Arg Arg Tyr Gln Lys Lys Ala Thr Ala
155 160
aatcctgtgt t.agaagcagc t.gtgggggtc ccagtggaga tgagcctccc ccatgcctcc
agcagcctga ccctcgtgcc ctgtctcagg cgttctctag atcctttcct ctgtttccct. 650
ctctcgctgg caaaagtatg atctaattga aacaagactg aaggatcaat aaacagccat 710
ctgccccttc 720
<210> 16
<211> 164
<212> PRT
<213> Homo sapiens
<400> 16
Met tlla Ser Pro His Gln Glu Pro Lvs
1 5
Pro Gly Asp Leu lle Glu Ile Phe Arg (_eu GIy 'Tyr Glu His Trp Ala
10 15 2.0 25
Leu 'Tyr Ile Gly Asp Gly 'ryr Val Ile His Leu Ala Pro Pro Ser Glu
30 35 40
Tyr Pro Gly AIa Gly Ser Ser Ser Val Phe Ser Val Leu Ser Asn Ser
=15 50 55
Ala Glu V'al Lys Arg Glu Arg Leu Glu Asp Val Val Gly Gly Cys Cys
60 65 70
hyr Arg Val Asn Asn Ser Leu Asp His Glu Tyr Gln Pro Arg Pro Val
75 80 85
Glu Val Ile Ile Ser Ser Ala Lys Glu Met Val Gly Gln Lys Met Lys
90 95 100 105
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
17/28
Tyr Ser Ile Val Ser Arg Asn Cys Glu His Phe Val Thr Gln Leu Arg
110 II5 120
Tyr Gly Lys Ser Arg Cys Lys Gln Val Glu Lys Ala Lys Val Glu Val
125 130 135
Gly Val Ala Thr Ala Leu Gly Ile Leu Val Val Ala Gly Cys Ser Phe
140 145 150
Ala Ile Arg Arg Tyr Gln Lys Lys Ala Thr Ala
155 160
<210>17
<211>566
<212>DNA
<213>Homo sapiens
<400> 17
gatagccagc cgcggctgcc ct.t.gcgcttc ccgagctggc ggggtccgtg gtgcgggatc 60
gagattgcgg get atg gcg ccg aag gtt ttt cgt cag tac tgg gat at.c 109
Met :ala Pro Lys Val Phe .arg Gln '('yr Trp Asp Ile
1 5 10
ccc gat ggc acc gat tgc cac cgc aaa gcc tac agc acc acc agt att 1~7
Pro Asp Gly Thr Asp Cys His Arg Lys Ala Tyr Ser Thr Thr Ser Ile
15 20 25
gcc age gte get ggc ctg acc gcc get gec tac aga gtc aca ctc aat 205
2 5 Ala Ser Val Ala Gly Leu Thr Ala Ala Ala Tyr Arg Val Thr Leu Asn
3U 35 40
ect ecg gge acc ttc ct.t gaa gga gtg get aag gtt gga caa tac acg 253
Pro Pro Gly Thr Phe Leu Glu Gly Val Ala Lys Val Gly Gln Tyr Thr
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
18/28
45 50 55 60
ttc actgcagetgetgtc ggggccgtgtt.tggcctcaccacctgcatc 301
Phe ThrAlaAlaAlaVal GlyAlaValPheGlyLeuThrThrCysIle
65 70 75
agc gcccatgtccgcgag aagcccgacgaccccctgaactacttcctc 349
Ser AlaHisValArgGlu LysProAspAspProLeuAsnTyrPheLeu
80 85 90
ggt ggctgcgccggaggc ctgactctgggagcacgcacgcacaactac 397
Gly GlyCys.~11aGlyGly LeuThrLeuGlyAlaArgThrHisAsnTyr
95 l0U 105
ggg attggcgccgccgcc t.gcgtgtacttt.ggcatagcggcctccctg 445
Gly IleGlyAlaAlaAla CysValTyrPheGIyIleAlaAlaSerLeu
110 115 120
gtc aagatgggccggctg gagggct,gggaggtgtttgcaaaacccaag 493
Val LysMetGlyArgLeu GluGlyTrpGluValPheAlaLysProLys
125 130 135 140
gtg tgag gccgggacct agaaataaat 550
ccctgtgcct ccagcctgca
gaatgcgtcc
Val
tctgtgtctg t.gtgtg 566
<210> 18
<211> 141
<212> PRT
<213> Homo sapiens
<400> 18
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
19/28
Met tlla Pro Lys Val Phe Arg Gln Tyr Trp Asp Ile
1 5 10
Pro Asp Gly Thr Asp Cys His Arg Lys Ala Tyr Ser Thr Thr Ser Ile
15 20 25
Ala Ser Val Ala Gly Leu Thr Ala Ala Ala Tyr Arg Val Thr Leu Asn
30 35 40
Pro Pro Gly Thr Phe Leu Glu Gly Val Ala Lvs Val Gly Gln Tyr Thr
45 50 55 60
Phe Thr Ala Ala Ala Val Gly Ala Val Phe Gly Len Thr Thr Cys Ile
65 70 75
Ser Ala His Val Arg Glu Lys Pro Asp Asp Pro Leu Asn Tyr Phe Leu
80 85 90
Gly Gly Cys Ala Gly Gly Leu Thr Leu Gly Ala Arg Thr His Asn Tyr
95 100 105
Gly Ile Gly Ala Ala Ala Cys Val Tyr Phe Gly Ile Ala Ala Ser Leu
110 115 120
Val Lys Met Gly Arg Leu Glu Gly Trp Glu Val Phe Ala Lys Pro Lys
125 130 135 1=10
Val
<210> 19
<211> 1078
<212> DNA
2 5 <213> Homo sapiens
<400> 19
agtc atg get gcc gcc gtc get get gcc ggt gca ggg gaa ccc cag tcc 49
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
20128
Met Ala Ala Ala Val Ala Ala Ala Gly Ala Gly Glu Pro Gln Ser
1 5 10 1~
ccg gac gaa ttg ctc ccg aaa ggc gac gcg gag aag cct gag gag gag 97
Pro Asp Glu Leu Leu Pro Lys Gly Asp Ala Glu Lys Pro Glu Glu Glu
20 25 30
ctg gag gag gac gac gat gag gag cta gat gag acc ctg tcg gag aga 145
Leu Glu Glu Asp Asp Asp Glu Glu Leu Asp Glu Thr Leu Ser Glu Arg
35 40 45
cta tgg ggc ctg acg gag atg ttt ccg gag agg gtc cgg tcc gcg gcc 193
Leu Trp Glv Leu Thr Glu Met. Phe Pro Glu Arg Val Arg Ser .ala Ala
50 ~~ 60
gga gcc act ttt gat ctt tcc ctc ttt gtg get cag aaa atg tac agg 241
Gly Ala Thr Phe Asp Leu Ser Leu Phe Val Ala Gln Lys Met Tyr Arg
65 70 75
ttt tcc agg gca gcc ttg tgg att ggg acc act tcc ttt atg atc ctg 289
Phe Ser Arg Ala Ala Leu Trp Ile Gly Thr Thr Ser Phe Met Ile Leu
80 85 90 95
gtt ctt, ccc gtt gtc ttt. gag acg gag aag tt.g caa atg gag caa cag 337
Val Leu Pro Val Val Phe Glu Thr Glu Lys Leu Gln Met Glu Gln Gln
100 105 110
cag caa ctg cag cag cgg cag ata ctt cta gga cct aac aca ggg ctc 385
Gln Gln Leu Gln Gln Arg Gln Ile Leu Leu Gly Pro Asn Thr Gly Leu
115 120 125
tca gga gga atg cca ggg get cta ccc tca ctt cct gga aag atc 430
Ser Gly Gly liet Pro Gly Ala Leu Pro Ser Leu Pro Gly Lys Ile
130 135 140
tagattgtta ttgctgtttg agctgtctca gtgggataag tttgaaattc aagtgtttga 490
actgctgata atttggattt tt.tttttttt ttttaacttt ggcacattga tctatctaaa 550
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
21/28
cccggtgggg agaattatccccacattgtctcatggaaagactcaacttgcaactgtgcc610
ctccacacta tccttacttctgtctccactctgataccagagtgcagccatgcagacggt670
tattccagct ctggtcacccgactcctttcaccaaattgctcctaactggaagatctcac730
tttccccttg tggggtaggaaccgatgccagtgggagggatgtgcccctgaccattaacg790
actgttttttttttttttttttaaagaatggagttgttggggcgggacatgcacacaatg850
tgaaacagac aaaatgcattacacctgtagtgtaaagtggccactatgaatccctatgta910
tgagaggagg gaggcaggctgcagcttcagccacagaatggggactatggaagacagcag970
gagctcattt cctctgcacatttcggctgttagacctgtgtgtgtgtttaaaaaaagaga1030
agtcagtgct cactttttgtatttaaatattaaaaatgattccaactg 1078
<210> 20
<211> 142
<212> PRT
<213> Homo sapiens
<400> 20
Met Ala Ala Ala Val Ala Ala Ala Gly Ala Gly Glu Pro Gln Ser
1 5 10 1~
Pro Asp Glu Leu Leu Pro Lys Gly Asp Ala Glu Lys Pro Glu Glu Glu
20 25 30
Leu Glu Glu Asp Asp Asp Glu Glu Leu Asp Glu Thr Leu Ser Glu Arg
35 40 45
Leu Trp Gly Leu Thr Glu Met Phe Pro Glu Arg Val Arg Ser Ala Ala
50 55 60
Gly Ala Thr Phe Asp Leu Ser Leu Phe Val Ala Gln Lys Met Tyr Arg
65 70 75
Phe Ser Arg Ala Ala Leu Trp Ile Gly Thr Thr Ser Phe Met Ile Leu
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
22/28
80 85 90 95
Val Leu Pro Val Val Phe Glu Thr Glu Lys Leu Gln Met Glu Gln Gln
100 105 110
Gln Gln Leu Gln Gln Arg Gln Ile Leu Leu Gly Pro Asn Thr Gly Leu
115 120 125
Ser Gly Gly Met Pro Gly Ala Leu Pro Ser Leu Pro Gly Lys Ile
130 135 140
<210> 21
<211> 1310
<212> DNA
<213> Homo sapiens
<400> 21
actcatcctg ggctcaggta agagggcccg agct.cggagg cggcacatcc aggggggacg 60
ccaagggagc aggacggagc c atg gac ccc gcc agg aaa gca ggt gcc cag 111
Met Asp Pro Ala Arg Lys .ala Gly Ala Gln
1 5 10
gcc atg atc tgg act gca ggc tgg ctg ctg ctg ctg ctg ctt cgc gga 159
Ala Met Ile Trp Thr Ala Gly Trp Leu Leu Leu Leu Leu Leu Arg Gly
15 20 25
gga gcg cag gcc ctg gag tgc tac agc tgc gtg cag aaa gca gat gac 207
Gly Ala Gln .Ala Leu Glu Cys Tyr Ser Cys Val Gln Lys Ala Asp Asp
30 35 40
gga tgc tcc ccg aac aag at.g aag aca gtg aag tgc gcg ccg ggc gt.g 255
Gly Cys Ser Pro Asn Lys Met Lys Thr Val Lys Cys Ala Pro Gly Val
45 50 55
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
23!28
gac gtc tgc acc gag gcc gtg ggg gcg gtg gag acc atc cac gga caa 303
Asp 4'al Cys Thr Glu Ala Val Gly Ala Val Glu Thr Ile His Gly Gln
60 65 70
ttc tcg ctg gca gtg cgg ggt tgc ggt tcg gga ctc ccc ggc aag aat 351
Phe Ser Leu Ala Val Arg Gly Cys Gly Ser Gly Leu Pro Gly Lys Asn
75 80 85 90
gac cgc ggc ctg gat ctt cac ggg ctt ctg gcg ttc atc cag ctg cag 399
Asp Arg Gly Leu Asp Leu His Gly Leu Leu Ala Phe Ile Gln Leu Gln
95 100 105
caa tgc get cag gat cgc tgc aac gcc aag ctc aac ctc acc tcg cgg 447
Gln Cys Ala Gln Asp Arg Cys Asn Ala Lys Leu Asn Leu Thr Ser Arg
110 115 120
gcg ctc gac ccg gca ggt aat gag agt gca tac ccg ccc aac ggc gtg 495
Ala Leu Asp Pro Ala Gly Asn Glu Ser Ala Tyr Pro Pro Asn Gly Val
125 130 135
gag tgc tac agc tgt gtg ggc ctg agc cgg gag gcg tgc cag ggt aca 543
Glu Cys Tyr Ser Cys Val Gly Leu Ser Arg Glu Ala Cys Gln Gly Thr
140 145 150
tcg ccg ccg gtc gtg agc tgc tac aac gcc agc gat cat gtc tac aag 59I
Ser Pro Pro Val Val Ser Cys Tyr Asn Ala Ser Asp His Val Tyr Lys
155 160 165 170
ggc tgc ttc gac ggc aac gtc acc ttg acg gca get aat gtg act gtg 639
Gly Cys Phe Asp GIy Asn Val Thr Leu Thr Ala Ala Asn Val Thr Val
I75 180 185
2 5 tcc ttg cct gtc cgg ggc tgt gtc cag gat gaa ttc tgc act cgg gat 687
Ser Leu Pro Val Arg Gly Cys Val Gln Asp Glu Phe Cys Thr Arg Asp
190 195 200
gga gta aca ggc cca ggg ttc acg ctc agt ggc tcc tgt tgc cag ggg 735
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
24/28
Gly Val Thr Gly Pro Gly Phe Thr Leu Ser Gly Ser Cys Cys Gln Gly
205 210 215
tcc cgc tgt aac tct gac ctc cgc aac aag acc tac ttc tcc cct cga 783
Ser Arg Cys Asn Ser Asp Leu Arg Asn Lys Thr Tyr Phe Ser Pro Arg
220 225 230
atc cca ccc ctt gtc cgg ctg ccc cct cca gag ccc acg act gtg gcc 831
Ile Pro Pro Leu Val Arg Leu Pro Pro Pro Glu Pro Thr Thr Val Ala
235 240 245 250
tca acc aca tct gtc acc act tct acc tcg gcc cca gtg aga ccc aca 879
Ser Thr Thr Ser Val Thr Thr Ser Thr Ser Ala Pro Val Arg Pro 'fhr
255 260 265
tcc acc acc aaa ccc atg cca gcg cca acc agt cag act ccg aga cag 927
Ser Thr Thr Lys Pro Met Pro Ala Pro Thr Ser Gln Thr Pro Arg Gln
270 275 280
gga gta gaa cac gag gcc t.cc cgg gat gag gag ccc agg ttg act gga 975
Gly Val Glu His Glu Ala Ser Arg Asp Glu Glu Pro Arg Leu Thr Gly
285 290 295
ggc gce get gge eac cag gac cgc agc aat. tca ggg cag tat. cct gca 1023
Gly Ala Ala Gly His Gln tlsp Arg Ser Asn Ser Gly Gln Tyr Pro Ala
300 305 310
aaa ggg ggg cee eag cag ccc cat aat aaa ggc tgt gtg get ecc aca 1071
Lys Gly Gly Pro Gln Gln Pro His Asn Lys Gly Cys Val Ala Pro Thr
315 320 325 330
get gga ttg gca gce ctt etg ttg gcc gtg get get ggt gtc cta ctg 1119
tlla Gly Leu Ala Ala Leu Leu Leu Ala Val Ala Ala Glv Val Leu Leu
335 340 345
t gagcttctcc acctggaaat. ttccctctca cctacttctc tggccctggg 1170
tacccctctt ctcatcactt cctgttccca ccactggact gggctggccc agcccctgtt 1230
CA 02308120 2000-04-10
WO 99/18202 PGT/JP98/04474
25128
tttccaacat tccccagtat ccccagcttc tgctgcgctg gtttgcggct ttgggaaata 1290
aaataccgtt gtatatattc 1310
<210> 22
<211> 346
<212> PRT
<213> Homo sapiens
<400> 22
Met Asp Pro Ala Arg Lys Ala Gly Ala Gln
1 5 10
Ala Stet Ile Trp Thr Ala Gly Trp Leu Leu Leu Leu Leu Leu Arg Gly
20 25
15 Gly Ala Gln Ala Leu Glu Cys Tyr Ser Cys Val Gln Lys Ala Asp Asp
30 35 4U
Gly Cys Ser Pro Asn Lys Met Lys Thr Val Lys Cys Ala Pro Gly Val
45 50 55
Asp Val Cys Thr Glu Ala Val Gly tlla Val Glu Thr Ile His Glv Gln
60 65 70
Phe Ser Leu Ala Val Arg Gly Cys Gly Ser Gly Leu Pro Gly Lys Asn
75 80 85 90
Asp Arg Gly Leu Asp Leu His Gly Leu Leu Ala Phe Ile Gln Leu Gln
95 100 105
Gln Cys Ala Gln Asp Arg Cys Asn Ala Lys Leu Asn Leu Thr Ser Arg
110 115 120
Ala Leu Asp Pro Ala Gly Asn Glu Ser Ala Tyr Pro Pro Asn Gly Val
125 130 135
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
26/28
Glu CysTyrSerCysValGlyLeuSerArgGluAlaCysGlnGlyThr
140 145 150
Ser ProProValValSerCysTyrAsnAlaSerAspHisValTyrLys
155 160 165 170
Gly CysPheAspGlyAsnValThrLeuThrAlaAlaAsnValThrVal
175 180 185
Ser LeuProValArgGlyCysValGlnAspGluPheCysThrArgAsp
190 195 200
Gly ValThrGlyProGlyPheThrLeuSerGlySerCysCysGlnGly
205 210 215
Ser ArgCysAsnSer:lspLeuArgAsnLysThrTyrPheSerProArg
220 225 230
Ile ProProLeuValArgLeuProProProGluProThrThrValAla
235 240 245 250
Ser ThrThrSerValThrThrSerThrSerAlaProValArgProThr
255 260 265
Ser ThrThrLysProMetProAlaProThrSerGlnThrProArgGln
270 275 280
Gly ValGluHisGluAlaSerArgAspGluGluProArgl.euThrGly
285 290 295
Gly AlaAlaGlyHisGlnAspArgSerAsnSerGlyGlnTyrProAla
300 305 310
Lys GlyGlyProGlnGlnProHisAsnLysGlyCysValAlaProThr
315 320 325 330
Ala GlyLeuAlaAlaLeuLeuLeuAlaValAlaAlaGlyValLeuLeu
335 340 345
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
27/28
<210> 23
<211> 781
<212> DNA
<213~ Homo sapiens
<400> 23
caattcccgt tgttgcgttgcgtttccttc ctct.ttcactccgcgctcac ggcggcggcc60
aaagcggcgg cgacggcggcgcgagaacga cccggcggccagttctcttc ctcctgcgca120
cctgccctgc tcggtcagtcagtcggcggc cggcgcccggcttgtgctca gacctcgcgc1$0
ttgcggcgcccaggcccagcggccgtagct agcgt.ctggcctgagaacct cggcgctccg240
gcggcgcggg caccacgagcggagcctcgc agcggctccagaggaggcag gcgagtgagc300
gagtccgagg ggtggccggggcaggtggtg gcgccgcgaag atg gtc gcc 356
aag caa
Met Val Ala Lys
Gln
1 5
agg atc atg gcc aac atc acc cag =104
cgt aac cgc
gag
aag
cac
agc
aag
Arg Ile Arg Met Ala Asn lle Thr Gln
Asn Arg
Glu
Lys
His
Ser
Lys
10 15 20
ggc aac gtc gcc aag gaa gag aag gcg =la2
acc tct
rcg
aga
aat
gcc
ccc
Gly Asn Val Ala Lys r Ser Arg Asn Glu Glu Lvs Ala
Th Ala Pro Ser
25 30 35
gta gga ccc tgg tta ttg get ctc ttc att ttt gtt gtc tgt ggt tct
Val Gly Pro Trp Leu Leu Ala Leu Phe Ile Phe Val Val Cys Gly Ser
40 45 50
gca att ttc cag att att caa agt atc agg atg ggc atg t gaagtgactg a50
Ala Ile Phe Gln Ile Ile Gln Ser Ile Arg Met Gly Met
55 60 65
accttaagat gtttccattc tcctgtgaat tttaacttga actcattcct gatgtttgat 610
accctggttg aaaacaattc agtaaagcat. cctgcctcag aatgactttc ctatcatgct. 670
CA 02308120 2000-04-10
WO 99/18202 PCT/JP98/04474
28/28
tcatgtgtca ttccaaggtt tcttcatgag tcattccaag ttttctagtc cataccacag 730
tgccttgcaa aaaacaccac atgaataaag caataaaatt tgattgttaa g 781
<210> 24
<211> 66
<212> PRT
<213> Homo sapiens
Met Val Ala Lvs Gln
1 5
Arg Ile Arg Met Ala Asn Glu Lys His Ser Lys Asn Ile Thr Gln Arg
10 15 20
Gly Asn Val Ala Lys Thr Ser Arg Asn Ala Pro Glu Glu Lys Ala Ser
25 30 35
Val GIy Pro Trp Leu Leu Ala Leu Phe Ile Phe Val Val Cys Gly Ser
40 45 50
Ala Ile Phe Gln Ile Ile Gln Ser Ile Arg Met Gly Met
~5 60 65
