U.S. patent application number 10/678274 was filed with the patent office on 2004-05-20 for human pelota homolog.
This patent application is currently assigned to SmithKline Beecham Corp.. Invention is credited to Hansbury, Michael Joseph, Jackson, Jeffrey Richard.
Application Number | 20040096888 10/678274 |
Document ID | / |
Family ID | 25400394 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040096888 |
Kind Code |
A1 |
Jackson, Jeffrey Richard ;
et al. |
May 20, 2004 |
Human pelota homolog
Abstract
68772 polypeptides and polynucleotides and methods for producing
such polypeptides by recombinant techniques are disclosed. Also
disclosed are methods for utilizing 68772 polypeptides and
polynucleotides in the design of protocols for the treatment of
proliferative diseases such as leukemias, solid tumor cancers and
metastases; chronic inflammatory proliferative diseases such as
psoriasis and rheumatoid arthritis; proliferative cardiovascular
diseases such as restenosis; prolifertive ocular disorders such as
diabetic retinopathy; and benign hyperproliferative diseases such
as hemangiomas, among others, and diagnostic assays for such
conditions.
Inventors: |
Jackson, Jeffrey Richard;
(Collegeville, PA) ; Hansbury, Michael Joseph;
(Collingswood, NJ) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
SmithKline Beecham Corp.
Philadelphia
PA
19101
|
Family ID: |
25400394 |
Appl. No.: |
10/678274 |
Filed: |
October 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10678274 |
Oct 6, 2003 |
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10023895 |
Dec 21, 2001 |
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6657047 |
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10023895 |
Dec 21, 2001 |
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09265642 |
Mar 10, 1999 |
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6342584 |
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09265642 |
Mar 10, 1999 |
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09145947 |
Sep 2, 1998 |
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5925539 |
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09145947 |
Sep 2, 1998 |
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08892715 |
Jul 15, 1997 |
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5922853 |
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Current U.S.
Class: |
435/6.16 ;
435/183; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C07K 14/4702 20130101;
A61P 9/08 20180101; A61P 43/00 20180101; A61P 29/00 20180101; A61P
35/02 20180101; A61P 35/00 20180101; A61P 9/00 20180101; A61K 38/00
20130101; A61P 27/02 20180101; A61P 3/10 20180101; A61P 17/06
20180101; A61P 35/04 20180101; A61P 17/00 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/183; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/00 |
Claims
What is claimed:
1. An isolated polynucleotide comprising a nucleotide sequence that
has at least 80% identity over its entire length to a nucleotide
sequence encoding the 68772 polypeptide of SEQ ID NO:2; or a
nucleotide sequence complementary to said polynucleotide.
2. The polynucleotide of claim 1 wherein said polynucleotide
comprises the nucleotide sequence contained in SEQ ID NO:1 encoding
the 68772 polypeptide of SEQ ID NO:2.
3. The polynucleotide of claim 1 wherein said polynucleotide
comprises a nucleotide sequence that is at least 80% identical to
that of SEQ ID NO:1 over its entire length.
4. The polynucleotide of claim 3 which is polynucleotide of SEQ ID
NO:1.
5. The polynucleotide of claim 1 which is DNA or RNA.
6. A DNA or RNA molecule comprising an expression system, wherein
said expression system is capable of producing a 68772 polypeptide
comprising an amino acid sequence, which has at least 80% identity
with the polypeptide of SEQ ID NO:2 when said expression system is
present in a compatible host cell.
7. A host cell comprising the expression system of claim 6.
8. A process for producing a 68772 polypeptide comprising culturing
a host cell of claim 7 under conditions sufficient for the
production of said polypeptide and recovering the polypeptide from
the culture.
9. A process for producing a cell which produces a 68772
polypeptide thereof comprising transforming or transfecting a host
cell with the expression system of claim 6 such that the host cell,
under appropriate culture conditions, produces a 68772
polypeptide.
10. A 68772 polypeptide comprising an amino acid sequence which is
at least 80% identical to the amino acid sequence of SEQ ID NO:2
over its entire length.
11. The polypeptide of claim 10 which comprises the amino acid
sequence of SEQ ID NO:2.
12. An antibody immunospecific for the 68772 polypeptide of claim
10.
13. A method for the treatment of a subject in need of enhanced
activity or expression of 68772 polypeptide of claim 10 comprising:
(a) administering to the subject a therapeutically effective amount
of an agonist to said polypeptide; and/or (b) providing to the
subject an isolated polynucleotide comprising a nucleotide sequence
that has at least 80% identity to a nucleotide sequence encoding
the 68772 polypeptide of SEQ ID NO:2 over its entire length; or a
nucleotide sequence complementary to said nucleotide sequence in a
form so as to effect production of said polypeptide activity in
vivo.
14. A method for the treatment of a subject having need to inhibit
activity or expression of 68772 polypeptide of claim 10 comprising:
(a) administering to the subject a therapeutically effective amount
of an antagonist to said polypeptide; and/or (b) administering to
the subject a nucleic acid molecule that inhibits the expression of
the nucleotide sequence encoding said polypeptide; and/or (c)
administering to the subject a therapeutically effective amount of
a polypeptide that competes with said polypeptide for its ligand,
substrate, or receptor.
15. A process for diagnosing a disease or a susceptibility to a
disease in a subject related to expression or activity of 68772
polypeptide of claim 10 in a subject comprising: (a) determining
the presence or absence of a mutation in the nucleotide sequence
encoding said 68772 polypeptide in the genome of said subject;
and/or (b) analyzing for the presence or amount of the 68772
polypeptide expression in a sample derived from said subject.
16. A method for identifying compounds which inhibit (antagonize)
or agonize the 68772 polypeptide of claim 10 which comprises: (a)
contacting a candidate compound with cells which express the 68772
polypeptide; and (b) observing the binding, or stimulation or
inhibition of a functional response; or comparing the ability of
the cells (or cell membrane) which were contacted with the
candidate compounds with the same cells which were not contacted
for 68772 polypeptide activity.
17. An agonist identified by the method of claim 16.
18. An antagonist identified by the method of claim 16.
19. An isolated 68772 polynucleotide comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence having at least 80% identity to a nucleotide sequence
encoding the 68772 polypeptide expressed by the cDNA insert
deposited at the ATCC with Deposit Number ATCC 98438; and (b) a
nucleotide sequence complementary to the nucleotide sequence of
(a).
20. A recombinant host cell produced by a method of claim 9 or a
membrane thereof expressing a 68772 polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/023,895, filed Dec. 21, 2001, which is a divisional of U.S.
application Ser. No. 09/265, 642, filed Mar. 10, 1999 (now U.S.
Pat. No. 6,342,584), which is a divisional of 09/145,947, filed
Sep. 2, 1998 (now U.S. Pat. No. 5,925,539), which is a divisional
of 08/892,715, filed Jul. 15, 1997 (now U.S. Pat. No.
5,922,853).
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by them and to the use of such polynucleotides
and polypeptides, and to their production. More particularly, the
polynucleotides and polypeptides of the present invention relate to
Pelota family, hereinafter referred to as 68772. The invention also
relates to inhibiting or activating the action of such
polynucleotides and polypeptides.
BACKGROUND OF THE INVENTION
[0003] Regulation of the cell cycle is controlled by a family of
cyclins, cyclin dependent kinases (CDKs), CDK regulatory kinases,
and phosphatases. See, Lees, E., Curr. Opin. Cell. Biol. 1995,
7:773-780; Piwinica-Worms, H., J. Lab. Clin. Med. 1996,
128:350-354. Progression from the G2 phase to the M phase of the
cell cycle requires the activity of cdc25 phosphatase. In
Drosophila, mutations in the pelota gene have the same phenotype as
mutations in the twine and/or string genes, which are cdc25
homologs, Eberhart, C. G. and Wasserman. S. A., Devel. 1995,
121:3477-3486. Specific cell cycle effects of pelota mutations are
seen in both meiosis and mitosis, including G2/M arrest between
mitotic and meiotic cell division, and disruption of nuclear
envelope breakdown and spindle formation. Regulation of pelota
offers a means of controlling a critical event in the cell
cycle.
[0004] This indicates that the Pelota family has an established,
proven history as therapeutic targets. Clearly there is a need for
identification and characterization of further members of Pelota
family which can play a role in preventing, ameliorating or
correcting dysfunctions or diseases, including, but not limited to,
proliferative diseases such as leukemias, solid tumor cancers and
metastases; chronic inflammatory proliferative diseases such as
psoriasis and rheumatoid arthritis; proliferative cardiovascular
diseases such as restenosis; proliferative ocular disorders such as
diabetic retinopathy; and benign hyperproliferative diseases such
as hemangiomas.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention relates to 68772 polypeptides
and recombinant materials and methods for their production.
[0006] Another aspect of the invention relates to methods for using
such 68772 polypeptides and polynucleotides. Such uses include the
treatment of proliferative diseases such as leukemias, solid tumor
cancers and metastases; chronic inflammatory proliferative diseases
such as psoriasis and rheumatoid arthritis; proliferative
cardiovascular diseases such as restenosis; proliferative ocular
disorders such as diabetic retinopathy; and benign
hyperproliferative diseases such as hemangiomas, among others. In
still another aspect, the invention relates to methods to identify
agonists and antagonists using the materials provided by the
invention, and treating conditions associated with 68772 imbalance
with the identified compounds. Yet another aspect of the invention
relates to diagnostic assays for detecting diseases associated with
inappropriate 68772 activity or levels.
DESCRIPTION OF THE INVENTION
[0007] Definitions
[0008] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0009] "68772" refers, among others, generally to a polypeptide
having the amino acid sequence set forth in SEQ ID NO:2 or an
allelic variant thereof.
[0010] "68772 activity or 68772 polypeptide activity" or
"biological activity of the 68772 or 68772 polypeptide" refers to
the metabolic or physiologic function of said 68772 including
similar activities or improved activities or these activities with
decreased undesirable side-effects. Also included are antigenic and
immunogenic activities of said 68772.
[0011] "68772 gene" refers to a polynucleotide having the
nucleotide sequence set forth in SEQ ID NO:1 or allelic variants
thereof and/or their complements.
[0012] "Antibodies" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
[0013] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance occurs in
nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the
term is employed herein.
[0014] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0015] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cystine, formation of pyroglutamate, formylation, gamma-
carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, proteolytic
processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. See,
for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and
Wold, F., Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al., "Analysis for protein modifications and nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al.,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann
NY Acad Sci (1992) 663:48-62.
[0016] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic variant, or it may be a variant that is not known to
occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis.
[0017] "Identity" is a measure of the identity of nucleotide
sequences or amino acid sequences. In general, the sequences are
aligned so that the highest order match is obtained. "Identity" per
se has an art-recognized meaning and can be calculated using
published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed.,
Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA,
PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje,
G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). While
there exist a number of methods to measure identity between two
polynucleotide or polypeptide sequences, the term "identity" is
well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J
Applied Math (1988) 48:1073). Methods commonly employed to
determine identity or similarity between two sequences include, but
are not limited to, those disclosed in Guide to Huge Computers,
Martin J. Bishop, ed., Academic Press, San Diego, 1994, and
Carillo, H., and Lipton, D., SIAM J Applied Math (1988)
48:1073.
[0018] Methods to determine identity and similarity are codified in
computer programs. Preferred computer program methods to determine
identity and similarity between two sequences include, but are not
limited to, GCS program package (Devereux, J., et al., Nucleic
Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul,
S. F. et al., J Molec Biol (1990) 215:403).
[0019] As an illustration, by a polynucleotide having a nucleotide
sequence having at least, for example, 95% "identity" to a
reference nucleotide sequence of SEQ ID NO:1 is intended that the
nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence of SEQ ID NO: 1. In other words, to
obtain a polynucleotide having a nucleotide sequence at least 95%
identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5 or 3 terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0020] Similarly, by a polypeptide having an amino acid sequence
having at least, for example, 95% "identity" to a reference amino
acid sequence of SEQ ID NO:2 is intended that the amino acid
sequence of the polypeptide is identical to the reference sequence
except that the polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the reference amino
acid of SEQ ID NO: 2. In other words, to obtain a polypeptide
having an amino acid sequence at least 95% identical to a reference
amino acid sequence, up to 5% of the amino acid residues in the
reference sequence may be deleted or substituted with another amino
acid, or a number of amino acids up to 5% of the total amino acid
residues in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0021] Polypeptides of the Invention
[0022] In one aspect, the present invention relates to 68772
polypeptides (or 68772 proteins). The 68772 polypeptides include
the polypeptide of SEQ ID NO:2; as well as polypeptides comprising
the amino acid sequence of SEQ ID NO: 2; and polypeptides
comprising the amino acid sequence which have at least 80% identity
to that of SEQ ID NO:2 over its entire length, and still more
preferably at least 90% identity, and even still more preferably at
least 95% identity to SEQ ID NO:
[0023] Furthermore, those with at least 97-99% are highly
preferred. Also included within 68772 polypeptides are polypeptides
having the amino acid sequence which have at least 80% identity to
the polypeptide having the amino acid sequence of SEQ ID NO:2 over
its entire length, and still more preferably at least 90% identity,
and still more preferably at least 95% identity to SEQ ID NO:2.
Furthermore, those with at least 97-99% are highly preferred.
Preferably 68772 polypeptide exhibit at least one biological
activity of 68772.
[0024] The 68772 polypeptides may be in the form of the "mature"
protein or may be a part of a larger protein such as a fusion
protein. It is often advantageous to include an additional amino
acid sequence which contains secretory or leader sequences,
prosequences, sequences which aid in purification such as multiple
histidine residues, or an additional sequence for stability during
recombinant production.
[0025] Fragments of the 68772 polypeptides are also included in the
invention. A fragment is a polypeptide having an amino acid
sequence that entirely is the same as part, but not all, of the
amino acid sequence of the aforementioned 68772 polypeptides. As
with 68772 polypeptides, fragments may be "free-standing," or
comprised within a larger polypeptide of which they form a part or
region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments from about amino acid number 1-20,
21-40, 41-60, 61-80, 81-100, and 101 to the end of 68772
polypeptide. In this context "about" includes the particularly
recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino
acid at either extreme or at both extremes.
[0026] Preferred fragments include, for example, truncation
polypeptides having the amino acid sequence of 68772 polypeptides,
except for deletion of a continuous series of residues that
includes the amino terminus, or a continuous series of residues
that includes the carboxyl terminus or deletion of two continuous
series of residues, one including the amino terminus and one
including the carboxyl terminus Also preferred are fragments
characterized by structural or functional attributes such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions. Other
preferred fragments are biologically active fragments. Biologically
active fragments are those that mediate 68772 activity, including
those with a similar activity or an improved activity, or with a
decreased undesirable activity. Also included are those that are
antigenic or immunogenic in an animal, especially in a human.
[0027] Preferably, all of these polypeptide fragments retain the
biological activity of the 68772, including antigenic activity.
[0028] Variants of the defined sequence and fragments also form
part of the present invention. Preferred variants are those that
vary from the referents by conservative amino acid
substitutions--i.e., those that substitute a residue with another
of like characteristics. Typical such substitutions are among Ala,
Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp
and Glu; among Asn and Gln; and among the basic residues Lys and
Arg; or aromatic residues Phe and Tyr.
[0029] Particularly preferred are variants in which several, 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination.
[0030] The 68772 polypeptides of the invention can be prepared in
any suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[0031] Polynucleotides of the Invention
[0032] Another aspect of the invention relates to 68772
polynucleotides. 68772 polynucleotides include isolated
polynucleotides, which encode the 68772 polypeptides and fragments,
and polynucleotides closely related thereto. More specifically,
68772 polynucleotide of the invention include a polynucleotide
comprising the nucleotide sequence set forth in SEQ ID NO: 1
encoding a 68772 polypeptide of SEQ ID NO: 2, and polynucleotide
having the particular sequence of SEQ ID NO: 1. 68772
polynucleotides further include a polynucleotide comprising a
nucleotide sequence that has at least 80% identity over its entire
length to a nucleotide sequence encoding the 68772 polypeptide of
SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence
that is at least 80% identical to that of SEQ ID NO: 1 over its
entire length. In this regard, polynucleotides at least 90%
identical are particularly preferred, and those with at least 95%
are especially preferred. Furthermore those with at least 97% are
highly preferred and those with at least 98-99% are most highly
preferred, with at least 99% being the most preferred. Also
included under 68772 polynucleotides are a nucleotide sequence
which has sufficient identity to a nucleotide sequence contained in
SEQ ID NO: 1 or contained in the cDNA insert in the plasmid
deposited with the ATCC Deposit number ATCC 98438 to hybridize
under conditions useable for amplification or for use as a probe or
marker. Moreover, 68772 polynucleotide include a nucleotide
sequence having at least 80% identity to a nucleotide sequence
encoding the 68772 polypeptide expressed by the cDNA insert
deposited at the ATCC with Deposit Number ATCC 98438, and a
nucleotide sequence comprising at least 15 contiguous nucleotides
of such cDNA insert. In this regard, polynucleotides at least 90%
identical are particularly preferred, and those with at least 95%
are especially preferred. Furthermore, those with at least 97% are
highly preferred and those with at least 98-99% are most highly
preferred, with at least 99% being the most preferred. The
invention also provides polynucleotides which are complementary to
all the above 68772 polynucleotides.
[0033] A deposit containing a human 68772 cDNA has been deposited
with the American Type Culture Collection (ATCC), 12301 Park Lawn
Drive, Rockville, Md. 20852, USA, on May 28, 1997, and assigned
ATCC Deposit Number ATCC 98438. The deposited material (clone) is
SOLR containing UniZap (Stratagene, La Jolla, Calif.) that further
contains the full length 68772 cDNA, referred to as "Human pelota
cDNA clone from a human T-cell library, ATG-1030" upon deposit. The
cDNA insert is within Eco RI, Xho I site(s) in the vector. The
nucleotide sequence of the polynucleotides contained in the
deposited material, as well as the amino acid sequence of the
polypeptide encoded thereby, are controlling in the event of any
conflict with any description of sequences herein.
[0034] The deposit has been made under the terms of the Budapest
Treaty on the international recognition of the deposit of
micro-organisms for purposes of patent procedure. The strain will
be irrevocably and without restriction or condition released to the
public upon the issuance of a patent. The deposit is provided
merely as convenience to those of skill in the art and is not
admission that a deposit is required for enablement, such as that
required under 35 U.S.C. .sctn. 112.
[0035] 68772 of the invention is structurally related to other
proteins of the Pelota family, as shown by the results of
sequencing the cDNA of Table 1 (SEQ ID NO:1) encoding human 68772.
The cDNA sequence of SEQ ID NO: 1 contains an open reading frame
(nucleotide number 250 to 1407) encoding a polypeptide of 385 amino
acids of SEQ ID NO:2.
[0036] Amino acid sequence of Table 2 (SEQ ID NO:2) has about 65%
identity (using Gap in GCG (Needleman Wunsch)) in 395 amino acid
residues with Drosophila melanogaster pelota (Eberhart and
Wasserman, Devel. 121:3477-3486, 1995).
[0037] Furthermore, 68772 (SEQ ID NO:2) is 36% identical to
Saccharomyces cerevisiae DOM34 over 387 amino acid residues (Lalo
et al., Compets Rendus de l'Academie des Sciences 316:367-373,
1993). Nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 63%
identity (using Gap in GCG (Needleman Wunsch)) in 1186 nucleotide
residues with Drosophila melanogaster pelota (Eberhart and
Wasserman, Devel. 121:3477-3486, 1995). Furthermore, 68772 (SEQ ID
NO:1) is 45% identical to Saccharomyces cerevisiae DOM34 over 1176
nucleotide base residues (Lalo et al., Compets Rendus de l'Academie
des Sciences 316:367-373, 1993) Thus 68772 polypeptides and
polynucleotides of the present invention are expected to have,
inter alia, similar biological functions/properties to their
homologous polypeptides and polynucleotides, and their utility is
obvious to anyone skilled in the art.
1TABLE 1.sup.a CCCGGGCGCTGCAGTGTTCCCCGAGCCTGTTAGACG-
CAGCGCGCCGGGAGACTGAGAGAGGAAAGGATA GAGGAAGTGCTGCCCTAGGCTGCA-
TGAGTCGAAGCAAGCGTGTTTCCTTCCCGCCAGGCAAGTGCCCTT
AGAAACCGGGCCCCGCCCCCTTCCTGGCCTGCATTCCCATCCCCTCTCCCGGGGCGGAGGTGAGGACCT
CCTTGGTTCCTTTGGTTCTGTCAGTGAGCCCCTTCCTTGGCCATGAAGCTCGTGAGGAAGA-
ACATCGAG AAGGACAATGCGGGCCAGGTGACCCTGGTCCCCGAGGAGCCTGAGGACA-
TGTGGCACACTTACAACCTC GTGCAGGTGGGCGACAGCCTGCGCGGCTCCACCATCC-
GCAAGGTACAGACAGAGTCCTCCACGGGCAGC GTGGGCAGCAACCGGGTCCGCACTA-
CCCTCACTCTCTGCGTGGAGGCCATCGACTTCGACTCTCAAGCC
TGCCAGCTGCGGGTTAAGGGGACCAACATCCAAGAGAATGAGTATGTCAAGATGGGGGCTTACCACACC
ATCGAGCTGGAGCCCAACCGCCAGTTCACCCTGGCCAAGAAGCAGTGGGATAGTGTGGTAC-
TGGAGCGC ATCGAGCAGGCCTGTGACCCAGCCTGGAGCGCTGATGTGGCGGCTGTGG-
TCATGCAGGAAGGCCTCGCC CATATCTGCTTAGTCACTCCCAGCATGACCCTCACTC-
GGGCCAAGGTGGAGGTGAACATCCCTAGGAAA AGGAAAGGCAATTGCTCTCAGCATG-
ACCGGGCCTTGGAGCGGTTCTATGAACAGGTGGTCCAGGCTATC
CAGCGCCACATACACTTTGATGTTGTAAAGTGCATCCTGGTGGCCAGCCCAGGATTTGTGAGGGAGCAG
TTCTGCGACTACATGTTTCAACAAGCAGTGAAGACCGACAACAAACTGCTCCTGGAAAACC-
GGTCCAAA TTTCTTCAGGTACATGCCTCCTCCGGACACAAGTACTCCCTGAAAGAGG-
CCCTTTGTGACCCTACTGTG GCTAGCCGCCTTTCAGACACTAAAGCTGCTGGGGAAG-
TCAAAGCCTTGGATGACTTCTATAAAATGTTA CAGCATGAACCGGATCGAGCTTTCT-
ATGGACTCAAGCAGGTGGAGAAGGCCAATGAAGCCATGGCAATT
GACACATTGCTCATCAGCGATGAGCTCTTCAGGCATCAGGATGTAGCCACACGGAGCCGGTATGTGAGG
CTGGTGGACAGTGTGAAAGAGAATGCAGGCACCGCTAGGATATTCTCTAGTCTTCACGTTT-
CTGGGGAA CAGCTCAGCCAGTTGACTGGGGTAGCTGCCATTCTCCGCTTCCCTGTTC-
CCGAACTTTCTGACCAAGAG GGTGATTCCAGTTCTGAAGAGCATTAATGATTGAAAC-
TTAAAATTGAGACAATCTTGTGTTTCCTAAAC TGTTACAGTACATTTCTCAGCATCC-
TTGTGACAGAAAGCTGCAAGAAGGGCACTTTTTGATTCATACAG
GGATTTCTTATGTCTTTGGCTACACTAGATATTTTGTGATTGGCAAGACATGTATTTAAACAATAAACT
AAAAGGAAATAATCTCCACGTACTACCAAAAAAAAAAAAAAAAAA .sup.aA nucleotide
sequence of a human 68772. SEQ ID NO: 1.
[0038]
2TABLE 2.sup.b MKLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVG-
DSLRASTIRKVQTESSTGSVGSNRVRTTLTLCV EAIDFDSQACQLRVKGTNIQENEY-
VKMGAYHTIELEPNRQFTLAKKQWDSVVLERIEQACDPAWSADVA
AVVMQEGLAHICLVTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDVVKCILV
ASPGFVREQFCDYMFQQAVKTDNKLLLENRSKFLQVHASSGHKYSLKEALCDPTVASRLSD-
TKAAGEVK ALDDFYKMLQHEPDRAFYGLKQVEKANEAMAIDTLLISDELFRHQDVAT-
RSRYVRLVDSVKENAGTARI FSSLHVSGEQLSQLTGVAAILRFPVPELSDQEGDSSS- EED.
.sup.bAn amino acid of sequence of a human 68772. SEQ ID NO: 2.
[0039] One polynucleotide of the present invention encoding 68772
may be obtained using standard cloning and screening, from a cDNA
library derived from mRNA in cells of human activated T-Cells,
pancreas tumor, colon carcinoma, placenta, chondrosarcoma, hypoxic
synoviocytes, osteroclastoma, tonsils, promyelocyte, heart,
stimulated endothelial cells, and breast lymph node cells using the
expressed sequence tag (EST) analysis (Adams, M. D., et al Science
(1991) 252:1651-1656; Adams, M. D. et al., Nature, (1992)
355:632-634; Adams, M. D., et al., Nature (1995)377 Supp:3-174).
Polynucleotides of the invention can also be obtained from natural
sources such as genomic DNA libraries or can be synthesized using
well known and commercially available techniques.
[0040] The nucleotide sequence encoding 68772 polypeptide of SEQ ID
NO:2 may be identical to the polypeptide encoding sequence
contained in Table 1 (nucleotide number 250 to 1407 of SEQ ID NO:
1), or it may be a sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes the polypeptide of
SEQ ID NO:2.
[0041] When the polynucleotides of the invention are used for the
recombinant production of 68772 polypeptide, the polynucleotide may
include the coding sequence for the mature polypeptide or a
fragment thereof, by itself, the coding sequence for the mature
polypeptide or fragment in reading frame with other coding
sequences, such as those encoding a leader or secretory sequence, a
pre-, or pro- or prepro-protein sequence, or other fusion peptide
portions. For example, a marker sequence, which facilitates
purification of the fused polypeptide, can be encoded. In certain
preferred embodiments of this aspect of the invention, the marker
sequence is a hexa-histidine peptide, as provided in the pQE vector
(Qiagen, Inc.) and described in Gentz et al., Proc Natl Acad Sci
USA. (1989) 86:821-824, or is an HA tag. The polynucleotide may
also contain noncoding 5' and 3' sequences, such as transcribed,
non-translated sequences, splicing and polyadenylation signals,
ribosome binding sites and sequences that stabilize mRNA.
[0042] Further preferred embodiments are polynucleotides encoding
68772 variants comprise the amino acid sequence 68772 polypeptide
of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5,1-3, 1-2 or 1
amino acid residues are substituted, deleted or added, in any
combination.
[0043] The present invention further relates to polynucleotides
that hybridize to the herein above-described sequences. In this
regard, the present invention especially relates to
polynucleotides, which hybridize under stringent conditions to the
herein above-described polynucleotides. As herein used, the term
"stringent conditions" means hybridization will occur only if there
is at least 80%, and preferably at least 90%, and more preferably
at least 95%, yet even more preferably 97-99% identity between the
sequences.
[0044] Polynucleotides of the invention, which are identical or
sufficiently identical to a nucleotide sequence contained in SEQ ID
NO: 1 or a fragment thereof or to the cDNA insert in the plasmid
deposited at the ATCC with Deposit Number ATCC 98438 or a fragment
thereof, may be used as hybridization probes for cDNA and genomic
DNA, to isolate fill-length cDNAs and genomic clones encoding 68772
polypeptide and to isolate cDNA and genomic clones of other genes
that have a high sequence similarity to the 68772 gene. Such
hybridization techniques are known to those of skill in the
art.
[0045] Typically these nucleotide sequences are 80% identical,
preferably 90% identical, more preferably 95% identical to that of
the referent. The probes generally will comprise at least 15
nucleotides. Preferably, such probes will have at least 30
nucleotides and may have at least 50 nucleotides. Particularly
preferred probes will range between 30 and 50 nucleotides.
[0046] In one embodiment, to obtain a polynucleotide encoding 68772
polypeptide comprises the steps of screening an appropriate library
under stringent hybridization conditions with a labeled probe
having 15 the SEQ ID NO: 1 or a fragment thereof; and isolating
full-length cDNA and genomic clones containing said polynucleotide
sequence. Thus in another aspect, 68772 polynucleotides of the
present invention further include a nucleotide sequence comprising
a nucleotide sequence that hybridize under stringent condition to a
nucleotide sequence having SEQ ID NO: 1 or a fragment thereof. Also
included with 68772 polypeptides are polypeptide comprising amino
acid sequence encoded by nucleotide sequence obtained by the above
hybridization condition. Such hybridization techniques are well
known to those of skill in the art. Stringent hybridization
conditions are as defined above or, alternatively, conditions under
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 microgram/ml denatured, sheared salmon
sperm DNA, followed by washing the filters in 0.1.times.SSC at
about 65.degree. C.
[0047] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to animal and human
disease.
[0048] Vectors, Host Cells, Expression
[0049] The present invention also relates to vectors, which
comprise a polynucleotide or polynucleotides of the present
invention, and host cells which are genetically engineered with
vectors of the invention and to the production of polypeptides of
the invention by recombinant techniques. Cell-free translation
systems can also be employed to produce such proteins using RNAs
derived from the DNA constructs of the present invention.
[0050] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis et
al., BASIC METHODS IN MOLECULAR BIOLOGY(1986) and Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium
phosphate transfection, DEAE-dextan mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection.
[0051] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, E. coli,
Streptomyces and Bacillus subtilis cells; fungal cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa,
C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant
cells.
[0052] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well-known and routine techniques, such as, for example,
those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
[0053] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0054] If the 68772 polypeptide is to be expressed for use in
screening assays, generally, it is preferred that the polypeptide
be produced at the surface of the cell. In this event, the cells
may be harvested prior to use in the screening assay. It 68772
polypeptide is secreted into the medium, the medium can be
recovered in order to recover and purify the polypeptide; if
produced intracellularly, the cells must first be lysed before the
polypeptide is recovered. 68772 polypeptides can be recovered and
purified from recombinant cell cultures by well-known methods
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Most preferably, high
performance liquid chromatography is employed for purification.
Well-known techniques for refolding proteins may be employed to
regenerate active conformation when the polypeptide is denatured
during isolation and or purification.
[0055] Diagnostic Assays
[0056] This invention also relates to the use of 68772
polynucleotides for use as diagnostic reagents. Detection of a
mutated form of 68772 gene associated with a dysfunction will
provide a diagnostic tool that can add to or define a diagnosis of
a disease or susceptibility to a disease which results from
under-expression, over-expression or altered expression of 68772.
Individuals carrying mutations in the 68772 gene may be detected at
the DNA level by a variety of techniques.
[0057] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled 68772 nucleotide sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase digestion or by differences in melting temperatures.
[0058] DNA sequence differences may also be detected by alterations
in electrophoretic mobility of DNA fragments in gels, with or
without denaturing agents, or by direct DNA sequencing. See, e.g.,
Myers et al., Science (1985) 230:1242. Sequence changes at specific
locations may also be revealed by nuclease protection assays, such
as RNase and S1 protection or the chemical cleavage method. See
Cotton et al., Proc Natl Acad Sci USA. (1985) 85: 4397-4401. In
another embodiment an array of oligonucleotides probes comprising
68772 nucleotide sequence or fragments thereof can be constructed
to conduct efficient screening of e.g., genetic mutations. Array
technology methods are well known and have general applicability
and can be used to address a variety of questions in molecular
genetics including gene expression, genetic linkage, and genetic
variability. (See for example: M. Chee et al., Science, Vol 274, pp
610-613 (1996)).
[0059] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to proliferative diseases such as
leukemias, solid tumor cancers and metastases; chronic inflammatory
proliferative diseases such as psoriasis and rheumatoid arthritis;
proliferative cardiovascular diseases such as restenosis;
prolifertive ocular disorders such as diabetic retinopathy; and
benign hyperproliferative diseases such as hemangiomas through
detection of mutation in the 68772 gene by the methods
described.
[0060] In addition, proliferative diseases such as leukemias, solid
tumor cancers and metastases; chronic inflammatory proliferative
diseases such as psoriasis and rheumatoid arthritis; proliferative
cardiovascular diseases such as restenosis; prolifertive ocular
disorders such as diabetic retinopathy; and benign
hyperproliferative diseases such as hemangiomas, can be diagnosed
by methods comprising determining from a sample derived from a
subject an abnormally decreased or increased level of 68772
polypeptide or 68772 mRNA. Decreased or increased expression can be
measured at the RNA level using any of the methods well known in
the art for the quantitation of polynucleotides, such as, for
example, PCR, RT-PCR, RNase protection, Northern blotting and other
hybridization methods. Assay techniques that can be used to
determine levels of a protein, such as an 68772 polypeptide, in a
sample derived from a host are well-known to those of skill in the
art. Such assay methods include radioimmunoassay,
competitive-binding assays, Western Blot analysis and ELISA
assays.
[0061] Thus in another aspect, the present invention relates to a
diagnostic kit for a disease or susceptibility to a disease,
particularly proliferative diseases such as leukemias, solid tumor
cancers and metastases; chronic inflammatory proliferative diseases
such as psoriasis and rheumatoid arthritis; proliferative
cardiovascular diseases such as restenosis; prolifertive ocular
disorders such as diabetic retinopathy; and benign
hyperproliferative diseases such as hemangiomas, which
comprises:
[0062] (a) a 68772 polynucleotide, preferably the nucleotide
sequence of SEQ ID NO: 1, or a fragment thereof;
[0063] (b) a nucleotide sequence complementary to that of (a); (c)
a 68772 polypeptide, preferably the polypeptide of SEQ ID NO: 2, or
a fragment thereof; or
[0064] (d) an antibody to a 68772 polypeptide, preferably to the
polypeptide of SEQ D NO: 2.
[0065] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0066] Chromosome Assays
[0067] The nucleotide sequences of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. The mapping of relevant
sequences to chromosomes according to the present invention is an
important first step in correlating those sequences with gene
associated disease. Once a sequence has been mapped to a precise
chromosomal location, the physical position of the sequence on the
chromosome can be correlated with genetic map data. Such data are
found, for example, in V. McKusick, Mendelian Inheritance in Man
(available on line through Johns Hopkins University Welch Medical
Library). The relationship between genes and diseases that have
been mapped to the same chromosomal region are then identified
through linkage analysis (coinheritance of physically adjacent
genes).
[0068] The differences in the cDNA or genomic sequence between
affected and unaffected individuals can also be determined. If a
mutation is observed in some or all of the affected individuals but
not in any normal individuals, then the mutation is likely to be
the causative agent of the disease.
[0069] Antibodies
[0070] The polypeptides of the invention or their fragments or
analogs thereof, or cells expressing them can also be used as
immunogens to produce antibodies immunospecific for the 68772
polypeptides. The term "immunospecific" means that the antibodies
have substantially greater affinity for the polypeptides of the
invention than their affinity for other related polypeptides in the
prior art.
[0071] Antibodies generated against the 68772 polypeptides can be
obtained by administering the polypeptides or epitope-bearing
fragments, analogs or cells to an animal, preferably a nonhuman,
using routine protocols. For preparation of monoclonal antibodies,
any technique which provides antibodies produced by continuous cell
line cultures can be used.
[0072] Examples include the hybridoma technique (Kohler, G. and
Milstein, C., Nature (1975) 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
(1983) 4:72) and the EBV-hybridoma technique (Cole et al.,
MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss,
Inc., 1985).
[0073] Techniques for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can also be adapted to produce single
chain antibodies to polypeptides of this invention. Also,
transgenic mice, or other organisms including other mammals, may be
used to express humanized antibodies.
[0074] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or to purify the
polypeptides by affinity chromatography.
[0075] Antibodies against 68772 polypeptides may also be employed
to treat proliferative diseases such as leukemias, solid tumor
cancers and metastases; chronic inflammatory proliferative diseases
such as psoriasis and rheumatoid arthritis; proliferative
cardiovascular diseases such as restenosis; prolifertive ocular
disorders such as diabetic retinopathy; and benign
hyperproliferative diseases such as hemangiomas, among others.
[0076] Vaccines
[0077] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with 68772 polypeptide, or a fragment
thereof, adequate to produce antibody and/or T cell immune response
to protect said animal from proliferative diseases such as
leukemias, solid tumor cancers and metastases; chronic inflammatory
proliferative diseases such as psoriasis and rheumatoid arthritis;
proliferative cardiovascular diseases such as restenosis;
prolifertive ocular disorders such as diabetic retinopathy; and
benign hyperproliferative diseases such as hemangiomas, among
others. Yet another aspect of the invention relates to a method of
inducing immunological response in a mammal which comprises,
delivering 68772 polypeptide via a vector directing expression of
68772 polynucleotide in vivo in order to induce such an
immunological response to produce antibody to protect said animal
from diseases.
[0078] Further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to a 68772 polypeptide wherein the composition
comprises a 68772 polypeptide or 68772 gene. The vaccine
formulation may further comprise a suitable carrier. Since 68772
polypeptide may be broken down in the stomach, it is preferably
administered parenterally (including subcutaneous, intramuscular,
intravenous, intradermal etc. injection). Formulations suitable for
parenteral administration include aqueous and non-aqueous sterile
injection solutions which may contain antioxidants, buffers,
bacteriostats and solutes which render the formulation instonic
with the blood of the recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents or
thickening agents. The formulations may be presented in unit-dose
or multi-dose containers, for example, sealed ampoules and vials
and may be stored in a freeze-dried condition requiring only the
addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation may also include adjuvant systems for
enhancing the immunogenicity of the formulation, such as oil-in
water systems and other systems known in the art. The dosage will
depend on the specific activity of the vaccine and can be readily
determined by routine experimentation.
[0079] Screening Assays
[0080] The 68772 polypeptide of the present invention may be
employed in a screening process for compounds which activate
(agonists) or inhibit activation of (antagonists, or otherwise
called inhibitors) the 68772 polypeptide of the present invention.
Thus, polypeptides of the invention may also be used to assess
identify agonist or antagonists from, for example, cells, cell-free
preparations, chemical libraries, and natural product mixtures.
These agonists or antagonists may be natural or modified
substrates, ligands, receptors, enzymes, etc., as the case may be,
of the polypeptide of the present invention; or may be structural
or functional mimetics of the polypeptide of the present invention.
See Coligan et al., Current Protocols in Immunology 1(2):Chapter 5
(1991).
[0081] 68772 polypeptides are responsible for many biological
functions, including many pathologies. Accordingly, it is desirous
to find compounds and drugs which stimulate 68772 polypeptide on
the one hand and which can inhibit the function of 68772
polypeptide on the other hand. In general, agonists are employed
for therapeutic and prophylactic purposes for such conditions as
proliferative diseases such as leukemias, solid tumor cancers and
metastases; chronic inflammatory proliferative diseases such as
psoriasis and rheumatoid arthritis; proliferative cardiovascular
diseases such as restenosis; prolifertive ocular disorders such as
diabetic retinopathy; and benign hyperproliferative diseases such
as hemangiomas.
[0082] Antagonists may be employed for a variety of therapeutic and
prophylactic purposes for such conditions as proliferative diseases
such as leukemias, solid tumor cancers and metastases; chronic
inflammatory proliferative diseases such as psoriasis and
rheumatoid arthritis; proliferative cardiovascular diseases such as
restenosis; prolifertive ocular disorders such as diabetic
retinopathy; and benign hyperproliferative diseases such as
hemangiomas.
[0083] In general, such screening procedures may involve using
appropriate cells which express the 68772 polypeptide or respond to
68772 polypeptide of the present invention. Such cells include
cells from mammals, yeast Drosophila or E. coli. Cells which
express the 68772 polypeptide (or cell membrane containing the
expressed polypeptide) or respond to 68772 polypeptide are then
contacted with a test compound to observe binding, or stimulation
or inhibition of a functional response. The ability of the cells
which were contacted with the candidate compounds is compared with
the same cells which were not contacted for 68772 activity.
[0084] The assays may simply test binding of a candidate compound
wherein adherence to the cells bearing the 68772 polypeptide is
detected by means of a label directly or indirectly associated with
the candidate compound or in an assay involving competition with a
labeled competitor. Further, these assays may test whether the
candidate compound results in a signal generated by activation of
the 68772 polypeptide, using detection systems appropriate to the
cells bearing the 68772 polypeptide. Inhibitors of activation are
generally assayed in the presence of a known agonist and the effect
on activation by the agonist by the presence of the candidate
compound is observed.
[0085] The 68772 cDNA, protein and antibodies to the protein may
also be used to configure assays for detecting the effect of added
compounds on the production of 68772 mRNA and protein in cells. For
example, an ELISA may be constructed for measuring secreted or cell
associated levels of 68772 protein using monoclonal and polyclonal
antibodies by standard methods known in the art, and this can be
used to discover agents which may inhibit or enhance the production
of 68772 (also called antagonist or agonist, respectively) from
suitably manipulated cells or tissues. Standard methods for
conducting screening assays are well understood in the art.
[0086] The 68772 protein may be used to identify membrane bound or
soluble receptors, if any, through standard receptor binding
techniques known in the art. These include, but are not limited to,
ligand binding and crosslinking assays in which the 68772 is
labeled with a radioactive isotope (eg 125I), chemically modified
(eg biotinylated), or fused to a peptide sequence suitable for
detection or purification, and incubated with a source of the
putative receptor (cells, cell membranes, cell supernatants, tissue
extracts, bodily fluids). Other methods include biophysical
techniques such as surface plasmon resonance and spectroscopy. In
addition to being used for purification and cloning of the
receptor, these binding assays can be used to identify agonists and
antagonists of 68772 which compete with the binding of 68772 to its
receptors, if any. Standard methods for conducting screening assays
are well understood in the art.
[0087] Examples of potential 68772 polypeptide antagonists include
antibodies or, in some cases, oligonucleotides or proteins which
are closely related to the ligands, substrates, receptors, enzymes,
etc., as the case may be, of the 68772 polypeptide, e.g., a
fragment of the ligands, substrates, receptors, enzymes, etc.; or
small molecules which bind to the polypeptide of the present
invention but do not elicit a response, so that the activity of the
polypeptide is prevented.
[0088] Thus in another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for 68772 polypeptides; or
compounds which decrease or enhance the production of 68772
polypeptides, which comprises:
[0089] (a) a 68772 polypeptide, preferably that of SEQ ID NO:2;
[0090] (b) a recombinant cell expressing a 68772 polypeptide,
preferably that of SEQ ID NO:2;
[0091] (c) a cell membrane expressing a 68772 polypeptide;
preferably that of SEQ ID NO: 2; or
[0092] (d) antibody to a 68772 polypeptide, preferably that of SEQ
ID NO: 2.
[0093] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0094] Prophylactic and Therapeutic Methods
[0095] This invention provides methods of treating abnormal
conditions such as, proliferative diseases such as leukemias, solid
tumor cancers and metastases; chronic inflammatory proliferative
diseases such as psoriasis and rheumatoid arthritis; proliferative
cardiovascular diseases such as restenosis; prolifertive ocular
disorders such as diabetic retinopathy; and benign
hyperproliferative diseases such as hemangiomas, related to both an
excess of and insufficient amounts of 68772 polypeptide
activity.
[0096] If the activity of 68772 polypeptide is in excess, several
approaches are available. One approach comprises administering to a
subject an inhibitor compound (antagonist) as hereinabove described
along with a pharmaceutically acceptable carrier in an amount
effective to inhibit the function of the 68772 polypeptide, such
as, for example, by blocking the binding of ligands, substrates,
enzymes, receptors, etc., or by inhibiting a second signal, and
thereby alleviating the abnormal condition. In another approach,
soluble forms of 68772 polypeptides still capable of binding the
ligand, substrate, enzymes, receptors, etc. in competition with
endogenous 68772 polypeptide may be administered. Typical
embodiments of such competitors comprise fragments of the 68772
polypeptide.
[0097] In still another approach, expression of the gene encoding
endogenous 68772 polypeptide can be inhibited using expression
blocking techniques. Known such techniques involve the use of
antisense sequences, either internally generated or separately
administered. See, for example, O'Connor, J Neurochem (1991) 56:560
in Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988). Alternatively,
oligonucleotides which form triple helices with the gene can be
supplied. See, for example, Lee et al., Nucleic Acids Res (1979)
6:3073; Cooney et al., Science (1988) 241:456; Dervan et al.,
Science (1991) 251:1360. These oligomers can be administered per se
or the relevant oligomers can be expressed in vivo.
[0098] For treating abnormal conditions related to an
under-expression of 68772 and its activity, several approaches are
also available. One approach comprises administering to a subject a
therapeutically effective amount of a compound which activates
68772 polypeptide, i.e., an agonist as described above, in
combination with a pharmaceutically acceptable carrier, to thereby
alleviate the abnormal condition. Alternatively, gene therapy may
be employed to effect the endogenous production of 68772 by the
relevant cells in the subject. For example, a polynucleotide of the
invention may be engineered for expression in a replication
defective retroviral vector, as discussed above. The retroviral
expression construct may then be isolated and introduced into a
packaging cell transduced with a retroviral plasmid vector
containing
[0099] RNA encoding a polypeptide of the present invention such
that the packaging cell now produces infectious viral particles
containing the gene of interest. These producer cells may be
administered to a subject for engineering cells in vivo and
expression of the polypeptide in vivo. For overview of gene
therapy, see Chapter 20, Gene Therapy and other Molecular
Genetic-based Therapeutic Approaches, (and references cited
therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS
Scientific Publishers Ltd (1996). Another approach is to administer
therapeutic amount of 68772 polypeptides in combination with a
suitable pharmaceutical carrier.
[0100] Formulation and Administration
[0101] Peptides, such as the soluble form of 68772 polypeptides,
and agonists and antagonist peptides or small molecules, may be
formulated in combination with a suitable pharmaceutical carrier.
Such formulations comprise a therapeutically effective amount of
the polypeptide or compound, and a pharmaceutically acceptable
carrier or excipient. Such carriers include but are not limited to,
saline, buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. Formulation should suit the mode of
administration, and is well within the skill of the art. The
invention further relates to pharmaceutical packs and kits
comprising one or more containers filled with one or more of the
ingredients of the aforementioned compositions of the
invention.
[0102] Polypeptides and other compounds of the present invention
may be employed alone or in conjunction with other compounds, such
as therapeutic compounds.
[0103] Preferred forms of systemic administration of the
pharmaceutical compositions include injection, typically by
intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, or intraperitoneal, can be used.
Alternative means for systemic administration include transmucosal
and transdermal administration using penetrants such as bile salts
or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral
administration may also be possible. Administration of these
compounds may also be topical and/or localized, in the form of
salves, pastes, gels and the like.
[0104] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. Suitable dosages, however, are in the range
of 0.1-100 .mu.g/kg of subject. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0105] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
EXAMPLES
[0106] The examples below are carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
illustrate, but do not limit the invention.
Example 1
[0107] The full-length clone (68772) was identified through
searches of the Human Genome Sciences database.
[0108] Northern blotting of multiple tissue human RNA blots
performed using 68772 (human pelota) as a probe, detected a message
of approximately 1.9 kb in several cancer cell lines: HL-60,
HelaS3, K-562, MOLT-4, Raji, SW480, A549, and G361. The message was
also found in fetal liver, peripheral blood lymphocytes, and also
weakly expressed in bone marrow and thymus. No apparent message was
detected in brain, spleen, appendix, lymph node, heart, placenta,
lung, liver, skeletal muscle, kidney, or pancreas.
[0109] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
Sequence CWU 1
1
2 1 1632 DNA human 1 cccgggcgct gcagtgttcc ccgagcctgt tagacgcagc
gcgccgggag actgagagag 60 gaaaggatag aggaagtgct gccctaggct
gcatgagtcg aagcaagcgt gtttccttcc 120 cgccaggcaa gtgcccttag
aaaccgggcc ccgccccctt cctggcctgc attcccatcc 180 cctctcccgg
ggcggaggtg aggacctcct tggttccttt ggttctgtca gtgagcccct 240
tccttggcca tgaagctcgt gaggaagaac atcgagaagg acaatgcggg ccaggtgacc
300 ctggtccccg aggagcctga ggacatgtgg cacacttaca acctcgtgca
ggtgggcgac 360 agcctgcgcg cctccaccat ccgcaaggta cagacagagt
cctccacggg cagcgtgggc 420 agcaaccggg tccgcactac cctcactctc
tgcgtggagg ccatcgactt cgactctcaa 480 gcctgccagc tgcgggttaa
ggggaccaac atccaagaga atgagtatgt caagatgggg 540 gcttaccaca
ccatcgagct ggagcccaac cgccagttca ccctggccaa gaagcagtgg 600
gatagtgtgg tactggagcg catcgagcag gcctgtgacc cagcctggag cgctgatgtg
660 gcggctgtgg tcatgcagga aggcctcgcc catatctgct tagtcactcc
cagcatgacc 720 ctcactcggg ccaaggtgga ggtgaacatc cctaggaaaa
ggaaaggcaa ttgctctcag 780 catgaccggg ccttggagcg gttctatgaa
caggtggtcc aggctatcca gcgccacata 840 cactttgatg ttgtaaagtg
catcctggtg gccagcccag gatttgtgag ggagcagttc 900 tgcgactaca
tgtttcaaca agcagtgaag accgacaaca aactgctcct ggaaaaccgg 960
tccaaatttc ttcaggtaca tgcctcctcc ggacacaagt actccctgaa agaggccctt
1020 tgtgacccta ctgtggctag ccgcctttca gacactaaag ctgctgggga
agtcaaagcc 1080 ttggatgact tctataaaat gttacagcat gaaccggatc
gagctttcta tggactcaag 1140 caggtggaga aggccaatga agccatggca
attgacacat tgctcatcag cgatgagctc 1200 ttcaggcatc aggatgtagc
cacacggagc cggtatgtga ggctggtgga cagtgtgaaa 1260 gagaatgcag
gcaccgctag gatattctct agtcttcacg tttctgggga acagctcagc 1320
cagttgactg gggtagctgc cattctccgc ttccctgttc ccgaactttc tgaccaagag
1380 ggtgattcca gttctgaaga ggattaatga ttgaaactta aaattgagac
aatcttgtgt 1440 ttcctaaact gttacagtac atttctcagc atccttgtga
cagaaagctg caagaagggc 1500 actttttgat tcatacaggg atttcttatg
tctttggcta cactagatat tttgtgattg 1560 gcaagacatg tatttaaaca
ataaactaaa aggaaataat ctccacgtac taccaaaaaa 1620 aaaaaaaaaa aa 1632
2 385 PRT human 2 Met Lys Leu Val Arg Lys Asn Ile Glu Lys Asp Asn
Ala Gly Gln Val 1 5 10 15 Thr Leu Val Pro Glu Glu Pro Glu Asp Met
Trp His Thr Tyr Asn Leu 20 25 30 Val Gln Val Gly Asp Ser Leu Arg
Ala Ser Thr Ile Arg Lys Val Gln 35 40 45 Thr Glu Ser Ser Thr Gly
Ser Val Gly Ser Asn Arg Val Arg Thr Thr 50 55 60 Leu Thr Leu Cys
Val Glu Ala Ile Asp Phe Asp Ser Gln Ala Cys Gln 65 70 75 80 Leu Arg
Val Lys Gly Thr Asn Ile Gln Glu Asn Glu Tyr Val Lys Met 85 90 95
Gly Ala Tyr His Thr Ile Glu Leu Glu Pro Asn Arg Gln Phe Thr Leu 100
105 110 Ala Lys Lys Gln Trp Asp Ser Val Val Leu Glu Arg Ile Glu Gln
Ala 115 120 125 Cys Asp Pro Ala Trp Ser Ala Asp Val Ala Ala Val Val
Met Gln Glu 130 135 140 Gly Leu Ala His Ile Cys Leu Val Thr Pro Ser
Met Thr Leu Thr Arg 145 150 155 160 Ala Lys Val Glu Val Asn Ile Pro
Arg Lys Arg Lys Gly Asn Cys Ser 165 170 175 Gln His Asp Arg Ala Leu
Glu Arg Phe Tyr Glu Gln Val Val Gln Ala 180 185 190 Ile Gln Arg His
Ile His Phe Asp Val Val Lys Cys Ile Leu Val Ala 195 200 205 Ser Pro
Gly Phe Val Arg Glu Gln Phe Cys Asp Tyr Met Phe Gln Gln 210 215 220
Ala Val Lys Thr Asp Asn Lys Leu Leu Leu Glu Asn Arg Ser Lys Phe 225
230 235 240 Leu Gln Val His Ala Ser Ser Gly His Lys Tyr Ser Leu Lys
Glu Ala 245 250 255 Leu Cys Asp Pro Thr Val Ala Ser Arg Leu Ser Asp
Thr Lys Ala Ala 260 265 270 Gly Glu Val Lys Ala Leu Asp Asp Phe Tyr
Lys Met Leu Gln His Glu 275 280 285 Pro Asp Arg Ala Phe Tyr Gly Leu
Lys Gln Val Glu Lys Ala Asn Glu 290 295 300 Ala Met Ala Ile Asp Thr
Leu Leu Ile Ser Asp Glu Leu Phe Arg His 305 310 315 320 Gln Asp Val
Ala Thr Arg Ser Arg Tyr Val Arg Leu Val Asp Ser Val 325 330 335 Lys
Glu Asn Ala Gly Thr Ala Arg Ile Phe Ser Ser Leu His Val Ser 340 345
350 Gly Glu Gln Leu Ser Gln Leu Thr Gly Val Ala Ala Ile Leu Arg Phe
355 360 365 Pro Val Pro Glu Leu Ser Asp Gln Glu Gly Asp Ser Ser Ser
Glu Glu 370 375 380 Asp 385
* * * * *