U.S. patent application number 11/294997 was filed with the patent office on 2006-03-30 for novel serine threonine kinase member, h2520-59.
This patent application is currently assigned to AMGEN INC.. Invention is credited to Alex J. Bowers, John F. Boylan.
Application Number | 20060067926 11/294997 |
Document ID | / |
Family ID | 36147341 |
Filed Date | 2006-03-30 |
United States Patent
Application |
20060067926 |
Kind Code |
A1 |
Boylan; John F. ; et
al. |
March 30, 2006 |
Novel serine threonine kinase member, h2520-59
Abstract
The present invention relates to a serine threonine kinase. The
invention also relates to nucleic acids encoding the kinase,
vectors, host cells, antibodies and recombinant methods for
producing the h2520-59 polypeptide. In addition, the invention
discloses therapeutic, diagnostic and research utilities for
h2520-59 and related products.
Inventors: |
Boylan; John F.; (Thousand
Oaks, CA) ; Bowers; Alex J.; (Simi Valley,
CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 S. WACKER DRIVE, SUITE 6300
SEARS TOWER
CHICAGO
IL
60606
US
|
Assignee: |
AMGEN INC.
Thousand Oaks
CA
|
Family ID: |
36147341 |
Appl. No.: |
11/294997 |
Filed: |
December 6, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10117766 |
Apr 5, 2002 |
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11294997 |
Dec 6, 2005 |
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09909474 |
Jul 19, 2001 |
6881542 |
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10117766 |
Apr 5, 2002 |
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60219204 |
Jul 19, 2000 |
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Current U.S.
Class: |
424/94.5 ;
435/194; 435/320.1; 435/325; 435/6.14; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12N 9/1205 20130101; C12N 9/6424 20130101; C12Q 2600/136 20130101;
A61K 38/00 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
424/094.5 ;
435/006; 435/069.1; 435/194; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; A61K 38/48 20060101 A61K038/48; C12N 9/12 20060101
C12N009/12 |
Claims
1-24. (canceled)
25. An antibody produced by immunizing an animal with a peptide
comprising an amino acid sequence of SEQ ID NO: 2.
26. An antibody or fragment thereof that specifically binds the
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO: 2.
27. The antibody of claim 26 that is a monoclonal antibody.
28. A hybridoma that produces a monoclonal antibody that binds to a
peptide comprising an amino acid sequence of SEQ ID NO: 2.
29. A method of detecting or quantitating the amount of h2520-59
polypeptide in a sample comprising contacting a sample suspected of
containing h2520-59 polypeptide with the anti-h2520-59 antibody or
antibody fragment of any of claims 25, 26, or 27 and detecting the
binding of said antibody or antibody fragment.
30. A selective binding agent or fragment thereof that specifically
binds at least one polypeptide wherein said polypeptide comprises
the amino acid sequence set forth in SEQ ID NO: 2.
31. (canceled)
32. The selective binding agent of claim 30 that is a humanized
antibody.
33. The selective binding agent of claim 30 that is a human
antibody or fragment thereof.
34. The selective binding agent of claim 30 that is a polyclonal
antibody or fragment thereof.
35. The selective binding agent of claim 30 that is a monoclonal
antibody or fragment thereof.
36. The selective binding agent of claim 30 that is a chimeric
antibody or fragment thereof.
37. The selective binding agent of claim 30 that is a
complementarity determining region (CDR)-grafted antibody or
fragment thereof.
38. The selective binding agent of claim 30 that is an
anti-idiotypic antibody or fragment thereof.
39. The selective binding agent of claim 30 which is an antibody
variable region fragment.
40. The variable region fragment of claim 39 which is a Fab or a
Fab' fragment.
41. A selective binding agent or fragment thereof comprising at
least one complementarity determining region with specificity for a
polypeptide having the amino acid sequence of SEQ ID NO: 2.
42. The selective binding agent of claim 30 which is bound to a
detectable label.
43. The selective binding agent of claim 30 which antagonizes
h2520-59 polypeptide biological activity.
44. A method for treating, preventing, or ameliorating a disease,
condition, or disorder comprising administering to a patient an
effective amount of a selective binding agent according to claim
30.
45. A selective binding agent produced by immunizing an animal with
a polypeptide comprising the amino acid sequence of SEQ ID NO:
2.
46. A hybridoma that produces a selective binding agent capable of
binding a polypeptide comprising the amino acid sequence set forth
in SEQ ID NO: 2.
47-78. (canceled)
79. A selective binding agent that specifically binds a first
polypeptide with an amino acid sequence comprising the amino acid
sequence set forth in SEQ ID NO: 2, and antagonizes the binding of
the first polypeptide to an activating transcription factor-4
(ATF-4) polypeptide.
80. The selective binding agent of claim 79 that is an antibody or
fragment thereof.
81. The selective binding agent of claim 79 that is a humanized
antibody.
82. The selective binding agent of claim 79 that is a human
antibody or fragment thereof.
83. The selective binding agent of claim 79 that is a polyclonal
antibody or fragment thereof.
84. The selective binding agent of claim 79 that is a monoclonal
antibody or fragment thereof.
85. The selective binding agent of claim 79 that is a chimeric
antibody or fragment thereof.
86. The selective binding agent of claim 79 that is a
complementarity determining region (CDR)-grafted antibody or
fragment thereof.
87. The selective binding agent of claim 79 that is an
anti-idiotypic antibody or fragment thereof.
88. The selective binding agent of claim 79 which is an antibody
variable region fragment.
89. The selective binding agent of claim 88 wherein the variable
region fragment is a Fab or a Fab' fragment.
90. The selective binding agent of claim 79 which is bound to a
detectable label.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/909,474 filed Jul. 19, 2001, which claims
benefit of U.S. Provisional Application Ser. No. 60/219,204 filed
Jul. 19, 2000, all of which are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel human serine
threonine kinase family member (h2520-59), and uses thereof. The
invention also relates to vectors, host cells, selective binding
agents, such as antibodies, and methods for producing h2520-59
polypeptides. Also provided for are methods for the diagnosis,
treatment, amelioration and/or prevention of diseases associated
with h2520-59 polypeptides.
BACKGROUND OF THE INVENTION
[0003] Technical advances in identification, cloning, expression
and manipulation of nucleic acid molecules and deciphering of the
human genome have greatly accelerated discovery of novel
therapeutics based upon deciphering of the human genome. Rapid
nucleic acid sequencing techniques can now generate sequence
information at unprecedented rates and, coupled with computational
analyses, allow the assembly of overlapping sequences into the
partial and entire genomes as well as identification of
polypeptide-encoding regions. A comparison of a predicted amino
acid sequence against a database compilation of known amino acid
sequences allows one to determine the extent of homology to
previously identified sequences and/or structural landmarks. The
cloning and expression of a polypeptide-encoding region of a
nucleic acid molecule provides a polypeptide product for structural
and functional analyses. The manipulation of nucleic acid molecules
and encoded polypeptides to create variants and derivatives thereof
may confer advantageous properties on a product for use as a
therapeutic.
[0004] In spite of significant technical advances in genome
research over the past decade, the potential for development of
novel therapeutics based on the human genome is still largely
unrealized. Many genes encoding potentially beneficial polypeptide
therapeutics, or those encoding polypeptides which may act as
"targets" for therapeutic molecules, have still not been
identified.
[0005] Accordingly, it is an object of the invention to identify
novel polypeptides and nucleic acid molecules encoding the same
which have diagnostic or therapeutic benefit.
[0006] Protein phosphorylation at specific amino acid residues is
an important biological theme involved in the regulation of most
cellular processes including cell cycle progression and division,
signal transduction, and apoptosis. Site-specific phosphorylation
can either activate or inactivate protein functions helping to link
the extracellular environmental information to intracellular
processes. Protein kinases represent a large and diverse group of
enzymes with current estimates of around 2,000 members. Included in
this family are many subgroups encoding oncogenes, growth factor
receptors, signal transduction intermediates, apoptosis related
kinases, and cyclin dependent kinases. Given the importance and
diversity of protein kinase function, it is not surprising that
alterations in phosphorylation are associated with many disease
states such as cancer, diabetes, arthritis, and hypertension.
[0007] Serine-threonine kinases (ser/thr kinases) are a large
sub-family of protein kinases which specifically phosphorylate
serine and threonine residues. All ser/thr kinase family members
contain a 250 amino acid catalytic domain which enzymatically
transfers a phosphate group from an ATP molecule to a hydroxyl
group on a serine or threonine side chain of a protein. (Hanks et
al., Science 241: 42-52, 1988).
[0008] A number of ser/thr kinase family members are involved in
tumor growth or cellular transformation by either increasing
cellular proliferation or decreasing the rate of apoptosis. For
example, the mitogen-activated protein kinases (MAPKs) are ser/thr
kinases which act as intermediates within the signaling cascades of
both growth/survival factors, such as EGF, and death receptors,
such as the TNF receptor. Expression of ser/thr kinases, such as
protein kinase A, protein kinase B and protein kinase C, have been
shown be elevated in some tumor cells. Further, cyclin dependent
kinases (cdk) are ser/thr kinases that play an important role in
cell cycle regulation. Increased expression or activation of these
kinases may cause uncontrolled cell proliferation leading to tumor
growth. (See Cross et al., Exp. Cell Res. 256: 34-41, 2000).
[0009] Thus, identification of members of the ser/thr kinase family
has led to a better understanding of cell proliferation,
differentiation and survival. Identification of the novel ser/thr
kinase gene and polypeptide, as described herein, will further
clarify the understanding of these processes and facilitate the
development of therapies for pathological conditions which involve
cellular hyperproliferation and other biological processes.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a novel serine/threonine
kinase family and uses thereof. More specifically, the present
invention relates to novel h2520-59 nucleic acid molecules and
encoded polypeptides, and uses thereof.
[0011] The invention provides for an isolated nucleic acid molecule
comprising a nucleotide sequence selected from the group consisting
of:
[0012] (a) the nucleotide sequence set forth in SEQ ID NO: 1;
[0013] (b) the h2520-59 encoding portion of SEQ ID NO: 1 comprising
nucleotides 49-1122 of SEQ ID NO 1;
[0014] (c) a nucleotide sequence encoding the polypeptide set forth
in SEQ ID NO: 2;
[0015] (d) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of (a) or (b) or
(c), wherein the encoded polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2; and
[0016] (e) a nucleotide sequence complementary to any of
(a)-(d).
[0017] The invention also provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0018] (a) a nucleotide sequence encoding a polypeptide that
exhibits at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99
percent identity to the polypeptide set forth in SEQ ID NO: 2,
wherein the polypeptide has an activity of the encoded polypeptide
set forth in SEQ ID NO: 2 as determined using a computer program
selected from the group consisting of GAP, BLASTP, BLASTN, FASTA,
BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm;
[0019] (b) a nucleotide sequence encoding an allelic variant or
splice variant of the nucleotide sequence set forth in SEQ ID NO:
1, wherein the encoded polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2;
[0020] (c) a nucleotide sequence of SEQ ID NO: 1, (a), or (b)
encoding a polypeptide fragment of at least about 25 amino acid
residues, wherein the polypeptide has an activity of the
polypeptide set forth in SEQ ID NO: 2;
[0021] (d) a nucleotide sequence encoding a polypeptide that has a
substitution and/or deletion of 1 to 358 amino acid residues set
forth in SEQ ID NO: 2 wherein the encoded polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2;
[0022] (e) a nucleotide sequence of SEQ ID NO: 1, or (a)-(d)
comprising a fragment of at least about 16 nucleotides;
[0023] (f) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of any of (a)-(e),
wherein the encoded polypeptide has an activity of the polypeptide
set forth in SEQ ID NO: 2; and
[0024] (g) a nucleotide sequence complementary to any of
(a)-(f).
[0025] The invention further provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0026] (a) a nucleotide sequence encoding a polypeptide set forth
in SEQ ID NO: 2 with at least one conservative amino acid
substitution, wherein the encoded polypeptide has an activity of
the polypeptide set forth in SEQ ID NO: 2;
[0027] (b) a nucleotide sequence encoding a polypeptide set forth
in SEQ ID NO: 2 with at least one amino acid insertion, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
SEQ ID NO: 2;
[0028] (c) a nucleotide sequence encoding a polypeptide set forth
in SEQ ID NO: 2 with at least one amino acid deletion, wherein the
encoded polypeptide has an activity of the polypeptide set forth in
SEQ ID NO: 2;
[0029] (d) a nucleotide sequence encoding a polypeptide set forth
in SEQ ID NO: 2 which has a C- and/or N-terminal truncation,
wherein the encoded polypeptide has an activity of the polypeptide
set forth in SEQ ID NO: 2;
[0030] (e) a nucleotide sequence encoding a polypeptide set forth
in SEQ ID NO: 2 with at least one modification selected from the
group consisting of amino acid substitutions, amino acid
insertions, amino acid deletions, C-terminal truncation, and
N-terminal truncation, wherein the polypeptide has an activity of
the encoded polypeptide set forth in SEQ ID NO: 2;
[0031] (f) a nucleotide sequence of (a)-(e) comprising a fragment
of at least about 16 nucleotides;
[0032] (g) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of any of (a)-(f),
wherein the encoded polypeptide has an activity of the polypeptide
set forth in SEQ ID NO: 2; and
[0033] (h) a nucleotide sequence complementary to any of
(a)-(e).
[0034] The invention also provides for an expression vector
comprising the isolated nucleic acid molecules set forth herein;
recombinant host cells (eukaryotic and/or prokaryotic) that
comprise the vector; the process for producing a h2520 polypeptide
comprising culturing the host cell under suitable conditions to
express the polypeptide and optionally isolating the polypeptide
from the culture; and the isolated polypeptide produced by this
process. The nucleic acid molecule used in this process may also
comprise promoter DNA other than the promoter DNA for the native
h2520-59 polypeptide operatively linked to the nucleotide sequence
encoding the h2520-59 polypeptide.
[0035] The invention also provides for a nucleic acid molecule as
described in the previous paragraphs wherein the percent identity
is determined using a computer program selected from the group
consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit,
and the Smith-Waterman algorithm.
[0036] The present invention provides a process for identifying
candidate inhibitors and/or stimulators of h2520-59 polypeptide
activity or production comprising exposing a host cell to the
candidate inhibitors and/or stimulators, measuring h2520
polypeptide activity or production in the host cell, and comparing
this activity with control cells (i.e., cells not exposed to the
candidate inhibitor and/or stimulator). In a related aspect, the
invention provides for the inhibitors and/or stimulators identified
by any of the preceding methods.
[0037] The invention also provides for an isolated polypeptide
comprising the amino acid sequence set forth in SEQ ID NO: 2.
[0038] The invention provides for an isolated polypeptide
comprising the amino acid sequence selected from the group
consisting of:
[0039] (a) the mature amino acid sequence set forth in SEQ ID NO: 2
comprising a mature amino terminus at residue 1, and optionally
further comprising an amino-terminal methionine;
[0040] (b) an amino acid sequence for an ortholog of SEQ ID NO: 2,
wherein the polypeptide has an activity of the polypeptide set
forth in SEQ ID NO: 2;
[0041] (c) an amino acid sequence that exhibits at least about 70,
75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identity to the amino
acid sequence of SEQ ID NO: 2, wherein the polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2 as determined
using a computer program selected from the group consisting of GAP,
BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the
Smith-Waterman algorithm;
[0042] (d) a fragment of the amino acid sequence set forth in SEQ
ID NO: 2 comprising at least about 25 amino acid residues, wherein
the polypeptide has an activity of the polypeptide set forth in SEQ
ID NO: 2; and
[0043] (e) an amino acid sequence for an allelic variant or splice
variant of either the amino acid sequence set forth in SEQ ID NO:
2, or at least one of (a)-(c), wherein the polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2.
[0044] The invention further provides for an isolated polypeptide
comprising the amino acid sequence selected from the group
consisting of:
[0045] (a) the amino acid sequence set forth in SEQ ID NO: 2 with
at least one conservative amino acid substitution, wherein the
polypeptide has an activity of the polypeptide set forth in SEQ ID
NO: 2;
[0046] (b) the amino acid sequence set forth in SEQ ID NO: 2 with
at least one amino acid insertion, wherein the polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2;
[0047] (c) the amino acid sequence set forth in SEQ ID NO: 2 with
at least one amino acid deletion, wherein the polypeptide has an
activity of the polypeptide set forth in SEQ ID NO: 2;
[0048] (d) the amino acid sequence set forth in SEQ ID NO: 2 which
has a C- and/or N-terminal truncation, wherein the polypeptide has
an activity of the polypeptide set forth in SEQ ID NO: 2; and
[0049] (e) the amino acid sequence set forth in SEQ ID NO: 2, with
at least one modification selected from the group consisting of
amino acid substitutions, amino acid insertions, amino acid
deletions, C-terminal truncation, and N-terminal truncation,
wherein the polypeptide has an activity of the polypeptide set
forth in SEQ ID NO: 2.
[0050] Analogs of h2520-59 are provided for in the present
invention which result from conservative and non-conservative amino
acid substitutions of the h2520-59 polypeptide of SEQ ID NO: 2.
Such analogs include a h2520-59 polypeptide wherein the amino acid
corresponding to position 31 of SEQ ID NO: 2 is valine, isoleucine,
methionine, leucine, phenylalanine, alanine, or norleucine; a
h2520-59 polypeptide wherein the amino acid corresponding to
position 60 of SEQ ID NO: 2 is threonine or serine; a h2520-59
polypeptide wherein the amino acid corresponding to position 229 of
SEQ ID NO: 2 is glutamic acid or aspartic acid; a h2520-59
polypeptide wherein the amino acid corresponding to position 258 of
SEQ ID NO: 2 is histidine, asparagine, glutamine, lysine, or
arginine; a h2520-59 polypeptide wherein the amino acid
corresponding to position 283 of SEQ ID NO: 2 is glycine, proline,
or alanine; and a h2520-59 polypeptide wherein the amino acid
corresponding to position 314 of SEQ ID NO: 2 is tryptophan,
tyrosine, or phenylalanine.
[0051] The present invention also provides for an isolated
polypeptide encoded by the nucleic acid molecules set forth
herein.
[0052] The present invention further provides for an antibody or
fragment thereof that specifically binds an h2520-59 polypeptide as
set forth herein. This antibody can be polyclonal or monoclonal,
and can be produced by immunizing an animal with a peptide
comprising an amino acid sequence of SEQ ID NO: 2.
[0053] Also provided is the hybridoma that produces a monoclonal
antibody that binds to a peptide comprising an amino acid sequence
of SEQ ID NO: 2.
[0054] The present invention also provides for a method of
detecting or quantitating the amount of h2520-59 polypeptide in a
sample comprising contacting a sample suspected of containing
h2520-59 polypeptide with the anti-h2520-59 antibody or antibody
fragment set forth herein and detecting the binding of said
antibody or antibody fragment.
[0055] Additionally provided by the invention are selective binding
agents or fragments thereof that are capable of specifically
binding the h2520-59 polypeptides, derivatives, variants, and
fragments (preferably having sequences of at least about 25 amino
acids) thereof. These selective binding agents may be antibodies
such as humanized antibodies, human antibodies, polyclonal
antibodies, monoclonal antibodies, chimeric antibodies,
complementarity determining region (CDR)-grafted antibodies,
anti-idiotypic antibodies, and fragments thereof. Furthermore, the
selective binding agents may be antibody variable region fragments,
such as Fab or Fab' fragments, or fragments thereof, and may
comprise at least one complementarity determining region with
specificity for a h2520-59 polypeptide set forth herein. The
selective binding agent may also be bound to a detectable label,
such as a radiolabel, a fluorescent label, an enzyme label, or any
other label known in the art. Further, the selective binding agent
may antagonize h2520-59 polypeptide biological activity, and/or be
produced by immunizing an animal with a h2520-59 polypeptide as set
forth herein.
[0056] The present invention also provides for a hybridoma that
produces a selective binding agent capable of binding h2520-59
polypeptide as set forth herein.
[0057] Also provided is a method for treating, preventing, or
ameliorating a disease, condition, or disorder comprising
administering to a patient an effective amount of a selective
binding agent as set forth herein. An effective amount, or a
therapeutically effective amount, is an amount sufficient to result
in a detectable change in the course or magnitude of the disease,
condition or disorder, such as the intensity or duration of
presentment of any symptom associated therewith.
[0058] Pharmaceutical compositions comprising the above-described
nucleic acid molecules, polypeptides, or selective binding agents
and one or more pharmaceutically acceptable formulation agents are
also encompassed by the invention. The pharmaceutically acceptable
formulation agent may be a carrier, adjuvant, solubilizer,
stabilizer, or anti-oxidant. The nucleic acid molecules of the
present invention may be contained in viral vectors. The
pharmaceutical compositions are used to provide therapeutically
effective amounts of the nucleic acid molecules or polypeptides of
the present invention.
[0059] Also provided are derivatives of the h2520-59 polypeptides
of the present invention. These polypeptides may be covalently
modified with a water-soluble polymer wherein the water-soluble
polymer is selected from the group consisting of polyethylene
glycol, monomethoxy-polyethylene glycol, dextran, cellulose,
poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol
homopolymers, polypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols, and polyvinyl alcohol.
[0060] The present invention also provides for fusion polypeptides
comprising the polypeptide sequences set forth herein fused to a
heterologous amino acid sequence, which may be an IgG constant
domain or fragment thereof.
[0061] Methods for treating, preventing or ameliorating a medical
condition, such as cancer, in a mammal resulting from increased
levels of h2520-59 polypeptide are also included in the present
invention. These methods include administering to a patient a
therapeutically effective amount of an antagonist selected from the
group consisting of selective binding agents, small molecules,
peptides, peptide derivatives and antisense oligonucleotides. The
cancer may include lung cancer, colon cancer or breast cancer.
[0062] Methods for treating, preventing or ameliorating a medical
condition in a mammal resulting from decreased levels of h2520-59
polypeptide are also included in the present invention. These
methods comprise administering to a patient a therapeutically
effective amount of a h2520-59 polypeptide; a nucleic acid molecule
encoding a h2520-59 polypeptide; or a nucleic acid molecule
comprising elements that regulate or modulate the expression of a
h2520-59 polypeptide. Examples of these methods include gene
therapy and cell therapy and are further described herein.
[0063] The invention encompasses methods of diagnosing a
pathological condition or a susceptibility to a pathological
condition in a subject caused by or resulting from abnormal levels
of h2520-59 polypeptide comprising determining the presence or
amount of expression of the h2520-59 polypeptide in a biological,
tissue, or cellular sample; and comparing the level of said
polypeptide in a biological, tissue, or cellular sample from either
normal subjects or the subject at a different time, wherein
susceptibility to a pathological condition is based on the presence
or amount of expression of the polypeptide.
[0064] The invention also provides for devices comprising a
membrane suitable for implantation to administer a h2520-59
polypeptide, wherein h2520-59 polypeptide or cells which can
secrete said peptide may be, encapsulated in the membrane. The said
membrane is permeable to the h2520-59 polypeptide; preferably, the
membrane is impermeable to detrimental materials such as materials
larger than the polypeptide.
[0065] The present invention also provides a method of identifying
compounds which bind to a h2520-59 polypeptide. The method
comprises contacting a h2520-59 polypeptide with a test molecule
and determining the extent of binding of the test molecule to the
polypeptide. The method may further comprise determining whether
such test molecules are agonists or antagonists of a h2520-59
polypeptide. The present invention further provides a method of
testing the impact of molecules on the expression of h2520-59
polypeptide or on the activity of h2520-59 polypeptide.
[0066] The present invention further provides a method of
modulating levels of a h2520-polypeptide in an animal comprising
administering to the animal the nucleic acid molecule set forth
herein.
[0067] A transgenic non-human animal comprising a nucleic acid
molecule encoding a h2520-59 polypeptide is also encompassed by the
invention. The h2520-59 nucleic acid molecule is introduced into
the animal in a manner that allows expression and increased levels
of the h2520-59 polypeptide, which may include increased
circulating levels. The transgenic non-human animal is preferably a
mammal.
[0068] The present invention provides for a diagnostic reagent
comprising a detectably labeled polynucleotide encoding the amino
acid sequence set out in SEQ ID NO: 2, or a fragment, variant or
homolog thereof, including allelic variants and spliced variants
thereof. The detectably labeled polynucleotide may be a
first-strand cDNA, DNA, or RNA.
[0069] The invention also provides a method for detecting the
presence of h2520-59 nucleic acid molecules in a biological sample
comprising the steps of:
[0070] (a) providing a biological sample suspected of containing
h2520-59 nucleic acid molecules;
[0071] (b) contacting the biological sample with a diagnostic
reagent under conditions wherein the diagnostic reagent will
hybridize with h2520-59 nucleic acid molecules contained in said
biological sample;
[0072] (c) detecting hybridization between h2520-59 nucleic acid
molecules in the biological sample and the diagnostic reagent;
and
[0073] (d) comparing the level of hybridization between the
biological sample and diagnostic reagent with the level of
hybridization between a known concentration of h2520-59 nucleic
acid molecules and the diagnostic reagent.
[0074] The invention also provides a method for detecting the
presence of h2520-59 nucleic acid molecules in a tissue or cellular
sample comprising the steps of:
[0075] (a) providing a tissue or cellular sample suspected of
containing h2520-59 nucleic acid molecules;
[0076] (b) contacting the tissue or cellular sample with a
diagnostic reagent under conditions wherein the diagnostic reagent
will hybridize with h2520-59 nucleic acid molecules;
[0077] (c) detecting hybridization between h2520-59 nucleic acid
molecules in the tissue or cellular sample and the diagnostic
reagent; and
[0078] (d) comparing the level of hybridization between the tissue
or cellular sample and diagnostic reagent with the level of
hybridization between a known concentration of h2520-59 nucleic
acid molecules and the diagnostic reagent.
[0079] Interestingly, h2520-59 polypeptide was highly expressed in
a wide range of primary human tumor cells. Therefore, the present
polypeptide, and cognate nucleic acids, have demonstrated utility
in distinguishing transformed cells from the non-transformed
cellular background.
[0080] In another aspect of the present invention, the h2520-59
polypeptides may be used for identifying receptors or binding
partners thereof ("h2520-59 receptors" or "h2520-59 binding
partners"). Various forms of "expression cloning" have been
extensively used to clone receptors for proteins or co-factors.
See, for example, Simonsen et al., Trends in Pharmacological
Sciences, 15: 437-441, 1994, and Tartaglia et al., Cell,
83:1263-1271, 1995. The isolation of the h2520-59 receptor(s) or
h2520-59 binding partner(s) is useful for identifying or developing
novel agonists and antagonists of the h2520-59
polypeptide-signaling pathway.
[0081] The present invention also provides for agonists and
antagonists of h2520-59 polypeptide activity. Such agonists and
antagonists include soluble h2520-59 ligand(s), anti-h2520-59
selective binding agents (such as h2520-59 antibodies and
derivatives thereof), small molecules, peptides or peptide
derivatives capable of binding h2520-59 polypeptides, or antisense
oligonucleotides, any of which can be used for potentially treating
one or more diseases or disorders, such as those recited
herein.
[0082] In certain embodiments, a h2520-59 polypeptide agonist or
antagonist may be a protein, peptide, carbohydrate, lipid, or small
molecular weight molecule which interacts with a h2520-59
polypeptide to regulate its activity.
[0083] Also provided in the present invention is a polynucleotide
described above attached to a solid support, as well as an array of
polynucleotides comprising at least one polynucleotide as described
above.
[0084] The h2520-59 polypeptides and nucleic acid molecules of the
present invention may be used to treat, prevent, ameliorate,
diagnose and/or detect diseases and disorders, including those
recited herein. Expression analysis in biological, cellular or
tissue samples suggests that h2520-59 polypeptide may play a role
in the diagnosis and/or treatment of hyperproliferative diseases
such as immune disorders, angiogenesis and vasculogenesis, wound
healing, diabetes mellitus, psoriasis, liver disease, inflammation
and cancer. This expression can de detected with a diagnostic agent
such as a h2520-59 nucleic acid.
BRIEF DESCRIPTION OF THE FIGURES
[0085] FIG. 1 depicts a nucleic acid sequence (SEQ ID NO: 1) which
encodes the human h2520-59 polypeptide sequence (SEQ ID NO: 2).
[0086] FIG. 2 presents an alignment of the predicted amino acid
sequence of the h2520-59 polypeptide (SEQ ID NO: 2) with the
following polypeptide sequences: GENBANK BAB15597, Accession No:
AK026945, (SEQ ID NO: 8), sequence number 1367 from WO 00/55350
(SEQ ID NO: 9), sequence number 1102 from WO 00/55350 (SEQ ID NO:
10), and JJ503-KS polypeptide (sequence number 9 from WO 00/08180;
SEQ ID NO: 11) using the Pileup Program (Wisconsin GCC Program
Package ver. 8.1).
[0087] FIG. 3 presents an alignment of the h2520-59 polypeptide
(SEQ ID NO: 2) with the following known polypeptides which share
high identity: SKIP3 (GENBANK Accession No. AF250311), NIPK
(GENBANK Accession No. AB020967), dog C5FW (GENBANK Accession No.
X99144), and Drosophila Tribbles (GENBANK Accession No. AF204688),
as well as a highly related human family member, SKIP1 (GENBANK
Accession No. AF250310) using the Pileup Program (Wisconsin GCC
Program Package ver. 8.1). Amino acids that are identical between
all five proteins are marked in gray, and the consensus sequence is
indicated in bold below the alignments.
[0088] FIG. 4 presents an alignment of the h2520-59 polypeptide
(SEQ ID NO: 2) kinase-like domain with known kinase domains of sthe
following known functional kinases: protein kinase C-alpha
(PKC-.alpha.) (SEQ ID NO: 36; GENBANK Accession No. P17252), serine
threonine-specific protein kinase (STK2) (SEQ ID NO: 37; GENBANK
Accession No. 178885), and 5'-AMP-activated protein kinase (AMPK)
(SEQ ID NO: 38; GENBANK Accession No. P54646). Protein kinase
subdomains are denoted in the alignment. Amino acids that are
identical between all four sequences are marked in gray and the
consensus sequence is indicated in bold below the alignments.
h2520-59 has significant homology to the kinase substrate binding
domains of known kinases (subdomains VIA-XI) but is highly variant
within the ATP binding domains (subdomains I-V).
[0089] FIG. 5 demonstrates Northern blot analysis of h2520-59 mRNA
expression in various human normal tissue and human tumor cell
lines. h2520-59 mRNA was highly expressed in normal human liver
tissue and multiple human tumor cell lines.
[0090] FIG. 6 presents Northern blot analysis of h2520-59 in
primary human lung and colon tumors. h2520-59 mRNA was upregulated
in both tumor types. Panel A represents a Northern blot on lung
tumor mRNA isolated from the following: lane 1, well differentiated
lung cell adenocarcinoma from a 48 year old male; lane 2,
moderately well differentiated lung cell adenocarcinoma from a 56
year old male; lane 3, moderately differentiated lung cell
carcinoma from a 74 year old female; lane 4, pooled normal lung
tissue from two donors; lane 5, pooled normal lung tissue five
donors. Panel B represents a Northern blot of colon tumor mRNA and
normal colon mRNA isolated from the following: lane 1, moderately
differentiated colon cell adenocarcinoma from a 58 year old male;
lane 2, well differentiated colon cell adenocarcinoma from a 63
year old female; lane 3 well differentiated colon cell
adenocarcinoma from a 35 year old male; lane 4, pooled normal colon
tissue from ten donors; lane 5, normal lung tissue from one
donor.
[0091] FIG. 7 presents results from real-time RT-PCR experiments.
h2520-59 mRNA was overexpressed in multiple primary human tumors as
compared to normal tissue. Real-time RT-PCR was performed on total
RNA samples from either normal human tissue or primary human tumor
tissues. Normal tissue includes samples from both non-disease
containing organs as well as normal tumor-adjacent tissue. All
values were normalized against 18S rRNA relative transcript levels.
P-value confidence was calculated using an unpaired comparison
t-test of the mean difference of normal and tumor 2520-59
transcript levels from each organ; n=2.
[0092] FIG. 8 presents results from in situ hybridization (ISH)
experiments. ISH detected the presence of h2520-59 mRNA in human
tumors. h2520-59 mRNA was clearly present over tumor cells in a
human lung adenocarcinoma (A) and a poorly differentiated human
colon adenocarcinoma (C), but not detectable in normal human lung
(B) or colon (D) or in stromal elements and infiltrating
lymphocytes within the tumor sections. Arrows indicate tumor cells
expressing high levels of h2520-59 mRNA. In a section of a human
breast tumor metastasis to brain (E), h2520-59 mRNA ISH signal is
present over tumor areas (T) but not over adjacent areas of
non-malignant brain tissue. For all panels, a bright field H&E
stain is on the left and h2520-59 mRNA ISH photographed under dark
field illumination is on the right.
[0093] FIG. 9 presents in situ hybridization (ISH) and
bromodeoxyuridine (BrdU) labeling of h2520-59 mRNA in human tumor
xenografts grown subcutaneously in nude mice. Both HT-29 colon
carcinoma (A & B) and PC-3 prostate carcinoma (C & D)
xenografts expressed h2520-59 mRNA. Low magnification insets show
the distribution of signal in the tumor mass with the boxed areas
shown at higher magnification in the large panels. h2520-59 mRNA
expression did not correlate with proliferation as measured by BrdU
labeling (dark stained nuclei indicated by arrowheads in B &
D). In the HT-29 xenograft, h2520-59 mRNA ISH signal (arrowheads)
was present over cells proximal to an area of apoptosis (black
arrows). In the PC3 xenograft, h2520-59 mRNA was expressed
throughout the tumor. Panels A & C are h2520-59 mRNA ISH
photographed under dark field illumination. Panels B & D are
adjacent sections immunostained for BrdU; low magnification inserts
were H&E stained.
[0094] FIG. 10 demonstrates relative h2520-59 mRNA and protein
expression under hypoxic conditions over time. h2520-59 mRNA
transcript and protein expression accumulated over time under
hypoxic growth conditions. HT-29 or PC-3 cells were grown under
hypoxia or normoxia for 0, 24, 48 and 72 hours. Panel A displays
TaqMan PCR analysis of h2520-59 mRNA expression. All values were
normalized against 18S rRNA levels, n=3. Panel B depicts Western
blot analysis of h2520-59 polypeptide expression. Western blots
were probed with anti-2520-59 polyclonal antibody from direct cell
lysates.
[0095] FIG. 11 presents Western blots demonstrating results of
yeast two-hybrid studies where h2520-59 participated in the
proteolysis of activating transcription factor 4 (ATF4) and
co-immunoprecipitated with ATF4. Panel A depicts Western blots of
lysates of U2-0S cells transfected with expression vectors encoding
h2520-59-myc, and ATF4-HA and treated with the proteosome inhibitor
MG-132 as indicated. h2520-59-myc was detected with anti-h2520-59
antibody, and ATF4-HA was detected with anti-HA antibody. Panel B
depicts Immunoprecipitation of ATF4-HA with anti-HA antibody from
U2-0S cells transfected with expression vectors encoding
h2520-59-myc and ATF4-HA as indicated. h2520-59-myc was detected
with anti-myc antibody. * indicates detection of the heavy chain of
the immunoprecipitation antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0096] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described therein. All references cited in this application
are expressly incorporated by reference herein.
Definitions
[0097] The term "h2520-59 nucleic acid molecule" or
"polynucleotide" refers to a nucleic acid molecule comprising or
consisting of a nucleotide sequence set forth in SEQ ID NO: 1, a
nucleotide sequence encoding the polypeptide set forth in SEQ ID
NO: 2, or the nucleic acid sequence of the DNA insert in American
Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, Va. 20110-2209, deposit No. PTA-1759, deposited on Apr.
25, 2000, and nucleic acids molecules as defined herein.
[0098] The term "h2520-59 polypeptide" refers to a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, and related
polypeptides. Related polypeptides include: h2520-59 polypeptide
allelic variants, h2520-59 polypeptide orthologs, h2520-59
polypeptide splice variants, h2520-59 polypeptide variants and
h2520-59 polypeptide derivatives. The h2520-59 polypeptides may be
mature polypeptides, as defined herein, and may or may not have an
amino terminal methionine residue, depending on the method by which
they are prepared.
[0099] The term "h2520-59 polypeptide allelic variant" refers to
the polypeptide encoded by one of several possible naturally
occurring alternate forms of a gene occupying a given locus on a
chromosome of an organism or a population of organisms.
[0100] The term "h2520-59 polypeptide derivatives" refers to a
polypeptide having an amino acid sequence as set forth in SEQ ID
NO: 2, h2520-59 polypeptide allelic variants, h2520-59 polypeptide
orthologs, h2520-59 polypeptide splice variants, or h2520-59
polypeptide variants, as defined herein, that have been chemically
modified.
[0101] The term "h2520-59 polypeptide fragment" refers to a
polypeptide that comprises a truncation at the amino terminus (with
or without a leader sequence) and/or a truncation at the carboxy
terminus of the polypeptide whose sequence is set forth in SEQ ID
NO: 2, h2520-59 polypeptide allelic variants, h2520-59 polypeptide
orthologs, h2520-59 polypeptide splice variants and/or a h2520-59
polypeptide variant having one or more amino acid additions or
substitutions or internal deletions (wherein the resulting
polypeptide is at least 6 amino acids or more in length) as
compared to the h2520-59 polypeptide amino acid sequence set forth
in SEQ ID NO: 2. h2520-59 polypeptide fragments may result from
alternative RNA splicing or from in vivo protease activity. In
preferred embodiments, truncations comprise about 10 amino acids,
or about 20 amino acids, or about 50 amino acids, or about 75 amino
acids, or about 100 amino acids, or more than about 100 amino
acids. The polypeptide fragments so produced will comprise about 25
contiguous amino acids, or about 50 amino acids, or about 75 amino
acids, or about 100 amino acids, or about 150 amino acids, or about
200 amino acids. Such h2520-59 polypeptide fragments may optionally
comprise an amino terminal methionine residue. It will be
appreciated that such fragments can be used, for example, to
generate antibodies to h2520-59 polypeptides.
[0102] The term "h2520-59 fusion polypeptide" refers to a fusion of
one or more amino acids (such as a heterologous peptide or
polypeptide) at the amino or carboxy terminus of the polypeptide
set forth in SEQ ID NO: 2, h2520-59 polypeptide allelic variants,
h2520-59 polypeptide orthologs, h2520-59 polypeptide splice
variants, or h2520-59 polypeptide variants having one or more amino
acid deletions, substitutions or internal additions as compared to
the h2520-59 polypeptide amino acid sequence set forth in SEQ ID
NO: 2.
[0103] The term "h2520-59 polypeptide ortholog" refers to a
polypeptide from another species that corresponds to the h2520-59
polypeptide amino acid sequence set forth in SEQ ID NO: 2. For
example, mouse and human h2520-59 polypeptides are considered
orthologs of each other.
[0104] The term "h2520-59 polypeptide splice variant" refers to a
nucleic acid molecule, usually RNA, which is generated by
alternative processing of intron sequences in an RNA primary
transcript containing the non-contiguous coding region of the
h2520-59 polypeptide amino acid sequence set forth in SEQ ID NO:
2.
[0105] The term "h2520-59 polypeptide variants" refers to h2520-59
polypeptides comprising amino acid sequences having one or more
amino acid sequence substitutions, deletions (such as internal
deletions and/or h2520-59 polypeptide fragments), and/or additions
(such as internal additions and/or h2520-59 fusion polypeptides) as
compared to the h2520-59 polypeptide amino acid sequence set forth
in SEQ ID NO: 2 (with or without a leader sequence). Variants may
be naturally occurring (e.g., h2520-59 polypeptide allelic
variants, h2520-59 polypeptide orthologs and h2520-59 polypeptide
splice variants) or may be artificially constructed. Such h2520-59
polypeptide variants may be prepared from the corresponding nucleic
acid molecules having a DNA sequence that varies accordingly from
the DNA sequence set forth in SEQ ID NO: 1. In preferred
embodiments, the variants have from 1 to 3, or from 1 to 5, or from
1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from
1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino
acid substitutions, insertions, additions and/or deletions, wherein
the substitutions may be conservative, or non-conservative, or any
combination thereof.
[0106] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody, and additionally capable of being used in an animal
to produce antibodies capable of binding to an epitope of each
antigen. An antigen may have one or more epitopes.
[0107] The term "biologically active h2520-59 polypeptides" refers
to h2520-59 polypeptides having at least one activity
characteristic of the polypeptide comprising the amino acid
sequence of SEQ ID NO: 2.
[0108] The terms "effective amount" and "therapeutically effective
amount" each refer to the amount of a h2520-59 polypeptide or
h2520-59 nucleic acid molecule used to support an observable level
of one or more biological activities of the h2520-59 polypeptides
set forth herein.
[0109] The term "expression vector" refers to a vector which is
suitable for use in a host cell and contains nucleic acid sequences
which direct and/or control the expression of heterologous nucleic
acid sequences. Expression includes, but is not limited to,
processes such as transcription, translation, and RNA splicing, if
introns are present.
[0110] The term "host cell" is used to refer to a cell which has
been transformed with a nucleic acid sequence and then of
expressing a selected gene of interest. The term includes the
progeny of the parent cell, whether or not the progeny is identical
in morphology or in genetic make-up to the original parent, so long
as the selected gene is present.
[0111] The term "identity," as known in the art, refers to a
relationship between the sequences of two or more polypeptide
molecules or two or more nucleic acid molecules, as determined by
comparing the sequences. In the art, "identity" also means the
degree of sequence relatedness between nucleic acid molecules or
polypeptides, as the case may be, as determined by the match
between strings of two or more nucleotide or two or more amino acid
sequences. "Identity" measures the percent of identical matches
between the smaller of two or more sequences with gap alignments
(if any) addressed by a particular mathematical model or computer
program (i.e., "algorithms").
[0112] The term "similarity" is a related concept, but in contrast
to "identity," refers to a measure of similarity which includes
both identical matches and conservative substitution matches. If
two polypeptide sequences have, for example, 10/20 identical amino
acids, and the remainder are all non-conservative substitutions,
then the percent identity and similarity would both be 50%. If, in
the same example, there are five more positions where there are
conservative substitutions, then the percent identity remains 50%,
but the percent similarity would be 75% ( 15/20). Therefore, in
cases where there are conservative substitutions, the degree of
percent similarity between two polypeptides will be higher than the
percent identity between those two polypeptides.
[0113] The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule of the invention that (1) has been separated
from at least about 50 percent of proteins, lipids, carbohydrates
or other materials with which it is naturally found when total DNA
is isolated from the source cells, (2) is not linked to all or a
portion of a polynucleotide to which the "isolated nucleic acid
molecule" is linked in nature, (3) is operably linked to a
polynucleotide which it is not linked to in nature, or (4) does not
occur in nature as part of a larger polynucleotide sequence.
Preferably, the isolated nucleic acid molecule of the present
invention is substantially free from any other contaminating
nucleic acid molecule(s) or other contaminants that are found in
its natural environment that would interfere with its use in
polypeptide production or its therapeutic, diagnostic, prophylactic
or research use.
[0114] The term "isolated polypeptide" refers to a polypeptide of
the present invention that (1) has been separated from at least
about 50 percent of polynucleotides, lipids, carbohydrates or other
materials with which it is naturally found when isolated from the
source cell, (2) is not linked (by covalent or noncovalent
interaction) to all or a portion of a polypeptide to which the
"isolated polypeptide" is linked in nature, (3) is operably linked
(by covalent or noncovalent interaction) to a polypeptide with
which it is not linked in nature, or (4) does not occur in nature.
Preferably, the isolated polypeptide is substantially free from any
other contaminating polypeptides or other contaminants that are
found in its natural environment that would interfere with its
therapeutic, diagnostic, prophylactic or research use.
[0115] The term "mature h2520-59 polypeptide" refers to a h2520-59
polypeptide lacking a leader sequence. A mature h2520-59
polypeptide may also include other modifications such as
proteolytic processing of the amino terminus (with or without a
leader sequence) and/or the carboxy terminus, cleavage of a smaller
polypeptide from a larger precursor, N-linked and/or O-linked
glycosylation, and the like. An exemplary mature h2520-59
polypeptide is depicted by amino acid residue 1 through amino acid
residue 358 of SEQ ID NO: 2.
[0116] The terms "nucleic acid sequence" or "nucleic acid molecule"
refer to a DNA or RNA sequence. The terms encompass molecules
formed from any of the known base analogs of DNA and RNA such as,
but not limited to, 4-acetylcytosine, 8-hydroxy-N-6-methyladenine,
aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,
N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5-methoxycarbonyl-methyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, 2-thiocytosine, and 2,6-diaminopurine.
[0117] The term "naturally occurring" or "native" when used in
connection with biological materials such as nucleic acid
molecules, polypeptides, host cells, and the like, refers to
materials which are found in nature and are not manipulated by man.
Similarly, "non-naturally occurring" or "non-native" as used herein
refers to a material that is not found in nature or that has been
structurally modified or synthesized by man.
[0118] The term "operably linked" is used herein to refer to a
method of flanking sequences wherein the flanking sequences so
described are configured or assembled so as to perform their usual
function. Thus, a flanking sequence operably linked to a coding
sequence may be capable of effecting the replication, transcription
and/or translation of the coding sequence. For example, a coding
sequence is operably linked to a promoter when the promoter is
capable of directing transcription of that coding sequence. A
flanking sequence need not be contiguous with the coding sequence,
so long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence, and the promoter
sequence can still be considered "operably linked" to the coding
sequence.
[0119] The terms "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier" as used herein refer to one or
more formulation materials suitable for accomplishing or enhancing
the delivery of the h2520-59 polypeptide, h2520-59 nucleic acids
molecule, or h2520-59 selective binding agent as a pharmaceutical
composition.
[0120] The term "selective binding agent" refers to a molecule or
molecules having specificity for a h2520-59 polypeptide. As used
herein the terms "specific" and "specifically" refer to the ability
of the selective binding agents to bind to human h2520-59
polypeptides and not to bind to human non-h2520-59 polypeptides. It
will be appreciated, however, that the selective binding agents may
also bind orthologs of the polypeptide set forth in SEQ ID NO: 2,
that is, interspecies versions thereof, such as mouse and rat
polypeptides.
[0121] The term "transduction" is used to refer to the transfer of
nucleic acid from one bacterium to another. "Transduction" also
refers to the acquisition and transfer of eukaryotic cellular
sequences by viruses such as retroviruses.
[0122] The term "transfection" is used to refer to the uptake of
foreign or exogenous DNA by a cell, and a cell has been
"transfected" when the exogenous DNA has been introduced inside the
cell membrane. A number of transfection techniques are well known
in the art and are disclosed herein. See, for example, Graham et
al., Virology, 52: 456, 1973; Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratories (New York,
1989); Davis et al., Basic Methods in Molecular Biology Elsevier,
1986; and Chu et al., Gene, 13: 197, 1981. Such techniques can be
used to introduce one or more exogenous DNA moieties into suitable
host cells.
[0123] The term "transformation" as used herein refers to a change
in a cell's genetic characteristics, and a cell has been
transformed when it has been modified to contain new DNA. For
example, a cell is transformed where it is genetically modified
from its native state. Following transfection or transduction, the
transforming DNA may recombine with that of the cell by physically
integrating into a chromosome of the cell, it may be maintained
transiently as an episomal element without being replicated, or it
may replicate independently as a plasmid. A cell is considered to
have been stably transformed when the DNA is replicated with the
division of the cell.
[0124] The term "vector" is used to refer to any molecule (e.g.,
nucleic acid, plasmid, or virus) used to transfer coding
information to a host cell.
Relatedness of Nucleic Acid Molecules and/or Polypeptides
[0125] It is understood that related nucleic acid molecules include
allelic or splice variants of the nucleic acid molecule of SEQ ID
NO: 1, and include sequences which are complementary to any of the
above nucleotide sequences. Related nucleic acid molecules also
include a nucleotide sequence encoding a polypeptide comprising or
consisting essentially of a substitution, modification, addition
and/or deletion of one or more amino acid residues compared to the
polypeptide in SEQ ID NO: 2.
[0126] Fragments include molecules which encode a polypeptide of at
least about 25 amino acid residues, or about 50, or about 75, or
about 100, or greater than about 100 amino acid residues of the
polypeptide of SEQ ID NO: 2.
[0127] In addition, related h2520-59 nucleic acid molecules include
those molecules which comprise nucleotide sequences which hybridize
under moderately or highly stringent conditions as defined herein
with the fully complementary sequence of the nucleic acid molecule
of SEQ ID NO: 1, or of a molecule encoding a polypeptide, which
polypeptide comprises the amino acid sequence as shown in SEQ ID
NO: 2, or of a nucleic acid fragment as defined herein, or of a
nucleic acid fragment encoding a polypeptide as defined herein.
Hybridization probes may be prepared using the h2520-59 sequences
provided herein to screen cDNA, genomic or synthetic DNA libraries
for related sequences. Regions of the DNA and/or amino acid
sequence of a h2520-59 polypeptide that exhibit significant
identity to known sequences are readily determined using sequence
alignment algorithms as described herein and those regions may be
used to design probes for screening.
[0128] The term "highly stringent conditions" refers to those
conditions that are designed to permit hybridization of DNA strands
whose sequences are highly complementary, and to exclude
hybridization of significantly mismatched DNAs. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of "highly stringent conditions" for
hybridization and washing are 0.015 M sodium chloride and 0.0015 M
sodium citrate at 65-68.degree. C.; or 0.015 M sodium chloride,
0.0015 M sodium citrate, and 50% formamide at 42.degree. C. See
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. (1989) and
Anderson et al., Nucleic Acid Hybridization: a Practical Approach,
Ch. 4, IRL Press Limited (Oxford, England).
[0129] More stringent conditions (such as higher temperature, lower
ionic strength, higher formamide, or other denaturing agent) may
also be used; however, the degree of hybridization will be
affected. Other agents may be included in the hybridization and
washing buffers for the purpose of reducing non-specific and/or
background hybridization. Examples are 0.1% bovine serum albumin,
0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium
dodecylsulfate (NaDodSO.sub.4 or SDS), ficoll, Denhardt's solution,
sonicated salmon sperm DNA (or another non-complementary DNA), and
dextran sulfate, although other suitable agents can also be used.
The concentration and types of these additives can be changed
without substantially affecting the stringency of the hybridization
conditions. Hybridization experiments are usually carried out at pH
6.8-7.4; however, at typical ionic strength conditions, the rate of
hybridization is nearly independent of pH. (See Anderson et al.,
Nucleic Acid Hybridization: a Practical Approach, Ch. 4, IRL Press
Limited (Oxford, England)).
[0130] Factors affecting the stability of DNA duplex include base
composition, length, and degree of base pair mismatch.
Hybridization conditions can be adjusted by one skilled in the art
in order to accommodate these variables and allow DNAs of different
sequence relatedness to form hybrids. The melting temperature of a
perfectly matched DNA duplex can be estimated by the following
equation: Tm(.degree. C.)=81.5+16.6(log[Na.sup.+])+0.41(%
G+C)-600/N-0.72(% formamide) where N is the length of the duplex
formed in nucleotides, [Na.sup.+] is the molar concentration of the
sodium ion in the hybridization or washing solution, and % G+C is
the percentage of (guanine+cytosine) bases in the hybrid. For
imperfectly matched hybrids, the melting temperature is reduced by
approximately 1.degree. C. for each 1% mismatch.
[0131] The term "moderately stringent conditions" refers to
conditions under which a DNA duplex with a greater degree of base
pair mismatching than could occur under "highly stringent
conditions" is able to form. Examples of typical "moderately
stringent conditions" are 0.015 M sodium chloride and 0.0015 M
sodium citrate at 50-65.degree. C.; or 0.015 M sodium chloride,
0.0015 M sodium citrate, and 20% formamide at 37-50.degree. C. By
way of example, a "moderately stringent" condition of 50.degree. C.
in 0.015 M sodium ion will allow about a 21% mismatch.
[0132] It will be appreciated by those skilled in the art that
there is no absolute distinction between "highly" and "moderately"
stringent conditions. For example, at 0.015 M sodium ion (no
formamide), the melting temperature of perfectly matched long DNA
is about 71.degree. C. With a wash at 65.degree. C. (at the same
ionic strength), this would allow for approximately a 6% mismatch.
To capture more distantly related sequences, one skilled in the art
can simply lower the temperature or raise the ionic strength.
[0133] A good estimate of the melting temperature in 1.0 M NaCl*
for oligonucleotide probes up to about 20 nucleotides is given by:
*The sodium ion concentration in 6.times. salt sodium citrate (SSC)
is 1.0 M. See Suggs et al., Developmental Biology Using Purified
Genes, p. 683, Brown and Fox (eds.) (1981). Tm=2.degree. C. per A-T
base pair+4.degree. C. per G-C base pair
[0134] High stringency washing conditions for oligonucleotides are
usually at a temperature of 0-5.degree. C. below the Tm of the
oligonucleotide in 6.times.SSC, 0.1% SDS for longer
oligonucleotides.
[0135] In another embodiment, related nucleic acid molecules
comprise or consist of a nucleotide sequence that is about 70
percent (70%) identical to the nucleotide sequence as shown in SEQ
ID NO: 1, or comprise or consist essentially of a nucleotide
sequence encoding a polypeptide that is about 70 percent (70%)
identical to the polypeptide set forth in SEQ ID NO: 2. In
preferred embodiments, the nucleotide sequences are about 75
percent, or about 80 percent, or about 85 percent, or about 90
percent, or about 95, 96, 97, 98, or 99 percent identical to the
nucleotide sequence as shown in SEQ ID NO: 1, or the nucleotide
sequences encode a polypeptide that is about 75 percent, or about
80 percent, or about 85 percent, or about 90 percent, or about 95,
96, 97, 98, or 99 percent identical to the polypeptide sequence set
forth in SEQ ID NO: 2.
[0136] Differences in the nucleic acid sequence may result in
conservative and/or non-conservative modifications of the amino
acid sequence relative to the amino acid sequence of SEQ ID NO:
2.
[0137] Conservative modifications to the amino acid sequence of SEQ
ID NO: 2 (and the corresponding modifications to the encoding
nucleotides) will produce h2520-59 polypeptides having functional
and chemical characteristics similar to those of a naturally
occurring h2520-59 polypeptide. In contrast, substantial
modifications in the functional and/or chemical characteristics of
h2520-59 polypeptides may be accomplished by selecting
substitutions in the amino acid sequence of SEQ ID NO: 2 that
differ significantly in their effect on maintaining (a) the
structure of the molecular backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[0138] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
normative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described
(Cunningham and Wells, Science 244:1081-1085, 1989) for "alanine
scanning mutagenesis".
[0139] Conservative amino acid substitutions also encompass
non-naturally occurring amino acid residues which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics, and other
reversed or inverted forms of amino acid moieties.
[0140] Naturally occurring residues may be divided into classes
based on common side chain properties: [0141] 1) hydrophobic:
norleucine, Met, Ala, Val, Leu, Ile; [0142] 2) neutral hydrophilic:
Cys, Ser, Thr, Asn, Gln; [0143] 3) acidic: Asp, Glu; [0144] 4)
basic: His, Lys, Arg; [0145] 5) residues that influence chain
orientation: Gly, Pro; and [0146] 6) aromatic: Trp, Tyr, Phe.
[0147] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the human h2520-59 polypeptide that are homologous, or
similar, with non-human h2520-59 polypeptide orthologs, or into the
non-homologous regions of the molecule.
[0148] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of its hydrophobicity and charge
characteristics. They are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0149] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157: 105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within +2 is preferred, those which are
within .+-.1 are particularly preferred, and those within +0.5 are
even more particularly preferred.
[0150] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functionally
equivalent protein or peptide thereby created is intended, in part,
for use in immunological embodiments, as in the present case. The
greatest local average hydrophilicity of a protein, as governed by
the hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property
of the protein.
[0151] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0 1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, the substitution of amino acids whose
hydrophilicity values are within +2 is preferred, those which are
within +1 are particularly preferred, and those within .+-.0.5 are
even more particularly preferred. One may also identify epitopes
from primary amino acid sequences on the basis of hydrophilicity.
These regions are also referred to as "epitopic core regions."
[0152] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
h2520-59 polypeptide, or to increase or decrease the affinity of
the h2520-59 polypeptides for their substrates, described
herein.
[0153] Exemplary amino acid substitutions are set forth in Table I.
TABLE-US-00001 TABLE I Conservative Amino Acid Substitutions
Original Exemplary Preferred Residues Substitutions Substitutions
Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu
Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn,
Gln, Lys, Mg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine Leu
Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino- Arg
butyric Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Leu
Ala, Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe
Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala,
Norleucine
[0154] A skilled artisan will be able to determine suitable
variants of the polypeptide set forth in SEQ ID NO: 2 using well
known techniques. For identifying suitable areas of the molecule
that may be changed without destroying activity, one skilled in the
art may target areas not likely to be important for activity. For
example, when similar polypeptides with similar activities from the
same species or from other species are known, one skilled in the
art may compare the amino acid sequence of a h2520-59 polypeptide
to such similar polypeptides. With such a comparison, one can
identify residues and portions of the molecules that are conserved
among similar polypeptides. It will be appreciated that changes in
areas of a h2520-59 polypeptide that are not conserved relative to
such similar polypeptides would be less likely to adversely affect
the biological activity and/or structure of the h2520-59
polypeptide. One skilled in the art would also know that, even in
relatively conserved regions, one may substitute chemically similar
amino acids for the naturally occurring residues while retaining
activity (conservative amino acid residue substitutions).
Therefore, even areas that may be important for biological activity
or for structure may be subject to conservative amino acid
substitutions without destroying the biological activity or without
adversely affecting the polypeptide structure.
[0155] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a h2520-59 polypeptide that correspond to amino acid
residues which are important for activity or structure in similar
polypeptides. One skilled in the art may opt for chemically similar
amino acid substitutions for such predicted important amino acid
residues of h2520-59 polypeptides.
[0156] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of such
information, one skilled in the art may predict the alignment of
amino acid residues of a h2520-59 polypeptide with respect to its
three-dimensional structure. One skilled in the art may choose not
to make radical changes to amino acid residues predicted to be on
the surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each desired amino acid residue. The variants
can then be screened using activity assays known to those skilled
in the art. Such variants could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided,
either alone or in combination with other mutations.
[0157] The h2520-59 polypeptide analogs of the invention can be
determined by comparing the amino acid sequence of h2520-59
polypeptide with related family members. Exemplary h2520-59
polypeptide-related family members may include, but are not limited
to, GENBANK BAB15597, Accession No. BAB15597 (SEQ ID NO: 8),
sequence number 1367 in WO 00/55350 (SEQ ID NO: 9), sequence number
1102 in WO 00/55350 (SEQ ID NO: 10), and JJ503-KS polypeptide
(sequence number 9 in WO 00/08180; SEQ ID NO: 11). This comparison
can be accomplished by using a Pileup alignment (Wisconsin GCG
Program Package, ver. 8.1; as shown in FIG. 2) or an equivalent
(overlapping) comparison with multiple family members within
conserved and non-conserved regions. As shown in FIG. 2, the
predicted amino acid sequence of a h2520-59 polypeptide (SEQ ID NO:
2) is aligned with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and
SEQ ID NO: 11. The public sequence, GENBANK BAB15597, Accession No.
AK026945 (SEQ ID NO: 8), was entered in GENBANK on Sep. 29, 2000
with no function described, but is 100% aligned with h2520-59 (SEQ
ID NO: 2). The other sequences displayed in FIG. 2 (SEQ ID NO:
9-11) are partially aligned with h2520-59 (SEQ ID NO: 2).
[0158] Other h2520-59 polypeptide analogs can be identified using
these or other methods known to those of skill in the art. These
overlapping sequences provide guidance for conservative and
non-conservative amino acids substitutions resulting in additional
h2520-59 analogs. It will be appreciated that these amino acid
substitutions can consist of naturally occurring or non-naturally
occurring amino acids. For example, as depicted in FIG. 2,
alignment of the amino acids of these related polypeptides
indicates potential h2520-59 analogs may have the Val residue at
position 31 of SEQ ID NO: 2 substituted with an Ile, Met, Leu, Phe,
Ala, or norleucine residue; the Thr residue at position 60 of SEQ
ID NO: 2 substituted with a Ser residue; the Glu residue at
position 229 of SEQ ID NO: 2 substituted with an Asp residue; the
His residue at position 258 of SEQ ID NO: 2 substituted with an
Asn, Gln, Lys, or Arg residue; the Gly residue at position 283 of
SEQ ID NO: 2 substituted with a Pro or Ala residue; and the Trp
residue at position 314 of SEQ ID NO: 2 substituted with a Tyr or
Phe residue.
[0159] A number of scientific publications have been devoted to the
prediction of secondary structure. See Chou et al., Biochemistry,
13(2): 222-245, 1974; Chou et al., Biochemistry, 113(2): 211-222,
1974; Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:
45-148, 1978; Chou et al., Ann. Rev. Biochem., 47: 251-276 and Chou
et al., Biophys. J., 26: 367-384, 1979. Moreover, computer programs
are currently available to assist with predicting secondary
structure. One method of predicting secondary structure is based
upon homology modeling. For example, two polypeptides or proteins
which have a sequence identity of greater than 30%, or similarity
greater than 40%, often have similar structural topologies. The
recent growth of the protein structural data base (PDB) has
provided enhanced predictability of secondary structure, including
the potential number of folds within a polypeptide's or protein's
structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247, 1999.
It has been suggested (Brenner et al., Curr. Opin. Struct. Biol.,
7(3): 369-376, 1997) that there are a limited number of folds in a
given polypeptide or protein and that once a critical number of
structures have been resolved, structural prediction becomes
dramatically more accurate.
[0160] Additional methods of predicting secondary structure include
"threading" (Jones et al., Current Opin. Struct. Biol., 7(3):
377-87 (1997); Sippl et al., Structure, 4(1):15-9 (1996)), "profile
analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et
al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage"
(See Home, supra, and Brenner, supra 1997).
[0161] Preferred h2520-59 polypeptide variants include
glycosylation variants wherein the number and/or type of
glycosylation sites has been altered compared to the amino acid
sequence set forth in SEQ ID NO: 2. In one embodiment, h2520-59
polypeptide variants comprise a greater or a lesser number of
N-linked glycosylation sites than the amino acid sequence set forth
in SEQ ID NO: 2. An N-linked glycosylation site is characterized by
the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid
residue designated as X may be any amino acid residue except
proline. The substitution of amino acid residues to create this
sequence provides a potential new site for the addition of an
N-linked carbohydrate chain. Alternatively, substitutions which
eliminate this sequence will remove an existing N-linked
carbohydrate chain. Also provided is a rearrangement of N-linked
carbohydrate chains wherein one or more N-linked glycosylation
sites (typically those that are naturally occurring) are eliminated
and one or more new N-linked sites are created. Additional
preferred h2520-59-like variants include cysteine variants, wherein
one or more cysteine residues are deleted from or substituted for
another amino acid (e.g., serine) as compared to the amino acid
sequence set forth in SEQ ID NO: 2. Cysteine variants are useful
when h2520-59 polypeptides must be refolded into a biologically
active conformation such as after the isolation of insoluble
inclusion bodies. Cysteine variants generally have fewer cysteine
residues than the native protein, and typically have an even number
to minimize interactions resulting from unpaired cysteines.
[0162] In addition, the polypeptide comprising the amino acid
sequence of SEQ ID NO: 2 or a h2520-59 polypeptide variant may be
fused to a homologous polypeptide to form a homodimer or to a
heterologous polypeptide to form a heterodimer. Heterologous
peptides and polypeptides include, but are not limited to: an
epitope to allow for the detection and/or isolation of a h2520-59
fusion polypeptide; a transmembrane receptor protein or a portion
thereof, such as an extracellular domain, or a transmembrane and
intracellular domain; a ligand or a portion thereof which binds to
a transmembrane receptor protein; an enzyme or portion thereof
which is catalytically active; a polypeptide or peptide which
promotes oligomerization, such as a leucine zipper domain; a
polypeptide or peptide which increases stability, such as an
immunoglobulin constant region; and a polypeptide which has a
therapeutic activity different from the polypeptide comprising the
amino acid sequence set forth in SEQ ID NO: 2 or a h2520-59
polypeptide variant.
[0163] Fusions can be made either at the amino terminus or at the
carboxy terminus of the polypeptide comprising the amino acid
sequence set forth in SEQ ID NO: 2 or a h2520-59 polypeptide
variant. Fusions may be direct with no linker or adapter molecule
or indirect using a linker or adapter molecule. A linker or adapter
molecule may be one or more amino acid residues, typically from 20
to about 50 amino acid residues. A linker or adapter molecule may
also be designed with a cleavage site for a DNA restriction
endonuclease in an encoding polynucleotide or for a protease to
allow for the separation of the fused moieties. It will be
appreciated that once constructed, the fusion polypeptides can be
derivatized according to the methods described herein.
[0164] In a further embodiment of the invention, the polypeptide
comprising the amino acid sequence of SEQ ID NO: 2 or a h2520-59
polypeptide variant is fused to one or more domains of an Fc region
of human IgG. Antibodies comprise two functionally independent
parts, a variable domain known as "Fab," which binds antigens, and
a constant domain known as "Fc," which is involved in effector
functions such as complement activation and attack by phagocytic
cells. An Fe has a long serum half-life, whereas an Fab is
short-lived. Capon et al., Nature, 337: 525-31 (1989). When
constructed together with a therapeutic protein, an Fc domain can
provide longer half-life or incorporate such functions as Fc
receptor binding, protein A binding, complement fixation and
perhaps even placental transfer. Id. Table II summarizes the use of
certain Fc fusions known in the art. TABLE-US-00002 TABLE II Fc
Fusion with Therapeutic Proteins Form of Fusion Therapeutic Fc
partner implications Reference IgGl N-terminus Hodgkin's disease;
U.S. Patent No. of CD30-L anaplastic lymphoma; 5,480,981 T-cell
leukemia Murine IL-10 anti-inflammatory; Zheng et al., J.
Fc.gamma.2a transplant rejection Immunol., 154: 5590- 600, 1995
IgG1 TNF septic shock Fisher et al., N. receptor Engl. J. Med.,
334: 1697-1702, 1996; Van Zee et al., , J. Immunol., 156: 2221- 30,
1996 IgG, IgA, TNF inflammation, U.S. Pat. No. IgM, or receptor
autoimmune disorders 5,808,029 IgE (excluding the first domain)
IgG1 CD4 AIDS Capon et al., Nature, receptor 337: 525-31, 1989
IgG1, N-terminus anti-cancer, antiviral Harvill et al., IgG3 of
IL-2 Immunotech., 1: 95- 105, 1995 IgG1 C-terminus osteoarthritis;
WO 97/23614, of OPG bone density published July 3, 1997 IgG1
N-terminus anti-obesity PCT/US 97/23183, of leptin filed December
11, 1997 Human Ig CTLA-4 autoimmune disorders Linsley, J. Exp.
C.gamma.1 Med., 174: 561-9, 1991
[0165] In one example, all or a portion of the human IgG hinge, CH2
and CH3 regions may be fused at either the N-terminus or C-terminus
of the h2520-59 polypeptides using methods known to the skilled
artisan. The resulting h2520-59 fusion polypeptide may be purified
by use of a Protein A affinity column. Peptides and proteins fused
to an Fc region have been found to exhibit a substantially greater
half-life in vivo than the unfused counterpart. Also, a fusion to
an Fc region allows for dimerization/multimerization of the fusion
polypeptide. The Fc region may be a naturally occurring Fc region,
or may be altered to improve certain qualities, such as therapeutic
qualities, circulation time, reduce aggregation, etc.
[0166] Identity and similarity of related nucleic acid molecules
and polypeptides can be readily calculated by known methods. Such
methods include, but are not limited to, those described in
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 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM
J., Applied Math., 48:1073, 1988.
[0167] Preferred methods to determine identity and/or similarity
are designed to give the largest match between the sequences
tested. Methods to determine identity and similarity are described
in publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., Nucl. Acids. Res., 12:387, 1984; Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410,
1990)). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBI) and other sources
(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra). The well-known Smith-Waterman algorithm
may also be used to determine identity.
[0168] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, in a preferred
embodiment, the selected alignment method (GAP program) will result
in an alignment that spans at least 50 contiguous amino acids of
the target polypeptide.
[0169] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span," as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
1/10 times the gap opening penalty), as well as a comparison matrix
such as PAM 250 or BLOSUM 62 are used in conjunction with the
algorithm. A standard comparison matrix (see Dayhoff et al., Atlas
of Protein Sequence and Structure, vol. 5, supp. 3 (1978) for the
PAM 250 comparison matrix; Henikoff et al., Proc. Natl. Acad. Sci
USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is
also used by the algorithm.
[0170] Preferred parameters for a polypeptide sequence comparison
include the following: [0171] Algorithm: Needleman et al., J. Mol.
Biol., 48, 443-453, 1970; [0172] Comparison matrix: BLOSUM 62 from
Henikoff et al., Proc. Natl. Acad. Sci. USA, 89: 10915-10919,
1992); [0173] Gap Penalty: 12 [0174] Gap Length Penalty: 4 [0175]
Threshold of Similarity: 0
[0176] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for
polypeptide comparisons (along with no penalty for end gaps) using
the GAP algorithm.
[0177] Preferred parameters for nucleic acid molecule sequence
comparisons include the following: [0178] Algorithm: Needleman et
al., J. Mol. Biol., 48: 443-453, 1970; [0179] Comparison matrix:
matches=+10, mismatch=0 [0180] Gap Penalty: 50 [0181] Gap Length
Penalty: 3
[0182] The GAP program is also useful with the above parameters.
The aforementioned parameters are the default parameters for
nucleic acid molecule comparisons.
[0183] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, thresholds of similarity,
etc. may be used by those of skill in the art, including those set
forth in the Program Manual, Wisconsin Package, Version 9,
September, 1997. The particular choices to be made will be apparent
to those of skill in the art and will depend on the specific
comparison to be made, such as DNA-to-DNA, protein-to-protein,
protein-to-DNA, and additionally, whether the comparison is between
given pairs of sequences (in which case GAP or BestFit are
generally preferred) or between one sequence and a large database
of sequences (in which case FASTA or BLASTA are preferred).
Synthesis
[0184] It will be appreciated by those skilled in the art that the
nucleic acid and polypeptide molecules described herein may be
produced by recombinant and other means.
Nucleic Acid Molecules
[0185] The nucleic acid molecules encode a polypeptide comprising
the amino acid sequence of a h2520-59 polypeptide and can readily
be obtained in a variety of ways including, without limitation,
chemical synthesis, cDNA or genomic library screening, expression
library screening and/or PCR amplification of cDNA.
[0186] Recombinant DNA methods used herein are generally those set
forth in Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989), and/or Ausubel et al., eds., Current Protocols in Molecular
Biology, Green Publishers Inc. and Wiley and Sons, NY (1994). The
present invention provides for nucleic acid molecules as described
herein and methods for obtaining such molecules.
[0187] Where a gene encoding the amino acid sequence of a h2520-59
polypeptide has been identified from one species, all or a portion
of that gene may be used as a probe to identify orthologs or
related genes from the same species. The probes or primers may be
used to screen cDNA libraries from various tissue sources believed
to express the h2520-59 polypeptide. In addition, part or all of a
nucleic acid molecule having the sequence set forth in SEQ ID NO: 1
may be used to screen a genomic library to identify and isolate a
gene encoding the amino acid sequence of a h2520-59 polypeptide.
Typically, conditions of moderate or high stringency will be
employed for screening to minimize the number of false positives
obtained from the screening.
[0188] Nucleic acid molecules encoding the amino acid sequence of
h2520-59 polypeptides may also be identified by expression cloning
which employs the detection of positive clones based upon a
property of the expressed protein. Typically, nucleic acid
libraries are screened by the binding of an antibody or other
binding partner (e.g., receptor, ligand, or co-factor) to cloned
proteins which are expressed and displayed on a host cell surface.
The antibody or binding partner is modified with a detectable label
to identify those cells expressing the desired clone.
[0189] Recombinant expression techniques conducted in accordance
with the descriptions set forth below may be followed to produce
these polynucleotides and to express the encoded polypeptides. For
example, by inserting a nucleic acid sequence which encodes the
amino acid sequence of a h2520-59 polypeptide into an appropriate
vector, one skilled in the art can readily produce large quantities
of the desired nucleotide sequence. The sequences can then be used
to generate detection probes or amplification primers.
Alternatively, a polynucleotide encoding the amino acid sequence of
a h2520-59 polypeptide can be inserted into an expression vector.
By introducing the expression vector into an appropriate host, the
encoded h2520-59 polypeptide may be produced in large amounts.
[0190] Another method for obtaining a suitable nucleic acid
sequence is the polymerase chain reaction (PCR). In this method,
cDNA is prepared from poly(A)+RNA or total RNA using the enzyme
reverse transcriptase. Two oligonucleotide primers, typically
complementary to two separate regions of cDNA encoding the amino
acid sequence of a h2520-59 polypeptide, are then added to the cDNA
along with a polymerase such as Taq polymerase, and the polymerase
amplifies the cDNA region between the two primers.
[0191] Another means of preparing a nucleic acid molecule encoding
the amino acid sequence of a h2520-59 polypeptide is chemical
synthesis using methods well known to the skilled artisan such as
those described by Engels et al., (Angew. Chem. Intl. Ed., 28:
716-734, 1989). These methods include, inter alia, the
phosphotriester, phosphoramidite, and H-phosphonate methods for
nucleic acid synthesis. A preferred method for such chemical
synthesis is polymer-supported synthesis using standard
phosphoramidite chemistry. Typically, the DNA encoding the amino
acid sequence of a h2520-59 polypeptide will be several hundred
nucleotides in length. Nucleic acids larger than about 100
nucleotides can be synthesized as several fragments using these
methods. The fragments can then be ligated together to form the
full-length nucleotide sequence of a h2520-59 polypeptide. Usually,
the DNA fragment encoding the amino terminus of the polypeptide
will have an ATG, which encodes a methionine residue. This
methionine may or may not be present on the mature form of the
h2520-59 polypeptide, depending on whether the polypeptide produced
in the host cell is designed to be secreted from that cell. Other
methods known to the skilled artisan may be used as well.
[0192] In certain embodiments, nucleic acid variants contain codons
which have been altered for the optimal expression of a h2520-59
polypeptide in a given host cell. Particular codon alterations will
depend upon the h2520-59 polypeptide(s) and host cell(s) selected
for expression. Such "codon optimization" can be carried out by a
variety of methods, for example, by selecting codons which are
preferred for use in highly expressed genes in a given host cell.
Computer algorithms which incorporate codon frequency tables such
as "Ecohigh.cod" for codon preference of highly expressed bacterial
genes may be used and are provided by the University of Wisconsin
Package Version 9.0, Genetics Computer Group, Madison, Wis. Other
useful codon frequency tables include "Celegans_high.cod,"
"Celegans_low.cod," "Drosophila_high.cod," "Human_high.cod,"
"Maize_high.cod," and "Yeast_high.cod."
Vectors and Host Cells
[0193] A nucleic acid molecule encoding the amino acid sequences of
h2520-59 polypeptide may be inserted into an appropriate expression
vector using standard ligation techniques. The vector is typically
selected to be functional in the particular host cell employed
(i.e., the vector is compatible with the host cell machinery such
that amplification of the gene and/or expression of the gene can
occur). A nucleic acid molecule encoding the amino acid sequence of
h2520-59 polypeptide may be amplified/expressed in prokaryotic,
yeast, insect (baculovirus systems), and/or eukaryotic host cells.
Selection of the host cell will depend in part on whether a
h2520-59 polypeptide is to be post-translationally modified (e.g.,
glycosylated and/or phosphorylated). If so, yeast, insect, or
mammalian host cells are preferable. For a review of expression
vectors, see Meth. Enz., v. 185, D. V. Goeddel, ed. Academic Press
Inc., San Diego, Calif. (1990).
[0194] Typically, expression vectors used in any of the host cells
will contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences,
collectively referred to as "flanking sequences" in certain
embodiments, will typically include one or more of the following
nucleotide sequences: a promoter, one or more enhancer sequences,
an origin of replication, a transcriptional termination sequence, a
complete intron sequence containing a donor and acceptor splice
site, a sequence encoding a leader sequence for polypeptide
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed below.
[0195] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide molecule located at the 5' or 3'
end of the h2520-59 polypeptide coding sequence; the
oligonucleotide sequence encodes polyHis (such as hexaHis), or
another "tag" such as FLAG, HA (hemaglutinin influenza virus) or
myc for which commercially available antibodies exist. This tag is
typically fused to the polypeptide upon expression of the
polypeptide, and can serve as a means for affinity purification of
the h2520-59 polypeptide from the host cell. Affinity purification
can be accomplished, for example, by column chromatography using
antibodies against the tag as an affinity matrix. Optionally, the
tag can subsequently be removed from the purified h2520-59
polypeptide by various means such as using certain peptidases for
cleavage.
[0196] Flanking sequences may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of flanking sequences from more than one source), or
synthetic, or the flanking sequences may be native sequences which
normally function to regulate h2520-59 polypeptide expression. As
such, the source of a flanking sequence may be any prokaryotic or
eukaryotic organism, any vertebrate or invertebrate organism, or
any plant, provided that the flanking sequences is in, and can be
activated by, the host cell machinery.
[0197] The flanking sequences useful in the vectors of this
invention may be obtained by any of several methods well known in
the art. Typically, flanking sequences useful herein other than the
endogenous h2520-59 gene flanking sequences will have been
previously identified by mapping and/or by restriction endonuclease
digestion and can thus be isolated from the proper tissue source
using the appropriate restriction endonucleases. In some cases, the
full nucleotide sequence of one or more flanking sequence may be
known. Here, the flanking sequence may be synthesized using the
methods described herein for nucleic acid synthesis or cloning.
[0198] Where all or only a portion of the flanking sequence is
known, it may be obtained using PCR and/or by screening a genomic
library with suitable oligonucleotide and/or flanking sequence
fragments from the same or another species. Where the flanking
sequence is not known, a fragment of DNA containing a flanking
sequence may be isolated from a larger piece of DNA that may
contain, for example, a coding sequence or even another gene or
genes. Isolation may be accomplished by restriction endonuclease
digestion to produce the proper DNA fragment followed by isolation
using agarose gel purification, Qiagen.RTM. column chromatography
(Chatsworth, Calif.), or other methods known to the skilled
artisan. The selection of suitable enzymes to accomplish this
purpose will be readily apparent to one of ordinary skill in the
art.
[0199] An origin of replication is typically a part of those
prokaryotic expression vectors purchased commercially, and the
origin aids in the amplification of the vector in a host cell.
Amplification of the vector to a certain copy number can, in some
cases, be important for the optimal expression of the h2520-59
polypeptide. If the vector of choice does not contain an origin of
replication site, one may be chemically synthesized based on a
known sequence, and ligated into the vector. For example, the
origin of replication from the plasmid pBR322 (Product No. 303-3s,
New England Biolabs, Beverly, Mass.) is suitable for most
gram-negative bacteria and various origins (e.g., SV40, polyoma,
adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses
such as HPV or BPV) are useful for cloning vectors in mammalian
cells. Generally, the origin of replication component is not needed
for mammalian expression vectors (for example, the SV40 origin is
often used only because it contains the early promoter).
[0200] A transcription termination sequence is typically located 3'
of the end of a polypeptide coding region and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described herein.
[0201] A selectable marker gene element encodes a protein necessary
for the survival and growth of a host cell grown in a selective
culture medium. Typical selection marker genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, tetracycline, or kanamycin for prokaryotic host cells,
(b) complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex media. Preferred
selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene. A
neomycin resistance gene may also be used for selection in
prokaryotic and eukaryotic host cells.
[0202] Other selection genes may be used to amplify the gene which
will be expressed. Amplification is the process wherein genes which
are in greater demand for the production of a protein critical for
growth are reiterated within the chromosome(s) of successive
generations of recombinant cells. Examples of suitable selectable
markers for mammalian cells include dihydrofolate reductase (DHFR)
and thymidine kinase. The mammalian cell transformants are placed
under selection pressure which only the transformants are uniquely
adapted to survive by virtue of the selection gene present in the
vector. Selection pressure is imposed by culturing the transformed
cells under conditions in which the concentration of selection
agent in the medium is successively changed, thereby leading to the
amplification of both the selection gene and the DNA that encodes
h2520-59 polypeptide. As a result, increased quantities of h2520-59
polypeptide are synthesized from the amplified DNA.
[0203] A ribosome binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3', to the promoter and 5' to the
coding sequence of the h2520-59 polypeptide to be expressed. The
Shine-Dalgarno sequence is varied but is typically a polypurine
(i.e., having a high A-G content). Many Shine-Dalgarno sequences
have been identified, each of which can be readily synthesized
using methods set forth herein and used in a prokaryotic
vector.
[0204] A leader, or signal, sequence may be used to direct a
h2520-59 polypeptide out of the host cell. Typically, a nucleotide
sequence encoding the signal sequence is positioned in the coding
region of the h2520-59 nucleic acid molecule, or directly at the 5'
end of the h2520-59 polypeptide coding region. Many signal
sequences have been identified, and any of those that are
functional in the selected host cell may be used in conjunction
with the h2520-59 nucleic acid molecule. Therefore, a signal
sequence may be homologous (naturally occurring) or heterologous to
the h2520-59 gene or cDNA. Additionally, a signal sequence may be
chemically synthesized using methods described herein. In most
cases, the secretion of a h2520-59 polypeptide from the host cell
via the presence of a signal peptide will result in the removal of
the signal peptide from the secreted h2520-59 polypeptide. The
signal sequence may be a component of the vector, or it may be a
part of a h2520-59 nucleic acid molecule that is inserted into the
vector.
[0205] Included within the scope of this invention is the use of
either a nucleotide sequence encoding a native h2520-59 polypeptide
signal sequence joined to a h2520-59 polypeptide coding region or a
nucleotide sequence encoding a heterologous signal sequence joined
to a h2520-59 polypeptide coding region. The heterologous signal
sequence selected should be one that is recognized and processed,
i.e., cleaved by a signal peptidase, by the host cell. For
prokaryotic host cells that do not recognize and process the native
h2520-59 signal sequence, the signal sequence is substituted by a
prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, or heat-stable
enterotoxin II leaders. For yeast secretion, the native h2520-59
polypeptide signal sequence may be substituted by the yeast
invertase, alpha factor, or acid phosphatase leaders. In mammalian
cell expression the native signal sequence is satisfactory,
although other mammalian signal sequences may be used.
[0206] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various presequences to improve glycosylation or yield. For
example, one may alter the peptidase cleavage site of a particular
signal peptide, or add presequences, which also may affect
glycosylation. The final protein product may have, in the -1
position (relative to the first amino acid of the mature protein),
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the N-terminus. Alternatively,
use of some enzyme cleavage sites may result in a slightly
truncated form of the desired h2520-59 polypeptide, if the enzyme
cuts at such area within the mature polypeptide.
[0207] In many cases, transcription of a nucleic acid molecule is
increased by the presence of one or more introns in the vector;
this is particularly true where a polypeptide is produced in
eukaryotic host cells, especially mammalian host cells. The introns
used may be naturally occurring within the h2520-59 gene,
especially where the gene used is a full-length genomic sequence or
a fragment thereof. Where the intron is not naturally occurring
within the coding region (as for most cDNAs), the intron(s) may be
obtained from another source. The position of the intron with
respect to flanking sequences and the h2520-59 gene is generally
important, as the intron must be transcribed to be effective. Thus,
when a h2520-59 cDNA molecule is being transcribed, the preferred
position for the intron is 3' to the transcription start site, and
5' to the polyA transcription termination sequence. Preferably, the
intron or introns will be located on one side or the other (i.e.,
5' or 3') of the cDNA such that it does not interrupt the coding
sequence. Any intron from any source, including viral, prokaryotic
and eukaryotic (plant or animal) organisms, may be used to practice
this invention, provided that it is compatible with the host
cell(s) into which it is inserted. Also included herein are
synthetic introns. Optionally, more than one intron may be used in
the vector.
[0208] The expression and cloning vectors of the present invention
will each typically contain a promoter that is recognized by the
host organism and operably linked to the molecule encoding a
h2520-59 polypeptide. Promoters are untranscribed sequences
typically located upstream (5' to the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription of the structural gene. Promoters are conventionally
grouped into one of two classes, inducible promoters and
constitutive promoters. In this context, inducible promoters
include repressible/derepressible promoters and conventional
inducible promoters. Inducible promoters initiate increased levels
of transcription from DNA under their control in response to some
change in culture conditions, such as the presence or absence of a
nutrient or a change in temperature. Constitutive promoters, on the
other hand, initiate continual gene product production; that is,
there is little or no control over gene expression. A large number
of promoters, recognized by a variety of potential host cells, are
well known. A suitable promoter is operably linked to the DNA
encoding a h2520-59 polypeptide by, e.g., removing the promoter
from the source DNA by restriction enzyme digestion and inserting
the desired promoter sequence into the vector. The native h2520-59
promoter sequence may be used to direct amplification and/or
expression of a h2520-59 nucleic acid molecule. A heterologous
promoter is preferred, however, if it permits greater transcription
and higher yields of the expressed protein as compared to the
native promoter, and if it is compatible with the host cell system
that has been selected for use.
[0209] Promoters suitable for use with prokaryotic hosts include
the beta-lactamase and lactose promoter systems; alkaline
phosphatase, a tryptophan (trp) promoter system; and hybrid
promoters such as the tac promoter. Other known bacterial promoters
are also suitable. Their sequences have been published, thereby
enabling one skilled in the art to ligate them to the desired DNA
sequence(s), using linkers or adapters as needed to supply any
useful restriction sites.
[0210] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include, but are not limited to, those
obtained from the genomes of viruses such as polyoma virus, fowl
pox virus, adenovirus (such as Adenovirus 2), bovine papilloma
virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus,
hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other
suitable mammalian promoters include heterologous mammalian
promoters, e.g., heat-shock promoters and the actin promoter.
[0211] Additional promoters which may be of interest in controlling
h2520-59 gene transcription include, but are not limited to: the
SV40 early promoter region (Benoist and Chambon, Nature, 290:
304-310, 1981); the CMV promoter; the promoter contained in the 3'
long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell,
22: 787-797, 1980); the herpes simplex thymidine kinase promoter
(Wagner et al., Proc. Natl. Acad. Sci. USA, 78: 144-1445, 1981);
the regulatory sequences of the metallothionein gene (Brinster et
al., Nature, 296: 39-42, 1982); prokaryotic expression vectors such
as the beta-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl.
Acad. Sci. USA, 75: 3727-3731, 1978); or the tac promoter (DeBoer,
et al., Proc. Natl. Acad. Sci. USA, 80: 21-25, 1983). Also of
interest are the following animal transcriptional control regions,
which exhibit tissue specificity and have been utilized in
transgenic animals: the elastase I gene control region, which is
active in pancreatic acinar cells (Swift et al., Cell, 38: 639-646,
1984; Ornitz et al., Cold Spring Harbor Symp. Quant. Biol., 50:
399-409, 1986; MacDonald, Hepatology, 7: 425-515, 1987); the
insulin gene control region, which is active in pancreatic beta
cells (Hanahan, Nature, 315: 115-122, 1985); the immunoglobulin
gene control region, which is active in lymphoid cells (Grosschedl
et al., Cell, 38: 647-658 (1984); Adames et al., Nature, 318:
533-538 (1985); Alexander et al., Mol. Cell. Biol., 7: 1436-1444,
1987); the mouse mammary tumor virus control region, which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., Cell, 45: 485-495, 1986); the albumin gene control region,
which is active in liver (Pinkert et al., Genes and Devel., 1:
268-276, 1987); the alphafetoprotein gene control region, which is
active in liver (Krumlauf et al., Mol. Cell. Biol., 5: 1639-1648,
1985; Hammer et al., Science, 235: 53-58, 1987); the alpha
1-antitrypsin gene control region, which is active in the liver
(Kelsey et al., Genes and Devel., 1: 161-171, 1987); the
beta-globin gene control region, which is active in myeloid cells
(Mogram et al., Nature, 315: 338-340, 1985; Kollias et al., Cell,
46: 89-94, 1986); the myelin basic protein gene control region,
which is active in oligodendrocyte cells in the brain (Readhead et
al., Cell, 48: 703-712, 1987); the myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, Nature,
314: 283-286, 1985); and the gonadotropic releasing hormone gene
control region, which is active in the hypothalamus (Mason et al.,
Science, 234: 1372-1378, 1986).
[0212] An enhancer sequence may be inserted into the vector to
increase the transcription of a DNA encoding a h2520-59 polypeptide
of the present invention by higher eukaryotes. Enhancers are
cis-acting elements of DNA, usually about 10-300 bp in length, that
act on the promoter to increase its transcription. Enhancers are
relatively orientation- and position-independent. They have been
found 5' and 3' to the transcription unit. Several enhancer
sequences available from mammalian genes are known (e.g., globin,
elastase, albumin, alpha-feto-protein and insulin). Typically,
however, an enhancer from a virus will be used. The SV40 enhancer,
the cytomegalovirus early promoter enhancer, the polyoma enhancer,
and adenovirus enhancers are exemplary enhancing elements for the
activation of eukaryotic promoters. While an enhancer may be
spliced into the vector at a position 5' or 3' to a h2520-59
nucleic acid molecule, it is typically located at a site 5' from
the promoter.
[0213] Expression vectors of the invention may be constructed from
a starting vector such as a commercially available vector. Such
vectors may or may not contain all of the desired flanking
sequences. Where one or more of the desired flanking sequences are
not already present in the vector, they may be individually
obtained and ligated into the vector. Methods used for obtaining
each of the flanking sequences are well known to one skilled in the
art.
[0214] Preferred vectors for practicing this invention are those
which are compatible with bacterial, insect, and mammalian host
cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1
(Invitrogen Company, Carlsbad, Calif.), pBSII (Stratagene Company,
La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia
Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.),
pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No.
WO90/14363) and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).
[0215] Additional suitable vectors include, but are not limited to,
cosmids, plasmids, or modified viruses, but it will be appreciated
that the vector system must be compatible with the selected host
cell. Such vectors include, but are not limited to plasmids such as
Bluescript.RTM. plasmid derivatives (a high copy number ColE1-based
phagemid, Stratagene Cloning Systems Inc., La Jolla Calif.), PCR
cloning plasmids designed for cloning Taq-amplified PCR products
(e.g., TOPO.TM. TA Cloning.RTM. Kit, PCR2.1.RTM. plasmid
derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeast,
or virus vectors such as a baculovirus expression system (pBacPAK
plasmid derivatives, Clontech, Palo Alto, Calif.).
[0216] After the vector has been constructed and a nucleic acid
molecule encoding a h2520-59 polypeptide has been inserted into the
proper site of the vector, the completed vector may be inserted
into a suitable host cell for amplification and/or polypeptide
expression. The transformation of an expression vector for a
h2520-59 polypeptide into a selected host cell may be accomplished
by well-known methods such as transfection, infection, calcium
chloride-mediated transformation, electroporation, microinjection,
lipofection or the DEAE-dextran method or other known techniques.
The method selected will in part be a function of the type of host
cell to be used. These methods and other suitable methods are
well-known to the skilled artisan and are set forth, for example,
in Sambrook et al., supra.
[0217] Host cells may be prokaryotic host cells (such as E. coli)
or eukaryotic host cells (such as yeast, insect, or vertebrate
cells). The host cell, when cultured under appropriate conditions,
may synthesize a h2520-59 polypeptide which can subsequently be
collected from the culture medium (if the host cell secretes it
into the medium) or directly from the host cell producing it (if it
is not secreted). The selection of an appropriate host cell will
depend upon various factors, such as desired expression levels,
polypeptide modifications that are desirable or necessary for
activity, such as glycosylation or phosphorylation, and ease of
folding into a biologically active molecule.
[0218] A number of suitable host cells are known in the art and
many are available from the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209.
Examples include, but are not limited to, mammalian cells, such as
Chinese hamster ovary cells (CHO) (ATCC No. CCL61); CHO DHFR-cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220, 1980;
ATCC No. CRL9096), human embryonic kidney (HEK) 293 or 293T cells
(ATCC No. CRL1573); or 3T3 cells (ATCC No. CCL92). The selection of
suitable mammalian host cells and methods for transformation,
culture, amplification, screening, product production and
purification are known in the art. Other suitable mammalian cell
lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 (ATCC No.
CRL1651) cell lines and the CV-1 cell line (ATCC No. CCL70).
Further exemplary mammalian host cells include primate cell lines
and rodent cell lines, including transformed cell lines. Normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, as well as primary explants, are also suitable.
Candidate cells may be genotypically deficient in the selection
gene, or may contain a dominantly acting selection gene. Other
suitable mammalian cell lines include, but are not limited to,
mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines
derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell
lines, which are also available from the ATCC. Each of these cell
lines is known by and available to those skilled in the art of
protein expression.
[0219] Similarly useful as host cells suitable for the present
invention are bacterial cells. For example, the various strains of
E. coli (e.g., HB101, (ATCC No. 33694) DH5.alpha., DH10, and MC1061
(ATCC No. 53338)) are well-known as host cells in the field of
biotechnology. Various strains of B. subtilis, Pseudomonas spp.,
other Bacillus spp., Streptomyces spp., and the like may also be
employed in this method.
[0220] Many strains of yeast cells known to those skilled in the
art are also available as host cells for expression of the
polypeptides of the present invention. Preferred yeast cells
include, for example, Saccharomyces cerevisiae and Pichia
pastoris.
[0221] Additionally, where desired, insect cell systems may be
utilized in the methods of the present invention. Such systems are
described for example in Kitts et al., Biotechniques, 14: 810-817,
1993; Luckow, Curr. Opin. Biotechnol., 4: 564-572, 1993; and
Luckow, et al., J. Virol., 67: 4566-4579, 1993. Preferred insect
cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, Calif.).
[0222] One may also use transgenic animals to express glycosylated
h2520-59 polypeptides. For example, one may use a transgenic
milk-producing animal (a cow or goat, for example) and obtain the
present glycosylated polypeptide in the animal milk. One may also
use plants to produce h2520-59 polypeptides.
Polypeptide Production
[0223] Host cells comprising a h2520-59 polypeptide expression
vector may be cultured using standard media well known to the
skilled artisan. The media will usually contain all nutrients
necessary for the growth and survival of the cells. Suitable media
for culturing E. coli cells include, for example, Luria Broth (LB)
and/or Terrific Broth (TB). Suitable media for culturing eukaryotic
cells include, Roswell Park Memorial Institute medium 1640 (RPMI
1640), Minimal Essential Medium (MEM), and/or Dulbecco's Modified
Eagle Medium (DMEM), all of which may be supplemented with serum
and/or growth factors as indicated for the particular cell line
being cultured. A suitable medium for insect cultures is Grace's
medium supplemented with yeastolate, lactalbumin hydrolysate,
and/or fetal calf serum, as necessary.
[0224] Typically, an antibiotic or other compound useful for
selective growth of transformed cells is added as a supplement to
the media. The compound to be used will be dictated by the
selectable marker element present on the plasmid with which the
host cell was transformed. For example, where the selectable marker
element is kanamycin resistance, the compound added to the culture
medium will be kanamycin. Other compounds for selective growth
include ampicillin, tetracycline, and neomycin.
[0225] The amount of a h2520-59 polypeptide produced by a host cell
can be evaluated using standard methods known in the art. Such
methods include, without limitation, Western blot analysis,
SDS-polyacrylamide gel electrophoresis, non-denaturing gel
electrophoresis, chromatographic separation such as High
Performance Liquid Chromatography (HPLC), immunodetection such as
immunoprecipitation, and/or activity assays such as DNA binding gel
shift assays.
[0226] If a h2520-59 polypeptide has been designed to be secreted
from the host cells, the majority of polypeptide may be found in
the cell culture medium. If however, the h2520-59 polypeptide is
not secreted from the host cells, it will be present in the
cytoplasm and/or the nucleus (for eukaryotic host cells) or in the
cytosol (for bacterial host cells).
[0227] For a h2520-59 polypeptide situated in the host cell
cytoplasm and/or the nucleus (for eukaryotic host cells) or in the
cytosol (for bacterial host cells), intracellular material
(including inclusion bodies) can be extracted from the host cell
using any standard technique known to the skilled artisan. For
example, the host cells can be lysed to release the contents of the
periplasm/cytoplasm by osmotic shock French press, homogenization,
enzymatic disruption, exposure to detergents or chaotropes, and/or
sonication followed by centrifugation.
[0228] If a h2520-59 polypeptide has formed inclusion bodies in the
cytosol, the inclusion bodies can often bind to the inner and/or
outer cellular membranes and thus will be found primarily in the
pellet material after centrifugation. The pellet material can then
be treated at pH extremes or with a chaotropic agent such as a
detergent, guanidine, guanidine derivatives, urea, or urea
derivatives in the presence of a reducing agent such as
dithiothreitol at alkaline pH or tris carboxyethyl phosphine at
acid pH to release, break apart, and solubilize the inclusion
bodies. The h2520-59 polypeptide in its now soluble form can then
be analyzed using gel electrophoresis, immunoprecipitation or the
like. If it is desired to isolate the h2520-59 polypeptide,
isolation may be accomplished using standard methods such as those
described herein and in Marston et al., Meth. Enzymol.,
182:264-275, 1990.
[0229] In some cases, a h2520-59 polypeptide may not be
biologically active upon isolation. Various methods for "refolding"
or converting the polypeptide to its tertiary structure and
generating disulfide linkages can be used to restore biological
activity. Such methods include exposing the solubilized polypeptide
to a pH usually above 7 and in the presence of a particular
concentration of a. The selection of chaotrope is very similar to
the choices used for inclusion body solubilization, but usually the
chaotrope is used at a lower concentration and is not necessarily
the same as chaotropes used for the solubilization. In most cases
the refolding/oxidation solution will also contain a reducing agent
or the reducing agent plus its oxidized form in a specific ratio to
generate a particular redox potential allowing for disulfide
shuffling to occur in the formation of the protein's cysteine
bridge(s). Some of the commonly used redox couples include
cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cuprous
chloride, dithiothreitol (DTT)/dithiane DTT, and 2-2mercaptoethanol
(.beta.ME)/dithio-.beta.(ME). A cosolvent may be used to increase
the efficiency of the refolding, and the more common reagents used
for this purpose include glycerol, polyethylene glycol of various
molecular weights, arginine and the like.
[0230] If inclusion bodies are not formed to a significant degree
upon expression of a h2520-59 polypeptide, then the polypeptide
will be found primarily in the supernatant after centrifugation of
the cell homogenate. The polypeptide may be further isolated from
the supernatant using methods such as those described herein or
otherwise known in the art.
[0231] The purification of a h2520-59 polypeptide from solution can
be accomplished using a variety of techniques. If the polypeptide
has been synthesized such that it contains a tag such as
Hexahistidine (h2520-59 polypeptide/hexaHis) or other small peptide
such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc
(Invitrogen, Carlsbad, Calif.) at either its carboxyl or amino
terminus, it may be purified in a one-step process by passing the
solution through an affinity column where the column matrix has a
high affinity for the tag.
[0232] For example, polyhistidine binds with great affinity and
specificity to nickel; thus an affinity column of nickel (such as
the Qiagen.RTM. nickel columns) can be used for purification of
h2520-59 polypeptide/polyHis. See for example, Ausubel et al.,
eds., Current Protocols in Molecular Biology, Section 10.11.8, John
Wiley & Sons, New York (1993).
[0233] Additionally, the h2520-59 polypeptide may be purified
through use of a monoclonal antibody which is capable of
specifically recognizing and binding to the h2520-59
polypeptide.
[0234] Suitable procedures for purification thus include, without
limitation, affinity chromatography, immunoaffinity chromatography,
ion exchange chromatography, molecular sieve chromatography, High
Performance Liquid Chromatography (HPLC), elctrophoresis (including
native gel elctrophoresis) followed by gel elution, and preparative
isoelectric focusing ("Isoprime" machine/technique, Hoefer
Scientific, San Francisco, Calif.). In some cases, two or more of
these purification techniques may be combined to achieve increased
purity.
[0235] The h2520-59 polypeptides may also be prepared by chemical
synthesis methods (such as solid-phase peptide synthesis) using
techniques known in the art, such as those set forth by Merrifield
et al., J. Am. Chem. Soc., 85:2149, 1963, Houghten et al., Proc.
Natl. Acad. Sci. USA, 82:5132, 1985, and Stewart and Young, Solid
Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984.
Such polypeptides may be synthesized with or without a methionine
on the amino terminus. Chemically synthesized h2520-59 polypeptides
may be oxidized using methods set forth in these references to form
disulfide bridges. Chemically synthesized h2520-59 polypeptides are
expected to have comparable biological activity to the
corresponding h2520-59 polypeptides produced recombinantly or
purified from natural sources, and thus may be used interchangeably
with a recombinant or natural h2520-59 polypeptide.
[0236] Another means of obtaining a h2520-59 polypeptide is via
purification from biological samples such as source tissues and/or
fluids in which the h2520-59 polypeptide is naturally found. Such
purification can be conducted using methods for protein
purification as described herein or as otherwise known in the art.
The presence of the h2520-59 polypeptide during purification may be
monitored, for example, using an antibody prepared against
recombinantly produced h2520-59 polypeptide or peptide fragments
thereof.
[0237] A number of additional methods for producing nucleic acids
and polypeptides are known in the art, and the methods can be used
to produce polypeptides having specificity for h2520-59. See, for
example, Roberts et al., Proc. Natl. Acad. Sci. U.S.A.,
94:12297-12303, 1997, which describes the production of fusion
proteins between an mRNA and its encoded peptide. See also Roberts,
Curr. Opin. Chem. Biol., 3:268-273, 1999. Additionally, U.S. Pat.
No. 5,824,469 describes methods of obtaining oligonucleotides
capable of carrying out a specific biological function. The
procedure involves generating a heterogeneous pool of
oligonucleotides, each having a 5' randomized sequence, a central
preselected sequence, and a 3' randomized sequence. The resulting
heterogeneous pool is introduced into a population of cells that do
not exhibit the desired biological function. Subpopulations of the
cells are then screened for those which exhibit a predetermined
biological function. From that subpopulation, oligonucleotides
capable of carrying out the desired biological function are
isolated.
[0238] U.S. Pat. Nos. 5,763,192; 5,814,476; 5,723,323; and
5,817,483 describe processes for producing peptides or
polypeptides. This is done by producing stochastic genes or
fragments thereof, and then introducing these genes into host cells
which produce one or more proteins encoded by the stochastic genes.
The host cells are then screened to identify those clones producing
peptides or polypeptides having the desired activity.
[0239] Another method for producing peptides or polypeptides is
described in PCT/US98/20094 (WO99/15650) filed by Athersys, Inc.
Known as "Random Activation of Gene Expression for Gene Discovery"
(RAGE-GD), the process involves the activation of endogenous gene
expression or over-expression of a gene by in situ recombination
methods. For example, expression of an endogenous gene is activated
or increased by integrating a regulatory sequence into the target
cell which is capable of activating expression of the gene by
non-homologous or illegitimate recombination. The target DNA is
first subjected to radiation, and a genetic promoter inserted. The
promoter randomly locates a break at the front 5' end of a gene,
initiating transcription of the gene. This results in expression of
the desired peptide or polypeptide.
[0240] It will be appreciated that these methods can also be used
to create comprehensive h2520-59-like protein expression libraries,
which can subsequently be used for high throughput phenotypic
screening in a variety of assays, such as biochemical assays,
cellular assays, and whole organism assays (e.g., plant, mouse,
etc.).
Chemical Derivatives
[0241] Chemically modified derivatives of the h2520-59 polypeptides
may be prepared by one skilled in the art, given the disclosures
set forth below herein. h2520-59 polypeptide derivatives are
modified in a manner that is different, either in the type or
location of the molecules naturally attached to the polypeptide.
Derivatives may include molecules formed by the deletion of one or
more naturally attached chemical groups. The polypeptide comprising
the amino acid sequence of SEQ ID NO: 2, or a h2520-59 polypeptide
variant, may be modified by the covalent attachment of one or more
polymers. For example, the polymer selected is typically water
soluble so that the protein to which it is attached does not
precipitate in an aqueous environment, such as a physiological
environment. Included within the scope of suitable polymers is a
mixture of polymers. Preferably, for therapeutic use of the
end-product preparation, the polymer will be pharmaceutically
acceptable.
[0242] The polymers each may be of any molecular weight and may be
branched or unbranched. The polymers each typically have an average
molecular weight of between about 2 kDa to about 100 kDa (the term
"about" indicating that in preparations of a water soluble polymer,
some molecules will weigh more, some less, than the stated
molecular weight). The average molecular weight of each polymer is
preferably between about 5 kDa and about 50 kDa, more preferably
between about 12 kDa and about 40 kDa and most preferably between
about 20 kDa to about 35 kDa. Suitable water-soluble polymers or
mixtures thereof include, but are not limited to, N-linked or
O-linked carbohydrates; sugars; phosphates; polyethylene glycol
(PEG) (including the forms of PEG that have been used to derivatize
proteins, including mono-(C1-C10) alkoxy- or aryloxy-polyethylene
glycol), monomethoxy-polyethylene glycol; dextran (such as low
molecular weight dextran of, for example, about 6 kD), cellulose;
or other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, a polypropylene
oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g.,
glycerol) and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules which may be used
to prepare covalently attached multimers of the polypeptide
comprising the amino acid sequence of SEQ ID NO: 2 or a h2520-59
polypeptide variant.
[0243] In general, chemical derivatization may be performed under
any suitable condition used to react a protein with an activated
polymer molecule. Methods for preparing chemical derivatives of
polypeptides will generally comprise the steps of (a) reacting the
polypeptide with the activated polymer molecule (such as a reactive
ester or aldehyde derivative of the polymer molecule) under
conditions whereby the polypeptide comprising the amino acid
sequence of SEQ ID NO: 2, or a h2520-59 polypeptide variant becomes
attached to one or more polymer molecules, and (b) obtaining the
reaction product(s). The optimal reaction conditions will be
determined based on known parameters and the desired result. For
example, the larger the ratio of polymer molecules:protein, the
greater the percentage of attached polymer molecule. In one
embodiment, the h2520-59 polypeptide derivative may have a single
polymer molecule moiety at the amino terminus. (See, for example,
U.S. Pat. No. 5,234,784).
[0244] The pegylation of the polypeptide may be specifically
carried out by any of the pegylation reactions known in the art, as
described for example in the following references: Francis et al.,
Focus on Growth Factors, 3:4-10 (1992); EP 0154316; EP 0401384 and
U.S. Pat. No. 4,179,337. For example, pegylation may be carried out
via an acylation reaction or an alkylation reaction with a reactive
polyethylene glycol molecule (or an analogous reactive
water-soluble polymer) as described herein. For the acylation
reactions, the polymer(s) selected should have a single reactive
ester group. For reductive alkylation, the polymer(s) selected
should have a single reactive aldehyde group. A reactive aldehyde
is, for example, polyethylene glycol propionaldehyde, which is
water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof
(see U.S. Pat. No. 5,252,714).
[0245] In another embodiment, h2520-59 polypeptides may be
chemically coupled to biotin, and the biotin/h2520-59 polypeptide
molecules which are conjugated are then allowed to bind to avidin,
resulting in tetravalent avidin/biotin/h2520-59 polypeptide
molecules. h2520-59 polypeptides may also be covalently coupled to
dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting
conjugates precipitated with anti-DNP or anti-TNP-IgM to form
decameric conjugates with a valency of 10.
[0246] Generally, conditions which may be alleviated or modulated
by the administration of the present h2520-59 polypeptide
derivatives include those described herein for h2520-59
polypeptides. However, the h2520-59 polypeptide derivatives
disclosed herein may have additional activities, enhanced or
reduced biological activity, or other characteristics, such as
increased or decreased half-life, as compared to the
non-derivatized molecules.
Genetically Engineered Non-Human Animals
[0247] Additionally included within the scope of the present
invention are non-human animals such as mice, rats, rabbits, or
other rodents, goats, or sheep, or other farm animals, in which the
gene (or genes) encoding the native h2520-59 polypeptide has (have)
been disrupted ("knocked out") such that the level of expression of
this gene or genes is (are) significantly decreased or completely
abolished. Such animals may be prepared using techniques and
methods such as those described in U.S. Pat. No. 5,557,032.
[0248] The present invention further includes non-human animals
such as mice, rats, rabbits, or other rodents, goats, sheep, or
other farm animals, in which either the native form of the h2520-59
gene(s) for that animal or a heterologous h2520-59 gene(s) is (are)
over-expressed by the animal, thereby creating a "transgenic"
animal. Such transgenic animals may be prepared using well-known
methods such as those described in U.S. Pat. No. 5,489,743 and PCT
Application No. WO 94/28122.
[0249] The present invention further includes non-human animals in
which the promoter for one or more of the h2520-59 polypeptides of
the present invention is either activated or inactivated (e.g., by
using homologous recombination methods) to alter the level of
expression of one or more of the native h2520-59 polypeptides.
[0250] These non-human animals may be used for drug candidate
screening. In such screening, the impact of a drug candidate on the
animal is measured; for example, drug candidates may decrease or
increase the expression of the h2520-59 gene. In certain
embodiments, the amount of h2520-59 polypeptide that is produced is
measured after the exposure of the animal to the drug candidate.
Additionally, in certain embodiments, one may detect the actual
impact of the drug candidate on the animal. For example, the
overexpression of a particular gene may result in, or be associated
with, a disease or pathological condition. In such cases, one may
test a drug candidate's ability to decrease expression of the gene
or its ability to prevent, inhibit, or eliminate a pathological
condition. In other examples, the production of a particular
metabolic product such as a fragment of a polypeptide may result
in, or be associated with, a disease or pathological condition. In
such cases, one may test a drug candidate's ability to decrease the
production of such a metabolic product or its ability to prevent,
inhibit, or eliminate a pathological condition.
Microarray
[0251] It will be appreciated that DNA microarray technology can be
utilized in accordance with the present invention. DNA microarrays
are miniature, high density arrays of nucleic acids positioned on a
solid support, such as glass. Each cell or element within the array
has numerous copies of a single species of DNA which acts as a
target for hybridization for its cognate mRNA. In expression
profiling using DNA microarray technology, mRNA is first extracted
from a cell or tissue sample and then converted enzymatically to
fluorescently labeled cDNA. This material is hybridized to the
microarray and unbound cDNA is removed by washing. The expression
of discrete genes represented on the array is then visualized by
quantitating the amount of labeled cDNA which is specifically bound
to each target DNA. In this way, the expression of thousands of
genes can be quantitated in a high-throughput, parallel manner from
a single sample of biological material.
[0252] This high-throughput expression profiling has a broad range
of applications with respect to the h2520-59 molecules of the
invention, including, but not limited to: the identification and
validation of h2520-59 disease-related genes as targets for
therapeutics; molecular toxicology of h2520-59 molecules and
inhibitors thereof; stratification of populations and generation of
surrogate markers for clinical trials; and the enhancement of
h2520-59-related small molecule drug discovery by aiding in the
identification of selective compounds in high-throughput screens
(HTS).
Selective Binding Agents
[0253] As used herein, the term "selective binding agent" refers to
a molecule which has specificity for one or more h2520-59
polypeptides. Suitable selective binding agents include, but are
not limited to, antibodies and derivatives thereof, polypeptides,
and small molecules. Suitable selective binding agents may be
prepared using methods known in the art. It will be appreciated
that the term "selective binding agent" can also refer to a
molecule which has specificity for transcription factors, such as
activating transcription factor 4 (ATF4), thereby inhibiting the
interaction of ATF4 with h2520-59 (see Example 7). The invention
thus further relates to methods of inhibiting the interaction
between ATF4 and h2520-59 by administering a selective binding
agent having specificity for at least one of ATF4 and h2520-59. The
invention also relates to methods of treating, preventing, or
ameliorating a disease, condition, or disorder comprising
administering to a patient an effective amount of a selective
binding agent having specificity for at least one of ATF4 and
h2520-59.
[0254] An exemplary h2520-59 polypeptide selective binding agent of
the present invention is capable of binding a certain portion of
the h2520-59 polypeptide, thereby inhibiting the binding of the
polypeptide to the h2520-59 polypeptide receptor(s).
[0255] Selective binding agents, such as antibodies and antibody
fragments that bind h2520-59 polypeptides, are within the scope of
the present invention. The antibodies may be polyclonal, including
monospecific polyclonal, monoclonal (MAbs), recombinant, chimeric,
humanized such as CDR-grafted, human, single chain, and/or
bispecific, as well as fragments, variants or derivatives thereof.
Antibody fragments include those portions of the antibody which
bind to an epitope on the h2520-59 polypeptide. Examples of such
fragments include Fab and F(ab') fragments generated by enzymatic
cleavage of full-length antibodies. Other binding fragments include
those generated by recombinant DNA techniques, such as the
expression of recombinant plasmids containing nucleic acid
sequences encoding antibody variable regions.
[0256] Polyclonal antibodies directed toward a h2520-59 polypeptide
generally are produced in animals (e.g., rabbits or mice) by means
of multiple subcutaneous, intramuscular, or intraperitoneal
injections of h2520-59 polypeptide and an adjuvant. It may be
useful to conjugate a h2520-59 polypeptide to a carrier protein
that is immunogenic in the species to be immunized, such as keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor. Also, aggregating agents such as alum are used
to enhance the immune response. After immunization, the animals are
bled and the serum is assayed for anti-h2520-59 polypeptide
antibody titer.
[0257] Monoclonal antibodies directed toward h2520-59 polypeptide
are produced using any method which provides for the production of
antibody molecules by continuous cell lines in culture. Examples of
suitable methods for preparing monoclonal antibodies include the
hybridoma methods of Kohler et al. (Nature, 256: 495-497, 1975) and
the human B-cell hybridoma method of Kozbor (J. Immunol., 133:
3001, 1984; Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New
York, 1987). Also provided by the invention are hybridoma cell
lines which produce monoclonal antibodies reactive with h2520-59
polypeptides.
[0258] Monoclonal antibodies of the invention may be modified for
use as therapeutics. One embodiment is a "chimeric" antibody in
which a portion of the heavy and/or light chain is identical with
or homologous to a corresponding sequence in antibodies derived
from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is/are
identical with or homologous to a corresponding sequence in
antibodies derived from another species or belonging to another
antibody class or subclass. Also included are fragments of such
antibodies, so long as they exhibit the desired biological
activity. See U.S. Pat. No. 4,816,567 and Morrison et al., Proc.
Natl. Acad. Sci. U.S.A., 81: 6851-6855 (1985).
[0259] In another embodiment, a monoclonal antibody of the
invention is a "humanized" antibody. Methods for humanizing
non-human antibodies are well known in the art. (see U.S. Pat. Nos.
5,585,089, and 5,693,762). Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. Humanization can be performed, for example, using
methods described in the art (Jones et al., Nature 321: 522-525,
1986; Riechmann et al., Nature, 332: 323-327, 1988; Verhoeyen et
al., Science 239:1534-1536, 1988), by substituting at least a
portion of a rodent complementarity-determining region (CDR) for
the corresponding region of a human antibody.
[0260] Also encompassed by the invention are human antibodies which
bind h2520-59 polypeptide, fragments, variants and/or derivatives.
Using transgenic animals (e.g., mice) that are capable of producing
a repertoire of human antibodies in the absence of endogenous
immunoglobulin production, such antibodies are produced by
immunization with a h2520-59 antigen (i.e., having at least 6
contiguous amino acids), optionally conjugated to a carrier. See,
for example, Jakobovits et al., Proc. Natl. Acad. Sci. U.S.A., 90:
2551-2555, 1993; Jakobovits et al., Nature 362: 255-258, 1993;
Bruggermann et al., Year in Immunol., 7: 33, 1993. In one method,
such transgenic animals are produced by incapacitating the
endogenous loci encoding the heavy and light immunoglobulin chains
therein, and inserting nucleic acids encoding human heavy and light
chain proteins into the genome thereof. Partially modified animals,
that is those having less than the full complement of
modifications, are then cross-bred to obtain an animal having all
of the desired immune system modifications. When administered an
immunogen, these transgenic animals produce antibodies with human
variable regions, including human (rather than, e.g., murine) amino
acid sequences, including variable regions which are immunospecific
for these antigens. See PCT Application Nos. PCT/US96/05928 and
PCT/US93/06926. Additional methods are described in U.S. Pat. No.
5,545,807, PCT Application Nos. PCT/US91/245, PCT/GB89/01207, and
in EP 546073B1 and EP 546073A1. Human antibodies may also be
produced by the expression of recombinant DNA in host cells or by
expression in hybridoma cells as described herein.
[0261] In an alternative embodiment, human antibodies can be
produced from phage-display libraries (Hoogenboom et al., J. Mol.
Biol. 227: 381, 1991; Marks et al., J. Mol. Biol. 222: 581, 1991).
These processes mimic immune selection through the display of
antibody repertoires on the surface of filamentous bacteriophage,
and subsequent identification of phage by their binding to an
antigen of choice. One such technique is described in PCT
Application No. PCT/US98/17364, filed in the name of Adams et al.,
which describes the isolation of high affinity and functionally
agonistic antibodies for MPL- and msk-receptors using such an
approach.
[0262] Chimeric, CDR grafted, and humanized antibodies are
typically produced by recombinant methods. Nucleic acids encoding
the antibodies are introduced into host cells and expressed using
materials and procedures described herein or known in the art. In a
preferred embodiment, the antibodies are produced in mammalian host
cells, such as CHO cells. Monoclonal (e.g., human) antibodies may
be produced by the expression of recombinant DNA in host cells or
by expression in hybridoma cells as described herein.
[0263] The anti-h2520-59 antibodies of the invention may be
employed in any known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual
of Techniques, pp. 147-158, CRC Press, Inc., 1987) for the
detection and quantitation of h2520-59 polypeptides. The antibodies
will bind h2520-59 polypeptides with an affinity which is
appropriate for the assay method being employed.
[0264] For diagnostic applications, in certain embodiments,
anti-h2520-59 antibodies typically will be labeled with a
detectable moiety. The detectable moiety can be any one which is
capable of producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety may be a radioisotope,
such as .sup.3H, .sup.14C, .sup.32P, .sup.35S, or .sup.125I, a
fluorescent or chemiluminescent compound, such as fluorescein
isothiocyanate, rhodamine, or luciferin; or an enzyme, such as
alkaline phosphatase, b-galactosidase, or horseradish peroxidase
(Bayer et al., Meth. Enzymol., 184: 138-163, 1990).
[0265] Competitive binding assays rely on the ability of a labeled
standard (e.g., a h2520-59 polypeptide, or an immunologically
reactive portion thereof) to compete with the test sample analyte
(a h2520-59 polypeptide) for binding with a limited amount of
anti-h2520-59 antibody. The amount of a h2520-59 polypeptide in the
test sample is inversely proportional to the amount of standard
that becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies typically are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte which remain unbound.
[0266] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected and/or quantitated. In a
sandwich assay, the test sample analyte is typically bound by a
first antibody which is immobilized on a solid support, and
thereafter a second antibody binds to the analyte, thus forming an
insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110.
The second antibody may itself be labeled with a detectable moiety
(direct sandwich assays) or may be measured using an
anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assays). For example, one type of
sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in
which case the detectable moiety is an enzyme.
[0267] The selective binding agents, including anti-h2520-59
antibodies, are also useful for in vivo imaging. An antibody
labeled with a detectable moiety may be administered to an animal,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host is assayed. The antibody may be
labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0268] Selective binding agents of the invention, including
anti-h2520-59 antibodies, may be used as therapeutics. These
therapeutic agents are generally agonists or antagonists, in that
they either enhance or reduce, respectively, at least one of the
biological activities of a h2520-59 polypeptide. In one embodiment,
antagonist antibodies of the invention are antibodies or binding
fragments thereof which are capable of specifically binding to a
h2520-59 polypeptide and which are capable of inhibiting or
eliminating the functional activity of a h2520-59 polypeptide in
vivo or in vitro. In preferred embodiments, the selective binding
agent, e.g., an antagonist antibody, will inhibit the functional
activity of a h2520-59 polypeptide by at least about 50%, and
preferably by at least about 80%. In another embodiment, the
selective binging agent may be an antibody that is capable of
interacting with a h2520-59 binding partner (a ligand, co-factor,
or receptor) thereby inhibiting or eliminating h2520-59 activity in
vitro or in vivo. Selective binding agents, including agonist and
antagonist anti-h2520-59 antibodies, are identified by screening
assays which are well known in the art.
[0269] The invention also relates to a kit comprising h2520-59
selective binding agents (such as antibodies) and other reagents
useful for detecting h2520-59 polypeptide levels in biological
samples. Such reagents may include a secondary activity, a
detectable label, blocking serum, positive and negative control
samples, and detection reagents.
Assaying for Other Modulators of h2520-59 Polypeptide Activity
[0270] In some situations, it may be desirable to identify
molecules that are modulators, i.e., agonists or antagonists, of
the activity of a h2520-59 polypeptide. Natural or synthetic
molecules that modulate h2520-59 polypeptide may be identified
using one or more screening assays, such as those described herein.
Such molecules may be administered either in an ex vivo manner, or
in an in vivo manner by injection, or by oral delivery,
implantation device, or the like. "Test molecule(s)" refers to the
molecule(s) that is/are under evaluation for the ability to
modulate (i.e., increase or decrease) an activity of a h2520-59
polypeptide. Most commonly, a test molecule will interact directly
with a h2520-59 polypeptide. However, it is also contemplated that
a test molecule may also modulate h2520-59 polypeptide activity
indirectly, such as by affecting h2520-59 gene expression, or by
binding to a h2520-59 binding partner (e.g., receptor, co-factor,
or ligand). In one embodiment, a test molecule will bind to a
h2520-59 polypeptide with an affinity constant of at least about
10.sup.-6 M, preferably about 10.sup.-8 M, more preferably about
10.sup.-9 M, and even more preferably about 10.sup.-10 M.
[0271] Methods for identifying compounds which interact with
h2520-59 polypeptides are encompassed by the present invention. In
certain embodiments, an h2520-59 polypeptide is incubated with a
test molecule under conditions which permit the interaction of the
test molecule with a h2520-59 polypeptide, and the extent of the
interaction can be measured. The test molecule(s) can be screened
in a substantially purified form or in a crude mixture.
[0272] In certain embodiments, a h2520-59 polypeptide agonist or
antagonist may be a protein, peptide, carbohydrate, lipid, or small
molecular weight molecule which interacts with h2520-59 polypeptide
to regulate its activity. Molecules which regulate h2520-59
polypeptide expression include nucleic acids which are
complementary to nucleic acid encoding a h2520-59 polypeptide, or
are complementary to nucleic acids sequences which direct or
control the expression of h2520-59 polypeptide, and which act as
anti-sense regulators of expression.
[0273] Once a set of test molecules has been identified as
interacting with a h2520-59 polypeptide, the molecules may be
further evaluated for their ability to increase or decrease
h2520-59 polypeptide activity. The measurement of the interaction
of test molecules with h2520-59 polypeptides may be carried out in
several formats, including cell-based binding assays, membrane
binding assays, solution-phase assays and immunoassays. In general,
test molecules are incubated with a h2520-59 polypeptide for a
specified period of time, and h2520-59 polypeptide activity is
determined by one or more assays for measuring biological
activity.
[0274] The interaction of test molecules with h2520-59 polypeptides
may also be assayed directly using polyclonal or monoclonal
antibodies in an immunoassay. Alternatively, modified forms of
h2520-59 polypeptides containing epitope tags as described herein
may be used in immunoassays.
[0275] h2520-59 polypeptides displaying biological activity through
an interaction with a binding partner (e.g., a receptor, a ligand
or a co-factor), are assessed by a variety of in vitro assays that
may be used to measure the binding of a h2520-59 polypeptide to the
corresponding binding partner (such as a selective binding agent,
receptor, ligand, or co-factor). These assays are used to screen
test molecules for their ability to increase or decrease the rate
and/or the extent of binding of a h2520-59 polypeptide to its
binding partner. In one assay, a h2520-59 polypeptide is
immobilized in the wells of a microtiter plate. Radiolabeled
h2520-59 binding partner (for example, iodinated h2520-59 binding
partner) and the test molecule(s) are added either one at a time
(in either order) or simultaneously to the wells. After incubation,
the wells are washed and counted using a scintillation counter, to
determine the extent to which the binding partner bound to the
h2520-59 polypeptide. Typically, the molecules will be tested over
a range of concentrations, and a series of control wells lacking
one or more elements of the test assays is used for accuracy in the
evaluation of the results. An alternative to this method involves
reversing the "positions" of the proteins, i.e., immobilizing a
h2520-59 polypeptide binding partner to the microtiter plate wells,
incubating with the test molecule and radiolabeled h2520-59
polypeptide, and determining the extent of h2520-59 polypeptide
binding. See, for example, chapter 18, Current Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons, New
York, N.Y. (1995).
[0276] As an alternative to radiolabelling, a h2520-59 polypeptide
or its binding partner may be conjugated to biotin and the presence
of biotinylated protein is detected using streptavidin linked to an
enzyme, such as horseradish peroxidase (HRP) or alkaline
phosphatase (AP), that is detected calorimetrically, or by
fluorescent tagging of streptavidin. An antibody directed to a
h2520-59 polypeptide or to a h2520-59 binding partner and
conjugated to biotin may also be used and is detected after
incubation with enzyme-linked streptavidin linked to AP or HRP.
[0277] A h2520-59 polypeptide or a h2520-59 like binding partner
can also be immobilized by attachment to agarose beads, acrylic
beads or other types of such inert solid phase substrates. The
substrate-protein complex is placed in a solution containing the
complementary protein and the test compound. After incubation, the
beads are precipitated by centrifugation, and the amount of binding
between a h2520-59 polypeptide and its binding partner is assessed
using the methods described herein. Alternatively, the
substrate-protein complex is immobilized in a column, and the test
molecule and complementary protein are passed through the column.
The formation of a complex between a h2520-59 polypeptide and its
binding partner is then assessed using any of the techniques set
forth herein, i.e., radiolabelling, antibody binding or the
like.
[0278] Another in vitro assay that is useful for identifying a test
molecule that increases or decreases the formation of a complex
between a h2520-59 polypeptide and a h2520-59 binding partner is a
surface plasmon resonance detector system such as the BIAcore assay
system (Pharmacia, Piscataway, N.J.). The BIAcore system may be
carried out using the manufacturer's protocol. This assay
essentially involves the covalent binding of either h2520-59
polypeptide or a h2520-59 binding partner to a dextran-coated
sensor chip which is located in a detector. The test compound and
the other complementary protein is then injected, either
simultaneously or sequentially, into the chamber containing the
sensor chip. The amount of complementary protein that binds is
assessed based on the change in molecular mass which is physically
associated with the dextran-coated side of the sensor chip. The
change in molecular mass can be measured by the detector
system.
[0279] In some cases, it may be desirable to evaluate two or more
test compounds together for their ability to increase or decrease
the formation of a complex between a h2520-59 polypeptide and a
h2520-59 binding partner. In these cases, the assays set forth
herein can be readily modified by adding such additional test
compound(s) either simultaneous with, or subsequent to, the first
test compound. The remainder of the steps in the assay are set
forth herein.
[0280] In vitro assays such as those described herein may be used
advantageously to screen large numbers of compounds for effects on
complex formation by h2520-59 polypeptide and h2520-59 binding
partner. The assays may be automated to screen compounds generated
in phage display, synthetic peptide, and chemical synthesis
libraries.
[0281] Compounds which increase or decrease the formation of a
complex between a h2520-59 polypeptide and a h2520-59 binding
partner may also be screened in cell culture using cells and cell
lines expressing either h2520-59 polypeptide or h2520-59 binding
partner. Cells and cell lines may be obtained from any mammal, but
preferably will be from human or other primate, canine, or rodent
sources. The binding of a h2520-59 polypeptide to cells expressing
h2520-59 binding partner at the surface is evaluated in the
presence or absence of test molecules, and the extent of binding
may be determined by, for example, flow cytometry using a
biotinylated antibody to a h2520-59 binding partner. Cell culture
assays can be used advantageously to further evaluate compounds
that score positive in protein binding assays described herein.
[0282] Cell cultures can also be used to screen the impact of a
drug candidate. For example, drug candidates may decrease or
increase the expression of the h2520-59 gene. In certain
embodiments, the amount of h2520-59 polypeptide that is produced
may be measured after exposure of the cell culture to the drug
candidate. In certain embodiments, one may detect the actual impact
of the drug candidate on the cell culture. For example, the
overexpression of a particular gene may have a particular impact on
the cell culture. In such cases, one may test a drug candidate's
ability to increase or decrease the expression of the gene or its
ability to prevent or inhibit a particular impact on the cell
culture. In other examples, the production of a particular
metabolic product such as a fragment of a polypeptide may result
in, or be associated with, a disease or pathological condition. In
such cases, one may test a drug candidate's ability to decrease the
production of such a metabolic product in a cell culture.
[0283] A yeast two hybrid system (Chien et al., Proc. Natl. Acad.
Sci. USA, 88:9578-9583, 1991) can be used to identify novel
polypeptides that bind to, or interact with, h2520-59 polypeptides.
As an example, hybrid constructs comprising DNA encoding a
cytoplasmic domain of a h2520-59 polypeptide fused to a yeast
GAL4-DNA binding domain may be used as a two-hybrid bait plasmid.
Positive clones emerging from the screening may be characterized
further to identify interacting proteins.
Internalizing Proteins
[0284] The TAT protein sequence (from HIV) can be used to
internalize proteins into a cell by targeting the lipid bi-layer
component of the cell membrane. See, e.g., Falwell et al., Proc.
Natl. Acad. Sci. U.S.A. 91: 664-668, 1994. For example, an 11 amino
acid sequence (YGRKKRRQRRR; SEQ ID NO: 5) of the HIV TAT protein
(termed the "protein transduction domain", or TAT PTD) has been
shown to mediate delivery of large bioactive proteins such as
.beta.-galactosidase and p27Kip across the cytoplasmic membrane and
the nuclear membrane of a cell. See Schwarze et al., Science, 285:
1569-1572, 1999; and Nagahara et al., Nature Medicine, 4:
1449-1452, 1998. Schwarze et al., supra, demonstrated that cultured
cells acquired .beta.-galactosidase activity when exposed to a
fusion of the TAT PDT and .beta.-galactosidase. Injection of mice
with the TAT-.beta.-gal fusion proteins resulted in .beta.-gal
expression in a number of tissues, including liver, kidney, lung,
heart, and brain tissue.
[0285] It will thus be appreciated that the TAT protein sequence
may be used to internalize a desired protein or polypeptide into a
cell. In the context of the present invention, the TAT protein
sequence can be fused to another molecule such as a h2520-59
antagonist (i.e., anti-h2520-59 selective binding agent or small
molecule) and administered intracellularly to inhibit the activity
of the h2520-59 molecule. Where desired, the h2520-59 protein
itself, or a peptide fragment or modified form of h2520-59, may be
fused to such a protein transducer for administrating to cells
using the procedures described above.
Cell Source Identification Using h2520-59 Polypeptides
[0286] In accordance with certain embodiments of the invention, it
may be useful to be able to determine the source of a certain cell
type associated with a h2520-59 polypeptide. For example, it may be
useful to determine the origin of a disease or pathological
condition as an aid in selecting an appropriate therapy. h2520-59
polypeptide is specifically associated with transformed cells. In
certain embodiments, nucleic acids encoding a h2520-59 polypeptide
can be used as a probe to identify transformed cells by screening
the nucleic acids of the cells with such a probe. In other
embodiments, one may use anti-h2520-59 polypeptide antibodies to
test for the presence of a h2520-59 polypeptide in cells, to
determine if such cells are tumor-derived.
Diseases and Therapeutic Uses
[0287] In one aspect, the present invention provides reagents and
methods useful for treating diseases and conditions characterized
by aberrant levels of h2520-59 activity in a cell. A non-exclusive
list of acute and chronic diseases which are treated, diagnosed,
ameliorated, or prevented with the polypeptides, nucleic acids,
antibodies, and/or fragments thereof of the invention include
hyperproliferative pathological conditions such as immune
disorders, angiogenesis, vasculogenesis, wound healing, diabetes
mellitus including diabetes type I and type II, psoriasis, liver
diseases such as hepatitis and cirrhosis, osteoporosis,
inflammatory conditions such as osteoarthritis and rheumatoid
arthritis, pregnancy and cancer.
[0288] More specifically, the types of cancers and tumor cells that
are treated, diagnosed, ameliorated or prevented with h2520-59
polypeptides, nucleic acids, antibodies, and/or fragments thereof
include, but are not limited to, ACTH-producing tumors, acute
lymphocytic leukemias, acute nonlymphocytic leukemias, cancers of
the adrenal cortex, bladder cancer, brain cancer, breast cancer,
cervical cancer, chronic lymphocytic leukemias, chronic myelocytic
leukemias, colorectal cancer, cutaneous T-cell lymphomas,
endometrial cancer, esophageal cancer, Ewing's sarcoma, gallbladder
cancer, hairy cell leukemia, head and neck cancer, Hodgkin's
lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung
cancer (small and non-small cell), malignant peritoneal effusion,
malignant pleural effusion, melanoma, mesothelioma, multiple
myeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma,
osteosarcoma, ovarian cancer, ovarian (germ cell) cancer,
pancreatic cancer, penile cancer, prostate cancer, retinoblastoma,
skin cancer, soft tissue sarcoma, squamous cell carcinoma, stomach
cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms,
uterine cancer, vaginal cancer, cancer of the vulva, Wilms' tumor,
adenocarcinoma of the breast, prostate, and colon, all forms of
bronchogenic carcinoma of the lung, myeloid, melanoma, hepatoma,
neuroblastoma, papilloma, apudoma, choristoma, branchioma,
malignant carcinoid syndrome, carcinoid heart disease, carcinoma
(e.g. Walker, basal cell, basosquamous, Brown-Pearce, ductal,
Ehrlich tumor, Krebs 2, Merkel's cell, mucinous, non-small cell
lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic,
squamous cell, and transitional cell). Additional types of cancers
that may be treated include histiocytic disorders, leukemia,
histiocytosis malignant, Hodgkin's disease, immunoproliferative
small, non-Hodgkin's lymphoma, plasmacytoma, reticuloendotheliosis,
melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma,
fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma,
mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, chordoma,
craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma,
mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma,
teratoma, thymoma, and trophoblastic tumor. Further, the following
types of cancers may also be treated: adenoma, cholangioma,
cholesteatoma, cyclindroma, cystadenocarcinoma, cystadenoma,
granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma,
islet cell tumor, Leydig cell tumor, papilloma, Sertoli cell tumor,
theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma,
myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma,
ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma,
neuroblastoma, neuroepithelioma, neurofibroma, neuroma,
paraganglioma, and paraganglioma nonchromaffin. The types of
cancers that may be treated also include, but are not limited to,
the following: angiokeratoma, angiolymphoid hyperplasia with
eosinophilia, angioma sclerosing, angiomatosis, glomangioma,
hemangioendothelioma, hemangioma, hemangiopericytoma,
hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma,
pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes,
fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma,
liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian
carcinoma, rhabdomyosarcoma, sarcoma, neoplasms, nerofibromatosis,
and cervical dysplasia.
[0289] The invention further provides compositions and methods
useful for treatment of other conditions in which cells have become
immortalized or hyperproliferative due to abnormally high
expression of h2520-59.
[0290] Other diseases or disorders caused or mediated by
undesirable levels of h2520-59 polypeptide are contemplated by the
therapeutic and diagnostic methods of the invention. By way of
illustration, such undesirable levels include excessively elevated
levels and sub-normal levels.
h2520-59 Compositions and Administration
[0291] Therapeutic compositions within the scope of the present
invention include h2520-59 pharmaceutical compositions that may
comprise a therapeutically effective amount of a h2520-59
polypeptide or a h2520-59 nucleic acid molecule in admixture with a
pharmaceutically or physiologically acceptable formulation agent
selected for suitability with the mode of administration to a human
or non-human animal such as a mammal. Pharmaceutical compositions
may comprise a therapeutically effective amount of one or more
h2520-59 selective binding agents in admixture with a
pharmaceutically or physiologically acceptable formulation agent
selected for suitability with the mode of administration.
[0292] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0293] The pharmaceutical composition may contain formulation
materials for modifying, maintaining or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or
penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine or lysine); antimicrobials;
antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, other organic acids); bulking agents (such as
mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides and other carbohydrates (such as glucose, mannose, or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring; flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (sucrose or
sorbitol); tonicity enhancing agents (such as alkali metal halides
(preferably sodium or potassium chloride, mannitol, or sorbitol);
delivery vehicles; diluents; excipients and/or pharmaceutical
adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A.
R. Gennaro, ed., Mack Publishing Company, 1990).
[0294] The optimal pharmaceutical composition will be determined by
one skilled in the art depending upon, for example, the intended
route of administration, delivery format, and desired dosage. See,
for example, Remington's Pharmaceutical Sciences, supra. Such
compositions may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of the h2520-59
molecule.
[0295] The primary vehicle or carrier in a pharmaceutical
composition may be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier may be water for injection,
physiological saline solution or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute therefor. In one
embodiment of the present invention, h2520-59 polypeptide
compositions may be prepared for storage by mixing the selected
composition having the desired degree of purity with optional
formulation agents (Remington's Pharmaceutical Sciences, supra) in
the form of a lyophilized cake or an aqueous solution. Further, the
h2520-59 polypeptide product may be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0296] The h2520-59 pharmaceutical compositions can be selected for
parenteral delivery. Alternatively, the compositions may be
selected for inhalation or for delivery through the digestive
tract, such as orally, or through other delivery routes known in
the art. The preparation of such pharmaceutically acceptable
compositions is within the skill of the art.
[0297] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at slightly lower pH, typically within a pH range of from about 5
to about 8.
[0298] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be in the
form of a pyrogen-free, parenterally acceptable aqueous solution
comprising the desired h2520-59 molecule in a pharmaceutically
acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which a h2520-59 molecule
is formulated as a sterile, isotonic solution, properly preserved.
Yet another preparation can involve the formulation of the desired
molecule with an agent, such as injectable microspheres,
bio-erodible particles, polymeric compounds (polylactic acid,
polyglycolic acid), beads, or liposomes, that provides for the
controlled or sustained release of the product which may then be
delivered via a depot injection. Hyaluronic acid may also be used,
and this may have the effect of promoting sustained duration in the
circulation. Other suitable means for the introduction of the
desired molecule include implantable drug delivery devices.
[0299] In one embodiment, a pharmaceutical composition may be
formulated for inhalation. For example, a h2520-59-like molecule
may be formulated as a dry powder for inhalation. h2520-59
polypeptide or h2520-59 nucleic acid molecule inhalation solutions
may also be formulated with a propellant for aerosol delivery. In
yet another embodiment, solutions may be nebulized. Pulmonary
administration is further described in PCT Application No.
PCT/US94/001875, which describes pulmonary delivery of chemically
modified proteins.
[0300] It is also contemplated that certain formulations may be
administered orally. In one embodiment of the present invention,
h2520-59 molecules which are administered in this fashion can be
formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. For
example, a capsule may be designed to release the active portion of
the formulation at the point in the gastrointestinal tract when
bioavailability is maximized and pre-systemic degradation is
minimized. Additional agents can be included to facilitate
absorption of the h2520-59 molecule. Diluents, flavorings, low
melting point waxes, vegetable oils, lubricants, suspending agents,
tablet disintegrating agents, and binders may also be employed.
[0301] Another pharmaceutical composition may involve an effective
quantity of h2520-59 molecules in a mixture with non-toxic
excipients which are suitable for the manufacture of tablets. By
dissolving the tablets in sterile water, or other appropriate
vehicle, solutions can be prepared in unit dose form. Suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0302] Additional h2520-59 pharmaceutical compositions will be
evident to those skilled in the art, including formulations
involving h2520-59 polypeptides in sustained- or
controlled-delivery formulations. Techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art. See
for example, PCT/US93/00829 which describes controlled release of
porous polymeric microparticles for the delivery of pharmaceutical
compositions. Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g., films or microcapsules. Sustained release matrices
may include polyesters, hydrogels, polylactides (U.S. Pat. No.
3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556, 1983),
poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.
Mater. Res., 15:167-277, 1981; Langer et al., Chem. Tech.,
12:98-105, 1982), ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
compositions also include liposomes, which can be prepared by any
of several methods known in the art. See, e.g., Eppstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688-3692, 1985; EP 36,676; EP
88,046; EP 143,949.
[0303] The h2520-59 pharmaceutical composition to be used for in
vivo administration typically must be sterile. This may be
accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this
method may be conducted either prior to or following lyophilization
and reconstitution. The composition for parenteral administration
may be stored in lyophilized form or in solution. In addition,
parenteral compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0304] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0305] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0306] An effective amount of a h2520-59 pharmaceutical composition
to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
thus vary depending, in part, upon the molecule delivered, the
indication for which the h2520-59 molecule is being used, the route
of administration, and the size (body weight, body surface or organ
size) and condition (the age and general health) of the patient.
Accordingly, the clinician may titer the dosage and modify the
route, of administration to obtain the optimal therapeutic effect.
A typical dosage may range from about 0.1 mg/kg up to about 100
mg/kg or more, depending on the factors mentioned above. In other
embodiments, the dosage may range from 0.1 mg/kg up to about 100
mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about 100
mg/kg.
[0307] The frequency of dosing will depend upon the pharmacokinetic
parameters of the h2520-59 molecule in the formulation used.
Typically, a clinician will administer the composition until a
dosage is reached that achieves the desired effect. The composition
may therefore be administered as a single dose, or as two or more
doses (which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via implantation
device or catheter. Further refinement of the appropriate dosage is
routinely made by those of ordinary skill in the art and is within
the ambit of tasks routinely performed by them. Appropriate dosages
may be ascertained through use of appropriate dose-response data,
which is routinely obtained.
[0308] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g. orally, through
injection by intravenous, intraperitoneal, intracerebral
(intra-parenchymal), intracerebroventricular, intramuscular,
intra-ocular, intraarterial, intraportal, or intralesional routes,
by sustained release systems or by implantation devices. Where
desired, the compositions may be administered by bolus injection or
continuously by infusion, or by implantation device.
[0309] Alternatively or additionally, the composition may be
administered locally via implantation of a membrane, sponge, or
another appropriate material onto which the desired molecule has
been absorbed or encapsulated. Where an implantation device is
used, the device may be implanted into any suitable tissue or
organ, and delivery of the desired molecule may be via diffusion,
timed-release bolus, or continuous administration.
[0310] In some cases, it may be desirable to use h2520-59
pharmaceutical compositions in an ex vivo manner. In such
instances, cells, tissues, or organs that have been removed from
the patient are exposed to h2520-59 pharmaceutical compositions
after which the cells, tissues and/or organs are subsequently
implanted back into the patient.
[0311] In other cases, a h2520-59 polypeptide can be delivered by
implanting certain cells that have been genetically engineered,
using methods such as those described herein, to express and
secrete the polypeptide. Such cells may be animal or human cells,
and may be autologous, heterologous, or xenogeneic. Optionally, the
cells may be immortalized. In order to decrease the chance of an
immunological response, the cells may be encapsulated to avoid
infiltration of surrounding tissues. The encapsulation materials
are typically biocompatible, semi-permeable polymeric enclosures or
membranes that allow the release of the protein product(s) but
prevent the destruction of the cells by the patient's immune system
or by other detrimental factors from the surrounding tissues.
[0312] Additional embodiments of the present invention relate to
cells and methods (e.g., homologous recombination and/or other
recombinant production methods) for both the in vitro production of
therapeutic polypeptides and for the production and delivery of
therapeutic polypeptides by gene therapy or cell therapy.
Homologous and other recombination methods may be used to modify a
cell that contains a normally transcriptionally silent h2520-59
gene, or an underexpressed gene, and thereby produce a cell which
expresses therapeutically efficacious amounts of h2520-59
polypeptides.
[0313] Homologous recombination is a technique originally developed
for targeting genes to induce or correct mutations in
transcriptionally active genes (Kucherlapati et al., Prog. Nucl.
Acid Res. & Mol. Biol, 36:301, 1989). The basic technique was
developed as a method for introducing specific mutations into
specific regions of the mammalian genome (Thomas et al., Cell,
44:419-428, 1986; Thomas and Capecchi, Cell, 51:503-512, 1987;
Doetschman et al., Proc. Natl. Acad. Sci. U.S.A., 85:8583-8587,
1988) or to correct specific mutations within defective genes
(Doetschman et al., Nature, 330:576-578, 1987). Exemplary
homologous recombination techniques are described in U.S. Pat. No.
5,272,071 (EP 9193051, EP Publication No. 505500; PCT/US90/07642,
International Publication No. WO 91/09955).
[0314] Through homologous recombination, the DNA sequence to be
inserted into the genome can be directed to a specific region of
the gene of interest by attaching it to targeting DNA. The
targeting DNA is a nucleotide sequence that is complementary
(homologous) to a region of the genomic DNA. Small pieces of
targeting DNA that are complementary to a specific region of the
genome are put in contact with the parental strand during the DNA
replication process. It is a general property of DNA that has been
inserted into a cell to hybridize, and therefore, recombine with
other pieces of endogenous DNA through shared homologous regions.
If this complementary strand is attached to an oligonucleotide that
contains a mutation or a different sequence or an additional
nucleotide, it too is incorporated into the newly synthesized
strand as a result of the recombination. As a result of the
proofreading function, it is possible for the new sequence of DNA
to serve as the template. Thus, the transferred DNA is incorporated
into the genome.
[0315] Attached to these pieces of targeting DNA are regions of DNA
which may interact with or control the expression of a h2520-59
polypeptide, e.g., flanking sequences. For example, a
promoter/enhancer element, a suppressor, or an exogenous
transcription modulatory element is inserted in the genome of the
intended host cell in proximity and orientation sufficient to
influence the transcription of DNA encoding the desired h2520-59
polypeptide. The control element controls a portion of the DNA
present in the host cell genome. Thus, the expression of the
desired h2520-59 polypeptide may be achieved not by transfection of
DNA that encodes the h2520-59 gene itself, but rather by the use of
targeting DNA (containing regions of homology with the endogenous
gene of interest) coupled with DNA regulatory segments that provide
the endogenous gene sequence with recognizable signals for
transcription of a h2520-59 polypeptide.
[0316] In an exemplary method, the expression of a desired targeted
gene in a cell (i.e., a desired endogenous cellular gene) is
altered via homologous recombination into the cellular genome at a
preselected site, by the introduction of DNA which includes at
least a regulatory sequence, an exon and a splice donor site. These
components are introduced into the chromosomal (genomic) DNA in
such a manner that this, in effect, results in the production of a
new transcription unit (in which the regulatory sequence, the exon
and the splice donor site present in the DNA construct are
operatively linked to the endogenous gene). As a result of the
introduction of these components into the chromosomal DNA, the
expression of the desired endogenous gene is altered.
[0317] Altered gene expression, as described herein, encompasses
activating (or causing to be expressed) a gene which is normally
silent (unexpressed) in the cell as obtained, as well as increasing
the expression of a gene which is not expressed at physiologically
significant levels in the cell as obtained. The embodiments further
encompass changing the pattern of regulation or induction such that
it is different from the pattern of regulation or induction that
occurs in the cell as obtained, and reducing (including
eliminating) the expression of a gene which is expressed in the
cell as obtained.
[0318] One method by which homologous recombination can be used to
increase, or cause, h2520-59 polypeptide production from a cell's
endogenous h2520-59 gene involves first using homologous
recombination to place a recombination sequence from a
site-specific recombination system (e.g., Cre/loxP, FLP/FRT) (Sauer
et al., Curr. Opin. Biotech., 5:521-527, 1994; Sauer et al., Meth.
Enzymol., 225:890-900, 1993) upstream (that is, 5' to) of the
cell's endogenous genomic h2520-59 polypeptide coding region. A
plasmid containing a recombination site homologous to the site that
was placed just upstream of the genomic h2520-59 polypeptide coding
region is introduced into the modified cell line along with the
appropriate recombinase enzyme. This recombinase causes the plasmid
to integrate, via the plasmid's recombination site, into the
recombination site located just upstream of the genomic h2520-59
polypeptide coding region in the cell line (Baubonis and Sauer,
Nucl. Acids Res., 21:2025-2029, 1993; O'Gorman et al., Science,
251:1351-1355, 1991). Any flanking sequences known to increase
transcription (e.g., enhancer/promoter, intron, translational
enhancer), if properly positioned in this plasmid, would integrate
in such a manner as to create a new or modified transcriptional
unit resulting in de novo or increased h2520-59 polypeptide
production from the cell's endogenous h2520-59 gene.
[0319] A further method to use the cell line in which the
site-specific recombination sequence had been placed just upstream
of the cell's endogenous genomic h2520-59 polypeptide coding region
is to use homologous recombination to introduce a second
recombination site elsewhere in the cell line's genome. The
appropriate recombinase enzyme is then introduced into the
two-recombination-site cell line, causing a recombination event
(deletion, inversion, translocation) (Sauer et al., Curr. Opin.
Biotech., supra; Sauer, Meth. Enzymol., supra) that would create a
new or modified transcriptional unit resulting in de novo or
increased h2520-59 polypeptide production from the cell's
endogenous h2520-59 gene.
[0320] An additional approach for increasing, or causing, the
expression of h2520-59 polypeptide from a cell's endogenous
h2520-59 gene involves increasing, or causing, the expression of a
gene or genes (e.g., transcription factors) and/or decreasing the
expression of a gene or genes (e.g., transcriptional repressors) in
a manner which results in de novo or increased h2520-59 polypeptide
production from the cell's endogenous h2520-59 gene. This method
includes the introduction of a non-naturally occurring polypeptide
(e.g., a polypeptide comprising a site specific DNA binding domain
fused to a transcriptional factor domain) into the cell such that
de novo or increased h2520-59 polypeptide production from the
cell's endogenous h2520-59 gene results.
[0321] The present invention further relates to DNA constructs
useful in the method of altering expression of a target gene. In
certain embodiments, the exemplary DNA constructs comprise: (a) one
or more targeting sequences; (b) a regulatory sequence; (c) an
exon; and (d) an unpaired splice-donor site. The targeting sequence
in the DNA construct directs the integration of elements (a)-(d)
into a target gene in a cell such that the elements (b)-(d) are
operatively linked to sequences of the endogenous target gene. In
another embodiment, the DNA constructs comprise: (a) one or more
targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a
splice-donor site, (e) an intron, and (f) a splice-acceptor site,
wherein the targeting sequence directs the integration of elements
(a)-(f) such that the elements of (b)-(f) are operatively linked to
the endogenous gene. The targeting sequence is homologous to the
preselected site in the cellular chromosomal DNA with which
homologous recombination is to occur. In the construct, the exon is
generally 3' of the regulatory sequence and the splice-donor site
is 3' of the exon.
[0322] If the sequence of a particular gene is known, such as the
nucleic acid sequence of h2520-59 polypeptide presented herein, a
piece of DNA that is complementary to a selected region of the gene
can be synthesized or otherwise obtained, such as by appropriate
restriction of the native DNA at specific recognition sites
bounding the region of interest. This piece serves as a targeting
sequence(s) upon insertion into the cell in that it will hybridize
to its homologous region within the genome. It is conventionally
believed that if this hybridization occurs during DNA replication,
this piece of DNA, and any additional sequence attached thereto,
will act as an Okazaki fragment and will be incorporated into the
newly synthesized daughter strand of DNA. The present invention,
therefore, includes nucleotides encoding a h2520-59 polypeptide,
which nucleotides may be used as targeting sequences.
[0323] h2520-59 polypeptide cell therapy, e.g., the implantation of
cells producing h2520-59 polypeptides, is also contemplated. This
embodiment involves implanting cells capable of synthesizing and
secreting a biologically active form of h2520-59 polypeptide. Such
h2520-59 polypeptide-producing cells can be cells that are natural
producers of h2520-59 polypeptides or may be recombinant cells
whose ability to produce h2520-59 polypeptides has been augmented
by transformation with a gene encoding the desired h2520-59
polypeptide or with a gene augmenting the expression of h2520-59
polypeptide. Such a modification may be accomplished by means of a
vector suitable for delivering the gene as well as promoting its
expression and secretion. In order to minimize a potential
immunological reaction in patients being administered a h2520-59
polypeptide, as may occur with the administration of a polypeptide
of a foreign species, it is preferred that the natural cells
producing h2520-59 polypeptide be of human origin and produce human
h2520-59 polypeptide. Likewise, it is preferred that the
recombinant cells producing h2520-59 polypeptide be transformed
with an expression vector containing a gene encoding a human
h2520-59 polypeptide.
[0324] Implanted cells may be encapsulated to avoid the
infiltration of surrounding tissue. Human or non-human animal cells
may be implanted in patients in biocompatible, semipermeable
polymeric enclosures or membranes that allow the release of
h2520-59 polypeptide, but prevent the destruction of the cells by
the recombination), tissue-specific promoter, enhancer(s) or
silencer(s), DNA molecules capable of providing a selective
advantage over the parent cell, DNA molecules useful as labels to
identify transformed cells, negative selection systems,
cell-specific binding agents (as, for example, for cell targeting),
cell-specific internalization factors, and transcription factors to
enhance expression by a vector as well as factors to enable vector
manufacture.
[0325] A gene therapy DNA construct can then be introduced into
cells (either ex vivo or in vivo) using viral or non-viral vectors.
One means for introducing the gene therapy DNA construct is by
means of viral vectors as described herein. Certain vectors, such
as retroviral vectors, will deliver the DNA construct to the
chromosomal DNA of the cells, and the gene can integrate into the
chromosomal DNA. Other vectors will function as episomes, and the
gene therapy DNA construct will remain unintegrated.
[0326] In yet other embodiments, regulatory elements can be
included for the controlled expression of the h2520-59 gene in the
target cell. Such elements are turned on in response to an
appropriate effector. In this way, a therapeutic polypeptide can be
expressed when desired. One conventional control means involves the
use of small molecule dimerizers or rapalogs [as described in WO
96/41865 (PCT/US96/099486); WO 97/31898 (PCT/US97/03137) and WO
97/31899 (PCT/US95/03157)] used to dimerize chimeric proteins which
contain a small-molecule binding domain and a domain capable of
initiating biological process, such as a DNA-binding protein or
transcriptional activation protein. The dimerization of the
proteins can be used to initiate transcription of the
transgene.
[0327] An alternative regulation technology uses a method of
storing proteins expressed from the gene of interest inside the
cell as an aggregate or cluster. The gene of interest is expressed
as a fusion protein that includes a conditional aggregation domain
which results in the retention of the aggregated protein in the
endoplasmic reticulum. The stored proteins are stable and inactive
inside the cell. The proteins can be released, however, by
administering a drug (e.g., small molecule ligand) that removes the
conditional aggregation domain and thereby specifically breaks
apart the aggregates or clusters so that the proteins may be
secreted from the cell. See, Aridor and Balch, Science 287:816-817,
2000; Rivera et al., Science 287:826-830, 2000.
[0328] Other suitable control means or gene switches include, but
are not limited to, the following systems. Mifepristone (RU486) is
used as a progesterone antagonist. The binding of a modified
progesterone receptor ligand-binding domain to the progesterone
antagonist activates transcription by forming a dimer of two
transcription factors which then pass into the nucleus to bind DNA.
The ligand-binding domain is modified to eliminate the ability of
the receptor to bind to the natural ligand. The modified steroid
hormone receptor system is further described in U.S. Pat. No.
5,364,791; WO 96/40911; and WO 97/10337.
[0329] Yet another control system uses ecdysone (a fruit fly
steroid hormone) which binds to and activates an ecdysone receptor
(cytoplasmic receptor). The receptor then translocates to the
nucleus to bind a specific DNA response element (promoter from
ecdysone-responsive gene). The ecdysone receptor includes a
transactivation domain/DNA-binding domain/ligand-binding domain to
initiate transcription. The ecdysone system is further described in
U.S. Pat. No. 5,514,578; WO 97/38117; WO 96/37609; and WO
93/03162.
[0330] Another control means uses a positive
tetracycline-controllable transactivator. This system involves a
mutated tet repressor protein DNA-binding domain (mutated tet R-4
amino acid changes which resulted in a reverse
tetracycline-regulated transactivator protein, i.e., it binds to a
tet operator in the presence of tetracycline) linked to a
polypeptide which activates transcription. Such systems are
described in U.S. Pat. Nos. 5,464,758, 5,650,298 and 5,654,168.
[0331] Additional expression control systems and nucleic acid
constructs are described in U.S. Pat. Nos. 5,741,679 and 5,834,186,
to Innovir Laboratories Inc.
[0332] In vivo gene therapy may be accomplished by introducing the
gene encoding a h2520-59 polypeptide into cells via local injection
of a h2520-59 nucleic acid molecule or by other appropriate viral
or non-viral delivery vectors. See, Hefti, Neurobiology,
25:1418-1435, 1994. For example, a nucleic acid molecule encoding a
h2520-59 polypeptide may be contained in an adeno-associated virus
(AAV) vector for delivery to the targeted cells (e.g., Johnson,
International Publication No. WO 95/34670; International
Application No. PCT/US95/07178). The recombinant AAV genome
typically contains AAV inverted terminal repeats flanking a DNA
sequence encoding a h2520-59 polypeptide operably linked to
functional promoter and polyadenylation sequences.
[0333] Alternative suitable viral vectors include, but are not
limited to, retrovirus, adenovirus, herpes simplex virus,
lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus,
alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma
virus vectors. U.S. Pat. No. 5,672,344 describes an in vivo
viral-mediated gene transfer system involving a recombinant
neurotrophic HSV-1 vector. U.S. Pat. No. 5,399,346 provides
examples of a process for providing a patient with a therapeutic
protein by the delivery of human cells which have been treated in
vitro to insert a DNA segment encoding a therapeutic protein.
Additional methods and materials for the practice of gene therapy
techniques are described in U.S. Pat. No. 5,631,236 involving
adenoviral vectors; U.S. Pat. No. 5,672,510 involving retroviral
vectors; and U.S. Pat. No. 5,635,399 involving retroviral vectors
expressing cytokines.
[0334] Nonviral delivery methods include, but are not limited to,
liposome-mediated transfer, naked DNA delivery (direct injection),
receptor-mediated transfer (ligand-DNA complex), electroporation,
calcium phosphate precipitation, and microparticle bombardment
(e.g., gene gun). Gene therapy materials and methods may also
include the use of inducible promoters, tissue-specific
enhancer-promoters, DNA sequences designed for site-specific
integration, DNA sequences capable of providing a selective
advantage over the parent cell, labels to identify transformed
cells, negative selection systems and expression control systems
(safety measures), cell-specific binding agents (for cell
targeting), cell-specific internalization factors, and
transcription factors to enhance expression by a vector as well as
methods of vector manufacture. Such additional methods and
materials for the practice of gene therapy techniques are described
in U.S. Pat. No. 4,970,154 involving electroporation techniques;
WO96/40958 involving nuclear ligands; U.S. Pat. No. 5,679,559
describing a lipoprotein-containing system for gene delivery; U.S.
Pat. No. 5,676,954 involving liposome carriers; U.S. Pat. No.
5,593,875 concerning methods for calcium phosphate transfection;
and U.S. Pat. No. 4,945,050 wherein biologically active particles
are propelled at cells at a speed whereby the particles penetrate
the surface of the cells and become incorporated into the interior
of the cells.
[0335] It is also contemplated that h2520-59 gene therapy or cell
therapy can further include the delivery of one or more additional
polypeptide(s) in the same or a different cell(s). Such cells may
be separately introduced into the patient, or the cells may be
contained in a single implantable device, such as the encapsulating
membrane described above, or the cells may be separately modified
by means of viral vectors.
[0336] A means to increase endogenous h2520-59 polypeptide
expression in a cell via gene therapy is to insert one or more
enhancer element(s) into the h2520-59 polypeptide promoter, where
the enhancer element(s) can serve to increase transcriptional
activity of the h2520-59 gene. The enhancer element(s) used will be
selected based on the tissue in which one desires to activate the
gene(s); enhancer elements known to confer promoter activation in
that tissue will be selected. For example, if a gene encoding a
h2520-59 polypeptide is to be "turned on" in T-cells, the lck
promoter enhancer element may be used. Here, the functional portion
of the transcriptional element to be added may be inserted into a
fragment of DNA containing the h2520-59 polypeptide promoter (and
optionally, inserted into a vector and/or 5' and/or 3' flanking
sequence(s), etc.) using standard cloning techniques. This
construct, known as a "homologous recombination construct," can
then be introduced into the desired cells either ex vivo or in
vivo.
[0337] Gene therapy also can be used to decrease h2520-59
polypeptide expression by modifying the nucleotide sequence of the
endogenous promoter(s). Such modification is typically accomplished
via homologous recombination methods. For example, a DNA molecule
containing all or a portion of the promoter of the h2520-59 gene(s)
selected for inactivation can be engineered to remove and/or
replace pieces of the promoter that regulate transcription. For
example the TATA box and/or the binding site of a transcriptional
activator of the promoter may be deleted using standard molecular
biology techniques; such deletion can inhibit promoter activity
thereby repressing the transcription of the corresponding h2520-59
gene. The deletion of the TATA box or the transcription activator
binding site in the promoter may be accomplished by generating a
DNA construct comprising all or the relevant portion of the
h2520-59 polypeptide promoter(s) (from the same or a related
species as the h2520-59 gene(s) to be regulated) in which one or
more of the TATA box and/or transcriptional activator binding site
nucleotides are mutated via substitution, deletion and/or insertion
of one or more nucleotides. As a result, the TATA box and/or
activator binding site has decreased activity or is rendered
completely inactive. The construct will typically contain at least
about 500 bases of DNA that correspond to the native (endogenous)
5' and 3' DNA sequences adjacent to the promoter segment that has
been modified. The construct may be introduced into the appropriate
cells (either ex vivo or in vivo) either directly or via a viral
vector as described herein. Typically, the integration of the
construct into the genomic DNA of the cells will be via homologous
recombination, where the 5' and 3' DNA sequences in the promoter
construct can serve to help integrate the modified promoter region
via hybridization to the endogenous chromosomal DNA.
Additional Uses of h2520-59 Nucleic Acids and Polypeptides
[0338] Nucleic acid molecules of the present invention (including
those that do not themselves encode biologically active
polypeptides) may be used to map the locations of the h2520-59 gene
and related genes on chromosomes. Mapping may be done by techniques
known in the art, such as PCR amplification and in situ
hybridization.
[0339] The full coding region of the h2520-59 gene is contained in
the human chromosome 20 sequence as listed in GENBANK Accession No.
AL034548. The human 2520-59 gene is specifically localized to
chromosome 20p12.2-13 134952-152220 which includes intron/exon
boundaries.
[0340] h2520-59 RNA levels are elevated in a wide range of human
primary tumors. Expression has been observed in brain, colon, lung,
skin, bone marrow, prostate, kidney, testis, uterus, and cervix
cancers. (See Example 2 and FIGS. 5-7). Based on the presence of a
putative kinase catalytic domain in the amino acid sequence,
h2520-59 polypeptide may play a role in maintaining transformed
phenotypes. (See Example 1).
[0341] h2520-59 nucleic acid molecules (and related molecules that
do not themselves encode biologically active polypeptides), may be
useful as hybridization probes in diagnostic assays to test, either
qualitatively or quantitatively, for the presence of a h2520-59 DNA
or corresponding RNA in mammalian tissue or bodily fluid samples.
h2520-59 may serve as a diagnosis/prognosis marker or assay for a
wide variety of human cancers. Monitoring changes in the expression
of h2520-59 during cancer treatment may be used as a surrogate
marker to monitor tumor growth and treatment success.
[0342] The h2520-59 polypeptides may be used (simultaneously or
sequentially) in combination with one or more cytokines, growth
factors, antibiotics, anti-inflammatories, and/or chemotherapeutic
agents as is appropriate for the indication being treated. h2520-59
may be useful as an inhibitor. In addition, peptide inhibitors
designed from h2520-59 polypeptide may be used as therapeutics or
identifying substances which modulate h2520-59 polypeptide
activity.
[0343] Other methods may also be employed where it is desirable to
inhibit the activity of one or more h2520-59 polypeptides. Such
inhibition may be effected by nucleic acid molecules which are
complementary to and hybridize to expression control sequences
(triple helix formation) or to h2520-59 mRNA. For example,
antisense DNA or RNA molecules, which have a sequence that is
complementary to at least a portion of the selected h2520-59
gene(s), can be introduced into the cell. Antisense probes may be
designed by available techniques using the sequence of h2520-59
polypeptide disclosed herein. Typically, each such antisense
molecule will be complementary to the start site (5' end) of each
selected h2520-59 gene. When the antisense molecule then hybridizes
to the corresponding h2520-59 mRNA, translation of this mRNA is
prevented or reduced. Antisense inhibitors provide information
relating to the decrease or absence of a h2520-59 polypeptide in a
cell or organism.
[0344] Alternatively, gene therapy may be employed to create a
dominant-negative inhibitor of one or more h2520-59 polypeptides.
In this situation, the DNA encoding a mutant polypeptide of each
selected h2520-59 polypeptide can be prepared and introduced into
the cells of a patient using either viral or non-viral methods as
described herein. Each such mutant is typically designed to compete
with endogenous polypeptide in its biological role. Particularly,
h2520-59 contains a kinase domain that may be useful in designing
dominant negative gene therapy for treatment in a wide variety of
tumors
[0345] In addition, a h2520-59 polypeptide, may be used as an
immunogen, that is, the polypeptide contains at least one epitope
to which antibodies may be raised. Selective binding agents that
bind to a h2520-59 polypeptide (as described herein) may be used
for in vivo and in vitro diagnostic purposes, including, but not
limited to, use in labeled form to detect the presence of h2520-59
polypeptide in a body fluid or cell sample. The antibodies may also
be used to prevent, treat, or diagnose a number of diseases and
disorders, including those recited herein. The antibodies may bind
to a h2520-59 polypeptide so as to diminish or block at least one
activity characteristic of a h2520-59 polypeptide, or may bind to a
polypeptide to increase at least one activity characteristic of a
h2520-59 polypeptide (including by increasing the pharmacokinetics
of the h2520-59 polypeptide).
[0346] cDNA encoding h2520-59 polypeptide in E. coli strain TOP10
was deposited with the ATCC on Apr. 25, 2000 and has Accession No.
PTA-1759.
Hypoxia and Solid Tumors
[0347] Regions of hypoxia are a hallmark of solid tumors. Tumor
cells modulate the regulation of specific genes allowing adaptation
and survival in the harsh hypoxic environment. h2520-59, a novel
human kinase-like gene, is overexpressed in multiple human tumors
and regulated under hypoxia.
[0348] Signaling pathways that drive tumor progression are varied
and complex. As the size of a tumor increases, nutrients and oxygen
become limiting within the tumor microenvironment. In response to
localized nutrient deficiency, increased acidity, and hypoxia,
specific signaling pathways are activated in tumor cells to
regulate the transcription of genes promoting cell survival
(reviewed in Lal et al., J Natl Cancer Inst 93:1337-1343, 2001;
Hockel and Vopel, J Natl Cancer Inst 93:266-276, 2001; and
Fafournoux et al., Biochem J 351:1-12, 2000). Some of these
transcription factors include the hypoxia-inducible factor 1-alpha
(HIF-1.alpha.) (reviewed in Semenza, Genes & Develop
14:1983-1991, 2000), as well as stress and nutrient deficiency
regulated genes, CHOP/GADD153, C/EBP.alpha., C/EBP.beta.
(Fafournoux, 2000, supra), the proto-oncogene c-myc (Yao et al., J
Natl Cancer Inst 87:117-122, 1995), and AP-1 transcription factors
c-jun (Ausserer et al., Mol Cell Biol 14:5032-5042, 1994) and fos
(Yao et al., Mol Cell Biol 14:5997-6003, 1994). Regulation of these
transcription factors occurs at several levels, including
upregulation of gene expression by the HIF-1 complex, modulation of
protein stability, and selective binding to specific heterodimer
partners such as members of the AP-1 and activating transcription
factor/cyclic AMP response element binding protein (ATF/CREB) basic
region-leucine zipper (bZIP) transcription factor families.
Interestingly, specific members of the bZIP transcription factor
family have not been implicated in tumor progression but are
associated with responses to stress pathways, such as low levels of
oxygen, the antioxidant response and general cellular stress. One
such bZIP family member is activating transcription factor 4 (ATF4)
(Estes et al., Exp Cell Res 220: 47-54, 1995; Gachon et al., FEBS
Letters 502:57-62, 2001; He et al., J Biol Chem 276:20858-20865,
2001).
[0349] Human ATF4, also known as CREB2, TAXREB67 and C/ATF, is a
member of the ATF/CREB family of bZIP transcription factors (for
review, see Hai and Hartman, Gene 273:1-11, 2001). ATF4
transactivates the transcription of specific genes through binding
to the cAMP response element (CRE) (Karpinski et al., Proc Natl
Acad Sci USA 89:4820-4824, 1992). Multiple gene promoters have been
identified as being transactivated by ATF4, including CHOP/GADD153
(Fawcett et al., Biochem J 339:135-141, 1999), Interleukin-2 CD28
response element (CD28RE) (Butscher et al., J Biol Chem
273:552-560, 1998), and most recently the adenomatous polyposis
coli (APC) tumor suppressor binding protein RP1 (Wadle et al.,
Oncogene 20:5920-5929, 2001). The transcriptional selectivity of
ATF4 is modulated by the formation of heterodimers with multiple
C/EBP bZIP proteins including: CHOP/GADD153 (Gachon et al., 2001,
supra), C/EBP.alpha. (Nishizawa and Nagata, FEBS Letters 299:36-38,
1992), C/EBP.beta., CRP2 (Vallejo et al., Proc Natl Acad Sci USA
90:4679-4683, 1993), and C/EBP.gamma. (Vinson et al., Genes &
Develop 7:1047-1058, 1993) as well as AP-1 family members (fos and
jun proteins) (Hai and Curran, Proc Natl Acad Sci USA 88:3720-3724,
1991; Kato et al., Mol & Cell Endocrinol 154:151-159, 1999).
ATF4 also associates with Tax, a human T cell leukemia virus type I
transactivator (Reddy et al., Oncogene 14:2785-2792, 1997), CREB
binding protein (CBP) (Liang and Hai, J Biol Chem 272:24088-24095,
1997), and Zip kinase (Kawai et al., Mol & Cell Biol
18:1642-1651, 1998). Additionally, protein stability plays a role
in modulating ATF4 function. For example, ATF4 binding with
.beta.TrCP, a F-box protein which is part of the E3 ubiquitin
ligase complex, leads to ubiquitin mediated degradation of ATF4
(Lassot et al., Mol & Cell Biol 21:2192-2202, 2001). Although
many of these factors which are known to participate in the
regulation of ATF4 have been identified, it is still not understood
why ATF4 participates in multiple stress response pathways in
normal cells and has yet not been implicated as being crucial for
tumor cell progression. h2520-59 has been shown to bind ATF4 (see
Example 7).
[0350] The Drosophila tribbles gene was recently identified as a
developmental cell-cycle brake that blocks mitotic progression in
the mesoderm during early Drosophila gastrulation (Seher et al.,
Curr Biol 10:623-629, 2000; Mata et al., Cell 101:511-522, 2000;
Grosshans and Wieschaus, Cell 101:523-531, 2000). The tribbles
protein has a high homology to serine/threonine kinases, including
h2520-59 (see Example 1 and FIG. 3). Although tribbles contains
almost all of the consensus amino acids which correspond to the
kinase catalytic core, it is highly divergent through the consensus
ATP binding pocket (Seher et al., 2000, supra). The function of the
tribbles protein is to slow cell cycle progression by inducing the
degradation of the CDC25 activators of mitosis orthologs, string
and twine, during Drosophila gastrulation (Mata et al., 2000,
supra; Grosshans and Wieschaus, 2000, supra).
[0351] Drosophila tribbles has been shown to interact with and
activate the proteolysis of a Drosophila CREB-C/EBP basic
region-leucine zipper transcription factor family member, slbo,
using similar overexpression and co-immunoprecipitation studies
(Rorth et al., Molecular Cell 6:23-30, 2000). It appears that a
h2520-59-bZIP transcription factor interaction has been conserved
in Drosophila and humans in both binding ability and activation of
proteolysis. This is consistent with the importance of protein
stability in the regulation of the h2520-59 family of kinase-like
proteins.
[0352] The mammalian orthologs of Drosophila tribbles (dog: C5FW,
rat: NIPK, human: C8FW) have been identified in multiple species,
and like tribbles, they all share homology to h2520-59 (see Example
1 and FIG. 3). In dog thyroid cells, the levels of C5FW protein
have been shown to increase upon stimulation with thyrotropin or
epidermal growth factor (Wilkin et al., Eur J Biochem 248:660-668,
1997). Expression of NIPK (Neuronal cell death Inducible Putative
Kinase) is induced when rat neuronal PC6-3 cells are deprived of
NGF (Mayumi-Matsuda et al., Biochem & Biophys Res Comm
258:260-264, 1999). A human partial protein, C8FW, was identified
as a binding partner of 12-LOX in epidermoid carcinoma A431 cells
(Tang et al., Biochem 39:3185-3191, 2000). Like tribbles protein,
C5FW, NIPK and C8FW proteins all appear to contain the consensus
serine/threonine kinase catalytic core, but lack a consensus ATP
binding pocket. No functional data has been reported for these
proteins in mammalian cells, and little is known regarding their
regulated expression.
[0353] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the invention in any way.
EXAMPLE 1
Human h2520-59 DNA and Protein
A. Cloning, Sequencing, and Localization of h2520-59 DNA
[0354] Materials and methods for cDNA cloning and analysis are
described in Sambrook et al., supra.
[0355] A Hidden Markov Model built against known serine/threonine
catalytic cores was used to query the Celera human genomic database
to identify potential kinases. This search identified an EST
sequence, reference no. GA 6736448-150850-151365, as set out in
(SEQ ID NO: 12) as a putative serine threonine (ser/thr)
kinase.
[0356] Polymerase chain reaction (PCR) primers were designed based
on the identified sequence to generate a 200 base pair product. The
forward primer was 5'-TGG TGC TGG AGA ACC TGG AGG-3' (SEQ ID NO: 3)
and the reverse primer was 5'-CGA GTC CTG GAA GGG GTA GTG-3' (SEQ
ID NO: 4). These primers were then used to screen the human lung
Rapid-Screen.TM. cDNA Library Panel (Origene Technologies,
Rockville, Md.) according to the manufacturer's instructions for
the full-length h2520-59 cDNA. PCR was carried out with 40 nM of
both the forward and reverse primers, 20 .mu.l of H2O, 5 .mu.l of
the cDNA library, and 1 Ready-To-Go PCR bead (Amersham Pharmacia
Biotech). The PCR reaction conditions were 94.degree. C. for 3
minutes, followed by 30 cycles of 30 seconds at 94.degree. C., 45
seconds at 58.degree. C., 1 minute at 72.degree. C., and a
subsequent incubation for 7 minutes at 72.degree. C. at the end of
the 30th cycle. The PCR reactions were analyzed on a 2% agarose gel
and positive reactions contained a 200 base pair band.
[0357] The corresponding subplates within the lung Rapid-Screen.TM.
cDNA Library Panel were screened using the PCR conditions described
above. The positive wells were then screened according to the
Rapid-Screen cDNA Library Panel using the above-described PCR
conditions. The plasmid DNA from the positive wells was prepared
with the Qiagen Spin Mini-Prep Kit according to the manufacturer's
instruction. The positive full-length h2520-59 clone was three-way
sequence walked for confirmation. The sequences of both strands of
the cDNA insert were verified by standard sequencing techniques.
The sequence obtained was found to have high homology to a later
filed GENBANK submission for a sequence which was labeled "SKIP3"
(see GENBANK Accession no. AF250311; SEQ ID NO: 30).
[0358] The cDNA sequence encoding the h2520-59 polypeptide is shown
in FIG. 1 (SEQ ID NO: 1). The h2520-59 gene is 2059 nucleotides in
length with a 1074 nucleotide coding region. This coding region
encodes a 358 amino acid polypeptide (SEQ ID NO: 2).
[0359] The full coding region of h2520-59 is contained within the
human chromosome 20 sequence described in GENBANK Accession No.
AL34548 and is specifically localized to chromosome p2012.2-13
134952-152220, including intron/exon boundaries.
B. h2520-59 Protein
[0360] The alignment of the deduced amino acid sequence with known
ser/thr kinases determined h2520-59 polypeptide contained a
putative kinase domain about three quarters toward the C-terminus.
Sequence homology of the putative kinase domain revealed 26%
homology with other members of the ser/thr kinase family such as
RIP1, Rat Death Domain, Cdk2, and C8FW within the kinase
domain.
[0361] The h2520-59 amino acid sequence was determined to be
closely related to several previously reported proteins including
NIPK (described by Mayumi-Matsuda et al., supra). NIPK (SEQ ID NO:
31) is 72% identical to the h2520-59 amino acid sequence and may
represent the rat ortholog. Other related proteins are dog C5FW
(SEQ ID NO: 32; 43.3%), a recent human full length C8FW GENBANK
deposit, SKIP1 (SEQ ID NO: 33; 40.6%), and Drosophila tribbles (SEQ
ID NO: 34; 21%). The highest area of conservation among this family
of proteins occurs in the carboxy-terminal half of the proteins. In
contrast, the tribbles amino terminal protein region is highly
variant to the other family members, suggesting that some functions
of tribbles may vary from its vertebrate counterparts.
EXAMPLE 2
Expression of h2520-59 in Cells and Tissues
A. Matched cDNA Pair Expression Analysis
[0362] To determine if h2520-59 mRNA was elevated in tumor cells as
compared to normal cells, matched cDNA pair expression analysis was
performed. Selected matched cDNA pair libraries (Clontech
Laboratories, Palo Alto, Calif.) were screened by PCR. The PCR
reactions were performed under the conditions described in Example
I. The PCR reactions were analyzed on a 4-20% acrylamide TBE gel.
The matched cDNA pairs consisted of corresponding tumor and normal
cDNA libraries isolated from the same individual. These pairs
allowed for the determination of elevated gene expression in tumor
tissues as compared to normal tissue from the same patient.
Prostate, lung, colon, ovary and uterus tissues were screened. The
h2520-59 RNA expression was elevated in lung squamous cell
carcinoma from a 73-year-old male (lot no. 9090813), colon
adenocarcinoma from a 61-year-old female (lot no. 9080438), colon
adenocarcinoma from a 75-year-old male (lot no. 9110789), colon
adenocarcinoma from a 58-year-old male (lot no. 9100415), colon
adenocarcinoma from a 79-year-old female (lot no. 9100394), colon
adenocarcinoma from a 35-year-old female (lot no. 9100396), colon
adenocarcinoma from an individual of unknown age and gender (lot
no. 9100395), and ovarian serous cystadenocarcinoma for a
61-year-old female (lot no. 9090814).
B. Northern Blot Analysis
[0363] Differential tissue expression patterns of h2520-59 mRNA
determined by the matched cDNA pair analysis were verified by
Northern blot analysis. The probe was generated by performing PCR,
as described in Example 1, on the Lung Marathon-Ready.TM. cDNA
library (Clontech, Palo Alto, Calif.). The resulting 200-base-pair
product was isolated from an agarose gel and TOPO TA cloned using
the TA Cloning Kit.RTM. according to the manufacturer's
instructions (Invitrogen, Carlsbad Calif.). The 200-base-pair cDNA
was radiolabeled with dCTP using a random primer labeling kit
(Boehringer Mannheim, Indianapolis, Ind.).
[0364] Northern blot analysis was performed with human multiple
tissue Northern blot and cancer cell line multiple tissue Northern
blots (Clontech, Palo Alto, Calif.) as well as the human lung and
colon single tumor Multi-type NBA blots (Biochain, San Leandro,
Calif.; lot no. 8910072) which contained tumor samples paired with
normal controls and were normalized to the total amount of mRNA
loaded (NBA) (See FIGS. 5 and 6). The blots were prehybridized in
buffer containing 5.times.SSPE, 50% formamide, 5.times. Denhardt's
solution, 0.5% SDS and 100 g/ml of denatured salmon sperm DNA for 3
hours at 42.degree. C. The blots were then hybridized in the
above-described buffer containing 50 ng/ml of .sup.32P-labeled
probe for 20 hours at 42.degree. C. The blots were washed 3 times
in 2.times.SSC/0.05% SDS for 15 minutes at room temperature.
Subsequently, the blots were washed 3 times in 0.01.times.SSC/0.01%
SDS for 20 minutes at 50.degree. C. Radioactive blots were exposed
overnight to a phosphorimaging screen and visualized with a
phosphorimager (Molecular Dynamics, Sunnyvale, Calif.) at 100
micron resolution.
[0365] The lung tumor multi-type blot contained mRNA from poorly-
to moderately-differentiated squamous cell carcinoma,
poorly-differentiated squamous cell carcinoma, bronchio-alveolar
carcinoma, and normal tissue. h2520-59 mRNA was overexpressed in
poorly- to moderately-differentiated squamous cell carcinoma and
bronchio-alveolar carcinoma as compared to the normal lung tissue
(control).
[0366] h2520-59 mRNA expression was also observed in brain, colon,
lung, skin, bone marrow, prostate, kidney, testis, uterus and
cervix cancer tissues using the human Rapid Scan Panel according to
the manufacturer's instructions (Origene, Rockville, Md.). This
panel was screened using the PCR primers and conditions described
in Example 1.
[0367] In summary, Northern blot analysis of h2520-59 mRNA
expression in primary human tumor tissue showed that h2520-59 was
overexpressed in specific tumor samples including, poorly- to
moderately-differentiated squamous lung cell carcinoma,
poorly-differentiated squamous lung cell carcinoma, and colon
adenocarcinoma, while having a lower relative expression in normal
human tissues.
C. Real-Time RT-PCR
[0368] Expanding upon the normal/tumor h2520-59 Northern blot
analysis, real-time RT-PCR of h2520-59 across a wide range of
tumors from multiple tissues was performed. Total RNA samples from
normal human and primary human tumor tissues were purchased
directly from suppliers; Biochain (San Leandro, Calif.), Clontech
(Palo Alto, Calif.), and Ambion (Austin, Tex.). Total RNA samples
were adjusted to 5 ng/ml. Real-time PCR was carried out using
h2520-59 primers 5'-CGGCTACCACATCCAAGGA-3' (SEQ ID NO: 16),
5'-GCTGGAATTACCGCGGCT-3' (SEQ ID NO: 17), and a probe of
5'-ETGCTGGCACCAGACTTGCCCTCX-3' (SEQ ID NO: 18), where E represents
6-FAM and X represents TAMRA. 6-FAM and TAMRA are fluorescent tags
for Taqman analysis. For normalization, the primers to 18S rRNA
were 5'-AATCCTTGAAGGAAATGACATTGAG-3' (SEQ ID NO: 19),
5'-TCCTTGTTTTTAACTGTTGTGGCTT-3' (SEQ ID NO: 20), and a probe of
5'-ETTGTTTCAAATTCAGCGGCTTTGATTCAGX-3' (SEQ ID NO: 21), where E
represents 6-FAM and X represents TAMRA. Real-time RT-PCR was
performed in a 50 ml reaction with 25 ng sample total RNA using the
One Step RT-PCR master mix reagent (Applied Biosystems, Foster
City, Calif.) on an ABI Prism 7700 sequence detector (Perkin Elmer,
Wellesley, Mass.). Samples were run in duplicate and normalized to
the relative transcript level of 18S rRNA. P-value confidence was
calculated using an unpaired comparison t-test of the mean
difference of the 18S rRNA normalized normal and tumor h2520-59
transcript levels from each organ.
[0369] Real-time RT-PCR indicated that h2520-59 was widely
overexpressed in many primary tumor types in comparison to normal
tissues (see FIG. 7). The breast tumors in the analysis had the
highest relative overexpression of h2520-59 as well as the broadest
expression of h2520-59 of the patient samples tested. These breast
tumor types included infiltrating and noninfiltrating ductal
carcinomas, lobular carcinoma, and mucinous adenocarcinoma. As
determined by nonlinear PCR and Northern blot analysis, real-time
RT-PCR of colorectal tumors also showed a broad overexpression of
h2520-59 in tumor types including well to moderately differentiated
adenocarcinoma, and colon and rectum adenocarcinomas with
metastases to the lymph nodes. Lung tumors overexpressing h2520-59
included only squamous cell carcinomas and a giant cell carcinoma
although multiple lung adenocarcinomas and keratinizing squamous
cell carcinomas were examined. In comparison with breast, colon,
and lung tumors, uterine and ovarian tumors express h2520-59 at
lower relative levels. Within the uterine-ovarian tumor sample
group, the highest h2520-59 expressing tumor types included uterine
moderately to poorly differentiated adenocarcinomas, a uterine
malignant mixed Mullerian tumor, and ovarian adenocarcinomas.
D. In Situ Hybridization
[0370] To better understand the expression pattern of h2520-59 in
primary human tumors, in situ hybridization (ISH) was used to
analyze h2520-59 expression in human tumor xenografts (see FIG. 9).
Human colorectal adenocarcinoma cells (HT-29) and human prostate
adenocarcinoma cells (PC-3) were grown subcutaneously as xenografts
in nu/nu mice, injected with 5.times.10.sup.6 cells into the right
flank, to a volume of 500 mm.sup.3. Tumors were harvested,
sectioned, stained and probed by ISH for h2520-59 expression. A
[.gamma.-.sup.33P]-labeled antisense RNA probe corresponding to
full-length h2520-59 was hybridized to 5 .mu.m thick sections of
paraffin-embedded tissue followed by RNase digestion and a high
stringency wash as previously described. Signal was detected by
emulsion autoradiography; sections were counterstained with
hematoxylin and eosin and photographed using darkfield
illumination. ISH revealed the localized expression of h2520-59 to
specific tumor cells, while surrounding normal tissues show only
background hybridization.
[0371] The expression pattern of h2520-59 was also determined by
ISH in human tumor sections and normal controls (see FIG. 8).
h2520-59 localized to specific areas in both lung and colon tumors
with only minimal background hybridization to normal lung and colon
tissue. No detectable signal appeared over stromal elements,
infiltrating lymphocytes or adjacent normal tissues in the tumor
samples. This data correlated with the primary tumor/normal
Northern and real-time RT-PCR analysis and indicated that h2520-59
overexpression was not due to highly expressing infiltrating
non-cancerous cells, but rather was localized to specific cancer
cell clusters within each tumor. In addition to primary tumors,
h2520-59 expression was localized in tumor cells in metastatic
breast carcinoma lesions in the brain, indicating a potential
requirement for h2520-59 in late stage metastatic disease. Overall,
the transcription of h2520-59 mRNA was upregulated in tumor
sections of human lung cancer, colon cancer, and breast cancer
cells that had metastasized to the brain.
E. Hypoxia and BrdU Analysis
[0372] While h2520-59 was highly expressed in a large percentage of
PC-3 cells, the expression of h2520-59 was more localized in the
HT-29 xenograft. Although the h2520-59 ISH patterns of both the
primary human tumors and the tumor cell xenografts showed a
specific localized pattern of hybridization, h2520-59 expression in
the HT-29 xenograft appeared to be proximal to a region of cell
death (see FIG. 9). Therefore, to determine if h2520-59 expression
correlated with cellular proliferation, bromodeoxyuridine (BrdU)
analysis was carried out.
[0373] HT-29 and PC-3 human tumor cells were grown as xenografts in
nu/nu mice as done for the ISH experiment. However, one hour prior
to sacrifice, each animal received an intravenous injection of BrdU
(Aldrich Chemical Co., Milwaukee, Wis.). Paraffin sections adjacent
to those used for ISH were incubated with a rat anti-BrdU antibody
(Harlan Sera Lab) followed by biotinylated rabbit anti-rat
secondary antibody (DAKO Corp. Carpenteria, Calif.), then
peroxidase-linked avidin-biotin complex (Vector Labs, Burlingame,
Calif.) detected with diaminobenzidine/H.sub.2O.sub.2. Sections
were counterstained with hematoxylin.
[0374] Expression of h2520-59 was very high in this region and the
cells were neither proliferating nor undergoing apoptosis, but
instead were located proximal to an apoptotic region. Previously,
HT-29 cells have been shown to undergo apoptosis when exposed to
hypoxia (Yao et al., J Natl Cancer Inst 87:117-122, 1995), and it
was hypothesized that the peri-apoptotic region in the HT-29
xenograft may have contained cells undergoing a hypoxic response.
To test this hypothesis, HT-29 and PC-3 cells were cultured under
hypoxic conditions for 0, 24, 48 and 72 hours to determine if
h2520-59 is upregulated at the transcript or protein level under
hypoxia. Hypoxic growth was induced in a Bactron Anaerobic Chamber
(Sheldon Manufacturing Inc.) under 0.5% O.sub.2, 5% CO.sub.2 and
94.5% N.sub.2 at 37.degree. C. The MG-132 proteasome inhibitor
(Calbiochem, San Diego Calif.) was dosed for 18 hours at 10
.mu.M.
[0375] For both HT-29 and PC-3 cells, h2520-59 mRNA expression
increased after 72 hours under hypoxia. h2520-59 protein levels
began to increase by 48 hours for both HT-29 and PC-3 cells and
remained elevated at 72, hours (see FIG. 10). This was in contrast
to h2520-59 protein levels from cells under normoxic conditions
which maintained h2520-59 protein at a steady level. Interestingly,
many genes that are known to be regulated under hypoxia, such as
HIF-1.alpha., VEGF, NDRG1, and carbonic anhydrase (Lal et al., J
Natl Cancer Inst 93:1337-1343, 2001), have been shown to be
upregulated at both the mRNA and protein levels within 24 hours of
exposure to hypoxic conditions. The relatively late initiation of
expression and protein accumulation of h2520-59 under hypoxia
indicates that h2520-59 may be downstream in the hypoxic response
pathway relative to the classical hypoxia activated genes.
[0376] In the HT-29 xenograft, BrdU positive cells localized distal
from the h2520-59 positive cells, indicating that h2520-59
expression does not correlate with cell proliferation. Also for the
HT-29 xenograft, the area of cell death undergoing apoptosis was
identified by both the nuclear staining pattern by hematoxilyn and
eosin (H&E) and the hematoxilyn counterstain on the BrdU
sections. Thus, h2520-59 expression in the HT-29 xenograft was
localized to a peri-apoptotic region.
F. Western Blot Analysis
[0377] Expression of the h2520-59 polypeptide was detected by
Western blot analysis using three antibodies raised against each of
three short peptide fragments (ELDDNLDTERPVQKRARSGPQPRLC, SEQ ID
NO: 13; GPYVLLEPEEGGRAYQALHCPTGTE, SEQ ID NO: 14;
RSHLWEAAQVVPDGLGLDEAREEEC, SEQ ID NO: 15) generally corresponding
to amino acids 20-43, 69-93, and 326-349, respectively, of the
h2520-59 polypeptide sequence (SEQ ID NO: 2). The three antibodies
were mixed in equal concentrations by volume for use as a Western
blot probe. Western blots were carried out using standard
techniques on lysates from the following human cell lines, most
available from the ATCC with one noted exception: osteosarcoma
cells (U-2 OS, ATCC Accession No. HTB-96), U-2 OS cells transfected
with h2520-59 (positive control), lung carcinoma cells (A549, ATCC
Accession No. CCL-185), colorectal carcinoma cells (Colo 205, ATCC
Accession No. CCL-222; and HCT 116, ATCC Accession No. CCL-247),
fibrosarcoma cells (HT-1080, ATCC Accession No. CCL-121), breast
adenocarcinoma cells (MDA-MB-231, ATCC Accession No. HTB-26;
MDA-MB-468, ATCC Accession No. HTB-132; and SK-BR-3, ATCC Accession
No. HTB-30), pancreatic carcinoma cells (MiaPaca-2, ATCC Accession
No. CRL-1420), prostate adenocarcinoma cells (PC-3, ATCC Accession
No. CRL-1435), ovarian adenocarcinoma cells (SKOV-3, ATCC Accession
No. HTB-77), human non-fetal skin fibroblast cells (AG01523A,
Coriell Cell Repositories, Camden, N.J.), and a fibrocystic breast
cell line (MCF 10A, ATCC Accession No. CRL-10317). The presence of
the h2520-59 polypeptide was detected in two of the breast cancer
cell lines, MDA-MB-468 and SKBR-3, as well as in the positive
control. Non-transfected U2-OS cells did not express h2520-59.
EXAMPLE 3
Production of h2520-59 Polypeptides
A. Bacterial Expression
[0378] PCR is used to amplify template DNA sequences encoding a
h2520-59 polypeptide using primers corresponding to the 5' and 3'
ends of the sequence. The 5' end primer has the sequence:
5'-CGGGGCGAGATGCGAGCCAC-3' (SEQ ID NO: 6) and the 3' end primer has
the sequence: 5'-AGGGTGGTCCTAGCCATACA-3' (SEQ ID NO: 7). The
amplified DNA products may be modified to contain restriction
enzyme sites to allow for insertion into expression vectors. PCR
products are gel purified and inserted into expression vectors
using standard recombinant DNA methodology. An exemplary vector,
such as pAMG21 (ATCC No. 98113) containing the lux promoter and a
gene encoding kanamycin resistance is digested with BamHI and NdeI
for directional cloning of inserted DNA. The ligated mixture is
transformed into an E. coli host strain by electroporation and
transformants are selected for kanamycin resistance. Plasmid DNA
from selected colonies is isolated and subjected to DNA sequencing
to confirm the presence of the insert.
[0379] Transformed host cells are incubated in 2.times.YT medium
containing 30 mg/ml kanamycin at 30.degree. C. prior to induction.
Gene expression is induced by the addition of
N-(3-oxohexanoyl)-dl-homoserine lactone to a final concentration of
30 ng/ml followed by incubation at either 30.degree. C. or
37.degree. C. for six hours. The expression of h2520-59 polypeptide
is evaluated by centrifugation of the culture, resuspension and
lysis of the bacterial pellets, and analysis of host cell proteins
by SDS-polyacrylamide gel electrophoresis.
[0380] Inclusion bodies containing h2520-59 polypeptide are
purified as follows. Bacterial cells are pelleted by centrifugation
and resuspended in water. The cell suspension is lysed by
sonication and pelleted by centrifugation at 195,000.times.g for 5
to 10 minutes. The supernatant is discarded, and the pellet is
washed and transferred to a homogenizer. The pellet is homogenized
in 5 ml of a Percoll solution (75% liquid Percoll, 0.15M NaCl)
until uniformly suspended and then diluted and centrifuged at
21,600.times.g for 30 minutes. Gradient fractions containing the
inclusion bodies are recovered and pooled. The isolated inclusion
bodies are analyzed by SDS-PAGE.
[0381] A single band on an SDS polyacrylamide gel corresponding to
E. coli-produced h2520-59 polypeptide is excised from the gel, and
the N-terminal amino acid sequence is determined essentially as
described by Matsudaira et al. (J. Biol. Chem. 262:10-35,
1987).
B. Mammalian Cell Production
[0382] PCR is used to amplify template DNA sequences encoding a
h2520-59 polypeptide using primers corresponding to the 5' and 3'
ends of the sequence. The primer sequences corresponding to the 5'
and 3' ends are described above. The amplified DNA products may be
modified to contain restriction enzyme sites to allow for insertion
into expression vectors. PCR products are gel purified and inserted
into expression vectors using standard recombinant DNA methodology.
An exemplary expression vector, pCEP4 (Invitrogep, Carlsbad,
Calif.), which contains an Epstein-Barr virus origin of
replication, may be used for the expression of h2520-59 in
293-EBNA-1 (Epstein-Barr virus nuclear antigen) cells. Amplified
and gel-purified PCR products are ligated into the pCEP4 vector and
lipofected into 293-EBNA cells. The transfected cells are selected
in 100 mg/ml hygromycin and the resulting drug-resistant cultures
are grown to confluence. The cells are then cultured in serum-free
medium for 72 hours. The conditioned medium is removed and h2520-59
polypeptide expression is analyzed by SDS-PAGE.
[0383] h2520-59 polypeptide expression is detected by silver
staining. Alternatively, h2520-59 polypeptide is produced as a
fusion protein with an epitope tag, such as an IgG constant domain
or a FLAG epitope, which may be detected by Western blot analysis
using antibodies to the tag peptide.
[0384] h2520-59 polypeptides is excised from an SDS-polyacrylamide
gel, or h2520-59 fusion proteins are purified by affinity
chromatography to the epitope tag, and subjected to N-terminal
amino acid sequence analysis as described herein.
EXAMPLE 4
Production of Anti-h2520-59 Polypeptide Antibodies
[0385] Polyclonal antibodies were generated by immunizing rabbits
with synthetic peptides (KLH coupled) corresponding to three
different regions within h2520-59, amino acids 20-43, 69-93 and
326-349. Antibody purification was performed using ImmunoPure
Protein A/G (Pierce, Rockford Ill.). The three purified antibodies
were used to probe Western blots in an equal-volume ratio.
[0386] Antibodies to h2520-59 polypeptides also are obtained by the
immunization of animals with purified protein or with h2520-59
peptides produced by biological or chemical synthesis. Suitable
procedures for generating antibodies include those described in
Hudson and Hay, Practical Immunology, 2nd Edition, Blackwell
Scientific Publications (1980).
[0387] In one procedure for the production of antibodies, animals
(typically mice or rabbits) are injected with a h2520-59 antigen
(such as a h2520-59 polypeptide), and those with sufficient serum
titer levels, as determined by ELISA, are selected for hybridoma
production. Spleens of immunized animals are collected and prepared
as single cell suspensions from which splenocytes are recovered.
The splenocytes are fused to mouse myeloma cells (such as
Sp2/0-Ag14 cells; ATCC No. CRL1581), allowed to incubate in DMEM
with 200 U/ml penicillin, 200 mg/ml streptomycin sulfate, and 4 mM
glutamine, then incubated in HAT selection medium (Hypoxanthine;
Aminopterin; Thymidine). After selection, the tissue culture
supernatants are taken from each well containing a hybridoma and
tested for anti-h2520-59 antibody production by ELISA.
[0388] Alternative procedures for obtaining anti-h2520-59
antibodies may also be employed, such as the immunization of
transgenic mice harboring human Ig loci for the production of human
antibodies, and the screening of synthetic antibody libraries, such
as those generated by mutagenesis of an antibody variable
domain.
EXAMPLE 5
Biological Activity of h2520-59 Polypeptides
[0389] Analysis of the deduced h2520-59 amino acid sequence
indicated that the polypeptide contains a putative ser/thr kinase
domain toward the C-terminus. To determine if h2520-59 polypeptide
exhibits ser/thr kinase enzymatic activity, phosphorylation studies
are performed. These studies are carried out within host cells
stably expressing the h2520-59 nucleotide as described in Example
3.
[0390] As described in Papst et al. (J. Biol. Chem. 273:
15077-15084, 1998), COS cells stably expressing h2520-59 nucleic
acids are grown in phosphate-free medium containing 10% fetal
bovine serum and 150 .mu.Ci/ml of [.sup.32P]orthophosphate (30
Ci/mmol). After a 3 hour incubation, the radiolabeled cells are
lysed in lysis buffer (25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5%
sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 .mu.M PMSF, 50
.mu.g/ml aprotinin, 50 .mu.M leupeptin). The lysates are
immunoprecipitated with either a mouse phosphoserine or mouse
phosphothreonine monoclonal antibody (Calbiochem, San Diego,
Calif.). The immunoprecipitates are separated on a 7.5%
SDS-polyacrylamide gel and the radiolabeled proteins are visualized
by autoradiography.
[0391] If h2520-59 polypeptide exhibits ser/thr kinase activity,
those cells overexpressing h2520-59 polypeptide will have elevated
levels of phosphorylated proteins as compared to untransfected COS
cells. Immunoprecipitation with antibodies specific for
phosphoserine or phosphothreonine will demonstrate that h2520-59
polypeptide phosphorylates serine and threonine residues.
EXAMPLE 6
Kinase Activity of h2520-59 Polypeptides
[0392] Much like the Drosophila tribbles, rat NIPK and dog CSFW,
h2520-59 has significant sequence homology to several consensus
kinase subdomains (see FIG. 4), but varies to a considerable degree
at the predicted ATP binding subdomains. The region of highest
homology includes subdomains VIA-XI of the consensus protein kinase
domain (Hanks and Hunter, FASEB 9:576-596, 1995). h2520-59 is
highly variant throughout subdomains I-V, most notably in the ATP
binding pocket, including the glycine rich loop in subdomain I, the
nearly invariant lysine in subdomain II, the glutamic acid in
subdomain III, and the valine-methionine motif of subdomain V. The
kinase catalytic core of h2520-59 in subdomain VIB is not an exact
match with the consensus, RDLKxxN (SEQ ID NO: 35), but instead
replaces the asparagine with the basic residue arginine. Also,
h2520-59 does not appear to contain either the DFG ATP orientation
and transfer motif of subdomain VII nor the serine/threonine
phosphorylation activation site of subdomain VIII. Overall,
h2520-59 contains the classic substrate binding domains of a
protein kinase, but lacks the ATP binding and kinase activation
domains.
[0393] To determine the ability for h2520-59 to act as a kinase, we
used h2520-59-myc from transiently transfected U2-0S cell lysates
in in vitro phosphorylation kinase experiments with .alpha.-casein,
.beta.-casein, myelin basic protein, or histone H1 as potential
substrates of many known serine/threonine kinases. The h2520-59 myc
tagged mammalian expression vector was generated by first, PCR
modification of full-length h2520-59 with primers
5'-GCCCTTACGACCATGGGAGATGCGAGCC-3' (SEQ ID NO: 22) and
5'-ATCTGCGGCCGCGCCATACAGAACCACTTC-3' (SEQ ID NO: 23) to insert NcoI
and NotI restriction sites into the 5' and 3' ends of the full
length h2520-59 cDNA respectively. T/A cloning (Invitrogen,
Carlsbad Calif.) was used to anneal and amplify the fragment as per
the manufacturer's directions. The modified product was digested
with NcoI and NotI and subcloned into pCMV-myc-cyto (Invitrogen,
Carlsbad Calif.).
[0394] Immunoprecipitated h2520-59-myc was combined with purified
recombinant substrate and [.gamma.-.sup.32P] ATP and a kinase
buffer (e.g., 40 mM Hepes-HCl pH 8.0, 2.0 mM DTT, 0.1 mM EGTA, 5 mM
magnesium acetate) for 30 minutes. The 32P incorporated into the
substrate was then separated from free 32P-ATP by SDS-PAGE, and the
incorporated 32P was detected by visualization after exposure to
x-ray film. Under the conditions described above, no particular
substrate phosphorylation or autophosphorylation of h2520-59 has
yet been observed. Additional experimental conditions are being
tested.
[0395] Kinase activity of h2520-59 is also measured by the
phosphorylation of a substrate using conventional techniques known
in the art, such as the quantitation of the incorporated
radioactivity (gamma phosphates) in the substrate using a gamma
radioisotope counter. Phosphorylation of specific amino acid
residues within the substrate is determined by phosphoaminoacid
analysis of the hydrolyzed protein as described by Boyle et al.,
Methods. Enzymol. 201:110-148, 1991.
[0396] Methods for finding additional family members are found in
references as follows: Hanks and Quinn, Methods. Enzymol.
200:38-62, 1991; Hardie et al, The Protein Kinase Facts Book, pp
7-47, 1995; Hanks and Hunter, FASEB 9(8): 576-96, 1995.
Alternatively, the serine/threonine kinase activity of h2520-59 can
be determined by using a Phospho-Serine/Threonine Assay Kit
(Luminex Corporation, Austin, Tex.; Upstate Biotechnology, Waltham,
Mass.). These assays utilize myelin basic protein (MBP) as a
substrate covalently linked to a fluorescent bead set. After the
kinase reaction, phosphorylated MBP is detected by adding a mixture
of two phosphoserine/phosphothreonine monoclonal antibodies
followed by a biotinylated secondary antibody and a
streptavidin-phycoerythrin conjugate. Mean fluorescence intensity
is determined according to manufacturer's instructions.
EXAMPLE 7
Activating Transcription Factor-4 (ATF-4) is a Binding Partner for
h2520-59
[0397] In order to determine how h2520-59 functions in human cells,
yeast-two-hybrid analysis was carried out using full-length
h2520-59 fused to a Gal-4 DNA binding domain as the bait and a cDNA
library from normal human liver cells fused to the Gal-4 activation
domain as the prey. Saccharomyces cerevisiae AH109 was transformed
with pGBKT7-H2520-59 and mated to MatchMaker Y187 pretransformed
liver cDNA library (Clontech, Palo Alto, Calif.).
[0398] For the yeast-2-hybrid pGBKT7-H2520-59 GAL4 DNA binding
domain fusion, h2520-59 was PCR modified from pCMV-h2520-59-myc
with the primers 5'-TGGCCACCATGGCATATGCGAGCCACCCCT-3' (SEQ ID NO:
24) and 5'-TTTTGGATCTGGTCGACCGCCATACAGAAC-3' (SEQ ID NO: 25) to
insert NdeI and SalI sites into the 5' and 3' ends of the full
length h2520-59 cDNA respectively. T/A cloning (Invitrogen,
Carlsbad Calif.) was then used to anneal and amplify the fragment
as per the manufacturer's directions. The modified product was then
digested with NdeI and SalI and subcloned into pGBKT7 (Clontech,
Palo Alto Calif.). pCMV-HA-ATF4 was generated by first full length
PCR cloning ATF4 from the human liver Marathon cDNA library
(Clontech, Palo Alto Calif.) using the primers
5'-TTCGAATTAAGCACATTCCTC-3' (SEQ ID NO: 26) and
5'-ATGACCGAAATGAGCTTCCT-3' (SEQ ID NO: 27). The PCR fragment was
T/A cloned and amplified using the primers
5'-GCCCTTTTCGTCGACAGCACATTCCTCGAT-3' (SEQ ID NO: 28) and
5'-GAATTCGGCGGCCGCCTAGGGGACCCTTTT-3' (SEQ ID NO: 29) to insert SalI
and NotI restriction sites into the 5' and 3' ends of the ATF4 cDNA
respectively. The PCR fragment was T/A cloned and amplified then
digested with SalI and NotI and subcloned into pCMV-HA (Clontech,
Palo Alto Calif.). pCMV-HA-ATF5 was generated by using the isolated
positive yeast-2-hybrid clone from the pACT2 activation domain
library that consisted of a shortened full length ATF5 that was
missing the first 171 nucleotides from the 5' end of the gene.
pACT2-ATF5 was digested with EcoRI and BglII and subcloned into
pCMV-HA (Clontech, Palo Alto Calif.).
[0399] A human liver cDNA library was chosen due to the increased
h2520-59 expression in normal human liver seen by Northern blot
analysis. X-.beta.-gal positive transformants were selected
according to the manufacturer's instructions. Positive colonies
(200) were screened and the library inserts were sequenced. Sixty
percent of the positive colonies contained the full length coding
sequence for basic region-leucine zipper transcription factor
ATF4/CREB2 (GENBANK Accession No. NM001675); 30% contained a 5'
truncated ATF5/ATFx (GENBANK Accession No. NM012068) that was
missing the first 171 nucleotides of the gene but was otherwise in
frame and intact; and 10% contained cryptic sequence.
[0400] Transient overexpression and co-immunoprecipitation of
h2520-59 and ATF4 in U2-0S cells was performed to confirm the
yeast-two-hybrid result and investigate the interaction of h2520-59
and ATF4. h2520-59 is expressed throughout the HT-29 and PC-3 tumor
samples. Cells were transfected with h2520-59 fused to the myc
epitope (h2520-59-myc), ATF4 fused to the HA epitope (ATF4-HA), or
h2520-59-myc and ATF4-HA in combination. The h2520-59 myc tagged
mammalian expression vector was generated as described in Example
6. For all transient transfections, 2.times.10.sup.5 cells were
plated per well of a six well plate and grown for 24 hours. 2.25
.mu.g of the indicated plasmid DNA was transfected for 24 hours
with FuGENE transfection reagent (Roche Diagnostics, Indianapolis
Ind.) according to the manufacturer's instructions. Cells were
grown an additional 24 hours and harvested. Transfected cells were
treated with the proteasome inhibitor, MG132, as indicated, and
co-transfection of h2520-59 with ATF4 resulted in reduced
expression of both proteins by Western blot. Treatment with the MG
132 confirmed that this reduction in h2520-59 and ATF4 was a result
of proteolysis. Results were replicated with a second proteasome
inhibitor, lactacystin.
[0401] Cells were harvested by washing one time with PBS and
incubating with TG buffer+1% IGEPAL [TG buffer: 1% triton X-100,
10% glycerol, 20 mM Hepes, pH 7.2, 100 mM NaCl, 10 mM NaF, 10 mM
Na3VO4, 1 tablet Complete Protease Inhibitor (Roche Diagnostics,
Indianapolis Ind.) per 25 ml and Pefabloc (Roche Diagnostics,
Indianapolis Ind.)] for 10 minutes at 4.degree. C. Cells were
scraped and cell debris was pelleted at 3,000.times.g for 5
minutes. The supernatant was incubated with 6 ?g of the indicated
antibody for one hour at 4.degree. C., 50 .mu.l of ImmunoPure
Protein A/G (Pierce, Rockford Ill.) was added and incubated for 30
minutes at 4.degree. C. The reaction was washed three times in TG
buffer+1 M NaCl and one time in TG buffer alone. The precipitations
were heated at 100.degree. C. for 5 minutes with 100 .mu.l sample
loading buffer (125 mM Tris HCl, pH 6.8, 5% SDS, 20% glycerol, 12%
bromophenol blue, 5% 2-mercaptoethanol), centrifuged and loaded
directly onto 12% SDS-PAGE gels to resolve total protein. Proteins
were transferred to Protran nitrocellulose (Schleicher &
Schuell) blocked for 1 hour in 5% powdered milk+1.times.PBS+0.2%
Tween. 6 .mu.g of the indicated antibody was incubated with the
blot overnight at 4.degree. C. The blots were developed using the
Vectastain kit (Vector Laboratories, Burlingame, Calif.) as per the
manufacturer's instructions and the Renaissance Western blot
chemiluminescence reagent kit (NEN, Boston, Mass.).
[0402] Although there was a decrease in protein levels of h2520-59
and ATF4 in the above experiments, co-immunoprecipitation of
h2520-59-ATF4 transfections revealed that a low level of h2520-59
and ATF4 bind to each other under transient transfection conditions
in U2-0S cells (see FIG. 11).
[0403] While the present invention has been described in terms of
the preferred embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations which come within the scope of the invention as claimed.
Sequence CWU 1
1
38 1 2059 DNA Homo sapiens CDS (49)..(1122) 1 gctctgagcc ccggcggcgc
ccgggcccac gcggaacgac ggggcgag atg cga gcc 57 Met Arg Ala 1 acc cct
ctg gct gct cct gcg ggt tcc ctg tcc agg aag aag cgg ttg 105 Thr Pro
Leu Ala Ala Pro Ala Gly Ser Leu Ser Arg Lys Lys Arg Leu 5 10 15 gag
ttg gat gac aac tta gat acc gag cgt ccc gtc cag aaa cga gct 153 Glu
Leu Asp Asp Asn Leu Asp Thr Glu Arg Pro Val Gln Lys Arg Ala 20 25
30 35 cga agt ggg ccc cag ccc aga ctg ccc ccc tgc ctg ttg ccc ctg
agc 201 Arg Ser Gly Pro Gln Pro Arg Leu Pro Pro Cys Leu Leu Pro Leu
Ser 40 45 50 cca cct act gct cca gat cgt gca act gct gtg gcc act
gcc tcc cgt 249 Pro Pro Thr Ala Pro Asp Arg Ala Thr Ala Val Ala Thr
Ala Ser Arg 55 60 65 ctt ggg ccc tat gtc ctc ctg gag ccc gag gag
ggc ggg cgg gcc tac 297 Leu Gly Pro Tyr Val Leu Leu Glu Pro Glu Glu
Gly Gly Arg Ala Tyr 70 75 80 cgg gcc ctg cac tgc cct aca ggc act
gag tat acc tgc aag gtg tac 345 Arg Ala Leu His Cys Pro Thr Gly Thr
Glu Tyr Thr Cys Lys Val Tyr 85 90 95 ccc gtc cag gaa gcc ctg gcc
gtg ctg gag ccc tac gcg cgg ctg ccc 393 Pro Val Gln Glu Ala Leu Ala
Val Leu Glu Pro Tyr Ala Arg Leu Pro 100 105 110 115 ccg cac aag cat
gtg gct cgg ccc act gag gtc ctg gct ggt acc cag 441 Pro His Lys His
Val Ala Arg Pro Thr Glu Val Leu Ala Gly Thr Gln 120 125 130 ctc ctc
tac gcc ttt ttc act cgg acc cat ggg gac atg cac agc ctg 489 Leu Leu
Tyr Ala Phe Phe Thr Arg Thr His Gly Asp Met His Ser Leu 135 140 145
gtg cga agc cgc cac cgt atc cct gag cct gag gct gcc gtg ctc ttc 537
Val Arg Ser Arg His Arg Ile Pro Glu Pro Glu Ala Ala Val Leu Phe 150
155 160 cgc cag atg gcc acc gcc ctg gcg cac tgt cac cag cac ggt ctg
gtc 585 Arg Gln Met Ala Thr Ala Leu Ala His Cys His Gln His Gly Leu
Val 165 170 175 ctg cgt gat ctc aag ctg tgt cgc ttt gtc ttc gct gac
cgt gag agg 633 Leu Arg Asp Leu Lys Leu Cys Arg Phe Val Phe Ala Asp
Arg Glu Arg 180 185 190 195 aag aag ctg gtg ctg gag aac ctg gag gac
tcc tgc gtg ctg act ggg 681 Lys Lys Leu Val Leu Glu Asn Leu Glu Asp
Ser Cys Val Leu Thr Gly 200 205 210 cca gat gat tcc ctg tgg gac aag
cac gcg tgc cca gcc tac gtg gga 729 Pro Asp Asp Ser Leu Trp Asp Lys
His Ala Cys Pro Ala Tyr Val Gly 215 220 225 cct gag ata ctc agc tca
cgg gcc tca tac tcg ggc aag gca gcc gat 777 Pro Glu Ile Leu Ser Ser
Arg Ala Ser Tyr Ser Gly Lys Ala Ala Asp 230 235 240 gtc tgg agc ctg
ggc gtg gcg ctc ttc acc atg ctg gcc ggc cac tac 825 Val Trp Ser Leu
Gly Val Ala Leu Phe Thr Met Leu Ala Gly His Tyr 245 250 255 ccc ttc
cag gac tcg gag cct gtc ctg ctc ttc ggc aag atc cgc cgc 873 Pro Phe
Gln Asp Ser Glu Pro Val Leu Leu Phe Gly Lys Ile Arg Arg 260 265 270
275 ggg gcc tac gcc ttg cct gca ggc ctc tcg gcc cct gcc cgc tgt ctg
921 Gly Ala Tyr Ala Leu Pro Ala Gly Leu Ser Ala Pro Ala Arg Cys Leu
280 285 290 gtt cgc tgc ctc ctt cgt cgg gag cca gct gaa cgg ctc aca
gcc aca 969 Val Arg Cys Leu Leu Arg Arg Glu Pro Ala Glu Arg Leu Thr
Ala Thr 295 300 305 ggc atc ctc ctg cac ccc tgg ctg cga cag gac ccg
atg ccc tta gcc 1017 Gly Ile Leu Leu His Pro Trp Leu Arg Gln Asp
Pro Met Pro Leu Ala 310 315 320 cca acc cga tcc cat ctc tgg gag gct
gcc cag gtg gtc cct gat gga 1065 Pro Thr Arg Ser His Leu Trp Glu
Ala Ala Gln Val Val Pro Asp Gly 325 330 335 ctg ggg ctg gac gaa gcc
agg gaa gag gag gga gac aga gaa gtg gtt 1113 Leu Gly Leu Asp Glu
Ala Arg Glu Glu Glu Gly Asp Arg Glu Val Val 340 345 350 355 ctg tat
ggc taggaccacc ctactacacg ctcagctgcc aacagtggat 1162 Leu Tyr Gly
tgagtttggg ggtagctcca agccttctcc tgcctctgaa ctgagccaaa ccttcagtgc
1222 cttccagaag ggagaaaggc agaagcctgt gtggagtgtg ctgtgtacac
atctgctttg 1282 ttccacacac atgcagttcc tgcttgggtg cttatcaggt
gccaagccct gttctcggtg 1342 ctgggagtac agcagtgagc aaaggagaca
atattccctg ctcacagaga tgacaaactg 1402 gcatccttga gctgacaaca
cttttccatg accataggtc actgtctaca ctgggtacac 1462 tttgtaccag
tgtcggcctc cactgatgct ggtgctcagg cacctctgtc caaggacaat 1522
ccctttcaca aacaaaccag ctgcctttgt atcttgtacc ttttcagaga aagggaggta
1582 tccctgtgcc aaaggctcca ggcctctccc ctgcaactca ggacccaagc
ccagctcact 1642 ctgggaactg tgttcccagc atctctgtcc tcttgattaa
gagattctcc ttccaggcct 1702 aagcctggga tttgggccag agataagaat
ccaaactatg aggctagttc ttgtctaact 1762 caagactgtt ctggaatgag
ggtccaggcc tgtcaaccat ggggcttctg acctgagcac 1822 caaggttgag
ggacaggatt aggcagggtc tgtcctgtgg ccacctggaa agtcccaggt 1882
gggactcttc tggggacact tggggtccac aatcccaggt ccatactcta ggttttggat
1942 accatgagta tgtatgttta cctgtgccta ataaaggaga attatgaaat
aaaaaaaaaa 2002 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaa 2059 2 358 PRT Homo sapiens 2 Met Arg Ala Thr
Pro Leu Ala Ala Pro Ala Gly Ser Leu Ser Arg Lys 1 5 10 15 Lys Arg
Leu Glu Leu Asp Asp Asn Leu Asp Thr Glu Arg Pro Val Gln 20 25 30
Lys Arg Ala Arg Ser Gly Pro Gln Pro Arg Leu Pro Pro Cys Leu Leu 35
40 45 Pro Leu Ser Pro Pro Thr Ala Pro Asp Arg Ala Thr Ala Val Ala
Thr 50 55 60 Ala Ser Arg Leu Gly Pro Tyr Val Leu Leu Glu Pro Glu
Glu Gly Gly 65 70 75 80 Arg Ala Tyr Arg Ala Leu His Cys Pro Thr Gly
Thr Glu Tyr Thr Cys 85 90 95 Lys Val Tyr Pro Val Gln Glu Ala Leu
Ala Val Leu Glu Pro Tyr Ala 100 105 110 Arg Leu Pro Pro His Lys His
Val Ala Arg Pro Thr Glu Val Leu Ala 115 120 125 Gly Thr Gln Leu Leu
Tyr Ala Phe Phe Thr Arg Thr His Gly Asp Met 130 135 140 His Ser Leu
Val Arg Ser Arg His Arg Ile Pro Glu Pro Glu Ala Ala 145 150 155 160
Val Leu Phe Arg Gln Met Ala Thr Ala Leu Ala His Cys His Gln His 165
170 175 Gly Leu Val Leu Arg Asp Leu Lys Leu Cys Arg Phe Val Phe Ala
Asp 180 185 190 Arg Glu Arg Lys Lys Leu Val Leu Glu Asn Leu Glu Asp
Ser Cys Val 195 200 205 Leu Thr Gly Pro Asp Asp Ser Leu Trp Asp Lys
His Ala Cys Pro Ala 210 215 220 Tyr Val Gly Pro Glu Ile Leu Ser Ser
Arg Ala Ser Tyr Ser Gly Lys 225 230 235 240 Ala Ala Asp Val Trp Ser
Leu Gly Val Ala Leu Phe Thr Met Leu Ala 245 250 255 Gly His Tyr Pro
Phe Gln Asp Ser Glu Pro Val Leu Leu Phe Gly Lys 260 265 270 Ile Arg
Arg Gly Ala Tyr Ala Leu Pro Ala Gly Leu Ser Ala Pro Ala 275 280 285
Arg Cys Leu Val Arg Cys Leu Leu Arg Arg Glu Pro Ala Glu Arg Leu 290
295 300 Thr Ala Thr Gly Ile Leu Leu His Pro Trp Leu Arg Gln Asp Pro
Met 305 310 315 320 Pro Leu Ala Pro Thr Arg Ser His Leu Trp Glu Ala
Ala Gln Val Val 325 330 335 Pro Asp Gly Leu Gly Leu Asp Glu Ala Arg
Glu Glu Glu Gly Asp Arg 340 345 350 Glu Val Val Leu Tyr Gly 355 3
21 DNA Artificial Sequence PCR Primer 3 tggtgctgga gaacctggag g 21
4 21 DNA Artificial Sequence PCR Primer 4 cgagtcctgg aaggggtagt g
21 5 11 PRT Artificial Sequence HIV TAT peptide 5 Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg 1 5 10 6 20 DNA Artificial Sequence PCR
Primer 6 cggggcgaga tgcgagccac 20 7 20 DNA Artificial Sequence PCR
Primer 7 agggtggtcc tagccataca 20 8 358 PRT Homo sapiens 8 Met Arg
Ala Thr Pro Leu Ala Ala Pro Ala Gly Ser Leu Ser Arg Lys 1 5 10 15
Lys Arg Leu Glu Leu Asp Asp Asn Leu Asp Thr Glu Arg Pro Val Gln 20
25 30 Lys Arg Ala Arg Ser Gly Pro Gln Pro Arg Leu Pro Pro Cys Leu
Leu 35 40 45 Pro Leu Ser Pro Pro Thr Ala Pro Asp Arg Ala Thr Ala
Val Ala Thr 50 55 60 Ala Ser Arg Leu Gly Pro Tyr Val Leu Leu Glu
Pro Glu Glu Gly Gly 65 70 75 80 Arg Ala Tyr Gln Ala Leu His Cys Pro
Thr Gly Thr Glu Tyr Thr Cys 85 90 95 Lys Val Tyr Pro Val Gln Glu
Ala Pro Ala Val Leu Glu Pro Tyr Ala 100 105 110 Arg Leu Pro Pro His
Lys His Val Ala Arg Pro Thr Glu Val Leu Ala 115 120 125 Gly Thr Gln
Leu Leu Tyr Ala Phe Phe Thr Arg Thr His Gly Asp Met 130 135 140 His
Ser Leu Val Arg Ser Arg His Arg Ile Pro Glu Pro Glu Ala Ala 145 150
155 160 Val Leu Phe Arg Gln Met Ala Thr Ala Leu Ala His Cys His Gln
His 165 170 175 Gly Leu Val Leu Arg Asp Leu Lys Leu Cys Arg Phe Val
Phe Ala Asp 180 185 190 Arg Glu Arg Lys Lys Leu Val Leu Glu Asn Leu
Glu Asp Ser Cys Val 195 200 205 Leu Thr Gly Pro Asp Asp Ser Leu Trp
Asp Lys His Ala Cys Pro Ala 210 215 220 Tyr Val Gly Pro Glu Ile Leu
Ser Ser Arg Ala Ser Tyr Ser Gly Lys 225 230 235 240 Ala Ala Asp Val
Trp Ser Leu Gly Val Ala Leu Phe Thr Met Leu Ala 245 250 255 Gly His
Tyr Pro Phe Gln Asp Ser Glu Pro Val Leu Leu Phe Gly Lys 260 265 270
Ile Arg Arg Gly Ala Tyr Ala Leu Pro Ala Gly Leu Ser Ala Pro Ala 275
280 285 Arg Cys Leu Val Arg Cys Leu Leu Arg Arg Glu Pro Ala Glu Arg
Leu 290 295 300 Thr Ala Thr Gly Ile Leu Leu His Pro Trp Leu Arg Gln
Asp Pro Met 305 310 315 320 Pro Leu Ala Pro Thr Arg Ser His Leu Trp
Glu Ala Ala Gln Val Val 325 330 335 Pro Asp Gly Leu Gly Leu Asp Glu
Ala Arg Glu Glu Glu Gly Asp Arg 340 345 350 Glu Val Val Leu Tyr Gly
355 9 153 PRT Homo sapiens misc_feature (136) Xaa = unknown or
other 9 Leu Arg Phe Ala Ser Pro Gly Pro Gly Ala Gly Arg Ala Arg Asp
Ser 1 5 10 15 Gln Arg Lys Trp Arg Arg Leu Arg Ala Arg Pro Leu Leu
Gly Pro Gly 20 25 30 Gln Gly Trp Ser Trp Ala Gly Ile Pro Ser Ser
Ala Ala Ala Gln Arg 35 40 45 Ala Gly Pro Pro Ala Gly Ala Leu Glu
Ala Leu Ser Pro Gly Gly Ala 50 55 60 Arg Ala His Ala Glu Arg Arg
Gly Glu Met Arg Ala Thr Pro Leu Ala 65 70 75 80 Ala Pro Ala Gly Ser
Leu Ser Arg Lys Lys Arg Leu Glu Leu Asp Asp 85 90 95 Asn Leu Asp
Thr Glu Arg Pro Val Gln Lys Arg Ala Arg Ser Gly Pro 100 105 110 Gln
Pro Arg Leu Pro Pro Cys Leu Leu Pro Leu Ser Pro Pro Thr Ala 115 120
125 Pro Asp Arg Ala Thr Ala Val Xaa Thr Xaa Ser Arg Xaa Xaa Xaa Tyr
130 135 140 Val Leu Leu Glu Ala Arg Arg Xaa Ala 145 150 10 233 PRT
Homo sapiens 10 Gly Pro Gly Trp Tyr Pro Ala Pro Leu Arg Leu Phe His
Ser Asp Pro 1 5 10 15 Trp Gly His Ala Gln Pro Gly Ala Lys Arg His
Arg Ile Pro Glu Pro 20 25 30 Glu Ala Ala Val Leu Phe Arg Gln Met
Ala Thr Ala Leu Ala His Cys 35 40 45 His Gln His Gly Leu Val Leu
Arg Asp Leu Lys Leu Cys Arg Phe Val 50 55 60 Phe Ala Asp Arg Glu
Arg Lys Lys Leu Val Leu Glu Asn Leu Glu Asp 65 70 75 80 Ser Cys Val
Leu Thr Gly Pro Asp Asp Ser Leu Trp Asp Lys His Ala 85 90 95 Cys
Pro Ala Tyr Val Gly Pro Glu Ile Leu Ser Ser Arg Ala Ser Tyr 100 105
110 Ser Gly Lys Ala Ala Asp Val Trp Ser Leu Gly Val Ala Leu Phe Thr
115 120 125 Met Leu Ala Gly His Tyr Pro Phe Gln Asp Ser Glu Pro Val
Leu Leu 130 135 140 Phe Gly Lys Ile Arg Arg Gly Ala Tyr Ala Leu Pro
Ala Gly Leu Ser 145 150 155 160 Ala Pro Ala Arg Cys Leu Val Arg Cys
Leu Leu Arg Arg Glu Pro Ala 165 170 175 Glu Arg Leu Thr Ala Thr Gly
Ile Leu Leu His Pro Trp Leu Arg Gln 180 185 190 Asp Pro Met Pro Leu
Ala Pro Thr Arg Ser His Leu Trp Glu Ala Ala 195 200 205 Gln Val Val
Pro Asp Gly Leu Gly Leu Asp Glu Ala Arg Glu Glu Glu 210 215 220 Gly
Asp Arg Glu Val Val Leu Tyr Gly 225 230 11 360 PRT Homo sapiens 11
Gly Gln Gly Trp Ser Trp Ala Gly Ile Pro Ser Ser Ala Ala Ala Gln 1 5
10 15 Arg Ala Gly Pro Pro Ala Gly Ala Leu Glu Ala Leu Ser Pro Gly
Gly 20 25 30 Ala Arg Ala His Ala Glu Arg Arg Gly Glu Met Arg Ala
Thr Pro Leu 35 40 45 Ala Ala Pro Ala Gly Ser Leu Ser Arg Lys Lys
Arg Leu Glu Leu Asp 50 55 60 Asp Asn Leu Asp Thr Glu Arg Pro Val
Gln Lys Arg Ala Arg Ser Gly 65 70 75 80 Pro Gln Pro Arg Leu Pro Pro
Cys Leu Leu Pro Leu Ser Pro Pro Thr 85 90 95 Ala Pro Asp Arg Ala
Thr Ala Val Ala Thr Ala Ser Arg Leu Gly Pro 100 105 110 Tyr Val Leu
Leu Glu Pro Glu Glu Gly Gly Arg Ala Tyr Gln Ala Leu 115 120 125 His
Cys Pro Thr Gly Thr Glu Tyr Thr Cys Lys Val Tyr Pro Val Gln 130 135
140 Glu Ala Leu Ala Val Leu Glu Pro Tyr Ala Arg Leu Pro Pro His Lys
145 150 155 160 His Val Ala Arg Pro Thr Glu Val Leu Ala Gly Thr Gln
Leu Leu Tyr 165 170 175 Ala Phe Phe Thr Arg Thr His Gly Asp Met His
Ser Leu Val Arg Ser 180 185 190 Arg His Arg Ile Pro Glu Pro Glu Ala
Ala Val Leu Phe Arg Gln Met 195 200 205 Ala Thr Ala Leu Ala His Cys
His Gln His Gly Leu Val Leu Arg Asp 210 215 220 Leu Lys Leu Cys Arg
Phe Val Phe Ala Asp Arg Glu Arg Lys Lys Leu 225 230 235 240 Val Leu
Glu Asn Leu Glu Asp Ser Cys Val Leu Thr Gly Pro Asp Asp 245 250 255
Ser Leu Trp Asp Lys His Ala Cys Pro Ala Tyr Val Gly Pro Glu Ile 260
265 270 Leu Ser Ser Arg Ala Ser Tyr Ser Gly Lys Ala Ala Asp Val Trp
Ser 275 280 285 Leu Gly Val Ala Leu Phe Thr Met Leu Ala Gly His Tyr
Pro Phe Gln 290 295 300 Asp Ser Glu Pro Val Leu Leu Phe Gly Lys Ile
Arg Arg Gly Ala Tyr 305 310 315 320 Ala Leu Pro Ala Gly Leu Ser Ala
Pro Ala Arg Cys Leu Val Arg Cys 325 330 335 Leu Leu Arg Arg Glu Pro
Ala Glu Arg Leu Thr Ala Thr Gly Ile Leu 340 345 350 Leu His Pro Trp
Leu Arg Gln Asp 355 360 12 510 DNA Homo sapiens 12 ccttctgttt
ctccccatgt cccaggaaga agctggtgct ggagaacctg gaggactcct 60
gcgtgctgac tgggccagat gattccctgt gggacaagca cgcgtgccca gcctacgtgg
120 gacctgagat actcagctca cgggcctcat actcgggcaa ggcagccgat
gtctggagcc 180 tgggcgtggc gctcttcacc atgctggccg gccactaccc
cttccaggac tcggagcctg 240 tcctgctctt cggcaagatc cgccgcgggg
cctacgcctt gcctgcaggc ctctcggccc 300 ctgcccgctg tctggttcgc
tgcctccttc gtcgggagcc agctgaacgg ctcacagcca 360 caggcatcct
cctgcacccc tggctgcgac aggacccgat gcccttagcc ccaacccgat 420
cccatctctg ggaggctgcc caggtggtcc ctgatggact ggggctggac gaagccaggg
480 aagaggaggg agacagagaa gtggttctgt 510 13 25 PRT Homo sapiens 13
Glu Leu Asp Asp Asn Leu Asp Thr Glu Arg Pro Val Gln Lys Arg Ala 1 5
10 15 Arg Ser Gly Pro Gln Pro Arg Leu Cys 20 25 14 25 PRT Homo
sapiens 14 Gly Pro Tyr Val Leu Leu Glu Pro Glu Glu Gly Gly Arg Ala
Tyr Gln 1
5 10 15 Ala Leu His Cys Pro Thr Gly Thr Glu 20 25 15 25 PRT Homo
sapiens 15 Arg Ser His Leu Trp Glu Ala Ala Gln Val Val Pro Asp Gly
Leu Gly 1 5 10 15 Leu Asp Glu Ala Arg Glu Glu Glu Cys 20 25 16 19
DNA Artificial Sequence PCR Primer 16 cggctaccac atccaagga 19 17 18
DNA Artificial Sequence PCR Primer 17 gctggaatta ccgcggct 18 18 24
DNA Artificial Sequence PCR Primer 18 ntgctggcac cagacttgcc ctcn 24
19 25 DNA Artificial Sequence PCR Primer 19 aatccttgaa ggaaatgaca
ttgag 25 20 25 DNA Artificial Sequence PCR Primer 20 tccttgtttt
taactgttgt ggctt 25 21 31 DNA Artificial Sequence PCR Primer 21
nttgtttcaa attcagcggc tttgattcag n 31 22 28 DNA Artificial Sequence
PCR Primer 22 gcccttacga ccatgggaga tgcgagcc 28 23 30 DNA
Artificial Sequence PCR Primer 23 atctgcggcc gcgccataca gaaccacttc
30 24 30 DNA Artificial Sequence PCR Primer 24 tggccaccat
ggcatatgcg agccacccct 30 25 30 DNA Artificial Sequence PCR Primer
25 ttttggatct ggtcgaccgc catacagaac 30 26 21 DNA Artificial
Sequence PCR Primer 26 ttcgaattaa gcacattcct c 21 27 20 DNA
Artificial Sequence PCR Primer 27 atgaccgaaa tgagcttcct 20 28 30
DNA Artificial Sequence PCR Primer 28 gcccttttcg tcgacagcac
attcctcgat 30 29 30 DNA Artificial Sequence PCR Primer 29
gaattcggcg gccgcctagg ggaccctttt 30 30 360 PRT Homo sapiens 30 Met
Arg Ala Thr Pro Leu Ala Ala Pro Ala Gly Ser Leu Ser Arg Lys 1 5 10
15 Lys Arg Leu Glu Leu Asp Asp Asn Leu Asp Thr Glu Arg Pro Val Gln
20 25 30 Lys Arg Ala Arg Ser Gly Pro Gln Pro Arg Leu Pro Pro Cys
Leu Leu 35 40 45 Pro Leu Ser Pro Pro Thr Ala Pro Asp Arg Ala Thr
Ala Val Ala Thr 50 55 60 Ala Ser Arg Leu Gly Pro Tyr Val Leu Leu
Glu Pro Glu Glu Gly Gly 65 70 75 80 Arg Ala Tyr Gln Ala Leu His Cys
Pro Thr Gly Thr Glu Tyr Thr Cys 85 90 95 Lys Val Tyr Pro Val Gln
Glu Ala Leu Ala Val Leu Glu Pro Tyr Ala 100 105 110 Arg Val Pro Pro
His Lys His Val Ala Arg Pro Thr Glu Val Leu Ala 115 120 125 Gly Thr
Gln Leu Leu Tyr Ala Phe Phe Thr Arg Thr His Gly Asp Met 130 135 140
His Ser Leu Val Arg Ser Arg His Arg Ile Pro Glu Pro Glu Ala Ala 145
150 155 160 Val Leu Phe Arg Gln Met Ala Thr Ala Leu Ala His Cys His
Gln His 165 170 175 Gly Leu Val Leu Arg Asp Leu Lys Leu Cys Arg Phe
Val Phe Ala Asp 180 185 190 Arg Asp Arg Glu Lys Lys Lys Leu Val Leu
Glu Asn Leu Glu Asp Ser 195 200 205 Cys Val Leu Thr Gly Pro Asp Asp
Ser Leu Trp Asp Lys His Ala Cys 210 215 220 Pro Ala Tyr Val Gly Pro
Glu Ile Leu Ser Ser Arg Ala Ser Tyr Ser 225 230 235 240 Gly Lys Ala
Ala Asp Val Trp Ser Leu Gly Val Ala Leu Phe Thr Met 245 250 255 Leu
Ala Gly His Tyr Pro Phe Gln Asp Ser Glu Pro Val Leu Leu Phe 260 265
270 Gly Lys Ile Arg Arg Gly Ala Tyr Ala Leu Pro Ala Gly Leu Ser Ala
275 280 285 Pro Ala Arg Cys Leu Val Arg Cys Leu Leu Arg Arg Glu Pro
Ala Glu 290 295 300 Arg Leu Thr Ala Thr Gly Ile Leu Leu His Pro Trp
Leu Arg Gln Asp 305 310 315 320 Pro Met Pro Leu Ala Pro Thr Arg Ser
His Leu Trp Glu Ala Ala Gln 325 330 335 Val Val Pro Asp Gly Leu Gly
Leu Asp Glu Ala Arg Glu Glu Glu Gly 340 345 350 Asp Arg Glu Val Val
Leu Tyr Gly 355 360 31 349 PRT Rattus sp. 31 Met Arg Ala Thr Ser
Leu Ala Ala Ser Ala Asp Val Pro Cys Arg Lys 1 5 10 15 Lys Pro Leu
Glu Phe Asp Asp Asn Ile Asp Val Glu Cys Pro Val Leu 20 25 30 Lys
Arg Val Arg Asp Glu Pro Glu Pro Gly Pro Thr Pro Ser Leu Pro 35 40
45 Pro Ala Ser Asp Leu Ser Pro Ala Val Ala Pro Ala Thr Arg Leu Gly
50 55 60 Pro Tyr Ile Leu Leu Glu Arg Glu Gln Gly Asn Cys Thr Tyr
Arg Ala 65 70 75 80 Leu His Cys Pro Thr Gly Thr Glu Tyr Thr Cys Lys
Val Tyr Pro Ala 85 90 95 Ser Glu Ala Gln Ala Val Leu Ala Pro Tyr
Ala Arg Leu Pro Thr His 100 105 110 Gln His Val Ala Arg Pro Thr Glu
Val Leu Leu Gly Ser Gln Leu Leu 115 120 125 Tyr Thr Phe Phe Thr Lys
Thr His Gly Asp Leu His Ser Leu Val Arg 130 135 140 Ser Arg Arg Gly
Ile Pro Glu Pro Glu Ala Ala Ala Leu Phe Arg Gln 145 150 155 160 Met
Ala Ser Ala Val Ala His Cys His Lys His Gly Leu Ile Leu Arg 165 170
175 Asp Leu Lys Leu Arg Arg Phe Val Phe Ser Asn Cys Glu Arg Thr Lys
180 185 190 Leu Val Leu Glu Asn Leu Glu Asp Ala Cys Val Met Thr Gly
Pro Asp 195 200 205 Asp Ser Leu Trp Asp Lys His Ala Cys Pro Ala Tyr
Val Gly Pro Glu 210 215 220 Ile Leu Ser Ser Arg Pro Ser Tyr Ser Gly
Arg Ala Ala Asp Val Trp 225 230 235 240 Ser Leu Gly Val Ala Leu Phe
Thr Met Leu Ala Gly Arg Tyr Pro Phe 245 250 255 Gln Asp Ser Glu Pro
Ala Leu Leu Phe Gly Lys Ile Arg Arg Gly Thr 260 265 270 Phe Ala Leu
Pro Glu Gly Leu Ser Ala Ser Ala Arg Cys Leu Ile Arg 275 280 285 Cys
Leu Leu Arg Arg Glu Pro Ser Glu Arg Leu Val Ala Leu Gly Ile 290 295
300 Leu Leu His Pro Trp Leu Arg Glu Asp Cys Ser Gln Val Ser Pro Pro
305 310 315 320 Arg Ser Asp Arg Arg Glu Met Asp Gln Val Val Pro Asp
Gly Pro Gln 325 330 335 Leu Glu Glu Ala Glu Glu Gly Glu Val Gly Leu
Tyr Gly 340 345 32 343 PRT C. familiaris 32 Met Asn Ile His Arg Ser
Thr Pro Ile Thr Ile Ala Arg Tyr Gly Arg 1 5 10 15 Ser Arg Asn Lys
Thr Gln Asp Phe Glu Glu Leu Ser Ser Ile Arg Ser 20 25 30 Ala Glu
Pro Ser Gln Ser Phe Ser Pro Asn Leu Gly Ser Pro Ser Pro 35 40 45
Pro Glu Thr Pro Asn Leu Ser His Cys Val Ser Cys Ile Gly Lys Tyr 50
55 60 Leu Leu Leu Glu Pro Leu Glu Gly Asp His Val Phe Arg Ala Val
His 65 70 75 80 Leu His Ser Gly Glu Glu Leu Val Cys Lys Val Phe Asp
Ile Ser Cys 85 90 95 Tyr Gln Glu Ser Leu Ala Pro Cys Phe Cys Leu
Ser Ala His Ser Asn 100 105 110 Ile Asn Gln Ile Thr Glu Ile Ile Leu
Gly Glu Thr Lys Ala Tyr Val 115 120 125 Phe Phe Glu Arg Ser Tyr Gly
Asp Met His Ser Phe Val Arg Thr Cys 130 135 140 Lys Lys Leu Arg Glu
Glu Glu Ala Ala Arg Leu Phe Tyr Gln Ile Ala 145 150 155 160 Ser Ala
Val Ala His Cys His Asp Gly Gly Leu Val Leu Arg Asp Leu 165 170 175
Lys Leu Arg Lys Phe Ile Phe Lys Asp Glu Glu Arg Thr Arg Val Lys 180
185 190 Leu Glu Ser Leu Glu Asp Ala Tyr Ile Leu Arg Gly Asp Asp Asp
Ser 195 200 205 Leu Ser Asp Lys His Gly Cys Pro Ala Tyr Val Ser Pro
Glu Ile Leu 210 215 220 Asn Thr Ser Gly Ser Tyr Ser Gly Lys Ala Ala
Asp Val Trp Ser Leu 225 230 235 240 Gly Val Met Leu Tyr Thr Met Leu
Val Gly Arg Tyr Pro Phe His Asp 245 250 255 Ile Glu Pro Ser Ser Leu
Phe Ser Lys Ile Arg Arg Gly Gln Phe Asn 260 265 270 Ile Pro Glu Thr
Leu Ser Pro Lys Ala Lys Cys Leu Ile Arg Ser Ile 275 280 285 Leu Arg
Arg Glu Pro Ser Glu Arg Leu Thr Ser Gln Glu Ile Leu Asp 290 295 300
His Pro Trp Phe Ser Thr Asp Phe Ser Val Ser Asn Ser Gly Tyr Gly 305
310 315 320 Ala Lys Glu Val Ser Asp Gln Leu Val Pro Asp Val Asn Met
Glu Glu 325 330 335 Asn Leu Asp Pro Phe Phe Asn 340 33 372 PRT Homo
sapiens 33 Met Arg Val Gly Pro Val Arg Ser Ala Met Ser Gly Ala Ser
Gln Pro 1 5 10 15 Arg Gly Pro Ala Leu Leu Phe Pro Ala Thr Arg Gly
Val Pro Ala Lys 20 25 30 Arg Leu Leu Asp Ala Asp Asp Ala Ala Ala
Val Ala Ala Lys Cys Pro 35 40 45 Arg Leu Ser Glu Cys Ser Ser Pro
Pro Asp Tyr Leu Ser Pro Pro Gly 50 55 60 Ser Pro Cys Ser Pro Gln
Pro Pro Pro Ala Ala Pro Gly Ala Gly Gly 65 70 75 80 Gly Ser Gly Ser
Ala Pro Gly Pro Ser Arg Ile Ala Asp Tyr Leu Leu 85 90 95 Leu Pro
Leu Ala Glu Arg Glu His Val Ser Arg Ala Leu Cys Ile His 100 105 110
Thr Gly Arg Glu Leu Arg Cys Lys Val Phe Pro Ile Lys His Tyr Gln 115
120 125 Asp Lys Ile Arg Pro Tyr Ile Gln Leu Pro Ser His Ser Asn Ile
Thr 130 135 140 Gly Ile Val Glu Val Ile Leu Gly Glu Thr Lys Ala Tyr
Val Phe Phe 145 150 155 160 Glu Lys Ser Phe Gly Asp Met His Ser Tyr
Val Arg Ser Arg Lys Arg 165 170 175 Leu Arg Glu Glu Glu Ala Ala Arg
Leu Phe Lys Gln Ile Val Ser Ala 180 185 190 Val Ala His Cys His Gln
Ser Ala Ile Val Leu Gly Asp Leu Lys Leu 195 200 205 Arg Lys Phe Val
Phe Ser Thr Glu Glu Arg Thr Gln Leu Arg Leu Glu 210 215 220 Ser Leu
Glu Asp Thr His Ile Met Lys Gly Glu Asp Asp Ala Leu Ser 225 230 235
240 Asp Lys His Gly Cys Pro Ala Tyr Val Ser Pro Glu Ile Leu Asn Thr
245 250 255 Thr Gly Thr Tyr Ser Gly Lys Ala Ala Asp Val Trp Ser Leu
Gly Val 260 265 270 Met Leu Tyr Thr Leu Leu Val Gly Arg Tyr Pro Phe
His Asp Ser Asp 275 280 285 Pro Ser Ala Leu Phe Ser Lys Ile Arg Arg
Gly Gln Phe Cys Ile Pro 290 295 300 Glu His Ile Ser Pro Lys Ala Arg
Cys Leu Ile Arg Ser Leu Leu Arg 305 310 315 320 Arg Glu Pro Ser Glu
Arg Leu Thr Ala Pro Glu Ile Leu Leu His Pro 325 330 335 Trp Phe Glu
Ser Val Leu Glu Pro Gly Tyr Ile Asp Ser Glu Ile Gly 340 345 350 Thr
Ser Asp Gln Ile Val Pro Glu Tyr Gln Glu Asp Ser Asp Ile Ser 355 360
365 Ser Phe Phe Cys 370 34 484 PRT Drosophila melanogaster 34 Met
Asp Asn Ser Ser Gly Gln Asn Ser Arg Thr Ala Ser Ser Ala Ser 1 5 10
15 Thr Ser Lys Ile Val Asn Tyr Ser Ser Pro Val Ser Pro Gly Val Ala
20 25 30 Ala Ala Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Gly Met
Ser Ser 35 40 45 Ser Gln Glu Asp Thr Val Leu Gly Leu Phe Thr Pro
Lys Lys Glu Phe 50 55 60 Pro Asn Ala Lys Met Leu Gln Thr Ile Arg
Glu Lys Leu Met Thr Pro 65 70 75 80 Gly Gly Ala Cys Asp Leu Leu Ala
Leu Gly Ile Ala Ala Glu Pro Thr 85 90 95 Asp Gln Gln Pro Val Lys
Leu Ile Gln Gln Arg Tyr Leu Ile Ser Ala 100 105 110 Gln Pro Ser His
Ile Ser Ala Ala Val Ala Ala Lys Thr Pro Ala Ser 115 120 125 Tyr Arg
His Leu Val Asp Leu Thr Ala Ser Asn Leu Arg Cys Val Asp 130 135 140
Ile Phe Thr Gly Glu Gln Phe Leu Cys Arg Ile Val Asn Glu Pro Leu 145
150 155 160 His Lys Val Gln Arg Ala Tyr Phe Gln Leu Gln Gln His Asp
Glu Glu 165 170 175 Leu Arg Arg Ser Thr Ile Tyr Gly His Pro Leu Ile
Arg Pro Val His 180 185 190 Asp Ile Ile Pro Leu Thr Lys Asp Arg Thr
Tyr Ile Leu Ile Ala Pro 195 200 205 Val Pro Gln Glu Arg Asp Ser Thr
Gly Gly Val Thr Gly Val Tyr Glu 210 215 220 Asn Leu His Thr Tyr Ile
Arg His Ala Lys Arg Leu Cys Glu Thr Glu 225 230 235 240 Ala Arg Ala
Ile Phe His Gln Ile Cys Gln Thr Val Gln Val Cys His 245 250 255 Arg
Asn Gly Ile Ile Leu Arg Asp Leu Lys Leu Lys Arg Phe Tyr Phe 260 265
270 Ile Asp Glu Ala Arg Thr Lys Leu Gln Tyr Glu Ser Leu Glu Gly Ser
275 280 285 Met Ile Leu Asp Gly Glu Asp Asp Thr Leu Ser Asp Lys Ile
Gly Cys 290 295 300 Pro Leu Tyr Thr Ala Pro Glu Leu Leu Cys Pro Gln
Gln Thr Tyr Lys 305 310 315 320 Gly Lys Pro Ala Asp Met Trp Ser Leu
Gly Val Ile Leu Tyr Thr Met 325 330 335 Leu Val Gly Gln Tyr Pro Phe
Tyr Glu Lys Ala Asn Cys Asn Leu Ile 340 345 350 Thr Val Ile Arg His
Gly Asn Val Gln Ile Pro Leu Thr Leu Ser Lys 355 360 365 Ser Val Arg
Trp Leu Leu Leu Ser Leu Leu Arg Lys Asp Tyr Thr Glu 370 375 380 Arg
Met Thr Ala Ser His Ile Phe Leu Thr Pro Trp Leu Arg Glu Gln 385 390
395 400 Arg Pro Phe His Met Tyr Leu Pro Val Asp Val Glu Val Ala Glu
Asp 405 410 415 Trp Ser Asp Ala Glu Glu Asp Glu Gly Thr Ala Ala Asp
Ala Met Asp 420 425 430 Asp Asp Glu Glu Gly Leu Cys Pro Leu Gly Asp
Lys His Glu Tyr Glu 435 440 445 Asp Ile Gly Val Glu Pro Leu Asp Tyr
Thr Arg Ser Thr Leu Gln Met 450 455 460 Ala Gln Asn Ala Asn Gly Leu
Ser Thr Glu Pro Glu Pro Asp Thr Asp 465 470 475 480 Val Asp Met Gly
35 7 PRT Unknown Consensus kinase domain 35 Arg Asp Leu Lys Xaa Xaa
Asn 1 5 36 672 PRT Homo sapiens 36 Met Ala Asp Val Phe Pro Gly Asn
Asp Ser Thr Ala Ser Gln Asp Val 1 5 10 15 Ala Asn Arg Phe Ala Arg
Lys Gly Ala Leu Arg Gln Lys Asn Val His 20 25 30 Glu Val Lys Asp
His Lys Phe Ile Ala Arg Phe Phe Lys Gln Pro Thr 35 40 45 Phe Cys
Ser His Cys Thr Asp Phe Ile Trp Gly Phe Gly Lys Gln Gly 50 55 60
Phe Gln Cys Gln Val Cys Cys Phe Val Val His Lys Arg Cys His Glu 65
70 75 80 Phe Val Thr Phe Ser Cys Pro Gly Ala Asp Lys Gly Pro Asp
Thr Asp 85 90 95 Asp Pro Arg Ser Lys His Lys Phe Lys Ile His Thr
Tyr Gly Ser Pro 100 105 110 Thr Phe Cys Asp His Cys Gly Ser Leu Leu
Tyr Gly Leu Ile His Gln 115 120 125 Gly Met Lys Cys Asp Thr Cys Asp
Met Asn Val His Lys Gln Cys Val 130 135 140 Ile Asn Val Pro Ser Leu
Cys Gly Met Asp His Thr Glu Lys Arg Gly 145 150 155 160 Arg Ile Tyr
Leu Lys Ala Glu Val Ala Asp Glu Lys Leu His Val Thr 165 170 175 Val
Arg Asp Ala Lys Asn Leu Ile Pro Met Asp Pro Asn Gly Leu Ser 180 185
190 Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro Asp Pro Lys Asn Glu Ser
195 200 205 Lys Gln Lys Thr Lys Thr Ile Arg Ser Thr Leu Asn Pro Gln
Trp Asn 210 215 220 Glu Ser Phe Thr Phe Lys Leu Lys Pro Ser Asp Lys
Asp Arg Arg Leu 225 230 235
240 Ser Val Glu Ile Trp Asp Trp Asp Arg Thr Thr Arg Asn Asp Phe Met
245 250 255 Gly Ser Leu Ser Phe Gly Val Ser Glu Leu Met Lys Met Pro
Ala Ser 260 265 270 Gly Trp Tyr Lys Leu Leu Asn Gln Glu Glu Gly Glu
Tyr Tyr Asn Val 275 280 285 Pro Ile Pro Glu Gly Asp Glu Glu Gly Asn
Met Glu Leu Arg Gln Lys 290 295 300 Phe Glu Lys Ala Lys Leu Gly Pro
Ala Gly Asn Lys Val Ile Ser Pro 305 310 315 320 Ser Glu Asp Arg Lys
Gln Pro Ser Asn Asn Leu Asp Arg Val Lys Leu 325 330 335 Thr Asp Phe
Asn Phe Leu Met Val Leu Gly Lys Gly Ser Phe Gly Lys 340 345 350 Val
Met Leu Ala Asp Arg Lys Gly Thr Glu Glu Leu Tyr Ala Ile Lys 355 360
365 Ile Leu Lys Lys Asp Val Val Ile Gln Asp Asp Asp Val Glu Cys Thr
370 375 380 Met Val Glu Lys Arg Val Leu Ala Leu Leu Asp Lys Pro Pro
Phe Leu 385 390 395 400 Thr Gln Leu His Ser Cys Phe Gln Thr Val Asp
Arg Leu Tyr Phe Val 405 410 415 Met Glu Tyr Val Asn Gly Gly Asp Leu
Met Tyr His Ile Gln Gln Val 420 425 430 Gly Lys Phe Lys Glu Pro Gln
Ala Val Phe Tyr Ala Ala Glu Ile Ser 435 440 445 Ile Gly Leu Phe Phe
Leu His Lys Arg Gly Ile Ile Tyr Arg Asp Leu 450 455 460 Lys Leu Asp
Asn Val Met Leu Asp Ser Glu Gly His Ile Lys Ile Ala 465 470 475 480
Asp Phe Gly Met Cys Lys Glu His Met Met Asp Gly Val Thr Thr Arg 485
490 495 Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Ile Ala
Tyr 500 505 510 Gln Pro Tyr Gly Lys Ser Val Asp Trp Trp Ala Tyr Gly
Val Leu Leu 515 520 525 Tyr Glu Met Leu Ala Gly Gln Pro Pro Phe Asp
Gly Glu Asp Glu Asp 530 535 540 Glu Leu Phe Gln Ser Ile Met Glu His
Asn Val Ser Tyr Pro Lys Ser 545 550 555 560 Leu Ser Lys Glu Ala Val
Ser Ile Cys Lys Gly Leu Met Thr Lys His 565 570 575 Pro Ala Lys Arg
Leu Gly Cys Gly Pro Glu Gly Glu Arg Asp Val Arg 580 585 590 Glu His
Ala Phe Phe Arg Arg Ile Asp Trp Glu Lys Leu Glu Asn Arg 595 600 605
Glu Ile Gln Pro Pro Phe Lys Pro Lys Val Cys Gly Lys Gly Ala Glu 610
615 620 Asn Phe Asp Lys Phe Phe Thr Arg Gly Gln Pro Val Leu Thr Pro
Pro 625 630 635 640 Asp Gln Leu Val Ile Ala Asn Ile Asp Gln Ser Asp
Phe Glu Gly Phe 645 650 655 Ser Tyr Val Asn Pro Gln Phe Val His Pro
Ile Leu Gln Ser Ala Val 660 665 670 37 841 PRT Homo sapiens 37 Met
Pro Leu Ala Ala Tyr Cys Tyr Leu Arg Val Val Gly Lys Gly Ser 1 5 10
15 Tyr Gly Glu Val Thr Leu Val Lys His Arg Arg Asp Gly Lys Gln Tyr
20 25 30 Val Ile Lys Lys Leu Asn Leu Arg Asn Ala Ser Ser Arg Glu
Arg Arg 35 40 45 Ala Ala Glu Gln Glu Ala Gln Leu Leu Ser Gln Leu
Lys His Pro Asn 50 55 60 Ile Val Thr Tyr Lys Glu Ser Trp Glu Gly
Gly Asp Gly Leu Leu Tyr 65 70 75 80 Ile Val Met Gly Phe Cys Glu Gly
Gly Asp Leu Tyr Arg Lys Leu Lys 85 90 95 Glu Gln Lys Gly Gln Leu
Leu Pro Glu Asn Gln Val Val Glu Trp Phe 100 105 110 Val Gln Ile Ala
Met Ala Leu Gln Tyr Leu His Glu Lys His Ile Leu 115 120 125 His Arg
Asp Leu Lys Thr Gln Asn Val Phe Leu Thr Arg Thr Asn Ile 130 135 140
Ile Lys Val Gly Asp Leu Gly Ile Ala Arg Val Leu Glu Asn His Cys 145
150 155 160 Asp Met Ala Ser Thr Leu Ile Gly Thr Pro Tyr Tyr Met Ser
Pro Glu 165 170 175 Leu Phe Ser Asn Lys Pro Tyr Asn Tyr Lys Ser Asp
Val Trp Ala Leu 180 185 190 Gly Cys Cys Val Tyr Glu Met Ala Thr Leu
Lys His Ala Phe Asn Ala 195 200 205 Lys Asp Met Asn Ser Leu Val Tyr
Arg Ile Ile Glu Gly Lys Leu Pro 210 215 220 Ala Met Pro Arg Asp Tyr
Ser Pro Glu Leu Ala Glu Leu Ile Arg Thr 225 230 235 240 Met Leu Ser
Lys Arg Pro Glu Glu Arg Pro Ser Val Arg Ser Ile Leu 245 250 255 Arg
Gln Pro Tyr Ile Lys Arg Gln Ile Ser Phe Phe Leu Glu Ala Thr 260 265
270 Lys Ile Lys Thr Ser Lys Asn Asn Ile Lys Asn Gly Asp Ser Gln Ser
275 280 285 Lys Pro Phe Ala Thr Val Val Ser Gly Glu Ala Glu Ser Asn
His Glu 290 295 300 Val Ile His Pro Gln Pro Leu Ser Ser Glu Gly Ser
Gln Thr Tyr Ile 305 310 315 320 Met Gly Glu Gly Lys Cys Leu Ser Gln
Glu Lys Pro Arg Ala Ser Gly 325 330 335 Leu Leu Lys Ser Pro Ala Ser
Leu Lys Ala His Thr Cys Lys Gln Asp 340 345 350 Leu Ser Asn Thr Thr
Glu Leu Ala Thr Ile Ser Ser Val Asn Ile Asp 355 360 365 Ile Leu Pro
Ala Lys Gly Arg Asp Ser Val Ser Asp Gly Phe Val Gln 370 375 380 Glu
Asn Gln Pro Arg Tyr Leu Asp Ala Ser Asn Glu Leu Gly Gly Ile 385 390
395 400 Cys Ser Ile Ser Gln Val Glu Glu Glu Met Leu Gln Asp Asn Thr
Lys 405 410 415 Ser Ser Ala Gln Pro Glu Asn Leu Ile Pro Met Trp Ser
Ser Asp Ile 420 425 430 Val Thr Gly Glu Lys Asn Glu Pro Val Lys Pro
Leu Gln Pro Leu Ile 435 440 445 Lys Glu Gln Lys Pro Lys Asp Gln Ser
Leu Ala Leu Ser Pro Lys Leu 450 455 460 Glu Cys Ser Gly Thr Ile Leu
Ala His Ser Asn Leu Arg Leu Leu Gly 465 470 475 480 Ser Ser Asp Ser
Pro Ala Ser Ala Ser Arg Val Ala Gly Ile Thr Gly 485 490 495 Val Cys
His His Ala Gln Asp Gln Val Ala Gly Glu Cys Ile Ile Glu 500 505 510
Lys Gln Gly Arg Ile His Pro Asp Leu Gln Pro His Asn Ser Gly Ser 515
520 525 Glu Pro Ser Leu Ser Arg Gln Arg Arg Gln Lys Arg Arg Glu Gln
Thr 530 535 540 Glu His Arg Gly Glu Lys Arg Gln Val Arg Arg Asp Leu
Phe Ala Phe 545 550 555 560 Gln Glu Ser Pro Pro Arg Phe Leu Pro Ser
His Pro Ile Val Gly Lys 565 570 575 Val Asp Val Thr Ser Thr Gln Lys
Glu Ala Glu Asn Gln Arg Arg Val 580 585 590 Val Thr Gly Ser Val Ser
Ser Ser Arg Ser Ser Glu Met Ser Ser Ser 595 600 605 Lys Asp Arg Pro
Leu Ser Ala Arg Glu Arg Arg Arg Leu Lys Gln Ser 610 615 620 Gln Glu
Glu Met Ser Ser Ser Gly Pro Ser Val Arg Lys Ala Ser Leu 625 630 635
640 Ser Val Ala Gly Pro Gly Lys Pro Gln Glu Glu Asp Gln Pro Leu Pro
645 650 655 Ala Arg Arg Leu Ser Ser Asp Cys Ser Val Thr Gln Glu Arg
Lys Gln 660 665 670 Ile His Cys Leu Ser Glu Asp Glu Leu Ser Ser Ser
Thr Ser Ser Thr 675 680 685 Asp Lys Ser Asp Gly Asp Tyr Gly Glu Gly
Lys Gly Gln Thr Asn Glu 690 695 700 Ile Asn Ala Leu Val Gln Leu Met
Thr Gln Thr Leu Lys Leu Asp Ser 705 710 715 720 Lys Glu Ser Cys Glu
Asp Val Pro Val Ala Asn Pro Val Ser Glu Phe 725 730 735 Lys Leu His
Arg Lys Tyr Arg Asp Thr Leu Ile Leu His Gly Lys Val 740 745 750 Ala
Glu Glu Ala Glu Glu Ile His Phe Lys Glu Leu Pro Ser Ala Ile 755 760
765 Met Pro Gly Ser Glu Lys Ile Arg Arg Leu Val Glu Val Leu Arg Thr
770 775 780 Asp Val Ile Arg Gly Leu Gly Val Gln Leu Leu Glu Gln Val
Tyr Asp 785 790 795 800 Leu Leu Glu Glu Glu Asp Glu Phe Asp Arg Glu
Val Arg Leu Arg Glu 805 810 815 His Met Gly Glu Lys Tyr Thr Thr Tyr
Ser Val Lys Ala Arg Gln Leu 820 825 830 Lys Phe Phe Glu Glu Asn Met
Asn Phe 835 840 38 552 PRT Homo sapiens 38 Met Ala Glu Lys Gln Lys
His Asp Gly Arg Val Lys Ile Gly His Tyr 1 5 10 15 Val Leu Gly Asp
Thr Leu Gly Val Gly Thr Phe Gly Lys Val Lys Ile 20 25 30 Gly Glu
His Gln Leu Thr Gly His Lys Val Ala Val Lys Ile Leu Asn 35 40 45
Arg Gln Lys Ile Arg Ser Leu Asp Val Val Gly Lys Ile Lys Arg Glu 50
55 60 Ile Gln Asn Leu Lys Leu Phe Arg His Pro His Ile Ile Lys Leu
Tyr 65 70 75 80 Gln Val Ile Ser Thr Pro Thr Asp Phe Phe Met Val Met
Glu Tyr Val 85 90 95 Ser Gly Gly Glu Leu Phe Asp Tyr Ile Cys Lys
His Gly Arg Val Glu 100 105 110 Glu Met Glu Ala Arg Arg Leu Phe Gln
Gln Ile Leu Ser Ala Val Asp 115 120 125 Tyr Cys His Arg His Met Val
Val His Arg Asp Leu Lys Pro Glu Asn 130 135 140 Val Leu Leu Asp Ala
His Met Asn Ala Lys Ile Ala Asp Phe Gly Leu 145 150 155 160 Ser Asn
Met Met Ser Asp Gly Glu Phe Leu Arg Thr Ser Cys Gly Ser 165 170 175
Pro Asn Tyr Thr Ala Pro Glu Val Ile Ser Gly Arg Leu Tyr Ala Gly 180
185 190 Pro Glu Val Asp Ile Trp Ser Cys Gly Val Ile Leu Tyr Ala Leu
Leu 195 200 205 Cys Gly Thr Leu Pro Phe Asp Asp Glu His Val Pro Thr
Leu Phe Lys 210 215 220 Lys Ile Arg Gly Gly Val Phe Tyr Ile Pro Glu
Tyr Leu Asn Arg Ser 225 230 235 240 Val Ala Thr Leu Leu Met His Met
Leu Gln Val Asp Pro Leu Lys Arg 245 250 255 Ala Thr Ile Lys Asp Ile
Arg Glu His Glu Trp Phe Lys Gln Gly Leu 260 265 270 Pro Ser Tyr Leu
Phe Pro Glu Asp Pro Ser Tyr Asp Ala Asn Val Ile 275 280 285 Asp Asp
Glu Ala Val Lys Glu Val Cys Glu Lys Phe Glu Cys Thr Glu 290 295 300
Ser Glu Val Met Asn Ser Leu Tyr Ser Gly Asp Pro Gln Asp Gln Leu 305
310 315 320 Ala Val Ala Tyr His Leu Ile Ile Asp Asn Arg Arg Ile Met
Asn Gln 325 330 335 Ala Ser Glu Phe Tyr Leu Ala Ser Ser Pro Pro Ser
Gly Ser Phe Met 340 345 350 Asp Asp Ser Ala Met His Ile Pro Pro Gly
Leu Lys Pro His Pro Glu 355 360 365 Arg Met Pro Pro Leu Ile Ala Asp
Ser Pro Lys Ala Arg Cys Pro Leu 370 375 380 Asp Ala Leu Asn Thr Thr
Lys Pro Lys Ser Leu Ala Val Lys Lys Ala 385 390 395 400 Lys Trp Arg
Gln Gly Ile Arg Ser Gln Ser Lys Pro Tyr Asp Ile Met 405 410 415 Ala
Glu Val Tyr Arg Ala Met Lys Gln Leu Asp Phe Glu Trp Lys Val 420 425
430 Val Asn Ala Tyr His Leu Arg Val Arg Arg Lys Asn Pro Val Thr Gly
435 440 445 Asn Tyr Val Lys Met Ser Leu Gln Leu Tyr Leu Val Asp Asn
Arg Ser 450 455 460 Tyr Leu Leu Asp Phe Lys Ser Ile Asp Asp Glu Val
Val Glu Gln Arg 465 470 475 480 Ser Gly Ser Ser Thr Pro Gln Arg Ser
Cys Ser Ala Ala Gly Leu His 485 490 495 Arg Pro Arg Ser Ser Phe Asp
Ser Thr Thr Ala Glu Ser His Ser Leu 500 505 510 Ser Gly Ser Leu Thr
Gly Ser Leu Thr Gly Ser Thr Leu Ser Ser Val 515 520 525 Ser Pro Arg
Leu Gly Ser His Thr Met Asp Phe Phe Glu Met Cys Ala 530 535 540 Ser
Leu Ile Thr Thr Leu Ala Arg 545 550
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