U.S. patent application number 12/010210 was filed with the patent office on 2008-12-25 for antibodies to chemokine alpha-5.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Yi Li, Jian Ni, Craig A. Rosen, Steven M. Ruben, Ying-Fei Wei.
Application Number | 20080317756 12/010210 |
Document ID | / |
Family ID | 22069070 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080317756 |
Kind Code |
A1 |
Wei; Ying-Fei ; et
al. |
December 25, 2008 |
Antibodies to Chemokine Alpha-5
Abstract
The present invention relates to a novel CK.alpha.-5 protein
which is a member of the alpha chemokine family. In particular,
isolated nucleic acid molecules are provided encoding the human
CK.alpha.-5 protein. CK.alpha.-5 polypeptides are also provided, as
are vectors, host cells and recombinant methods for producing the
same. Antibodies and fragments thereof that specifically bind to
CK.alpha.-5 polypeptides are also disclosed, as are compositions
and methods for making such antibodies. The invention further
relates to screening methods for identifying agonists and
antagonists of CK.alpha.-5 activity. Also provided are diagnostic
methods for detecting immune system-related disorders and
therapeutic methods for treating immune system-related
disorders.
Inventors: |
Wei; Ying-Fei; (San Mateo,
CA) ; Li; Yi; (Sunnyvale, CA) ; Ni; Jian;
(Rockville, MD) ; Rosen; Craig A.; (Laytonsville,
MD) ; Ruben; Steven M.; (Olney, MD) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Human Genome Sciences, Inc.
|
Family ID: |
22069070 |
Appl. No.: |
12/010210 |
Filed: |
January 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11224076 |
Sep 13, 2005 |
7320869 |
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12010210 |
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10114482 |
Apr 3, 2002 |
7060261 |
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11224076 |
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09195106 |
Nov 18, 1998 |
6395514 |
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10114482 |
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60066369 |
Nov 21, 1997 |
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Current U.S.
Class: |
424/139.1 ;
435/6.11; 435/6.12; 435/7.2; 436/94; 514/1.1; 514/44A |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/522 20130101; C12N 2799/026 20130101; A61K 38/00 20130101;
C07K 14/521 20130101; Y10T 436/143333 20150115; C07K 2319/00
20130101; A61P 37/00 20180101; Y02A 50/423 20180101 |
Class at
Publication: |
424/139.1 ;
435/7.2; 435/6; 436/94; 514/12; 514/44 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/566 20060101 G01N033/566; C12Q 1/68 20060101
C12Q001/68; A61K 31/7105 20060101 A61K031/7105; G01N 33/53 20060101
G01N033/53; A61K 38/17 20060101 A61K038/17 |
Claims
1-28. (canceled)
29. A method of screening for a CK.alpha.-5 agonist comprising: (a)
combining (i) a cell expressing a receptor, wherein the receptor
activity is enhanced by CK.alpha.-5 polypeptide, (ii) CK.alpha.-5
polypeptide, and (iii) a test compound; (b) assaying a calcium
mobilization or a chemotactic activity of the cell expressing the
receptor; and (c) comparing the calcium mobilization or the
chemotactic activity of the cell with a standard level of calcium
mobilization or a standard level of chemotactic activity, wherein
an increase in calcium mobilization or chemotactic activity
compared to the standard level indicates the test compound is an
agonist of CK.alpha.-5 activity.
30. A method of screening for a CK.alpha.-5 antagonist comprising:
a) combining (i) a cell expressing a receptor, wherein the receptor
activity is enhanced by CK.alpha.-5 polypeptide, (ii) CK.alpha.-5
polypeptide, and (iii) a test compound; b) assaying a calcium
mobilization or a chemotactic activity of the cell expressing the
receptor; and c) comparing the calcium mobilization or the
chemotactic activity of the cell with a standard level of calcium
mobilization or a standard level of chemotactic activity, wherein a
decrease in calcium mobilization or chemotactic activity compared
to the standard level indicates the test compound is an antagonist
of CK.alpha.-5 activity.
31. A method of detecting CK.alpha.-5 nucleic acid in a sample
comprising: (a) contacting the sample with a nucleic acid probe
under nucleic acid hybridization conditions, wherein the nucleic
acid probe comprises SEQ ID NO: 1 or a fragment thereof, a
complement of SEQ ID NO: 1 or a fragment thereof, and wherein the
fragment comprises at least 20 contiguous nucleic acids; and (b)
detecting hybridization of the probe and the CK.alpha.-5 nucleic
acid.
32. The method of claim 31, further comprising amplifying the
CK.alpha.-5 nucleic acid prior to the contacting and detecting
steps.
33. The method of claim 31, wherein the fragment comprises at least
20 continuous nucleic acids of nucleic acid residues 622-1303 of
SEQ ID NO: 1.
34. The method of claim 31, wherein the fragment comprises at least
20 contiguous nucleic acid residues complementary to nucleic acid
residues of 622-1303 of SEQ ID NO: 1.
35. A method of assaying an expression level of CK.alpha.-5 nucleic
acid in a biological sample comprising: (a) contacting the sample
with a hybridization probe under nucleic acid hybridization
conditions, wherein the hybridization probe comprises SEQ ID NO: 1
or a fragment thereof, a complement of SEQ ID NO: 1 or a fragment
thereof, and wherein the fragment comprises at least 20 contiguous
nucleic acids; (b) detecting a level of hybridization probes,
wherein the level of hybridization probes corresponds to the
hybridization probes that are hybridized to the CK.alpha.-5 nucleic
acid; and (c) comparing the level of hybridization probes with a
standard level of hybridization probes.
36. The method of claim 35, wherein the biological sample is
selected from the group consisting of: serum, plasma, urine,
synovial fluid, spinal fluid, immune tissue, and skeletal
tissue.
37. The method of claim 35, wherein the CK.alpha.-5 nucleic acid
comprises mRNA.
38. The method of claim 35, further comprising amplification of
CK.alpha.-5 nucleic acid prior to the contacting, detecting and
comparing steps.
39. A method of treating an individual in need of CK.alpha.-5
polypeptide activity comprising administering to the individual a
therapeutically effective amount of an agonist of CK.alpha.-5.
40. A method of treating an individual in need of CK.alpha.-5
polypeptide activity comprising administering to the individual a
therapeutically effective amount of a polypeptide selected from the
group consisting of SEQ ID NO: 2, a fragment comprising amino acid
residues 28-254 of SEQ ID NO: 2, a fragment comprising amino acid
residues 28-205 of SEQ ID NO: 2, and a combination thereof.
41. A method of treating an individual in need of a decreased level
of CK.alpha.-5 polypeptide activity comprising administering to the
individual a therapeutically effective amount of an antagonist of
CK.alpha.-5.
42. The method of claim 41, wherein the antagonist comprises an
isolated antibody or fragment thereof that specifically binds to a
protein selected from the group consisting of: (a) a protein whose
amino acid sequence consists of amino acid residues 1 to 254 of SEQ
ID NO: 2; (b) a protein whose amino acid sequence consists of amino
acid residues 28 to 254 of SEQ ID NO: 2; (c) a protein whose amino
acid sequence consists of amino acid residues 28 to 205 of SEQ ID
NO: 2; (d) a protein whose amino acid sequence consists of a
portion of SEQ ID NO: 2, wherein said portion is at least 30
contiguous amino acid residues in length; and (e) a protein whose
amino acid sequence consists of a portion of SEQ ID NO: 2, wherein
said portion is at least 50 contiguous amino acid residues in
length.
43. The method of claim 41, wherein the antagonist comprises
antisense RNA.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/114,482, filed Apr. 3, 2002, which is a divisional of U.S.
application Ser. No. 09/195,106, filed Nov. 18, 1998 (now U.S. Pat.
No. 6,395,514, issued May 28, 2002), which is a non-provisional of
and claims benefit under 35 U.S.C. .sctn. 119(e) of U.S.
Provisional Application No. 60/066,369, filed Nov. 21, 1997, which
is hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel human gene encoding
a polypeptide which is a member of the chemokine family. More
specifically, isolated nucleic acid molecules are provided encoding
a human polypeptide named Chemokine Alpha-5, hereinafter referred
to as "CK.alpha.-5". CK.alpha.-5 polypeptides are also provided, as
are vectors, host cells and recombinant methods for producing the
same. Also provided are diagnostic methods for detecting disorders
related to the immune system, and therapeutic methods for treating
such disorders. The invention further relates to screening methods
for identifying agonists and antagonists of CK.alpha.-5
activity.
BACKGROUND OF THE INVENTION
[0003] The ability to control the migration and "trafficking" of
various cell types is controlled by a subset of factors, or
proteins, among which chemokines are an example.
[0004] Chemokines, also referred to as intercrine cytokines, are a
subfamily of structurally and functionally related chemotactic
cytokines. These molecules are usually 8-10 kd in size, however,
larger membrane bound chemokine polypeptides have been identified.
In general, chemokines exhibit 20% to 75% homology at the amino
acid level and are characterized by four conserved cysteine
residues that form two disulfide bonds. Based on the arrangement of
the first two cysteine residues, chemokines have been classified
into two subfamilies, alpha and beta. In the alpha subfamily, the
first two cysteines are separated by one amino acid and hence are
referred to as the "C--X--C" subfamily. In the beta subfamily, the
two cysteines are in an adjacent position and are, therefore,
referred to as the "C--C" subfamily. Thus far, at least sixteen
different members of this family have been identified in
humans.
[0005] The intercrine cytokines exhibit a wide variety of
functions. A hallmark feature is their ability to elicit
chemotactic migration of distinct cell types, including monocytes,
neutrophils, T lymphocytes, basophils and fibroblasts. Many
chemokines have proinflammatory activity and are involved in
multiple steps during an inflammatory reaction. These activities
include stimulation of histamine release, lysosomal enzyme and
leukotriene release, increased adherence of target immune cells to
endothelial cells, enhanced binding of complement proteins, induced
expression of granulocyte adhesion molecules and complement
receptors, and respiratory burst. In addition to their involvement
in inflammation, certain chemokines have been shown to exhibit
other activities. For example, macrophage inflammatory protein 1
(MIP-1) is able to suppress hematopoietic stem cell proliferation,
platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell
growth, Interleukin-8 (IL-8) promotes proliferation of
keratinocytes, and GRO is an autocrine growth factor for melanoma
cells.
[0006] In light of the diverse biological activities, it is not
surprising that chemokines have been implicated in a number of
physiological and disease conditions, including lymphocyte
trafficking, wound healing, hematopoietic regulation and
immunological disorders such as allergy, asthma and arthritis.
[0007] Members of the "C--C" branch exert their effects on the
following cells: eosinophils which destroy parasites to lessen
parasitic infection and cause chronic inflammation in the airways
of the respiratory system; macrophages which suppress tumor
formation in vertebrates; and basophils which release histamine
which plays a role in allergic inflammation. However, members of
one branch may exert an effect on cells which are normally
responsive to the other branch of chemokines and, therefore, no
precise role can be attached to the members of the branches.
[0008] While members of the C--C branch act predominantly on
mononuclear cells and members of the C--X--C branch act
predominantly on neutrophils a distinct chemoattractant property
cannot be assigned to a chemokine based on this guideline. Some
chemokines from one family show characteristics of the other.
[0009] The polypeptide of the present invention has the conserved
cysteine residues of the "C--X--C" region, and has amino acid
sequence homology to known chemokines.
[0010] Thus, there is a need for polypeptides that function as
regulators of the migration of distinct cell types and of their
roles in dysfunction and disease, since disturbances of such
regulation may be involved in disorders relating to hemostasis,
angiogenesis, tumor metastasis, cellular migration and ovulation,
as well as neurogenesis. Therefore, there is a need for
identification and characterization of such human polypeptides
which can play a role in detecting, preventing, ameliorating or
correcting such disorders.
SUMMARY OF THE INVENTION
[0011] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding at least a portion
of the CK.alpha.-5 polypeptide having the complete amino acid
sequence shown in SEQ ID NO:2 or the complete amino acid sequence
encoded by a cDNA clone deposited as plasmid DNA as ATCC.RTM.
Deposit Number 209231 on Aug. 29, 1997. The ATCC.RTM. is located at
10801 University Boulevard, Manassas, Va. 20110-2209. The
nucleotide sequence determined by sequencing the deposited
CK.alpha.-5 clone, which is shown in FIG. 1 (SEQ ID NO:1), contains
an open reading frame encoding a complete polypeptide of 254 amino
acid residues, including an initiation codon encoding an N terminal
methionine at nucleotide positions 542-544. Nucleic acid molecules
of the invention include those encoding the complete amino acid
sequence excepting the N-terminal methionine shown in SEQ ID NO:2,
or the complete amino acid sequence excepting the N-terminal
methionine encoded by a cDNA clone in ATCC.RTM. Deposit Number
209231, which molecules also can encode additional amino acids
fused to the N-terminus of the CK.alpha.-5 amino acid sequence.
[0012] The polypeptide of the present invention has amino acid
sequence homology to known chemokines, including the conserved
C--X--C cysteine pattern characteristic of the alpha subfamily of
chemokines beginning with the first cysteine from the amino
terminus in SEQ ID NO:2.
[0013] CK.alpha.-5 also lacks the ELR motif found in some alpha
chemokines immediately preceding the first cysteine residue, which
is known to be required for the neutrophil and endothelial cell
chemotactic activity as well as the angiogenic activity of
IL-8.
[0014] The encoded polypeptide has a predicted leader sequence of
27 amino acids underlined in FIG. 1; and the amino acid sequence of
the predicted mature CK.alpha.-5 protein is also shown in FIG. 1 as
amino acid residues 28-254 and as residues 28-254 in SEQ ID NO:2.
Further, the polypeptide is predicted to consist of an
extracellular domain having the sequence from residues 28 to 205 in
FIG. 1 (28 to 205 in SEQ ID NO:2), a transmembrane domain having a
sequence from residue 206 to 227 in FIG. 1 (206 to 227 in SEQ ID
NO:2), and an intracellular domain having a sequence from residue
228 to 254 in FIG. 1 (228 to 254 in SEQ ID NO:2).
[0015] Thus, one aspect of the invention provides an isolated
polynucleotide comprising a nucleotide sequence selected from the
group consisting of: (a) a nucleotide sequence encoding the
CK.alpha.-5 polypeptide having the complete amino acid sequence in
FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding the
CK.alpha.-5 polypeptide having the complete amino acid sequence in
FIG. 1 (SEQ ID NO:2) excepting the N-terminal methionine (i.e.,
positions 2 to 254 of SEQ ID NO:2); (c) a nucleotide sequence
encoding the predicted mature CK.alpha.-5 polypeptide having the
amino acid sequence at positions 28-254 in FIG. 1 (28-254 in SEQ ID
NO:2); (d) a nucleotide sequence encoding the predicted
extracellular domain of the CK.alpha.-5 polypeptide having the
amino acid sequence at positions 28-205 in FIG. 1 (28-205 in SEQ ID
NO:2); (e) a nucleotide sequence encoding the CK.alpha.-5
polypeptide having the complete amino acid sequence encoded by the
cDNA clone contained in ATCC.RTM. Deposit No. 209231; (f) a
nucleotide sequence encoding the CK.alpha.-5 polypeptide having the
complete amino acid sequence excepting the N terminal methionine
encoded by the cDNA clone contained in ATCC.RTM. Deposit No.
209231; (g) a nucleotide sequence encoding the mature CK.alpha.-5
polypeptide encoded by the cDNA clone contained in ATCC.RTM.
Deposit No. 209231; (h) a nucleotide sequence encoding the
extracellular domain of CK.alpha.-5 encoded by the cDNA clone
contained in ATCC.RTM. Deposit No. 209231; and (i) a nucleotide
sequence complementary to any of the nucleotide sequences in (a),
(b), (c), (d), (e), (f), (g) or (h) above.
[0016] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above,
or a polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e), (f),
(g), (h) or (i), above. This polynucleotide which hybridizes does
not hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A
residues or of only T residues. An additional nucleic acid
embodiment of the invention relates to an isolated nucleic acid
molecule comprising a polynucleotide which encodes the amino acid
sequence of an epitope-bearing portion of a CK.alpha.-5 polypeptide
having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g)
or (h), above.
[0017] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of CK.alpha.-5 polypeptides or peptides
by recombinant techniques.
[0018] The invention further provides an isolated CK.alpha.-5
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of the full-length
CK.alpha.-5 polypeptide having the complete amino acid sequence
shown in SEQ ID NO:2 or the complete amino acid sequence encoded by
the cDNA clone contained in the ATCC.RTM. Deposit No. 209231; (b)
the amino acid sequence of the full-length CK.alpha.-5 polypeptide
having the complete amino acid sequence shown in SEQ ID NO:2
excepting the N-terminal methionine (i.e., positions 2 to 254 of
SEQ ID NO:2) or the complete amino acid sequence excepting the N
terminal methionine encoded by the cDNA clone contained in the
ATCC.RTM. Deposit No. 209231; (c) the amino acid sequence of the
mature CK.alpha.-5 polypeptide having the amino acid sequence shown
in SEQ ID NO:2 as residues 28-254 or the amino acid sequence of the
mature CK.alpha.-5 encoded by the cDNA clone contained in the
ATCC.RTM. Deposit No. 209231; and (d) the amino acid sequence of
the extracellular domain of the CK.alpha.-5 polypeptide having the
amino acid sequence shown in SEQ ID NO:2 as residues 28-205 or the
amino acid sequence of the extracellular domain of CK.alpha.-5
encoded by the cDNA clone contained in the ATCC.RTM. Deposit No.
209231. The polypeptides of the present invention also include
polypeptides having an amino acid sequence at least 80% identical,
more preferably at least 90% identical, and still more preferably
95%, 96%, 97%, 98% or 99% identical to those described in (a), (b),
(c) or (d) above, as well as polypeptides having an amino acid
sequence with at least 90% similarity, and more preferably at least
95% similarity, to those above. Polynucleotides encoding
polypeptides having an amino acid sequence at least 80% identical,
more preferably at least 90% identical, and still more preferably
95%, 96%, 97%, 98% or 99% identical to those described in (a), (b),
(c) or (d) above, as well as polypeptides having an amino acid
sequence with at least 90% similarity, and more preferably at least
95% similarity, to those above are also provided.
[0019] An additional embodiment of this aspect of the invention
relates to a peptide or polypeptide which comprises the amino acid
sequence of an epitope-bearing portion of a CK.alpha.-5 polypeptide
having an amino acid sequence described in (a), (b), (c) or (d),
above. Peptides or polypeptides having the amino acid sequence of
an epitope-bearing portion of a CK.alpha.-5 polypeptide of the
invention include portions of such polypeptides with at least six
(6) or seven (7), preferably at least nine (9), and more preferably
at least about 30 amino acids to about 50 amino acids, although
epitope-bearing polypeptides of any length up to and including the
entire amino acid sequence of a polypeptide of the invention
described above also are included in the invention.
[0020] In another embodiment, the invention provides an isolated
antibody that binds specifically to a CK.alpha.-5 polypeptide
having an amino acid sequence described in (a), (b), (c) or (d)
above. The invention further provides methods for isolating
antibodies that bind specifically to a CK.alpha.-5 polypeptide
having an amino acid sequence as described herein. Such antibodies
are useful diagnostically or therapeutically as described
below.
[0021] The invention also provides for pharmaceutical compositions
comprising CK.alpha.-5 polypeptides, particularly human CK.alpha.-5
polypeptides, which may be employed, for instance, to stimulate
wound healing, to treat solid tumors, microbial infections,
autoimmune diseases, liver cirrhosis, osteoarthritis and pulmonary
fibrosis. Methods of treating individuals in need of CK.alpha.-5
polypeptides are also provided.
[0022] The invention further provides compositions comprising a
CK.alpha.-5 polynucleotide or a CK.alpha.-5 polypeptide for
administration to cells in vitro, to cells ex vivo and to cells in
vivo, or to a multicellular organism. In certain particularly
preferred embodiments of this aspect of the invention, the
compositions comprise a CK.alpha.-5 polynucleotide for expression
of a CK.alpha.-5 polypeptide in a host organism for treatment of
disease. Particularly preferred in this regard is expression in a
human patient for treatment of a dysfunction associated with
aberrant endogenous activity of a CK.alpha.-5
[0023] The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a
biological activity of the CK.alpha.-5 polypeptide, which involves
contacting a receptor which is enhanced by the CK.alpha.-5
polypeptide with the candidate compound in the presence of a
CK.alpha.-5 polypeptide, assaying calcium mobilization or
chemotactic activity of the cell expressing the receptor in the
presence of the candidate compound and the CK.alpha.-5 polypeptide,
and comparing the receptor activity to a standard level of
activity, the standard being assayed when contact is made between
the receptor and the CK.alpha.-5 polypeptide in the absence of the
candidate compound. In this assay, an increase in calcium
mobilization or chemotaxis over the standard indicates that the
candidate compound is an agonist of CK.alpha.-5 activity and a
decrease in calcium mobilization or chemotaxis compared to the
standard indicates that the compound is an antagonist of
CK.alpha.-5 activity.
[0024] It has been discovered that CK.alpha.-5 is expressed not
only in neutrophils but also in monocytes, macrophages, liver,
lung, testes, cerebellum and pineal gland. Therefore, nucleic acids
of the invention are useful as hybridization probes for
differential identification of the tissue(s) or cell type(s)
present in a biological sample. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). In addition, for a number of disorders
of the above tissues or cells, particularly of the immune system,
significantly higher or lower levels of CK.alpha.-5 gene expression
may be detected in certain tissues (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to a "standard" CK.alpha.-5 gene expression
level, i.e., the CK.alpha.-5 expression level in healthy tissue
from an individual not having the immune system disorder. Thus, the
invention provides a diagnostic method useful during diagnosis of
such a disorder, which involves: (a) assaying CK.alpha.-5 gene
expression level in cells or body fluid of an individual; (b)
comparing the CK.alpha.-5 gene expression level with a standard
CK.alpha.-5 gene expression level, whereby an increase or decrease
in the assayed CK.alpha.-5 gene expression level compared to the
standard expression level is indicative of disorder in the immune
system.
[0025] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of
CK.alpha.-5 activity in the body comprising administering to such
an individual a composition comprising a therapeutically effective
amount of an isolated CK.alpha.-5 polypeptide of the invention or
an agonist thereof.
[0026] A still further aspect of the invention is related to a
method for treating an individual in need of a decreased level of
CK.alpha.-5 activity in the body comprising, administering to such
an individual a composition comprising a therapeutically effective
amount of a CK.alpha.-5 antagonist. Preferred antagonists for use
in the present invention are CK.alpha.-5-specific antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIG. 1A-C shows the nucleotide sequence (SEQ ID NO:1) and
deduced amino acid sequence (SEQ ID NO:2) of CK.alpha.-5. The
predicted leader sequence of about 27 amino acids is underlined.
Note that the methionine residue at the beginning of the leader
sequence in FIG. 1 is shown in position number (positive).sub.1,
the leader positions in the corresponding sequence of SEQ ID NO:2
are designated with positive position numbers. Thus, the leader
sequence positions 1 to 27 in FIG. 1 correspond to positions -1 to
27 in SEQ ID NO:2.
[0028] FIG. 2 shows the regions of identity between the amino acid
sequence of the CK.alpha.-5 protein and translation product of the
rat mRNA for MIP1-.beta. (SEQ ID NO:3), as determined by Bestfit
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711) using the default parameters.
[0029] FIG. 3 shows an analysis of the CK.alpha.-5 amino acid
sequence. Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index--Jameson-Wolf" graph, the positive peaks indicate locations
of the highly antigenic regions of the CK.alpha.-5 protein, i.e.,
regions from which epitope-bearing peptides of the invention can be
obtained.
DETAILED DESCRIPTION
[0030] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a CK.alpha.-5
polypeptide having the amino acid sequence shown in SEQ ID NO:2,
which was determined by sequencing cloned cDNAs. The nucleotide
sequence shown in FIG. 1 (SEQ ID NO:1) was obtained by sequencing
two cDNA clones: HMSCJ62 and HNFEM05. HMSCJ62 contains the
full-length sequence shown in FIG. 1 and is deposited at Human
Genome Sciences, Inc., 9410 Key West Ave., Rockville, Md. 20850.
HNFEM05 contains all but the first 360 nucleotides (including all
of the coding region) and was deposited as American Type Culture
Collection ("ATCC.RTM.") Deposit No. 209231 on Aug. 29, 1997. The
ATCC.RTM. is located at 10801 University Boulevard, Manassas, Va.
20110-2209.
[0031] The deposited clones are contained in the pBluescript SK(-)
plasmid (Stratagene, La Jolla, Calif.).
[0032] The polypeptide of the present invention has amino acid
sequence homology to known chemokines, including the conserved
C--X--C cysteine pattern characteristic of the alpha subfamily of
chemokines beginning with the first cysteine from the amino
terminus in SEQ ID NO:2. The CK.alpha.-5 protein of the present
invention also shares sequence homology with other chemokines
including in particular the translation product of the human mRNA
for MIP1-.beta. (SEQ ID NO:3) as shown in FIG. 2.
[0033] Of the known members of the alpha chemokine family, the
majority contain an ELR motif (e.g., IL-8, ENA-78, GCP2,
GRO-.alpha., PBP, CTAP-III and NAP-2) and others lack the ELR motif
(IP-10, PF4 and MIG). CK.alpha.-5 lacks the ELR motif immediately
preceding the first cysteine residue. It has been clearly shown
that this ELR motif is required for the neutrophil and endothelial
cell chemotactic activity as well as the angiogenic activity of
IL-8 (Strieter et al., J. Biol. Chem., 270:27348-27357 (1995)). In
addition, it has been shown that the ELR-C--X--C chemokines have in
vitro and in vivo angiogenic activity, whereas the C--X--C
chemokines lacking the ELR motif are actually angiostatic (Strieter
et al., supra; Angiolillo et al., J. Exp. Med., 182:155-162 (1995);
and Koch et al., Science, 258:1798-1801 (1992)). In terms of a
possible role of such factors in tumor angiogenesis, Smith et al.,
J. Exp. Med., 179:1409-1415 (1994), has reported increased IL-8
levels in bronchogeneic carcinoma tumor tissues which appear to be
produced from the tumor cells.
Nucleic Acid Molecules
[0034] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as above. Therefore, as is known in the art for any DNA sequence
determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90%
identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced
DNA molecule. The actual sequence can be more precisely determined
by other approaches including manual DNA sequencing methods well
known in the art. As is also known in the art, a single insertion
or deletion in a determined nucleotide sequence compared to the
actual sequence will cause a frame shift in translation of the
nucleotide sequence such that the predicted amino acid sequence
encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the
sequenced DNA molecule, beginning at the point of such an insertion
or deletion.
[0035] By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
[0036] Using the information provided herein, such as the
nucleotide sequence in FIG. 1 (SEQ ID NO:1), a nucleic acid
molecule of the present invention encoding a CK.alpha.-5
polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in FIG. 1 (SEQ ID NO:1) was discovered in cDNA libraries
derived primarily from immune-system tissues. Clone HMSCJ62 was
isolated from a monocyte cDNA library and HNFEM05 was isolated from
a neutrophil cDNA library.
[0037] Additional clones of the same gene were also identified in
cDNA libraries from the following human tissues: colon and
osteoclastoma.
[0038] The determined nucleotide sequence of the CK.alpha.-5 cDNA
of FIG. 1 (SEQ ID NO:1) contains an open reading frame encoding a
protein of 254 amino acid residues, with an initiation codon at
nucleotide positions 542-544 of the nucleotide sequence in FIG. 1
(SEQ ID NO:1). The amino acid sequence of the CK.alpha.-5 protein
shown in SEQ ID NO:2 is about 51% similar and 28% identical to the
rat Macrophage Inflammatory Protein-1.beta. (FIG. 2), which can be
accessed on GenBank as Accession No. gi|459148.
[0039] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, the actual
complete CK.alpha.-5 polypeptide encoded by the deposited cDNA,
which comprises about 254 amino acids, may be somewhat longer or
shorter. More generally, the actual open reading frame may be
anywhere in the range of .+-.20 amino acids, more likely in the
range of .+-.10 amino acids, of that predicted from the methionine
codon at the N-terminus shown in FIG. 1 (SEQ ID NO:1).
Leader and Mature Sequences
[0040] The amino acid sequence of the complete CK.alpha.-5 protein
includes a leader sequence and a mature protein, as shown in SEQ ID
NO:2. More in particular, the present invention provides nucleic
acid molecules encoding a mature form of the CK.alpha.-5 protein.
Thus, according to the signal hypothesis, once export of the
growing protein chain across the rough endoplasmic reticulum has
been initiated, proteins secreted by mammalian cells have a signal
or secretory leader sequence which is cleaved from the complete
polypeptide to produce a secreted "mature" form of the protein.
Most mammalian cells and even insect cells cleave secreted proteins
with the same specificity. However, in some cases, cleavage of a
secreted protein is not entirely uniform, which results in two or
more mature species of the protein. Further, it has long been known
that the cleavage specificity of a secreted protein is ultimately
determined by the primary structure of the complete protein, that
is, it is inherent in the amino acid sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence
encoding the mature CK.alpha.-5 polypeptide having the amino acid
sequence encoded by the cDNA clone identified as ATCC.RTM. Deposit
No. 209231. By the "mature CK.alpha.-5 polypeptide having the amino
acid sequence encoded by the cDNA clone in ATCC.RTM. Deposit No.
209231" is meant the mature form(s) of the CK.alpha.-5 protein
produced by expression in a mammalian cell (e.g., COS cells, as
described below) of the complete open reading frame encoded by the
human DNA sequence of the clone contained in the deposited
vector.
[0041] In addition, methods for predicting whether a protein has a
secretory leader as well as the cleavage point for that leader
sequence are available. For instance, the method of McGeoch (Virus
Res. 3:271-286 (1985)) uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje (Nucleic Acids Res.
14:4683-4690 (1986)) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2 where
+1 indicates the amino terminus of the mature protein. The accuracy
of predicting the cleavage points of known mammalian secretory
proteins for each of these methods is in the range of 75-80% (von
Heinje, supra). However, the two methods do not always produce the
same predicted cleavage point(s) for a given protein.
[0042] In the present case, the deduced amino acid sequence of the
complete CK.alpha.-5 polypeptide was analyzed by a computer program
"PSORT", available from Dr. Kenta Nakai of the Institute for
Chemical Research, Kyoto University (see K. Nakai and M. Kanehisa,
Genomics 14:897-911 (1992)), which is an expert system for
predicting the cellular location of a protein based on the amino
acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are
incorporated." The computation analysis above predicted one
potential cleavage site within the complete amino acid sequence
shown in SEQ ID NO:2; that is, between residues 27 and 28 in FIG. 1
and residues 27 and 28 in SEQ ID NO:2.
[0043] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0044] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically. By "isolated" the inventors
intend to specifically exclude very large pieces of genetic
material (200 kb+) such as isolated chromosomes and yeast
artificial chromosomes (YACs).
[0045] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) with
an initiation codon at positions 542-544 of the nucleotide sequence
shown in FIG. 1 (SEQ ID NO:1).
[0046] Also included are DNA molecules comprising the coding
sequence for the predicted mature CK.alpha.-5 protein shown at
positions 28-254 of SEQ ID NO:2.
[0047] In addition, isolated nucleic acid molecules of the
invention include DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode a CK.alpha.-5
protein. Of course, the genetic code and species-specific codon
preferences are well known in the art. Thus, it would be routine
for one skilled in the art to generate the degenerate variants
described above, for instance, to optimize codon expression for a
particular host (e.g., change codons in the human mRNA to those
preferred by a bacterial host such as E. coli).
[0048] In another aspect, the invention provides isolated nucleic
acid molecules encoding the CK.alpha.-5 polypeptide having an amino
acid sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC.RTM. Deposit No. 209231 on Aug. 29, 1997.
[0049] Preferably, this nucleic acid molecule will encode the
mature polypeptide encoded by the above-described deposited cDNA
clone.
[0050] The invention further provides an isolated nucleic acid
molecule having the nucleotide sequence shown in FIG. 1 (SEQ ID
NO:1) or the nucleotide sequence of the CK.alpha.-5 cDNA contained
in the above-described deposited clone, or a nucleic acid molecule
having a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as
probes for gene mapping, by in situ hybridization with chromosomes,
and for detecting expression of the CK.alpha.-5 gene in human
tissue, for instance, by Northern blot analysis.
[0051] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein as well as to fragments of the isolated nucleic acid
molecules described herein. In particular, the invention provides a
polynucleotide comprising a nucleotide sequence representing the
portion of SEQ ID NO:1 which consists of positions 1-1303,
542-1303, 622-1303, 625-1303, 628-1303, 631-1303; 634-1303,
637-1303, 640-1303, 643-1303, 646-1303, 649-1303, 652-1303,
652-787, 652-817, 652-847, 652-877, 652-907, 652-937, 652-967,
652-997, 652-1027, 652-1057, 652-1087, 652-1117, 652-1147,
652-1177, 652-1207, 652-1237, or 652-1267 of SEQ ID NO:1.
[0052] In addition, the following nucleic acid molecules having
nucleotide sequences related to extensive portions of SEQ ID NO:1
have been identified: HNFCO09R (SEQ ID NO:4) from clone HNFCO09;
HNEDS26R (SEQ ID NO:5) from clone HNEDS26R; HOABD47R (SEQ ID NO:6)
from clone HOABD47; HTEIR41R (SEQ ID NO:7) from clone HTEIR41;
HMNAD36R (SEQ ID NO:8) from clone HMNAD36; and HTEDQ07RA (SEQ ID
NO:20) from clone HTEDQ07.
[0053] The following publicly available expressed sequence tags
("ESTs"), which relate to portions of SEQ ID NO:1, have also been
identified: GenBank Accession No. AA130776 (SEQ ID NO:9); GenBank
Accession No. AA290712 (SEQ ID NO:10); GenBank Accession No.
AA366329 (SEQ ID NO:11); GenBank Accession No. AA121716 (SEQ ID
NO:12); GenBank Accession No. AA146672 (SEQ ID NO:13); GenBank
Accession No. AA149359 (SEQ ID NO:14); GenBank Accession No.
AA569970 (SEQ ID NO:21); and GenBank Accession No. AA994552 (SEQ ID
NO:22).
[0054] Polynucleotides of the invention preferably are not, and do
not comprise, one or more of the forgoing sequences 1-14 and
20-22.
[0055] Further, the invention includes a polynucleotide comprising
any portion of at least about 17 nucleotides, preferably at least
about 20, 25, or 30, and most preferably at least about 50
nucleotides, of SEQ ID NO:1 from nucleotide 542 to 1303.
[0056] The invention also includes a polynucleotide comprising any
portion of at least about 500, preferably about 600 nucleotides, of
SEQ ID NO:1, preferably from residue 542 to 1,303. Particularly
preferred nucleotide fragments in this regard include the fragment
from nucleotide 622 to 1,303 in SEQ ID NO:1.
[0057] More generally, by a fragment of an isolated nucleic acid
molecule having the nucleotide sequence of the deposited cDNA or
the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is intended
fragments at least about 15 nt, and more preferably at least about
20 nt, still more preferably at least about 30 nt, and even more
preferably, at least about 40 nt in length which are useful as
diagnostic probes and primers as discussed herein. Of course,
larger fragments 50-1,000 nt in length are also useful according to
the present invention as are fragments corresponding to most, if
not all, of the nucleotide sequence of the deposited cDNA or as
shown in FIG. 1 (SEQ ID NO:1). By a fragment at least 20 nt in
length, for example, is intended fragments which include 20 or more
contiguous bases from the nucleotide sequence of the deposited cDNA
or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1).
Preferred nucleic acid fragments of the present invention include
nucleic acid molecules encoding epitope-bearing portions of the
CK.alpha.-5 polypeptide as identified from the Jameson-Wolf
antigenic index shown in FIG. 3 and described in more detail
below.
[0058] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in
ATCC.RTM. Deposit No. 209231. By "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0059] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of
the reference polynucleotide. These are useful as diagnostic probes
and primers as discussed above and in more detail below.
[0060] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in FIG. 1
(SEQ ID NO:1)). Of course, a polynucleotide which hybridizes only
to a poly A sequence (such as the 3' terminal poly(A) tract of the
CK.alpha.-5 cDNA shown in FIG. 1 (SEQ ID NO:1)), or to a
complementary stretch of T (or U) residues, would not be included
in a polynucleotide of the invention used to hybridize to a portion
of a nucleic acid of the invention, since such a polynucleotide
would hybridize to any nucleic acid molecule containing a poly (A)
stretch or the complement thereof (e.g., practically any
double-stranded cDNA clone).
[0061] As indicated, nucleic acid molecules of the present
invention which encode a CK.alpha.-5 polypeptide may include, but
are not limited to those encoding the amino acid sequence of the
mature polypeptide, by itself, and the coding sequence for the
mature polypeptide and additional sequences, such as those encoding
the about 27 amino acid leader or secretory sequence, such as a
pre-, or pro- or prepro-protein sequence; the coding sequence of
the mature polypeptide, with or without the aforementioned
additional coding sequences.
[0062] Also encoded by nucleic acids of the invention are the above
protein sequences together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
[0063] Thus, the sequence encoding the polypeptide may be fused to
a marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. The
"HA" tag is another peptide useful for purification which
corresponds to an epitope derived from the influenza hemagglutinin
protein, which has been described by Wilson et al., Cell 37: 767
(1984). As discussed below, other such fusion proteins include the
CK.alpha.-5 fused to Fc at the N- or C-terminus.
Variant and Mutant Polynucleotides
[0064] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the CK.alpha.-5 protein.
Variants may occur naturally, such as a natural allelic variant. By
an "allelic variant" is intended one of several alternate forms of
a gene occupying a given locus on a chromosome of an organism.
Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0065] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the CK.alpha.-5 protein or portions thereof. Also
especially preferred in this regard are conservative
substitutions.
[0066] Most highly preferred are nucleic acid molecules encoding
the mature protein having the amino acid sequence shown in SEQ ID
NO:2 or the mature CK.alpha.-5 amino acid sequence encoded by the
deposited cDNA clone either of which may be modified so as to
encode an amino terminal methionine or other N- or C-terminal
fusion peptides or polypeptides.
[0067] Further embodiments include an isolated polynucleotide
comprising a nucleotide sequence at least 90% identical, and more
preferably at least 95%, 96%, 97%, 98% or 99% identical to a
nucleic acid sequence selected from the group consisting of: (a) a
nucleotide sequence encoding the CK.alpha.-5 polypeptide shown as
SEQ ID NO:2; (b) a nucleotide sequence encoding the CK.alpha.-5
polypeptide shown as residues 1-254 of SEQ ID NO:2; (c) a
nucleotide sequence encoding the predicted mature CK.alpha.-5 shown
as residues 28-254 in SEQ ID NO:2; (d) a nucleotide sequence
encoding the predicted extracellular domain of the CK.alpha.-5
polypeptide shown as residues 28-205 in SEQ ID NO:2; (e) a
nucleotide sequence encoding the CK.alpha.-5 polypeptide having the
complete amino acid sequence encoded by the cDNA clone contained in
ATCC.RTM. Deposit No. 209231; (f) a nucleotide sequence encoding
the CK.alpha.-5 polypeptide having the complete amino acid sequence
excepting the N terminal methionine encoded by the cDNA clone
contained in ATCC.RTM. Deposit No. 209231; (g) a nucleotide
sequence encoding the mature CK.alpha.-5 polypeptide having the
amino acid sequence encoded by the cDNA clone contained in
ATCC.RTM. Deposit No. 209231; (h) a nucleotide sequence encoding
the extracellular domain of the CK.alpha.-5 polypeptide having the
amino acid sequence encoded by the cDNA clone contained in
ATCC.RTM. Deposit No. 209231; and (i) a nucleotide sequence
complementary to any of the nucleotide sequences in (a), (b), (c),
(d), (e), (O), (g) or (h) above.
[0068] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above,
or a polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e), (f),
(g), (h) or (i), above. This polynucleotide which hybridizes does
not hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A
residues or of only T residues. An additional nucleic acid
embodiment of the invention relates to an isolated nucleic acid
molecule comprising a polynucleotide which encodes the amino acid
sequence of an epitope-bearing portion of a CK.alpha.-5 polypeptide
having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g)
or (h) above.
[0069] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of CK.alpha.-5 polypeptides or peptides
by recombinant techniques.
[0070] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a CK.alpha.-5 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the CK.alpha.-5 polypeptide. In other
words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0071] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the nucleotide sequence shown in FIG. 1 or to the
nucleotides sequence of the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics
2:482-489 (1981), to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for
instance, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0072] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) or to the
nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having CK.alpha.-5 activity. This
is because even where a particular nucleic acid molecule does not
encode a polypeptide having CK.alpha.-5 activity, one of skill in
the art would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having CK.alpha.-5
activity include, inter alia, (1) isolating the CK.alpha.-5 gene or
allelic variants thereof in a cDNA library; (2) in situ
hybridization (e.g., "FISH") to metaphase chromosomal spreads to
provide precise chromosomal location of the CK.alpha.-5 gene, as
described in Verma et al., Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York (1988); and Northern Blot
analysis for detecting CK.alpha.-5 mRNA expression in specific
tissues.
[0073] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) or to the
nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having CK.alpha.-5 protein activity. By "a
polypeptide having CK.alpha.-5 activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the extracellular portion of the complete CK.alpha.-5
protein. One such activity believed to be possessed by such
polypeptides is the ability to modulate colony formation of bone
marrow progenitor cells, as does Human Chemokine HCC-1. An in vitro
colony forming assay for measuring the extent of inhibition of
myeloid progenitor cells is described in Young et al., The Journal
of Immunology 155:2661-2667 (1995). Briefly, the assay involves
collecting human or mouse bone marrow cells and plating the same on
agar, adding one or more growth factors and either (1) transfected
host cell-supernatant containing CK.alpha.-5 protein (or a
candidate polypeptide) or (2) nontransfected host cell-supernatant
control, and measuring the effect on colony formation by murine and
human CFU-granulocyte-macrophages (CFU-GM), by human burst-forming
unit-erythroid (BFU-E), or by human CFU
granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM). Such
activity is useful for protecting myeloid progenitor cells during
chemotherapy.
[0074] CK.alpha.-5 protein modulates immune system cell
proliferation and differentiation in a dose-dependent manner in the
above-described assay. Thus, "a polypeptide having CK.alpha.-5
protein activity" includes polypeptides that also exhibit any of
the same myeloid progenitor modulation activities in the
above-described assays in a dose-dependent manner. Although the
degree of dose-dependent activity need not be identical to that of
the CK.alpha.-5 protein, preferably, "a polypeptide having
CK.alpha.-5 protein activity" will exhibit substantially similar
dose-dependence in a given activity as compared to the CK.alpha.-5
protein (i.e., the candidate polypeptide will exhibit greater
activity or not more than about 25-fold less and, preferably, not
more than about tenfold less activity relative to the reference
CK.alpha.-5 protein).
[0075] Like other CXC chemokines, CK.alpha.-5 exhibits activity on
leukocytes including for example monocytes, lymphocytes and
neutrophils. For this reason CK.alpha.-5 is active in directing the
proliferation, differentiation and migration of these cell types.
Such activity is useful for immune enhancement or suppression,
myeloprotection, stem cell mobilization, acute and chronic
inflammatory control and treatment of leukemia. Assays for
measuring such activity are known in the art. For example, see
Peters et al., Immun. Today 17:273 (1996); Young et al., J. Exp.
Med. 182:1111 (1995); Caux et al., Nature 390:258 (1992); and
Santiago-Schwarz et al., Adv. Exp. Med. Biol. 378:7 (1995)." The
assays and protocols from each of the forgoing journal articles are
incorporated herein by reference.
[0076] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in FIG. 1 (SEQ ID NO:1) will encode a polypeptide "having
CK.alpha.-5 protein activity." In fact, since degenerate variants
of these nucleotide sequences all encode the same polypeptide, this
will be clear to the skilled artisan even without performing the
above described comparison assay. It will be further recognized in
the art that, for such nucleic acid molecules that are not
degenerate variants, a reasonable number will also encode a
polypeptide having CK.alpha.-5 protein activity. This is because
the skilled artisan is fully aware of amino acid substitutions that
are either less likely or not likely to significantly effect
protein function (e.g., replacing one aliphatic amino acid with a
second aliphatic amino acid), as further described below.
Vectors and Host Cells
[0077] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of CK.alpha.-5 polypeptides or fragments thereof by
recombinant techniques. The vector may be, for example, a phage,
plasmid, viral or retroviral vector. Retroviral vectors may be
replication competent or replication defective. In the latter case,
viral propagation generally will occur only in complementing host
cells.
[0078] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0079] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0080] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
[0081] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc., supra; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic
vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from
Stratagene; and pSVK3, pBPV, pMSG and pSVL available from
Pharmacia. Other suitable vectors will be readily apparent to the
skilled artisan.
[0082] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0083] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to stabilize
and purify proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0084] The CK.alpha.-5 protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include: products purified from natural sources, including bodily
fluids, tissues and cells, whether directly isolated or cultured;
products of chemical synthetic procedures; and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non-glycosylated. In addition,
polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated
processes. Thus, it is well known in the art that the N-terminal
methionine encoded by the translation initiation codon generally is
removed with high efficiency from any protein after translation in
all eukaryotic cells. While the N-terminal methionine on most
proteins also is efficiently removed in most prokaryotes, for some
proteins this prokaryotic removal process is inefficient, depending
on the nature of the amino acid to which the N-terminal methionine
is covalently linked.
Polypeptides and Fragments
[0085] The invention further provides an isolated CK.alpha.-5
polypeptide having the amino acid sequence encoded by the deposited
cDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide or
polypeptide comprising a portion of the above polypeptides.
Variant and Mutant Polypeptides
[0086] To improve or alter the characteristics of CK.alpha.-5
polypeptides, protein engineering may be employed. Recombinant DNA
technology known to those skilled in the art can be used to create
novel mutant proteins or "muteins including single or multiple
amino acid substitutions, deletions, additions or fusion proteins.
Such modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
N-Terminal and C-Terminal Deletion Mutants
[0087] For instance, for many proteins, including the extracellular
domain of a membrane associated protein or the mature form(s) of a
secreted protein, it is known in the art that one or more amino
acids may be deleted from the N-terminus or C-terminus without
substantial loss of biological function. For instance, Ron et al.,
J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins
that had heparin binding activity even if 3, 8, or 27
amino-terminal amino acid residues were missing. In the present
case, since the protein of the invention is a member of the
chemokine polypeptide family, deletions of N-terminal amino acids
up to the first "Cys" required for formation of a disulfide bridge
may retain some biological activity such as receptor binding or
modulation of target cell activities. Polypeptides having further
N-terminal deletions including the cysteine residue at position 38
in FIG. 1 (position 11 in SEQ ID NO:2) would not be expected to
retain such biological activities because it is known that this
residue in a chemokine-related polypeptide is required for forming
a disulfide bridge to provide structural stability which is needed
for receptor binding and signal transduction.
[0088] However, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification of loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or mature form of the protein generally will
be retained when less than the majority of the residues of the
complete or mature protein are removed from the N-terminus. Whether
a particular polypeptide lacking N-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
[0089] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the CK.alpha.-5 protein
shown in SEQ ID NO:2, up to the cysteine residue at position number
38, and polynucleotides encoding such polypeptides. In particular,
the present invention provides polypeptides comprising the amino
acid sequence of residues n-205 of SEQ ID NO:2, where n is an
integer in the range of 24-38, and Cys-38 is the position of the
first residue from the N-terminus of the extracellular domain of
the CK.alpha.-5 polypeptide (shown in SEQ ID NO:2) believed to be
required for receptor binding activity of the CK.alpha.-5
protein.
[0090] More in particular, the invention provides polypeptides
comprising an amino acid sequence selected from the group
consisting of: 24 to 205, 25 to 205, 26 to 205, 27 to 205, 28 to
205, 29 to 205, 30 to 205, 31 to 205, 32 to 205, 33 to 205, 34 to
205, 35 to 205, 36 to 205, 37 to 205, and 38 to 205, all of SEQ ID
NO:2. Polynucleotides encoding such polypeptides are also
provided.
[0091] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance, Interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein
(Dobeli et al., J. Biotechnology 7:199-216 (1988). In the present
case, since the protein of the invention is a member of the
chemokine polypeptide family, deletions of C-terminal amino acids
up to the cysteine at position 82 of SEQ ID NO:2 may retain some
biological activity such as receptor binding or modulation of
target cell activities. Polypeptides having further C-terminal
deletions including the cysteine residue at position 82 of FIG. 1
(82 of SEQ ID NO:2) would not be expected to retain such biological
activities because it is known that this residue in a
chemokine-related polypeptide is required for forming a disulfide
bridge to provide structural stability which is needed for receptor
binding and signal transduction.
[0092] However, even if deletion of one or more amino acids from
the C-terminus of a protein results in modification of loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or mature form of the protein generally will
be retained when less than the majority of the residues of the
complete or mature protein are removed from the C-terminus. Whether
a particular polypeptide lacking C-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
[0093] Accordingly, the present invention further provides
polypeptides having one or more residues from the carboxy terminus
of the amino acid sequence of the CK.alpha.-5 shown in SEQ ID NO:2,
up to the cysteine residue at position 82 of SEQ ID NO:2, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides having the amino acid
sequence of residues (24)-m of the amino acid sequence in SEQ ID
NO:2, where m is any integer in the range of 82 to 205, and residue
cysteine-82 is the position of the first residue from the
C-terminus of the complete CK.alpha.-5 polypeptide (shown in SEQ ID
NO:2) believed to be required for receptor binding and target cell
modulation activities of the CK.alpha.-5 protein.
[0094] More in particular, the invention provides polypeptides
comprising an amino acid sequence selected from the group
consisting of: 24 to 82, 24 to 83, 24 to 84, 24 to 85, 24 to 86, 24
to 87, 24 to 88, 24 to 89, 24 to 90, 24 to 91, 24 to 92, 24 to 93,
24 to 94, 24 to 95, 24 to 96, 24 to 97, 24 to 98, 24 to 99, 24 to
100, 24 to 101, 24 to 102, 24 to 103, 24 to 104, 24 to 105, 24 to
106, 24 to 107, 24 to 108, 24 to 109, 24 to 110, 24 to 111, 24 to
112, 24 to 113, 24 to 114, 24 to 115, 24 to 116, 24 to 117, 24 to
118, 24 to 119, 24 to 120, 24 to 121, 24 to 122, 24 to 123, 24 to
124, 24 to 125, 24 to 126, 24 to 127, 24 to 128, 24 to 129, 24 to
130, 24 to 131, 24 to 132, 24 to 133, 24 to 134, 24 to 135, 24 to
136, 24 to 137, 24 to 138, 24 to 139, 24 to 140, 24 to 141, 24 to
142, 24 to 143, 24 to 144, 24 to 145, 24 to 146, 24 to 147, 24 to
148, 24 to 149, 24 to 150, 24 to 151, 24 to 152, 24 to 153, 24 to
154, 24 to 155, 24 to 156, 24 to 157, 24 to 158, 24 to 159, 24 to
160, 24 to 161, 24 to 162, 24 to 163, 24 to 164, 24 to 165, 24 to
166, 24 to 167, 24 to 168, 24 to 169, 24 to 170, 24 to 171, 24 to
172, 24 to 173, 24 to 174, 24 to 175, 24 to 176, 24 to 177, 24 to
178, 24 to 179, 24 to 180, 24 to 181, 24 to 182, 24 to 183, 24 to
184, 24 to 185, 24 to 186, 24 to 187, 24 to 188, 24 to 189, 24 to
190, 24 to 191, 24 to 192, 24 to 193, 24 to 194, 24 to 195, 24 to
196, 24 to 196, 24 to 197, 24 to 198, 24 to 199, 24 to 200, 24 to
201, 24 to 202, 24 to 203, 24 to 204, and 24 to 205, all of SEQ ID
NO:2. Polynucleotides encoding these polypeptides also are
provided.
[0095] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
the soluble form (extracellular domain), which may be described
generally as having residues n-m of SEQ ID NO:2, where n and m are
integers as described above. Particularly preferred are
polypeptides comprising an amino acid sequence selected from the
group consisting of: 38 to 82, 38 to 83, 37 to 84, 37 to 85, 37 to
86, 37 to 87, 37 to 88, 37 to 89, 36 to 90, 36 to 91, 36 to 92, 36
to 93, 36 to 94, 36 to 95, 36 to 96, 36 to 97, 36 to 98, 36 to 99,
36 to 100, 35 to 101, 35 to 102, 35 to 103, 35 to 104, 35 to 105,
35 to 106, 35 to 107, 35 to 108, 35 to 109, 35 to 110, 35 to 111,
34 to 112, 34 to 113, 34 to 114, 34 to 115, 34 to 116, 34 to 117,
34 to 118, 34 to 119, 34 to 120, 34 to 121, 34 to 122, 34 to 123,
34 to 124, 34 to 125, 34 to 126, 34 to 127, 33 to 128, 33 to 129,
33 to 130, 33 to 131, 33 to 132, 33 to 133, 33 to 134, 33 to 135,
33 to 136, 33 to 137, 33 to 138, 33 to 139, 33 to 140, 33 to 141,
33 to 142, 33 to 143, 33 to 144, 33 to 145, 33 to 146, 33 to 147,
33 to 148, 33 to 149, 33 to 150, 33 to 151, 33 to 152, 33 to 153,
33 to 154, 33 to 155, 33 to 156, 33 to 157, 33 to 158, 33 to 159,
33 to 160, 33 to 161, 33 to 162, 33 to 163, 33 to 164, 33 to 165,
33 to 166, 33 to 167, 33 to 168, 33 to 169, 33 to 170, 33 to 171,
33 to 172, 33 to 173, 33 to 174, 33 to 175, 33 to 176, 33 to 177,
33 to 178, 33 to 179, 33 to 180, 33 to 181, 33 to 182, 33 to 183,
33 to 184, 32 to 185, 32 to 186, 32 to 187, 32 to 188, 32 to 189,
32 to 190, 32 to 191, 32 to 192, 32 to 193, 32 to 194, 32 to 195,
32 to 196, 31 to 197, 31 to 198, 31 to 199, 31 to 200, 31 to 201,
31 to 202, 31 to 203, 31 to 204, and 31 to 205, all of SEQ ID NO:2.
Polynucleotides encoding these polypeptides also are provided.
[0096] Also included are a nucleotide sequence encoding a
polypeptide consisting of a portion of the complete CK.alpha.-5
amino acid sequence encoded by the cDNA clone contained in
ATCC.RTM. Deposit No. 209231, where this portion excludes from 23
to about 37 amino acids from the amino terminus of the
extracellular domain of the CK.alpha.-5 polypeptide encoded by the
cDNA clone contained in ATCC.RTM. Deposit No. 209231, or from 49 to
about 172 amino acids from the carboxy terminus, or any combination
of the above amino terminal and carboxy terminal deletions, of the
extracellular domain encoded by the cDNA clone contained in
ATCC.RTM. Deposit No. 209231. Polynucleotides encoding all of the
above deletion mutant polypeptide forms also are provided.
Other Mutants
[0097] In addition to terminal deletion forms of the protein
discussed above, it also will be recognized by one of ordinary
skill in the art that some amino acid sequences of the CK.alpha.-5
polypeptide can be varied without significant effect of the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity.
[0098] Thus, the invention further includes variations of the
CK.alpha.-5 polypeptide which show substantial CK.alpha.-5
polypeptide activity or which include regions of CK.alpha.-5
protein such as the protein portions discussed below. Such mutants
include deletions, insertions, inversions, repeats, and type
substitutions selected according to general rules known in the art
so as have little effect on activity. For example, guidance
concerning how to make phenotypically silent amino acid
substitutions is provided in Bowie, J. U. et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main approaches for studying the
tolerance of an amino acid sequence to change. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
[0099] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described in Bowie, J.
U. et al., supra, and the references cited therein. Typically seen
as conservative substitutions are those shown in Table 1.
[0100] Thus, the fragment, derivative or analog of the polypeptide
of SEQ D NO:2, or that encoded by the deposited cDNA, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the above
form of the polypeptide, such as an IgG Fe fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the above form of the polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are deemed to be
within the scope of those skilled in the art from the teachings
herein
[0101] Thus, the CK.alpha.-5 of the present invention may include
one or more amino acid substitutions, deletions or additions,
either from natural mutations or human manipulation. As indicated,
changes are preferably of a minor nature, such as conservative
amino acid substitutions that do not significantly affect the
folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0102] Amino acids in the CK.alpha.-5 protein of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as
receptor binding or in vitro or in vitro proliferative
activity.
[0103] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).
[0104] Replacement of amino acids can also change the selectivity
of the binding of a ligand to cell surface receptors. For example,
Ostade et al., Nature 361:266-268 (1993) describes certain
mutations resulting in selective binding of TNF-.alpha. to only one
of the two known types of TNF receptors. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
[0105] As described above CK.alpha.-5 lacks an ELR motif. By making
a specific mutation in CK.alpha.-5 to include an ELR motif in the
position where such a motif is typically found in related
chemokines, CK.alpha.-5 will act as an antagonist, thus possessing
angiogenic activity. Accordingly, polypeptides of the present
invention include CK.alpha.-5-ELR mutants. Such CK.alpha.-5-EILR
mutants are comprised of the full-length or preferably the mature
CK.alpha.-5 protein. Further, since CK.alpha.-5 is a member of the
chemokine-related protein family, to modulate rather than
completely eliminate biological activities of CK.alpha.-5
preferably mutations are made in sequences encoding amino acids in
the CK.alpha.-5 extracellular domain, i.e., in positions 28 to 205
of SEQ ID NO:2, more preferably in residues within this region
which are not conserved in all members of the chemokine family. As
is known in the art, the four spatially conserved cysteines present
in all chemokines, positions 38, 40, 68 and 82 in CK.alpha.-5, are
required for the formation of two disulfide bridges. Thus, it is
preferable not to alter any of the four cysteine residues located
at positions 38, 40, 68 and 82 in SEQ ID NO:2. Also forming part of
the present invention are isolated polynucleotides comprising
nucleic acid sequences which encode the above CK.alpha.-5
mutants.
[0106] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the CK.alpha.-5
polypeptide can be substantially purified by the one-step method
described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides
of the invention also can be purified from natural or recombinant
sources using anti-CK.alpha.-5 antibodies of the invention in
methods which are well known in the art of protein
purification.
[0107] The invention further provides an isolated CK.alpha.-5
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of the full-length
CK.alpha.-5 polypeptide having the complete amino acid sequence
shown in SEQ ID NO:2 or the complete amino acid sequence encoded by
the cDNA clone contained in the ATCC.RTM. Deposit No. 209231; (b)
the amino acid sequence of the full-length CK.alpha.-5 polypeptide
having the complete amino acid sequence shown in SEQ ID NO:2
excepting the N-terminal methionine (i.e., positions 2 to 254 of
SEQ ID NO:2) or the complete amino acid sequence excepting the N
terminal methionine encoded by the cDNA clone contained in the
ATCC.RTM. Deposit No. 209231; (c) the amino acid sequence of the
mature CK.alpha.-5 polypeptide having the amino acid sequence shown
in SEQ ID NO:2 as residues 28-254 or the amino acid sequence of the
mature CK.alpha.-5 encoded by the cDNA clone contained in the
ATCC.RTM. Deposit No. 209231; and (d) the amino acid sequence of
the extracellular domain of the CK.alpha.-5 polypeptide having the
amino acid sequence shown in SEQ ID NO:2 as residues 28-205 or the
amino acid sequence of the extracellular domain of the CK.alpha.-5
polypeptide encoded by the cDNA clone contained in the ATCC.RTM.
Deposit No. 209231.
[0108] Further polypeptides of the present invention include
polypeptides which have at least 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above. The polypeptides of
the invention also comprise those which are at least 80% identical,
more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to the
polypeptide encoded by the deposited cDNA or to the polypeptide of
SEQ ID NO:2, and also include portions of such polypeptides with at
least 30 amino acids and more preferably at least 50 amino acids.
Polynucleotides encoding such variant polypeptides are also
provided.
[0109] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489, 1981) to find the best segment of
similarity between two sequences.
[0110] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
CK.alpha.-5 polypeptide is intended that the amino acid sequence of
the polypeptide is identical to the reference sequence except that
the polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the reference amino acid of
the CK.alpha.-5 polypeptide. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to
a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0111] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in SEQ ID NO:2 or to the amino acid
sequence encoded by deposited cDNA clone can be determined
conventionally using known computer programs such the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0112] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0113] As described in detail below, the polypeptides of the
present invention can also be used to raise polyclonal and
monoclonal antibodies, which are useful in assays for detecting
CK.alpha.-5 protein expression as described below or as agonists
and antagonists capable of enhancing or inhibiting CK.alpha.-5
protein function. Further, such polypeptides can be used in the
yeast two-hybrid system to "capture" CK.alpha.-5 protein binding
proteins which are also candidate agonists and antagonists
according to the present invention. The yeast two hybrid system is
described in Fields and Song, Nature 340:245-246 (1989).
Epitope-Bearing Portions
[0114] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0115] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) "Antibodies that react with predetermined sites on
proteins," Science, 219:660-666. Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Antigenic epitope-bearing peptides and
polypeptides of the invention are therefore useful to raise
antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et
al., Cell 37:767-778 (1984) at 777.
[0116] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate
CK.alpha.-5-specific antibodies include: a polypeptide comprising
amino acid residues from about Tyr-39 to about Ser-51 in SEQ ID
NO:2; a polypeptide comprising amino acid residues from about
Cys-82 to about Val-90 in SEQ ID NO:2; a polypeptide comprising
amino acid residues from about Thr-91 to about Pro-110 in SEQ ID
NO:2; a polypeptide comprising amino acid residues from about
Ser-120 to about Ile-145; a polypeptide comprising amino acid
residues from about Asp4 to about Ser-9; a polypeptide comprising
amino acid residues from about Lys-42 to about Pro-50; a
polypeptide comprising amino acid residues from about Gly-83 to
about Pro-88; a polypeptide comprising amino acid residues from
about Glu-128 to about Asp-133; a polypeptide comprising amino acid
residues from about Ser-159 to about Thr-170; a polypeptide
comprising amino acid residues from about Glu-184 to about Pro-198;
a polypeptide comprising amino acid residues from about Lys-228 to
about Asp-240; and a polypeptide comprising amino acid residues
from about Pro-250 to about Asn-253. These polypeptide fragments
have been determined to bear antigenic epitopes of the CK.alpha.-5
protein by the analysis of the Jameson-Wolf antigenic index, as
shown in FIG. 3, above.
[0117] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. See, e.g.,
Houghten, R. A. (1985) "General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids." Proc. Natl. Acad. Sci. USA 82:5131-5135; this "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
[0118] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al, Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2547-2354 (1985).
Immunogenic epitope-bearing peptides of the invention, i.e., those
parts of a protein that elicit an antibody response when the whole
protein is the immunogen, are identified according to methods known
in the art. See, for instance, Geysen et al., supra. Further still,
U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method
of detecting or determining the sequence of monomers (amino acids
or other compounds) which is a topological equivalent of the
epitope (i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
Fusion Proteins
[0119] As one of skill in the art will appreciate, CK.alpha.-5
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EP A 394,827;
Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG part can
also be more efficient in binding and neutralizing other molecules
than the monomeric CK.alpha.-5 protein or protein fragment alone
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).
Antibodies
[0120] CK.alpha.-5-protein specific antibodies for use in the
present invention can be raised against the intact CK.alpha.-5
protein or an antigenic polypeptide fragment thereof, which may be
presented together with a carrier protein, such as an albumin, to
an animal system (such as rabbit or mouse) or, if it is long enough
(at least about 25 amino acids), without a carrier.
[0121] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab')2
fragments) which are capable of specifically binding to CK.alpha.-5
protein. Fab and F(ab')2 fragments lack the Fc fragment of intact
antibody, clear more rapidly from the circulation, and may have
less non-specific tissue binding of an intact antibody (Wahl et
al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are
preferred.
[0122] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
CK.alpha.-5 protein or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of CK.alpha.-5 protein is prepared and purified to
render it substantially free of natural contaminants. Such a
preparation is then introduced into an animal in order to produce
polyclonal antisera of greater specific activity.
[0123] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or CK.alpha.-5 protein binding
fragments thereof). Such monoclonal antibodies can be prepared
using hybridoma technology (Kohler et al., Nature 256:495 (1975);
Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur.
J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp.
563-681). In general, such procedures involve immunizing an animal
(preferably a mouse) with a CK.alpha.-5 protein antigen or, more
preferably, with a CK.alpha.-5 protein-expressing cell. Suitable
cells can be recognized by their capacity to bind anti-CK.alpha.-5
protein antibody. Such cells may be cultured in any suitable tissue
culture medium; however, it is preferable to culture cells in
Earle's modified Eagle's medium supplemented with 10% fetal bovine
serum (inactivated at about 56.degree. C.), and supplemented with
about 10 g/l of nonessential amino acids, about 1,000 U/ml of
penicillin, and about 100 .mu.g/ml of streptomycin. The splenocytes
of such mice are extracted and fused with a suitable myeloma cell
line. Any suitable myeloma cell line may be employed in accordance
with the present invention; however, it is preferable to employ the
parent myeloma cell line (SP2O), available from the American Type
Culture Collection, Rockville, Md. After fusion, the resulting
hybridoma cells are selectively maintained in HAT medium, and then
cloned by limiting dilution as described by Wands et al.
(Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained
through such a selection are then assayed to identify clones which
secrete antibodies capable of binding the CK.alpha.-5 protein
antigen.
[0124] Alternatively, additional antibodies capable of binding to
the CK.alpha.-5 protein antigen may be produced in a two-step
procedure through the use of anti-idiotypic antibodies. Such a
method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
CK.alpha.-5-protein specific antibodies are used to immunize an
animal, preferably a mouse. The splenocytes of such an animal are
then used to produce hybridoma cells, and the hybridoma cells are
screened to identify clones which produce an antibody whose ability
to bind to the CK.alpha.-5 protein-specific antibody can be blocked
by the CK.alpha.-5 protein antigen. Such antibodies comprise
anti-idiotypic antibodies to the CK.alpha.-5 protein-specific
antibody and can be used to immunize an animal to induce formation
of further CK.alpha.-5 protein-specific antibodies.
[0125] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, CK.alpha.-5 protein-binding fragments
can be produced through the application of recombinant DNA
technology or through synthetic chemistry.
[0126] For in vivo use of anti-CK.alpha.-5 in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. See, for review, Morrison, Science 229:1202 (1985); Oi et al.,
BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).
Immune System-Related Disorders
Diagnosis
[0127] The present inventors have discovered that CK.alpha.-5 is
expressed in neutrophils and monocytes. For a number of immune
system-related disorders, substantially altered (increased or
decreased) levels of CK.alpha.-5 gene expression can be detected in
immune system tissue or other cells or bodily fluids (e.g., sera,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to a "standard"
CK.alpha.-5 gene expression level, that is, the CK.alpha.-5
expression level in immune system tissues or bodily fluids from an
individual not having the immune system disorder. Thus, the
invention provides a diagnostic method useful during diagnosis of
an immune system disorder, which involves measuring the expression
level of the gene encoding the CK.alpha.-5 protein in immune system
tissue or other cells or body fluid from an individual and
comparing the measured gene expression level with a standard
CK.alpha.-5 gene expression level, whereby an increase or decrease
in the gene expression level compared to the standard is indicative
of an immune system disorder.
[0128] In particular, it is believed that certain tissues in
mammals with cancer, particularly osteosarcoma, express
significantly altered levels of the CK.alpha.-5 protein and mRNA
encoding the CK.alpha.-5 protein when compared to a corresponding
"standard" level. Further, it is believed that altered levels of
the CK.alpha.-5 protein can be detected in certain body fluids
(e.g., sera, plasma, urine, and spinal fluid) from mammals with
such a cancer when compared to sera from mammals of the same
species not having the cancer.
[0129] Thus, the invention provides a diagnostic method useful
during diagnosis of an immune system disorder, including cancers of
the skeletal and immune systems, which involves measuring the
expression level of the gene encoding the CK.alpha.-5 protein in
immune or skeletal system tissue or other cells or body fluid from
an individual and comparing the measured gene expression level with
a standard CK.alpha.-5 gene expression level, whereby an increase
or decrease in the gene expression level compared to the standard
is indicative of an immune system disorder.
[0130] Where a diagnosis of a disorder in the immune system,
including diagnosis of a tumor, has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting enhanced or
depressed CK.alpha.-5 gene expression will experience a worse
clinical outcome relative to patients expressing the gene at a
level nearer the standard level.
[0131] By "assaying the expression level of the gene encoding the
CK.alpha.-5 protein" is intended qualitatively or quantitatively
measuring or estimating the level of the CK.alpha.-5 protein or the
level of the mRNA encoding the CK.alpha.-5 protein in a first
biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the CK.alpha.-5 protein level or mRNA level
in a second biological sample). Preferably, the CK.alpha.-5 protein
level or mRNA level in the first biological sample is measured or
estimated and compared to a standard CK.alpha.-5 protein level or
mRNA level, the standard being taken from a second biological
sample obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having a disorder of the immune system. As will be appreciated in
the art, once a standard CK.alpha.-5 protein level or mRNA level is
known, it can be used repeatedly as a standard for comparison.
[0132] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains CK.alpha.-5 protein or mRNA. As
indicated, biological samples include body fluids (such as sera,
plasma, urine, synovial fluid and spinal fluid) which contain free
CK.alpha.-5 protein, immune and skeletal system tissue, and other
tissue sources found to express complete mature CK.alpha.-5 or a
CK.alpha.-5 receptor. Methods for obtaining tissue biopsies and
body fluids from mammals are well known in the art. Where the
biological sample is to include mRNA, a tissue biopsy is the
preferred source.
[0133] The present invention is useful for diagnosis or treatment
of various immune system-related disorders in mammals, preferably
humans. Such disorders include tumors, cancers, sarcomas such as
osteosarcoma, interstitial lung disease (such as Langerhans cell
granulomatosis) and any disregulation of immune cell function
including, but not limited to, autoimmunity, arthritis, leukemias,
lymphomas, immunosuppression, immunity, humoral immunity,
inflammatory bowel disease, myelo suppression, and the like.
[0134] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the CK.alpha.-5 protein are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0135] Assaying CK.alpha.-5 protein levels in a biological sample
can occur using antibody-based techniques. For example, CK.alpha.-5
protein expression in tissues can be studied with classical
immunohistological methods (Jalkanen, M., et al., J. Cell. Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting CK.alpha.-5 protein gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0136] In addition to assaying CK.alpha.-5 protein levels in a
biological sample obtained from an individual, CK.alpha.-5 protein
can also be detected in vivo by imaging. Antibody labels or markers
for in vivo imaging of CK.alpha.-5 protein include those detectable
by X-radiography, NMR or ESR. For X-radiography, suitable labels
include radioisotopes such as barium or cesium, which emit
detectable radiation but are not overtly harmful to the subject.
Suitable markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma.
[0137] A CK.alpha.-5 protein-specific antibody or antibody fragment
which has been labeled with an appropriate detectable imaging
moiety, such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined for immune system disorder. It will be understood in
the art that the size of the subject and the imaging system used
will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of .sup.99mTc. The labeled
antibody or antibody fragment will then preferentially accumulate
at the location of cells which contain CK.alpha.-5 protein. In vivo
tumor imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982)).
Treatment
[0138] As noted above, CK.alpha.-5 polynucleotides and polypeptides
are useful for diagnosis of conditions involving abnormally high or
low expression of CK.alpha.-5 activities. Given the cells and
tissues where CK.alpha.-5 is expressed as well as the activities
modulated by CK.alpha.-5, it is readily apparent that a
substantially altered (increased or decreased) level of expression
of CK.alpha.-5 in an individual compared to the standard or
"normal" level produces pathological conditions related to the
bodily system(s) in which CK.alpha.-5 is expressed and/or is
active.
[0139] It will also be appreciated by one of ordinary skill that,
since the CK.alpha.-5 protein of the invention is a member of the
chemokine family the mature secreted form of the protein may be
released in soluble form from the cells which express the
CK.alpha.-5 by proteolytic cleavage. Therefore, when CK.alpha.-5 is
added from an exogenous source to cells, tissues or the body of an
individual, the protein will exert its physiological activities on
its target cells of that individual. Also, cells expressing this
transmembrane protein may be added to cells, tissues or the body of
an individual and these added cells will bind to cells expressing
receptor for CK.alpha.-5, whereby the cells expressing CK.alpha.-5
can cause actions, e.g., cell stimulation, on the receptor-bearing
target cells.
[0140] Therefore, it will be appreciated that conditions caused by
a decrease in the standard or normal level of CK.alpha.-5 activity
in an individual, particularly disorders of the immune system, can
be treated by administration of CK.alpha.-5 polypeptide in the form
of the soluble extracellular domain or cells expressing the
complete protein. Thus, the invention also provides a method of
treatment of an individual in need of an increased level of
CK.alpha.-5 activity comprising administering to such an individual
a pharmaceutical composition comprising an amount of an isolated
CK.alpha.-5 polypeptide of the invention, effective to increase the
CK.alpha.-5 activity level in such an individual.
[0141] Since CK.alpha.-5 lacks an ELR motif, it also should be an
angiostatic factor rather than an angiogenic factor. In addition,
since CK.alpha.-5 inhibits endothelial cell function, it will have
a wide range of anti-inflammatory activities. CK.alpha.-5 may be
employed as an anti-neovascularizing agent to treat solid tumors by
stimulating the invasion and activation of host defense cells,
e.g., cytotoxic T cells and macrophages and by inhibiting the
angiogenesis of tumors. Those of skill in the art will recognize
other non-cancer indications where blood vessel proliferation is
not wanted. They may also be employed to enhance host defenses
against resistant chronic and acute infections, for example,
myobacterial infections via the attraction and activation of
microbicidal leukocytes. CK.alpha.-5 may also be employed to
inhibit T-cell proliferation by the inhibition of IL-2 biosynthesis
for the treatment of T-cell mediated auto-immune diseases and
lymphocytic leukemias. CK.alpha.-5 may also be employed to
stimulate wound healing, both via the recruitment of debris
clearing and connective tissue promoting inflammatory cells and
also via its control of excessive TGF-mediated fibrosis. In this
same manner, CK.alpha.-5 may also be employed to treat other
fibrotic disorders, including liver cirrhosis, osteoarthritis and
pulmonary fibrosis. CK.alpha.-5 also increases the presence of
eosinophils which have the distinctive function of killing the
larvae of parasites that invade tissues, as in schistosomiasis,
trichinosis and ascariasis. It may also be employed to regulate
hematopoiesis, by regulating the activation and differentiation of
various hematopoietic progenitor cells, for example, to release
mature leukocytes from the bone marrow following chemotherapy,
i.e., in stem cell mobilization. CK.alpha.-5 may also be employed
to treat sepsis. Also, by making a specific mutation to include an
ELR motif, CK.alpha.-5-ELR will have angiogenic activities which
are useful for treating all of the disease states where
angiogenesis would be beneficial, i.e., to promote wound healing,
re-vascularization of damaged limbs from injury or disease, and
others known to those of skill in the art.
Formulations
[0142] The CK.alpha.-5 polypeptide composition will be formulated
and dosed in a fashion consistent with good medical practice,
taking into account the clinical condition of the individual
patient (especially the side effects of treatment with CK.alpha.-5
polypeptide alone), the site of delivery of the CK.alpha.-5
polypeptide composition, the method of administration, the
scheduling of administration, and other factors known to
practitioners. The "effective amount" of CK.alpha.-5 polypeptide
for purposes herein is thus determined by such considerations.
[0143] As a general proposition, the total pharmaceutically
effective amount of CK.alpha.-5 polypeptide administered
parenterally per dose will be in the range of about 1 g/kg/day to
10 mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the CK.alpha.-5 polypeptide is typically administered
at a dose rate of about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour,
either by 1-4 injections per day or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed. The length of treatment needed to
observe changes and the interval following treatment for responses
to occur appears to vary depending on the desired effect.
[0144] Pharmaceutical compositions containing the CK.alpha.-5 of
the invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0145] The CK.alpha.-5 polypeptide is also suitably administered by
sustained-release systems. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or microcapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,
U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277
(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (R. Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
CK.alpha.-5 polypeptide compositions also include liposomally
entrapped CK.alpha.-5 polypeptide. Liposomes containing CK.alpha.-5
polypeptide are prepared by methods known per se: DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985);
Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP
52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.
Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324. Ordinarily, the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater
than about 30 mol. percent cholesterol, the selected proportion
being adjusted for the optimal CK.alpha.-5 polypeptide therapy.
[0146] For parenteral administration, in one embodiment, the
CK.alpha.-5 polypeptide is formulated generally by mixing it at the
desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
[0147] Generally, the formulations are prepared by contacting the
CK.alpha.-5 polypeptide uniformly and intimately with liquid
carriers or finely divided solid carriers or both. Then, if
necessary, the product is shaped into the desired formulation.
Preferably the carrier is a parenteral carrier, more preferably a
solution that is isotonic with the blood of the recipient. Examples
of such carrier vehicles include water, saline, Ringer's solution,
and dextrose solution. Non-aqueous vehicles such as fixed oils and
ethyl oleate are also useful herein, as well as liposomes.
[0148] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0149] The CK.alpha.-5 polypeptide is typically formulated in such
vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml,
preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of
CK.alpha.-5 polypeptide salts. CK.alpha.-5 polypeptide to be used
for therapeutic administration must be sterile. Sterility is
readily accomplished by filtration through sterile filtration
membranes (e.g., 0.2 micron membranes). Therapeutic CK.alpha.-5
polypeptide 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. CK.alpha.-5 polypeptide ordinarily will be stored in unit
or multi-dose containers, for example, sealed ampoules or vials, as
an aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous
CK.alpha.-5 polypeptide solution, and the resulting mixture is
lyophilized. The infusion solution is prepared by reconstituting
the lyophilized CK.alpha.-5 polypeptide using bacteriostatic
Water-for-Injection.
[0150] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
Agonists and Antagonists--Assays and Molecules
[0151] The invention also provides a method of screening compounds
to identify those which enhance or block the action of CK.alpha.-5
on cells, such as its interaction with CK.alpha.-5-binding
molecules such as receptor molecules. An agonist is a compound
which increases the natural biological functions of CK.alpha.-5 or
which functions in a manner similar to CK.alpha.-5, while
antagonists decrease or eliminate such functions.
[0152] In another aspect of this embodiment the invention provides
a method for identifying a receptor protein or other ligand-binding
protein which binds specifically to a CK.alpha.-5 polypeptide. For
example, a cellular compartment, such as a membrane or a
preparation thereof, may be prepared from a cell that expresses a
molecule that binds CK.alpha.-5. The preparation is incubated with
labeled CK.alpha.-5 CK.alpha.-5 and complexes of CK.alpha.-5 bound
to the receptor or other binding protein are isolated and
characterized according to routine methods known in the art.
Alternatively, the CK.alpha.-5 polypeptide may be bound to a solid
support so that binding molecules solubilized from cells are bound
to the column and then eluted and characterized according to
routine methods.
[0153] In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
CK.alpha.-5, such as a molecule of a signaling or regulatory
pathway modulated by CK.alpha.-5. The preparation is incubated with
labeled CK.alpha.-5 in the absence or the presence of a candidate
molecule which may be a CK.alpha.-5 agonist or antagonist. The
ability of the candidate molecule to bind the binding molecule is
reflected in decreased binding of the labeled ligand. Molecules
which bind gratuitously, i.e., without inducing the effects of
CK.alpha.-5 on binding the CK.alpha.-5 binding molecule, are most
likely to be good antagonists. Molecules that bind well and elicit
effects that are the same as or closely related to CK.alpha.-5 are
agonists.
[0154] CK.alpha.-5-like effects of potential agonists and
antagonists may by measured, for instance, by determining activity
of a second messenger system following interaction of the candidate
molecule with a cell or appropriate cell preparation, and comparing
the effect with that of CK.alpha.-5 or molecules that elicit the
same effects as CK.alpha.-5. Second messenger systems that may be
useful in this regard include but are not limited to AMP guanylate
cyclase, ion channel or phosphoinositide hydrolysis second
messenger systems.
[0155] Another example of an assay for CK.alpha.-5 antagonists is a
competitive assay that combines CK.alpha.-5 and a potential
antagonist with membrane-bound CK.alpha.-5 receptor molecules or
recombinant CK.alpha.-5 receptor molecules under appropriate
conditions for a competitive inhibition assay. CK.alpha.-5 can be
labeled, such as by radioactivity, such that the number of
CK.alpha.-5 molecules bound to a receptor molecule can be
determined accurately to assess the effectiveness of the potential
antagonist.
[0156] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
receptor molecule, without inducing CK.alpha.-5-induced activities,
thereby preventing the action of CK.alpha.-5 by excluding
CK.alpha.-5 from binding.
[0157] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56:
560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression." CRC Press, Boca Raton, Fla. (1988). Triple helix
formation is discussed in, for instance Lee et al., Nucleic Acids
Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);
and Dervan et al., Science 251: 1360 (1991). The methods are based
on binding of a polynucleotide to a complementary DNA or RNA. For
example, the 5' coding portion of a polynucleotide that encodes the
mature polypeptide of the present invention may be used to design
an antisense RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is designed to be complementary to
a region of the gene involved in transcription thereby preventing
transcription and the production of CK.alpha.-5. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into CK.alpha.-5 polypeptide. The
oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of CK.alpha.-5 protein.
[0158] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described above.
[0159] The antagonists may be employed for instance to inhibit the
chemotaxis and activation of macrophages and their precursors, and
of neutrophils, basophils, B lymphocytes and some T-cell subsets,
e.g., activated and CD8 cytotoxic T cells and natural killer cells,
in certain auto-immune and chronic inflammatory and infective
diseases. Examples of auto-immune diseases include multiple
sclerosis, and insulin-dependent diabetes. The antagonists may also
be employed to treat infectious diseases including silicosis,
sarcoidosis, idiopathic pulmonary fibrosis by preventing the
recruitment and activation of mononuclear phagocytes. They may also
be employed to treat idiopathic hyper-eosinophilic syndrome by
preventing eosinophil production and migration. Endotoxic shock may
also be treated by the antagonists by preventing the migration of
macrophages and their production of the human chemokine
polypeptides of the present invention. The antagonists may also be
employed for treating atherosclerosis, by preventing monocyte
infiltration in the artery wall. The antagonists may also be
employed to treat histamine-mediated allergic reactions and
immunological disorders including late phase allergic reactions,
chronic urticaria, and atopic dermatitis by inhibiting
chemokine-induced mast cell and basophil degranulation and release
of histamine. IgE-mediated allergic reactions such as allergic
asthma, rhinitis, and eczema may also be treated. The antagonists
may also be employed to treat chronic and acute inflammation by
preventing the attraction of monocytes to a wound area. They may
also be employed to regulate normal pulmonary macrophage
populations, since chronic and acute inflammatory pulmonary
diseases are associated with sequestration of mononuclear
phagocytes in the lung. Antagonists may also be employed to treat
rheumatoid arthritis by preventing the attraction of monocytes into
synovial fluid in the joints of patients. Monocyte influx and
activation plays a significant role in the pathogenesis of both
degenerative and inflammatory arthropathies. The antagonists may be
employed to interfere with the deleterious cascades attributed
primarily to IL-1 and TNF, which prevents the biosynthesis of other
inflammatory cytokines. In this way, the antagonists may be
employed to prevent inflammation. The antagonists may also be
employed to inhibit prostaglandin-independent fever induced by
chemokines. The antagonists may also be employed to treat cases of
bone marrow failure, for example, aplastic anemia and
myelodysplastic syndrome. The antagonists may also be employed to
treat asthma and allergy by preventing eosinophil accumulation in
the lung. The antagonists may also be employed to treat
subepithelial basement membrane fibrosis which is a prominent
feature of the asthmatic lung. Antibodies against CK.alpha.-5 may
be employed to bind to and inhibit CK.alpha.-5 activity to treat
ARDS, by preventing infiltration of neutrophils into the lung after
injury. Any of the above antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
hereinafter described.
Gene Mapping
[0160] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0161] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a CK.alpha.-5
protein gene. This can be accomplished using a variety of well
known techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose.
[0162] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Fluorescence
in situ hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes from
the cDNA as short as 50 or 60 bp. For a review of this technique,
see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,
Pergamon Press, New York (1988).
[0163] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0164] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0165] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1(a)
Expression and Purification of "His-tagged" CK.alpha.-5 in E.
coli
[0166] The bacterial expression vector pQE60 is used for bacterial
expression in this example. (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibiotic
resistance ("Ampr") and contains a bacterial origin of replication
("ori"), an IPTG inducible promoter, a ribosome binding site
("RBS"), six codons encoding histidine residues that allow affinity
purification using nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin sold by QIAGEN, Inc., supra, and suitable single
restriction enzyme cleavage sites. These elements are arranged such
that an inserted DNA fragment encoding a polypeptide expresses that
polypeptide with the six His residues (i.e., a "6.times.His tag")
covalently linked to the carboxyl terminus of that polypeptide.
[0167] The DNA sequence encoding the desired portion CK.alpha.-5
protein lacking the hydrophobic leader sequence and transmembrane
domain is amplified from the deposited cDNA clone using PCR
oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the CK.alpha.-5 protein and to
sequences in the deposited construct 3' to the cDNA coding
sequence. Additional nucleotides containing restriction sites to
facilitate cloning in the pQE60 vector are added to the 5' and 3'
sequences, respectively.
[0168] For cloning the mature protein, the 5' primer has the
sequence 5' GCGCCATGGAATGGCAACGAGGGCAGC 3' (SEQ ID NO: 15)
containing the underlined NcoI restriction site. One of ordinary
skill in the art would appreciate, of course, that the point in the
protein coding sequence where the 5' primer begins may be varied to
amplify a DNA segment encoding any desired portion of the complete
protein shorter or longer than the mature form. The 3' primer has
the sequence 5' CGCAAGCTTTTATGTGGCTGATGTCCTGGC 3' (SEQ ID NO: 16)
containing the underlined HindIII restriction.
[0169] The amplified CK.alpha.-5 DNA fragment and the vector pQE60
are digested with NcoI and HindIII and the digested DNAs are then
ligated together. Insertion of the CK.alpha.-5 DNA into the
restricted pQE60 vector places the CK.alpha.-5 protein coding
region downstream from the IPTG-inducible promoter and in-frame
with an initiating AUG and the six histidine codons.
[0170] Alternatively, a preferred bacterial expression vector
"pHE4-5" containing an ampicillin resistance gene may be used in
this example. pHE4-5/MPIFD23 vector plasmid DNA contains a filler
insert between unique restriction enzyme sites NdeI and Asp718 and
was deposited with the American Type Culture Collection, 12301 Park
Lawn Drive, Rockville, Md. 20852, on Sep. 30, 1997 and given
Accession No. 209311. Using 5' and 3' primers described herein with
restriction enzyme sites for NdeI and Asp 718 substituted for the
NcoI and HindIII sites in the respective primers, a suitable
CK.alpha.-5 encoding DNA fragment for subcloning into pHE4-5 can be
amplified. The stuffer DNA insert in pHE4-5/MPIFD23 should be
removed prior to ligating the CK.alpha.-5 fragment to pHE4-5.
pHE4-5 contains a strong bacterial promoter allowing for high
yields of most heterologous proteins.
[0171] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E.
coli strain M15/rep4, containing multiple copies of the plasmid
pREP4, which expresses the lac repressor and confers kanamycin
resistance ("Kanr"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing CK.alpha.-5 protein, is available
commercially from QIAGEN, Inc., supra. Transformants are identified
by their ability to grow on LB plates in the presence of ampicillin
and kanamycin. Plasmid DNA is isolated from resistant colonies and
the identity of the cloned DNA confirmed by restriction analysis,
PCR and DNA sequencing.
[0172] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-.beta.-D-thiogalactopyranoside ("IPTG") is then added to
a final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0173] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the CK.alpha.-5 is
loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity
resin column (available from QIAGEN, Inc., supra). Proteins with a
6.times.His tag bind to the Ni-NTA resin with high affinity and can
be purified in a simple one-step procedure (for details see: The
QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the
supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8,
the column is first washed with 10 volumes of 6 M guanidine-HCl, pH
8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and
finally the CK.alpha.-5 is eluted with 6 M guanidine-HCl, pH 5.
[0174] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM immidazole.
Immidazole is removed by a final dialyzing step against PBS or 50
mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified
protein is stored at 4.degree. C. or frozen at -80.degree. C.
[0175] The following alternative method may be used to purify
CK.alpha.-5 expressed in E coli when it is present in the form of
inclusion bodies. Unless otherwise specified, all of the following
steps are conducted at 4-10.degree. C.
[0176] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
[0177] The cells ware then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0178] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the CK.alpha.-5 polypeptide-containing supernatant is incubated
at 4.degree. C. overnight to allow further GuHCl extraction.
[0179] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0180] To clarify the refolded CK.alpha.-5 polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
[0181] Fractions containing the CK.alpha.-5 polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the CK.alpha.-5 polypeptide (determined, for instance,
by 16% SDS-PAGE) are then pooled.
[0182] The resultant CK.alpha.-5 polypeptide exhibits greater than
95% purity after the above refolding and purification steps. No
major contaminant bands are observed from Commassie blue stained
16% SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 ng/ml according to
LAL assays.
Example 2
Cloning and Expression of CK.alpha.-5 Protein in a Baculovirus
Expression System
[0183] In this illustrative example, the plasmid shuttle vector pA2
is used to insert the cloned DNA encoding complete protein,
including its naturally associated secretory signal (leader)
sequence, into a baculovirus to express the mature CK.alpha.-5
protein, using standard methods as described in Summers et al., A
Manual of Methods for Baculovirus Vectors and Insect Cell Culture
Procedures, Texas Agricultural Experimental Station Bulletin No.
1555 (1987). This expression vector contains the strong polyhedrin
promoter of the Autographa californica nuclear polyhedrosis virus
(AcMNPV) followed by convenient restriction sites such as BamHI,
Xba I and Asp718. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For easy selection
of recombinant virus, the plasmid contains the beta-galactosidase
gene from E. coli under control of a weak Drosophila promoter in
the same orientation, followed by the polyadenylation signal of the
polyhedrin gene. The inserted genes are flanked on both sides by
viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate a viable virus that express the
cloned polynucleotide.
[0184] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0185] The cDNA sequence encoding the full length CK.alpha.-5
protein in the deposited clone, including the AUG initiation codon
and the naturally associated leader sequence shown in SEQ ID NO:2,
is amplified using PCR oligonucleotide primers corresponding to the
5' and 3' sequences of the gene. The 5' primer has the sequence
5'CGCGGATCCGCCATCATGGGACGGGACTTGCGG 3' (SEQ ID NO: 17) containing
the underlined BamHI restriction enzyme site, an efficient signal
for initiation of translation in eukaryotic cells, as described by
Kozak, M., J. Mol. Biol. 196:947-950 (1987). The 3' primer has the
sequence 5' GCGTCTAGATCAGGTATTAGAGTCAGG 3' (SEQ ID NO:18)
containing the underlined XbaI restriction site. Those of ordinary
skill in the art will recognize that other primers could be used to
generate nucleic acid fragments encoding shorter polypeptides. For
example, the 5' primer above (SEQ ID NO:17) with the following 3'
primer could be used to amplify the nucleic acid fragment encoding
the soluble extracellular domain:
[0186] 5' GCGTCTAGATTATGTGGCTGATGTCCTGGC 3' (SEQ ID NO:19). The 3'
primer contains the underlined XbaI site.
[0187] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with BamHI and XbaI
and again is purified on a 1% agarose gel.
[0188] The plasmid is digested with the restriction enzymes BamHI
and XbaI and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.).
[0189] Fragment and the dephosphorylated plasmid are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human CK.alpha.-5 gene by digesting DNA from individual
colonies using BamHI and XbaI and then analyzing the digestion
product by gel electrophoresis. The sequence of the cloned fragment
is confirmed by DNA sequencing. This plasmid is designated herein
pA2GPCK.alpha.-5.
[0190] Five .mu.g of the plasmid pA2GPCK.alpha.-5 is co-transfected
with 1.0 .mu.g of a commercially available linearized baculovirus
DNA ("BaculoGold.TM. baculovirus DNA", Pharmingen, San Diego,
Calif.), using the lipofection method described by Felgner et al.,
Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). One .mu.g of
BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid
pA2GPCK.alpha.-5 are mixed in a sterile well of a microtiter plate
containing 50 .mu.l of serum-free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards, 10 .mu.l Lipofectin plus 90
.mu.l Grace's medium are added, mixed and incubated for 15 minutes
at room temperature. Then the transfection mixture is added
drop-wise to Sf9 insect cells (ATCC.RTM. CRL 1711) seeded in a 35
mm tissue culture plate with 1 ml Grace's medium without serum. The
plate is then incubated for 5 hours at 27.degree. C. The
transfection solution is then removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf serum is
added. Cultivation is then continued at 27.degree. C. for four
days.
[0191] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10). After appropriate incubation, blue stained plaques are
picked with the tip of a micropipettor (e.g., Eppendorf). The agar
containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 .mu.l of Grace's medium and the
suspension containing the recombinant baculovirus is used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants
of these culture dishes are harvested and then they are stored at
4.degree. C. The recombinant virus is called V-CK.alpha.-5.
[0192] To verify the expression of the CK.alpha.-5 gene Sf9 cells
are grown in Grace's medium supplemented with 10% heat-inactivated
FBS. The cells are infected with the recombinant baculovirus
V-CK.alpha.-5 at a multiplicity of infection ("MOI") of about 2. If
radiolabeled proteins are desired, 6 hours later the medium is
removed and is replaced with SF900 II medium minus methionine and
cysteine (available from Life Technologies Inc., Rockville, Md.).
After 42 hours, 5 .mu.Ci of .sup.35S-Methionine and 5 .mu.Ci
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
[0193] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the mature form or extracellular domain of the
CK.alpha.-5 protein and thus the cleavage point and length of the
naturally associated secretory signal peptide.
Example 3
Cloning and Expression of CK.alpha.-5 in Mammalian Cells
[0194] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC.RTM. 37152), pSV2dhfr (ATCC.RTM.
37146) and pBC12MI (ATCC.RTM. 67109). Mammalian host cells that
could be used include, human Hela, 293, H9 and Jurkat cells, mouse
NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells,
mouse L cells and Chinese hamster ovary (CHO) cells.
[0195] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[0196] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins.
[0197] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a)
Cloning and Expression in COS Cells
[0198] The expression plasmid, pCK.alpha.-5HA, is made by cloning a
portion of the cDNA encoding the extracellular domain of the
CK.alpha.-5 protein into the expression vector pcDNAI/Amp or
pcDNAIII (which can be obtained from Invitrogen, Inc.).
[0199] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker.
[0200] A DNA fragment encoding the soluble extracellular domain of
the CK.alpha.-5 polypeptide is cloned into the polylinker region of
the vector so that recombinant protein expression is directed by
the CMV promoter. The plasmid construction strategy is as follows.
The CK.alpha.-5 cDNA of the deposited clone is amplified using
primers that contain convenient restriction sites, much as
described above for construction of vectors for expression of
CK.alpha.-5 in E. coli. Suitable primers include the following,
which are used in this example. The 5' primer, containing the
underlined BamHI site, a Kozak sequence, and an AUG start codon,
has the following sequence:
5' CGCGGATCCGCCATCATGGGACGGGACTTGCGG 3' (SEQ ID NO:17). The 3'
primer, containing the underlined XbaI site has the following
sequence: 5' GCGTCTAGATCAGGTATTAGAGTCAGG 3' (SEQ ID NO:18).
[0201] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with BamHI and XbaI and then ligated. The ligation
mixture is transformed into E. Coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037), and the transformed culture is plated on
ampicillin media plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis or other
means for the presence of the fragment encoding the extracellular
domain of the CK.alpha.-5 polypeptide
[0202] For expression of recombinant CK.alpha.-5, COS cells are
transfected with an expression vector, as described above, using
DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under
conditions for expression of CK.alpha.-5 by the vector.
[0203] Expression of the CK.alpha.-5-HA fusion protein is detected
by radiolabeling and immunoprecipitation, using methods described
in, for example Harlow et al., Antibodies: A Laboratory Manual, 2nd
Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). To this end, two days after transfection, the cells are
labeled by incubation in media containing .sup.35S-cysteine for 8
hours. The cells and the media are collected, and the cells are
washed and the lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson et al. cited above. Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE and autoradiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
[0204] The vector pC4 is used for the expression of CK.alpha.-5
polypeptide in this example. Plasmid pC4 is a derivative of the
plasmid pSV2-dhfr (ATCC.RTM. Accession No. 37146). The plasmid
contains the mouse DHFR gene under control of the SV40 early
promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate activity that are transfected with these plasmids can
be selected by growing the cells in a selective medium (alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic
agent methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented (see,
e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R.
T., 1978, J Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C.
1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and
Sydenham, M. A. 1991, Biotechnology 9:64-68). Cells grown in
increasing concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach may be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained which contain
the amplified gene integrated into one or more chromosome(s) of the
host cell.
[0205] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rouse
Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology,
March 1985:438-447) plus a fragment isolated from the enhancer of
the immediate early gene of human cytomegalovirus (CMV) (Boshart et
al., Cell 41:521-530 (1985)). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: BamHI, Xba I, and Asp718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and
similar systems can be used to express the CK.alpha.-5 polypeptide
in a regulated way in mammalian cells (Gossen, M., & Bujard, H.
1992, Proc. Natl. Acad. Sci. USA 89:5547-5551). For the
polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well. Stable cell
lines carrying a gene of interest integrated into the chromosomes
can also be selected upon co-transfection with a selectable marker
such as gpt, G418 or hygromycin. It is advantageous to use more
than one selectable marker in the beginning, e.g., G418 plus
methotrexate.
[0206] The plasmid pC4 is digested with the restriction enzymes
BamHI and XbaI and then dephosphorylated using calf intestinal
phosphatase by procedures known in the art. The vector is then
isolated from a 1% agarose gel.
[0207] The DNA sequence encoding the extracellular domain of the
CK.alpha.-5 polypeptide is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the desired
portion of the gene. The 5' and 3' primers are the same as those
used in Example 3(a) above.
[0208] The amplified fragment is digested with the endonucleases
BamHI and XbaI and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
[0209] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using lipofectin
(Felgner et al., supra). The plasmid pSV2-neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that
confers resistance to a group of antibiotics including G418. The
cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
After 2 days, the cells are trypsinized and seeded in hybridoma
cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After
about 10-14 days single clones are trypsinized and then seeded in
6-well petri dishes or 10 ml flasks using different concentrations
of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (I .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Example 4
Tissue distribution of CK.alpha.-5 mRNA Expression
[0210] Northern blot analysis is carried out to examine CK.alpha.-5
gene expression in human tissues, using methods described by, among
others, Sambrook et al., cited above. A cDNA probe containing the
entire nucleotide sequence of the CK.alpha.-5 protein (SEQ ID NO:1)
is labeled with .sup.32P using the Rediprime.TM. DNA labeling
system (Amersham Life Science), according to manufacturer's
instructions. After labeling, the probe is purified using a CHROMA
SPIN-100.TM. column (Clontech Laboratories, Inc.), according to
manufacturer's protocol number PT1200-1. The purified labeled probe
is then used to examine various human tissues for CK.alpha.-5
mRNA.
[0211] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are obtained
from Clontech and are examined with the labeled probe using
ExpressHyb.TM. hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots are mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures.
[0212] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[0213] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
[0214] Further, the sequence listing in both hard copy and
electronic form submitted herewith is incorporated herein by
reference.
Sequence CWU 1
1
2212309DNAHomo
sapiensCDS(542)..(1303)sig_peptide(542)..(622)mat_peptide(623)..(1303)
1cggcgactct ctccaccggg ccgcccggga ggctcatgca gcgcggctgg gtcccgcggc
60gcccggatcg gggaagtgaa agtgcctcgg aggaggaggg ccggtccggc agtgcagccg
120cctcacaggt cggcggacgg gccaggcggg cggcctcctg aaccgaaccg
aatcggctcc 180tcgggccgtc gtcctcccgc ccctcctcgc ccgccgccgg
agttttcttt cggtttcttc 240caagattcct ggccttccct cgacggagcc
gggcccagtg cgggggcgca gggcgcggga 300gctccacctc ctcggctttc
cctgcgtcca gaggctggca tggcgcgggc cgagtactga 360gcgcacggtc
ggggcacagc agggccggtg ggtgcagctg gctcgcgcct cctctccggc
420cgccgtctcc tccggtcccc ggcgaaagcc attgagacac cagctggacg
tcacgcgccg 480gagcatgtct gggagtcaga gcgaggtggc tccatccccg
cagagtccgc ggagccccga 540g atg gga cgg gac ttg cgg ccc ggg tcc cgc
gtg ctc ctg ctc ctg ctt 589Met Gly Arg Asp Leu Arg Pro Gly Ser Arg
Val Leu Leu Leu Leu Leu-25 -20 -15ctg ctc ctg ctg gtg tac ctg act
cag cca ggc aat ggc aac gag ggc 637Leu Leu Leu Leu Val Tyr Leu Thr
Gln Pro Gly Asn Gly Asn Glu Gly-10 -5 -1 1 5agc gtc act gga agt tgt
tat tgt ggt aaa aga att tct tcc gac tcc 685Ser Val Thr Gly Ser Cys
Tyr Cys Gly Lys Arg Ile Ser Ser Asp Ser10 15 20ccg cca tcg gtt cag
ttc atg aat cgt ctc cgg aaa cac ctg aga gct 733Pro Pro Ser Val Gln
Phe Met Asn Arg Leu Arg Lys His Leu Arg Ala25 30 35tac cat cgg tgt
cta tac tac acg agg ttc cag ctc ctt tcc tgg agc 781Tyr His Arg Cys
Leu Tyr Tyr Thr Arg Phe Gln Leu Leu Ser Trp Ser40 45 50gtg tgt gga
ggc aac aag gac cca tgg gtt cag gaa ttg atg agc tgt 829Val Cys Gly
Gly Asn Lys Asp Pro Trp Val Gln Glu Leu Met Ser Cys55 60 65ctt gat
ctc aaa gaa tgt gga cat gct tac tcg ggg att gtg gcc cac 877Leu Asp
Leu Lys Glu Cys Gly His Ala Tyr Ser Gly Ile Val Ala His70 75 80
85cag aag cat tta ctt cct acc agc ccc cca act tct cag gcc tca gag
925Gln Lys His Leu Leu Pro Thr Ser Pro Pro Thr Ser Gln Ala Ser
Glu90 95 100ggg gca tct tca gat atc cac acc cct gcc cag atg ctc ctg
tcc acc 973Gly Ala Ser Ser Asp Ile His Thr Pro Ala Gln Met Leu Leu
Ser Thr105 110 115ttg cag tcc act cag cgc ccc acc ctc cca gta gga
tca ctg tcc tcg 1021Leu Gln Ser Thr Gln Arg Pro Thr Leu Pro Val Gly
Ser Leu Ser Ser120 125 130gac aaa gag ctc act cgt ccc aat gaa acc
acc att cac act gcg ggc 1069Asp Lys Glu Leu Thr Arg Pro Asn Glu Thr
Thr Ile His Thr Ala Gly135 140 145cac agt ctg gca gtt ggg cct gag
gct ggg gag aac cag aag cag ccg 1117His Ser Leu Ala Val Gly Pro Glu
Ala Gly Glu Asn Gln Lys Gln Pro150 155 160 165gaa aaa aat gct ggt
ccc aca gcc agg aca tca gcc aca gtg ccg gtc 1165Glu Lys Asn Ala Gly
Pro Thr Ala Arg Thr Ser Ala Thr Val Pro Val170 175 180ctg tgc ctc
ctg gcc atc atc ttc atc ctc acc gca gcc ctt tcc tat 1213Leu Cys Leu
Leu Ala Ile Ile Phe Ile Leu Thr Ala Ala Leu Ser Tyr185 190 195gtg
ctg tgc aag agg agg agg ggg cag tca ccg cag tcc tct cca gat 1261Val
Leu Cys Lys Arg Arg Arg Gly Gln Ser Pro Gln Ser Ser Pro Asp200 205
210ctg ccg gtt cat tat ata cct gtg gca cct gac tct aat acc 1303Leu
Pro Val His Tyr Ile Pro Val Ala Pro Asp Ser Asn Thr215 220
225tgagccaaga atggaagctt gtgaggagac ggactctatg ttgcccaggc
tgttatggaa 1363ctcctgagtc aagtgatcct cccaccttgg cctctgaagg
tgcgaggatt ataggcgtca 1423cctaccacat ccagcctaca cgtatttgtt
aatatctaac ataggactaa ccagccactg 1483ccctctctta ggcccctcat
ttaaaaacgg ttatactata aaatctgctt ttcacactgg 1543gtgataataa
cttggacaaa ttctatgtgt attttgtttt gttttgcttt gctttgtttt
1603gagacggagt ctcgctctgt catccaggct ggagtgcagt ggcatgatct
cggctcactg 1663caacccccat ctcccaggtt caagcgattc tcctgcctcc
tcctaagtag ctgggactac 1723aggtgctcac caccacaccc ggctaatttt
ttgtattttt agtagagacg gggtttcacc 1783atgttgacca ggctggtctc
gaactcctga cctggtgatc tgcccaccca ggcctcccaa 1843agtgctggga
ttaaaggtgt gagccaccat gcctggccct atgtgtgttt tttaactact
1903aaaaattatt tttgtaatga ttgagtcttc tttatggaaa caactggcct
cagcccttgc 1963gcccttactg tgattcctgg cttcattttt tgctgatggt
tccccctcgt cccaaatctc 2023tctcccagta caccagttgt tcctccccca
cctcagccct ctcctgcatc ctcctgtacc 2083cgcaacgaag gcctgggctt
tcccaccctc cctccttagc aggtgccgtg ctgggacacc 2143atacgggttg
gtttcacctc ctcagtccct tgcctacccc agtgagagtc tgatcttgtt
2203tttattgtta ttgcttttat tattattgct tttattatca ttaaaactct
agttcttgtt 2263ttgtctctca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa
23092254PRTHomo sapiens 2Met Gly Arg Asp Leu Arg Pro Gly Ser Arg
Val Leu Leu Leu Leu Leu-25 -20 -15Leu Leu Leu Leu Val Tyr Leu Thr
Gln Pro Gly Asn Gly Asn Glu Gly-10 -5 -1 1 5Ser Val Thr Gly Ser Cys
Tyr Cys Gly Lys Arg Ile Ser Ser Asp Ser10 15 20Pro Pro Ser Val Gln
Phe Met Asn Arg Leu Arg Lys His Leu Arg Ala25 30 35Tyr His Arg Cys
Leu Tyr Tyr Thr Arg Phe Gln Leu Leu Ser Trp Ser40 45 50Val Cys Gly
Gly Asn Lys Asp Pro Trp Val Gln Glu Leu Met Ser Cys55 60 65Leu Asp
Leu Lys Glu Cys Gly His Ala Tyr Ser Gly Ile Val Ala His70 75 80
85Gln Lys His Leu Leu Pro Thr Ser Pro Pro Thr Ser Gln Ala Ser Glu90
95 100Gly Ala Ser Ser Asp Ile His Thr Pro Ala Gln Met Leu Leu Ser
Thr105 110 115Leu Gln Ser Thr Gln Arg Pro Thr Leu Pro Val Gly Ser
Leu Ser Ser120 125 130Asp Lys Glu Leu Thr Arg Pro Asn Glu Thr Thr
Ile His Thr Ala Gly135 140 145His Ser Leu Ala Val Gly Pro Glu Ala
Gly Glu Asn Gln Lys Gln Pro150 155 160 165Glu Lys Asn Ala Gly Pro
Thr Ala Arg Thr Ser Ala Thr Val Pro Val170 175 180Leu Cys Leu Leu
Ala Ile Ile Phe Ile Leu Thr Ala Ala Leu Ser Tyr185 190 195Val Leu
Cys Lys Arg Arg Arg Gly Gln Ser Pro Gln Ser Ser Pro Asp200 205
210Leu Pro Val His Tyr Ile Pro Val Ala Pro Asp Ser Asn Thr215 220
225392PRTRattus norvegicus 3Met Lys Leu Cys Val Ser Ala Phe Ser Leu
Leu Leu Leu Val Ala Ala1 5 10 15Phe Cys Asp Ser Val Leu Ser Ala Pro
Ile Gly Ser Asp Pro Pro Thr20 25 30Ser Cys Cys Phe Ser Tyr Thr Ser
Arg Lys Ile His Arg Asn Phe Val35 40 45Met Asp Tyr Tyr Glu Thr Ser
Ser Leu Cys Ser Gln Pro Ala Val Val50 55 60Phe Leu Thr Lys Lys Gly
Arg Gln Ile Cys Ala Asp Pro Ser Glu Pro65 70 75 80Trp Val Asn Glu
Tyr Val Asn Asp Leu Glu Leu Asn85 904321DNAHomo
sapiensmisc_feature(16)a, t, g, or c 4ggcagagacg ggactngcgg
cccgggtccc gcgtnnctnc tgctcctgct tctgctcctg 60ctggtgtacc taactcagcc
aggcaatggc aacgagggca gcgtcactgg nagttgttat 120tgnggtaaaa
gaatttcttc cgactccccg ccatcggttc agttcatgaa tcgtctccgg
180aaacacctga gaagcttacc atcggtgtct gatactacac gaggttccag
ctcctttcct 240ggagcgtgtn tggnaggcaa caaggaaccc atgggttnca
gggattngat gagctgtnct 300tgatntncaa aggaatgtgg g 3215361DNAHomo
sapiensmisc_feature(51)a, t, g, or c 5atgacactcc aaaacatggc
accttggtaa ttacgaaaac agcagaaaat ntggacatgc 60ttactcgggg attntggccc
accagaagcn tttacttcct accagccccc caatttttna 120ggcctcagag
ggggcatctt ccagatatcc acacccctgc ccagatgctc ctttnccacc
180ttgncagtcc acttcagcgc cccaccctcc cagtaggatc actgtcctcg
gacaaagagc 240tcactcgtcc caatgaaacc accatttcac actgcgggcc
acagtttggg cagctgggcc 300tgnaggctgg gggagaacca ggaagncagc
cggnaaaaaa tggntggttc ccacagncag 360g 3616183DNAHomo
sapiensmisc_feature(17)..(18)a, t, g, or c 6aggaattaat gacctgnntt
aatctnaaag antgtggaca tgcttactcg gggattgtgg 60cccaccaaaa gcatttnctt
cctaccagcc ccccaatttt taaggcctca angggggcat 120ctncagatat
ccacacccct gcccagntga tcctgtncca ccttgacagt gcnacttcag 180agg
1837334DNAHomo sapiensmisc_feature(23)a, t, g, or c 7ggcacgagct
cgcgcctcct ctncggccgc cgtctcctcc ggtccccggc gaaagcatnt 60gagacancag
ctggacgtca cgcgccggag catgtctggg agtcagagcg aggtggctcc
120atcccngcag agtccgcgga gccccgagat gggacgggac ttgcggcccg
ggtnccgcgt 180gctcctgctc ctgcttctgc ttcctgctgg tgtacctgac
tcaagccagg caatgggcaa 240acgaggggca ncgtcaattg gaagttggtt
aattgtggta aaaggaattt ttttnccgac 300ttcccggnca atnggtttca
agtttcantg aatc 3348498DNAHomo sapiensmisc_feature(338)a, t, g, or
c 8aattcggcac gaggcggccc ctccttgcag gtgcctccag gaccctctct
ggtccagcct 60gaactccttc ccggctcacc gagcccatct ccgggttccg tttgctctca
ttccccgaaa 120cttcatgact ccgtctcacc tactggggag tttcccagtc
ccaggagaaa gggctctttc 180ttggtggcaa cttcacgcct ctggaatgag
gaaggagccg gggaaggaaa gaaggccgcc 240gcagctaaag cccaggtctc
tcctccgcag ttccagcttc ctttcctgga gcgtgtgttg 300aggcaacaag
gacccatggg ttcaggaatt tgatgagntg ttttgattca aaggtgagat
360tttncaatgn ttttaagttt gcanccaagg tttagcagtt tcanctnana
tttttttaac 420tnagcttttc cttgggccga aatctagact tttttnanct
gtccaagatt aggtngaggg 480gncctaccna ttggccaa 4989387DNAHomo
sapiensmisc_feature(376)a, t, g, or c 9ctgactcagc caggcaatgg
caacgagggc agcgtcactg gaagttgtta ttgtggtaaa 60agaatttctt ccgactcccc
gccatcggtt cagttcatga atcgtctccg gaaacacctg 120agagcttacc
atcggtgtct atactacacg aggttccagc tcctttcctg gagcgtgtgt
180ggaggcaaca aggacccatg ggttcaggaa ttgatgagct gtcttgatct
caaagaatgt 240ggacatgctt actcggggat tgtggcccac cagaaagcat
ttacttccta ccaagccccc 300caatttctca ggcctcagag ggggcatctt
cagatatcca cacccctgcc cagatgctcc 360tgtccacctt gcagtncact cagcgcc
38710406DNAHomo sapiens 10ggcgaaacga ttgagacacc agctggacgt
cacgcgccgg agcatgtctg ggagtcagag 60cgaggtggct ccatccccgc agagtccgcg
gacgcccgag atgggacggg acttgcggcc 120cgggtcccgc gtgctcctgc
tcctgcttct gctcctgctg gtgtacctga ctcagccagg 180caatggcaac
gagggcagcg tcactggaag ttgttattgt ggtaaaagaa tttcttccga
240ctccccgcca tcggttcagt tcatgaatcg tctccggaaa cacctgagag
cttaccatcg 300gtgtctatac tacacgaggt tccaggctcc tttcctggag
cgtgtgtggg ggcaacaagg 360acccatgggt tcaggaattg atgagctgtc
ttgatctcaa agaatg 40611248DNAHomo sapiensmisc_feature(7)a, t, g, or
c 11gcttctnctc ctgctggtgt acctnantca gccaggcaat ggcaacgagg
gcagcgtcac 60tggaagttgt tattgtggta aaagaatttn ttccgactcc ccgccatcgg
ttcagttcat 120gaatcgtctc cggaaacacc tgagagctta ccatcggtgt
ctatactaca cgaggttcca 180gctcctttcc tggagcgtgt gtgggggcaa
caaggaccca tgggttcagg aattgatgag 240ctgtcttt 24812425DNAHomo
sapiensmisc_feature(244)a, t, g, or c 12ttactcgggg attgtggccc
accagaagca tttacttcct accagccccc caacttctca 60ggcctcagag ggggcatctt
cagatatcca cacccctgcc cagatgctcc tgtccacctt 120gcagtccact
cagcgcccca ccctcccagt aggatcactg tcctcggaca aagagctcac
180tcgtcccaat gaaaccacca ttcacactgc gggccacagt ctggcagttg
ggcctgaggc 240tggnggagaa ccagaagcag ccggaaaaaa atgctggtcc
cacagccagg acatcagcca 300caagtgcngg tcctgtgcct cctggccatc
atcttcatcc tcacccgcag ccctttccta 360tgtgctgtga agagngagga
gggggcagtc acgcagtcct tctccagatt ctggccggtt 420canta
42513209DNAHomo sapiens 13tttcttccga ctccccgcca tcggttcagt
tcatgaatcg tctccggaaa cacctgagag 60cttaccatcg gtgtctatac tacacgaggt
tccagctcct ttcctggagc gtgtgtggag 120gcaacaagga cccatgggtt
caggaattga tgagctgtct tgatctcaaa gaatgtggac 180atgcttactc
ggggattgtg gcccaccag 20914405DNAHomo sapiensmisc_feature(159)a, t,
g, or c 14agatatccac acccctgccc agatgctcct gtccaccttg cagtccactc
agcgccccac 60cctcccagta ggatcactgt cctcggacaa agagctcact cgtcccaatg
aaaccaccat 120tcacactgcg ggccacagtc tggcagctgg gcctgaggnt
ggggagaacc agaagcagcc 180ggaaaaaaat gctggtccca cagccaggac
atcagccaca gtgccagtcc tgtgcctcct 240ggccatcatc ttcatcctca
ccgcagccct ttcctatgtg ctgtgaagat tgaggagggg 300gcagtcaccg
cagtcctctc cagatctgcc cggttcatta tatacctggt ggcacctgac
360tctaatacct tgagccaaga atggaagctt gtgaggagac ggact
4051527DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15gcgccatgga atggcaacga gggcagc
271630DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 16cgcaagcttt tatgtggctg atgtcctggc
301724DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17cgcggatccg ccatcatggg acgg 241827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18gcgtctagat caggtattag agtcagg 271930DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19gcgtctagat tatgtggctg atgtcctggc 3020489DNAHomo
sapiensmisc_feature(9)a, t, g, or c 20attcggcana gtgaaggctg
gggagaacca gaagcagccg gaaaaaaatg ctggtcccac 60agccaggaca tcagccacag
tgccagtcct gtgcctcctg gncatcatct tcatcctcac 120cgcagccctt
tcctatgtgc tgtgcaagag gaggaggggg cagtnaccgc agtcctcttc
180cagatctgcc ggttcattat atacctgtgg gnaactgatt ctaataactg
aggccaagaa 240tnggaaggtt tnttaggaga ngggattttt tntttcccag
gtttttatgg gaatttctga 300gttaaagggt tccttccaat ttgggntttn
taagggnngn ggggttttaa ggggngaant 360nncaaatnna ggttaaaggg
nttttttaan antttnaaaa aaggggttta nnnnnatnnn 420nnnttttttg
gggnnnnttt taaaagggtt ttttttaaaa nngggttttn naaanggggg 480aaaaaanna
48921395DNAHomo sapiens 21tttttttttt tttttttttt tttttttttt
tttttttttt caggggtcag tctcctttat 60tacacttttt gtaagaataa gccacagttt
accctcacaa acttccattc ttggctcagg 120tattagagtc aggtgccaca
ggtatataat gaaccggcag atctggagag gactgcggtg 180actgccccct
cctcctcttg cacagcacat aggaaagggc tgcggtgagg atgaagatga
240tggccaggag gcacaggacc ggcactgtgg ctgatgtcct ggctgtggga
ccagcatttt 300tttccggctg cttctggttc tccccagcct caggcccaac
tgccagactg tggcccgcag 360tgtgaatggt ggtttcattg ggacgagtga gctct
39522339DNAHomo sapiens 22ttttcagggg tcagtctcct ttattacact
ttttgtaaga ataagccaca gtttaccctc 60acaaacttcc attcttggct caggtattag
agtcaggtgc cacaggtata taatgaaccg 120gcagatctgg agaggactgc
ggtgactgcc ccctcctcct cttgcacagc acataggaaa 180gggctgcggt
gaggatgaag atgatggcca ggaggacagg accggcactg tggctgattt
240cctggctgtg ggaccagcat ttttttccgg ctgcttctgg ttctccccag
cctcaggccc 300aactgccaga ctgtggcccg cagtgtgaat ggtggtttc 339
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