U.S. patent application number 14/158063 was filed with the patent office on 2014-06-12 for antibodies that bind novel pcsk9 variants.
This patent application is currently assigned to Bristol-Myers Squibb Company. The applicant listed for this patent is Bristol-Myers Squibb Company. Invention is credited to Jian Chen, John N. Feder, Bowman Miao, Gabriel A. Mintier, Rex Arnold Parker.
Application Number | 20140161808 14/158063 |
Document ID | / |
Family ID | 39721706 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140161808 |
Kind Code |
A1 |
Mintier; Gabriel A. ; et
al. |
June 12, 2014 |
ANTIBODIES THAT BIND NOVEL PCSK9 VARIANTS
Abstract
The present invention provides novel polynucleotides encoding
PCSK9b and PCSK9c polypeptides, fragments and homologues thereof.
Also provided are vectors, host cells, antibodies, and recombinant
and synthetic methods for producing said polypeptides. The
invention further relates to diagnostic and therapeutic methods for
applying these novel PCSK9b and PCSK9c polypeptides to the
diagnosis, treatment, and/or prevention of various diseases and/or
disorders related to these polypeptides. The invention further
relates to screening methods for identifying agonists and
antagonists of the polynucleotides and polypeptides of the present
invention.
Inventors: |
Mintier; Gabriel A.;
(Hightstown, NJ) ; Chen; Jian; (Princeton, NJ)
; Feder; John N.; (Belle Mead, NJ) ; Miao;
Bowman; (Churchville, PA) ; Parker; Rex Arnold;
(Titusville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bristol-Myers Squibb Company |
Princeton |
NJ |
US |
|
|
Assignee: |
Bristol-Myers Squibb
Company
Princeton
NJ
|
Family ID: |
39721706 |
Appl. No.: |
14/158063 |
Filed: |
January 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13720000 |
Dec 19, 2012 |
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14158063 |
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13336371 |
Dec 23, 2011 |
8354264 |
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13720000 |
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12903658 |
Oct 13, 2010 |
8105804 |
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13336371 |
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12456798 |
Jun 23, 2009 |
7846706 |
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12903658 |
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Current U.S.
Class: |
424/139.1 ;
530/387.9 |
Current CPC
Class: |
C07K 16/40 20130101;
C12N 9/6424 20130101; A61K 38/00 20130101; C12Y 304/21061 20130101;
C12Y 302/01008 20130101; Y10T 436/143333 20150115; C07K 2317/76
20130101; C07K 2317/41 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. An isolated antibody or antigen binding fragment thereof that
binds to a polypeptide comprising SEQ ID NO:2.
2. The isolated antibody of claim 1, wherein said antibody is a
monoclonal antibody.
3. The isolated antibody of claim 1, wherein said antibody is a
polyclonal antibody.
4. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody or antigen binding fragment of
claim 1 and a pharmaceutically acceptable carrier.
5. An isolated antibody or antigen binding fragment thereof that
binds to a polypeptide comprising SEQ ID NO:4.
6. The isolated antibody of claim 5, wherein said antibody is a
monoclonal antibody.
7. The isolated antibody of claim 5, wherein said antibody is a
polyclonal antibody.
8. A pharmaceutical composition comprising a therapeutically
effective amount of the antibody or antigen binding fragment of
claim 5 and a pharmaceutically acceptable carrier.
Description
[0001] This application is a divisional application of
nonprovisional application U.S. Ser. No. 13/336,371, filed Dec. 23,
2011, which is a divisional application of nonprovisional
application U.S. Ser. No. 12/903,658, filed Oct. 13, 2010, now
granted U.S. Pat. No. 8,105,804, which is a divisional application
of U.S. Ser. No. 12/456,798, filed Jun. 23, 2009, now granted U.S.
Pat. No. 7,846,706, which is a divisional application of U.S. Ser.
No. 11/824,461, filed Jun. 28, 2007, now granted U.S. Pat. No.
7,572,618, which claims the benefit of provisional application U.S.
Ser. No. 60/818,234 filed Jun. 30, 2006. The entire teachings of
the referenced application are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention provides novel polynucleotides
encoding PCSK9b and PCSK9c polypeptides, fragments and homologues
thereof. Also provided are vectors, host cells, antibodies, and
recombinant and synthetic methods for producing said polypeptides.
The invention further relates to diagnostic and therapeutic methods
for applying these novel PCSK9b and PCSK9c polypeptides to the
diagnosis, treatment, and/or prevention of various diseases and/or
disorders related to these polypeptides. The invention further
relates to screening methods for identifying agonists and
antagonists of the polynucleotides and polypeptides of the present
invention.
BACKGROUND OF THE INVENTION
[0003] Atherosclerosis is a disease of the arteries responsible for
coronary heart disease (CVD) that underlies most deaths in
industrialized countries (Lusis, 2000). Several risk factors for
CHD have now been well established: dyslipidemias, hypertension,
diabetes, smoking, poor diet, inactivity and stress. The most
clinically relevant and common dyslipidemias are characterized by
an increase in beta-lipoproteins (VLDL and LDL particles) with
hypercholesterolemia in the absence or presence of
hypertriglyceridemia (Fredrickson et al, 1967). An isolated
elevation of LDL cholesterol is one of the most common risk factors
for CVD. Twin studies (Austin et al, 1987) and family data
(Perusse, 1989; Rice et al, 1991) have shown the importance of
genetic factors in the development of the disease, particularly
when its complications occur early in life. Mendelian forms of
hypercholesterolemia have been identified: at first the autosomal
dominant form (ADH) (Khachadurian, 1964) and later the autosomal
recessive form (ARH), initially described as "pseudohomozygous type
II hyperlipoproteinemia" (Morganroth et al, 1967).
[0004] ADH is a heterogeneous genetic disorder. Its most frequent
and archetypal form is Familial Hypercholesterolemia (FH) with a
frequency of 1 in 500 for heterozygotes and 1 per million for
homozygotes (Goldstein et al, 1973). The disease is co-dominant
with homozygotes being affected earlier and more severely than
heterozygotes. FH is caused by mutations in the gene that encodes
the LDL receptor (Goldstein & Brown, 1978) (LDLR at
19p13.1-p13.3) (MIN 143890). It is characterized by a selective
increase of LDL cholesterol levels in plasma giving rise to tendon
and skin xanthomas, arcus corneae and cardiovascular deposits
leading to progressive and premature atherosclerosis, CHD and
mortality (occurring before 55 years). The second form of ADH is
Familial Defective apo B-100 (FDB) caused by mutations in the
apolipoprotein B gene (APOB at 2p23-p24), encoding the ligand of
the LDL receptor (Inneraty et al, 1987) (MIN 144010). The existence
of a greater level of genetic heterogeneity in ADH (Saint-Jore et
al, 2000) has been reported and the implication of a third locus
named HCHOLA3 (formerly FH3) has been detected and mapped at
1p34.1-p32 in a French family (Varret et al, 1999) (MIM 603776).
These results were confirmed by Hunt et al. in a large Utah kindred
(Hunt et al, 2000).
[0005] PCSK9, for Proprotein Convertase Subtilisin/Kexin type 9
(also referred to as HCHOLA3, NARC-1, or FH3) is a protease
belonging to the proteinase K subfamily of the secretory subtilase
family (Naureckiene et al., Arch. of Biochem. And Biophy.,
420:55-57 (2003)). PCSK9 has been shown to play a role in
cholesterol homeostasis by regulating apolipoprotein receptor
secretion. It may also have a role in the differentiation of brain
cortical neurons (Seidah et al., PNAS 100(3):928-933 (2003)).
[0006] The wild type PCSK9 gene contains 12 exons. The translated
protein contains a signal peptide in the NH2-terminus, and in cells
and tissues an about 74 kDa zymogen (precursor) form of the
full-length protein is found in the endoplasmic reticulum. During
initial processing in the cell, the about 14 kDa prodomain peptide
is autocatalytically cleaved to yield a mature about 60 kDa protein
containing the catalytic domain and a C-terminal domain often
referred to as the cysteine-histidine rich domain (CHRD) (FIG. 1).
This about 60 kDa form of PCSK9 is secreted from liver cells. The
secreted form of PCSK9 appears to be the physiologically active
species, although an intracellular functional role of the about 60
kDa form has not been ruled out.
[0007] Several mutant forms of PCSK9 are known, including S127R,
N157K, F216L, R218S, and D374Y, with S127R, F216L, and D374Y being
linked to autosomal dominant hypercholesterolemia (ADH). Benjannet
et al. (J. Biol. Chem., 279(47):48865-48875 (2004)) demonstrated
that the S127R and D374Y mutations result in a significant decrease
in the level of pro-PCSK9 processed in the ER to form the active
secreted zymogen. As a consequence it is believed that wild-type
PCSK9 increases the turnover rate of the LDL receptor causing
inhibition of LDL clearance (Maxwell et al., PNAS, 102(6):2069-2074
(2005); Benjannet et al., and Lalanne et al), while PCSK9 autosomal
dominant mutations result in increased levels of LDLR, increased
clearance of circulating LDL, and a corresponding decrease in
plasma cholesterol levels (Rashid et al., PNAS, 102(15):5374-5379
(2005).
[0008] Lalanne et al. demonstrated that LDL catabolism was impaired
and apolipoprotein B-containing lipoprotein synthesis was enhanced
in two patients harboring S127R mutations in PCSK9 (J. Lipid
Research, 46:1312-1319 (2005)). Sun et al. also provided evidence
that mutant forms of PCSK9 are also the cause of unusually severe
dominant hypercholesterolaemia as a consequence of its affect of
increasing apolipoprotein B secretion (Sun et al, Hum. Mol. Genet.,
14(9):1161-1169 (2005)). These results were consistent with earlier
results which demonstrated adenovirus-mediated overexpression of
PCSK9 in mice results in severe hypercholesteromia due to durastic
decreases in the amount of LDL receptor Dubuc et al., Thromb. Vasc.
Biol., 24:1454-1459 (2004), in addition to results demonstrating
mutant forms of PCSK9 also reduce the level of LDL receptor (Park
et al., J. Biol. Chem., 279:50630-50638 (2004). The overexpression
of PCSK9 in cell lines, including liver-derived cells, and in
livers of mice in vivo, results in a pronounced reduction in LDLR
protein levels and LDLR functional activity without changes in LDLR
mRNA level (Maxwell K. N., Breslow J. L., Proc. Nat. Amer. Sci.,
101:7100-7105 (2004); Benjannet S. et al., J. Bio. Chem. 279:
48865-48875 (2004)).
[0009] Using the above examples, it is clear the availability of
novel forms of PCSK9 provide an opportunity for the identification
of PCSK9 agonists, as well as, in the identification of PCSK9
inhibitors. All of which might be therapeutically useful under
different circumstances.
[0010] 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, in
addition to their use in the production of PCSK9b and PCSK9c
polypeptides or peptides using recombinant techniques. Synthetic
methods for producing the polypeptides and polynucleotides of the
present invention are provided. Also provided are diagnostic
methods for detecting diseases, disorders, and/or conditions
related to the PCSK9b and PCSK9c polypeptides and polynucleotides,
and therapeutic methods for treating such diseases, disorders,
and/or conditions. The invention further relates to screening
methods for identifying binding partners of the polypeptides.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention provides isolated nucleic acid
molecules, that comprise, or alternatively consist of, a
polynucleotide encoding the PCSK9b protein having the amino acid
sequence shown in FIGS. 1A-C (SEQ ID NO:2), respectively, or the
amino acid sequence encoded by the cDNA clone, PCSK9b (also
referred to as PCSK9-b), deposited as ATCC.RTM. Deposit Number
PTA-7622 on May 10, 2006.
[0012] The present invention provides isolated nucleic acid
molecules, that comprise, or alternatively consist of, a
polynucleotide encoding the PCSK9c protein having the amino acid
sequence shown in FIGS. 1A-D (SEQ ID NO:4), respectively, or the
amino acid sequence encoded by the cDNA clone, PCSK9c (also
referred to as PCSK9-c), deposited as ATCC.RTM. Deposit Number
PTA-7622 on May 10, 2006.
[0013] 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, in
addition to their use in the production of PCSK9b or PCSK9c
polypeptides or peptides using recombinant techniques. Synthetic
methods for producing the polypeptides and polynucleotides of the
present invention are provided. Also provided are diagnostic
methods for detecting diseases, disorders, and/or conditions
related to the PCSK9b or PCSK9c polypeptides and polynucleotides,
and therapeutic methods for treating such diseases, disorders,
and/or conditions. The invention further relates to screening
methods for identifying binding partners of the polypeptides.
[0014] The invention further provides an isolated PCSK9b or PCSK9c
polypeptide having an amino acid sequence encoded by a
polynucleotide described herein.
[0015] The invention further relates to a polynucleotide encoding a
polypeptide fragment of SEQ ID NO:2 or SEQ ID NO:4, or a
polypeptide fragment encoded by the cDNA sequence included in the
deposited clone, which is hybridizable to SEQ ID NO:1 or SEQ ID
NO:3.
[0016] The invention further relates to a polynucleotide encoding a
polypeptide domain of SEQ ID NO:2 or SEQ ID NO:4 or a polypeptide
domain encoded by the cDNA sequence included in the deposited
clone, which is hybridizable to SEQ ID NO:1 or SEQ ID NO:3.
[0017] The invention further relates to a polynucleotide encoding a
polypeptide epitope of SEQ ID NO:2 or SEQ ID NO:4 or a polypeptide
epitope encoded by the cDNA sequence included in the deposited
clone, which is hybridizable to SEQ ID NO:1 or SEQ ID NO:3.
[0018] The invention further relates to a polynucleotide encoding a
polypeptide of SEQ ID NO:2 or SEQ ID NO:4 or the cDNA sequence
included in the deposited clone, which is hybridizable to SEQ ID
NO:1 or SEQ ID NO:3, having biological activity.
[0019] The invention further relates to a polynucleotide which is a
variant of SEQ ID NO:1 or SEQ ID NO:3.
[0020] The invention further relates to a polynucleotide which is
an allelic variant of SEQ ID NO:1 or SEQ ID NO:3.
[0021] The invention further relates to a polynucleotide which
encodes a species homologue of the SEQ ID NO:2 or SEQ ID NO:4.
[0022] The invention further relates to a polynucleotide which
represents the complimentary sequence (antisense) of SEQ ID NO:1 or
SEQ ID NO:4.
[0023] The invention further relates to a polynucleotide capable of
hybridizing under stringent conditions to any one of the
polynucleotides specified herein, wherein said polynucleotide does
not hybridize under stringent conditions to a nucleic acid molecule
having a nucleotide sequence of only A residues or of only T
residues.
[0024] The invention further relates to an isolated nucleic acid
molecule of SEQ ID NO:2 or SEQ ID NO:4, wherein the polynucleotide
fragment comprises a nucleotide sequence encoding a subtilisin
protease K proteinase.
[0025] The invention further relates to an isolated nucleic acid
molecule of SEQ ID NO:1 or SEQ ID NO:3, wherein the polynucleotide
fragment comprises a nucleotide sequence encoding the sequence
identified as SEQ ID NO:2 or SEQ ID NO:4 or the polypeptide encoded
by the cDNA sequence included in the deposited clone, which is
hybridizable to SEQ ID NO:1 or SEQ ID NO:3.
[0026] The invention further relates to an isolated nucleic acid
molecule of SEQ ID NO:1 or SEQ ID NO:3, wherein the polynucleotide
fragment comprises the entire nucleotide sequence of SEQ ID NO:1 or
SEQ ID NO:3 or the cDNA sequence included in the deposited clone,
which is hybridizable to SEQ ID NO:1 or SEQ ID NO:3.
[0027] The invention further relates to an isolated nucleic acid
molecule of SEQ ID NO:1 or SEQ ID NO:3, wherein the nucleotide
sequence comprises sequential nucleotide deletions from either the
C-terminus or the N-terminus.
[0028] The invention further relates to an isolated polypeptide
comprising an amino acid sequence that comprises a polypeptide
fragment of SEQ ID NO:2 or SEQ ID NO:4 or the encoded sequence
included in the deposited clone.
[0029] The invention further relates to a polypeptide fragment of
SEQ ID NO:2 or SEQ ID NO:4 or the encoded sequence included in the
deposited clone, having biological activity.
[0030] The invention further relates to a polypeptide domain of SEQ
ID NO:2 or SEQ ID NO:4 or the encoded sequence included in the
deposited clone.
[0031] The invention further relates to a polypeptide epitope of
SEQ ID NO:2 or SEQ ID NO:4 or the encoded sequence included in the
deposited clone.
[0032] The invention further relates to a full length protein of
SEQ ID NO:2 or SEQ ID NO:4 or the encoded sequence included in the
deposited clone.
[0033] The invention further relates to a variant of SEQ ID NO:2 or
SEQ ID NO:4.
[0034] The invention further relates to an allelic variant of SEQ
ID NO:2 or SEQ ID NO:4.
[0035] The invention further relates to a species homologue of SEQ
ID NO:2 or SEQ ID NO:4.
[0036] The invention further relates to the isolated polypeptide of
SEQ ID NO:2 or SEQ ID NO:4, wherein the full length protein
comprises sequential amino acid deletions from either the
C-terminus or the N-terminus.
[0037] The invention further relates to an isolated antibody that
binds specifically to the isolated polypeptide of SEQ ID NO:2 or
SEQ ID NO:4.
[0038] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition, comprising
administering to a mammalian subject a therapeutically effective
amount of the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 or the
polynucleotide of SEQ ID NO:1 or SEQ ID NO:3.
[0039] The invention further relates to a method of diagnosing a
pathological condition or a susceptibility to a pathological
condition in a subject comprising the steps of: (a) determining the
presence or absence of a mutation in the polynucleotide of SEQ ID
NO:1 or SEQ ID NO:3; and (b) diagnosing a pathological condition or
a susceptibility to a pathological condition based on the presence
or absence of said mutation.
[0040] The invention further relates to a method of diagnosing a
pathological condition or a susceptibility to a pathological
condition in a subject comprising the steps of: (a) determining the
presence or amount of expression of the polypeptide of SEQ ID NO:2
or SEQ ID NO:4 in a biological sample; and (b) diagnosing a
pathological condition or a susceptibility to a pathological
condition based on the presence or amount of expression of the
polypeptide.
[0041] The invention further relates to a method for identifying a
binding partner to the polypeptide of SEQ ID NO:2 or SEQ ID NO:4
comprising the steps of: (a) contacting the polypeptide of SEQ ID
NO:2 or SEQ ID NO:4 with a binding partner; and (b) determining
whether the binding partner effects an activity of the
polypeptide.
[0042] The invention further relates to a gene corresponding to the
cDNA sequence of SEQ ID NO:1 or SEQ ID NO:3.
[0043] The invention further relates to a method of identifying an
activity in a biological assay, wherein the method comprises the
steps of: (a) expressing SEQ ID NO:1 or SEQ ID NO:3 in a cell; (b)
isolating the supernatant; (c) detecting an activity in a
biological assay; and (d) identifying the protein in the
supernatant having the activity.
[0044] The invention further relates to a process for making
polynucleotide sequences encoding gene products having altered
activity selected from the group consisting of SEQ ID NO:2 or SEQ
ID NO:4 activity comprising the steps of: (a) shuffling a
nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; (b) expressing
the resulting shuffled nucleotide sequences; and (c) selecting for
altered activity selected from the group consisting of SEQ ID NO:2
or SEQ ID NO:4 activity as compared to the activity selected from
the group consisting of SEQ ID NO:2 or SEQ ID NO:4 activity of the
gene product of said unmodified nucleotide sequence.
[0045] The invention further relates to a shuffled polynucleotide
sequence produced by a shuffling process, wherein said shuffled DNA
molecule encodes a gene product having enhanced tolerance to an
inhibitor of any one of the activities selected from the group
consisting of SEQ ID NO:2 or SEQ ID NO:4 activity.
[0046] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition with the polypeptide
provided as SEQ ID NO:2 or SEQ ID NO:4, in addition to, its
encoding nucleic acid, wherein the medical condition is a
reproductive disorder.
[0047] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition with the polypeptide
provided as SEQ ID NO:2 or SEQ ID NO:4, in addition to, its
encoding nucleic acid, wherein the medical condition is a disorder
related to aberrant PCSK9 signaling and/or activity.
[0048] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition with the polypeptide
provided as SEQ ID NO:2 or SEQ ID NO:4, in addition to, its
encoding nucleic acid, wherein the medical condition is a
cardiovascular disorder, hypercholesterolemia, autosomal dominant
hypercholesterolemia; disorders associated with aberrant LDL
receptor function; disorders associated with apolipoprotein B;
disorders associated with autosomal recessive hypercholesterolemia;
disorders associated with elevated cholesterol; disorders
associated with elevated LDL; disorders associated with reduced
clearance rate of LDL in the liver; disorders associated with
elevated LDL apoB production; familial hypercholesterolemia; lipid
metabolism disorders; elevated LDL; cholesterol depositions; tendon
xanthomas; atheroma; premature arteriosclerosis, coronary heart
disease; famialial defective apolipoprotein B; statin
hypersensitivity; disordesr associated with accelerated LDLR
degradation, neural differentiation disorders.
[0049] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition with the polypeptide
provided as SEQ ID NO:2 or SEQ ID NO:4, in addition to, its
encoding nucleic acid, wherein the medical condition is a metabolic
disorder.
[0050] The invention further relates to a method for preventing,
treating, or ameliorating a medical condition with the polypeptide
provided as SEQ ID NO:2 or SEQ ID NO:4, in addition to, its
encoding nucleic acid, wherein the medical condition is a metabolic
disorder selected from the group consisting of: dyslipidemia,
diabetic dyslipidemia, mixed dyslipidemia, hypercholesteremia,
hypertriglyceridemia, type II diabetes mellitus, type I diabetes,
insulin resistance, hyperlipidemia, obesity, anorexia nervosa.
[0051] The invention further relates to a method of identifying a
compound that modulates the biological activity of PCSK9b and/or
PCSK9c, comprising the steps of: (a) combining a candidate
modulator compound with PCSK9b and/or PCSK9c having the sequence
set forth in SEQ ID NO:2 or SEQ ID NO:4; and (b) measuring an
effect of the candidate modulator compound on the activity of
PCSK9b and/or PCSK9c.
[0052] The invention further relates to a method of identifying a
compound that modulates the biological activity of PCSK9b and/or
PCSK9c, comprising the steps of: (a) combining a candidate
modulator compound with PCSK9b and/or PCSK9c having the sequence
set forth in SEQ ID NO:2 or SEQ ID NO:4; and (b) measuring an
effect of the candidate modulator compound on the activity of
PCSK9b and/or PCSK9c, wherein said method optionally includes the
addition of a suitable PCSK9 substrate either before, during, or
after addition of said candidate modulator compound.
[0053] The invention further relates to a method of identifying an
antagonist compound that modulates the biological activity of
PCSK9b and/or PCSK9c, comprising the steps of: (a) combining a
candidate modulator compound with PCSK9b and/or PCSK9c having the
sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 in the presence of
a suitable PCSK9 substrate; and (b) identifying antagonist
compounds by measuring an effect of the candidate modulator
compound on the activity of PCSK9b and/or PCSK9c, wherein said
identified antagonist compound decreases proteinase activity of
PCSK9b and/or PCSK9c.
[0054] The invention further relates to a method of identifying an
agonist compound that modulates the biological activity of PCSK9b
and/or PCSK9c, comprising the steps of: (a) combining a candidate
modulator compound with PCSK9b and/or PCSK9c having the sequence
set forth in SEQ ID NO:2 or SEQ ID NO:4 in the presence of a
suitable PCSK9 substrate; and (b) identifying agonist compounds by
measuring an effect of the candidate modulator compound on the
activity of PCSK9b and/or PCSK9c, wherein said identified agonist
compound increases proteinase activity of PCSK9b and/or PCSK9c.
[0055] The invention further relates to a method of identifying a
compound that modulates the biological activity of PCSK9b and/or
PCSK9c, comprising the steps of: (a) combining a candidate
modulator compound with a host cell expressing PCSK9b and/or PCSK9c
having the sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4; and
(b) measuring an effect of the candidate modulator compound on the
activity of the expressed PCSK9b and/or PCSK9c.
[0056] The invention further relates to a method of identifying an
antagonist compound that modulates the biological activity of
wild-type PCSK9, comprising the steps of: (a) combining a candidate
modulator compound with PCSK9b and/or PCSK9c having the sequence
set forth in SEQ ID NO:2 or SEQ ID NO:4 in the presence of a
suitable PCSK9 substrate; and (b) identifying antagonist compounds
by measuring an effect of the candidate modulator compound on the
activity of PCSK9, wherein said identified antagonist compound
decreases proteinase activity of PCSK9.
[0057] The invention further relates to a method of identifying an
agonist compound that modulates the biological activity of PCSK9,
comprising the steps of: (a) combining a candidate modulator
compound with PCSK9b and/or PCSK9c having the sequence set forth in
SEQ ID NO:2 or SEQ ID NO:4 in the presence of a suitable PCSK9
substrate; and (b) identifying agonist compounds by measuring an
effect of the candidate modulator compound on the activity of
PCSK9, wherein said identified agonist compound increases
proteinase activity of PCSK9.
[0058] The invention further relates to a method of identifying a
compound that modulates the biological activity of PCSK9,
comprising the steps of: (a) combining a candidate modulator
compound with a host cell expressing PCSK9b and/or PCSK9c having
the sequence as set forth in SEQ ID NO:2 or SEQ ID NO:4; and (b)
measuring an effect of the candidate modulator compound on the
activity of the expressed PCSK9b and/or PCSK9c.
[0059] The invention further relates to a method of screening for a
compound that is capable of modulating the biological activity of
PCSK9b and/or PCSK9c, comprising the steps of: (a) providing a host
cell described herein; (b) determining the biological activity of
PCSK9b and/or PCSK9c in the absence of a modulator compound; (c)
contacting the cell with the modulator compound; and (d)
determining the biological activity of PCSK9b and/or PCSK9c in the
presence of the modulator compound; wherein a difference between
the activity of PCSK9b and/or PCSK9c in the presence of the
modulator compound and in the absence of the modulator compound
indicates a modulating effect of the compound.
[0060] The invention further relates to a method of screening for a
compound that is capable of modulating the biological activity of
PCSK9, comprising the steps of: (a) providing a host cell
comprising PCSK9b and/or PCSK9c; (b) determining the biological
activity of PCSK9 in the absence of a modulator compound; (c)
contacting the cell with the modulator compound; and (d)
determining the biological activity of PCSK9 in the presence of the
modulator compound; wherein a difference between the activity of
PCSK9 in the presence of the modulator compound and in the absence
of the modulator compound indicates a modulating effect of the
compound.
[0061] The invention further relates to an N-terminal truncation of
PCSK9 (SEQ ID NO:5), wherein said N-terminal truncation results in
the deletion of anywhere between about 1 to about 218 amino acids
from the N-terminus of SEQ ID NO:5, and wherein said N-terminal
truncation results in elevated PCSK9 biological activity,
including, but not limited to decreased LDLR protein levels, and/or
decreased LDL uptake by LDLR.
[0062] The invention further relates to an N-terminal truncation of
PCSK9 (SEQ ID NO:5), wherein said N-terminal truncation results in
the deletion of anywhere between about 1 to about 218 amino acids
from the N-terminus of SEQ ID NO:5, including, but not limited to
decreased LDLR protein levels, and/or decreased LDL uptake by LDLR,
and wherein said elevated PCSK9 biological activity is at least
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 100%, or more than wildtype elevated
PCSK9 biological activity. In this context, the term "about" shall
be construed to mean anywhere between 1, 2, 3, 4, or 5 percent more
or less than the cited amount. Alternatively, said elevated PCSK9
biological activity may be at least about 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., or 10.times. more than wildtype PCSK9 biological
activity. In this context, the term "about" shall be construed to
mean anywhere between 0.1.times., 0.2.times., 0.3.times.,
0.4.times., 0.5.times., 0.6.times., 0.7.times., 0.8.times., or
0.9.times. more or less than the cited amount.
[0063] As used herein the terms "modulate" or "modulates" refer to
an increase or decrease in the amount, quality or effect of a
particular activity, DNA, RNA, or protein of PCSK9b and/or
PCSK9c.
BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
[0064] The file of this patent contains at least one Figure
executed in color. Copies of this patent with color Figure(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0065] FIGS. 1A-C show the polynucleotide sequence (SEQ ID NO:1)
and deduced amino acid sequence (SEQ ID NO:2) of the novel PCSK9
variant, PCSK9b of the present invention. The standard one-letter
abbreviation for amino acids is used to illustrate the deduced
amino acid sequence. The polynucleotide sequence contains a
sequence of 3175 nucleotides (SEQ ID NO:1), encoding a polypeptide
of 315 amino acids (SEQ ID NO:2). An analysis of the PCSK9b
polypeptide determined that it comprised the following features: a
catalytic domain located from about amino acid 10 to about amino
acid 256 of SEQ ID NO:2 denoted in italics, with the canonical
catalytic triad residing at amino acids D17, H57, and S217 of SEQ
ID NO:2 denoted by double underlining; and six conserved cysteine
residues located at amino acids C54, C86, C132, C154, C189, and
C206 of SEQ ID NO:2 denoted in bold, with disulfite bonds predicted
to form between the following cysteine pairs: C54 and C86, and C154
and C189; and a predicted Ca.sup.2+ ion binding domain predicted to
form between residues D191, P162, and V164 of SEQ ID NO:2, denoted
by an asterisk (*) below the amino acid residue.
[0066] FIGS. 2A-D show the polynucleotide sequence (SEQ ID NO:3)
and deduced amino acid sequence (SEQ ID NO:4) of the novel PCSK9
variant, PCSK9c of the present invention. The standard one-letter
abbreviation for amino acids is used to illustrate the deduced
amino acid sequence. The polynucleotide sequence contains a
sequence of 3756 nucleotides (SEQ ID NO:3), encoding a polypeptide
of 523 amino acids (SEQ ID NO:4). An analysis of the PCSK9c
polypeptide determined that it comprised the following features: a
catalytic domain located from about amino acid 10 to about amino
acid 256 of SEQ ID NO:4 denoted in italics, with the canonical
catalytic triad residing at amino acids D17, H57, and S217 of SEQ
ID NO:4 denoted by double underlining; and six conserved cysteine
residues located at amino acids C54, C86, C132, C154, C189, and
C206 of SEQ ID NO:4 denoted in bold, with disulfide bonds predicted
to form between the following cysteine pairs: C54 and C86, and C154
and C189; and a predicted Ca.sup.2+ ion binding domain predicted to
form between residues D191, P162, and V164 of SEQ ID NO:4, denoted
by an asterisk (*) below the amino acid residue.
[0067] FIGS. 3A-C show the regions of identity and similarity
between the sequences of the encoded PCSK9b and PCSK9c polypeptides
of the present invention with the sequence of the human wild type
PCSK9 protein (PCSK9; GENBANK.RTM. Accession No:
gi|NM.sub.--174936; SEQ ID NO:5); as well as the sequence of a
known variant of the wild type PCSK9 polypeptide (PCSK9 variant;
GENBANK.RTM. Accession No: gi|AK124635; SEQ ID NO:6). The alignment
was performed using the CLUSTALW algorithm using default parameters
as described herein (VECTOR NTI.RTM. suite of programs). The darkly
shaded amino acids represent regions of matching identity. The
lightly shaded amino acids represent regions of matching
similarity. Dots (".cndot.") between residues indicate gapped
regions of non-identity for the aligned polypeptides. The location
of the conserved catalytic triad amino acids are denoted by an
asterisk (*).
[0068] FIG. 4 shows a table illustrating the percent identity and
percent similarity between the PCSK9b and PCSK9c polypeptides of
the present invention with PCSK9 and its variant. As shown, the
catalytic domain of both PCSK9b and PCSK9c shares 100% identity
with the catalytic domain of PCSK9. The percent identity and
percent similarity values were determined using the CLUSTALW
algorithm using default parameters as described herein (VECTOR
NTI.RTM. suite of programs).
[0069] FIG. 5 provides a schematic diagram of the PCSK9b and PCSK9c
variants in comparison to the wild type PCSK9. As shown, both
PCSK9b and PCSK9c variants start from original intron 3. PCSK9b has
a novel splicing site in exon 9 which causes a frame shift and
early truncation of the C-terminal domain as a consequence of an
in-frame stop codon.
[0070] FIG. 6 shows an expression profile of the human PCSK9b and
PCSK9c polypeptides. The figure illustrates the relative expression
level of PCSK9b and PCSK9c amongst various mRNA tissue sources. The
identity of each tissue is provided in Table IV in Example 3. As
shown, the PCSK9b and PCSK9c polypeptides were expressed
predominately in cerebellum of the brain and liver, at levels
approximately 3500 fold higher than the lowest expressed tissue.
Significant expression was observed in the lung, in pulmonary blood
vessels, and tissues of the GI tract. Expression data was obtained
by measuring the steady state PCSK9b and PCSK9c mRNA levels by
quantitative PCR using the PCR primer pair provided as SEQ ID NO:10
and 11, and TAQMAN.RTM. probe (SEQ ID NO:12) as described in
Example 3 herein.
[0071] FIG. 7 shows the results of overexpression of PCSK9b and
PCSK9c variants in HEK and CHO cells. Panel "A" shows the observed
expression level of PCSK9, while Panel "B" shows the observed
expression level of LDLR, after HEK cells were transiently
transfected with wild type PCSK9 (WT), PCSK9 mutant D374Y, and
PCSK9b and PCSK9c plasmids. PCSK9b variant plasmids are represented
as "#3114", "#3115", while the PCSK9c variant plasmids are
represented as "#3116" and "#3117"). Expression data was obtained
by measuring the steady state PCSK9b and PCSK9c mRNA levels by
quantitative PCR as described in Example 4 herein.
[0072] FIG. 8 shows the results of western blot analysis of both
cell lysates and conditioned media of HEK and CHO cells transiently
transfected with PCSK9b and PCSK9c variants using PCSK9 specific
antibody. Panel "A" shows the Western blot of PCSK9 in cell lysates
from PCSK9 transfected HEK cells using PCSK9 antibody, while Panel
"B" shows the Western blot of PCSK9 of conditioned media from CHO
cells transiently transfected with PCSK9 variants. PCSK9b variant
plasmids are represented as "#3114", "#3115", while the PCSK9c
variant plasmids are represented as "#3116" and "#3117"). Wildtype
PCSK9 is represented as "WT", while the PCSK9 mutant D374Y is
represented as "D374Y". As shown, both PCSK9b and PCSK9c variant
proteins were secreted by the transfected cells as evidenced by
their detection in conditioned media despite both variants lacking
a signal peptide. Western blots were performed according to the
method described in Example 4 herein.
[0073] FIG. 9 shows the results of experiments designed to assess
whether PCSK9b and PCSK9c variants are able to decrease LDL binding
to the LDLR. Panel A shows the results of DiI-LDL uptake in HepG2
cells transfected with PCSK9b and PCSK9c, while Panel B shows the
results of Western blot on cell lysates from CHO cells transfected
with PCSK9b or PCSK9c. Both PCSK9 and LDLR antibodies were used for
the Western Blots. PCSK9b variant plasmids are represented as
"3114", "3115"; the PCSK9c variant plasmids are represented as
"3116" and "3117"; wildtype PCSK9 is represented as "WT"; the PCSK9
mutant D374Y is represented as "D3"; vector only is represented as
"vec", and cells incubated in 10% FBS containing medium is
represented as "FBS", while cell incubated in 5% lipoprotein
deficient serum growth medium is represented as "LPDS". As shown in
panel "A", transient transfection of HepG2 cells with both PCSK9b
and PCSK9c variant proteins resulted in a decreased level of
DiI-LDL uptake that exceeded the level observed for wildtype PCSK9.
As shown in panel "B", transient transfection of CHO cells with the
PCSK9c variant resulted in decreased LDLR protein level, while
transient transfection of variant b did not appear to affect LDLR
protein level under these conditions. DiI-LDL uptake assays and
Western blots were performed according to the methods described in
Example 4 herein.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention may be understood more readily by
reference to the following detailed description of the preferred
embodiments of the invention and the Examples included herein.
[0075] The invention provides novel human sequences that encode
variants of PCSK9 thereof, PCSK9b and PCSK9c, in addition to
N-terminal truncated forms of PCSK9. PCSK9 has been implicated in
the incidence of a variety of diseases and/or disorders, including
hypercholesterolemia, its related cardiovascular disorders, in
addition to other disorders known in the art or described
herein.
[0076] In the present invention, "isolated" refers to material
removed from its original environment (e.g., the natural
environment if it is naturally occurring), and thus is altered "by
the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of
matter, or could be contained within a cell, and still be
"isolated" because that vector, composition of matter, or
particular cell is not the original environment of the
polynucleotide. The term "isolated" does not refer to genomic or
cDNA libraries, whole cell total or mRNA preparations, genomic DNA
preparations (including those separated by electrophoresis and
transferred onto blots), sheared whole cell genomic DNA
preparations or other compositions where the art demonstrates no
distinguishing features of the polynucleotide/sequences of the
present invention.
[0077] In specific embodiments, the polynucleotides of the
invention are about at least 15, at least 30, at least 50, at least
100, at least 125, at least 500, at least 837, at least 903, at
least 1000, or at least 1554 continuous nucleotides but are less
than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5
kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In this context,
about means 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer or
shorter at either the 5' or 3' end, or both. In a further
embodiment, polynucleotides of the invention comprise a portion of
the coding sequences, as disclosed herein, but do not comprise all
or a portion of any intron. In another embodiment, the
polynucleotides comprising coding sequences do not contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of
interest in the genome). In other embodiments, the polynucleotides
of the invention do not contain the coding sequence of more than
1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic
flanking gene(s).
[0078] As used herein, a "polynucleotide" refers to a molecule
having a nucleic acid sequence contained in SEQ ID NO:1 or SEQ ID
NO:3, or the cDNA contained within the clone deposited with the
ATCC.RTM.. For example, the polynucleotide can contain the
nucleotide sequence of the full length cDNA sequence, including the
5' and 3' untranslated sequences, the coding region, with or
without a signal sequence, the secreted protein coding region, as
well as fragments, epitopes, domains, and variants of the nucleic
acid sequence. Moreover, as used herein, a "polypeptide" refers to
a molecule having the translated amino acid sequence generated from
the polynucleotide as broadly defined.
[0079] In the present invention, the full length sequence
identified as SEQ ID NO:1 or SEQ ID NOP:3 was generated by
overlapping sequences contained in one or more clones (contig
analysis). Representative clones containing all of the sequence for
SEQ ID NO:1 and SEQ ID NO:3 was deposited with the American Type
Culture Collection ("ATCC.RTM."). As shown in Table I, each clone
is identified by a cDNA Clone ID (Identifier) and the ATCC.RTM.
Deposit Number. The ATCC.RTM. is located at 10801 University
Boulevard, Manassas, Va. 20110-2209, USA. The ATCC.RTM. deposit was
made pursuant to the terms of the Budapest Treaty on the
international recognition of the deposit of microorganisms for
purposes of patent procedure. The deposited clone for PCSK9b is
inserted in the pSport2 vector (Invitrogen). The deposited clone
for PCSK9c is inserted in the pSport1 vector (Invitrogen).
[0080] 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, preferably
a Model 3700, from Applied Biosystems, Inc.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
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.
[0081] Using the information provided herein, such as the
nucleotide sequence in FIGS. 1A-C (SEQ ID NO:1), a nucleic acid
molecule of the present invention encoding the PCSK9b polypeptide
may be obtained using standard cloning and screening procedures,
such as those for cloning cDNAs using mRNA as starting
material.
[0082] Using the information provided herein, such as the
nucleotide sequence in FIGS. 2A-D (SEQ ID NO:3), a nucleic acid
molecule of the present invention encoding the PCSK9c polypeptide
may be obtained using standard cloning and screening procedures,
such as those for cloning cDNAs using mRNA as starting
material.
[0083] A "polynucleotide" of the present invention also includes
those polynucleotides capable of hybridizing, under stringent
hybridization conditions, to sequences contained in SEQ ID NO:1,
the complement thereof, or the cDNA within the clone deposited with
the ATCC.RTM.. "Stringent hybridization conditions" refers to an
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.
[0084] Also contemplated are nucleic acid molecules that hybridize
to the polynucleotides of the present invention at lower stringency
hybridization conditions. Changes in the stringency of
hybridization and signal detection are primarily accomplished
through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt
conditions, or temperature. For example, lower stringency
conditions include an overnight incubation at 37 degree C. in a
solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl; 0.2M
NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C. with
1.times.SSPE, 0.1% SDS. In addition, to achieve even lower
stringency, washes performed following stringent hybridization can
be done at higher salt concentrations (e.g. 5.times.SSC).
[0085] Note that variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. Typical blocking reagents include Denhardt's reagent,
BLOTTO, heparin, denatured salmon sperm DNA, and commercially
available proprietary formulations. The inclusion of specific
blocking reagents may require modification of the hybridization
conditions described above, due to problems with compatibility.
[0086] Of course, a polynucleotide which hybridizes only to polyA+
sequences (such as any 3' terminal polyA+ tract of a cDNA shown in
the sequence listing), or to a complementary stretch of T (or U)
residues, would not be included in the definition of
"polynucleotide" 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 generated using oligo dT as a primer).
[0087] The polynucleotide of the present invention can be composed
of any polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. For example,
polynucleotides can be composed of single- and double-stranded DNA,
DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions. In addition,
the polynucleotide can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. A polynucleotide may
also contain one or more modified bases or DNA or RNA backbones
modified for stability or for other reasons. "Modified" bases
include, for example, tritylated bases and unusual bases such as
inosine. A variety of modifications can be made to DNA and RNA;
thus, "polynucleotide" embraces chemically, enzymatically, or
metabolically modified forms.
[0088] The polypeptide of the present invention can be composed of
amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres, and may contain amino acids
other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as posttranslational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, Proteins--Structure and
Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); Posttranslational Covalent Modification
of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);
Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
[0089] "SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ
ID NO:Y" refers to a polypeptide sequence, both sequences are
identified by an integer specified in Table I.
[0090] "A polypeptide having biological activity" refers to
polypeptides exhibiting activity similar, but not necessarily
identical to, an activity of a polypeptide of the present
invention, including mature forms, as measured in a particular
biological assay, with or without dose dependency. In the case
where dose dependency does exist, it need not be identical to that
of the polypeptide, but rather substantially similar to the
dose-dependence in a given activity as compared to the polypeptide
of the present invention (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, and most
preferably, not more than about three-fold less activity relative
to the polypeptide of the present invention.)
[0091] The term "organism" as referred to herein is meant to
encompass any organism referenced herein, though preferably to
eukaryotic organisms, more preferably to mammals, and most
preferably to humans.
[0092] The present invention encompasses the identification of
proteins, nucleic acids, or other molecules, that bind to
polypeptides and polynucleotides of the present invention (for
example, in a receptor-ligand interaction). The polynucleotides of
the present invention can also be used in interaction trap assays
(such as, for example, that described by Ozenberger and Young (Mol
Endocrinol., 9(10):1321-9, (1995); and Ann. N.Y. Acad. Sci., 7;
766:279-81, (1995)).
[0093] The polynucleotide and polypeptides of the present invention
are useful as probes for the identification and isolation of
full-length cDNAs and/or genomic DNA which correspond to the
polynucleotides of the present invention, as probes to hybridize
and discover novel, related DNA sequences, as probes for positional
cloning of this or a related sequence, as probe to "subtract-out"
known sequences in the process of discovering other novel
polynucleotides, as probes to quantify gene expression, and as
probes for microarrays.
[0094] In addition, polynucleotides and polypeptides of the present
invention may comprise one, two, three, four, five, six, seven,
eight, or more membrane domains.
[0095] Also, in preferred embodiments the present invention
provides methods for further refining the biological function of
the polynucleotides and/or polypeptides of the present
invention.
[0096] Specifically, the invention provides methods for using the
polynucleotides and polypeptides of the invention to identify
orthologs, homologs, paralogs, variants, and/or allelic variants of
the invention. Also provided are methods of using the
polynucleotides and polypeptides of the invention to identify the
entire coding region of the invention, non-coding regions of the
invention, regulatory sequences of the invention, and secreted,
mature, pro-, prepro-, forms of the invention (as applicable).
[0097] In preferred embodiments, the invention provides methods for
identifying the glycosylation sites inherent in the polynucleotides
and polypeptides of the invention, and the subsequent alteration,
deletion, and/or addition of said sites for a number of desirable
characteristics which include, but are not limited to, augmentation
of protein folding, inhibition of protein aggregation, regulation
of intracellular trafficking to organelles, increasing resistance
to proteolysis, modulation of protein antigenicity, and mediation
of intercellular adhesion.
[0098] In further preferred embodiments, methods are provided for
evolving the polynucleotides and polypeptides of the present
invention using molecular evolution techniques in an effort to
create and identify novel variants with desired structural,
functional, and/or physical characteristics.
[0099] The present invention further provides for other
experimental methods and procedures currently available to derive
functional assignments. These procedures include but are not
limited to spotting of clones on arrays, micro-array technology,
PCR based methods (e.g., quantitative PCR), anti-sense methodology,
gene knockout experiments, and other procedures that could use
sequence information from clones to build a primer or a hybrid
partner.
Polynucleotides and Polypeptides of the Invention
Features of the Polypeptide Encoded by Polynucleotide No:1
[0100] The polypeptide of this polynucleotide provided as SEQ ID
NO:2 (FIGS. 1A-C), encoded by the polynucleotide sequence according
to SEQ ID NO:1 (FIGS. 1A-C), and/or encoded by the polynucleotide
contained within the deposited clone, PCSK9b (also referred to as
PCSK9-b), is a variant of the human PCSK9 polypeptide (PCSK9;
GENBANK.RTM. Accession No: gi|NM.sub.--174936; SEQ ID NO:5). An
alignment of the PCSK9b polypeptide with PCSK9 in addition to a
known PCSK9 variant (PCSK9 variant; GENBANK.RTM. Accession No:
gi|AK124635; SEQ ID NO:6) is provided in FIGS. 3A-C. The percent
identity and similarity values between the PCSK9b polypeptide to
these polypeptides is provided in FIG. 4.
[0101] The determined nucleotide sequence of the PCSK9b cDNA in
FIGS. 1A-C (SEQ ID NO:1) contains an open reading frame encoding a
protein of about 315 amino acid residues, with a deduced molecular
weight of about 33.2 kDa. The amino acid sequence of the predicted
PCSK9b polypeptide is shown in FIGS. 1A-C (SEQ ID NO:2).
[0102] The PCSK9b polypeptide was predicted to comprise a catalytic
domain located from about amino acid 10 to about amino acid 256 of
SEQ ID NO:2, with the canonical catalytic triad residing at amino
acids D17, H57, and S217 of SEQ ID NO:2; and six conserved cysteine
residues located at amino acids C54, C86, C132, C154, C189, and
C206 of SEQ ID NO:2, with disulfite bonds predicted to form between
the following cysteine pairs: C54 and C86, and C154 and C189; and a
predicted Ca.sup.2+ ion binding domain predicted to form between
residues D191, P162, and V164 of SEQ ID NO:2. In this context, the
term "about" may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acids beyond the N-terminal and/or C-terminal boundaries
of the above referenced amino acid locations.
[0103] Since the PCSK9b polypeptide retains the catalytic triad of
the wild-type PCSK9 polypeptide, in addition to its conserved
cysteines, it is expected that the PCSK9b polypeptide retains at
least some PCSK9 biological activity, including but not limited to
proteinase activity, convertase activity, subtilisin-kexin
isozyme-1/site 1 protease activity, autocatalytic activity cleaving
the PCSK9, PCSK9b, PCSK9c, or other variants of PCSK9 between amino
acids corresponding to amino acids Gln-151 and Ser-152;
sterol-dependent gene expression regulatory activity (Maxwell et
al., J Lipid Res. 2003; 44: 2109-2119), insulin-dependent gene
expression regulatory activity (Shimomura et al., Proc Natl Acad
Sci USA. 1999; 96: 13656-13661), LXR transcription factor-dependent
gene expression regulatory activity (Repa et al., Genes Dev. 2000;
14: 2819-2830); LDL receptor protein regulatory activity (Maxwell
et al., Proc Natl Acad Sci USA. 2004; 101: 7100-7105);
statin-dependent upregulation activity (Dubuc et al., Arterioscler
Thromb Vasc Biol. 2004; 24: 1454-1459).
[0104] In confirmation of PCSK9b retaining PCSK9 biological
activity, DiI-LDL uptake assays were performed and PCSK9b was shown
to have PCSK9 activity. Surprisingly, PCSK9b was found to have
greater activity than wildtype PCSK9. The DiI-LDL uptake assay is a
standardized functional assay for the LDLR receptor, and wildtype
PCSK9 activity acts to reduce LDLR activity. Therefore DiI-LDL
uptake by cells can be used as a surrogate functional assay for
measuring PCSK9 activity. Transient expression of PCSK9b acted to
decrease the uptake of DiI-LDL in HepG2 cells, compared to vector
control, indicating that PCSK9b is competent to express PCSK9
functional activity on LDLR (FIG. 9). In this assay, PCSK9b showed
greater activity than wild-type PCSK9, though not as great as
PCSK9c. Accordingly, PCSK9b retains the ability of wildtype PCSK9
to modulate the functional activity on LDLR.
[0105] PCSK9b was shown to have about 25% more function than
wildtype PCSK9 as demonstrated in the LDL uptake assay (see FIG.
9A). Accordingly, the invention further relates to an N-terminal
truncation of PCSK9, such as PCSK9c for example (SEQ ID NO:2),
wherein said N-terminal truncation results in an elevation of PCSK9
biological activity, including, but not limited to decreased LDLR
protein levels and/or decreased LDL uptake by LDLR, and wherein
said elevated PCSK9 biological activity is at least about 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 100%, or more than wildtype elevated PCSK9
biological activity. In this context, the term "about" shall be
construed to mean anywhere between 1, 2, 3, 4, or 5 percent more or
less than the cited amount. Alternatively, said elevated PCSK9
biological activity may be at least about 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., or 10.times. more than wildtype PCSK9 biological
activity. In this context, the term "about" shall be construed to
mean anywhere between 0.1.times., 0.2.times., 0.3.times.,
0.4.times., 0.5.times., 0.6.times., 0.7.times., 0.8.times., or
0.9.times. more or less than the cited amount.
[0106] In preferred embodiments, the present invention also
encompasses a polynucleotide that comprises a polypeptide that
encodes at least about 279 contiguous amino acids of SEQ ID NO:2.
The present invention also encompasses a polynucleotide that
comprises at least about 837 contiguous nucleotides of SEQ ID NO:1.
In this context, the term "about" shall be construe to mean 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 more or less amino acids at either the N- or C-terminus, or
both, or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 nucleotides at either the 5 prime or 3 prime end,
or both. Preferably, the polypeptides and/or polypeptides encoded
by said polynucleotides retain biological activity.
[0107] In preferred embodiments, the present invention encompasses
a polynucleotide lacking the initiating start codon, in addition
to, the methinone of the resulting encoded polypeptide of PCSK9b.
Specifically, the present invention encompasses the polynucleotide
corresponding to nucleotides 253 thru 1194 of SEQ ID NO:1, and the
polypeptide corresponding to amino acids 2 thru 315 of SEQ ID NO:2.
Also encompassed are recombinant vectors comprising said encoding
sequence, and host cells comprising said vector.
[0108] As described herein, misense mutations of the wild-type
PCSK9 at amino acid locations S127R, F216L, and D374Y have been
shown to result in aberrant function of the PCSK9 protein resulting
in the incidence of hypercholesterolemia (Attie, A. D., Art.
Thromb. and Vasc. Biol., 2004; 24:1337). According, the same
mutations at the corresponding amino acid positions of the PCSK9b
polypeptide of the present invention would be useful in methods of
diagnosing patients susceptible to the incidence of
hypercholesterolemia. It should be noted that PCSK9b lacks a
corresponding amino acid for the S127R mutation on account of
alternative splicing (see alignment provided in FIGS. 3A-C).
[0109] In preferred embodiments, the present invention also
encompasses a polynucleotide of SEQ ID NO:2 wherein the serine at
amino acid position 47 is substituted with a leucine. The present
invention also encompasses a polypeptide of SEQ ID NO:2 lacking the
initiating start codon, in addition to, the methionine of the
resulting encoded polypeptide of PCSK9b, wherein serine at amino
acid position 47 is substituted with a leucine. Polynucleotides
encoding these polypeptides are also provided. Also encompassed are
recombinant vectors comprising said encoding sequence, and host
cells comprising said vector.
[0110] In preferred embodiments, the present invention also
encompasses a polynucleotide of SEQ ID NO:2 wherein the aspartic
acid at amino acid position 205 is substituted with a tyrosine. The
present invention also encompasses a polypeptide of SEQ ID NO:2
lacking the initiating start codon, in addition to, the methionine
of the resulting encoded polypeptide of PCSK9b, wherein the
aspartic acid at amino acid position 205 is substituted with a
tyrosine. Polynucleotides encoding these polypeptides are also
provided. Also encompassed are recombinant vectors comprising said
encoding sequence, and host cells comprising said vector.
[0111] Since the PCSK9b polypeptide represents a variant form of
the wild-type PCSK9 polypeptide (PCSK9; GENBANK.RTM. Accession No:
gi|NM.sub.--174936; SEQ ID NO:5), it is expected that the
expression pattern of the PCSK9b variant is the same or similar to
the PCSK9 polypeptide.
[0112] The wild-type PCSK9 polypeptide was determined to be
predominantly expressed in liver and neuronal tissue, and to a
lesser extent in kidney mesenchymal cells and intestinal epithelia
(Seidah et al., PNAS 100(3):928-933 (2003)).
[0113] Expression profiling designed to measure the steady state
mRNA levels encoding the PCSK9b and PCSK9c polypeptides confirmed
that they shared the predominate liver and neuronal expression
pattern of wild-type PCSK9. Specifically, PCSK9b and PCSK9c were
predominately expressed in liver and cerebellum at levels that were
over 3500 times that of the tissue with the lowest expression (the
heart). PCSK9b and PCSK9c were also expressed relatively highly in
the lung and its associated vasculature. PCSK9b and PCSK9c were
also expressed throughout the gastrointestinal tract (See FIG. 6).
The PCSK9b and PCSK9c expression in the cerebellum indicates that
PCSK9b and PCSK9c may function in neuronal differentiation.
[0114] The PCSK9b polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following diseases and/or disorders: autosomal
dominant hypercholesterolemia; disorders associated with aberrant
LDL receptor function; disorders associated with apolipoprotein B;
disorders associated with autosomal recessive hypercholesterolemia;
disorders associated with elevated cholesterol; disorders
associated with elevated LDL; disorders associated with reduced
clearance rate of LDL in the liver; disorders associated with
elevated LDL apoB production; familial hypercholesterolemia; lipid
metabolism disorders; elevated LDL; cholesterol depositions; tendon
xanthomas; atheroma; premature arteriosclerosis, coronary heart
disease; famialial defective apolipoprotein B; statin
hypersensitivity; disorders associated with accelerated LDLR
degradation.
[0115] The PCSK9b polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following diseases and/or disorders: neural
differentiation disorders.
[0116] The PCSK9b polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following cardiovascular diseases and/or
disorders: myocardio infarction, congestive heart failure,
arrthymias, cardiomyopathy, atherosclerosis, arterialsclerosis,
microvascular disease, embolism, thromobosis, pulmonary edema,
palpitation, dyspnea, angina, hypotension, syncope, heart murmur,
aberrant ECG, hypertrophic cardiomyopathy, the Marfan syndrome,
sudden death, prolonged QT syndrome, congenital defects, cardiac
viral infections, valvular heart disease, hypertension, among
others disclosed herein, particularly in the "Cardiovascular
Disorders" section and below.
[0117] Similarly, PCSK9b polynucleotides and polypeptides may be
useful for ameliorating cardiovascular diseases and symptoms which
result indirectly from various non-cardiovascular effects, which
include, but are not limited to, the following, obesity, smoking,
Down syndrome (associated with endocardial cushion defect); bony
abnormalities of the upper extremities (associated with atrial
septal defect in the Holt-Oram syndrome); muscular dystrophies
(associated with cardiomyopathy); hemochromatosis and glycogen
storage disease (associated with myocardial infiltration and
restrictive cardiomyopathy); congenital deafness (associated with
prolonged QT interval and serious cardiac arrhythmias); Raynaud's
disease (associated with primary pulmonary hypertension and
coronary vasospasm); connective tissue disorders, i.e., the Marfan
syndrome, Ehlers-Danlos and Hurler syndromes, and related disorders
of mucopolysaccharide metabolism (aortic dilatation, prolapsed
mitral valve, a variety of arterial abnormalities); acromegaly
(hypertension, accelerated coronary atherosclerosis, conduction
defects, cardiomyopathy); hyperthyroidism (heart failure, atrial
fibrillation); hypothyroidism (pericardial effusion, coronary
artery disease); rheumatoid arthritis (pericarditis, aortic valve
disease); scleroderma (cor pulmonale, myocardial fibrosis,
pericarditis); systemic lupus erythematosus (valvulitis,
myocarditis, pericarditis); sarcoidosis (arrhythmias,
cardiomyopathy); postmenopausal effects, Chlamydial infections,
polycystic ovary disease, thyroid disease, alcoholism, diet, and
exfoliative dermatitis (high-output heart failure), for
example.
[0118] Moreover, polynucleotides and polypeptides, including
fragments and/or antagonists thereof, have uses which include,
directly or indirectly, treating, preventing, diagnosing, and/or
prognosing the following, non-limiting, cardiovascular infections:
blood stream invasion, bacteremia, sepsis, Streptococcus pneumoniae
infection, group a streptococci infection, group b streptococci
infection, Enterococcus infection, nonenterococcal group D
streptococci infection, nonenterococcal group C streptococci
infection, nonenterococcal group G streptococci infection,
Streptoccus viridans infection, Staphylococcus aureus infection,
coagulase-negative staphylococci infection, gram-negative Bacilli
infection, Enterobacteriaceae infection, Pseudomonas spp.
Infection, Acinobacter spp. Infection, Flavobacterium
meningosepticum infection, Aeromonas spp. Infection,
Stenotrophomonas maltophilia infection, gram-negative coccobacilli
infection, Haemophilus influenza infection, Branhamella catarrhalis
infection, anaerobe infection, Bacteriodes fragilis infection,
Clostridium infection, fungal infection, Candida spp. Infection,
non-albicans Candida spp. Infection, Hansenula anomala infection,
Malassezia furfur infection, nontuberculous Mycobacteria infection,
Mycobacterium avium infection, Mycobacterium chelonae infection,
Mycobacterium fortuitum infection, spirochetal infection, Borrelia
burgdorferi infection, in addition to any other cardiovascular
disease and/or disorder (e.g., non-sepsis) implicated by the
causative agents listed above or elsewhere herein.
[0119] The PCSK9b polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following metabolic diseases and/or disorders:
dyslipidemia, diabetic dyslipidemia, mixed dyslipidemia,
hypercholesteremia, hypertriglyceridemia, type II diabetes
mellitus, type I diabetes, insulin resistance, hyperlipidemia,
obesity, and/or anorexia nervosa.
[0120] The present invention also encompasses therapeutic
combinations of a modulator of PCSK9b with a statin for the
treatment, prevention and/or amelioration of a disease or disorder
referenced herein, particularly dyslipidemia. Representative
statins include, but are not limited to, the following:
pravastatin, lovastatin, cerivastatin, simvastatin, pitivastatin,
atorvastatin or rousuvastatin.
[0121] The PCSK9b polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include modulating signal transduction activity, in various
cells, tissues, and organisms, and particularly in liver, brain,
mammalian adipose, omentum, spleen, inflammatory tissues,
macrophages, neutrophils, synovial histiomonocytes, neutrophils,
and epithelioid histiocytes.
[0122] The PCSK9b polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following hepatic disorders: hepatoblastoma,
jaundice, hepatitis, liver metabolic diseases and conditions that
are attributable to the differentiation of hepatocyte progenitor
cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebic abcess,
hydatid cyst, cystadenocarcinoma, adenoma, focal nodular
hyperplasia, hemangioma, hepatocellulae carcinoma,
cholangiocarcinoma, and angiosarcoma, granulomatous liver disease,
liver transplantation, hyperbilirubinemia, jaundice, parenchymal
liver disease, portal hypertension, hepatobiliary disease, hepatic
parenchyma, hepatic fibrosis, anemia, gallstones, cholestasis,
carbon tetrachloride toxicity, beryllium toxicity, vinyl chloride
toxicity, choledocholithiasis, hepatocellular necrosis, aberrant
metabolism of amino acids, aberrant metabolism of carbohydrates,
aberrant synthesis proteins, aberrant synthesis of glycoproteins,
aberrant degradation of proteins, aberrant degradation of
glycoproteins, aberrant metabolism of drugs, aberrant metabolism of
hormones, aberrant degradation of drugs, aberrant degradation of
drugs, aberrant regulation of lipid metabolism, aberrant regulation
of cholesterol metabolism, aberrant glycogenesis, aberrant
glycogenolysis, aberrant glycolysis, aberrant gluconeogenesis,
hyperglycemia, glucose intolerance, hyperglycemia, decreased
hepatic glucose uptake, decreased hepatic glycogen synthesis,
hepatic resistance to insulin, portal-systemic glucose shunting,
peripheral insulin resistance, hormonal abnormalities, increased
levels of systemic glucagon, decreased levels of systemic cortisol,
increased levels of systemic insulin, hypoglycemia, decreased
gluconeogenesis, decreased hepatic glycogen content, hepatic
resistance to glucagon, elevated levels of systemic aromatic amino
acids, decreased levels of systemic branched-chain amino acids,
hepatic encephalopathy, aberrant hepatic amino acid transamination,
aberrant hepatic amino acid oxidative deamination, aberrant ammonia
synthesis, aberant albumin secretion, hypoalbuminemia, aberrant
cytochromes b5 function, aberrant P450 function, aberrant
glutathione S-acyltransferase function, aberrant cholesterol
synthesis, and aberrant bile acid synthesis.
[0123] Moreover, polynucleotides and polypeptides, including
fragments and/or antagonists thereof, have uses which include,
directly or indirectly, treating, preventing, diagnosing, and/or
prognosing the following, non-limiting, hepatic infections: liver
disease caused by sepsis infection, liver disease caused by
bacteremia, liver disease caused by Pneomococcal pneumonia
infection, liver disease caused by Toxic shock syndrome, liver
disease caused by Listeriosis, liver disease caused by
Legionnaries' disease, liver disease caused by Brucellosis
infection, liver disease caused by Neisseria gonorrhoeae infection,
liver disease caused by Yersinia infection, liver disease caused by
Salmonellosis, liver disease caused by Nocardiosis, liver disease
caused by Spirochete infection, liver disease caused by Treponema
pallidum infection, liver disease caused by Brrelia burgdorferi
infection, liver disease caused by Leptospirosis, liver disease
caused by Coxiella burnetii infection, liver disease caused by
Rickettsia richettsii infection, liver disease caused by Chlamydia
trachomatis infection, liver disease caused by Chlamydia psittaci
infection, liver disease caused by hepatitis virus infection, liver
disease caused by Epstein-Barr virus infection in addition to any
other hepatic disease and/or disorder implicated by the causative
agents listed above or elsewhere herein.
[0124] The PCSK9b polynucleotides and polypeptides, including
fragments and/or antagonsists thereof, may have uses which include
identification of modulators of PCSK9b function including
antibodies (for detection or neutralization), naturally-occurring
modulators and small molecule modulators. Antibodies to domains of
the PCSK9b protein could be used as diagnostic agents of lipid
metabolic disorders, including hypercholesterolemia, among
others.
[0125] The expression level of PCSK9b also may be useful as a
biomarker for predicting which patients may be at risk of
developing hypercholesterolemia, and/or those patients which may be
at risk of being overly sensitive to statin therapy.
[0126] PCSK9b polypeptides and polynucleotides have additional uses
which include diagnosing diseases related to the over and/or under
expression of PCSK9b by identifying mutations in the PCSK9b gene by
using PCSK9b sequences as probes or by determining PCSK9b protein
or mRNA expression levels. PCSK9b polypeptides may be useful for
screening compounds that affect the activity of the protein. PCSK9b
peptides can also be used for the generation of specific antibodies
and as bait in yeast two hybrid screens to find proteins the
specifically interact with PCSK9b (described elsewhere herein).
[0127] In preferred embodiments, the present invention is also
directed to polynucleotides comprising, or alternatively consisting
of, a sequence encoding the following N-terminal PCSK9b deletion
polypeptides: M1-R315, S2-R315, P3-R315, W4-R315, K5-R315, D6-R315,
G7-R315, G8-R315, S9-R315, L10-R315, V11-R315, E12-R315, V13-R315,
Y14-R315, L15-R315, L16-R315, D17-R315, T18-R315, S19-R315,
I20-R315, Q21-R315, S22-R315, D23-R315, H24-R315, R25-R315,
E26-R315, I27-R315, E28-R315, G29-R315, R30-R315, V31-R315,
M32-R315, V33-R315, T34-R315, D35-R315, F36-R315, E37-R315,
N38-R315, V39-R315, P40-R315, E41-R315, E42-R315, D43-R315,
G44-R315, T45-R315, R46-R315, F47-R315, H48-R315, R49-R315,
Q50-R315, A51-R315, S52-R315, K53-R315, C54-R315, D55-R315,
S56-R315, H57-R315, G58-R315, T59-R315, H60-R315, L61-R315,
A62-R315, G63-R315, V64-R315, V65-R315, S66-R315, G67-R315,
R68-R315, D69-R315, A70-R315, G71-R315, V72-R315, A73-R315,
K74-R315, G75-R315, A76-R315, S77-R315, M78-R315, R79-R315,
S80-R315, L81-R315, R82-R315, V83-R315, L84-R315, N85-R315,
C86-R315, Q87-R315, G88-R315, K89-R315, G90-R315, T91-R315,
V92-R315, S93-R315, G94-R315, T95-R315, L96-R315, I97-R315,
G98-R315, L99-R315, E100-R315, F101-R315, I102-R315, R103-R315,
K104-R315, S105-R315, Q106-R315, L107-R315, V108-R315, Q109-R315,
P110-R315, V111-R315, G112-R315, P113-R315, L114-R315, V115-R315,
V116-R315, L117-R315, L118-R315, P119-R315, L120-R315, A121-R315,
G122-R315, G123-R315, Y124-R315, S125-R315, R126-R315, V127-R315,
L128-R315, N129-R315, A130-R315, A131-R315, C132-R315, Q133-R315,
R134-R315, L135-R315, A136-R315, R137-R315, A138-R315, G139-R315,
V140-R315, V141-R315, L142-R315, V143-R315, T144-R315, A145-R315,
A146-R315, G147-R315, N148-R315, F149-R315, R150-R315, D151-R315,
D152-R315, A153-R315, C154-R315, L155-R315, Y156-R315, S157-R315,
P158-R315, A159-R315, S160-R315, A161-R315, P162-R315, E163-R315,
V164-R315, I165-R315, T166-R315, V167-R315, G168-R315, A169-R315,
T170-R315, N171-R315, A172-R315, Q173-R315, D174-R315, Q175-R315,
P176-R315, V177-R315, T178-R315, L179-R315, G180-R315, T181-R315,
L182-R315, G183-R315, T184-R315, N185-R315, F186-R315, G187-R315,
R188-R315, C189-R315, V190-R315, D191-R315, L192-R315, F193-R315,
A194-R315, P195-R315, G196-R315, E197-R315, D198-R315, I199-R315,
I200-R315, G201-R315, A202-R315, S203-R315, S204-R315, D205-R315,
C206-R315, S207-R315, T208-R315, C209-R315, F210-R315, V211-R315,
S212-R315, Q213-R315, S214-R315, G215-R315, T216-R315, S217-R315,
Q218-R315, A219-R315, A220-R315, A221-R315, H222-R315, V223-R315,
A224-R315, G225-R315, I226-R315, A227-R315, A228-R315, M229-R315,
M230-R315, L231-R315, S232-R315, A233-R315, E234-R315, P235-R315,
E236-R315, L237-R315, T238-R315, L239-R315, A240-R315, E241-R315,
L242-R315, R243-R315, Q244-R315, R245-R315, L246-R315, I247-R315,
H248-R315, F249-R315, S250-R315, A251-R315, K252-R315, D253-R315,
V254-R315, I255-R315, N256-R315, E257-R315, A258-R315, W259-R315,
F260-R315, P261-R315, E262-R315, D263-R315, Q264-R315, R265-R315,
V266-R315, L267-R315, T268-R315, P269-R315, N270-R315, L271-R315,
V272-R315, A273-R315, A274-R315, L275-R315, P276-R315, P277-R315,
S278-R315, T279-R315, H280-R315, G281-R315, A282-R315, G283-R315,
P284-R315, F285-R315, C286-R315, R287-R315, L288-R315, A289-R315,
A290-R315, V291-R315, L292-R315, Q293-R315, D294-R315, C295-R315,
V296-R315, V297-R315, S298-R315, T299-R315, L300-R315, G301-R315,
A302-R315, Y303-R315, T304-R315, D305-R315, G306-R315, H307-R315,
S308-R315, and/or H309-R315 of SEQ ID NO:2. Polypeptide sequences
encoded by these polynucleotides are also provided. In addition,
the invention also encompasses polynucleotides encoding a
polypeptide that is at least as long as any one of the
aforementioned polypeptides. The present invention also encompasses
the use of these N-terminal PCSK9b deletion polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0128] In preferred embodiments, the present invention is also
directed to polynucleotides comprising, or alternatively consisting
of, a sequence encoding the following C-terminal PCSK9b deletion
polypeptides: M1-R315, M1-P314, M1-R313, M1-L312, M1-P311, M1-R310,
M1-H309, M1-S308, M1-H307, M1-G306, M1-D305, M1-T304, M1-Y303,
M1-A302, M1-G301, M1-L300, M1-T299, M1-S298, M1-V297, M1-V296,
M1-C295, M1-D294, M1-Q293, M1-L292, M1-V291, M1-A290, M1-A289,
M1-L288, M1-R287, M1-C286, M1-F285, M1-P284, M1-G283, M1-A282,
M1-G281, M1-H280, M1-T279, M1-S278, M1-P277, M1-P276, M1-L275,
M1-A274, M1-A273, M1-V272, M1-L271, M1-N270, M1-P269, M1-T268,
M1-L267, M1-V266, M1-R265, M1-Q264, M1-D263, M1-E262, M1-P261,
M1-F260, M1-W259, M1-A258, M1-E257, M1-N256, M1-I255, M1-V254,
M1-D253, M1-K252, M1-A251, M1-S250, M1-F249, M1-H248, M1-I247,
M1-L246, M1-R245, M1-Q244, M1-R243, M1-L242, M1-E241, M1-A240,
M1-L239, M1-T238, M1-L237, M1-E236, M1-P235, M1-E234, M1-A233,
M1-S232, M1-L231, M1-M230, M1-M229, M1-A228, M1-A227, M1-I226,
M1-G225, M1-A224, M1-V223, M1-H222, M1-A221, M1-A220, M1-A219,
M1-Q218, M1-S217, M1-T216, M1-G215, M1-S214, M1-Q213, M1-S212,
M1-V211, M1-F210, M1-C209, M1-T208, M1-S207, M1-C206, M1-D205,
M1-S204, M1-S203, M1-A202, M1-G201, M1-I200, M1-I199, M1-D198,
M1-E197, M1-G196, M1-P195, M1-A194, M1-F193, M1-L192, M1-D191,
M1-V190, M1-C189, M1-R188, M1-G187, M1-F186, M1-N185, M1-T184,
M1-G183, M1-L182, M1-T181, M1-G180, M1-L179, M1-T178, M1-V177,
M1-P176, M1-Q175, M1-D174, M1-Q173, M1-A172, M1-N171, M1-T170,
M1-A169, M1-G168, M1-V167, M1-T166, M1-I165, M1-V164, M1-E163,
M1-P162, M1-A161, M1-S160, M1-A159, M1-P158, M1-S157, M1-Y156,
M1-L155, M1-C154, M1-A153, M1-D152, M1-D151, M1-R150, M1-F149,
M1-N148, M1-G147, M1-A146, M1-A145, M1-T144, M1-V143, M1-L142,
M1-V141, M1-V140, M1-G139, M1-A138, M1-R137, M1-A136, M1-L135,
M1-R134, M1-Q133, M1-C132, M1-A131, M1-A130, M1-N129, M1-L128,
M1-V127, M1-R126, M1-S125, M1-Y124, M1-G123, M1-G122, M1-A121,
M1-L120, M1-P119, M1-L118, M1-L117, M1-V116, M1-V115, M1-L114,
M1-P113, M1-G112, M1-V111, M1-P110, M1-Q109, M1-V108, M1-L107,
M1-Q106, M1-S105, M1-K104, M1-R103, M1-I102, M1-F101, M1-E100,
M1-L99, M1-G98, M1-I97, M1-L96, M1-T95, M1-G94, M1-S93, M1-V92,
M1-T91, M1-G90, M1-K89, M1-G88, M1-Q87, M1-C86, M1-N85, M1-L84,
M1-V83, M1-R82, M1-L81, M1-S80, M1-R79, M1-M78, M1-S77, M1-A76,
M1-G75, M1-K74, M1-A73, M1-V72, M1-G71, M1-A70, M1-D69, M1-R68,
M1-G67, M1-S66, M1-V65, M1-V64, M1-G63, M1-A62, M1-L61, M1-H60,
M1-T59, M1-G58, M1-H57, M1-S56, M1-D55, M1-C54, M1-K53, M1-S52,
M1-A51, M1-Q50, M1-R49, M1-H48, M1-F47, M1-R46, M1-T45, M1-G44,
M1-D43, M1-E42, M1-E41, M1-P40, M1-V39, M1-N38, M1-E37, M1-F36,
M1-D35, M1-T34, M1-V33, M1-M32, M1-V31, M1-R30, M1-G29, M1-E28,
M1-I27, M1-E26, M1-R25, M1-H24, M1-D23, M1-S22, M1-Q21, M1-I20,
M1-S19, M1-T18, M1-D17, M1-L16, M1-L15, M1-Y14, M1-V13, M1-E12,
M1-V11, M1-L10, M1-S9, M1-G8, and/or M1-G7 of SEQ ID NO:2.
Polypeptide sequences encoded by these polynucleotides are also
provided. In addition, the invention also encompasses
polynucleotides encoding a polypeptide that is at least as long as
any one of the aforementioned polypeptides. The present invention
also encompasses the use of these C-terminal PCSK9b deletion
polypeptides as immunogenic and/or antigenic epitopes as described
elsewhere herein.
[0129] Alternatively, preferred polypeptides of the present
invention may comprise polypeptide sequences corresponding to, for
example, internal regions of the PCSK9b polypeptide (e.g., any
combination of both N- and C-terminal PCSK9b polypeptide deletions)
of SEQ ID NO:2. For example, internal regions could be defined by
the equation: amino acid NX to amino acid CX, wherein NX refers to
any N-terminal deletion polypeptide amino acid of PCSK9b (SEQ ID
NO:2), and where CX refers to any C-terminal deletion polypeptide
amino acid of PCSK9b (SEQ ID NO:2). Polynucleotides encoding these
polypeptides are also provided. The present invention also
encompasses the use of these polypeptides as an immunogenic and/or
antigenic epitope as described elsewhere herein.
[0130] The present invention also encompasses immunogenic and/or
antigenic epitopes of the PCSK9b polypeptide.
[0131] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:1 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides consisting of
a nucleotide sequence described by the general formula of a-b,
where a is any integer between 1 to 3161 of SEQ ID NO:1, b is an
integer between 15 to 3175, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:1, and where b
is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:2
[0132] The polypeptide of this polynucleotide provided as SEQ ID
NO:4 (FIGS. 2A-D), encoded by the polynucleotide sequence according
to SEQ ID NO:3 (FIGS. 2A-D), and/or encoded by the polynucleotide
contained within the deposited clone, PCSK9c (also referred to as
PCSK9-c), is a variant of the human PCSK9 polypeptide (PCSK9;
GENBANK.RTM. Accession No: gi|NM.sub.--174936; SEQ ID NO:5). An
alignment of the PCSK9c polypeptide with PCSK9 in addition to a
known PCSK9 variant (PCSK9 variant; GENBANK.RTM. Accession No:
gi|AK124635; SEQ ID NO:6) is provided in FIGS. 3A-C. The percent
identity and similarity values between the PCSK9c polypeptide to
these polypeptides is provided in FIG. 4.
[0133] The determined nucleotide sequence of the PCSK9c cDNA in
FIGS. 2A-D (SEQ ID NO:3) contains an open reading frame encoding a
protein of about 523 amino acid residues, with a deduced molecular
weight of about 55.2 kDa. The amino acid sequence of the predicted
PCSK9c polypeptide is shown in FIGS. 2A-D (SEQ ID NO:4).
[0134] The PCSK9c polypeptide was predicted to comprise a catalytic
domain located from about amino acid 10 to about amino acid 256 of
SEQ ID NO:4, with the canonical catalytic triad residing at amino
acids D17, H57, and S217 of SEQ ID NO:4; and six conserved cysteine
residues located at amino acids C54, C86, C132, C154, C189, and
C206 of SEQ ID NO:4, with disulfide bonds predicted to form between
the following cysteine pairs: C54 and C86, and C154 and C189; and a
predicted Ca.sup.2+ ion binding domain predicted to form between
residues D191, P162, and V164 of SEQ ID NO:4. In this context, the
term "about" may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acids beyond the N-terminal and/or C-terminal boundaries
of the above referenced amino acid locations.
[0135] Since the PCSK9c polypeptide retains the catalytic triad of
the wild-type PCSK9 polypeptide, in addition to its conserved
cysteines, it is expected that the PCSK9c polypeptide retains PCSK9
biological activity, including but not limited to proteinase
activity, convertase activity, subtilisin-kexin isozyme-1/site 1
protease activity, autocatalytic activity cleaving the PCSK9,
PCSK9b, PCSK9c, or other variants of PCSK9 between amino acids
corresponding to amino acids Gln-151 and Ser-152; sterol-dependent
gene expression regulatory activity (Maxwell et al., J Lipid Res.
2003; 44: 2109-2119), insulin-dependent gene expression regulatory
activity (Shimomura et al., Proc Natl Acad Sci USA. 1999; 96:
13656-13661), LXR transcription factor-dependent gene expression
regulatory activity (Repa et al., Genes Dev. 2000; 14: 2819-2830);
LDL receptor protein regulatory activity (Maxwell et al., Proc Natl
Acad Sci USA. 2004; 101: 7100-7105); statin-dependent upregulation
activity (Dubuc et al., Arterioscler Thromb Vasc Biol. 2004; 24:
1454-1459).
[0136] In confirmation of PCSK9c retaining PCSK9 biological
activity, DiI-LDL uptake assays were performed and PCSK9c was shown
to have PCSK9 activity. Surprisingly, PCSK9c was found to have
greater activity than wildtype PCSK9. The DiI-LDL uptake assay is a
standardized functional assay for the LDLR receptor, and wildtype
PCSK9 activity acts to reduce LDLR activity. Therefore DiI-LDL
uptake by cells can be used as a surrogate functional assay for
measuring PCSK9 activity. Transient expression of PCSK9c acted to
decrease the uptake of DiI-LDL in HepG2 cells, compared to vector
control, indicating that PCSK9c is competent to express PCSK9
functional activity on LDLR (FIG. 9). In this assay, PCSK9c showed
greater activity than both wild-type PCSK9 and PCSK9b. Accordingly,
PCSK9c retains the ability of wildtype PCSK9 to modulate the
functional activity on LDLR.
[0137] PCSK9c was shown to have about 50% more function than
wildtype PCSK9 as demonstrated in the LDL uptake assay (see FIG.
9A). Accordingly, the invention further relates to an N-terminal
truncation of PCSK9, such as PCSK9c for example (SEQ ID NO:4),
wherein said N-terminal truncation results in an elevation of PCSK9
biological activity, including, but not limited to decreased LDLR
protein levels and/or decreased LDL uptake by LDLR, and wherein
said elevated PCSK9 biological activity is at least about 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 100%, or more than wildtype elevated PCSK9
biological activity. In this context, the term "about" shall be
construed to mean anywhere between 1, 2, 3, 4, or 5 percent more or
less than the cited amount. Alternatively, said elevated PCSK9
biological activity may be at least about 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., or 10.times. more than wildtype PCSK9 biological
activity. In this context, the term "about" shall be construed to
mean anywhere between 0.1.times., 0.2.times., 0.3.times.,
0.4.times., 0.5.times., 0.6.times., 0.7.times., 0.8.times., or
0.9.times. more or less than the cited amount.
[0138] In preferred embodiments, the present invention also
encompasses a polynucleotide that comprises a polypeptide that
encodes at least about 301 contiguous amino acids of SEQ ID NO:4.
The present invention also encompasses a polypeptide that comprises
at least about 496 amino acids of SEQ ID NO:4. The present
invention also encompasses a polypeptide that comprises at least
about 518 amino acids of SEQ ID NO:4. The present invention also
encompasses a polynucleotide that comprises at least about 903
contiguous nucleotides of SEQ ID NO:3. The present invention also
encompasses a polynucleotide that comprises at least about 1488
contiguous nucleotides of SEQ ID NO:3. The present invention also
encompasses a polynucleotide that comprises at least about 1554
contiguous nucleotides of SEQ ID NO:3. In this context, the term
"about" shall be construe to mean 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 more or less amino acids
at either the N- or C-terminus, or both, or 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides at
either the 5 prime or 3 prime end, or both. Preferably, the
polypeptides and/or polypeptides encoded by said polynucleotides
retain biological activity.
[0139] In preferred embodiments, the present invention encompasses
a polynucleotide lacking the initiating start codon, in addition to
the methionine of the resulting encoded polypeptide of PCSK9c.
Specifically, the present invention encompasses the polynucleotide
corresponding to nucleotides 884 thru 2049 of SEQ ID NO:3, and the
polypeptide corresponding to amino acids 2 thru 523 of SEQ ID NO:4.
Also encompassed are recombinant vectors comprising said encoding
sequence, and host cells comprising said vector.
[0140] Since the PCSK9c polypeptide represents a variant form of
the wild-type PCSK9 polypeptide (PCSK9; GENBANK.RTM. Accession No:
gi|NM.sub.--174936; SEQ ID NO:5), it is expected that the
expression pattern of the PCSK9c variant is the same or similar to
the PCSK9 polypeptide.
[0141] As described herein, misense mutations of the wild-type
PCSK9 at amino acid locations S127R, F216L, and D374Y have been
shown to result in aberrant function of the PCSK9 protein resulting
in the incidence of hypercholesterolemia (Attie, A. D., Art.
Thromb. and Vasc. Biol., 2004; 24:1337). According, the same
mutations at the corresponding amino acid positions of the PCSK9c
polypeptide of the present invention would be useful in methods of
diagnosing patients susceptible to the incidence of
hypercholesterolemia. It should be noted that PCSK9c lacks a
corresponding amino acid for the S127R mutation on account of
alternative splicing (see alignment provided in FIGS. 3A-C).
[0142] In preferred embodiments, the present invention also
encompasses a polynucleotide of SEQ ID NO:4 wherein the serine at
amino acid position 47 is substituted with a leucine. The present
invention also encompasses a polypeptide of SEQ ID NO:4 lacking the
initiating start codon, in addition to, the methionine of the
resulting encoded polypeptide of PCSK9c, wherein serine at amino
acid position 47 is substituted with a leucine. Polynucleotides
encoding these polypeptides are also provided. Also encompassed are
recombinant vectors comprising said encoding sequence, and host
cells comprising said vector.
[0143] In preferred embodiments, the present invention also
encompasses a polynucleotide of SEQ ID NO:4 wherein the aspartic
acid at amino acid position 205 is substituted with a tyrosine. The
present invention also encompasses a polypeptide of SEQ ID NO:4
lacking the initiating start codon, in addition to, the methionine
of the resulting encoded polypeptide of PCSK9c, wherein aspartic
acid at amino acid position 205 is substituted with a tyrosine.
Polynucleotides encoding these polypeptides are also provided. Also
encompassed are recombinant vectors comprising said encoding
sequence, and host cells comprising said vector.
[0144] The wild-type PCSK9 polypeptide was determined to be
predominantly expressed in liver and neuronal tissue, and to a
lesser extent in kidney mesenchymal cells and intestinal epithelia
(Seidah et al., PNAS 100(3):928-933 (2003)).
[0145] Expression profiling designed to measure the steady state
mRNA levels encoding the PCSK9b and PCSK9c polypeptides confirmed
that they shared the predominate liver and neuronal expression
pattern of wild-type PCSK9. Specifically, PCSK9b and PCSK9c were
predominately expressed in liver and cerebellum at levels that were
over 3500 times that of the tissue with the lowest expression (the
heart). PCSK9b and PCSK9c were also expressed relatively highly in
the lung and its associated vasculature. PCSK9b and PCSK9c were
also expressed throughout the gastrointestinal tract (See FIG. 6).
The PCSK9b and PCSK9c expression in the cerebellum indicates that
PCSK9b and PCSK9c may function in neuronal differentiation.
[0146] The PCSK9c polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following diseases and/or disorders: autosomal
dominant hypercholesterolemia; disorders associated with aberrant
LDL receptor function; disorders associated with apolipoprotein B;
disorders associated with autosomal recessive hypercholesterolemia;
disorders associated with elevated cholesterol; disorders
associated with elevated LDL; disorders associated with reduced
clearance rate of LDL in the liver; disorders associated with
elevated LDL apoB production; familial hypercholesterolemia; lipid
metabolism disorders; elevated LDL; cholesterol depositions; tendon
xanthomas; atheroma; premature arteriosclerosis, coronary heart
disease; famialial defective apolipoprotein B; statin
hypersensitivity; disorders associated with accelerated LDLR
degradation.
[0147] The PCSK9c polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following diseases and/or disorders: neural
differentiation disorders.
[0148] The PCSK9c polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following cardiovascular diseases and/or
disorders: myocardio infarction, congestive heart failure,
arrthymias, cardiomyopathy, atherosclerosis, arterialsclerosis,
microvascular disease, embolism, thromobosis, pulmonary edema,
palpitation, dyspnea, angina, hypotension, syncope, heart murmur,
aberrant ECG, hypertrophic cardiomyopathy, the Marfan syndrome,
sudden death, prolonged QT syndrome, congenital defects, cardiac
viral infections, valvular heart disease, hypertension, among
others disclosed herein, particularly in the "Cardiovascular
Disorders" section and below.
[0149] Similarly, PCSK9c polynucleotides and polypeptides may be
useful for ameliorating cardiovascular diseases and symptoms which
result indirectly from various non-cardiovascular effects, which
include, but are not limited to, the following, obesity, smoking,
Down syndrome (associated with endocardial cushion defect); bony
abnormalities of the upper extremities (associated with atrial
septal defect in the Holt-Oram syndrome); muscular dystrophies
(associated with cardiomyopathy); hemochromatosis and glycogen
storage disease (associated with myocardial infiltration and
restrictive cardiomyopathy); congenital deafness (associated with
prolonged QT interval and serious cardiac arrhythmias); Raynaud's
disease (associated with primary pulmonary hypertension and
coronary vasospasm); connective tissue disorders, i.e., the Marfan
syndrome, Ehlers-Danlos and Hurler syndromes, and related disorders
of mucopolysaccharide metabolism (aortic dilatation, prolapsed
mitral valve, a variety of arterial abnormalities); acromegaly
(hypertension, accelerated coronary atherosclerosis, conduction
defects, cardiomyopathy); hyperthyroidism (heart failure, atrial
fibrillation); hypothyroidism (pericardial effusion, coronary
artery disease); rheumatoid arthritis (pericarditis, aortic valve
disease); scleroderma (cor pulmonale, myocardial fibrosis,
pericarditis); systemic lupus erythematosus (valvulitis,
myocarditis, pericarditis); sarcoidosis (arrhythmias,
cardiomyopathy); postmenopausal effects, Chlamydial infections,
polycystic ovary disease, thyroid disease, alcoholism, diet, and
exfoliative dermatitis (high-output heart failure), for
example.
[0150] Moreover, polynucleotides and polypeptides, including
fragments and/or antagonists thereof, have uses which include,
directly or indirectly, treating, preventing, diagnosing, and/or
prognosing the following, non-limiting, cardiovascular infections:
blood stream invasion, bacteremia, sepsis, Streptococcus pneumoniae
infection, group a streptococci infection, group b streptococci
infection, Enterococcus infection, nonenterococcal group D
streptococci infection, nonenterococcal group C streptococci
infection, nonenterococcal group G streptococci infection,
Streptoccus viridans infection, Staphylococcus aureus infection,
coagulase-negative staphylococci infection, gram-negative Bacilli
infection, Enterobacteriaceae infection, Psudomonas spp. Infection,
Acinobacter spp. Infection, Flavobacterium meningosepticum
infection, Aeromonas spp. Infection, Stenotrophomonas maltophilia
infection, gram-negative coccobacilli infection, Haemophilus
influenza infection, Branhamella catarrhalis infection, anaerobe
infection, Bacteriodes fragilis infection, Clostridium infection,
fungal infection, Candida spp. Infection, non-albicans Candida spp.
Infection, Hansenula anomala infection, Malassezia furfur
infection, nontuberculous Mycobacteria infection, Mycobacterium
avium infection, Mycobacterium chelonae infection, Mycobacterium
fortuitum infection, spirochetal infection, Borrelia burgdorferi
infection, in addition to any other cardiovascular disease and/or
disorder (e.g., non-sepsis) implicated by the causative agents
listed above or elsewhere herein.
[0151] The PCSK9c polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following metabolic diseases and/or disorders:
dyslipidemia, diabetic dyslipidemia, mixed dyslipidemia,
hypercholesteremia, hypertriglyceridemia, type II diabetes
mellitus, type I diabetes, insulin resistance, hyperlipidemia,
obesity, and/or anorexia nervosa.
[0152] The present invention also encompasses therapeutic
combinations of a modulator of PCSK9c with a statin for the
treatment, prevention and/or amelioration of a disease or disorder
referenced herein, particularly dyslipidemia. Representative
statins include, but are not limited to, the following:
pravastatin, lovastatin, cerivastatin, simvastatin, pitivastatin,
atorvastatin or rousuvastatin.
[0153] The PCSK9c polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include modulating signal transduction activity, in various
cells, tissues, and organisms, and particularly in liver, brain,
mammalian adipose, omentum, spleen, inflammatory tissues,
macrophages, neutrophils, synovial histiomonocytes, neutrophils,
and epithelioid histiocytes.
[0154] The PCSK9c polynucleotides and polypeptides of the present
invention, including modulators and/or fragments thereof, have uses
that include detecting, prognosing, treating, preventing, and/or
ameliorating the following hepatic disorders: hepatoblastoma,
jaundice, hepatitis, liver metabolic diseases and conditions that
are attributable to the differentiation of hepatocyte progenitor
cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebic abcess,
hydatid cyst, cystadenocarcinoma, adenoma, focal nodular
hyperplasia, hemangioma, hepatocellulae carcinoma,
cholangiocarcinoma, and angiosarcoma, granulomatous liver disease,
liver transplantation, hyperbilirubinemia, jaundice, parenchymal
liver disease, portal hypertension, hepatobiliary disease, hepatic
parenchyma, hepatic fibrosis, anemia, gallstones, cholestasis,
carbon tetrachloride toxicity, beryllium toxicity, vinyl chloride
toxicity, choledocholithiasis, hepatocellular necrosis, aberrant
metabolism of amino acids, aberrant metabolism of carbohydrates,
aberrant synthesis proteins, aberrant synthesis of glycoproteins,
aberrant degradation of proteins, aberrant degradation of
glycoproteins, aberrant metabolism of drugs, aberrant metabolism of
hormones, aberrant degradation of drugs, aberrant degradation of
drugs, aberrant regulation of lipid metabolism, aberrant regulation
of cholesterol metabolism, aberrant glycogenesis, aberrant
glycogenolysis, aberrant glycolysis, aberrant gluconeogenesis,
hyperglycemia, glucose intolerance, hyperglycemia, decreased
hepatic glucose uptake, decreased hepatic glycogen synthesis,
hepatic resistance to insulin, portal-systemic glucose shunting,
peripheral insulin resistance, hormonal abnormalities, increased
levels of systemic glucagon, decreased levels of systemic cortisol,
increased levels of systemic insulin, hypoglycemia, decreased
gluconeogenesis, decreased hepatic glycogen content, hepatic
resistance to glucagon, elevated levels of systemic aromatic amino
acids, decreased levels of systemic branched-chain amino acids,
hepatic encephalopathy, aberrant hepatic amino acid transamination,
aberrant hepatic amino acid oxidative deamination, aberrant ammonia
synthesis, aberant albumin secretion, hypoalbuminemia, aberrant
cytochromes b5 function, aberrant P450 function, aberrant
glutathione S-acyltransferase function, aberrant cholesterol
synthesis, and aberrant bile acid synthesis.
[0155] Moreover, polynucleotides and polypeptides, including
fragments and/or antagonists thereof, have uses which include,
directly or indirectly, treating, preventing, diagnosing, and/or
prognosing the following, non-limiting, hepatic infections: liver
disease caused by sepsis infection, liver disease caused by
bacteremia, liver disease caused by Pneomococcal pneumonia
infection, liver disease caused by Toxic shock syndrome, liver
disease caused by Listeriosis, liver disease caused by
Legionnaries' disease, liver disease caused by Brucellosis
infection, liver disease caused by Neisseria gonorrhoeae infection,
liver disease caused by Yersinia infection, liver disease caused by
Salmonellosis, liver disease caused by Nocardiosis, liver disease
caused by Spirochete infection, liver disease caused by Treponema
pallidum infection, liver disease caused by Brrelia burgdorferi
infection, liver disease caused by Leptospirosis, liver disease
caused by Coxiella burnetii infection, liver disease caused by
Rickettsia richettsii infection, liver disease caused by Chlamydia
trachomatis infection, liver disease caused by Chlamydia psittaci
infection, liver disease caused by hepatitis virus infection, liver
disease caused by Epstein-Barr virus infection in addition to any
other hepatic disease and/or disorder implicated by the causative
agents listed above or elsewhere herein.
[0156] The PCSK9c polynucleotides and polypeptides, including
fragments and/or antagonsists thereof, may have uses which include
identification of modulators of PCSK9c function including
antibodies (for detection or neutralization), naturally-occurring
modulators and small molecule modulators. Antibodies to domains of
the PCSK9c protein could be used as diagnostic agents of lipid
metabolic disorders, including hypercholesterolemia, among
others.
[0157] The expression level of PCSK9c also may be useful as a
biomarker for predicting which patients may be at risk of
developing hypercholesterolemia, and/or those patients which may be
at risk of being overly sensitive to statin therapy.
[0158] PCSK9c polypeptides and polynucleotides have additional uses
which include diagnosing diseases related to the over and/or under
expression of PCSK9c by identifying mutations in the PCSK9c gene by
using PCSK9c sequences as probes or by determining PCSK9c protein
or mRNA expression levels. PCSK9c polypeptides may be useful for
screening compounds that affect the activity of the protein. PCSK9c
peptides can also be used for the generation of specific antibodies
and as bait in yeast two hybrid screens to find proteins the
specifically interact with PCSK9c (described elsewhere herein).
[0159] In preferred embodiments, the present invention is also
directed to polynucleotides comprising, or alternatively consisting
of, a sequence encoding following N-terminal PCSK9c deletion
polypeptides: M1-Q523, S2-Q523, P3-Q523, W4-Q523, K5-Q523, D6-Q523,
G7-Q523, G8-Q523, S9-Q523, L10-Q523, V11-Q523, E12-Q523, V13-Q523,
Y14-Q523, L15-Q523, L16-Q523, D17-Q523, T18-Q523, S19-Q523,
I20-Q523, Q21-Q523, S22-Q523, D23-Q523, H24-Q523, R25-Q523,
E26-Q523, I27-Q523, E28-Q523, G29-Q523, R30-Q523, V31-Q523,
M32-Q523, V33-Q523, T34-Q523, D35-Q523, F36-Q523, E37-Q523,
N38-Q523, V39-Q523, P40-Q523, E41-Q523, E42-Q523, D43-Q523,
G44-Q523, T45-Q523, R46-Q523, F47-Q523, H48-Q523, R49-Q523,
Q50-Q523, A51-Q523, S52-Q523, K53-Q523, C54-Q523, D55-Q523,
S56-Q523, H57-Q523, G58-Q523, T59-Q523, H60-Q523, L61-Q523,
A62-Q523, G63-Q523, V64-Q523, V65-Q523, S66-Q523, G67-Q523,
R68-Q523, D69-Q523, A70-Q523, G71-Q523, V72-Q523, A73-Q523,
K74-Q523, G75-Q523, A76-Q523, S77-Q523, M78-Q523, R79-Q523,
S80-Q523, L81-Q523, R82-Q523, V83-Q523, L84-Q523, N85-Q523,
C86-Q523, Q87-Q523, G88-Q523, K89-Q523, G90-Q523, T91-Q523,
V92-Q523, S93-Q523, G94-Q523, T95-Q523, L96-Q523, I97-Q523,
G98-Q523, L99-Q523, E100-Q523, F101-Q523, I102-Q523, R103-Q523,
K104-Q523, S105-Q523, Q106-Q523, L107-Q523, V108-Q523, Q109-Q523,
P110-Q523, V111-Q523, G112-Q523, P113-Q523, L114-Q523, V115-Q523,
V116-Q523, L117-Q523, L118-Q523, P119-Q523, L120-Q523, A121-Q523,
G122-Q523, G123-Q523, Y124-Q523, S125-Q523, R126-Q523, V127-Q523,
L128-Q523, N129-Q523, A130-Q523, A131-Q523, C132-Q523, Q133-Q523,
R134-Q523, L135-Q523, A136-Q523, R137-Q523, A138-Q523, G139-Q523,
V140-Q523, V141-Q523, L142-Q523, V143-Q523, T144-Q523, A145-Q523,
A146-Q523, G147-Q523, N148-Q523, F149-Q523, R150-Q523, D151-Q523,
D152-Q523, A153-Q523, C154-Q523, L155-Q523, Y156-Q523, S157-Q523,
P158-Q523, A159-Q523, S160-Q523, A161-Q523, P162-Q523, E163-Q523,
V164-Q523, I165-Q523, T166-Q523, V167-Q523, G168-Q523, A169-Q523,
T170-Q523, N171-Q523, A172-Q523, Q173-Q523, D174-Q523, Q175-Q523,
P176-Q523, V177-Q523, T178-Q523, L179-Q523, G180-Q523, T181-Q523,
L182-Q523, G183-Q523, T184-Q523, N185-Q523, F186-Q523, G187-Q523,
R188-Q523, C189-Q523, V190-Q523, D191-Q523, L192-Q523, F193-Q523,
A194-Q523, P195-Q523, G196-Q523, E197-Q523, D198-Q523, I199-Q523,
I200-Q523, G201-Q523, A202-Q523, S203-Q523, S204-Q523, D205-Q523,
C206-Q523, S207-Q523, T208-Q523, C209-Q523, F210-Q523, V211-Q523,
S212-Q523, Q213-Q523, S214-Q523, G215-Q523, T216-Q523, S217-Q523,
Q218-Q523, A219-Q523, A220-Q523, A221-Q523, H222-Q523, V223-Q523,
A224-Q523, G225-Q523, I226-Q523, A227-Q523, A228-Q523, M229-Q523,
M230-Q523, L231-Q523, S232-Q523, A233-Q523, E234-Q523, P235-Q523,
E236-Q523, L237-Q523, T238-Q523, L239-Q523, A240-Q523, E241-Q523,
L242-Q523, 8243-Q523, Q244-Q523, R245-Q523, L246-Q523, I247-Q523,
H248-Q523, F249-Q523, S250-Q523, A251-Q523, K252-Q523, D253-Q523,
V254-Q523, I255-Q523, N256-Q523, E257-Q523, A258-Q523, W259-Q523,
F260-Q523, P261-Q523, E262-Q523, D263-Q523, Q264-Q523, R265-Q523,
V266-Q523, L267-Q523, T268-Q523, P269-Q523, N270-Q523, L271-Q523,
V272-Q523, A273-Q523, A274-Q523, L275-Q523, P276-Q523, P277-Q523,
S278-Q523, T279-Q523, H280-Q523, G281-Q523, A282-Q523, G283-Q523,
W284-Q523, Q285-Q523, L286-Q523, F287-Q523, C288-Q523, R289-Q523,
T290-Q523, V291-Q523, W292-Q523, S293-Q523, A294-Q523, H295-Q523,
S296-Q523, G297-Q523, P298-Q523, T299-Q523, R300-Q523, M301-Q523,
A302-Q523, T303-Q523, A304-Q523, I305-Q523, A306-Q523, R307-Q523,
C308-Q523, A309-Q523, P310-Q523, D311-Q523, E312-Q523, E313-Q523,
L314-Q523, L315-Q523, S316-Q523, C317-Q523, S318-Q523, S319-Q523,
F320-Q523, S321-Q523, R322-Q523, S323-Q523, G324-Q523, K325-Q523,
R326-Q523, R327-Q523, G328-Q523, E329-Q523, R330-Q523, M331-Q523,
E332-Q523, A333-Q523, Q334-Q523, G335-Q523, G336-Q523, K337-Q523,
L338-Q523, V339-Q523, C340-Q523, R341-Q523, A342-Q523, H343-Q523,
N344-Q523, A345-Q523, F346-Q523, G347-Q523, G348-Q523, E349-Q523,
G350-Q523, V351-Q523, Y352-Q523, A353-Q523, I354-Q523, A355-Q523,
R356-Q523, C357-Q523, C358-Q523, L359-Q523, L360-Q523, P361-Q523,
Q362-Q523, A363-Q523, N364-Q523, C365-Q523, S366-Q523, V367-Q523,
H368-Q523, T369-Q523, A370-Q523, P371-Q523, P372-Q523, A373-Q523,
E374-Q523, A375-Q523, S376-Q523, M377-Q523, G378-Q523, T379-Q523,
R380-Q523, V381-Q523, H382-Q523, C383-Q523, H384-Q523, Q385-Q523,
Q386-Q523, G387-Q523, H388-Q523, V389-Q523, L390-Q523, T391-Q523,
G392-Q523, C393-Q523, S394-Q523, S395-Q523, H396-Q523, W397-Q523,
E398-Q523, V399-Q523, E400-Q523, D401-Q523, L402-Q523, G403-Q523,
T404-Q523, H405-Q523, K406-Q523, P407-Q523, P408-Q523, V409-Q523,
L410-Q523, R411-Q523, P412-Q523, R413-Q523, G414-Q523, Q415-Q523,
P416-Q523, N417-Q523, Q418-Q523, C419-Q523, V420-Q523, G421-Q523,
H422-Q523, R423-Q523, E424-Q523, A425-Q523, S426-Q523, I427-Q523,
H428-Q523, A429-Q523, S430-Q523, C431-Q523, C432-Q523, H433-Q523,
A434-Q523, P435-Q523, G436-Q523, L437-Q523, E438-Q523, C439-Q523,
K440-Q523, V441-Q523, K442-Q523, E443-Q523, H444-Q523, G445-Q523,
I446-Q523, P447-Q523, A448-Q523, P449-Q523, Q450-Q523, E451-Q523,
Q452-Q523, V453-Q523, T454-Q523, V455-Q523, A456-Q523, C457-Q523,
E458-Q523, E459-Q523, G460-Q523, W461-Q523, T462-Q523, L463-Q523,
T464-Q523, G465-Q523, C466-Q523, S467-Q523, A468-Q523, L469-Q523,
P470-Q523, G471-Q523, T472-Q523, S473-Q523, H474-Q523, V475-Q523,
L476-Q523, G477-Q523, A478-Q523, Y479-Q523, A480-Q523, V481-Q523,
D482-Q523, N483-Q523, T484-Q523, C485-Q523, V486-Q523, V487-Q523,
R488-Q523, S489-Q523, R490-Q523, D491-Q523, V492-Q523, S493-Q523,
T494-Q523, T495-Q523, G496-Q523, S497-Q523, T498-Q523, S499-Q523,
E500-Q523, G501-Q523, A502-Q523, V503-Q523, T504-Q523, A505-Q523,
V506-Q523, A507-Q523, I508-Q523, C509-Q523, C510-Q523, R511-Q523,
S512-Q523, R513-Q523, H514-Q523, L515-Q523, A516-Q523, and/or
Q517-Q523 of SEQ ID NO:4. Polypeptide sequences encoded by these
polynucleotides are also provided. In addition, the invention also
encompasses polynucleotides encoding a polypeptide that is at least
as long as any one of the aforementioned polypeptides. The present
invention also encompasses the use of these N-terminal PCSK9c
deletion polypeptides as immunogenic and/or antigenic epitopes as
described elsewhere herein.
[0160] In preferred embodiments, the present invention is also
directed to polynucleotides comprising, or alternatively consisting
of, a sequence encoding the following C-terminal PCSK9c deletion
polypeptides: M1-Q523, M1-L522, M1-E521, M1-Q520, M1-S519, M1-A518,
M1-Q517, M1-A516, M1-L515, M1-H514, M1-R513, M1-S512, M1-R511,
M1-C510, M1-C509, M1-I508, M1-A507, M1-V506, M1-A505, M1-T504,
M1-V503, M1-A502, M1-G501, M1-E500, M1-S499, M1-T498, M1-S497,
M1-G496, M1-T495, M1-T494, M1-S493, M1-V492, M1-D491, M1-R490,
M1-S489, M1-R488, M1-V487, M1-V486, M1-C485, M1-T484, M1-N483,
M1-D482, M1-V481, M1-A480, M1-Y479, M1-A478, M1-G477, M1-L476,
M1-V475, M1-H474, M1-S473, M1-T472, M1-G471, M1-P470, M1-L469,
M1-A468, M1-S467, M1-C466, M1-G465, M1-T464, M1-L463, M1-T462,
M1-W461, M1-G460, M1-E459, M1-E458, M1-C457, M1-A456, M1-V455,
M1-T454, M1-V453, M1-Q452, M1-E451, M1-Q450, M1-P449, M1-A448,
M1-P447, M1-I446, M1-G445, M1-H444, M1-E443, M1-K442, M1-V441,
M1-K440, M1-C439, M1-E438, M1-L437, M1-G436, M1-P435, M1-A434,
M1-H433, M1-C432, M1-C431, M1-S430, M1-A429, M1-H428, M1-I427,
M1-S426, M1-A425, M1-E424, M1-R423, M1-H422, M1-G421, M1-V420,
M1-C419, M1-Q418, M1-N417, M1-P416, M1-Q415, M1-G414, M1-R413,
M1-P412, M1-R411, M1-L410, M1-V409, M1-P408, M1-P407, M1-K406,
M1-H405, M1-T404, M1-G403, M1-L402, M1-D401, M1-E400, M1-V399,
M1-E398, M1-W397, M1-H396, M1-S395, M1-S394, M1-C393, M1-G392,
M1-T391, M1-L390, M1-V389, M1-H388, M1-G387, M1-Q386, M1-Q385,
M1-H384, M1-C383, M1-H382, M1-V381, M1-R380, M1-T379, M1-G378,
M1-M377, M1-S376, M1-A375, M1-E374, M1-A373, M1-P372, M1-P371,
M1-A370, M1-T369, M1-H368, M1-V367, M1-S366, M1-C365, M1-N364,
M1-A363, M1-Q362, M1-P361, M1-L360, M1-L359, M1-C358, M1-C357,
M1-R356, M1-A355, M1-I354, M1-A353, M1-Y352, M1-V351, M1-G350,
M1-E349, M1-G348, M1-G347, M1-F346, M1-A345, M1-N344, M1-H343,
M1-A342, M1-R341, M1-C340, M1-V339, M1-L338, M1-K337, M1-G336,
M1-G335, M1-Q334, M1-A333, M1-E332, M1-M331, M1-R330, M1-E329,
M1-G328, M1-R327, M1-R326, M1-K325, M1-G324, M1-S323, M1-R322,
M1-S321, M1-F320, M1-S319, M1-S318, M1-C317, M1-S316, M1-L315,
M1-L314, M1-E313, M1-E312, M1-D311, M1-P310, M1-A309, M1-C308,
M1-R307, M1-A306, M1-I305, M1-A304, M1-T303, M1-A302, M1-M301,
M1-R300, M1-T299, M1-P298, M1-G297, M1-S296, M1-H295, M1-A294,
M1-S293, M1-W292, M1-V291, M1-T290, M1-R289, M1-C288, M1-F287,
M1-L286, M1-Q285, M1-W284, M1-G283, M1-A282, M1-G281, M1-H280,
M1-T279, M1-S278, M1-P277, M1-P276, M1-L275, M1-A274, M1-A273,
M1-V272, M1-L271, M1-N270, M1-P269, M1-T268, M1-L267, M1-V266,
M1-R265, M1-Q264, M1-D263, M1-E262, M1-P261, M1-F260, M1-W259,
M1-A258, M1-E257, M1-N256, M1-I255, M1-V254, M1-D253, M1-K252,
M1-A251, M1-S250, M1-F249, M1-H248, M1-I247, M1-L246, M1-R245,
M1-Q244, M1-R243, M1-L242, M1-E241, M1-A240, M1-L239, M1-T238,
M1-L237, M1-E236, M1-P235, M1-E234, M1-A233, M1-S232, M1-L231,
M1-M230, M1-M229, M1-A228, M1-A227, M1-I226, M1-G225, M1-A224,
M1-V223, M1-H222, M1-A221, M1-A220, M1-A219, M1-Q218, M1-S217,
M1-T216, M1-G215, M1-S214, M1-Q213, M1-S212, M1-V211, M1-F210,
M1-C209, M1-T208, M1-S207, M1-C206, M1-D205, M1-S204, M1-S203,
M1-A202, M1-G201, M1-I200, M1-I199, M1-D198, M1-E197, M1-G196,
M1-P195, M1-A194, M1-F193, M1-L192, M1-D191, M1-V190, M1-C189,
M1-R188, M1-G187, M1-F186, M1-N185, M1-T184, M1-G183, M1-L182,
M1-T181, M1-G180, M1-L179, M1-T178, M1-V177, M1-P176, M1-Q175,
M1-D174, M1-Q173, M1-A172, M1-N171, M1-T170, M1-A169, M1-G168,
M1-V167, M1-T166, M1-I165, M1-V164, M1-E163, M1-P162, M1-A161,
M1-S160, M1-A159, M1-P158, M1-S157, M1-Y156, M1-L155, M1-C154,
M1-A153, M1-D152, M1-D151, M1-R150, M1-F149, M1-N148, M1-G147,
M1-A146, M1-A145, M1-T144, M1-V143, M1-L142, M1-V141, M1-V140,
M1-G139, M1-A138, M1-R137, M1-A136, M1-L135, M1-R134, M1-Q133,
M1-C132, M1-A131, M1-A130, M1-N129, M1-L128, M1-V127, M1-R126,
M1-S125, M1-Y124, M1-G123, M1-G122, M1-A121, M1-L120, M1-P119,
M1-L118, M1-L117, M1-V116, M1-V115, M1-L114, M1-P113, M1-G112,
M1-V111, M1-P110, M1-Q109, M1-V108, M1-L107, M1-Q106, M1-S105,
M1-K104, M1-R103, M1-I102, M1-F101, M1-E100, M1-L99, M1-G98,
M1-I97, M1-L96, M1-T95, M1-G94, M1-S93, M1-V92, M1-T91, M1-G90,
M1-K89, M1-G88, M1-Q87, M1-C86, M1-N85, M1-L84, M1-V83, M1-R82,
M1-L81, M1-S80, M1-R79, M1-M78, M1-S77, M1-A76, M1-G75, M1-K74,
M1-A73, M1-V72, M1-G71, M1-A70, M1-D69, M1-R68, M1-G67, M1-S66,
M1-V65, M1-V64, M1-G63, M1-A62, M1-L61, M1-H60, M1-T59, M1-G58,
M1-H57, M1-S56, M1-D55, M1-C54, M1-K53, M1-S52, M1-A51, M1-Q50,
M1-R49, M1-H48, M1-F47, M1-R46, M1-T45, M1-G44, M1-D43, M1-E42,
M1-E41, M1-P40, M1-V39, M1-N38, M1-E37, M1-F36, M1-D35, M1-T34,
M1-V33, M1-M32, M1-V31, M1-R30, M1-G29, M1-E28, M1-I27, M1-E26,
M1-R25, M1-H24, M1-D23, M1-S22, M1-Q21, M1-I20, M1-S19, M1-T18,
M1-D17, M1-L16, M1-L15, M1-Y14, M1-V13, M1-E12, M1-V11, M1-L10,
M1-S9, M1-G8, and/or M1-G7 of SEQ ID NO:4. Polypeptide sequences
encoded by these polynucleotides are also provided. In addition,
the invention also encompasses polynucleotides encoding a
polypeptide that is at least as long as any one of the
aforementioned polypeptides. The present invention also encompasses
the use of these C-terminal PCSK9c deletion polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0161] Alternatively, preferred polypeptides of the present
invention may comprise polypeptide sequences corresponding to, for
example, internal regions of the PCSK9c polypeptide (e.g., any
combination of both N- and C-terminal PCSK9c polypeptide deletions)
of SEQ ID NO:4. For example, internal regions could be defined by
the equation: amino acid NX to amino acid CX, wherein NX refers to
any N-terminal deletion polypeptide amino acid of PCSK9c (SEQ ID
NO:4), and where CX refers to any C-terminal deletion polypeptide
amino acid of PCSK9c (SEQ ID NO:4). Polynucleotides encoding these
polypeptides are also provided. The present invention also
encompasses the use of these polypeptides as an immunogenic and/or
antigenic epitope as described elsewhere herein.
[0162] The present invention also encompasses immunogenic and/or
antigenic epitopes of the PCSK9c polypeptide.
[0163] Many polynucleotide sequences, such as EST sequences, are
publicly available and accessible through sequence databases. Some
of these sequences are related to SEQ ID NO:3 and may have been
publicly available prior to conception of the present invention.
Preferably, such related polynucleotides are specifically excluded
from the scope of the present invention. To list every related
sequence would be cumbersome. Accordingly, preferably excluded from
the present invention are one or more polynucleotides consisting of
a nucleotide sequence described by the general formula of a-b,
where a is any integer between 1 to 3742 of SEQ ID NO:3, b is an
integer between 15 to 3756, where both a and b correspond to the
positions of nucleotide residues shown in SEQ ID NO:3, and where b
is greater than or equal to a+14.
Features of the Polypeptide Encoded by Polynucleotide No:3
[0164] The invention further relates to an N-terminal truncation of
PCSK9 (SEQ ID NO:5), wherein said N-terminal truncation results in
the deletion of anywhere between about 1 to about 218 amino acids
from the N-terminus of SEQ ID NO:5, and wherein said N-terminal
truncation results in elevated PCSK9 biological activity,
including, but not limited to decreased LDLR protein levels, and/or
decreased LDL uptake by LDLR. Polynucleotides encoding such PCSK9
truncations are provided as SEQ ID NO:38.
[0165] The inventors are the first to discover that N-terminal
truncations of PCSK9 result in elevated levels of PCSK9 biological
activity. Such truncated forms may also greatly facilitate the
identification of small molecule modulators of PCSK9 as well.
Experiments designed to assess whether truncation of the N-terminus
of PCSK9 results in elevated biological activity have been
performed. For example, truncation of the N-terminus by 15 amino
acids resulted in significant increases in PCSK9 activity (data not
shown), although not as significant as that observed for the PCSK9b
and PCSK9c variants (see FIGS. 9A-B). Furthermore, truncation was
tolerated by PCSK9 up to a truncation of about 218 amino acids,
after which decreased levels of PCSK9 biological activity was
observed (data not shown).
[0166] The invention further relates to an N-terminal truncation of
PCSK9 (SEQ ID NO:5), wherein said N-terminal truncation results in
the deletion of anywhere between about 1 to about 218 amino acids
from the N-terminus of SEQ ID NO:5, including, but not limited to
decreased LDLR protein levels and/or decreased LDL uptake by LDLR,
and wherein said elevated PCSK9 biological activity is at least
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 100%, or more than wildtype elevated
PCSK9 biological activity. In this context, the term "about" shall
be construed to mean anywhere between 1, 2, 3, 4, or 5 percent more
or less than the cited amount. Alternatively, said elevated PCSK9
biological activity may be at least about 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., or 10.times. more than wildtype PCSK9 biological
activity. In this context, the term "about" shall be construed to
mean anywhere between 0.1.times., 0.2.times., 0.3.times.,
0.4.times., 0.5.times., 0.6.times., 0.7.times., 0.8.times., or
0.9.times. more or less than the cited amount.
[0167] Preferably, an N-terminal deletion mutant of PCSK9 (SEQ ID
NO:5) is at least about 15 amino acids, but less than about 218
amino acids, of SEQ ID NO:5. Truncated forms of PCSK9 may be
created using molecular biology techniques (see Example 13),
proteolytic cleavage, post-translational processing, chemical
synthesis, etc., among other methods known in the art.
[0168] The coding region of PCSK9 encoding polynucleotides is
represented by nucleotides 292 to 2367 of SEQ ID NO:38.
[0169] In preferred embodiments, the present invention is also
directed to polynucleotides comprising, or alternatively consisting
of, a sequence encoding the following N-terminal PCSK9c deletion
polypeptides: M1-Q692, G2-Q692, T3-Q692, V4-Q692, S5-Q692, S6-Q692,
R7-Q692, R8-Q692, S9-Q692, W10-Q692, W11-Q692, P12-Q692, L13-Q692,
P14-Q692, L15-Q692, L16-Q692, L17-Q692, L18-Q692, L19-Q692,
L20-Q692, L21-Q692, L22-Q692, L23-Q692, G24-Q692, P25-Q692,
A26-Q692, G27-Q692, A28-Q692, R29-Q692, A30-Q692, Q31-Q692,
E32-Q692, D33-Q692, E34-Q692, D35-Q692, G36-Q692, D37-Q692,
Y38-Q692, E39-Q692, E40-Q692, L41-Q692, V42-Q692, L43-Q692,
A44-Q692, L45-Q692, R46-Q692, S47-Q692, E48-Q692, E49-Q692,
D50-Q692, G51-Q692, L52-Q692, A53-Q692, E54-Q692, A55-Q692,
P56-Q692, E57-Q692, H58-Q692, G59-Q692, T60-Q692, T61-Q692,
A62-Q692, T63-Q692, F64-Q692, H65-Q692, R66-Q692, C67-Q692,
A68-Q692, K69-Q692, D70-Q692, P71-Q692, W72-Q692, R73-Q692,
L74-Q692, P75-Q692, G76-Q692, T77-Q692, Y78-Q692, V79-Q692,
V80-Q692, V81-Q692, L82-Q692, K83-Q692, E84-Q692, E85-Q692,
T86-Q692, H87-Q692, L88-Q692, S89-Q692, Q90-Q692, S91-Q692,
E92-Q692, R93-Q692, T94-Q692, A95-Q692, R96-Q692, R97-Q692,
L98-Q692, Q99-Q692, A100-Q692, Q101-Q692, A102-Q692, A103-Q692,
R104-Q692, R105-Q692, G106-Q692, Y107-Q692, L108-Q692, T109-Q692,
K110-Q692, I111-Q692, L112-Q692, H113-Q692, V114-Q692, F115-Q692,
H116-Q692, G117-Q692, L118-Q692, L119-Q692, P120-Q692, G121-Q692,
F122-Q692, L123-Q692, V124-Q692, K125-Q692, M126-Q692, S127-Q692,
G128-Q692, D129-Q692, L130-Q692, L131-Q692, E132-Q692, L133-Q692,
A134-Q692, L135-Q692, K136-Q692, L137-Q692, P138-Q692, H139-Q692,
V140-Q692, D141-Q692, Y142-Q692, I143-Q692, E144-Q692, E145-Q692,
D146-Q692, S147-Q692, S148-Q692, V149-Q692, F150-Q692, A151-Q692,
Q152-Q692, S153-Q692, I154-Q692, P155-Q692, W156-Q692, N157-Q692,
L158-Q692, E159-Q692, R160-Q692, I161-Q692, T162-Q692, P163-Q692,
P164-Q692, R165-Q692, Y166-Q692, R167-Q692, A168-Q692, D169-Q692,
E170-Q692, Y171-Q692, Q172-Q692, P173-Q692, P174-Q692, D175-Q692,
G176-Q692, G177-Q692, S178-Q692, L179-Q692, V180-Q692, E181-Q692,
V182-Q692, Y183-Q692, L184-Q692, L185-Q692, D186-Q692, T187-Q692,
S188-Q692, I189-Q692, Q190-Q692, S191-Q692, D192-Q692, H193-Q692,
R194-Q692, E195-Q692, I196-Q692, E197-Q692, G198-Q692, R199-Q692,
V200-Q692, M201-Q692, V202-Q692, T203-Q692, D204-Q692, F205-Q692,
E206-Q692, N207-Q692, V208-Q692, P209-Q692, E210-Q692, E211-Q692,
D212-Q692, G213-Q692, T214-Q692, R215-Q692, F216-Q692, H217-Q692,
R218-Q692, Q219-Q692, A220-Q692, S221-Q692, K222-Q692, C223-Q692,
D224-Q692, S225-Q692, H226-Q692, G227-Q692, T228-Q692, H229-Q692,
L230-Q692, A231-Q692, G232-Q692, V233-Q692, V234-Q692, S235-Q692,
G236-Q692, R237-Q692, D238-Q692, A239-Q692, G240-Q692, V241-Q692,
A242-Q692, K243-Q692, G244-Q692, A245-Q692, S246-Q692, M247-Q692,
R248-Q692, S249-Q692, L250-Q692, R251-Q692, V252-Q692, L253-Q692,
N254-Q692, C255-Q692, Q256-Q692, G257-Q692, K258-Q692, G259-Q692,
T260-Q692, V261-Q692, S262-Q692, G263-Q692, T264-Q692, L265-Q692,
I266-Q692, G267-Q692, L268-Q692, E269-Q692, F270-Q692, I271-Q692,
R272-Q692, K273-Q692, S274-Q692, Q275-Q692, L276-Q692, V277-Q692,
Q278-Q692, P279-Q692, V280-Q692, G281-Q692, P282-Q692, L283-Q692,
V284-Q692, V285-Q692, L286-Q692, L287-Q692, P288-Q692, L289-Q692,
A290-Q692, G291-Q692, G292-Q692, Y293-Q692, S294-Q692, R295-Q692,
V296-Q692, L297-Q692, N298-Q692, A299-Q692, A300-Q692, C301-Q692,
Q302-Q692, R303-Q692, L304-Q692, A305-Q692, R306-Q692, A307-Q692,
G308-Q692, V309-Q692, V310-Q692, L311-Q692, V312-Q692, T313-Q692,
A314-Q692, A315-Q692, G316-Q692, N317-Q692, F318-Q692, R319-Q692,
D320-Q692, D321-Q692, A322-Q692, C323-Q692, L324-Q692, Y325-Q692,
S326-Q692, P327-Q692, A328-Q692, S329-Q692, A330-Q692, P331-Q692,
E332-Q692, V333-Q692, I334-Q692, T335-Q692, V336-Q692, G337-Q692,
A338-Q692, T339-Q692, N340-Q692, A341-Q692, Q342-Q692, D343-Q692,
Q344-Q692, P345-Q692, V346-Q692, T347-Q692, L348-Q692, G349-Q692,
T350-Q692, L351-Q692, G352-Q692, T353-Q692, N354-Q692, F355-Q692,
G356-Q692, R357-Q692, C358-Q692, V359-Q692, D360-Q692, L361-Q692,
F362-Q692, A363-Q692, P364-Q692, G365-Q692, E366-Q692, D367-Q692,
I368-Q692, I369-Q692, G370-Q692, A371-Q692, S372-Q692, S373-Q692,
D374-Q692, C375-Q692, S376-Q692, T377-Q692, C378-Q692, F379-Q692,
V380-Q692, S381-Q692, Q382-Q692, S383-Q692, G384-Q692, T385-Q692,
S386-Q692, Q387-Q692, A388-Q692, A389-Q692, A390-Q692, H391-Q692,
V392-Q692, A393-Q692, G394-Q692, I395-Q692, A396-Q692, A397-Q692,
M398-Q692, M399-Q692, L400-Q692, S401-Q692, A402-Q692, E403-Q692,
P404-Q692, E405-Q692, L406-Q692, T407-Q692, L408-Q692, A409-Q692,
E410-Q692, L411-Q692, R412-Q692, Q413-Q692, R414-Q692, L415-Q692,
I416-Q692, H417-Q692, F418-Q692, S419-Q692, A420-Q692, K421-Q692,
D422-Q692, V423-Q692, I424-Q692, N425-Q692, E426-Q692, A427-Q692,
W428-Q692, F429-Q692, P430-Q692, E431-Q692, D432-Q692, Q433-Q692,
R434-Q692, V435-Q692, L436-Q692, T437-Q692, P438-Q692, N439-Q692,
L440-Q692, V441-Q692, A442-Q692, A443-Q692, L444-Q692, P445-Q692,
P446-Q692, S447-Q692, T448-Q692, H449-Q692, G450-Q692, A451-Q692,
G452-Q692, W453-Q692, Q454-Q692, L455-Q692, F456-Q692, C457-Q692,
R458-Q692, T459-Q692, V460-Q692, W461-Q692, S462-Q692, A463-Q692,
H464-Q692, S465-Q692, G466-Q692, P467-Q692, T468-Q692, R469-Q692,
M470-Q692, A471-Q692, T472-Q692, A473-Q692, V474-Q692, A475-Q692,
R476-Q692, C477-Q692, A478-Q692, P479-Q692, D480-Q692, E481-Q692,
E482-Q692, L483-Q692, L484-Q692, S485-Q692, C486-Q692, S487-Q692,
S488-Q692, F489-Q692, S490-Q692, R491-Q692, S492-Q692, G493-Q692,
K494-Q692, R495-Q692, R496-Q692, G497-Q692, E498-Q692, R499-Q692,
M500-Q692, E501-Q692, A502-Q692, Q503-Q692, G504-Q692, G505-Q692,
K506-Q692, L507-Q692, V508-Q692, C509-Q692, R510-Q692, A511-Q692,
H512-Q692, N513-Q692, A514-Q692, F515-Q692, G516-Q692, G517-Q692,
E518-Q692, G519-Q692, V520-Q692, Y521-Q692, A522-Q692, I523-Q692,
A524-Q692, R525-Q692, C526-Q692, C527-Q692, L528-Q692, L529-Q692,
P530-Q692, Q531-Q692, A532-Q692, N533-Q692, C534-Q692, S535-Q692,
V536-Q692, H537-Q692, T538-Q692, A539-Q692, P540-Q692, P541-Q692,
A542-Q692, E543-Q692, A544-Q692, S545-Q692, M546-Q692, G547-Q692,
T548-Q692, R549-Q692, V550-Q692, H551-Q692, C552-Q692, H553-Q692,
Q554-Q692, Q555-Q692, G556-Q692, H557-Q692, V558-Q692, L559-Q692,
T560-Q692, G561-Q692, C562-Q692, S563-Q692, S564-Q692, H565-Q692,
W566-Q692, E567-Q692, V568-Q692, E569-Q692, D570-Q692, L571-Q692,
G572-Q692, T573-Q692, H574-Q692, K575-Q692, P576-Q692, P577-Q692,
V578-Q692, L579-Q692, R580-Q692, P581-Q692, R582-Q692, G583-Q692,
Q584-Q692, P585-Q692, N586-Q692, Q587-Q692, C588-Q692, V589-Q692,
G590-Q692, H591-Q692, R592-Q692, E593-Q692, A594-Q692, S595-Q692,
I596-Q692, H597-Q692, A598-Q692, S599-Q692, C600-Q692, C601-Q692,
H602-Q692, A603-Q692, P604-Q692, G605-Q692, L606-Q692, E607-Q692,
C608-Q692, K609-Q692, V610-Q692, K611-Q692, E612-Q692, H613-Q692,
G614-Q692, I615-Q692, P616-Q692, A617-Q692, P618-Q692, Q619-Q692,
E620-Q692, Q621-Q692, V622-Q692, T623-Q692, V624-Q692, A625-Q692,
C626-Q692, E627-Q692, E628-Q692, G629-Q692, W630-Q692, T631-Q692,
L632-Q692, T633-Q692, G634-Q692, C635-Q692, S636-Q692, A637-Q692,
L638-Q692, P639-Q692, G640-Q692, T641-Q692, S642-Q692, H643-Q692,
V644-Q692, L645-Q692, G646-Q692, A647-Q692, Y648-Q692, A649-Q692,
V650-Q692, D651-Q692, N652-Q692, T653-Q692, C654-Q692, V655-Q692,
V656-Q692, R657-Q692, S658-Q692, R659-Q692, D660-Q692, V661-Q692,
S662-Q692, T663-Q692, T664-Q692, G665-Q692, S666-Q692, T667-Q692,
S668-Q692, E669-Q692, G670-Q692, A671-Q692, V672-Q692, T673-Q692,
A674-Q692, V675-Q692, A676-Q692, I677-Q692, C678-Q692, C679-Q692,
R680-Q692, S681-Q692, R682-Q692, H683-Q692, L684-Q692, A685-Q692,
and/or Q686-Q692 of SEQ ID NO:5. Polypeptide sequences encoded by
these polynucleotides are also provided as SEQ ID NO:38. In
addition, the invention also encompasses polynucleotides encoding a
polypeptide that is at least as long as any one of the
aforementioned polypeptides. The present invention also encompasses
the use of these N-terminal PCSK9c deletion polypeptides as
immunogenic and/or antigenic epitopes as described elsewhere
herein.
[0170] In preferred embodiments, the present invention is also
directed to polynucleotides comprising, or alternatively consisting
of, a sequence encoding the following C-terminal PCSK9c deletion
polypeptides: M1-Q692, M1-L691, M1-E690, M1-Q689, M1-S688, M1-A687,
M1-Q686, M1-A685, M1-L684, M1-H683, M1-R682, M1-S681, M1-R680,
M1-C679, M1-C678, M1-I677, M1-A676, M1-V675, M1-A674, M1-T673,
M1-V672, M1-A671, M1-G670, M1-E669, M1-S668, M1-T667, M1-S666,
M1-G665, M1-T664, M1-T663, M1-S662, M1-V661, M1-D660, M1-R659,
M1-S658, M1-R657, M1-V656, M1-V655, M1-C654, M1-T653, M1-N652,
M1-D651, M1-V650, M1-A649, M1-Y648, M1-A647, M1-G646, M1-L645,
M1-V644, M1-H643, M1-S642, M1-T641, M1-G640, M1-P639, M1-L638,
M1-A637, M1-S636, M1-C635, M1-G634, M1-T633, M1-L632, M1-T631,
M1-W630, M1-G629, M1-E628, M1-E627, M1-C626, M1-A625, M1-V624,
M1-T623, M1-V622, M1-Q621, M1-E620, M1-Q619, M1-P618, M1-A617,
M1-P616, M1-I615, M1-G614, M1-H613, M1-E612, M1-K611, M1-V610,
M1-K609, M1-C608, M1-E607, M1-L606, M1-G605, M1-P604, M1-A603,
M1-H602, M1-C601, M1-C600, M1-S599, M1-A598, M1-H597, M1-I596,
M1-S595, M1-A594, M1-E593, M1-R592, M1-H591, M1-G590, M1-V589,
M1-C588, M1-Q587, M1-N586, M1-P585, M1-Q584, M1-G583, M1-R582,
M1-P581, M1-R580, M1-L579, M1-V578, M1-P577, M1-P576, M1-K575,
M1-H574, M1-T573, M1-G572, M1-L571, M1-D570, M1-E569, M1-V568,
M1-E567, M1-W566, M1-H565, M1-S564, M1-S563, M1-C562, M1-G561,
M1-T560, M1-L559, M1-V558, M1-H557, M1-G556, M1-Q555, M1-Q554,
M1-H553, M1-C552, M1-H551, M1-V550, M1-R549, M1-T548, M1-G547,
M1-M546, M1-S545, M1-A544, M1-E543, M1-A542, M1-P541, M1-P540,
M1-A539, M1-T538, M1-H537, M1-V536, M1-S535, M1-C534, M1-N533,
M1-A532, M1-Q531, M1-P530, M1-L529, M1-L528, M1-C527, M1-C526,
M1-R525, M1-A524, M1-I523, M1-A522, M1-Y521, M1-V520, M1-G519,
M1-E518, M1-G517, M1-G516, M1-F515, M1-A514, M1-N513, M1-H512,
M1-A511, M1-R510, M1-C509, M1-V508, M1-L507, M1-K506, M1-G505,
M1-G504, M1-Q503, M1-A502, M1-E501, M1-M500, M1-R499, M1-E498,
M1-G497, M1-R496, M1-R495, M1-K494, M1-G493, M1-S492, M1-R491,
M1-S490, M1-F489, M1-S488, M1-S487, M1-C486, M1-S485, M1-L484,
M1-L483, M1-E482, M1-E481, M1-D480, M1-P479, M1-A478, M1-C477,
M1-R476, M1-A475, M1-V474, M1-A473, M1-T472, M1-A471, M1-M470,
M1-R469, M1-T468, M1-P467, M1-G466, M1-S465, M1-H464, M1-A463,
M1-S462, M1-W461, M1-V460, M1-T459, M1-R458, M1-C457, M1-F456,
M1-L455, M1-Q454, M1-W453, M1-G452, M1-A451, M1-G450, M1-H449,
M1-T448, M1-S447, M1-P446, M1-P445, M1-L444, M1-A443, M1-A442,
M1-V441, M1-L440, M1-N439, M1-P438, M1-T437, M1-L436, M1-V435,
M1-R434, M1-Q433, M1-D432, M1-E431, M1-P430, M1-F429, M1-W428,
M1-A427, M1-E426, M1-N425, M1-I424, M1-V423, M1-D422, M1-K421,
M1-A420, M1-S419, M1-F418, M1-H417, M1-I416, M1-L415, M1-R414,
M1-Q413, M1-R412, M1-L411, M1-E410, M1-A409, M1-L408, M1-T407,
M1-L406, M1-E405, M1-P404, M1-E403, M1-A402, M1-S401, M1-L400,
M1-M399, M1-M398, M1-A397, M1-A396, M1-I395, M1-G394, M1-A393,
M1-V392, M1-H391, M1-A390, M1-A389, M1-A388, M1-Q387, M1-S386,
M1-T385, M1-G384, M1-S383, M1-Q382, M1-S381, M1-V380, M1-F379,
M1-C378, M1-T377, M1-S376, M1-C375, M1-D374, M1-S373, M1-S372,
M1-A371, M1-G370, M1-I369, M1-I368, M1-D367, M1-E366, M1-G365,
M1-P364, M1-A363, M1-F362, M1-L361, M1-D360, M1-V359, M1-C358,
M1-R357, M1-G356, M1-F355, M1-N354, M1-T353, M1-G352, M1-L351,
M1-T350, M1-G349, M1-L348, M1-T347, M1-V346, M1-P345, M1-Q344,
M1-D343, M1-Q342, M1-A341, M1-N340, M1-T339, M1-A338, M1-G337,
M1-V336, M1-T335, M1-I334, M1-V333, M1-E332, M1-P331, M1-A330,
M1-S329, M1-A328, M1-P327, M1-S326, M1-Y325, M1-L324, M1-C323,
M1-A322, M1-D321, M1-D320, M1-R319, M1-F318, M1-N317, M1-G316,
M1-A315, M1-A314, M1-T313, M1-V312, M1-L311, M1-V310, M1-V309,
M1-G308, M1-A307, M1-R306, M1-A305, M1-L304, M1-R303, M1-Q302,
M1-C301, M1-A300, M1-A299, M1-N298, M1-L297, M1-V296, M1-R295,
M1-S294, M1-Y293, M1-G292, M1-G291, M1-A290, M1-L289, M1-P288,
M1-L287, M1-L286, M1-V285, M1-V284, M1-L283, M1-P282, M1-G281,
M1-V280, M1-P279, M1-Q278, M1-V277, M1-L276, M1-Q275, M1-S274,
M1-K273, M1-R272, M1-I271, M1-F270, M1-E269, M1-L268, M1-G267,
M1-I266, M1-L265, M1-T264, M1-G263, M1-S262, M1-V261, M1-T260,
M1-G259, M1-K258, M1-G257, M1-Q256, M1-C255, M1-N254, M1-L253,
M1-V252, M1-R251, M1-L250, M1-S249, M1-R248, M1-M247, M1-S246,
M1-A245, M1-G244, M1-K243, M1-A242, M1-V241, M1-G240, M1-A239,
M1-D238, M1-R237, M1-G236, M1-S235, M1-V234, M1-V233, M1-G232,
M1-A231, M1-L230, M1-H229, M1-T228, M1-G227, M1-H226, M1-S225,
M1-D224, M1-C223, M1-K222, M1-S221, M1-A220, M1-Q219, M1-R218,
M1-H217, M1-F216, M1-R215, M1-T214, M1-G213, M1-D212, M1-E211,
M1-E210, M1-P209, M1-V208, M1-N207, M1-E206, M1-F205, M1-D204,
M1-T203, M1-V202, M1-M201, M1-V200, M1-R199, M1-G198, M1-E197,
M1-I196, M1-E195, M1-R194, M1-H193, M1-D192, M1-S191, M1-Q190,
M1-I189, M1-S188, M1-T187, M1-D186, M1-L185, M1-L184, M1-Y183,
M1-V182, M1-E181, M1-V180, M1-L179, M1-S178, M1-G177, M1-G176,
M1-D175, M1-P174, M1-P173, M1-Q172, M1-Y171, M1-E170, M1-D169,
M1-A168, M1-R167, M1-Y166, M1-R165, M1-P164, M1-P163, M1-T162,
M1-I161, M1-R160, M1-E159, M1-L158, M1-N157, M1-W156, M1-P155,
M1-I154, M1-S153, M1-Q152, M1-A151, M1-F150, M1-V149, M1-S148,
M1-S147, M1-D146, M1-E145, M1-E144, M1-I143, M1-Y142, M1-D141,
M1-V140, M1-H139, M1-P138, M1-L137, M1-K136, M1-L135, M1-A134,
M1-L133, M1-E132, M1-L131, M1-L130, M1-D129, M1-G128, M1-S127,
M1-M126, M1-K125, M1-V124, M1-L123, M1-F122, M1-G121, M1-P120,
M1-L119, M1-L118, M1-G117, M1-H116, M1-F115, M1-V114, M1-H113,
M1-L112, M1-I111, M1-K110, M1-T109, M1-L108, M1-Y107, M1-G106,
M1-R105, M1-R104, M1-A103, M1-A102, M1-Q101, M1-A100, M1-Q99,
M1-L98, M1-R97, M1-R96, M1-A95, M1-T94, M1-R93, M1-E92, M1-S91,
M1-Q90, M1-S89, M1-L88, M1-H87, M1-T86, M1-E85, M1-E84, M1-K83,
M1-L82, M1-V81, M1-V80, M1-V79, M1-Y78, M1-T77, M1-G76, M1-P75,
M1-L74, M1-R73, M1-W72, M1-P71, M1-D70, M1-K69, M1-A68, M1-C67,
M1-R66, M1-H65, M1-F64, M1-T63, M1-A62, M1-T61, M1-T60, M1-G59,
M1-H58, M1-E57, M1-P56, M1-A55, M1-E54, M1-A53, M1-L52, M1-G51,
M1-D50, M1-E49, M1-E48, M1-S47, M1-R46, M1-L45, M1-A44, M1-L43,
M1-V42, M1-L41, M1-E40, M1-E39, M1-Y38, M1-D37, M1-G36, M1-D35,
M1-E34, M1-D33, M1-E32, M1-Q31, M1-A30, M1-R29, M1-A28, M1-G27,
M1-A26, M1-P25, M1-G24, M1-L23, M1-L22, M1-L21, M1-L20, M1-L19,
M1-L18, M1-L17, M1-L16, M1-L15, M1-P14, M1-L13, M1-P12, M1-W11,
M1-W10, M1-S9, M1-R8, and/or M1--R7 of SEQ ID NO:5. Polypeptide
sequences encoded by these polynucleotides are also provided as SEQ
ID NO:38. In addition, the invention also encompasses
polynucleotides encoding a polypeptide that is at least as long as
any one of the aforementioned polypeptides. The present invention
also encompasses the use of these C-terminal PCSK9c deletion
polypeptides as immunogenic and/or antigenic epitopes as described
elsewhere herein.
TABLE-US-00001 TABLE I Total ATCC .RTM. NT NT 5' NT AA Total
Deposit SEQ Seq of Start 3' NT Seq AA Gene CDNA No. Z and ID. of
Codon of ID of No. CloneID Date Vector No. X Clone of ORF ORF No. Y
ORF 1. PCSK9b (also PTA-7622 pSPORT2 1 3175 250 1194 2 315 referred
to as 05/10/06 PCSK9-b) 2. PCSK9c (also PTA-7622 pSPORT1 3 3756 881
2449 4 523 referred to as 05/10/06 PCSK9-c)
[0171] Table I summarizes the information corresponding to each
"Gene No." described above. The nucleotide sequence identified as
"NT SEQ ID NO:X" was assembled from partially homologous
("overlapping") sequences obtained from the "cDNA clone ID"
identified in Table I and, in some cases, from additional related
DNA clones. The overlapping sequences were assembled into a single
contiguous sequence of high redundancy (usually several overlapping
sequences at each nucleotide position), resulting in a final
sequence identified as SEQ ID NO:X.
[0172] The cDNA Clone ID was deposited on the date and given the
corresponding deposit number listed in "ATCC.RTM. Deposit No:Z and
Date." "Vector" refers to the type of vector contained in the cDNA
Clone ID.
[0173] "Total NT Seq. Of Clone" refers to the total number of
nucleotides in the clone contig identified by "Gene No." The
deposited clone may contain all or most of the sequence of SEQ ID
NO:X. The nucleotide position of SEQ ID NO:X of the putative start
codon (methionine) is identified as "5' NT of Start Codon of
ORF."
[0174] The translated amino acid sequence, beginning with the
methionine, is identified as "AA SEQ ID NO:Y" although other
reading frames can also be easily translated using known molecular
biology techniques. The polypeptides produced by these alternative
open reading frames are specifically contemplated by the present
invention.
[0175] The total number of amino acids within the open reading
frame of SEQ ID NO:Y is identified as "Total AA of ORF".
[0176] SEQ ID NO:X (where X may be any of the polynucleotide
sequences disclosed in the sequence listing) and the translated SEQ
ID NO:Y (where Y may be any of the polypeptide sequences disclosed
in the sequence listing) are sufficiently accurate and otherwise
suitable for a variety of uses well known in the art and described
further herein. For instance, SEQ ID NO:X is useful for designing
nucleic acid hybridization probes that will detect nucleic acid
sequences contained in SEQ ID NO:X or the cDNA contained in the
deposited clone. These probes will also hybridize to nucleic acid
molecules in biological samples, thereby enabling a variety of
forensic and diagnostic methods of the invention. Similarly,
polypeptides identified from SEQ ID NO:Y may be used, for example,
to generate antibodies which bind specifically to proteins
containing the polypeptides and the proteins encoded by the cDNA
clones identified in Table I.
[0177] Nevertheless, DNA sequences generated by sequencing
reactions can contain sequencing errors. The errors exist as
misidentified nucleotides, or as insertions or deletions of
nucleotides in the generated DNA sequence. The erroneously inserted
or deleted nucleotides may cause frame shifts in the reading frames
of the predicted amino acid sequence. In these cases, the predicted
amino acid sequence diverges from the actual amino acid sequence,
even though the generated DNA sequence may be greater than 99.9%
identical to the actual DNA sequence (for example, one base
insertion or deletion in an open reading frame of over 1000
bases).
[0178] Accordingly, for those applications requiring precision in
the nucleotide sequence or the amino acid sequence, the present
invention provides not only the generated nucleotide sequence
identified as SEQ ID NO:X and the predicted translated amino acid
sequence identified as SEQ ID NO:Y, but also a sample of plasmid
DNA containing a cDNA of the invention deposited with the
ATCC.RTM., as set forth in Table I. The nucleotide sequence of each
deposited clone can readily be determined by sequencing the
deposited clone in accordance with known methods. The predicted
amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a
particular clone can also be directly determined by peptide
sequencing or by expressing the protein in a suitable host cell
containing the deposited cDNA, collecting the protein, and
determining its sequence.
[0179] The present invention also relates to the genes
corresponding to SEQ ID NO:X, SEQ ID NO:Y, or the deposited clone.
The corresponding gene can be isolated in accordance with known
methods using the sequence information disclosed herein. Such
methods include preparing probes or primers from the disclosed
sequence and identifying or amplifying the corresponding gene from
appropriate sources of genomic material.
[0180] Also provided in the present invention are species homologs,
allelic variants, and/or orthologs. The skilled artisan could,
using procedures well-known in the art, obtain the polynucleotide
sequence corresponding to full-length genes (including, but not
limited to the full-length coding region), allelic variants, splice
variants, orthologs, and/or species homologues of genes
corresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone,
relying on the sequence from the sequences disclosed herein or the
clones deposited with the ATCC.RTM.. For example, allelic variants
and/or species homologues may be isolated and identified by making
suitable probes or primers which correspond to the 5', 3', or
internal regions of the sequences provided herein and screening a
suitable nucleic acid source for allelic variants and/or the
desired homologue.
[0181] The polypeptides of the invention can be prepared in any
suitable manner. Such polypeptides include isolated naturally
occurring polypeptides, recombinantly produced polypeptides,
synthetically produced polypeptides, or polypeptides produced by a
combination of these methods. Means for preparing such polypeptides
are well understood in the art.
[0182] The polypeptides may be in the form of the protein, or may
be a part of a larger protein, such as a fusion protein (see
below). It is often advantageous to include an additional amino
acid sequence which contains secretory or leader sequences,
pro-sequences, sequences which aid in purification, such as
multiple histidine residues, or an additional sequence for
stability during recombinant production.
[0183] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of a polypeptide, can be
substantially purified using techniques described herein or
otherwise known in the art, such as, for example, 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,
synthetic or recombinant sources using protocols described herein
or otherwise known in the art, such as, for example, antibodies of
the invention raised against the full-length form of the
protein.
[0184] The present invention provides a polynucleotide comprising,
or alternatively consisting of, the sequence identified as SEQ ID
NO:X, and/or a cDNA provided in ATCC.RTM. Deposit No. Z:. The
present invention also provides a polypeptide comprising, or
alternatively consisting of, the sequence identified as SEQ ID
NO:Y, and/or a polypeptide encoded by the cDNA provided in
ATCC.RTM. Deposit NO:Z. The present invention also provides
polynucleotides encoding a polypeptide comprising, or alternatively
consisting of the polypeptide sequence of SEQ ID NO:Y, and/or a
polypeptide sequence encoded by the cDNA contained in ATCC.RTM.
Deposit No:Z.
[0185] Preferably, the present invention is directed to a
polynucleotide comprising, or alternatively consisting of, the
sequence identified as SEQ ID NO:X, and/or a cDNA provided in
ATCC.RTM. Deposit No.: that is less than, or equal to, a
polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs,
0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000
basepairs, or 10,000 basepairs in length.
[0186] The present invention encompasses polynucleotides with
sequences complementary to those of the polynucleotides of the
present invention disclosed herein. Such sequences may be
complementary to the sequence disclosed as SEQ ID NO:X, the
sequence contained in a deposit, and/or the nucleic acid sequence
encoding the sequence disclosed as SEQ ID NO:Y.
[0187] The present invention also encompasses polynucleotides
capable of hybridizing, preferably under reduced stringency
conditions, more preferably under stringent conditions, and most
preferably under highly stringent conditions, to polynucleotides
described herein. Examples of stringency conditions are shown in
Table II below: highly stringent conditions are those that are at
least as stringent as, for example, conditions A-F; stringent
conditions are at least as stringent as, for example, conditions
G-L; and reduced stringency conditions are at least as stringent
as, for example, conditions M-R.
TABLE-US-00002 TABLE II Hybridization Stringency Polynucleotide
Hybrid Temperature and Wash Temperature Condition Hybrid.+-. Length
(bp) .dagger-dbl. Buffer.dagger. and Buffer .dagger. A DNA:DNA
>or equal to 50 65.degree. C.; 1xSSC -or- 65.degree. C.;
42.degree. C.; 1xSSC, 50% 0.3xSSC formamide B DNA:DNA <50 Tb*;
1xSSC Tb*; 1xSSC C DNA:RNA >or equal to 50 67.degree. C.; 1xSSC
-or- 67.degree. C.; 45.degree. C.; 1xSSC, 50% 0.3xSSC formamide D
DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA >or equal to 50
70.degree. C.; 1xSSC -or- 70.degree. C.; 50.degree. C.; 1xSSC, 50%
0.3xSSC formamide F RNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA
>or equal to 50 65.degree. C.; 4xSSC -or- 65.degree. C.; 1xSSC
45.degree. C.; 4xSSC, 50% formamide H DNA:DNA <50 Th*; 4xSSC
Th*; 4xSSC I DNA:RNA >or equal to 50 67.degree. C.; 4xSSC -or-
67.degree. C.; 1xSSC 45.degree. C.; 4xSSC, 50% formamide J DNA:RNA
<50 Tj*; 4xSSC Tj*; 4xSSC K RNA:RNA >or equal to 50
70.degree. C.; 4xSSC -or- 67.degree. C.; 1xSSC 40.degree. C.;
6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC M
DNA:DNA >or equal to 50 50.degree. C.; 4xSSC -or- 50.degree. C.;
2xSSC 40.degree. C. 6xSSC, 50% formamide N DNA:DNA <50 Tn*;
6xSSC Tn*; 6xSSC O DNA:RNA >or equal to 50 55.degree. C.; 4xSSC
-or- 55.degree. C.; 2xSSC 42.degree. C.; 6xSSC, 50% formamide P
DNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA >or equal to 50
60.degree. C.; 4xSSC -or- 60.degree. C.; 2xSSC 45.degree. C.;
6xSSC, 50% formamide R RNA:RNA <50 Tr*; 4xSSC Tr*; 4xSSC
.dagger-dbl. - The "hybrid length" is the anticipated length for
the hybridized region(s) of the hybridizing polynucleotides. When
hybridizing a polynucleotide of unknown sequence, the hybrid is
assumed to be that of the hybridizing polynucleotide of the present
invention. When polynucleotides of known sequence are hybridized,
the hybrid length can be determined by aligning the sequences of
the polynucleotides and identifying the region or regions of
optimal sequence complementarity. Methods of aligning two or more
polynucleotide sequences and/or determining the percent identity
between two polynucleotide sequences are well known in the art
(e.g., MegAlign program of the DNASTAR .RTM. suite of programs,
etc). .dagger. - SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and
1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15M
NaCl and 15 mM sodium citrate) in the hybridization and wash
buffers; washes are performed for 15 minutes after hybridization is
complete. The hydridizations and washes may additionally include 5X
Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented
salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50%
formamide. *Tb - Tr: The hybridization temperature for hybrids
anticipated to be less than 50 base pairs in length should be
5-10.degree. C. less than the melting temperature Tm of the hybrids
there Tm is determined according to the following equations. For
hybrids less than 18 base pairs in length, Tm(.degree. C.) = 2(# of
A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49
base pairs in length, Tm(.degree. C.) = 81.5 +
16.6(log.sub.10[Na+]) + 0.41(% G + C) - (600/N), where N is the
number of bases in the hybrid, and [Na+] is the concentration of
sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165M).
.+-.The present invention encompasses the substitution of any one,
or more DNA or RNA hybrid partners with either a PNA, or a modified
polynucleotide. Such modified polynucleotides are known in the art
and are more particularly described elsewhere herein.
[0188] Additional examples of stringency conditions for
polynucleotide hybridization are provided, for example, in
Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols
in Molecular Biology, 1995, F. M., Ausubel et al., eds., John Wiley
and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby
incorporated by reference herein.
[0189] Preferably, such hybridizing polynucleotides have at least
70% sequence identity (more preferably, at least 80% identity; and
most preferably at least 90% or 95% identity) with the
polynucleotide of the present invention to which they hybridize,
where sequence identity is determined by comparing the sequences of
the hybridizing polynucleotides when aligned so as to maximize
overlap and identity while minimizing sequence gaps. The
determination of identity is well known in the art, and discussed
more specifically elsewhere herein.
[0190] The invention encompasses the application of PCR methodology
to the polynucleotide sequences of the present invention, the clone
deposited with the ATCC.RTM., and/or the cDNA encoding the
polypeptides of the present invention. PCR techniques for the
amplification of nucleic acids are described in U.S. Pat. No.
4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for
example, may include the following steps, of denaturation of
template nucleic acid (if double-stranded), annealing of primer to
target, and polymerization. The nucleic acid probed or used as a
template in the amplification reaction may be genomic DNA, cDNA,
RNA, or a PNA. PCR may be used to amplify specific sequences from
genomic DNA, specific RNA sequence, and/or cDNA transcribed from
mRNA. References for the general use of PCR techniques, including
specific method parameters, include Mullis et al., Cold Spring
Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR
Technology, Stockton Press, NY, 1989; Ehrlich et al., Science,
252:1643-1650, (1991); and "PCR Protocols, A Guide to Methods and
Applications", Eds., Innis et al., Academic Press, New York,
(1990).
Polynucleotide and Polypeptide Variants
[0191] The present invention also encompasses variants (e.g.,
allelic variants, orthologs, etc.) of the polynucleotide sequence
disclosed herein in SEQ ID NO:X, the complementary strand thereto,
and/or the cDNA sequence contained in the deposited clone.
[0192] The present invention also encompasses variants of the
polypeptide sequence, and/or fragments therein, disclosed in SEQ ID
NO:Y, a polypeptide encoded by the polynucleotide sequence in SEQ
ID NO:X, and/or a polypeptide encoded by a cDNA in the deposited
clone.
[0193] "Variant" refers to a polynucleotide or polypeptide
differing from the polynucleotide or polypeptide of the present
invention, but retaining essential properties thereof. Generally,
variants are overall closely similar, and, in many regions,
identical to the polynucleotide or polypeptide of the present
invention.
[0194] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide having a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence encoding a PCSK9b or
PCSK9c polypeptide having an amino acid sequence as shown in the
sequence listing and described in SEQ ID NO:2 or SEQ ID NO:4 or the
cDNA contained in ATCC.RTM. deposit No:Z; (b) a nucleotide sequence
encoding a mature PCSK9b or PCSK9c polypeptide having the amino
acid sequence as shown in the sequence listing and described in SEQ
ID NO:2 or SEQ ID NO:4 or the cDNA contained in ATCC.RTM. deposit
No:Z; (c) a nucleotide sequence encoding a biologically active
fragment of a PCSK9b or PCSK9c polypeptide having an amino acid
sequence shown in the sequence listing and described in SEQ ID NO:2
or SEQ ID NO:4 or the cDNA contained in ATCC.RTM. deposit No:Z; (d)
a nucleotide sequence encoding an antigenic fragment of a PCSK9b or
PCSK9c polypeptide having an amino acid sequence shown in the
sequence listing and described in SEQ ID NO:2 or SEQ ID NO:4 or the
cDNA contained in ATCC.RTM. deposit No:Z; (e) a nucleotide sequence
encoding a PCSK9b or PCSK9c polypeptide comprising the complete
amino acid sequence encoded by a human cDNA plasmid contained in
SEQ ID NO:2 or SEQ ID NO:4 or the cDNA contained in ATCC.RTM.
deposit No:Z; (f) a nucleotide sequence encoding a mature PCSK9b or
PCSK9c polypeptide having an amino acid sequence encoded by a human
cDNA plasmid contained in SEQ ID NO:2 or SEQ ID NO:4 or the cDNA
contained in ATCC.RTM. deposit No:Z; (g) a nucleotide sequence
encoding a biologically active fragment of a PCSK9b or PCSK9c
polypeptide having an amino acid sequence encoded by a human cDNA
plasmid contained in SEQ ID NO:2 or SEQ ID NO:4 or the cDNA
contained in ATCC.RTM. deposit No:Z; (h) a nucleotide sequence
encoding an antigenic fragment of a PCSK9b or PCSK9c polypeptide
having an amino acid sequence encoded by a human cDNA plasmid
contained in SEQ ID NO:X or the cDNA contained in ATCC.RTM. deposit
No:Z; and (i) a nucleotide sequence complimentary to any of the
nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h),
above.
[0195] The present invention is also directed to polynucleotide
sequences which comprise, or alternatively consist of, a
polynucleotide sequence which is at least about 80%, 85%, 90%, 91%,
92%, 93%, 93.6%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for
example, any of the nucleotide sequences in (a), (b), (c), (d),
(e), (f), (g), or (h), above. Polynucleotides encoded by these
nucleic acid molecules are also encompassed by the invention. In
another embodiment, the invention encompasses nucleic acid
molecules which comprise, or alternatively, consist of a
polynucleotide which hybridizes under stringent conditions, or
alternatively, under lower stringency conditions, to a
polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above.
Polynucleotides which hybridize to the complement of these nucleic
acid molecules under stringent hybridization conditions or
alternatively, under lower stringency conditions, are also
encompassed by the invention, as are polypeptides encoded by these
polypeptides.
[0196] Another aspect of the invention provides an isolated nucleic
acid molecule comprising, or alternatively, consisting of, a
polynucleotide having a nucleotide sequence selected from the group
consisting of: (a) a nucleotide sequence encoding a PCSK9b or
PCSK9c polypeptide having an amino acid sequence as shown in the
sequence listing and descried in Table I; (b) a nucleotide sequence
encoding a mature PCSK9b or PCSK9c polypeptide having the amino
acid sequence as shown in the sequence listing and descried in
Table I; (c) a nucleotide sequence encoding a biologically active
fragment of a PCSK9b or PCSK9c polypeptide having an amino acid
sequence as shown in the sequence listing and descried in Table I;
(d) a nucleotide sequence encoding an antigenic fragment of a
PCSK9b or PCSK9c polypeptide having an amino acid sequence as shown
in the sequence listing and descried in Table I; (e) a nucleotide
sequence encoding a PCSK9b or PCSK9c polypeptide comprising the
complete amino acid sequence encoded by a human cDNA in a cDNA
plasmid contained in the ATCC.RTM. Deposit and described in Table
I; (f) a nucleotide sequence encoding a mature PCSK9b or PCSK9c
polypeptide having an amino acid sequence encoded by a human cDNA
in a cDNA plasmid contained in the ATCC.RTM. Deposit and described
in Table I: (g) a nucleotide sequence encoding a biologically
active fragment of a PCSK9b or PCSK9c polypeptide having an amino
acid sequence encoded by a human cDNA in a cDNA plasmid contained
in the ATCC.RTM. Deposit and described in Table I; (h) a nucleotide
sequence encoding an antigenic fragment of a PCSK9b or PCSK9c
polypeptide having an amino acid sequence encoded by a human cDNA
in a cDNA plasmid contained in the ATCC.RTM. deposit and described
in Table I; and (i) a nucleotide sequence complimentary to any of
the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or
(h) above.
[0197] The present invention is also directed to nucleic acid
molecules which comprise, or alternatively, consist of, a
nucleotide sequence which is at least about 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any
of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g),
or (h), above.
[0198] The present invention encompasses polypeptide sequences
which comprise, or alternatively consist of, an amino acid sequence
which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%, 99.8%, or 99.9% identical to, the following non-limited
examples, the polypeptide sequence identified as SEQ ID NO:Y, the
polypeptide sequence encoded by a cDNA provided in the deposited
clone, and/or polypeptide fragments of any of the polypeptides
provided herein. Polynucleotides encoded by these nucleic acid
molecules are also encompassed by the invention. In another
embodiment, the invention encompasses nucleic acid molecules which
comprise, or alternatively, consist of a polynucleotide which
hybridizes under stringent conditions, or alternatively, under
lower stringency conditions, to a polynucleotide in (a), (b), (c),
(d), (e), (f), (g), or (h), above. Polynucleotides which hybridize
to the complement of these nucleic acid molecules under stringent
hybridization conditions or alternatively, under lower stringency
conditions, are also encompassed by the invention, as are
polypeptides encoded by these polypeptides.
[0199] The present invention is also directed to polypeptides which
comprise, or alternatively consist of, an amino acid sequence which
is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,
or 99.9% identical to, for example, the polypeptide sequence shown
in SEQ ID NO:Y, a polypeptide sequence encoded by the nucleotide
sequence in SEQ ID NO:X, a polypeptide sequence encoded by the cDNA
in cDNA plasmid:Z, and/or polypeptide fragments of any of these
polypeptides (e.g., those fragments described herein).
Polynucleotides which hybridize to the complement of the nucleic
acid molecules encoding these polypeptides under stringent
hybridization conditions or alternatively, under lower stringency
conditions, are also encompasses by the present invention, as are
the polypeptides encoded by these polynucleotides.
[0200] By a nucleic acid having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence of the
present invention, it is intended that the nucleotide sequence of
the nucleic acid is identical to the reference sequence except that
the nucleotide sequence may include up to five point mutations per
each 100 nucleotides of the reference nucleotide sequence encoding
the polypeptide. In other words, to obtain a nucleic acid 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. The
query sequence may be an entire sequence referenced in Table I, the
ORF (open reading frame), or any fragment specified as described
herein.
[0201] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide
sequence of the present invention can be determined conventionally
using known computer programs. A preferred method for determining
the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the CLUSTALW
computer program (Thompson, J. D., et al., Nucleic Acids Research,
2(22):4673-4680, (1994)), which is based on the algorithm of
Higgins, D. G., et al., Computer Applications in the Biosciences
(CABIOS), 8(2):189-191, (1992). In a sequence alignment the query
and subject sequences are both DNA sequences. An RNA sequence can
be compared by converting U's to T's. However, the CLUSTALW
algorithm automatically converts U's to T's when comparing RNA
sequences to DNA sequences. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
CLUSTALW alignment of DNA sequences to calculate percent identity
via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top
Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension
Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of
the subject nucleotide sequence, whichever is shorter. For multiple
alignments, the following CLUSTALW parameters are preferred: Gap
Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation
Penalty Range=8; End Gap Separation Penalty=Off; % Identity for
Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue
Gap=Off; and Transition Weighting=0. The pairwise and multple
alignment parameters provided for CLUSTALW above represent the
default parameters as provided with the ALIGNX.RTM. software
program (VECTOR NTI.RTM. suite of programs, version 6.0).
[0202] The present invention encompasses the application of a
manual correction to the percent identity results, in the instance
where the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions.
If only the local pairwise percent identity is required, no manual
correction is needed. However, a manual correction may be applied
to determine the global percent identity from a global
polynucleotide alignment. Percent identity calculations based upon
global polynucleotide alignments are often preferred since they
reflect the percent identity between the polynucleotide molecules
as a whole (i.e., including any polynucleotide overhangs, not just
overlapping regions), as opposed to, only local matching
polynucleotides. Manual corrections for global percent identity
determinations are required since the CLUSTALW program does not
account for 5' and 3' truncations of the subject sequence when
calculating percent identity. For subject sequences truncated at
the 5' or 3' ends, relative to the query sequence, the percent
identity is corrected by calculating the number of bases of the
query sequence that are 5' and 3' of the subject sequence, which
are not matched/aligned, as a percent of the total bases of the
query sequence. Whether a nucleotide is matched/aligned is
determined by results of the CLUSTALW sequence alignment. This
percentage is then subtracted from the percent identity, calculated
by the above CLUSTALW program using the specified parameters, to
arrive at a final percent identity score. This corrected score may
be used for the purposes of the present invention. Only bases
outside the 5' and 3' bases of the subject sequence, as displayed
by the CLUSTALW alignment, which are not matched/aligned with the
query sequence, are calculated for the purposes of manually
adjusting the percent identity score.
[0203] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
CLUSTALW alignment does not show a matched/alignment of the first
10 bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the CLUSTALW program. If
the remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by CLUSTALW
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are required for the purposes of the present invention.
[0204] In addition to the above method of aligning two or more
polynucleotide or polypeptide sequences to arrive at a percent
identity value for the aligned sequences, it may be desirable in
some circumstances to use a modified version of the CLUSTALW
algorithm which takes into account known structural features of the
sequences to be aligned, such as for example, the SWISS-PROT.RTM.
designations for each sequence. The result of such a modified
CLUSTALW algorithm may provide a more accurate value of the percent
identity for two polynucleotide or polypeptide sequences. Support
for such a modified version of CLUSTALW is provided within the
CLUSTALW algorithm and would be readily appreciated to one of skill
in the art of bioinformatics.
[0205] The variants may contain alterations in the coding regions,
non-coding regions, or both. Especially preferred are
polynucleotide variants containing alterations which produce silent
substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. Nucleotide
variants produced by silent substitutions due to the degeneracy of
the genetic code are preferred. Moreover, variants in which 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. Polynucleotide variants can be
produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the mRNA to
those preferred by a bacterial host such as E. coli).
[0206] Naturally occurring variants are called "allelic variants"
and refer to 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).) These allelic
variants can vary at either the polynucleotide and/or polypeptide
level and are included in the present invention. Alternatively,
non-naturally occurring variants may be produced by mutagenesis
techniques or by direct synthesis.
[0207] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides of the present invention. For
instance, one or more amino acids can be deleted from the
N-terminus or C-terminus of the protein without substantial loss of
biological function. The authors of Ron et al., J. Biol. Chem.
268:2984-2988 (1993), reported variant KGF proteins having heparin
binding activity even after deleting 3, 8, or 27 amino-terminal
amino acid residues. Similarly, Interferon gamma exhibited up to
ten times higher activity after deleting 8-10 amino acid residues
from the carboxy terminus of this protein (Dobeli et al., J.
Biotechnology 7:199-216 (1988)).
[0208] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J. Biol. Chem.
268:22105-22111 (1993)) conducted extensive mutational analysis of
human cytokine IL-1a. They used random mutagenesis to generate over
3,500 individual IL-1a mutants that averaged 2.5 amino acid changes
per variant over the entire length of the molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]." In
fact, only 23 unique amino acid sequences, out of more than 3,500
nucleotide sequences examined, produced a protein that
significantly differed in activity from wild-type.
[0209] Furthermore, even if deleting one or more amino acids from
the N-terminus or C-terminus of a polypeptide results in
modification or loss of one or more biological functions, other
biological activities may still be retained. For example, the
ability of a deletion variant to induce and/or to bind antibodies
which recognize the protein will likely be retained when less than
the majority of the residues of the protein are removed from the
N-terminus or C-terminus. Whether a particular polypeptide lacking
N- or C-terminal residues of a protein retains such immunogenic
activities can readily be determined by routine methods described
herein and otherwise known in the art.
[0210] Alternatively, such N-terminus or C-terminus deletions of a
polypeptide of the present invention may, in fact, result in a
significant increase in one or more of the biological activities of
the polypeptide(s). For example, biological activity of many
polypeptides are governed by the presence of regulatory domains at
either one or both termini. Such regulatory domains effectively
inhibit the biological activity of such polypeptides in lieu of an
activation event (e.g., binding to a cognate ligand or receptor,
phosphorylation, proteolytic processing, etc.). Thus, by
eliminating the regulatory domain of a polypeptide, the polypeptide
may effectively be rendered biologically active in the absence of
an activation event.
[0211] Thus, the invention further includes polypeptide variants
that show substantial biological activity. Such variants include
deletions, insertions, inversions, repeats, and 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 et al., Science 247:1306-1310 (1990), wherein the authors
indicate that there are two main strategies for studying the
tolerance of an amino acid sequence to change.
[0212] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, conserved
amino acids can be identified. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions where substitutions have been tolerated by natural
selection indicates that these positions are not critical for
protein function. Thus, positions tolerating amino acid
substitution could be modified while still maintaining biological
activity of the protein.
[0213] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (introduction
of single alanine mutations at every residue in the molecule) can
be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The
resulting mutant molecules can then be tested for biological
activity.
[0214] As the authors state, these two strategies 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 certain amino acid positions
in the protein. For example, most buried (within the tertiary
structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally
conserved.
[0215] The invention encompasses polypeptides having a lower degree
of identity but having sufficient similarity so as to perform one
or more of the same functions performed by the polypeptide of the
present invention. Similarity is determined by conserved amino acid
substitution. Such substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics (e.g., chemical properties). According to
Cunningham et al above, such conservative substitutions are likely
to be phenotypically silent. Additional guidance concerning which
amino acid changes are likely to be phenotypically silent are found
in Bowie et al., Science 247:1306-1310 (1990).
[0216] The invention encompasses polypeptides having a lower degree
of identity but having sufficient similarity so as to perform one
or more of the same functions performed by the polypeptide of the
present invention. Similarity is determined by conserved amino acid
substitution. Such substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics (e.g., chemical properties). According to
Cunningham et al above, such conservative substitutions are likely
to be phenotypically silent. Additional guidance concerning which
amino acid changes are likely to be phenotypically silent are found
in Bowie et al., Science 247:1306-1310 (1990).
[0217] Tolerated conservative amino acid substitutions of the
present invention involve replacement of the aliphatic or
hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the
hydroxyl residues Ser and Thr; replacement of the acidic residues
Asp and Glu; replacement of the amide residues Asn and Gln,
replacement of the basic residues Lys, Arg, and His; replacement of
the aromatic residues Phe, Tyr, and Trp, and replacement of the
small-sized amino acids Ala, Ser, Thr, Met, and Gly.
[0218] In addition, the present invention also encompasses the
conservative substitutions provided in Table III below.
TABLE-US-00003 TABLE III For Amino Acid Code Replace with any of:
Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys,
D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D- Ile, Orn, D-Orn
Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic
Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys,
S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,
Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn,
D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, .beta.-Ala, Acp
Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L
D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg,
D-homo-Arg, Met, D-Met, Ile, D- Ile, Orn, D-Orn Methionine M D-Met,
S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe,
Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or
5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,
L-1-thioazolidine-4-carboxylic acid, D- or L-1-
oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr,
Met, D-Met, Met(O), D-Met(O), L- Cys, D-Cys Threonine T D-Thr, Ser,
D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine
Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu,
Ile, D-Ile, Met, D-Met
[0219] Aside from the uses described above, such amino acid
substitutions may also increase protein or peptide stability. The
invention encompasses amino acid substitutions that contain, for
example, one or more non-peptide bonds (which replace the peptide
bonds) in the protein or peptide sequence. Also included are
substitutions that include amino acid residues other than naturally
occurring L-amino acids, e.g., D-amino acids or non-naturally
occurring or synthetic amino acids, e.g., .beta. or .gamma. amino
acids.
[0220] Both identity and similarity can be readily calculated by
reference to the following publications: Computational Molecular
Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Informatics Computer Analysis of
Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular
Biology, von Heinje, G., Academic Press, 1987; and Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991.
[0221] In addition, the present invention also encompasses
substitution of amino acids based upon the probability of an amino
acid substitution resulting in conservation of function. Such
probabilities are determined by aligning multiple genes with
related function and assessing the relative penalty of each
substitution to proper gene function. Such probabilities are often
described in a matrix and are used by some algorithms (e.g.,
BLAST.RTM., CLUSTALW, GAP.RTM., etc.) in calculating percent
similarity wherein similarity refers to the degree by which one
amino acid may substitute for another amino acid without lose of
function. An example of such a matrix is the PAM250 or BLOSUM62
matrix.
[0222] Aside from the canonical chemically conservative
substitutions referenced above, the invention also encompasses
substitutions which are typically not classified as conservative,
but that may be chemically conservative under certain
circumstances. Analysis of enzymatic catalysis for proteases, for
example, has shown that certain amino acids within the active site
of some enzymes may have highly perturbed pKa's due to the unique
microenvironment of the active site. Such perturbed pKa's could
enable some amino acids to substitute for other amino acids while
conserving enzymatic structure and function. Examples of amino
acids that are known to have amino acids with perturbed pKa's are
the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin,
the His-159 residue of Papain, etc. The conservation of function
relates to either anomalous protonation or anomalous deprotonation
of such amino acids, relative to their canonical, non-perturbed
pKa. The pKa perturbation may enable these amino acids to actively
participate in general acid-base catalysis due to the unique
ionization environment within the enzyme active site. Thus,
substituting an amino acid capable of serving as either a general
acid or general base within the microenvironment of an enzyme
active site or cavity, as may be the case, in the same or similar
capacity as the wild-type amino acid, would effectively serve as a
conservative amino substitution.
[0223] Besides conservative amino acid substitution, variants of
the present invention include, but are not limited to, the
following: (i) substitutions with one or more of the non-conserved
amino acid residues, where the substituted amino acid residues may
or may not be one encoded by the genetic code, or (ii) substitution
with one or more of amino acid residues having a substituent group,
or (iii) fusion of the mature polypeptide with another compound,
such as a compound to increase the stability and/or solubility of
the polypeptide (for example, polyethylene glycol), or (iv) fusion
of the polypeptide with additional amino acids, such as, for
example, an IgG Fc fusion region peptide, or leader or secretory
sequence, or a sequence facilitating purification. Such variant
polypeptides are deemed to be within the scope of those skilled in
the art from the teachings herein.
[0224] For example, polypeptide variants containing amino acid
substitutions of charged amino acids with other charged or neutral
amino acids may produce proteins with improved characteristics,
such as less aggregation. Aggregation of pharmaceutical
formulations both reduces activity and increases clearance due to
the aggregate's immunogenic activity. (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).)
[0225] Moreover, the invention further includes polypeptide
variants created through the application of molecular evolution
("DNA Shuffling") methodology to the polynucleotide disclosed as
SEQ ID NO:X, the sequence of the clone submitted in a deposit,
and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:Y.
Such DNA Shuffling technology is known in the art and more
particularly described elsewhere herein (e.g., WPC, Stemmer, PNAS,
91:10747, (1994)), and in the Examples provided herein).
[0226] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of the present
invention having an amino acid sequence which contains at least one
amino acid substitution, but not more than 50 amino acid
substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and still even more preferably, not more than 20
amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a peptide or polypeptide to
have an amino acid sequence which comprises the amino acid sequence
of the present invention, which contains at least one, but not more
than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In
specific embodiments, the number of additions, substitutions,
and/or deletions in the amino acid sequence of the present
invention or fragments thereof (e.g., the mature form and/or other
fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or
50-150, conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments
[0227] The present invention is directed to polynucleotide
fragments of the polynucleotides of the invention, in addition to
polypeptides encoded therein by said polynucleotides and/or
fragments.
[0228] In the present invention, a "polynucleotide fragment" refers
to a short polynucleotide having a nucleic acid sequence which: is
a portion of that contained in a deposited clone, or encoding the
polypeptide encoded by the cDNA in a deposited clone; is a portion
of that shown in SEQ ID NO:X or the complementary strand thereto,
or is a portion of a polynucleotide sequence encoding the
polypeptide of SEQ ID NO:Y. The nucleotide fragments of the
invention are preferably 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, at least about 50
nt, at least about 75 nt, or at least about 150 nt in length, or at
least about 875 nt in length, or at least about 837 nt in length,
or at least about 903 nt in length, or at least about 915 nt in
length, or at least about 930 nt in length, or at least about 945
nt in length, or at least about 1000 nt in length, or at least
about 1050 nt in length, or at least about 1100 nt in length, or at
least about 1150 nt in length, or at least about 1200 nt in length,
or at least about 1250 nt in length, or at least about 1300 nt in
length, or at least about 1350 nt in length, or at least about 1400
nt in length, or at least about 1450 nt in length, or at least
about 1500 nt in length, or at least about 1554 nt in length. A
fragment "at least 20 nt in length" for example, is intended to
include 20 or more contiguous bases from the cDNA sequence
contained in a deposited clone or the nucleotide sequence shown in
SEQ ID NO:X. In this context "about" includes the particularly
recited value, a value larger or smaller by several (25, 24, 23,
22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,
4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
These nucleotide fragments have uses that include, but are not
limited to, as diagnostic probes and primers as discussed herein.
Of course, larger fragments (e.g., 50, 150, 500, 600, 837, 903,
1554, 2000 nucleotides) are preferred.
[0229] Moreover, representative examples of polynucleotide
fragments of the invention, include, for example, fragments
comprising, or alternatively consisting of, a sequence from about
nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300,
301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700,
701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050,
1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,
1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,
1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,
1951-2000, or 2001 to the end of SEQ ID NO:X, or the complementary
strand thereto, or the cDNA contained in a deposited clone. In this
context "about" includes the particularly recited ranges, and
ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,
at either terminus or at both termini. Preferably, these fragments
encode a polypeptide which has biological activity. More
preferably, these polynucleotides can be used as probes or primers
as discussed herein. Also encompassed by the present invention are
polynucleotides which hybridize to these nucleic acid molecules
under stringent hybridization conditions or lower stringency
conditions, as are the polypeptides encoded by these
polynucleotides.
[0230] In the present invention, a "polypeptide fragment" refers to
an amino acid sequence which is a portion of that contained in SEQ
ID NO:Y or encoded by the cDNA contained in a deposited clone.
Protein (polypeptide) fragments may be "free-standing" or comprised
within a larger polypeptide of which the fragment forms a part or
region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments comprising, or alternatively
consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the
coding region. Moreover, polypeptide fragments can be about 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 279, 301,
325, 350, 375, 400, 425, 450, 475, 500, or 518 amino acids in
length. In this context "about" includes the particularly recited
ranges or values, and ranges or values larger or smaller by several
(25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1) amino acids, at either extreme or at
both extremes. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0231] Preferred polypeptide fragments include the full-length
protein. Further preferred polypeptide fragments include the
full-length protein having a continuous series of deleted residues
from the amino or the carboxy terminus, or both. For example, any
number of amino acids, ranging from 1-60, can be deleted from the
amino terminus of the full-length polypeptide. Similarly, any
number of amino acids, ranging from 1-30, can be deleted from the
carboxy terminus of the full-length protein. Furthermore, any
combination of the above amino and carboxy terminus deletions are
preferred. Similarly, polynucleotides encoding these polypeptide
fragments are also preferred.
[0232] Also preferred are polypeptide and polynucleotide fragments
characterized by structural or functional domains, such as
fragments that comprise alpha-helix and alpha-helix forming
regions, beta-sheet and beta-sheet-forming regions, turn and
turn-forming regions, coil and coil-forming regions, hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions.
Polypeptide fragments of SEQ ID NO:Y falling within conserved
domains are specifically contemplated by the present invention.
Moreover, polynucleotides encoding these domains are also
contemplated.
[0233] Other preferred polypeptide fragments are biologically
active fragments. Biologically active fragments are those
exhibiting activity similar, but not necessarily identical, to an
activity of the polypeptide of the present invention. The
biological activity of the fragments may include an improved
desired activity, or a decreased undesirable activity.
Polynucleotides encoding these polypeptide fragments are also
encompassed by the invention.
[0234] In a preferred embodiment, the functional activity displayed
by a polypeptide encoded by a polynucleotide fragment of the
invention may be one or more biological activities typically
associated with the full-length polypeptide of the invention.
Illustrative of these biological activities includes the fragments
ability to bind to at least one of the same antibodies which bind
to the full-length protein, the fragments ability to interact with
at least one of the same proteins which bind to the full-length,
the fragments ability to elicit at least one of the same immune
responses as the full-length protein (i.e., to cause the immune
system to create antibodies specific to the same epitope, etc.),
the fragments ability to bind to at least one of the same
polynucleotides as the full-length protein, the fragments ability
to bind to a receptor of the full-length protein, the fragments
ability to bind to a ligand of the full-length protein, and the
fragments ability to multimerize with the full-length protein.
However, the skilled artisan would appreciate that some fragments
may have biological activities which are desirable and directly
inapposite to the biological activity of the full-length protein.
The functional activity of polypeptides of the invention, including
fragments, variants, derivatives, and analogs thereof can be
determined by numerous methods available to the skilled artisan,
some of which are described elsewhere herein.
[0235] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NO:Y, or an epitope of the
polypeptide sequence encoded by a polynucleotide sequence contained
in ATCC.RTM. deposit No. Z or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ ID NO:X or
contained in ATCC.RTM. deposit No. Z under stringent hybridization
conditions or lower stringency hybridization conditions as defined
supra. The present invention further encompasses polynucleotide
sequences encoding an epitope of a polypeptide sequence of the
invention (such as, for example, the sequence disclosed in SEQ ID
NO:1 or SEQ ID NO:3), polynucleotide sequences of the complementary
strand of a polynucleotide sequence encoding an epitope of the
invention, and polynucleotide sequences which hybridize to the
complementary strand under stringent hybridization conditions or
lower stringency hybridization conditions defined supra.
[0236] The term "epitopes" as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope" as used herein, is defined as
a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). The term "antigenic epitope" as used herein, is defined as
a portion of a protein to which an antibody can immunospecifically
bind its antigen as determined by any method well known in the art,
for example, by the immunoassays described herein. Immunospecific
binding excludes non-specific binding but does not necessarily
exclude cross-reactivity with other antigens. Antigenic epitopes
need not necessarily be immunogenic.
[0237] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0238] In the present invention, antigenic epitopes preferably
contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, and, most
preferably, between about 15 to about 30 amino acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, or 100 amino acid residues in length, or longer.
Additional non-exclusive preferred antigenic epitopes include the
antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic epitopes are useful, for example, to raise antibodies,
including monoclonal antibodies that specifically bind the epitope.
Preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as any combination of two, three, four,
five or more of these antigenic epitopes. Antigenic epitopes can be
used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science
219:660-666 (1983)).
[0239] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic
epitopes. The polypeptides comprising one or more immunogenic
epitopes may be presented for eliciting an antibody response
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide
may be presented without a carrier. However, immunogenic epitopes
comprising as few as 8 to 10 amino acids have been shown to be
sufficient to raise antibodies capable of binding to, at the very
least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[0240] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0241] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides.
Such fusion proteins may facilitate purification and may increase
half-life in vivo. This has been shown 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. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of
an antigen across the epithelial barrier to the immune system has
been demonstrated for antigens (e.g., insulin) conjugated to an
FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT
Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG portion
disulfide bonds have also been found to be more efficient in
binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid
in detection and purification of the expressed polypeptide. For
example, a system described by Janknecht et al. allows for the
ready purification of non-denatured fusion proteins expressed in
human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the open reading
frame of the gene is translationally fused to an amino-terminal tag
consisting of six histidine residues. The tag serves as a matrix
binding domain for the fusion protein. Extracts from cells infected
with the recombinant vaccinia virus are loaded onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can
be selectively eluted with imidazole-containing buffers.
[0242] Additional fusion proteins of the invention may be generated
through the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco,
Biotechniques 24(2):308-13 (1998) (each of these patents and
publications are hereby incorporated by reference in its entirety).
In one embodiment, alteration of polynucleotides corresponding to
SEQ ID NO:X and the polypeptides encoded by these polynucleotides
may be achieved by DNA shuffling. DNA shuffling involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to generate variation in the polynucleotide sequence.
In another embodiment, polynucleotides of the invention, or the
encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. In another embodiment, one or
more components, motifs, sections, parts, domains, fragments, etc.,
of a polynucleotide encoding a polypeptide of the invention may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
Antibodies
[0243] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of SEQ ID NO:2,
and/or an epitope, of the present invention (as determined by
immunoassays well known in the art for assaying specific
antibody-antigen binding). Antibodies of the invention include, but
are not limited to, polyclonal, monoclonal, monovalent, bispecific,
heteroconjugate, multispecific, human, humanized or chimeric
antibodies, single chain antibodies, Fab fragments, F(ab')
fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to antibodies of the invention), and epitope-binding
fragments of any of the above. The term "antibody," as used herein,
refers to immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules that contain
an antigen binding site that immunospecifically binds an antigen.
The immunoglobulin molecules of the invention can be of any type
(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
Moreover, 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 protein. Fab and F(ab')2
fragments lack the Fc fragment of intact antibody, clear more
rapidly from the circulation of the animal or plant, and may have
less non-specific tissue binding than an intact antibody (Wahl et
al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are
preferred, as well as the products of a FAB or other immunoglobulin
expression library. Moreover, antibodies of the present invention
include chimeric, single chain, and humanized antibodies.
[0244] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat),
donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins, as described infra and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0245] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0246] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0247] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homologue of a polypeptide
of the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In specific embodiments, antibodies of the present invention
cross-react with murine, rat and/or rabbit homologues of human
proteins and the corresponding epitopes thereof. Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%,
less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to
a polypeptide of the present invention are also included in the
present invention. In a specific embodiment, the above-described
cross-reactivity is with respect to any single specific antigenic
or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or
more of the specific antigenic and/or immunogenic polypeptides
disclosed herein. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides which
hybridize to a polynucleotide of the present invention under
stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10-2 M, 10-2 M,
5.times.10-3 M, 10-3 M, 5.times.10-4 M, 10-4 M, 5.times.10-5 M,
10-5 M, 5.times.10-6 M, 10-6M, 5.times.10-7 M, 10-7 M, 5.times.10-8
M, 10-8 M, 5.times.10-9 M, 10-9 M, 5.times.10-10 M, 10-10 M,
5.times.10-11 M, 10-11 M, 5.times.10-12 M, 10-12 M, 5.times.10-13
M, 10-13 M, 5.times.10-14 M, 10-14 M, 5.times.10-15 M, or 10-15
M.
[0248] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85%, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[0249] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which do not prevent ligand
binding but prevent receptor activation. Receptor activation (i.e.,
signaling) may be determined by techniques described herein or
otherwise known in the art. For example, receptor activation can be
determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[0250] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
dimerization of the receptor. The antibodies may be specified as
agonists, antagonists or inverse agonists for biological activities
comprising the specific biological activities of the peptides of
the invention disclosed herein. The above antibody agonists can be
made using methods known in the art. See, e.g., PCT publication WO
96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood
92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678
(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman et al., Neuron 14(4):755-762 (1995); Muller et al.,
Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine
8(1):14-20 (1996) (which are all incorporated by reference herein
in their entireties).
[0251] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0252] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionucleotides, or toxins. See, e.g., PCT publications WO
92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[0253] The antibodies of the invention include derivatives that are
modified, i.e., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0254] The antibodies of the present invention may be generated by
any suitable method known in the art.
[0255] The antibodies of the present invention may comprise
polyclonal antibodies. Methods of preparing polyclonal antibodies
are known to the skilled artisan (Harlow, et al., Antibodies: A
Laboratory Manual, (Cold spring Harbor Laboratory Press, 2.sup.nd
ed. (1988); and Current Protocols, Chapter 2; which are hereby
incorporated herein by reference in its entirety). In a preferred
method, a preparation of the PCSK9b or PCSK9c 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. For example, a polypeptide of the invention can be
administered to various host animals including, but not limited to,
rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. The
administration of the polypeptides of the present invention may
entail one or more injections of an immunizing agent and, if
desired, an adjuvant. Various adjuvants may be used to increase the
immunological response, depending on the host species, and include
but are not limited to, Freund's (complete and incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art. For the purposes of the invention,
"immunizing agent" may be defined as a polypeptide of the
invention, including fragments, variants, and/or derivatives
thereof, in addition to fusions with heterologous polypeptides and
other forms of the polypeptides described herein.
[0256] Typically, the immunizing agent and/or adjuvant will be
injected in the mammal by multiple subcutaneous or intraperitoneal
injections, though they may also be given intramuscularly, and/or
through IV). The immunizing agent may include polypeptides of the
present invention or a fusion protein or variants thereof.
Depending upon the nature of the polypeptides (i.e., percent
hydrophobicity, percent hydrophilicity, stability, net charge,
isoelectric point etc.), it may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized. Such conjugation includes either chemical
conjugation by derivatizing active chemical functional groups to
both the polypeptide of the present invention and the immunogenic
protein such that a covalent bond is formed, or through
fusion-protein based methodology, or other methods known to the
skilled artisan. Examples of such immunogenic proteins include, but
are not limited to keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may
be used to increase the immunological response, depending on the
host species, including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Additional
examples of adjuvants which may be employed includes the MPL-TDM
adjuvant (monophosphoryl lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one
skilled in the art without undue experimentation.
[0257] The antibodies of the present invention may comprise
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et
al., Antibodies: A Laboratory Manual, (Cold spring Harbor
Laboratory Press, 2.sup.nd ed. (1988), by Hammerling, et al.,
Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., pp.
563-681 (1981); Kohler et al., Eur. J. Immunol. 6:511 (1976);
Kohler et al., Eur. J. Immunol. 6:292 (1976), or other methods
known to the artisan. Other examples of methods which may be
employed for producing monoclonal antibodies includes, but are not
limited to, the human B-cell hybridoma technique (Kosbor et al.,
1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad.
Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et
al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96). Such antibodies may be of any immunoglobulin
class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
The hybridoma producing the mAb of this invention may be cultivated
in vitro or in vivo. Production of high titers of mAbs in vivo
makes this the presently preferred method of production.
[0258] In a hybridoma method, a mouse, a humanized mouse, a mouse
with a human immune system, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro.
[0259] The immunizing agent will typically include polypeptides of
the present invention or a fusion protein thereof. Preferably, the
immunizing agent consists of an PCSK9b or PCSK9c polypeptide or,
more preferably, with a PCSK9b or PCSK9c polypeptide-expressing
cell. 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 degrees C.), and supplemented with about
10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin,
and about 100 ug/ml of streptomycin. Generally, either peripheral
blood lymphocytes ("PBLs") are used if cells of human origin are
desired, or spleen cells or lymph node cells are used if non-human
mammalian sources are desired. The lymphocytes are then fused with
an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, (1986), pp.
59-103) Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells of rodent, bovine and human
origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that
preferably contains one or more substances that inhibit the growth
or survival of the unfused, immortalized cells. For example, if the
parental cells lack the enzyme hypoxanthine guanine phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas
typically will include hypoxanthine, aminopterin, and thymidine
("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.
[0260] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. More preferred
are the parent myeloma cell line (SP2O) as provided by the
ATCC.RTM.. As inferred throughout the specification, human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63).
[0261] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the polypeptides of the present invention.
Preferably, the binding specificity of monoclonal antibodies
produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques are known in the art and within the skill
of the artisan. The binding affinity of the monoclonal antibody
can, for example, be determined by the Scatchard analysis of Munson
and Pollart, Anal. Biochem., 107:220 (1980).
[0262] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra, and/or according to Wands et al.
(Gastroenterology 80:225-232 (1981)). Suitable culture media for
this purpose include, for example, Dulbecco's Modified Eagle's
Medium and RPMI-1640. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0263] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-sepharose, hydroxyapatite chromatography, gel
exclusion chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
[0264] The skilled artisan would acknowledge that a variety of
methods exist in the art for the production of monoclonal
antibodies and thus, the invention is not limited to their sole
production in hydridomas. For example, the monoclonal antibodies
may be made by recombinant DNA methods, such as those described in
U.S. Pat. No. 4,816,567. In this context, the term "monoclonal
antibody" refers to an antibody derived from a single eukaryotic,
phage, or prokaryotic clone. The DNA encoding the monoclonal
antibodies of the invention can be readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of murine antibodies, or such chains
from human, humanized, or other sources). The hydridoma cells of
the invention serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transformed into host cells such as Simian COS cells, Chinese
hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. The DNA also
may be modified, for example, by substituting the coding sequence
for human heavy and light chain constant domains in place of the
homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et
al, supra) or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an
antibody of the invention, or can be substituted for the variable
domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent antibody.
[0265] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0266] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0267] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0268] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples described herein. In a
non-limiting example, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC.RTM.. Hybridomas are
selected and cloned by limited dilution. The hybridoma clones are
then assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide of the invention.
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[0269] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0270] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0271] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phage used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0272] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties). Examples
of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);
Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science
240:1038-1040 (1988).
[0273] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in which different portions of the antibody are derived
from different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a human
immunoglobulin constant region. Methods for producing chimeric
antibodies are known in the art. See e.g., Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et
al., (1989) J. Immunol. Methods 125:191-202; Cabilly et al.,
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);
U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are
incorporated herein by reference in their entirety. Humanized
antibodies are antibody molecules from non-human species antibody
that binds the desired antigen having one or more complementarity
determining regions (CDRs) from the non-human species and a
framework regions from a human immunoglobulin molecule. Often,
framework residues in the human framework regions will be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which
are incorporated herein by reference in their entireties.)
Antibodies can be humanized using a variety of techniques known in
the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and
5,585,089), veneering or resurfacing (EP 592,106; EP 519,596;
Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS
91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source that is non-human. These non-human
amino acid residues are often referred to as "import" residues,
which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the methods of
Winter and co-workers (Jones et al., Nature, 321:522-525 (1986);
Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possible some FR residues are substituted from
analogous sites in rodent antibodies.
[0274] In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin (Jones et
al., Nature, 321:522-525 (1986); Riechmann et al., Nature
332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596
(1992).
[0275] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety. The techniques of Cole et al., and Boerder et al.,
are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Riss, (1985); and Boerner et al., J. Immunol.,
147(1):86-95, (1991)).
[0276] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entirety. In addition, companies such as
Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0277] Similarly, human antibodies can be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and creation of
an antibody repertoire. This approach is described, for example, in
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,106, and in the following scientific publications:
Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al.,
Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol.,
14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996);
Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).
[0278] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/Technology 12:899-903 (1988)).
[0279] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol.
147(8):2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to neutralize polypeptide ligand. For example, such
anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or to bind its ligands/receptors, and thereby block
its biological activity.
[0280] Such anti-idiotypic antibodies capable of binding to the
PCSK9b or PCSK9c polypeptide can be produced in a two-step
procedure. Such a method makes use of the fact that antibodies are
themselves antigens, and therefore, it is possible to obtain an
antibody that binds to a second antibody. In accordance with this
method, 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 that produce an antibody whose ability to bind to
the protein-specific antibody can be blocked by the polypeptide.
Such antibodies comprise anti-idiotypic antibodies to the
protein-specific antibody and can be used to immunize an animal to
induce formation of further protein-specific antibodies.
[0281] The antibodies of the present invention may be bispecific
antibodies. Bispecific antibodies are monoclonal, Preferably human
or humanized, antibodies that have binding specificities for at
least two different antigens. In the present invention, one of the
binding specificities may be directed towards a polypeptide of the
present invention, the other may be for any other antigen, and
preferably for a cell-surface protein, receptor, receptor subunit,
tissue-specific antigen, virally derived protein, virally encoded
envelope protein, bacterially derived protein, or bacterial surface
protein, etc.
[0282] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities (Milstein and Cuello, Nature, 305:537-539
(1983). Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0283] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transformed into a suitable
host organism. For further details of generating bispecific
antibodies see, for example Suresh et al., Meth. In Enzym., 121:210
(1986).
[0284] Heteroconjugate antibodies are also contemplated by the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for the treatment of HIV infection (WO
91/00360; WO 92/20373; and EP03089). It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioester bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
Polynucleotides Encoding Antibodies
[0285] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID
NO:4.
[0286] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0287] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0288] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0289] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0290] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0291] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
[0292] More preferably, a clone encoding an antibody of the present
invention may be obtained according to the method described in the
Example section herein.
Methods of Producing Antibodies
[0293] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0294] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0295] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[0296] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0297] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0298] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0299] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0300] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0301] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0302] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIBTECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0303] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0304] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0305] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0306] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452 (1991), which are incorporated by
reference in their entireties.
[0307] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms can be made by fusing the polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337-11341 (1992) (said references incorporated by
reference in their entireties).
[0308] As discussed, supra, the polypeptides corresponding to a
polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 or
SEQ ID NO:4 may be fused or conjugated to the above antibody
portions to increase the in vivo half life of the polypeptides or
for use in immunoassays using methods known in the art. Further,
the polypeptides corresponding to SEQ ID NO:2 or SEQ ID NO:4 may be
fused or conjugated to the above antibody portions to facilitate
purification. One reported example describes 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 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many
cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and thus can result in, for example, improved
pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
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, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0309] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, 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. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"flag" tag.
[0310] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include 125I, 131I, 111In or 99Tc.
[0311] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologues
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0312] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, B-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator, an
apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,
International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or
an anti-angiogenic agent, e.g., angiostatin or endostatin; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0313] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0314] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0315] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0316] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
[0317] The present invention also encompasses the creation of
synthetic antibodies directed against the polypeptides of the
present invention. One example of synthetic antibodies is described
in Radrizzani, M., et al., Medicina, (Aires), 59(6):753-8, (1999)).
Recently, a new class of synthetic antibodies has been described
and are referred to as molecularly imprinted polymers (MIPs)
(Semorex, Inc.). Antibodies, peptides, and enzymes are often used
as molecular recognition elements in chemical and biological
sensors. However, their lack of stability and signal transduction
mechanisms limits their use as sensing devices. Molecularly
imprinted polymers (MIPs) are capable of mimicking the function of
biological receptors but with less stability constraints. Such
polymers provide high sensitivity and selectivity while maintaining
excellent thermal and mechanical stability. MIPs have the ability
to bind to small molecules and to target molecules such as organics
and proteins' with equal or greater potency than that of natural
antibodies. These "super" MIPs have higher affinities for their
target and thus require lower concentrations for efficacious
binding.
[0318] During synthesis, the MIPs are imprinted so as to have
complementary size, shape, charge and functional groups of the
selected target by using the target molecule itself (such as a
polypeptide, antibody, etc.), or a substance having a very similar
structure, as its "print" or "template." MIPs can be derivatized
with the same reagents afforded to antibodies. For example,
fluorescent `super` MIPs can be coated onto beads or wells for use
in highly sensitive separations or assays, or for use in high
throughput screening of proteins.
[0319] Moreover, MIPs based upon the structure of the
polypeptide(s) of the present invention may be useful in screening
for compounds that bind to the polypeptide(s) of the invention.
Such a MIP would serve the role of a synthetic "receptor" by
minimicking the native architecture of the polypeptide. In fact,
the ability of a MIP to serve the role of a synthetic receptor has
already been demonstrated for the estrogen receptor (Ye, L., Yu,
Y., Mosbach, K, Analyst., 126(6):760-5, (2001); Dickert, F, L.,
Hayden, O., Halikias, K, P, Analyst., 126(6):766-71, (2001)). A
synthetic receptor may either be mimicked in its entirety (e.g., as
the entire protein), or mimicked as a series of short peptides
corresponding to the protein (Rachkov, A., Minoura, N, Biochim,
Biophys, Acta., 1544(1-2):255-66, (2001)). Such a synthetic
receptor MIPs may be employed in any one or more of the screening
methods described elsewhere herein.
[0320] MIPs have also been shown to be useful in "sensing" the
presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X,
Biosens, Bioelectron., 16(3):179-85, (2001); Jenkins, A. L., Yin,
R., Jensen, J. L, Analyst., 126(6):798-802, (2001); Jenkins, A, L.,
Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001)). For
example, a MIP designed using a polypeptide of the present
invention may be used in assays designed to identify, and
potentially quantitate, the level of said polypeptide in a sample.
Such a MIP may be used as a substitute for any component described
in the assays, or kits, provided herein (e.g., ELISA, etc.).
[0321] A number of methods may be employed to create MIPs to a
specific receptor, ligand, polypeptide, peptide, organic molecule.
Several preferred methods are described by Esteban et al in J.
Anal, Chem., 370(7):795-802, (2001), which is hereby incorporated
herein by reference in its entirety in addition to any references
cited therein. Additional methods are known in the art and are
encompassed by the present invention, such as for example, Hart, B,
R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072-3, (2001); and
Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren,
B, J. Am. Chem, Soc., 123(10):2146-54, (2001); which are hereby
incorporated by reference in their entirety herein.
Uses for Antibodies Directed Against Polypeptides of the
Invention
[0322] The antibodies of the present invention have various
utilities. For example, such antibodies may be used in diagnostic
assays to detect the presence or quantification of the polypeptides
of the invention in a sample. Such a diagnostic assay may be
comprised of at least two steps. The first, subjecting a sample
with the antibody, wherein the sample is a tissue (e.g., human,
animal, etc.), biological fluid (e.g., blood, urine, sputum, semen,
amniotic fluid, saliva, etc.), biological extract (e.g., tissue or
cellular homogenate, etc.), a protein microchip (e.g., See Arenkov
P, et al., Anal Biochem., 278(2):123-131 (2000)), or a
chromatography column, etc. And a second step involving the
quantification of antibody bound to the substrate. Alternatively,
the method may additionally involve a first step of attaching the
antibody, either covalently, electrostatically, or reversibly, to a
solid support, and a second step of subjecting the bound antibody
to the sample, as defined above and elsewhere herein.
[0323] Various diagnostic assay techniques are known in the art,
such as competitive binding assays, direct or indirect sandwich
assays and immunoprecipitation assays conducted in either
heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A
Manual of Techniques, CRC Press, Inc., (1987), pp. 147-158). The
antibodies used in the diagnostic assays can be labeled with a
detectable moiety. The detectable moiety should be capable of
producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety may be a radioisotope, such as 2H,
14C, 32P, or 125I, a florescent or chemiluminescent compound, such
as fluorescein isothiocyanate, rhodamine, or luciferin, or an
enzyme, such as alkaline phosphatase, beta-galactosidase, green
fluorescent protein, or horseradish peroxidase. Any method known in
the art for conjugating the antibody to the detectable moiety may
be employed, including those methods described by Hunter et al.,
Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974);
Pain et al., J. Immunol. Metho., 40:219 (1981); and Nygren, J.
Histochem. And Cytochem., 30:407 (1982).
[0324] Antibodies directed against the polypeptides of the present
invention are useful for the affinity purification of such
polypeptides from recombinant cell culture or natural sources. In
this process, the antibodies against a particular polypeptide are
immobilized on a suitable support, such as a SEPHADEX.RTM. resin or
filter paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample containing the
polypeptides to be purified, and thereafter the support is washed
with a suitable solvent that will remove substantially all the
material in the sample except for the desired polypeptides, which
are bound to the immobilized antibody. Finally, the support is
washed with another suitable solvent that will release the desired
polypeptide from the antibody.
Immunophenotyping
[0325] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[0326] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
Assays for Antibody Binding
[0327] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0328] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0329] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0330] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0331] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., 3H or 125I) in the presence of increasing amounts
of an unlabeled second antibody.
Therapeutic Uses of Antibodies
[0332] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein. The treatment and/or prevention of
diseases, disorders, or conditions associated with aberrant
expression and/or activity of a polypeptide of the invention
includes, but is not limited to, alleviating symptoms associated
with those diseases, disorders or conditions. Antibodies of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0333] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0334] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0335] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0336] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10-2 M, 10-2 M, 5.times.10-3 M,
10-3 M, 5.times.10-4 M, 10-4 M, 5.times.10-5 M, 10-5 M,
5.times.10-6 M, 10-6 M, 5.times.10-7 M, 10-7 M, 5.times.10-8 M,
10-8 M, 5.times.10-9 M, 10-9 M, 5.times.10-10 M, 10-10 M,
5.times.10-11 M, 10-11 M, 5.times.10-12 M, 10-12 M, 5.times.10-13
M, 10-13 M, 5.times.10-14 M, 10-14 M, 5.times.10-15 M, and 10-15
M.
[0337] Antibodies directed against polypeptides of the present
invention are useful for inhibiting allergic reactions in animals.
For example, by administering a therapeutically acceptable dose of
an antibody, or antibodies, of the present invention, or a cocktail
of the present antibodies, or in combination with other antibodies
of varying sources, the animal may not elicit an allergic response
to antigens.
[0338] Likewise, one could envision cloning the gene encoding an
antibody directed against a polypeptide of the present invention,
said polypeptide having the potential to elicit an allergic and/or
immune response in an organism, and transforming the organism with
said antibody gene such that it is expressed (e.g., constitutively,
inducibly, etc.) in the organism. Thus, the organism would
effectively become resistant to an allergic response resulting from
the ingestion or presence of such an immune/allergic reactive
polypeptide. Moreover, such a use of the antibodies of the present
invention may have particular utility in preventing and/or
ameliorating autoimmune diseases and/or disorders, as such
conditions are typically a result of antibodies being directed
against endogenous proteins. For example, in the instance where the
polypeptide of the present invention is responsible for modulating
the immune response to auto-antigens, transforming the organism
and/or individual with a construct comprising any of the promoters
disclosed herein or otherwise known in the art, in addition, to a
polynucleotide encoding the antibody directed against the
polypeptide of the present invention could effective inhibit the
organisms immune system from eliciting an immune response to the
auto-antigen(s). Detailed descriptions of therapeutic and/or gene
therapy applications of the present invention are provided
elsewhere herein.
[0339] Alternatively, antibodies of the present invention could be
produced in a plant (e.g., cloning the gene of the antibody
directed against a polypeptide of the present invention, and
transforming a plant with a suitable vector comprising said gene
for constitutive expression of the antibody within the plant), and
the plant subsequently ingested by an animal, thereby conferring
temporary immunity to the animal for the specific antigen the
antibody is directed towards (See, for example, U.S. Pat. Nos.
5,914,123 and 6,034,298).
[0340] In another embodiment, antibodies of the present invention,
preferably polyclonal antibodies, more preferably monoclonal
antibodies, and most preferably single-chain antibodies, can be
used as a means of inhibiting gene expression of a particular gene,
or genes, in a human, mammal, and/or other organism. See, for
example, International Publication Number WO 00/05391, published
Feb. 3, 2000, to Dow Agrosciences LLC. The application of such
methods for the antibodies of the present invention are known in
the art, and are more particularly described elsewhere herein.
[0341] In yet another embodiment, antibodies of the present
invention may be useful for multimerizing the polypeptides of the
present invention. For example, certain proteins may confer
enhanced biological activity when present in a multimeric state
(i.e., such enhanced activity may be due to the increased effective
concentration of such proteins whereby more protein is available in
a localized location).
Antibody-Based Gene Therapy
[0342] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0343] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0344] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0345] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989). In specific embodiments, the
expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences
encoding both the heavy and light chains, or fragments thereof, of
the antibody.
[0346] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0347] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0348] In a specific embodiment, viral vectors that contains
nucleic acid sequences encoding an antibody of the invention are
used. For example, a retroviral vector can be used (see Miller et
al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors
contain the components necessary for the correct packaging of the
viral genome and integration into the host cell DNA. The nucleic
acid sequences encoding the antibody to be used in gene therapy are
cloned into one or more vectors, which facilitates delivery of the
gene into a patient. More detail about retroviral vectors can be
found in Boesen et al., Biotherapy 6:291-302 (1994), which
describes the use of a retroviral vector to deliver the mdr1 gene
to hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin.
Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114
(1993).
[0349] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0350] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0351] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0352] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0353] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0354] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as Tlymphocytes, Blymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0355] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0356] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0357] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[0358] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
Therapeutic/Prophylactic Administration and Compositions
[0359] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention,
preferably an antibody of the invention. In a preferred aspect, the
compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0360] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0361] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0362] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0363] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein,
ibid., pp. 317-327; see generally ibid.)
[0364] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)).
[0365] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0366] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0367] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0368] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0369] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0370] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0371] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0372] 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.
Optionally 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.
Diagnosis and Imaging with Antibodies
[0373] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases,
disorders, and/or conditions associated with the aberrant
expression and/or activity of a polypeptide of the invention. The
invention provides for the detection of aberrant expression of a
polypeptide of interest, comprising (a) assaying the expression of
the polypeptide of interest in cells or body fluid of an individual
using one or more antibodies specific to the polypeptide interest;
and (b) comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0374] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest; and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0375] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting 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;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0376] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of interest in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: (a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
(b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); (c)
determining background level; and (d) detecting the labeled
molecule in the subject, such that detection of labeled molecule
above the background level indicates that the subject has a
particular disease or disorder associated with aberrant expression
of the polypeptide of interest. Background level can be determined
by various methods including, comparing the amount of labeled
molecule detected to a standard value previously determined for a
particular system.
[0377] 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 99mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific 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).
[0378] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0379] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0380] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0381] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
Kits
[0382] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0383] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0384] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0385] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0386] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0387] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0388] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
Fusion Proteins
[0389] Any polypeptide of the present invention can be used to
generate fusion proteins. For example, the polypeptide of the
present invention, when fused to a second protein, can be used as
an antigenic tag. Antibodies raised against the polypeptide of the
present invention can be used to indirectly detect the second
protein by binding to the polypeptide. Moreover, because certain
proteins target cellular locations based on trafficking signals,
the polypeptides of the present invention can be used as targeting
molecules once fused to other proteins.
[0390] Examples of domains that can be fused to polypeptides of the
present invention include not only heterologous signal sequences,
but also other heterologous functional regions. The fusion does not
necessarily need to be direct, but may occur through linker
sequences.
[0391] Moreover, fusion proteins may also be engineered to improve
characteristics of the polypeptide of the present invention. 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 during purification from the host
cell or subsequent handling and storage. Peptide moieties may be
added to the polypeptide to facilitate purification. Such regions
may be removed prior to final preparation of the polypeptide.
Similarly, peptide cleavage sites can be introduced in-between such
peptide moieties, which could additionally be subjected to protease
activity to remove said peptide(s) from the protein of the present
invention. The addition of peptide moieties, including peptide
cleavage sites, to facilitate handling of polypeptides are familiar
and routine techniques in the art.
[0392] Moreover, polypeptides of the present invention, including
fragments, and specifically epitopes, can be combined with parts of
the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or
portions thereof (CH1, CH2, CH3, and any combination thereof,
including both entire domains and portions thereof), resulting in
chimeric polypeptides. These fusion proteins facilitate
purification and show an increased half-life in vivo. One reported
example describes 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 having disulfide-linked dimeric structures
(due to the IgG) can also be more efficient in binding and
neutralizing other molecules, than the monomeric secreted protein
or protein fragment alone. (Fountoulakis et al., J. Biochem.
270:3958-3964 (1995).)
[0393] Similarly, EP-A 0 464 533 (Canadian counterpart 2,045,869)
discloses fusion proteins comprising various portions of the
constant region of immunoglobulin molecules together with another
human protein or part thereof. In many cases, the Fc part in a
fusion protein is beneficial in therapy and diagnosis, and thus can
result in, for example, improved pharmacokinetic properties. (EP-A
0 232 262.) Alternatively, deleting the Fc part after the fusion
protein has been expressed, detected, and purified, would be
desired. For example, the Fc portion may hinder therapy and
diagnosis if the fusion protein is used as an 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);
K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
[0394] Moreover, the polypeptides of the present invention can be
fused to marker sequences (also referred to as "tags"). Due to the
availability of antibodies specific to such "tags", purification of
the fused polypeptide of the invention, and/or its identification
is significantly facilitated since antibodies specific to the
polypeptides of the invention are not required. Such purification
may be in the form of an affinity purification whereby an anti-tag
antibody or another type of affinity matrix (e.g., anti-tag
antibody attached to the matrix of a flow-thru column) that binds
to the epitope tag is present. In preferred embodiments, 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. Another
peptide tag useful for purification, the "HA" tag, corresponds to
an epitope derived from the influenza hemagglutinin protein.
(Wilson et al., Cell 37:767 (1984)).
[0395] The skilled artisan would acknowledge the existence of other
"tags" which could be readily substituted for the tags referred to
supra for purification and/or identification of polypeptides of the
present invention (Jones C., et al., J Chromatogr A. 707(1):3-22
(1995)). For example, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7
and 9E10 antibodies thereto (Evan et al., Molecular and Cellular
Biology 5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein
D (gD) tag and its antibody (Paborsky et al., Protein Engineering,
3(6):547-553 (1990), the FLAG.RTM.-peptide--i.e., the octapeptide
sequence DYKDDDDK (SEQ ID NO:56), (Hopp et al., Biotech.
6:1204-1210 (1988); the KT3 epitope peptide (Martin et al.,
Science, 255:192-194 (1992)); a-tubulin epitope peptide (Skinner et
al., J. Biol. Chem., 266:15136-15166, (1991)); the T7 gene 10
protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA,
87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP
epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).
[0396] The present invention also encompasses the attachment of up
to nine codons encoding a repeating series of up to nine arginine
amino acids to the coding region of a polynucleotide of the present
invention. The invention also encompasses chemically derivitizing a
polypeptide of the present invention with a repeating series of up
to nine arginine amino acids. Such a tag, when attached to a
polypeptide, has recently been shown to serve as a universal pass,
allowing compounds access to the interior of cells without
additional derivitization or manipulation (Wender, P., et al.,
unpublished data).
[0397] Protein fusions involving polypeptides of the present
invention, including fragments and/or variants thereof, can be used
for the following, non-limiting examples, subcellular localization
of proteins, determination of protein-protein interactions via
immunoprecipitation, purification of proteins via affinity
chromatography, functional and/or structural characterization of
protein. The present invention also encompasses the application of
hapten specific antibodies for any of the uses referenced above for
epitope fusion proteins. For example, the polypeptides of the
present invention could be chemically derivatized to attach hapten
molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of
monoclonal antibodies specific to such haptens, the protein could
be readily purified using immunoprecipitation, for example.
[0398] Polypeptides of the present invention, including fragments
and/or variants thereof, in addition to, antibodies directed
against such polypeptides, fragments, and/or variants, may be fused
to any of a number of known, and yet to be determined, toxins, such
as ricin, saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999;
886:233-5), or HC toxin (Tonukari N J, et al., Plant Cell. 2000
February; 12(2):237-248), for example. Such fusions could be used
to deliver the toxins to desired tissues for which a ligand or a
protein capable of binding to the polypeptides of the invention
exists.
[0399] The invention encompasses the fusion of antibodies directed
against polypeptides of the present invention, including variants
and fragments thereof, to said toxins for delivering the toxin to
specific locations in a cell, to specific tissues, and/or to
specific species. Such bifunctional antibodies are known in the
art, though a review describing additional advantageous fusions,
including citations for methods of production, can be found in P.
J. Hudson, Cuff. Opp. In. Imm. 11:548-557, (1999); this
publication, in addition to the references cited therein, are
hereby incorporated by reference in their entirety herein. In this
context, the term "toxin" may be expanded to include any
heterologous protein, a small molecule, radionucleotides, cytotoxic
drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell
or tissue-specific ligands, enzymes, of bioactive agents,
biological response modifiers, anti-fungal agents, hormones,
steroids, vitamins, peptides, peptide analogs, anti-allergenic
agents, anti-tubercular agents, anti-viral agents, antibiotics,
anti-protozoan agents, chelates, radioactive particles, radioactive
ions, X-ray contrast agents, monoclonal antibodies, polyclonal
antibodies and genetic material. In view of the present disclosure,
one skilled in the art could determine whether any particular
"toxin" could be used in the compounds of the present invention.
Examples of suitable "toxins" listed above are exemplary only and
are not intended to limit the "toxins" that may be used in the
present invention.
[0400] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
Vectors, Host Cells, and Protein Production
[0401] The present invention also relates to vectors containing the
polynucleotide of the present invention, host cells, and the
production of polypeptides 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.
[0402] 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.
[0403] The polynucleotide 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.
[0404] 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 (e.g., Saccharomyces cerevisiae
or Pichia pastoris (ATCC.RTM. Accession No. 201178)); 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.
[0405] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; PBLUESCRIPT.RTM.
vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,
pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and
pSVL available from Pharmacia. Preferred expression vectors for use
in yeast systems include, but are not limited to pYES2, pYD1,
pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from
Invitrogen, Carlsbad, Calif.). Other suitable vectors will be
readily apparent to the skilled artisan.
[0406] 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). It is
specifically contemplated that the polypeptides of the present
invention may in fact be expressed by a host cell lacking a
recombinant vector.
[0407] A polypeptide of this invention 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.
[0408] Polypeptides of the present invention, and preferably the
secreted form, can also be recovered from: 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.
[0409] In one embodiment, the yeast Pichia pastoris is used to
express the polypeptide of the present invention in a eukaryotic
system. Pichia pastoris is a methylotrophic yeast which can
metabolize methanol as its sole carbon source. A main step in the
methanol metabolization pathway is the oxidation of methanol to
formaldehyde using O2. This reaction is catalyzed by the enzyme
alcohol oxidase. In order to metabolize methanol as its sole carbon
source, Pichia pastoris must generate high levels of alcohol
oxidase due, in part, to the relatively low affinity of alcohol
oxidase for O2. Consequently, in a growth medium depending on
methanol as a main carbon source, the promoter region of one of the
two alcohol oxidase genes (AOX1) is highly active. In the presence
of methanol, alcohol oxidase produced from the AOX1 gene comprises
up to approximately 30% of the total soluble protein in Pichia
pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21
(1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F.,
et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous
coding sequence, such as, for example, a polynucleotide of the
present invention, under the transcriptional regulation of all or
part of the AOX1 regulatory sequence is expressed at exceptionally
high levels in Pichia yeast grown in the presence of methanol.
[0410] In one example, the plasmid vector pPIC9K is used to express
DNA encoding a polypeptide of the invention, as set forth herein,
in a Pichea yeast system essentially as described in "Pichia
Protocols: Methods in Molecular Biology" D. R. Higgins and J.
Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression
vector allows expression and secretion of a protein of the
invention by virtue of the strong AOX1 promoter linked to the
Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide
(i.e., leader) located upstream of a multiple cloning site.
[0411] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815,
as one skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG, as required.
[0412] In another embodiment, high-level expression of a
heterologous coding sequence, such as, for example, a
polynucleotide of the present invention, may be achieved by cloning
the heterologous polynucleotide of the invention into an expression
vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
[0413] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide sequences) that is operably associated with the
polynucleotides of the invention, and which activates, alters,
and/or amplifies endogenous polynucleotides. For example,
techniques known in the art may be used to operably associate
heterologous control regions (e.g., promoter and/or enhancer) and
endogenous polynucleotide sequences via homologous recombination,
resulting in the formation of a new transcription unit (see, e.g.,
U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No.
5,733,761, issued Mar. 31, 1998; International Publication No. WO
96/29411, published Sep. 26, 1996; International Publication No. WO
94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad.
Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989), the disclosures of each of which are
incorporated by reference in their entireties).
[0414] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller et al., Nature, 310:105-111
(1984)). For example, a polypeptide corresponding to a fragment of
a polypeptide sequence of the invention can be synthesized by use
of a peptide synthesizer. Furthermore, if desired, nonclassical
amino acids or chemical amino acid analogs can be introduced as a
substitution or addition into the polypeptide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4-diaminobutyric acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino
acids such as b-methyl amino acids, Ca-methyl amino acids,
Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0415] The invention encompasses polypeptides which are
differentially modified during or after translation, e.g., by
glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic synthesis in the presence of tunicamycin; etc.
[0416] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of prokaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein, the addition of epitope tagged peptide
fragments (e.g., FLAG.RTM., HA, GST, thioredoxin, maltose binding
protein, etc.), attachment of affinity tags such as biotin and/or
streptavidin, the covalent attachment of chemical moieties to the
amino acid backbone, N- or C-terminal processing of the
polypeptides ends (e.g., proteolytic processing), deletion of the
N-terminal methionine residue, etc.
[0417] Also provided by the invention are chemically modified
derivatives of the polypeptides of the invention which may provide
additional advantages such as increased solubility, stability and
circulating time of the polypeptide, or decreased immunogenicity
(see U.S. Pat. No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0418] The invention further encompasses chemical derivitization of
the polypeptides of the present invention, preferably where the
chemical is a hydrophilic polymer residue. Exemplary hydrophilic
polymers, including derivatives, may be those that include polymers
in which the repeating units contain one or more hydroxy groups
(polyhydroxy polymers), including, for example, poly(vinyl
alcohol); polymers in which the repeating units contain one or more
amino groups (polyamine polymers), including, for example,
peptides, polypeptides, proteins and lipoproteins, such as albumin
and natural lipoproteins; polymers in which the repeating units
contain one or more carboxy groups (polycarboxy polymers),
including, for example, carboxymethylcellulose, alginic acid and
salts thereof, such as sodium and calcium alginate,
glycosaminoglycans and salts thereof, including salts of hyaluronic
acid, phosphorylated and sulfonated derivatives of carbohydrates,
genetic material, such as interleukin-2 and interferon, and
phosphorothioate oligomers; and polymers in which the repeating
units contain one or more saccharide moieties (polysaccharide
polymers), including, for example, carbohydrates.
[0419] The molecular weight of the hydrophilic polymers may vary,
and is generally about 50 to about 5,000,000, with polymers having
a molecular weight of about 100 to about 50,000 being preferred.
The polymers may be branched or unbranched. More preferred polymers
have a molecular weight of about 150 to about 10,000, with
molecular weights of 200 to about 8,000 being even more
preferred.
[0420] For polyethylene glycol, the preferred molecular weight is
between about 1 kDa and about 100 kDa (the term "about" indicating
that in preparations of polyethylene glycol, some molecules will
weigh more, some less, than the stated molecular weight) for ease
in handling and manufacturing. Other sizes may be used, depending
on the desired therapeutic profile (e.g., the duration of sustained
release desired, the effects, if any on biological activity, the
ease in handling, the degree or lack of antigenicity and other
known effects of the polyethylene glycol to a therapeutic protein
or analog).
[0421] Additional preferred polymers which may be used to
derivatize polypeptides of the invention, include, for example,
poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers,
polysorbate and poly(vinyl alcohol), with PEG polymers being
particularly preferred. Preferred among the PEG polymers are PEG
polymers having a molecular weight of from about 100 to about
10,000. More preferably, the PEG polymers have a molecular weight
of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG
8,000, which have molecular weights of 2,000, 5,000 and 8,000,
respectively, being even more preferred. Other suitable hydrophilic
polymers, in addition to those exemplified above, will be readily
apparent to one skilled in the art based on the present disclosure.
Generally, the polymers used may include polymers that can be
attached to the polypeptides of the invention via alkylation or
acylation reactions.
[0422] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0423] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation
reaction to be performed, and the method of obtaining the selected
N-terminally pegylated protein. The method of obtaining the
N-terminally pegylated preparation (i.e., separating this moiety
from other monopegylated moieties if necessary) may be by
purification of the N-terminally pegylated material from a
population of pegylated protein molecules. Selective proteins
chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminus) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N-terminus with a
carbonyl group containing polymer is achieved.
[0424] As with the various polymers exemplified above, it is
contemplated that the polymeric residues may contain functional
groups in addition, for example, to those typically involved in
linking the polymeric residues to the polypeptides of the present
invention. Such functionalities include, for example, carboxyl,
amine, hydroxy and thiol groups. These functional groups on the
polymeric residues can be further reacted, if desired, with
materials that are generally reactive with such functional groups
and which can assist in targeting specific tissues in the body
including, for example, diseased tissue. Exemplary materials which
can be reacted with the additional functional groups include, for
example, proteins, including antibodies, carbohydrates, peptides,
glycopeptides, glycolipids, lectins, and nucleosides.
[0425] In addition to residues of hydrophilic polymers, the
chemical used to derivatize the polypeptides of the present
invention can be a saccharide residue. Exemplary saccharides which
can be derived include, for example, monosaccharides or sugar
alcohols, such as erythrose, threose, ribose, arabinose, xylose,
lyxose, fructose, sorbitol, mannitol and sedoheptulose, with
preferred monosaccharides being fructose, mannose, xylose,
arabinose, mannitol and sorbitol; and disaccharides, such as
lactose, sucrose, maltose and cellobiose. Other saccharides
include, for example, inositol and ganglioside head groups. Other
suitable saccharides, in addition to those exemplified above, will
be readily apparent to one skilled in the art based on the present
disclosure. Generally, saccharides which may be used for
derivitization include saccharides that can be attached to the
polypeptides of the invention via alkylation or acylation
reactions.
[0426] Moreover, the invention also encompasses derivitization of
the polypeptides of the present invention, for example, with lipids
(including cationic, anionic, polymerized, charged, synthetic,
saturated, unsaturated, and any combination of the above, etc.).
stabilizing agents.
[0427] The invention encompasses derivitization of the polypeptides
of the present invention, for example, with compounds that may
serve a stabilizing function (e.g., to increase the polypeptides
half-life in solution, to make the polypeptides more water soluble,
to increase the polypeptides hydrophilic or hydrophobic character,
etc.). Polymers useful as stabilizing materials may be of natural,
semi-synthetic (modified natural) or synthetic origin. Exemplary
natural polymers include naturally occurring polysaccharides, such
as, for example, arabinans, fructans, fucans, galactans,
galacturonans, glucans, mannans, xylans (such as, for example,
inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid,
pectins, including amylose, pullulan, glycogen, amylopectin,
cellulose, dextran, dextrin, dextrose, glucose, polyglucose,
polydextrose, pustulan, chitin, agarose, keratin, chondroitin,
dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and
various other natural homopolymer or heteropolymers, such as those
containing one or more of the following aldoses, ketoses, acids or
amines: erythose, threose, ribose, arabinose, xylose, lyxose,
allose, altrose, glucose, dextrose, mannose, gulose, idose,
galactose, talose, erythrulose, ribulose, xylulose, psicose,
fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose,
trehalose, maltose, cellobiose, glycine, serine, threonine,
cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic
acid, lysine, arginine, histidine, glucuronic acid, gluconic acid,
glucaric acid, galacturonic acid, mannuronic acid, glucosamine,
galactosamine, and neuraminic acid, and naturally occurring
derivatives thereof. Accordingly, suitable polymers include, for
example, proteins, such as albumin, polyalginates, and
polylactide-coglycolide polymers. Exemplary semi-synthetic polymers
include carboxymethylcellulose, hydroxymethylcellulose,
hydroxypropylmethylcellulose, methylcellulose, and
methoxycellulose. Exemplary synthetic polymers include
polyphosphazenes, hydroxyapatites, fluoroapatite polymers,
polyethylenes (such as, for example, polyethylene glycol (including
for example, the class of compounds referred to as PLURONIC.RTM.,
commercially available from BASF, Parsippany, N.J.),
polyoxyethylene, and polyethylene terephthlate), polypropylenes
(such as, for example, polypropylene glycol), polyurethanes (such
as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and
polyvinylpyrrolidone), polyamides including nylon, polystyrene,
polylactic acids, fluorinated hydrocarbon polymers, fluorinated
carbon polymers (such as, for example, polytetrafluoroethylene),
acrylate, methacrylate, and polymethylmethacrylate, and derivatives
thereof. Methods for the preparation of derivatized polypeptides of
the invention which employ polymers as stabilizing compounds will
be readily apparent to one skilled in the art, in view of the
present disclosure, when coupled with information known in the art,
such as that described and referred to in Unger, U.S. Pat. No.
5,205,290, the disclosure of which is hereby incorporated by
reference herein in its entirety.
[0428] Moreover, the invention encompasses additional modifications
of the polypeptides of the present invention. Such additional
modifications are known in the art, and are specifically provided,
in addition to methods of derivitization, etc., in U.S. Pat. No.
6,028,066, which is hereby incorporated in its entirety herein.
[0429] The polypeptides of the invention may be in monomers or
multimers (i.e., dimers, trimers, tetramers and higher multimers).
Accordingly, the present invention relates to monomers and
multimers of the polypeptides of the invention, their preparation,
and compositions (preferably, Therapeutics) containing them. In
specific embodiments, the polypeptides of the invention are
monomers, dimers, trimers or tetramers. In additional embodiments,
the multimers of the invention are at least dimers, at least
trimers, or at least tetramers.
[0430] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only polypeptides corresponding to the amino acid
sequence of SEQ ID NO:Y or encoded by the cDNA contained in a
deposited clone (including fragments, variants, splice variants,
and fusion proteins, corresponding to these polypeptides as
described herein). These homomers may contain polypeptides having
identical or different amino acid sequences. In a specific
embodiment, a homomer of the invention is a multimer containing
only polypeptides having an identical amino acid sequence. In
another specific embodiment, a homomer of the invention is a
multimer containing polypeptides having different amino acid
sequences. In specific embodiments, the multimer of the invention
is a homodimer (e.g., containing polypeptides having identical or
different amino acid sequences) or a homotrimer (e.g., containing
polypeptides having identical and/or different amino acid
sequences). In additional embodiments, the homomeric multimer of
the invention is at least a homodimer, at least a homotrimer, or at
least a homotetramer.
[0431] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the polypeptides
of the invention. In a specific embodiment, the multimer of the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In
additional embodiments, the heteromeric multimer of the invention
is at least a heterodimer, at least a heterotrimer, or at least a
heterotetramer.
[0432] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the polypeptides of the
invention. Such covalent associations may involve one or more amino
acid residues contained in the polypeptide sequence (e.g., that
recited in the sequence listing, or contained in the polypeptide
encoded by a deposited clone). In one instance, the covalent
associations are cross-linking between cysteine residues located
within the polypeptide sequences which interact in the native
(i.e., naturally occurring) polypeptide. In another instance, the
covalent associations are the consequence of chemical or
recombinant manipulation. Alternatively, such covalent associations
may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the
invention.
[0433] In one example, covalent associations are between the
heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are between the heterologous
sequence contained in an Fc fusion protein of the invention (as
described herein). In another specific example, covalent
associations of fusion proteins of the invention are between
heterologous polypeptide sequence from another protein that is
capable of forming covalently associated multimers, such as for
example, osteoprotegerin (see, e.g., International Publication
NO:WO 98/49305, the contents of which are herein incorporated by
reference in its entirety). In another embodiment, two or more
polypeptides of the invention are joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising
multiple polypeptides of the invention separated by peptide linkers
may be produced using conventional recombinant DNA technology.
[0434] Another method for preparing multimer polypeptides of the
invention involves use of polypeptides of the invention fused to a
leucine zipper or isoleucine zipper polypeptide sequence. Leucine
zipper and isoleucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins
(Landschulz et al., Science 240:1759, (1988)), and have since been
found in a variety of different proteins. Among the known leucine
zippers are naturally occurring peptides and derivatives thereof
that dimerize or trimerize. Examples of leucine zipper domains
suitable for producing soluble multimeric proteins of the invention
are those described in PCT application WO 94/10308, hereby
incorporated by reference. Recombinant fusion proteins comprising a
polypeptide of the invention fused to a polypeptide sequence that
dimerizes or trimerizes in solution are expressed in suitable host
cells, and the resulting soluble multimeric fusion protein is
recovered from the culture supernatant using techniques known in
the art.
[0435] Trimeric polypeptides of the invention may offer the
advantage of enhanced biological activity. Preferred leucine zipper
moieties and isoleucine moieties are those that preferentially form
trimers. One example is a leucine zipper derived from lung
surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters 344:191, (1994)) and in U.S. patent application Ser. No.
08/446,922, hereby incorporated by reference. Other peptides
derived from naturally occurring trimeric proteins may be employed
in preparing trimeric polypeptides of the invention.
[0436] In another example, proteins of the invention are associated
by interactions between FLAG.RTM. polypeptide sequence contained in
fusion proteins of the invention containing FLAG.RTM. polypeptide
sequence. In a further embodiment, associations proteins of the
invention are associated by interactions between heterologous
polypeptide sequence contained in FLAG.RTM. fusion proteins of the
invention and anti-FLAG.RTM. antibody.
[0437] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0438] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain (or hydrophobic or signal peptide) and which
can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety).
[0439] In addition, the polynucleotide insert of the present
invention could be operatively linked to "artificial" or chimeric
promoters and transcription factors. Specifically, the artificial
promoter could comprise, or alternatively consist, of any
combination of cis-acting DNA sequence elements that are recognized
by trans-acting transcription factors. Preferably, the cis acting
DNA sequence elements and trans-acting transcription factors are
operable in mammals. Further, the trans-acting transcription
factors of such "artificial" promoters could also be "artificial"
or chimeric in design themselves and could act as activators or
repressors to said "artificial" promoter.
Uses of the Polynucleotides
[0440] Each of the polynucleotides identified herein can be used in
numerous ways as reagents. The following description should be
considered exemplary and utilizes known techniques.
[0441] The polynucleotides of the present invention are useful for
chromosome identification. There exists an ongoing need to identify
new chromosome markers, since few chromosome marking reagents,
based on actual sequence data (repeat polymorphisms), are presently
available. Each polynucleotide of the present invention can be used
as a chromosome marker.
[0442] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the sequences shown in SEQ
ID NO:X. Primers can be selected using computer analysis so that
primers do not span more than one predicted exon in the genomic
DNA. These primers are then used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the SEQ ID NO:X will
yield an amplified fragment.
[0443] Similarly, somatic hybrids provide a rapid method of PCR
mapping the polynucleotides to particular chromosomes. Three or
more clones can be assigned per day using a single thermal cycler.
Moreover, sublocalization of the polynucleotides can be achieved
with panels of specific chromosome fragments. Other gene mapping
strategies that can be used include in situ hybridization,
prescreening with labeled flow-sorted chromosomes, and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0444] Precise chromosomal location of the polynucleotides can also
be achieved using fluorescence in situ hybridization (FISH) of a
metaphase chromosomal spread. This technique uses polynucleotides
as short as 500 or 600 bases; however, polynucleotides 2,000-4,000
bp are preferred. For a review of this technique, see Verma et al.,
"Human Chromosomes: a Manual of Basic Techniques" Pergamon Press,
New York (1988).
[0445] For chromosome mapping, the polynucleotides can be used
individually (to mark a single chromosome or a single site on that
chromosome) or in panels (for marking multiple sites and/or
multiple chromosomes). Preferred polynucleotides correspond to the
noncoding regions of the cDNAs because the coding sequences are
more likely conserved within gene families, thus increasing the
chance of cross hybridization during chromosomal mapping.
[0446] Once a polynucleotide has been mapped to a precise
chromosomal location, the physical position of the polynucleotide
can be used in linkage analysis. Linkage analysis establishes
coinheritance between a chromosomal location and presentation of a
particular disease. Disease mapping data are known in the art.
Assuming 1 megabase mapping resolution and one gene per 20 kb, a
cDNA precisely localized to a chromosomal region associated with
the disease could be one of 50-500 potential causative genes.
[0447] Thus, once coinheritance is established, differences in the
polynucleotide and the corresponding gene between affected and
unaffected organisms can be examined. First, visible structural
alterations in the chromosomes, such as deletions or
translocations, are examined in chromosome spreads or by PCR. If no
structural alterations exist, the presence of point mutations are
ascertained. Mutations observed in some or all affected organisms,
but not in normal organisms, indicates that the mutation may cause
the disease. However, complete sequencing of the polypeptide and
the corresponding gene from several normal organisms is required to
distinguish the mutation from a polymorphism. If a new polymorphism
is identified, this polymorphic polypeptide can be used for further
linkage analysis.
[0448] Furthermore, increased or decreased expression of the gene
in affected organisms as compared to unaffected organisms can be
assessed using polynucleotides of the present invention. Any of
these alterations (altered expression, chromosomal rearrangement,
or mutation) can be used as a diagnostic or prognostic marker.
[0449] Thus, the invention also provides a diagnostic method useful
during diagnosis of a disorder, involving measuring the expression
level of polynucleotides of the present invention in cells or body
fluid from an organism and comparing the measured gene expression
level with a standard level of polynucleotide expression level,
whereby an increase or decrease in the gene expression level
compared to the standard is indicative of a disorder.
[0450] By "measuring the expression level of a polynucleotide of
the present invention" is intended qualitatively or quantitatively
measuring or estimating the level of the polypeptide of the present
invention or the level of the mRNA encoding the polypeptide 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 polypeptide level or mRNA level in a
second biological sample). Preferably, the polypeptide level or
mRNA level in the first biological sample is measured or estimated
and compared to a standard polypeptide 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 organisms not having a
disorder. As will be appreciated in the art, once a standard
polypeptide level or mRNA level is known, it can be used repeatedly
as a standard for comparison.
[0451] By "biological sample" is intended any biological sample
obtained from an organism, body fluids, cell line, tissue culture,
or other source which contains the polypeptide of the present
invention or mRNA. As indicated, biological samples include body
fluids (such as the following non-limiting examples, sputum,
amniotic fluid, urine, saliva, breast milk, secretions,
interstitial fluid, blood, serum, spinal fluid, etc.) which contain
the polypeptide of the present invention, and other tissue sources
found to express the polypeptide of the present invention. Methods
for obtaining tissue biopsies and body fluids from organisms are
well known in the art. Where the biological sample is to include
mRNA, a tissue biopsy is the preferred source.
[0452] The method(s) provided above may preferably be applied in a
diagnostic method and/or kits in which polynucleotides and/or
polypeptides are attached to a solid support. In one exemplary
method, the support may be a "gene chip" or a "biological chip" as
described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174.
Further, such a gene chip with polynucleotides of the present
invention attached may be used to identify polymorphisms between
the polynucleotide sequences, with polynucleotides isolated from a
test subject. The knowledge of such polymorphisms (i.e. their
location, as well as, their existence) would be beneficial in
identifying disease loci for many disorders, including
proliferative diseases and conditions. Such a method is described
in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents
referenced supra are hereby incorporated by reference in their
entirety herein.
[0453] The present invention encompasses polynucleotides of the
present invention that are chemically synthesized, or reproduced as
peptide nucleic acids (PNA), or according to other methods known in
the art. The use of PNAs would serve as the preferred form if the
polynucleotides are incorporated onto a solid support, or gene
chip. For the purposes of the present invention, a peptide nucleic
acid (PNA) is a polyamide type of DNA analog and the monomeric
units for adenine, guanine, thymine and cytosine are available
commercially (Perceptive Biosystems). Certain components of DNA,
such as phosphorus, phosphorus oxides, or deoxyribose derivatives,
are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm,
R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.
Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D.
A. Driver, R. H. Berg, S. K. Kim, B Norden, and P. E. Nielsen,
Nature 365, 666 (1993), PNAs bind specifically and tightly to
complementary DNA strands and are not degraded by nucleases. In
fact, PNA binds more strongly to DNA than DNA itself does. This is
probably because there is no electrostatic repulsion between the
two strands, and also the polyamide backbone is more flexible.
Because of this, PNA/DNA duplexes bind under a wider range of
stringency conditions than DNA/DNA duplexes, making it easier to
perform multiplex hybridization. Smaller probes can be used than
with DNA due to the stronger binding characteristics of PNA:DNA
hybrids. In addition, it is more likely that single base mismatches
can be determined with PNA/DNA hybridization because a single
mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by
8.degree.-20.degree. C., vs. 4.degree.-16.degree. C. for the
DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA
means that hybridization can be done at low ionic strengths and
reduce possible interference by salt during the analysis.
[0454] In addition to the foregoing, a polynucleotide can be used
to control gene expression through triple helix formation or
antisense DNA or RNA. 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). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques,
preferred polynucleotides are usually oligonucleotides 20 to 40
bases in length and complementary to either the region of the gene
involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988);
and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself
(antisense--Okano, J. Neurochem. 56:560 (1991);
Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988).) Triple helix formation
optimally results in a shut-off of RNA transcription from DNA,
while antisense RNA hybridization blocks translation of an mRNA
molecule into polypeptide. Both techniques are effective in model
systems, and the information disclosed herein can be used to design
antisense or triple helix polynucleotides in an effort to treat or
prevent disease.
[0455] The present invention encompasses the addition of a nuclear
localization signal, operably linked to the 5' end, 3' end, or any
location therein, to any of the oligonucleotides, antisense
oligonucleotides, triple helix oligonucleotides, ribozymes, PNA
oligonucleotides, and/or polynucleotides, of the present invention.
See, for example, G. Cutrona, et al., Nat. Biotech., 18:300-303,
(2000); which is hereby incorporated herein by reference.
[0456] Polynucleotides of the present invention are also useful in
gene therapy. One goal of gene therapy is to insert a normal gene
into an organism having a defective gene, in an effort to correct
the genetic defect. The polynucleotides disclosed in the present
invention offer a means of targeting such genetic defects in a
highly accurate manner. Another goal is to insert a new gene that
was not present in the host genome, thereby producing a new trait
in the host cell. In one example, polynucleotide sequences of the
present invention may be used to construct chimeric RNA/DNA
oligonucleotides corresponding to said sequences, specifically
designed to induce host cell mismatch repair mechanisms in an
organism upon systemic injection, for example (Bartlett, R. J., et
al., Nat. Biotech, 18:615-622 (2000), which is hereby incorporated
by reference herein in its entirety). Such RNA/DNA oligonucleotides
could be designed to correct genetic defects in certain host
strains, and/or to introduce desired phenotypes in the host (e.g.,
introduction of a specific polymorphism within an endogenous gene
corresponding to a polynucleotide of the present invention that may
ameliorate and/or prevent a disease symptom and/or disorder, etc.).
Alternatively, the polynucleotide sequence of the present invention
may be used to construct duplex oligonucleotides corresponding to
said sequence, specifically designed to correct genetic defects in
certain host strains, and/or to introduce desired phenotypes into
the host (e.g., introduction of a specific polymorphism within an
endogenous gene corresponding to a polynucleotide of the present
invention that may ameliorate and/or prevent a disease symptom
and/or disorder, etc). Such methods of using duplex
oligonucleotides are known in the art and are encompassed by the
present invention (see EP1007712, which is hereby incorporated by
reference herein in its entirety).
[0457] The polynucleotides are also useful for identifying
organisms from minute biological samples. The United States
military, for example, is considering the use of restriction
fragment length polymorphism (RFLP) for identification of its
personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identifying personnel. This
method does not suffer from the current limitations of "Dog Tags"
which can be lost, switched, or stolen, making positive
identification difficult. The polynucleotides of the present
invention can be used as additional DNA markers for RFLP.
[0458] The polynucleotides of the present invention can also be
used as an alternative to RFLP, by determining the actual
base-by-base DNA sequence of selected portions of an organisms
genome. These sequences can be used to prepare PCR primers for
amplifying and isolating such selected DNA, which can then be
sequenced. Using this technique, organisms can be identified
because each organism will have a unique set of DNA sequences. Once
an unique ID database is established for an organism, positive
identification of that organism, living or dead, can be made from
extremely small tissue samples. Similarly, polynucleotides of the
present invention can be used as polymorphic markers, in addition
to, the identification of transformed or non-transformed cells
and/or tissues.
[0459] There is also a need for reagents capable of identifying the
source of a particular tissue. Such need arises, for example, when
presented with tissue of unknown origin. Appropriate reagents can
comprise, for example, DNA probes or primers specific to particular
tissue prepared from the sequences of the present invention. Panels
of such reagents can identify tissue by species and/or by organ
type. In a similar fashion, these reagents can be used to screen
tissue cultures for contamination. Moreover, as mentioned above,
such reagents can be used to screen and/or identify transformed and
non-transformed cells and/or tissues.
[0460] In the very least, the polynucleotides of the present
invention can be used as molecular weight markers on Southern gels,
as diagnostic probes for the presence of a specific mRNA in a
particular cell type, as a probe to "subtract-out" known sequences
in the process of discovering novel polynucleotides, for selecting
and making oligomers for attachment to a "gene chip" or other
support, to raise anti-DNA antibodies using DNA immunization
techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
[0461] Each of the polypeptides identified herein can be used in
numerous ways. The following description should be considered
exemplary and utilizes known techniques.
[0462] A polypeptide of the present invention can be used to assay
protein levels in a biological sample using antibody-based
techniques. For example, 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 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 (125I, I211), carbon (14C), sulfur
(35S), tritium (3H), indium (112In), and technetium (99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0463] In addition to assaying protein levels in a biological
sample, proteins can also be detected in vivo by imaging. Antibody
labels or markers for in vivo imaging of 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.
[0464] A protein-specific antibody or antibody fragment which has
been labeled with an appropriate detectable imaging moiety, such as
a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque
substance, or a material detectable by nuclear magnetic resonance,
is introduced (for example, parenterally, subcutaneously, or
intraperitoneally) into the mammal. 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 99mTc. The labeled antibody
or antibody fragment will then preferentially accumulate at the
location of cells which contain the specific 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).)
[0465] Thus, the invention provides a diagnostic method of a
disorder, which involves (a) assaying the expression of a
polypeptide of the present invention in cells or body fluid of an
individual; and (b) comparing the level of gene expression with a
standard gene expression level, whereby an increase or decrease in
the assayed polypeptide gene expression level compared to the
standard expression level is indicative of a disorder. With respect
to cancer, the presence of a relatively high amount of transcript
in biopsied tissue from an individual may indicate a predisposition
for the development of the disease, or may provide a means for
detecting the disease prior to the appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0466] Moreover, polypeptides of the present invention can be used
to treat, prevent, and/or diagnose disease. For example, patients
can be administered a polypeptide of the present invention in an
effort to replace absent or decreased levels of the polypeptide
(e.g., insulin), to supplement absent or decreased levels of a
different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD,
catalase, DNA repair proteins), to inhibit the activity of a
polypeptide (e.g., an oncogene or tumor suppressor), to activate
the activity of a polypeptide (e.g., by binding to a receptor), to
reduce the activity of a membrane bound receptor by competing with
it for free ligand (e.g., soluble TNF receptors used in reducing
inflammation), or to bring about a desired response (e.g., blood
vessel growth inhibition, enhancement of the immune response to
proliferative cells or tissues).
[0467] Similarly, antibodies directed to a polypeptide of the
present invention can also be used to treat, prevent, and/or
diagnose disease. For example, administration of an antibody
directed to a polypeptide of the present invention can bind and
reduce overproduction of the polypeptide. Similarly, administration
of an antibody can activate the polypeptide, such as by binding to
a polypeptide bound to a membrane (receptor).
[0468] At the very least, the polypeptides of the present invention
can be used as molecular weight markers on SDS-PAGE gels or on
molecular sieve gel filtration columns using methods well known to
those of skill in the art. Polypeptides can also be used to raise
antibodies, which in turn are used to measure protein expression
from a recombinant cell, as a way of assessing transformation of
the host cell. Moreover, the polypeptides of the present invention
can be used to test the following biological activities.
Gene Therapy Methods
[0469] Another aspect of the present invention is to gene therapy
methods for treating or preventing disorders, diseases and
conditions. The gene therapy methods relate to the introduction of
nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an
animal to achieve expression of a polypeptide of the present
invention. This method requires a polynucleotide which codes for a
polypeptide of the invention that operatively linked to a promoter
and any other genetic elements necessary for the expression of the
polypeptide by the target tissue. Such gene therapy and delivery
techniques are known in the art, see, for example, WO 90/11092,
which is herein incorporated by reference.
[0470] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a polynucleotide of the invention ex vivo, with the
engineered cells then being provided to a patient to be treated
with the polypeptide. Such methods are well-known in the art. For
example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993); Ferrantini et al., Cancer Research, 53:107-1112 (1993);
Ferrantini et al., J. Immunology 153: 4604-4615 (1994); Kaido, T.,
et al., Int. J. Cancer 60: 221-229 (1995); Ogura et al., Cancer
Research 50: 5102-5106 (1990); Santodonato, et al., Human Gene
Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy
4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38
(1996)), which are herein incorporated by reference. In one
embodiment, the cells which are engineered are arterial cells. The
arterial cells may be reintroduced into the patient through direct
injection to the artery, the tissues surrounding the artery, or
through catheter injection.
[0471] As discussed in more detail below, the polynucleotide
constructs can be delivered by any method that delivers injectable
materials to the cells of an animal, such as, injection into the
interstitial space of tissues (heart, muscle, skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[0472] In one embodiment, the polynucleotide of the invention is
delivered as a naked polynucleotide. The term "naked"
polynucleotide, DNA or RNA refers to sequences that are free from
any delivery vehicle that acts to assist, promote or facilitate
entry into the cell, including viral sequences, viral particles,
liposome formulations, lipofectin or precipitating agents and the
like. However, the polynucleotides of the invention can also be
delivered in liposome formulations and lipofectin formulations and
the like can be prepared by methods well known to those skilled in
the art. Such methods are described, for example, in U.S. Pat. Nos.
5,593,972, 5,589,466, and 5,580,859, which are herein incorporated
by reference.
[0473] The polynucleotide vector constructs of the invention used
in the gene therapy method are preferably constructs that will not
integrate into the host genome nor will they contain sequences that
allow for replication. Appropriate vectors include pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG
and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and
pRc/CMV2 available from Invitrogen. Other suitable vectors will be
readily apparent to the skilled artisan.
[0474] Any strong promoter known to those skilled in the art can be
used for driving the expression of polynucleotide sequence of the
invention. Suitable promoters include adenoviral promoters, such as
the adenoviral major late promoter; or heterologous promoters, such
as the cytomegalovirus (CMV) promoter; the respiratory syncytial
virus (RSV) promoter; inducible promoters, such as the MMT
promoter, the metallothionein promoter; heat shock promoters; the
albumin promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for the polynucleotides of the invention.
[0475] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0476] The polynucleotide construct of the invention can be
delivered to the interstitial space of tissues within the an
animal, including of muscle, skin, brain, lung, liver, spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas,
kidney, gall bladder, stomach, intestine, testis, ovary, uterus,
rectum, nervous system, eye, gland, and connective tissue.
Interstitial space of the tissues comprises the intercellular,
fluid, mucopolysaccharide matrix among the reticular fibers of
organ tissues, elastic fibers in the walls of vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within
connective tissue ensheathing muscle cells or in the lacunae of
bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery
to the interstitial space of muscle tissue is preferred for the
reasons discussed below. They may be conveniently delivered by
injection into the tissues comprising these cells. They are
preferably delivered to and expressed in persistent, non-dividing
cells which are differentiated, although delivery and expression
may be achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts. In vivo muscle cells are particularly competent
in their ability to take up and express polynucleotides.
[0477] For the naked nucleic acid sequence injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
mg/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0478] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
DNA constructs can be delivered to arteries during angioplasty by
the catheter used in the procedure.
[0479] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0480] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin, precipitating agents, etc. Such methods of delivery are
known in the art.
[0481] In certain embodiments, the polynucleotide constructs of the
invention are complexed in a liposome preparation. Liposomal
preparations for use in the instant invention include cationic
(positively charged), anionic (negatively charged) and neutral
preparations. However, cationic liposomes are particularly
preferred because a tight charge complex can be formed between the
cationic liposome and the polyanionic nucleic acid. Cationic
liposomes have been shown to mediate intracellular delivery of
plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA,
84:7413-7416 (1987), which is herein incorporated by reference);
mRNA (Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081
(1989), which is herein incorporated by reference); and purified
transcription factors (Debs et al., J. Biol. Chem., 265:10189-10192
(1990), which is herein incorporated by reference), in functional
form.
[0482] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
LIPOFECTIN.RTM., from GIBCO BRL, Grand Island, N.Y. (See, also,
Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7416 (1987),
which is herein incorporated by reference). Other commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0483] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication NO:WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0484] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0485] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0486] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology, 101:512-527
(1983), which is herein incorporated by reference. For example,
MLVs containing nucleic acid can be prepared by depositing a thin
film of phospholipid on the walls of a glass tube and subsequently
hydrating with a solution of the material to be encapsulated. SUVs
are prepared by extended sonication of MLVs to produce a
homogeneous population of unilamellar liposomes. The material to be
entrapped is added to a suspension of preformed MLVs and then
sonicated. When using liposomes containing cationic lipids, the
dried lipid film is resuspended in an appropriate solution such as
sterile water or an isotonic buffer solution such as 10 mM
Tris/NaCl, sonicated, and then the preformed liposomes are mixed
directly with the DNA. The liposome and DNA form a very stable
complex due to binding of the positively charged liposomes to the
cationic DNA. SUVs find use with small nucleic acid fragments. LUVs
are prepared by a number of methods, well known in the art.
Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos
et al., Biochim. Biophys. Acta, 394:483 (1975); Wilson et al.,
Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.
Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.
Commun, 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,
76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl.
Acad. Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV)
(Fraley et al., J. Biol. Chem., 255:10431 (1980); Szoka et al.,
Proc. Natl. Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al.,
Science, 215:166 (1982)), which are herein incorporated by
reference.
[0487] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ration will be about 3:1 to
about 1:3. Still more preferably, the ratio will be about 1:1.
[0488] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication NO:WO 94/9469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication NO:WO 94/9469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[0489] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding polypeptides of the invention. Retroviruses from
which the retroviral plasmid vectors may be derived include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis
virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
[0490] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy, 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO4 precipitation. In one alternative, the
retroviral plasmid vector may be encapsulated into a liposome, or
coupled to a lipid, and then administered to a host.
[0491] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding polypeptides
of the invention. Such retroviral vector particles then may be
employed, to transduce eukaryotic cells, either in vitro or in
vivo. The transduced eukaryotic cells will express polypeptides of
the invention.
[0492] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with polynucleotides of the invention contained in an
adenovirus vector. Adenovirus can be manipulated such that it
encodes and expresses polypeptides of the invention, and at the
same time is inactivated in terms of its ability to replicate in a
normal lytic viral life cycle. Adenovirus expression is achieved
without integration of the viral DNA into the host cell chromosome,
thereby alleviating concerns about insertional mutagenesis.
Furthermore, adenoviruses have been used as live enteric vaccines
for many years with an excellent safety profile (Schwartz et al.,
Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally, adenovirus
mediated gene transfer has been demonstrated in a number of
instances including transfer of alpha-1-antitrypsin and CFTR to the
lungs of cotton rats (Rosenfeld et al., Science, 252:431-434
(1991); Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore,
extensive studies to attempt to establish adenovirus as a causative
agent in human cancer were uniformly negative (Green et al. Proc.
Natl. Acad. Sci. USA, 76:6606 (1979)).
[0493] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155
(1992); Engelhardt et al., Human Genet. Ther., 4:759-769 (1993);
Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al.,
Nature, 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are
herein incorporated by reference. For example, the adenovirus
vector Ad2 is useful and can be grown in human 293 cells. These
cells contain the E1 region of adenovirus and constitutively
express E1a and E1b, which complement the defective adenoviruses by
providing the products of the genes deleted from the vector. In
addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and
Ad7) are also useful in the present invention.
[0494] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[0495] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, Curr. Topics in
Microbiol. Immunol., 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0496] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The
polynucleotide construct containing polynucleotides of the
invention is inserted into the AAV vector using standard cloning
methods, such as those found in Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press (1989). The
recombinant AAV vector is then transfected into packaging cells
which are infected with a helper virus, using any standard
technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include
adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses. Once the packaging cells are transfected and infected,
they will produce infectious AAV viral particles which contain the
polynucleotide construct of the invention. These viral particles
are then used to transduce eukaryotic cells, either ex vivo or in
vivo. The transduced cells will contain the polynucleotide
construct integrated into its genome, and will express the desired
gene product.
[0497] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding the polypeptide sequence of interest) via
homologous recombination (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; International Publication NO:WO 96/29411,
published Sep. 26, 1996; International Publication NO:WO 94/12650,
published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA,
86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438
(1989). This method involves the activation of a gene which is
present in the target cells, but which is not normally expressed in
the cells, or is expressed at a lower level than desired.
[0498] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the desired endogenous polynucleotide sequence
so the promoter will be operably linked to the endogenous sequence
upon homologous recombination.
[0499] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0500] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0501] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous sequence
is placed under the control of the promoter. The promoter then
drives the expression of the endogenous sequence.
[0502] The polynucleotides encoding polypeptides of the present
invention may be administered along with other polynucleotides
encoding angiogenic proteins. Angiogenic proteins include, but are
not limited to, acidic and basic fibroblast growth factors, VEGF-1,
VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor alpha and
beta, platelet-derived endothelial cell growth factor,
platelet-derived growth factor, tumor necrosis factor alpha,
hepatocyte growth factor, insulin like growth factor, colony
stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide
synthase.
[0503] Preferably, the polynucleotide encoding a polypeptide of the
invention contains a secretory signal sequence that facilitates
secretion of the protein. Typically, the signal sequence is
positioned in the coding region of the polynucleotide to be
expressed towards or at the 5' end of the coding region. The signal
sequence may be homologous or heterologous to the polynucleotide of
interest and may be homologous or heterologous to the cells to be
transfected. Additionally, the signal sequence may be chemically
synthesized using methods known in the art.
[0504] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., ALZA.RTM. minipumps), oral or suppositorial
solid (tablet or pill) pharmaceutical formulations, and decanting
or topical applications during surgery. For example, direct
injection of naked calcium phosphate-precipitated plasmid into rat
liver and rat spleen or a protein-coated plasmid into the portal
vein has resulted in gene expression of the foreign gene in the rat
livers. (Kaneda et al., Science, 243:375 (1989)).
[0505] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0506] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0507] Therapeutic compositions useful in systemic administration,
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[0508] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992), which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0509] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian. Therapeutic compositions of
the present invention can be administered to any animal, preferably
to mammals and birds. Preferred mammals include humans, dogs, cats,
mice, rats, rabbits sheep, cattle, horses and pigs, with humans
being particularly preferred.
Biological Activities
[0510] The polynucleotides or polypeptides, or agonists or
antagonists of the present invention can be used in assays to test
for one or more biological activities. If these polynucleotides and
polypeptides do exhibit activity in a particular assay, it is
likely that these molecules may be involved in the diseases
associated with the biological activity. Thus, the polynucleotides
or polypeptides, or agonists or antagonists could be used to treat
the associated disease.
Immune Activity
[0511] The polynucleotides or polypeptides, or agonists or
antagonists of the present invention may be useful in treating,
preventing, and/or diagnosing diseases, disorders, and/or
conditions of the immune system, by activating or inhibiting the
proliferation, differentiation, or mobilization (chemotaxis) of
immune cells. Immune cells develop through a process called
hematopoiesis, producing myeloid (platelets, red blood cells,
neutrophils, and macrophages) and lymphoid (B and T lymphocytes)
cells from pluripotent stem cells. The etiology of these immune
diseases, disorders, and/or conditions may be genetic, somatic,
such as cancer or some autoimmune diseases, disorders, and/or
conditions, acquired (e.g., by chemotherapy or toxins), or
infectious. Moreover, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention can be used as a
marker or detector of a particular immune system disease or
disorder.
[0512] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may be useful in treating,
preventing, and/or diagnosing diseases, disorders, and/or
conditions of hematopoietic cells. A polynucleotides or
polypeptides, or agonists or antagonists of the present invention
could be used to increase differentiation and proliferation of
hematopoietic cells, including the pluripotent stem cells, in an
effort to treat or prevent those diseases, disorders, and/or
conditions associated with a decrease in certain (or many) types
hematopoietic cells. Examples of immunologic deficiency syndromes
include, but are not limited to: blood protein diseases, disorders,
and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),
ataxia telangiectasia, common variable immunodeficiency, Digeorge
Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion
deficiency syndrome, lymphopenia, phagocyte bactericidal
dysfunction, severe combined immunodeficiency (SCIDs),
Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or
hemoglobinuria.
[0513] Moreover, a polynucleotides or polypeptides, or agonists or
antagonists of the present invention could also be used to modulate
hemostatic (the stopping of bleeding) or thrombolytic activity
(clot formation). For example, by increasing hemostatic or
thrombolytic activity, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention could be used to
treat or prevent blood coagulation diseases, disorders, and/or
conditions (e.g., afibrinogenemia, factor deficiencies, arterial
thrombosis, venous thrombosis, etc.), blood platelet diseases,
disorders, and/or conditions (e.g. thrombocytopenia), or wounds
resulting from trauma, surgery, or other causes. Alternatively, a
polynucleotides or polypeptides, or agonists or antagonists of the
present invention that can decrease hemostatic or thrombolytic
activity could be used to inhibit or dissolve clotting.
Polynucleotides or polypeptides, or agonists or antagonists of the
present invention are may also be useful for the detection,
prognosis, treatment, and/or prevention of heart attacks
(infarction), strokes, scarring, fibrinolysis, uncontrolled
bleeding, uncontrolled coagulation, uncontrolled complement
fixation, and/or inflammation.
[0514] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be useful in
treating, preventing, and/or diagnosing autoimmune diseases,
disorders, and/or conditions. Many autoimmune diseases, disorders,
and/or conditions result from inappropriate recognition of self as
foreign material by immune cells. This inappropriate recognition
results in an immune response leading to the destruction of the
host tissue. Therefore, the administration of a polynucleotides or
polypeptides, or agonists or antagonists of the present invention
that inhibits an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing autoimmune diseases, disorders, and/or
conditions.
[0515] Examples of autoimmune diseases, disorders, and/or
conditions that can be treated, prevented, and/or diagnosed or
detected by the present invention include, but are not limited to:
Addison's Disease, hemolytic anemia, antiphospholipid syndrome,
rheumatoid arthritis, dermatitis, allergic encephalomyelitis,
glomerulonephritis, Goodpasture's Syndrome, Graves' Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia,
Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura,
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,
Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,
Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and
autoimmune inflammatory eye disease.
[0516] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated, prevented, and/or diagnosed by polynucleotides or
polypeptides, or agonists or antagonists of the present invention.
Moreover, these molecules can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0517] A polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be used to treat,
prevent, and/or diagnose organ rejection or graft-versus-host
disease (GVHD). Organ rejection occurs by host immune cell
destruction of the transplanted tissue through an immune response.
Similarly, an immune response is also involved in GVHD, but, in
this case, the foreign transplanted immune cells destroy the host
tissues. The administration of a polynucleotides or polypeptides,
or agonists or antagonists of the present invention that inhibits
an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD.
[0518] Similarly, a polynucleotides or polypeptides, or agonists or
antagonists of the present invention may also be used to modulate
inflammation. For example, the polypeptide or polynucleotide or
agonists or antagonist may inhibit the proliferation and
differentiation of cells involved in an inflammatory response.
These molecules can be used to treat, prevent, and/or diagnose
inflammatory conditions, both chronic and acute conditions,
including chronic prostatitis, granulomatous prostatitis and
malacoplakia, inflammation associated with infection (e.g., septic
shock, sepsis, or systemic inflammatory response syndrome (SIRS)),
ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine induced lung injury, inflammatory bowel disease, Crohn's
disease, or resulting from over production of cytokines (e.g., TNF
or IL-1.)
Hyperproliferative Disorders
[0519] A polynucleotides or polypeptides, or agonists or
antagonists of the invention can be used to treat, prevent, and/or
diagnose hyperproliferative diseases, disorders, and/or conditions,
including neoplasms. A polynucleotides or polypeptides, or agonists
or antagonists of the present invention may inhibit the
proliferation of the disorder through direct or indirect
interactions. Alternatively, a polynucleotides or polypeptides, or
agonists or antagonists of the present invention may proliferate
other cells which can inhibit the hyperproliferative disorder.
[0520] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative diseases, disorders, and/or conditions can be
treated, prevented, and/or diagnosed. This immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, decreasing an
immune response may also be a method of treating, preventing,
and/or diagnosing hyperproliferative diseases, disorders, and/or
conditions, such as a chemotherapeutic agent.
[0521] Examples of hyperproliferative diseases, disorders, and/or
conditions that can be treated, prevented, and/or diagnosed by
polynucleotides or polypeptides, or agonists or antagonists of the
present invention include, but are not limited to neoplasms located
in the: colon, abdomen, bone, breast, digestive system, liver,
pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,
nervous (central and peripheral), lymphatic system, pelvic, skin,
soft tissue, spleen, thoracic, and urogenital.
[0522] Similarly, other hyperproliferative diseases, disorders,
and/or conditions can also be treated, prevented, and/or diagnosed
by a polynucleotides or polypeptides, or agonists or antagonists of
the present invention. Examples of such hyperproliferative
diseases, disorders, and/or conditions include, but are not limited
to: hypergammaglobulinemia, lymphoproliferative diseases,
disorders, and/or conditions, paraproteinemias, purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,
Gaucher's Disease, histiocytosis, and any other hyperproliferative
disease, besides neoplasia, located in an organ system listed
above.
[0523] One preferred embodiment utilizes polynucleotides of the
present invention to inhibit aberrant cellular division, by gene
therapy using the present invention, and/or protein fusions or
fragments thereof.
[0524] Thus, the present invention provides a method for treating
or preventing cell proliferative diseases, disorders, and/or
conditions by inserting into an abnormally proliferating cell a
polynucleotide of the present invention, wherein said
polynucleotide represses said expression.
[0525] Another embodiment of the present invention provides a
method of treating or preventing cell-proliferative diseases,
disorders, and/or conditions in individuals comprising
administration of one or more active gene copies of the present
invention to an abnormally proliferating cell or cells. In a
preferred embodiment, polynucleotides of the present invention is a
DNA construct comprising a recombinant expression vector effective
in expressing a DNA sequence encoding said polynucleotides. In
another preferred embodiment of the present invention, the DNA
construct encoding the polynucleotides of the present invention is
inserted into cells to be treated utilizing a retrovirus, or more
Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999
96: 324-326, which is hereby incorporated by reference). In a most
preferred embodiment, the viral vector is defective and will not
transform non-proliferating cells, only proliferating cells.
Moreover, in a preferred embodiment, the polynucleotides of the
present invention inserted into proliferating cells either alone,
or in combination with or fused to other polynucleotides, can then
be modulated via an external stimulus (i.e. magnetic, specific
small molecule, chemical, or drug administration, etc.), which acts
upon the promoter upstream of said polynucleotides to induce
expression of the encoded protein product. As such the beneficial
therapeutic affect of the present invention may be expressly
modulated (i.e. to increase, decrease, or inhibit expression of the
present invention) based upon said external stimulus.
[0526] Polynucleotides of the present invention may be useful in
repressing expression of oncogenic genes or antigens. By
"repressing expression of the oncogenic genes" is intended the
suppression of the transcription of the gene, the degradation of
the gene transcript (pre-message RNA), the inhibition of splicing,
the destruction of the messenger RNA, the prevention of the
post-translational modifications of the protein, the destruction of
the protein, or the inhibition of the normal function of the
protein.
[0527] For local administration to abnormally proliferating cells,
polynucleotides of the present invention may be administered by any
method known to those of skill in the art including, but not
limited to transfection, electroporation, microinjection of cells,
or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any other method described throughout the
specification. The polynucleotide of the present invention may be
delivered by known gene delivery systems such as, but not limited
to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems
(Yates et al., Nature 313:812 (1985)) known to those skilled in the
art. These references are exemplary only and are hereby
incorporated by reference. In order to specifically deliver or
transfect cells which are abnormally proliferating and spare
non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as described in the art and elsewhere herein) delivery
system known to those of skill in the art. Since host DNA
replication is required for retroviral DNA to integrate and the
retrovirus will be unable to self replicate due to the lack of the
retrovirus genes needed for its life cycle. Utilizing such a
retroviral delivery system for polynucleotides of the present
invention will target said gene and constructs to abnormally
proliferating cells and will spare the non-dividing normal
cells.
[0528] The polynucleotides of the present invention may be
delivered directly to cell proliferative disorder/disease sites in
internal organs, body cavities and the like by use of imaging
devices used to guide an injecting needle directly to the disease
site. The polynucleotides of the present invention may also be
administered to disease sites at the time of surgical
intervention.
[0529] By "cell proliferative disease" is meant any human or animal
disease or disorder, affecting any one or any combination of
organs, cavities, or body parts, which is characterized by single
or multiple local abnormal proliferations of cells, groups of
cells, or tissues, whether benign or malignant.
[0530] Any amount of the polynucleotides of the present invention
may be administered as long as it has a biologically inhibiting
effect on the proliferation of the treated cells. Moreover, it is
possible to administer more than one of the polynucleotide of the
present invention simultaneously to the same site. By "biologically
inhibiting" is meant partial or total growth inhibition as well as
decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose may be determined by assessing the
effects of the polynucleotides of the present invention on target
malignant or abnormally proliferating cell growth in tissue
culture, tumor growth in animals and cell cultures, or any other
method known to one of ordinary skill in the art.
[0531] The present invention is further directed to antibody-based
therapies which involve administering of anti-polypeptides and
anti-polynucleotide antibodies to a mammalian, preferably human,
patient for treating, preventing, and/or diagnosing one or more of
the described diseases, disorders, and/or conditions. Methods for
producing anti-polypeptides and anti-polynucleotide antibodies
polyclonal and monoclonal antibodies are described in detail
elsewhere herein. Such antibodies may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0532] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0533] In particular, the antibodies, fragments and derivatives of
the present invention are useful for treating, preventing, and/or
diagnosing a subject having or developing cell proliferative and/or
differentiation diseases, disorders, and/or conditions as described
herein. Such treatment comprises administering a single or multiple
doses of the antibody, or a fragment, derivative, or a conjugate
thereof.
[0534] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors,
for example, which serve to increase the number or activity of
effector cells which interact with the antibodies.
[0535] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of diseases,
disorders, and/or conditions related to polynucleotides or
polypeptides, including fragments thereof, of the present
invention. Such antibodies, fragments, or regions, will preferably
have an affinity for polynucleotides or polypeptides, including
fragments thereof. Preferred binding affinities include those with
a dissociation constant or Kd less than 5.times.10-6M, 10-6M,
5.times.10-7M, 10-7M, 5.times.10-8M, 10-8M, 5.times.10-9M, 10-9M,
5.times.10-10M, 10-10M, 5.times.10-11M, 10-11M, 5.times.10-12M,
10-12M, 5.times.10-13M, 10-13M, 5.times.10-14M, 10-14M,
5.times.10-15M, and 10-15M.
[0536] Moreover, polypeptides of the present invention may be
useful in inhibiting the angiogenesis of proliferative cells or
tissues, either alone, as a protein fusion, or in combination with
other polypeptides directly or indirectly, as described elsewhere
herein. In a most preferred embodiment, said anti-angiogenesis
effect may be achieved indirectly, for example, through the
inhibition of hematopoietic, tumor-specific cells, such as
tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer
Inst, 90(21):1648-53 (1998), which is hereby incorporated by
reference). Antibodies directed to polypeptides or polynucleotides
of the present invention may also result in inhibition of
angiogenesis directly, or indirectly (See Witte L, et al., Cancer
Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated
by reference)).
[0537] Polypeptides, including protein fusions, of the present
invention, or fragments thereof may be useful in inhibiting
proliferative cells or tissues through the induction of apoptosis.
Said polypeptides may act either directly, or indirectly to induce
apoptosis of proliferative cells and tissues, for example in the
activation of a death-domain receptor, such as tumor necrosis
factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related
apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See
Schulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998),
which is hereby incorporated by reference). Moreover, in another
preferred embodiment of the present invention, said polypeptides
may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or
through stimulating the expression of said proteins, either alone
or in combination with small molecule drugs or adjuvants, such as
apoptonin, galectins, thioredoxins, antiinflammatory proteins (See
for example, Mutat. Res. 400(1-2):447-55 (1998), Med Hypotheses.
50(5):423-33 (1998), Chem. Biol. Interact. April 24; 111-112:23-34
(1998), J Mol Med. 76(6):402-12 (1998), Int. J. Tissue React.
20(1):3-15 (1998), which are all hereby incorporated by
reference).
[0538] Polypeptides, including protein fusions to, or fragments
thereof, of the present invention are useful in inhibiting the
metastasis of proliferative cells or tissues. Inhibition may occur
as a direct result of administering polypeptides, or antibodies
directed to said polypeptides as described elsewhere herein, or
indirectly, such as activating the expression of proteins known to
inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr
Top Microbiol Immunol 1998; 231:125-41, which is hereby
incorporated by reference). Such therapeutic affects of the present
invention may be achieved either alone, or in combination with
small molecule drugs or adjuvants.
[0539] In another embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing polypeptides or
polypeptide antibodies associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs) to targeted cells
expressing the polypeptide of the present invention. Polypeptides
or polypeptide antibodies of the invention may be associated with
heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent
interactions.
[0540] Polypeptides, protein fusions to, or fragments thereof, of
the present invention are useful in enhancing the immunogenicity
and/or antigenicity of proliferating cells or tissues, either
directly, such as would occur if the polypeptides of the present
invention `vaccinated` the immune response to respond to
proliferative antigens and immunogens, or indirectly, such as in
activating the expression of proteins known to enhance the immune
response (e.g. chemokines), to said antigens and immunogens.
Cardiovascular Disorders
[0541] Polynucleotides or polypeptides, or agonists or antagonists
of the invention may be used to treat, prevent, and/or diagnose
cardiovascular diseases, disorders, and/or conditions, including
peripheral artery disease, such as limb ischemia.
[0542] Cardiovascular diseases, disorders, and/or conditions
include cardiovascular abnormalities, such as arterio-arterial
fistula, arteriovenous fistula, cerebral arteriovenous
malformations, congenital heart defects, pulmonary atresia, and
Scimitar Syndrome. Congenital heart defects include aortic
coarctation, cor triatriatum, coronary vessel anomalies, crisscross
heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of fallot, transposition of great vessels, double outlet
right ventricle, tricuspid atresia, persistent truncus arteriosus,
and heart septal defects, such as aortopulmonary septal defect,
endocardial cushion defects, Lutembacher's Syndrome, trilogy of
Fallot, ventricular heart septal defects.
[0543] Cardiovascular diseases, disorders, and/or conditions also
include heart disease, such as arrhythmias, carcinoid heart
disease, high cardiac output, low cardiac output, cardiac
tamponade, endocarditis (including bacterial), heart aneurysm,
cardiac arrest, congestive heart failure, congestive
cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart
hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy, right ventricular hypertrophy, post-infarction heart
rupture, ventricular septal rupture, heart valve diseases,
myocardial diseases, myocardial ischemia, pericardial effusion,
pericarditis (including constrictive and tuberculosis),
pneumopericardium, postpericardiotomy syndrome, pulmonary heart
disease, rheumatic heart disease, ventricular dysfunction,
hyperemia, cardiovascular pregnancy complications, Scimitar
Syndrome, cardiovascular syphilis, and cardiovascular
tuberculosis.
[0544] Arrhythmias include sinus arrhythmia, atrial fibrillation,
atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome,
bundle-branch block, sinoatrial block, long QT syndrome,
parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type
pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias
include paroxysmal tachycardia, supraventricular tachycardia,
accelerated idioventricular rhythm, atrioventricular nodal reentry
tachycardia, ectopic atrial tachycardia, ectopic junctional
tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia, Torsades de Pointes, and ventricular tachycardia.
[0545] Heart valve disease include aortic valve insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral
valve prolapse, tricuspid valve prolapse, mitral valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary
valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
[0546] Myocardial diseases include alcoholic cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic
subvalvular stenosis, pulmonary subvalvular stenosis, restrictive
cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion
injury, and myocarditis.
[0547] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[0548] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular diseases, disorders, and/or conditions, diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis,
erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,
hypertension, hypotension, ischemia, peripheral vascular diseases,
phlebitis, pulmonary veno-occlusive disease, Raynaud's disease,
CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior
vena cava syndrome, telangiectasia, atacia telangiectasia,
hereditary hemorrhagic telangiectasia, varicocele, varicose veins,
varicose ulcer, vasculitis, and venous insufficiency.
[0549] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0550] Arterial occlusive diseases include arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular
dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis
obliterans.
[0551] Cerebrovascular diseases, disorders, and/or conditions
include carotid artery diseases, cerebral amyloid angiopathy,
cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral arteriovenous malformation, cerebral artery diseases,
cerebral embolism and thrombosis, carotid artery thrombosis, sinus
thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural
hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral
infarction, cerebral ischemia (including transient), subclavian
steal syndrome, periventricular leukomalacia, vascular headache,
cluster headache, migraine, and vertebrobasilar insufficiency.
[0552] Embolisms include air embolisms, amniotic fluid embolisms,
cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromoboembolisms. Thrombosis include coronary
thrombosis, hepatic vein thrombosis, retinal vein occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and thrombophlebitis.
[0553] Ischemia includes cerebral ischemia, ischemic colitis,
compartment syndromes, anterior compartment syndrome, myocardial
ischemia, reperfusion injuries, and peripheral limb ischemia.
Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis,
Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and
Wegener's granulomatosis.
[0554] Polynucleotides or polypeptides, or agonists or antagonists
of the invention, are especially effective for the treatment of
critical limb ischemia and coronary disease.
[0555] Polypeptides may be administered using any method known in
the art, including, but not limited to, direct needle injection at
the delivery site, intravenous injection, topical administration,
catheter infusion, biolistic injectors, particle accelerators,
gelfoam sponge depots, other commercially available depot
materials, osmotic pumps, oral or suppositorial solid
pharmaceutical formulations, decanting or topical applications
during surgery, aerosol delivery. Such methods are known in the
art. Polypeptides of the invention may be administered as part of a
Therapeutic, described in more detail below. Methods of delivering
polynucleotides of the invention are described in more detail
herein.
Neurological Diseases
[0556] Nervous system diseases, disorders, and/or conditions, which
can be treated, prevented, and/or diagnosed with the compositions
of the invention (e.g., polypeptides, polynucleotides, and/or
agonists or antagonists), include, but are not limited to, nervous
system injuries, and diseases, disorders, and/or conditions which
result in either a disconnection of axons, a diminution or
degeneration of neurons, or demyelination. Nervous system lesions
which may be treated, prevented, and/or diagnosed in a patient
(including human and non-human mammalian patients) according to the
invention, include but are not limited to, the following lesions of
either the central (including spinal cord, brain) or peripheral
nervous systems: (1) ischemic lesions, in which a lack of oxygen in
a portion of the nervous system results in neuronal injury or
death, including cerebral infarction or ischemia, or spinal cord
infarction or ischemia; (2) traumatic lesions, including lesions
caused by physical injury or associated with surgery, for example,
lesions which sever a portion of the nervous system, or compression
injuries; (3) malignant lesions, in which a portion of the nervous
system is destroyed or injured by malignant tissue which is either
a nervous system associated malignancy or a malignancy derived from
non-nervous system tissue; (4) infectious lesions, in which a
portion of the nervous system is destroyed or injured as a result
of infection, for example, by an abscess or associated with
infection by human immunodeficiency virus, herpes zoster, or herpes
simplex virus or with Lyme disease, tuberculosis, syphilis; (5)
degenerative lesions, in which a portion of the nervous system is
destroyed or injured as a result of a degenerative process
including but not limited to degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis (ALS); (6) lesions associated with
nutritional diseases, disorders, and/or conditions, in which a
portion of the nervous system is destroyed or injured by a
nutritional disorder or disorder of metabolism including but not
limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke
disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease
(primary degeneration of the corpus callosum), and alcoholic
cerebellar degeneration; (7) neurological lesions associated with
systemic diseases including, but not limited to, diabetes (diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma,
or sarcoidosis; (8) lesions caused by toxic substances including
alcohol, lead, or particular neurotoxins; and (9) demyelinated
lesions in which a portion of the nervous system is destroyed or
injured by a demyelinating disease including, but not limited to,
multiple sclerosis, human immunodeficiency virus-associated
myelopathy, transverse myelopathy or various etiologies,
progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
[0557] In a preferred embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to protect neural cells from the damaging effects of cerebral
hypoxia. According to this embodiment, the compositions of the
invention are used to treat, prevent, and/or diagnose neural cell
injury associated with cerebral hypoxia. In one aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose neural cell injury associated with cerebral ischemia. In
another aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat, prevent, and/or diagnose neural cell injury
associated with cerebral infarction. In another aspect of this
embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose or prevent neural cell injury associated with a stroke. In
a further aspect of this embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat, prevent, and/or diagnose neural cell injury
associated with a heart attack.
[0558] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, compositions of the invention which elicit any of the
following effects may be useful according to the invention: (1)
increased survival time of neurons in culture; (2) increased
sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo. Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515
(1990)); increased sprouting of neurons may be detected by methods
known in the art, such as, for example, the methods set forth in
Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al.
(Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic
assay, antibody binding, Northern blot assay, etc., using
techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0559] In specific embodiments, motor neuron diseases, disorders,
and/or conditions that may be treated, prevented, and/or diagnosed
according to the invention include, but are not limited to,
diseases, disorders, and/or conditions such as infarction,
infection, exposure to toxin, trauma, surgical damage, degenerative
disease or malignancy that may affect motor neurons as well as
other components of the nervous system, as well as diseases,
disorders, and/or conditions that selectively affect neurons such
as amyotrophic lateral sclerosis, and including, but not limited
to, progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
Infectious Disease
[0560] A polypeptide or polynucleotide and/or agonist or antagonist
of the present invention can be used to treat, prevent, and/or
diagnose infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation and
differentiation of B and/or T cells, infectious diseases may be
treated, prevented, and/or diagnosed. The immune response may be
increased by either enhancing an existing immune response, or by
initiating a new immune response. Alternatively, polypeptide or
polynucleotide and/or agonist or antagonist of the present
invention may also directly inhibit the infectious agent, without
necessarily eliciting an immune response.
[0561] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated, prevented, and/or
diagnosed by a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention. Examples of viruses, include,
but are not limited to Examples of viruses, include, but are not
limited to the following DNA and RNA viruses and viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue,
EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus,
Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B,
and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. polynucleotides or polypeptides, or agonists
or antagonists of the invention, can be used to treat, prevent,
and/or diagnose any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g.,
hepatitis B). In an additional specific embodiment polynucleotides,
polypeptides, or agonists or antagonists of the invention are used
to treat patients nonresponsive to one or more other commercially
available hepatitis vaccines. In a further specific embodiment
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose AIDS.
[0562] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated, prevented, and/or diagnosed by
a polynucleotide or polypeptide and/or agonist or antagonist of the
present invention include, but not limited to, include, but not
limited to, the following Gram-Negative and Gram-positive bacteria
and bacterial families and fungi: Actinomycetales (e.g.,
Corynebacterium, Mycobacterium, Norcardia), Cryptococcus
neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia
(e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,
Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses,
E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E.
coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella
typhi, and Salmonella paratyphi), Serratia, Yersinia),
Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis,
Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Meisseria meningitidis, Pasteurellacea Infections (e.g.,
Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B),
Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis,
Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and
Streptococcal (e.g., Streptococcus pneumoniae and Group B
Streptococcus). These bacterial or fungal families can cause the
following diseases or symptoms, including, but not limited to:
bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis, uveitis), gingivitis, opportunistic infections (e.g.,
AIDS related infections), paronychia, prosthesis-related
infections, Reiter's Disease, respiratory tract infections, such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch
Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid,
pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B),
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,
Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections. Polynucleotides or polypeptides,
agonists or antagonists of the invention, can be used to treat,
prevent, and/or diagnose any of these symptoms or diseases. In
specific embodiments, polynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat, prevent, and/or
diagnose: tetanus, Diptheria, botulism, and/or meningitis type
B.
[0563] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by a polynucleotide or
polypeptide and/or agonist or antagonist of the present invention
include, but not limited to, the following families or class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and
Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium
falciparium, Plasmodium malariae and Plasmodium ovale). These
parasites can cause a variety of diseases or symptoms, including,
but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal disease (e.g., dysentery, giardiasis), liver disease,
lung disease, opportunistic infections (e.g., AIDS related),
malaria, pregnancy complications, and toxoplasmosis.
polynucleotides or polypeptides, or agonists or antagonists of the
invention, can be used totreat, prevent, and/or diagnose any of
these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose malaria.
[0564] Preferably, treatment or prevention using a polypeptide or
polynucleotide and/or agonist or antagonist of the present
invention could either be by administering an effective amount of a
polypeptide to the patient, or by removing cells from the patient,
supplying the cells with a polynucleotide of the present invention,
and returning the engineered cells to the patient (ex vivo
therapy). Moreover, the polypeptide or polynucleotide of the
present invention can be used as an antigen in a vaccine to raise
an immune response against infectious disease.
Regeneration
[0565] A polynucleotide or polypeptide and/or agonist or antagonist
of the present invention can be used to differentiate, proliferate,
and attract cells, leading to the regeneration of tissues. (See,
Science 276:59-87 (1997).) The regeneration of tissues could be
used to repair, replace, or protect tissue damaged by congenital
defects, trauma (wounds, burns, incisions, or ulcers), age, disease
(e.g. osteoporosis, osteocarthritis, periodontal disease, liver
failure), surgery, including cosmetic plastic surgery, fibrosis,
reperfusion injury, or systemic cytokine damage.
[0566] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue. Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[0567] Moreover, a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention may increase regeneration of
tissues difficult to heal. For example, increased tendon/ligament
regeneration would quicken recovery time after damage. A
polynucleotide or polypeptide and/or agonist or antagonist of the
present invention could also be used prophylactically in an effort
to avoid damage. Specific diseases that could be treated,
prevented, and/or diagnosed include of tendinitis, carpal tunnel
syndrome, and other tendon or ligament defects. A further example
of tissue regeneration of non-healing wounds includes pressure
ulcers, ulcers associated with vascular insufficiency, surgical,
and traumatic wounds.
[0568] Similarly, nerve and brain tissue could also be regenerated
by using a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention to proliferate and
differentiate nerve cells. Diseases that could be treated,
prevented, and/or diagnosed using this method include central and
peripheral nervous system diseases, neuropathies, or mechanical and
traumatic diseases, disorders, and/or conditions (e.g., spinal cord
disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated, prevented, and/or diagnosed using
the polynucleotide or polypeptide and/or agonist or antagonist of
the present invention.
Binding Activity
[0569] A polypeptide of the present invention may be used to screen
for molecules that bind to the polypeptide or for molecules to
which the polypeptide binds. The binding of the polypeptide and the
molecule may activate (agonist), increase, inhibit (antagonist), or
decrease activity of the polypeptide or the molecule bound.
Examples of such molecules include antibodies, oligonucleotides,
proteins (e.g., receptors), or small molecules.
[0570] Preferably, the molecule is closely related to the natural
ligand of the polypeptide, e.g., a fragment of the ligand, or a
natural substrate, a ligand, a structural or functional mimetic.
(See, Coligan et al., Current Protocols in Immunology 1(2):Chapter
5 (1991).) Similarly, the molecule can be closely related to the
natural receptor to which the polypeptide binds, or at least, a
fragment of the receptor capable of being bound by the polypeptide
(e.g., active site). In either case, the molecule can be rationally
designed using known techniques.
[0571] Preferably, the screening for these molecules involves
producing appropriate cells which express the polypeptide, either
as a secreted protein or on the cell membrane. Preferred cells
include cells from mammals, yeast, Drosophila, or E. coli. Cells
expressing the polypeptide (or cell membrane containing the
expressed polypeptide) are then preferably contacted with a test
compound potentially containing the molecule to observe binding,
stimulation, or inhibition of activity of either the polypeptide or
the molecule.
[0572] The assay may simply test binding of a candidate compound to
the polypeptide, wherein binding is detected by a label, or in an
assay involving competition with a labeled competitor. Further, the
assay may test whether the candidate compound results in a signal
generated by binding to the polypeptide.
[0573] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing a polypeptide, measuring polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule
activity or binding to a standard.
[0574] Preferably, an ELISA assay can measure polypeptide level or
activity in a sample (e.g., biological sample) using a monoclonal
or polyclonal antibody. The antibody can measure polypeptide level
or activity by either binding, directly or indirectly, to the
polypeptide or by competing with the polypeptide for a
substrate.
[0575] Additionally, the receptor to which a polypeptide of the
invention binds can be identified by numerous methods known to
those of skill in the art, for example, ligand panning and
FACS.RTM. sorting (Coligan, et al., Current Protocols in Immun.,
1(2), Chapter 5, (1991)). For example, expression cloning is
employed wherein polyadenylated RNA is prepared from a cell
responsive to the polypeptides, for example, NIH3T3 cells which are
known to contain multiple receptors for the FGF family proteins,
and SC-3 cells, and a cDNA library created from this RNA is divided
into pools and used to transfect COS cells or other cells that are
not responsive to the polypeptides. Transfected cells which are
grown on glass slides are exposed to the polypeptide of the present
invention, after they have been labeled. The polypeptides can be
labeled by a variety of means including iodination or inclusion of
a recognition site for a site-specific protein kinase.
[0576] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[0577] As an alternative approach for receptor identification, the
labeled polypeptides can be photoaffinity linked with cell membrane
or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE analysis and exposed to
X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and
subjected to protein microsequencing. The amino acid sequence
obtained from microsequencing would be used to design a set of
degenerate oligonucleotide probes to screen a cDNA library to
identify the genes encoding the putative receptors.
[0578] Moreover, the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of
polypeptides of the invention thereby effectively generating
agonists and antagonists of polypeptides of the invention. See
generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,
5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion
Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol.
16(2):76-82 (1998); Hansson, L. 0., et al., J. Mol. Biol.
287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques
24(2):308-13 (1998) (each of these patents and publications are
hereby incorporated by reference). In one embodiment, alteration of
polynucleotides and corresponding polypeptides of the invention may
be achieved by DNA shuffling. DNA shuffling involves the assembly
of two or more DNA segments into a desired polynucleotide sequence
of the invention molecule by homologous, or site-specific,
recombination. In another embodiment, polynucleotides and
corresponding polypeptides of the invention may be altered by being
subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections,
parts, domains, fragments, etc., of the polypeptides of the
invention may be recombined with one or more components, motifs,
sections, parts, domains, fragments, etc. of one or more
heterologous molecules. In preferred embodiments, the heterologous
molecules are family members. In further preferred embodiments, the
heterologous molecule is a growth factor such as, for example,
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF-I), transforming growth factor (TGF)-alpha, epidermal growth
factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone
morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins
A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth
differentiation factors (GDFs), nodal, MIS, inhibin-alpha,
TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived
neurotrophic factor (GDNF).
[0579] Other preferred fragments are biologically active fragments
of the polypeptides of the invention. Biologically active fragments
are those exhibiting activity similar, but not necessarily
identical, to an activity of the polypeptide. The biological
activity of the fragments may include an improved desired activity,
or a decreased undesirable activity.
[0580] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of the
polypeptide of the present invention. An example of such an assay
comprises combining a mammalian fibroblast cell, a the polypeptide
of the present invention, the compound to be screened and 3[H]
thymidine under cell culture conditions where the fibroblast cell
would normally proliferate. A control assay may be performed in the
absence of the compound to be screened and compared to the amount
of fibroblast proliferation in the presence of the compound to
determine if the compound stimulates proliferation by determining
the uptake of 3[H] thymidine in each case. The amount of fibroblast
cell proliferation is measured by liquid scintillation
chromatography which measures the incorporation of 3[H] thymidine.
Both agonist and antagonist compounds may be identified by this
procedure.
[0581] In another method, a mammalian cell or membrane preparation
expressing a receptor for a polypeptide of the present invention is
incubated with a labeled polypeptide of the present invention in
the presence of the compound. The ability of the compound to
enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system
following interaction of a compound to be screened and the receptor
is measured and the ability of the compound to bind to the receptor
and elicit a second messenger response is measured to determine if
the compound is a potential agonist or antagonist. Such second
messenger systems include but are not limited to, cAMP guanylate
cyclase, ion channels or phosphoinositide hydrolysis.
[0582] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat, prevent, and/or diagnose disease or to bring
about a particular result in a patient (e.g., blood vessel growth)
by activating or inhibiting the polypeptide/molecule. Moreover, the
assays can discover agents which may inhibit or enhance the
production of the polypeptides of the invention from suitably
manipulated cells or tissues. Therefore, the invention includes a
method of identifying compounds which bind to the polypeptides of
the invention comprising the steps of: (a) incubating a candidate
binding compound with the polypeptide; and (b) determining if
binding has occurred. Moreover, the invention includes a method of
identifying agonists/antagonists comprising the steps of: (a)
incubating a candidate compound with the polypeptide, (b) assaying
a biological activity, and (c) determining if a biological activity
of the polypeptide has been altered.
[0583] Also, one could identify molecules bind a polypeptide of the
invention experimentally by using the beta-pleated sheet regions
contained in the polypeptide sequence of the protein. Accordingly,
specific embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, the amino acid sequence of each beta
pleated sheet regions in a disclosed polypeptide sequence.
Additional embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, any combination or all of contained in
the polypeptide sequences of the invention. Additional preferred
embodiments of the invention are directed to polypeptides which
comprise, or alternatively consist of, the amino acid sequence of
each of the beta pleated sheet regions in one of the polypeptide
sequences of the invention. Additional embodiments of the invention
are directed to polypeptides which comprise, or alternatively
consist of, any combination or all of the beta pleated sheet
regions in one of the polypeptide sequences of the invention.
Drug Screening
[0584] Further contemplated is the use of the polypeptides of the
present invention, or the polynucleotides encoding these
polypeptides, to screen for molecules which modify the activities
of the polypeptides of the present invention. Such a method would
include contacting the polypeptide of the present invention with a
selected compound(s) suspected of having antagonist or agonist
activity, and assaying the activity of these polypeptides following
binding.
[0585] This invention is particularly useful for screening
therapeutic compounds by using the polypeptides of the present
invention, or binding fragments thereof, in any of a variety of
drug screening techniques. The polypeptide or fragment employed in
such a test may be affixed to a solid support, expressed on a cell
surface, free in solution, or located intracellularly. One method
of drug screening utilizes eukaryotic or prokaryotic host cells
which are stably transformed with recombinant nucleic acids
expressing the polypeptide or fragment. Drugs are screened against
such transformed cells in competitive binding assays. One may
measure, for example, the formulation of complexes between the
agent being tested and a polypeptide of the present invention.
[0586] Thus, the present invention provides methods of screening
for drugs or any other agents which affect activities mediated by
the polypeptides of the present invention. These methods comprise
contacting such an agent with a polypeptide of the present
invention or a fragment thereof and assaying for the presence of a
complex between the agent and the polypeptide or a fragment
thereof, by methods well known in the art. In such a competitive
binding assay, the agents to screen are typically labeled.
Following incubation, free agent is separated from that present in
bound form, and the amount of free or uncomplexed label is a
measure of the ability of a particular agent to bind to the
polypeptides of the present invention.
[0587] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the polypeptides of the present invention, and is described in
great detail in European Patent Application 84/03564, published on
Sep. 13, 1984, which is incorporated herein by reference herein.
Briefly stated, large numbers of different small peptide test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The peptide test compounds are reacted
with polypeptides of the present invention and washed. Bound
polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in
the aforementioned drug screening techniques. In addition,
non-neutralizing antibodies may be used to capture the peptide and
immobilize it on the solid support.
[0588] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding polypeptides of the present invention specifically compete
with a test compound for binding to the polypeptides or fragments
thereof. In this manner, the antibodies are used to detect the
presence of any peptide which shares one or more antigenic epitopes
with a polypeptide of the invention.
[0589] The human PCSK9b or PCSK9c polypeptides and/or peptides of
the present invention, or immunogenic fragments or oligopeptides
thereof, can be used for screening therapeutic drugs or compounds
in a variety of drug screening techniques. The fragment employed in
such a screening assay may be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. The
reduction or abolition of activity of the formation of binding
complexes between the ion channel protein and the agent being
tested can be measured. Thus, the present invention provides a
method for screening or assessing a plurality of compounds for
their specific binding affinity with a PCSK9b or PCSK9c
polypeptide, or a bindable peptide fragment, of this invention,
comprising providing a plurality of compounds, combining the PCSK9b
or PCSK9c polypeptide, or a bindable peptide fragment, with each of
a plurality of compounds for a time sufficient to allow binding
under suitable conditions and detecting binding of the PCSK9b or
PCSK9c polypeptide or peptide to each of the plurality of test
compounds, thereby identifying the compounds that specifically bind
to the PCSK9b or PCSK9c polypeptide or peptide.
[0590] Methods of identifying compounds that modulate the activity
of the novel human PCSK9b or PCSK9c polypeptides and/or peptides
are provided by the present invention and comprise combining a
potential or candidate compound or drug modulator of calpain
biological activity with an PCSK9b or PCSK9c polypeptide or
peptide, for example, the PCSK9b or PCSK9c amino acid sequence as
set forth in SEQ ID NO:2 or SEQ ID NO:4, and measuring an effect of
the candidate compound or drug modulator on the biological activity
of the PCSK9b or PCSK9c polypeptide or peptide. Such measurable
effects include, for example, physical binding interaction; the
ability to cleave a suitable calpain substrate; effects on native
and cloned PCSK9b or PCSK9c-expressing cell line; and effects of
modulators or other calpain-mediated physiological measures.
[0591] Another method of identifying compounds that modulate the
biological activity of the novel PCSK9b or PCSK9c polypeptides of
the present invention comprises combining a potential or candidate
compound or drug modulator of a calpain biological activity with a
host cell that expresses the PCSK9b or PCSK9c polypeptide and
measuring an effect of the candidate compound or drug modulator on
the biological activity of the PCSK9b or PCSK9c polypeptide. The
host cell can also be capable of being induced to express the
PCSK9b or PCSK9c polypeptide, e.g., via inducible expression.
Physiological effects of a given modulator candidate on the PCSK9b
or PCSK9c polypeptide can also be measured. Thus, cellular assays
for particular calpain modulators may be either direct measurement
or quantification of the physical biological activity of the PCSK9b
or PCSK9c polypeptide, or they may be measurement or quantification
of a physiological effect. Such methods preferably employ a PCSK9b
or PCSK9c polypeptide as described herein, or an overexpressed
recombinant PCSK9b or PCSK9c polypeptide in suitable host cells
containing an expression vector as described herein, wherein the
PCSK9b or PCSK9c polypeptide is expressed, overexpressed, or
undergoes upregulated expression.
[0592] Another aspect of the present invention embraces a method of
screening for a compound that is capable of modulating the
biological activity of a PCSK9b or PCSK9c polypeptide, comprising
providing a host cell containing an expression vector harboring a
nucleic acid sequence encoding a PCSK9b or PCSK9c polypeptide, or a
functional peptide or portion thereof (e.g., SEQ ID NO:2 or SEQ ID
NO:4); determining the biological activity of the expressed PCSK9b
or PCSK9c polypeptide in the absence of a modulator compound;
contacting the cell with the modulator compound and determining the
biological activity of the expressed PCSK9b or PCSK9c polypeptide
in the presence of the modulator compound. In such a method, a
difference between the activity of the PCSK9b or PCSK9c polypeptide
in the presence of the modulator compound and in the absence of the
modulator compound indicates a modulating effect of the
compound.
[0593] Essentially any chemical compound can be employed as a
potential modulator or ligand in the assays according to the
present invention. Compounds tested as calpain modulators can be
any small chemical compound, or biological entity (e.g., protein,
sugar, nucleic acid, lipid). Test compounds will typically be small
chemical molecules and peptides. Generally, the compounds used as
potential modulators can be dissolved in aqueous or organic (e.g.,
DMSO-based) solutions. The assays are designed to screen large
chemical libraries by automating the assay steps and providing
compounds from any convenient source. Assays are typically run in
parallel, for example, in microtiter formats on microtiter plates
in robotic assays. There are many suppliers of chemical compounds,
including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),
Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika
(Buchs, Switzerland), for example. Also, compounds may be
synthesized by methods known in the art.
[0594] High throughput screening methodologies are particularly
envisioned for the detection of modulators of the novel PCSK9b or
PCSK9c polynucleotides and polypeptides described herein. Such high
throughput screening methods typically involve providing a
combinatorial chemical or peptide library containing a large number
of potential therapeutic compounds (e.g., ligand or modulator
compounds). Such combinatorial chemical libraries or ligand
libraries are then screened in one or more assays to identify those
library members (e.g., particular chemical species or subclasses)
that display a desired characteristic activity. The compounds so
identified can serve as conventional lead compounds, or can
themselves be used as potential or actual therapeutics.
[0595] A combinatorial chemical library is a collection of diverse
chemical compounds generated either by chemical synthesis or
biological synthesis, by combining a number of chemical building
blocks (i.e., reagents such as amino acids). As an example, a
linear combinatorial library, e.g., a polypeptide or peptide
library, is formed by combining a set of chemical building blocks
in every possible way for a given compound length (i.e., the number
of amino acids in a polypeptide or peptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks.
[0596] The preparation and screening of combinatorial chemical
libraries is well known to those having skill in the pertinent art.
Combinatorial libraries include, without limitation, peptide
libraries (e.g. U.S. Pat. No. 5,010,175; Furka, 1991, Int. J. Pept.
Prot. Res., 37:487-493; and Houghton et al., 1991, Nature,
354:84-88). Other chemistries for generating chemical diversity
libraries can also be used. Nonlimiting examples of chemical
diversity library chemistries include, peptides (PCT Publication
No. WO 91/019735), encoded peptides (PCT Publication No. WO
93/20242), random bio-oligomers (PCT Publication No. WO 92/00091),
benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as
hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993,
Proc. Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides
(Hagihara et al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., 1992,
J. Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of
small compound libraries (Chen et al., 1994, J. Amer. Chem. Soc.,
116:2661), oligocarbamates (Cho et al., 1993, Science, 261:1303),
and/or peptidyl phosphonates (Campbell et al., 1994, J. Org. Chem.,
59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook,
all supra), peptide nucleic acid libraries (U.S. Pat. No.
5,539,083), antibody libraries (e.g., Vaughn et al., 1996, Nature
Biotechnology, 14(3):309-314) and PCT/US96/10287), carbohydrate
libraries (e.g., Liang et al., 1996, Science, 274-1520-1522) and
U.S. Pat. No. 5,593,853), small organic molecule libraries (e.g.,
benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; and U.S.
Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; and the like).
[0597] Devices for the preparation of combinatorial libraries are
commercially available (e.g., 357 MPS, 390 MPS, Advanced Chem Tech,
Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, a large number of combinatorial libraries are
commercially available (e.g., ComGenex, Princeton, N.J.; Asinex,
Moscow, Russia; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd.,
Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences,
Columbia, Md., and the like).
[0598] In one embodiment, the invention provides solid phase based
in vitro assays in a high throughput format, where the cell or
tissue expressing an ion channel is attached to a solid phase
substrate. In such high throughput assays, it is possible to screen
up to several thousand different modulators or ligands in a single
day. In particular, each well of a microtiter plate can be used to
perform a separate assay against a selected potential modulator,
or, if concentration or incubation time effects are to be observed,
every 5-10 wells can test a single modulator. Thus, a single
standard microtiter plate can assay about 96 modulators. If 1536
well plates are used, then a single plate can easily assay from
about 100 to about 1500 different compounds. It is possible to
assay several different plates per day; thus, for example, assay
screens for up to about 6,000-20,000 different compounds are
possible using the described integrated systems.
[0599] In another of its aspects, the present invention encompasses
screening and small molecule (e.g., drug) detection assays which
involve the detection or identification of small molecules that can
bind to a given protein, i.e., a PCSK9b or PCSK9c polypeptide or
peptide. Particularly preferred are assays suitable for high
throughput screening methodologies.
[0600] In such binding-based detection, identification, or
screening assays, a functional assay is not typically required. All
that is needed is a target protein, preferably substantially
purified, and a library or panel of compounds (e.g., ligands,
drugs, small molecules) or biological entities to be screened or
assayed for binding to the protein target. Preferably, most small
molecules that bind to the target protein will modulate activity in
some manner, due to preferential, higher affinity binding to
functional areas or sites on the protein.
[0601] An example of such an assay is the fluorescence based
thermal shift assay (3-Dimensional Pharmaceuticals, Inc., 3DP,
Exton, Pa.) as described in U.S. Pat. Nos. 6,020,141 and 6,036,920
to Pantoliano et al.; see also, J. Zimmerman, 2000, Gen. Eng. News,
20(8)). The assay allows the detection of small molecules (e.g.,
drugs, ligands) that bind to expressed, and preferably purified,
ion channel polypeptide based on affinity of binding determinations
by analyzing thermal unfolding curves of protein-drug or ligand
complexes. The drugs or binding molecules determined by this
technique can be further assayed, if desired, by methods, such as
those described herein, to determine if the molecules affect or
modulate function or activity of the target protein.
[0602] To purify a PCSK9b or PCSK9c polypeptide or peptide to
measure a biological binding or ligand binding activity, the source
may be a whole cell lysate that can be prepared by successive
freeze-thaw cycles (e.g., one to three) in the presence of standard
protease inhibitors. The PCSK9b or PCSK9c polypeptide may be
partially or completely purified by standard protein purification
methods, e.g., affinity chromatography using specific antibody
described infra, or by ligands specific for an epitope tag
engineered into the recombinant PCSK9b or PCSK9c polypeptide
molecule, also as described herein. Binding activity can then be
measured as described.
[0603] Compounds which are identified according to the methods
provided herein, and which modulate or regulate the biological
activity or physiology of the PCSK9b or PCSK9c polypeptides
according to the present invention are a preferred embodiment of
this invention. It is contemplated that such modulatory compounds
may be employed in treatment and therapeutic methods for treating a
condition that is mediated by the novel PCSK9b or PCSK9c
polypeptides by administering to an individual in need of such
treatment a therapeutically effective amount of the compound
identified by the methods described herein.
[0604] In addition, the present invention provides methods for
treating an individual in need of such treatment for a disease,
disorder, or condition that is mediated by the PCSK9b or PCSK9c
polypeptides of the invention, comprising administering to the
individual a therapeutically effective amount of the PCSK9b or
PCSK9c-modulating compound identified by a method provided
herein.
Antisense and Ribozyme (Antagonists)
[0605] In specific embodiments, antagonists according to the
present invention are nucleic acids corresponding to the sequences
contained in SEQ ID NO:X, or the complementary strand thereof,
and/or to nucleotide sequences contained a deposited clone. In one
embodiment, antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is
separately administered (see, for example, O'Connor, Neurochem.,
56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Raton, Fla. (1988). 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, 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, 251:1300 (1991). The methods are based on binding
of a polynucleotide to a complementary DNA or RNA.
[0606] For example, the use of c-myc and c-myb antisense RNA
constructs to inhibit the growth of the non-lymphocytic leukemia
cell line HL-60 and other cell lines was previously described.
(Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments
were performed in vitro by incubating cells with the
oligoribonucleotide. A similar procedure for in vivo use is
described in WO 91/15580. Briefly, a pair of oligonucleotides for a
given antisense RNA is produced as follows: A sequence
complimentary to the first 15 bases of the open reading frame is
flanked by an EcoR1 site on the 5 end and a HindIII site on the 3
end. Next, the pair of oligonucleotides is heated at 90.degree. C.
for one minute and then annealed in 2.times. ligation buffer (20 mM
TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM
ATP) and then ligated to the EcoR1/Hind III site of the retroviral
vector PMV7 (WO 91/15580).
[0607] 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 the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into receptor
polypeptide.
[0608] In one embodiment, the antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the
antisense nucleic acid of the invention. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in vertebrate cells. Expression
of the sequence encoding a polypeptide of the invention, or
fragments thereof, can be by any promoter known in the art to act
in vertebrate, preferably human cells. Such promoters can be
inducible or constitutive. Such promoters include, but are not
limited to, the SV40 early promoter region (Bernoist and Chambon,
Nature, 29:304-310 (1981), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell,
22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,
Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory
sequences of the metallothionein gene (Brinster et al., Nature,
296:39-42 (1982)), etc.
[0609] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a gene of interest. However, absolute complementarity, although
preferred, is not required. A sequence "complementary to at least a
portion of an RNA" referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex; in the case of double stranded antisense
nucleic acids of the invention, a single strand of the duplex DNA
may thus be tested, or triplex formation may be assayed. The
ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid
Generally, the larger the hybridizing nucleic acid, the more base
mismatches with a RNA sequence of the invention it may contain and
still form a stable duplex (or triplex as the case may be). One
skilled in the art can ascertain a tolerable degree of mismatch by
use of standard procedures to determine the melting point of the
hybridized complex.
[0610] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
Nature, 372:333-335 (1994). Thus, oligonucleotides complementary to
either the 5'- or 3'-non-translated, non-coding regions of a
polynucleotide sequence of the invention could be used in an
antisense approach to inhibit translation of endogenous mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in
accordance with the invention. Whether designed to hybridize to the
5'-, 3'- or coding region of mRNA, antisense nucleic acids should
be at least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific aspects the oligonucleotide is at least 10 nucleotides,
at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
[0611] Antisense oligonucleotides may be single or double stranded.
Double stranded RNA's may be designed based upon the teachings of
Paddison et al., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); and
International Publication Nos. WO 01/29058, and WO 99/32619; which
are hereby incorporated herein by reference.
[0612] SiRNA reagents are specifically contemplated by the present
invention. Such reagents are useful for inhibiting expression of
the polynucleotides of the present invention and may have
therapeutic efficacy. Several methods are known in the art for the
therapeutic treatment of disorders by the administration of siRNA
reagents. One such method is described by Tiscornia et al (PNAS,
100(4):1844-1848 (2003)); WO0409769, filed Jul. 18, 2003; and Reich
S J et al., Mol Vis. 2003 May 30; 9:210-6, which are incorporated
by reference herein in its entirety.
[0613] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652 (1987);
PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication NO:WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res., 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0614] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and
2,6-diaminopurine.
[0615] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0616] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0617] In yet another embodiment, the antisense oligonucleotide is
an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual b-units, the strands run parallel to each
other (Gautier et al., Nucl. Acids Res., 15:6625-6641 (1987)). The
oligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl.
Acids Res., 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue
(Inoue et al., FEBS Lett. 215:327-330 (1987)).
[0618] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(Nucl. Acids Res., 16:3209 (1988)), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.,
85:7448-7451 (1988)), etc.
[0619] While antisense nucleotides complementary to the coding
region sequence of the invention could be used, those complementary
to the transcribed untranslated region are most preferred.
[0620] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al,
Science, 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy mRNAs
corresponding to the polynucleotides of the invention, the use of
hammerhead ribozymes is preferred. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Haseloff
and Gerlach, Nature, 334:585-591 (1988). There are numerous
potential hammerhead ribozyme cleavage sites within each nucleotide
sequence disclosed in the sequence listing. Preferably, the
ribozyme is engineered so that the cleavage recognition site is
located near the 5' end of the mRNA corresponding to the
polynucleotides of the invention; i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional mRNA
transcripts.
[0621] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express the polynucleotides of the invention in vivo. DNA
constructs encoding the ribozyme may be introduced into the cell in
the same manner as described above for the introduction of
antisense encoding DNA. A preferred method of delivery involves
using a DNA construct "encoding" the ribozyme under the control of
a strong constitutive promoter, such as, for example, pol III or
pol II promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous messages and
inhibit translation. Since ribozymes unlike antisense molecules,
are catalytic, a lower intracellular concentration is required for
efficiency.
[0622] Antagonist/agonist compounds may be employed to inhibit the
cell growth and proliferation effects of the polypeptides of the
present invention on neoplastic cells and tissues, i.e. stimulation
of angiogenesis of tumors, and, therefore, retard or prevent
abnormal cellular growth and proliferation, for example, in tumor
formation or growth.
[0623] The antagonist/agonist may also be employed to prevent
hyper-vascular diseases, and prevent the proliferation of
epithelial lens cells after extracapsular cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the
present invention may also be desirous in cases such as restenosis
after balloon angioplasty.
[0624] The antagonist/agonist may also be employed to prevent the
growth of scar tissue during wound healing.
[0625] The antagonist/agonist may also be employed to treat,
prevent, and/or diagnose the diseases described herein.
[0626] Thus, the invention provides a method of treating or
preventing diseases, disorders, and/or conditions, including but
not limited to the diseases, disorders, and/or conditions listed
throughout this application, associated with overexpression of a
polynucleotide of the present invention by administering to a
patient (a) an antisense molecule directed to the polynucleotide of
the present invention, and/or (b) a ribozyme directed to the
polynucleotide of the present invention.
Biotic Associations
[0627] A polynucleotide or polypeptide and/or agonist or antagonist
of the present invention may increase the organisms ability, either
directly or indirectly, to initiate and/or maintain biotic
associations with other organisms. Such associations may be
symbiotic, nonsymbiotic, endosymbiotic, macrosymbiotic, and/or
microsymbiotic in nature. In general, a polynucleotide or
polypeptide and/or agonist or antagonist of the present invention
may increase the organisms ability to form biotic associations with
any member of the fungal, bacterial, lichen, mycorrhizal,
cyanobacterial, dinoflaggellate, and/or algal, kingdom, phylums,
families, classes, genuses, and/or species.
[0628] The mechanism by which a polynucleotide or polypeptide
and/or agonist or antagonist of the present invention may increase
the host organisms ability, either directly or indirectly, to
initiate and/or maintain biotic associations is variable, though
may include, modulating osmolarity to desirable levels for the
symbiont, modulating pH to desirable levels for the symbiont,
modulating secretions of organic acids, modulating the secretion of
specific proteins, phenolic compounds, nutrients, or the increased
expression of a protein required for host-biotic organisms
interactions (e.g., a receptor, ligand, etc.). Additional
mechanisms are known in the art and are encompassed by the
invention (see, for example, "Microbial Signalling and
Communication", eds., R. England, G. Hobbs, N. Bainton, and D. McL.
Roberts, Cambridge University Press, Cambridge, (1999); which is
hereby incorporated herein by reference).
[0629] In an alternative embodiment, a polynucleotide or
polypeptide and/or agonist or antagonist of the present invention
may decrease the host organisms ability to form biotic associations
with another organism, either directly or indirectly. The mechanism
by which a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention may decrease the host organisms
ability, either directly or indirectly, to initiate and/or maintain
biotic associations with another organism is variable, though may
include, modulating osmolarity to undesirable levels, modulating pH
to undesirable levels, modulating secretions of organic acids,
modulating the secretion of specific proteins, phenolic compounds,
nutrients, or the decreased expression of a protein required for
host-biotic organisms interactions (e.g., a receptor, ligand,
etc.). Additional mechanisms are known in the art and are
encompassed by the invention (see, for example, "Microbial
Signalling and Communication", eds., R. England, G. Hobbs, N.
Bainton, and D. McL. Roberts, Cambridge University Press,
Cambridge, (1999); which is hereby incorporated herein by
reference).
[0630] The hosts ability to maintain biotic associations with a
particular pathogen has significant implications for the overall
health and fitness of the host. For example, human hosts have
symbiosis with enteric bacteria in their gastrointestinal tracts,
particularly in the small and large intestine. In fact, bacteria
counts in feces of the distal colon often approach 10.sup.12 per
milliliter of feces. Examples of bowel flora in the
gastrointestinal tract are members of the Enterobacteriaceae,
Bacteriodes, in addition to a-hemolytic streptococci, E. coli,
Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli,
and yeasts. Such bacteria, among other things, assist the host in
the assimilation of nutrients by breaking down food stuffs not
typically broken down by the hosts digestive system, particularly
in the hosts bowel. Therefore, increasing the hosts ability to
maintain such a biotic association would help assure proper
nutrition for the host.
[0631] Aberrations in the enteric bacterial population of mammals,
particularly humans, has been associated with the following
disorders: diarrhea, ileus, chronic inflammatory disease, bowel
obstruction, duodenal diverticula, biliary calculous disease, and
malnutrition. A polynucleotide or polypeptide and/or agonist or
antagonist of the present invention are useful for treating,
detecting, diagnosing, prognosing, and/or ameliorating, either
directly or indirectly, and of the above mentioned diseases and/or
disorders associated with aberrant enteric flora population.
[0632] The composition of the intestinal flora, for example, is
based upon a variety of factors, which include, but are not limited
to, the age, race, diet, malnutrition, gastric acidity, bile salt
excretion, gut motility, and immune mechanisms. As a result, the
polynucleotides and polypeptides, including agonists, antagonists,
and fragments thereof, may modulate the ability of a host to form
biotic associations by affecting, directly or indirectly, at least
one or more of these factors.
[0633] Although the predominate intestinal flora comprises
anaerobic organisms, an underlying percentage represents aerobes
(e.g., E. coli). This is significant as such aerobes rapidly become
the predominate organisms in intraabdominal infections--effectively
becoming opportunistic early in infection pathogenesis. As a
result, there is an intrinsic need to control aerobe populations,
particularly for immune compromised individuals.
[0634] In a preferred embodiment, a polynucleotides and
polypeptides, including agonists, antagonists, and fragments
thereof, are useful for inhibiting biotic associations with
specific enteric symbiont organisms in an effort to control the
population of such organisms.
[0635] Biotic associations occur not only in the gastrointestinal
tract, but also on an in the integument. As opposed to the
gastrointestinal flora, the cutaneous flora is comprised almost
equally with aerobic and anaerobic organisms. Examples of cutaneous
flora are members of the gram-positive cocci (e.g., S. aureus,
coagulase-negative staphylococci, micrococcus, M. sedentarius),
gram-positive bacilli (e.g., Corynebacterium species, C.
minutissimum, Brevibacterium species, Propoionibacterium species,
P. acnes), gram-negative bacilli (e.g., Acinebacter species), and
fungi (Pityrosporum orbiculare). The relatively low number of flora
associated with the integument is based upon the inability of many
organisms to adhere to the skin. The organisms referenced above
have acquired this unique ability. Therefore, the polynucleotides
and polypeptides of the present invention may have uses which
include modulating the population of the cutaneous flora, either
directly or indirectly.
[0636] Aberrations in the cutaneous flora are associated with a
number of significant diseases and/or disorders, which include, but
are not limited to the following: impetigo, eethyma, blistering
distal dactulitis, pustules, folliculitis, cutaneous abscesses,
pitted keratolysis, trichomycosis axcillaris, dermatophytosis
complex, axillary odor, erthyrasma, cheesy foot odor, acne, tinea
versicolor, seborrheic dermititis, and Pityrosporum folliculitis,
to name a few. A polynucleotide or polypeptide and/or agonist or
antagonist of the present invention are useful for treating,
detecting, diagnosing, prognosing, and/or ameliorating, either
directly or indirectly, and of the above mentioned diseases and/or
disorders associated with aberrant cutaneous flora population.
[0637] Additional biotic associations, including diseases and
disorders associated with the aberrant growth of such associations,
are known in the art and are encompassed by the invention. See, for
example, "Infectious Disease", Second Edition, Eds., S. L.,
Gorbach, J. G., Bartlett, and N. R., Blacklow, W.B. Saunders
Company, Philadelphia, (1998); which is hereby incorporated herein
by reference).
Pheromones
[0638] In another embodiment, a polynucleotide or polypeptide
and/or agonist or antagonist of the present invention may increase
the organisms ability to synthesize and/or release a pheromone.
Such a pheromone may, for example, alter the organisms behavior
and/or metabolism.
[0639] A polynucleotide or polypeptide and/or agonist or antagonist
of the present invention may modulate the biosynthesis and/or
release of pheromones, the organisms ability to respond to
pheromones (e.g., behaviorally, and/or metabolically), and/or the
organisms ability to detect pheromones. Preferably, any of the
pheromones, and/or volatiles released from the organism, or
induced, by a polynucleotide or polypeptide and/or agonist or
antagonist of the invention have behavioral effects the
organism.
Other Activities
[0640] The polypeptide of the present invention, as a result of the
ability to stimulate vascular endothelial cell growth, may be
employed in treatment for stimulating re-vascularization of
ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions.
These polypeptide may also be employed to stimulate angiogenesis
and limb regeneration, as discussed above.
[0641] The polypeptide may also be employed for treating wounds due
to injuries, burns, post-operative tissue repair, and ulcers since
they are mitogenic to various cells of different origins, such as
fibroblast cells and skeletal muscle cells, and therefore,
facilitate the repair or replacement of damaged or diseased
tissue.
[0642] The polypeptide of the present invention may also be
employed stimulate neuronal growth and to treat, prevent, and/or
diagnose neuronal damage which occurs in certain neuronal disorders
or neuro-degenerative conditions such as Alzheimer's disease,
Parkinson's disease, and AIDS-related complex. The polypeptide of
the invention may have the ability to stimulate chondrocyte growth,
therefore, they may be employed to enhance bone and periodontal
regeneration and aid in tissue transplants or bone grafts.
[0643] The polypeptides of the present invention may be employed to
stimulate growth and differentiation of hematopoietic cells and
bone marrow cells when used in combination with other
cytokines.
[0644] The polypeptide of the invention may also be employed to
maintain organs before transplantation or for supporting cell
culture of primary tissues.
[0645] The polypeptide of the present invention may also be
employed for inducing tissue of mesodermal origin to differentiate
in early embryos.
[0646] The polypeptide or polynucleotides and/or agonist or
antagonists of the present invention may also increase or decrease
the differentiation or proliferation of embryonic stem cells,
besides, as discussed above, hematopoietic lineage.
[0647] The polypeptide or polynucleotides and/or agonist or
antagonists of the present invention may also be used to modulate
mammalian characteristics, such as body height, weight, hair color,
eye color, skin, percentage of adipose tissue, pigmentation, size,
and shape (e.g., cosmetic surgery). Similarly, polypeptides or
polynucleotides and/or agonist or antagonists of the present
invention may be used to modulate mammalian metabolism affecting
catabolism, anabolism, processing, utilization, and storage of
energy.
[0648] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may be used to change a mammal's mental
state or physical state by influencing biorhythms, caricadic
rhythms, depression (including depressive diseases, disorders,
and/or conditions), tendency for violence, tolerance for pain,
reproductive capabilities (preferably by Activin or Inhibin-like
activity), hormonal or endocrine levels, appetite, libido, memory,
stress, or other cognitive qualities.
[0649] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used to prepare individuals
for extraterrestrial travel, low gravity environments, prolonged
exposure to extraterrestrial radiation levels, low oxygen levels,
reduction of metabolic activity, exposure to extraterrestrial
pathogens, etc. Such a use may be administered either prior to an
extraterrestrial event, during an extraterrestrial event, or both.
Moreover, such a use may result in a number of beneficial changes
in the recipient, such as, for example, any one of the following,
non-limiting, effects: an increased level of hematopoietic cells,
particularly red blood cells which would aid the recipient in
coping with low oxygen levels; an increased level of B-cells,
T-cells, antigen presenting cells, and/or macrophages, which would
aid the recipient in coping with exposure to extraterrestrial
pathogens, for example; a temporary (i.e., reversible) inhibition
of hematopoietic cell production which would aid the recipient in
coping with exposure to extraterrestrial radiation levels; increase
and/or stability of bone mass which would aid the recipient in
coping with low gravity environments; and/or decreased metabolism
which would effectively facilitate the recipients ability to
prolong their extraterrestrial travel by any one of the following,
non-limiting means: (i) aid the recipient by decreasing their basal
daily energy requirements; (ii) effectively lower the level of
oxidative and/or metabolic stress in recipient (i.e., to enable
recipient to cope with increased extraterrestrial radiation levels
by decreasing the level of internal oxidative/metabolic damage
acquired during normal basal energy requirements; and/or (iii)
enabling recipient to subsist at a lower metabolic temperature
(i.e., cryogenic, and/or sub-cryogenic environment).
[0650] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used to increase the efficacy
of a pharmaceutical composition, either directly or indirectly.
Such a use may be administered in simultaneous conjunction with
said pharmaceutical, or separately through either the same or
different route of administration (e.g., intravenous for the
polynucleotide or polypeptide of the present invention, and orally
for the pharmaceutical, among others described herein.).
[0651] Polypeptide or polynucleotides and/or agonist or antagonists
of the present invention may also be used as a food additive or
preservative, such as to increase or decrease storage capabilities,
fat content, lipid, protein, carbohydrate, vitamins, minerals,
cofactors or other nutritional components.
[0652] Also preferred is a method of treatment of an individual in
need of an increased level of a protein activity, which method
comprises administering to such an individual a pharmaceutical
composition comprising an amount of an isolated polypeptide,
polynucleotide, or antibody of the claimed invention effective to
increase the level of said protein activity in said individual.
[0653] 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.
[0654] 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.
[0655] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, GENBANK.RTM. Accession numbers,
SWISS-PROT.RTM. Accession numbers, or other disclosures) in the
Background of the Invention, Detailed Description, Brief
Description of the Figures, and Examples is hereby incorporated
herein by reference in their entirety. Further, the hard copy of
the Sequence Listing submitted herewith, in addition to its
corresponding Computer Readable Form, are incorporated herein by
reference in their entireties.
REFERENCES
[0656] Lusis, A J. Atherosclerosis. Nature. 407, 233-241 (2000).
[0657] Fredrickson, D S., Levy, R I. & Lees, R S, N. Eng. J
Med. 276, 273-281 (1967). [0658] Austin, M A., King, M C., Bawol, R
D., Hulley, S B. & Friedman, G D. Am. J Epidemiol. 125, 308-318
(1987). [0659] Perusse, L. Arteriosclerosis. 9, 308-318 (1989).
[0660] Rice, T., Vogler, G P., Laskarzewski, P M., Perry, T S.
& Rao, D C. Hum. Biol. 63, 419-439 (1991). [0661] Khachadurian,
A K., Am. J Med. 37, 402-407, (1964). [0662] Morganroth, J., Levy,
R I., McMahon, A E. & Gotto, A M Jr. J Pediatr. 85, 639-643
(1974). [0663] Goldstein, J L. & Brown, M S., Johns Hopkins
Med. J 143, 8-16 (1978). [0664] Brown, M S. & Goldstein, J L.
Proc. Natl. Acad. Sci. USA. 96, 11041-11048 (1999). [0665] Varret,
M. et al. Am. J. Hum. Genet. 64, 1378-1387 (1999). [0666]
Innerarity, T L. et al. Proc. Natl. Acad. Sci. USA. 84, 6919-6923
(1987). [0667] Hunt, S C. et al. Arterioscler. Thromb. Vasc. Biol.
20, 1089-1093 (2000). [0668] Horton J. D., Cohen J. C., Hobbs H. H.
2007. Molecular biology of PCSK9: its role in LDL metabolism.
Trends in Biochem. Sci. 332(2): 71-77. [0669] Berge, K. E., Ose L,
Leren, T. P. 2006. Missense mutations in the PCSK9 gene are
associated with hypocholesterolemia and possibly increased response
to statin therapy. Arterioscl. Thromb. Vasc. Biol. 26(5):
1094-1100. [0670] Zhao Z, Tuakli-Wosornu, Y, Lagace T. A., Kinch
L., Grishin N. V., Horton J. D., Cohen J. C., Hobbs H. H. 2006.
Molecular characterization of loss-of-function mutations in PCSK
and identification of a compound heterozygote. Amer. J. Human Gen.
79: 514-523. [0671] Sun X.-M, Eden E. r., Tosi I., Neuwirth C. K.,
Wile D., Naoumova R. P., Soutar A. K. 2005. Evidence for effect of
mutant PCSK9 on apolipoprotein B secretion as the cause of
unusually severe dominant hypercholesterolemia. Hum Mol. Gen.
14(9). 1161-1169. [0672] Maxwell K. N., Breslow J. L. 2004.
Adenoviral-mediated expression of Pcsk9 in mice results in a
low-density lipoprotein receptor knockout phenotype. Proc. Nat.
Amer. Sci. 101: 7100-7105. [0673] Benjannet S, Rhainds, D.,
Essalmani R., Mayne J., Wickham L., Jin W., Asselin M.-C., Hamelin
J., Varret M., Allard D., Trillard M., Abifadel M., Tebon A., Attie
A. D. Rader D. J., Boileau C., Brissette L., Chretien M., Prat A.,
Seidah N. G. 2004. NARC-1/PCSK9 and its natural mutants. J. Bio.
Chem. 279: 48865-48875.
EXAMPLES
Example 1
Method of Cloning the Novel Human PCSK9b Polynucleotide of the
Present Invention
[0674] Using the sequence AK124635.1, a sense 80-mer oligo with
biotin on the 5' end was designed with the following sequence:
TABLE-US-00004 (SEQ ID NO: 7)
5'bGGCGCTTTCACCAGTGGCTGGGATGTGCTCTGTAGTTTCTGTGTGTT
AACTATAAGGTTGACTTTATGCTCATTCCCTCC-3'
[0675] One microliter (200 picograms) of the biotinylated oligo was
added to six microliters (six micrograms) of a anti-sense
single-stranded covalently closed circular brain cDNA library and
seven microliters of 100% formamide in a 0.2 ml PCR tube. The
mixture was heated in a thermal cycler to 95.degree. C. for 2
minutes. Fourteen microliters of 2.times. hybridization buffer (50%
formamide, 1.5M NaCl, 0.04M NaPO.sub.4 pH7.2, 5 mM EDTA, 0.2% SDS)
were added to the heated probe/cDNA library mixture and incubated
at 42.degree. C. for 24 hours. Hybrids between the biotinylated
oligo and the circular cDNA were isolated by diluting the
hybridization mixture to 228 microliters in a solution containing
1M NaCl, 10 mM Tris-HCl pH7.5, 1 mM EDTA, pH8.0 and adding 125
microliters of streptavidin magnetic beads. This solution was
incubated at 42.degree. C. for 60 minutes mixing every 5 minutes to
resuspend the beads. The beads were separated from the solution
with a magnet and washed three times in 200 microliters of
0.1.times.SSPE, 0.1% SDS at 45.degree. C.
[0676] The single stranded cDNAs were released from the
biotinylated oligo/streptavidin magnetic bead complex by adding 50
microliters of 0.1N NaOH and incubating at room temperature for 10
minutes. Six microliters of 3M Sodium Acetate were added along with
20 micrograms of glycogen and the solution was ethanol precipitated
with 120 microliters of 100% ethanol. The DNA was resuspended in 12
microliters of TE (10 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0).
[0677] The single stranded cDNA was converted into double strands
in a thermal cycler by mixing 5 microliters of the captured DNA
with 1.5 microliters 10 micromolar gene specific sense repair
primer with the following sequence: 5'-CTAGGCCTCCCTTTCCTTGT-3' (SEQ
ID NO:13), and 1.5 microliters of 10.times.PCR buffer. The mixture
was heated to 95.degree. C. for 20 seconds, then ramped down to
59.degree. C. At this time 15 microliters of a repair mix, that was
preheated to 70.degree. C., was added (repair mix contains 0.5
microliters of 10 mM dNTPs (2.5 mM each), 1.5 microliters of
10.times.PCR buffer, 12.75 microliters of water, and 0.25
microliters or 1.25 U of Taq polymerase). The solution was ramped
back to 73.degree. C. and incubated for 23 minutes. The repaired
DNA was ethanol precipitated and resuspended in 10 microliters of
TE. Two microliters were electroporated into E. coli DH12S cells
and resulting colonies were screened by PCR, using a primer pair
designed from the sequence AK124635.1, to identify the proper
cDNAs.
[0678] Oligos used to identify the cDNA by PCR:
TABLE-US-00005 PCSK9- 5'-CTAGGCCTCCCTTTCCTTGT-3' (SEQ ID NO: int3s
8) PCSK9- 5'-TTCCAAGGTGACATTTGTGG-3' (SEQ ID NO: int3a 9)
[0679] 95 colonies were screened, and one cDNA clone was positive
by PCR. The full-length sequence was obtained.
[0680] The full-length nucleotide sequence and the encoded
polypeptide for PCSK9b are shown in FIGS. 1A-C.
[0681] Analysis of this sequence indicated it was a variant of
proprotein convertase subtilisin/kexin type 9 (PCSK9).
Example 2
Method of Cloning the Novel Human PCSK9c Polynucleotide of the
Present Invention
[0682] A method nearly identical to the method described in Example
1 was utilized to clone the variant PCSK9c with the exceptions that
follow. From the sequence NM.sub.--174936.2, an anti-sense 80-mer
oligo with biotin on the 5' end was designed with the following
sequence:
TABLE-US-00006 (SEQ ID NO: 14).
5'bTGGCAGGCGGCGTTGAGGACGCGGCTGTACCCACCCGCCAGGGGCAG
CAGCACCACCAGTGGCCCCACAGGCTGGACCA-3'
and was hybridized to sense single-stranded covalently closed
circular brain/testis cDNA library as described above. Following
release from the biotinylated oligo/streptavidin magnetic bead
complex, the cDNA was precipitated as described above. The single
stranded cDNA was converted into double strands as described above
except that the following gene specific anti-sense repair primer
was used: 5'-GAGTAGAGGCAGGCATCGTC-3' (SEQ ID NO:17). Screening
colonies to identify proper cDNAs was performed with the following
PCR primers:
TABLE-US-00007 PCSK9- 5'-GCCTGGAGTTTATTCGGAAA-3' (SEQ ID NO: ex6s
15) PCSK9- 5'-GAGTAGAGGCAGGCATCGTC-3' (SEQ ID NO: ex6a 16)
[0683] 95 colonies were screened, and one cDNA clone was positive
by PCR. The full-length sequence of the cDNA was obtained. This
clone was also found to represent a variant of proprotein
convertase subtilisin/kexin type 9 (PCSK9).
[0684] The full-length nucleotide sequence and the encoded
polypeptide for PCSK9c are shown in FIGS. 1A-D.
Example 3
Method of Assessing the Expression Profile of the Novel PCSK9b and
PCSK9c Polypeptides of the Present Invention Using Expanded mRNA
Tissue and Cell Sources
[0685] Total RNA from tissues was isolated using the TRIZOL.RTM.
protocol (Invitrogen) and quantified by determining its absorbance
at 260 nM. An assessment of the 18s and 28s ribosomal RNA bands was
made by denaturing gel electrophoresis to determine RNA
integrity.
[0686] The specific sequence to be measured was aligned with
related genes found in GENBANK.RTM. to identity regions of
significant sequence divergence to maximize primer and probe
specificity. Gene-specific primers and probes were designed using
the ABI primer express software to amplify small amplicons (150
base pairs or less) to maximize the likelihood that the primers
function at 100% efficiency. All primer/probe sequences were
searched against Public Genbank databases to ensure target
specificity. Primer and probe sequences were designed to hybridize
to regions shared by both PCSK9b and PCSK9c, in addition to wild
type PCSK9. Primers and probes were obtained from ABI.
[0687] For PCSK9b and PCSK9c, the primer probe sequences were as
follows:
TABLE-US-00008 Forward 5'-CCTGCGCGTGCTCAACT-3' (SEQ ID NO: 10)
Primer Reverse 5'-CCGAATAAACTCCAGGCCTATG-3' (SEQ ID NO: Primer 11)
TAQMAN .RTM. 5'-CCGCTAACCGTGCCCTTCCCTTG-3' (SEQ ID NO: Probe
12)
DNA Contamination
[0688] To access the level of contaminating genomic DNA in the RNA,
the RNA was divided into 2 aliquots and one half was treated with
Rnase-free Dnase (Invitrogen). Samples from both the Dnase-treated
and non-treated were then subjected to reverse transcription
reactions with (RT+) and without (RT-) the presence of reverse
transcriptase. TAQMAN.RTM. assays were carried out with
gene-specific primers (see above) and the contribution of genomic
DNA to the signal detected was evaluated by comparing the threshold
cycles obtained with the RT+/RT- non-Dnase treated RNA to that on
the RT+/RT- Dnase treated RNA. The amount of signal contributed by
genomic DNA in the Dnased RT- RNA must be less that 10% of that
obtained with Dnased RT+ RNA. If not the RNA was not used in actual
experiments.
Reverse Transcription Reaction and Sequence Detection
[0689] 100 ng of Dnase-treated total RNA was annealed to 2.5 .mu.M
of the respective gene-specific reverse primer in the presence of
5.5 mM Magnesium Chloride by heating the sample to 72.degree. C.
for 2 min and then cooling to 55.degree. C. for 30 min. 1.25
U/.mu.l of MuLv reverse transcriptase and 500 .mu.M of each dNTP
was added to the reaction and the tube was incubated at 37.degree.
C. for 30 min. The sample was then heated to 90.degree. C. for 5
min to denature enzyme.
[0690] Quantitative sequence detection was carried out on an ABI
PRISM.RTM. 7700 by adding to the reverse transcribed reaction 2.5
.mu.M forward and reverse primers, 2.0 .mu.M of the TAQMAN.RTM.
probe, 500 .mu.M of each dNTP, buffer and 5 U AMPLITAQ GOLD.RTM..
The PCR reaction was then held at 94.degree. C. for 12 min,
followed by 40 cycles of 94.degree. C. for 15 sec and 60.degree. C.
for 30 sec.
Data Handling
[0691] The threshold cycle (Ct) of the lowest expressing tissue
(the highest Ct value) was used as the baseline of expression and
all other tissues were expressed as the relative abundance to that
tissue by calculating the difference in Ct value between the
baseline and the other tissues and using it as the exponent in
2.sup.(.DELTA.Ct)
[0692] The expanded expression profile of the PCSK9b and PCSK9c
polypeptides is provided in FIG. 6, and described elsewhere herein.
Table IV identifies the analyzed tissues.
TABLE-US-00009 TABLE IV Number Tissue 1 adipose_mesenteric_ileum 2
blood_vessel_cerebral 3 blood_vessel_pulmonary 4 blood_vessel_renal
5 brain_amygdala 6 brain_cerebellum 7
brain_cortex_cingulate_anterior 8 brain_cortex_cingulate_posterior
9 brain_cortex_frontal_medial 10 brain_cortex_temporal 11
brain_dorsal_raphe_nucleus 12 brain_hippocampus 13
brain_hypothalamus_anterior 14 brain_hypothalamus_posterior 15
brain_locus_coeruleus 16 brain_medulla_oblongata 17
brain_nucleus_accumbens 18 brain_substantia_nigra 19 breast 20
caecum 21 colon 22 duodenum 23 heart_left_atria 24
heart_left_ventricle 25 ileum 26 jejunum 27 kidney_cortex 28
kidney_medulla 29 kidney_pelvis 30 liver_parenchyma 31
lung_bronchus_primary 32 lung_bronchus_tertiary 33 lung_parenchyma
34 lymph_gland_tonsil 35 muscle_skeletal 36 oesophagus 37 pancreas
38 rectum 39 spinal_cord 40 stomach_antrum 41 stomach_body 42
stomach_pyloric_canal 43 testis
Example 4
Method of Characterizing the PCSK9b or PCSK9c Polypeptides of the
Present Invention
[0693] Expression analysis of PCSK9b and PCSK9c polynucleotides and
polypeptides using both quantitative PCR and Western blots, in
addition to the affect of PCSK9b and PCSK9c on LDL update by the
LDLR, was assessed according to the methods outlined herein.
Briefly:
Materials and Methods
[0694] Gene Cloning.
[0695] PCSK9 variant b was cloned from a human brain cDNA library,
and variant c was clone from a brain/testis cDNA library. Each
variant product was inserted in two expression vectors CD3 MYCHIS
and pEF-DESTS1 tagged with His and V5 or Myc epitopes at the
C-terminus (#3114 for PCSK9 variant b-pCD3 MYCHIS, 3115 for PCSK9
variant b-pEF-DESTS1, #3116 for PCSK9 variant c-pCD3 MYCHIS, #3117
for PCSK9 variant c-pEF-DESTS1). Wild type PCSK9 and PCSK9 mutant
D374 were cloned by PCR product insertion in pcDNA3 vector.
[0696] Cell Culture.
[0697] All cells lines including HepG2, Chinese hamster ovary cells
(CHO), and HEK293, were derived from American Type Cell Culture
(Manassa, Va.). Both HepG2 and HEK293 cells were grown in DMEM
medium (Invitrogen Gibco, Carlsbad, Calif.) supplemented with 10%
heat inactivated fetal bovine serum (FBS, Gibco), 1 mM sodium
pyruvate (Gibco), 2 mM Glutamax (Gibco), 100 ug/ml streptomycin
sulfate and 100 U/ml penicillin (Gibco). CHO cells were grown with
the F12 medium with Glutamax (Gibco) supplemented with 10% fetal
bovine serum.
[0698] DiI-LDL Uptake Assay.
[0699] 15,000 cells per well were plated in the 96-well poly D
lysine-coated plate with the above 10% FBS growth medium and grown
in 37.degree. C. incubator overnight. On day two, the medium was
changed to 5% lipoprotein deficient serum (LPDS) growth medium
supplemented with 50 .mu.M sodium mevalonate, and 100 ng/ml of an
HMG CoA reductase inhibitor. On day three, aliquots of
fluorescent-labeled LDL (DiI-LDL) (source: Biomedical Technologies,
Stoughton, Mass.) were added into the medium at a concentration of
10 ug/ml and continued to incubate for two more hours. Formaldehyde
was added to the cells at a concentration of 4% together with
Hoechst dye at 10 ug/ml. After 20 minute incubation at room
temperature, cells were washed with PBS three times and read on LJL
Biosystems (Molecular Device, Sunnyvale, Calif.) for DNA content at
the excitation wavelength of 360 nm, and emission wavelength of 460
nm. Cells were then lysed by NaOH (0.001 N) and SDS (0.001%).
DiI-LDL uptake was measured on LJL at the excitation wavelength of
540 nm, and emission wavelength of 580 nm. The DiI-LDL uptake data
were normalized to DNA content by taking the ratio of DiI-LDL
reading to Hoechst dye reading.
[0700] mRNA Quantification.
[0701] Total RNA isolation from cells was performed by the 6100
Nucleotide Acid PrepStation (ABI, Foster City, Calif.). Cells were
lysed by the Nucleic Acid Lysis Solution (ABI) following the
manufacturer's instruction. Lysed cells were loaded to the
instrument for RNA extraction based on the protocol suggested by
the manufacturer. RNA quantity was measured by the SPECTRAMAX.RTM.
Plus (Molecular Device, Sunnyvale, Calif.).
[0702] Quantitative RT-PCR:
[0703] cDNA was prepared by using iScript cDNA synthesis reagents
(Biorad, Hercules, and CA) following manufacturer instructions.
Quantitative PCR was performed using iTaq SYBR.RTM. Green Supermix
with ROX reagents (Bio-Rad) on an ABI PRISM.RTM. 7900HT Sequence
Detection System. Primer sequences for the assays were: wild type
PCSK9, forward TGTCTTTGCCCAGAGCATCC (SEQ ID NO:30), reverse
TATTCATCCGCCCGGTACC (SEQ ID NO:31); variant b, forward
AGATGTCATCAATGAGGCCT (SEQ ID NO:32), reverse AGCTGCCAACCTGCAAAAAC
(SEQ ID NO:33); variant b/c, forward CTCTGAGGTTGTGACTCGTGTGA (SEQ
ID NO:34), reverse AGCGTTCTCCACTCCACAAGA (SEQ ID NO:35); LDLR,
forward GAGAATGATCTGCAGCACCCA (SEQ ID NO:36), reverse
TGCTGATGACGGTGTCATAGG (SEQ ID NO:37). Gene expression levels were
normalized to GAPDH mRNA.
[0704] Western Blot Analysis.
[0705] Cells were plated in DMEM supplemented with 10% FBS on
6-well plates. On day two, 4 ug/well of plasmid DNA were
transfected by Lipofectamine 2000 (Invitrogen) following
manufacturer's suggestion. After 24 hr, the medium was switched to
DMEM media with 5% LPDS, 50 uM sodium mevalonate, and 100 ng/ml of
BMS-423526. After an additional 24 hr, the medium was removed and
the cells were lysed with the buffer containing 0.1% Triton X-100,
150 mM NaCl, 10 mM Tris-HCl (pH 7.4) plus Complete Protease
Inhibitor Cocktail (Roche). Protein concentration was assayed with
BioRad protein assay (BioRad). 10 ug of proteins from lysate or 15
ul from a total of 2 ml conditioned medium were loaded on 4-15%
Tris-HCl gels (Invitrogen). After protein transfer, membranes were
incubated with anti-hPCSK9 primary antibody or anti-LDLR primary
antibody, followed by IRdye 800 labeled anti-mouse IgG for PCSK9 or
IRdye 680 labeled anti-rabbit IgG for LDLR. Protein bands were
subsequently detected using the ODYSSEY.RTM. Infrared Imaging
System (Li-Cor, Lincoln, Nebr.) according to manufacturer's
instruction.
Results
[0706] Overexpression of PCSK9 Variants in Cells.
[0707] PCSK9 variant b and c were transfected into HEK and CHO as
well as HepG2 cells. Both variants were expressed in these cells
with mRNA levels comparable to that of wild type (full length)
PCSK9 and the gain-of-function mutant D374Y-PCSK9 (FIG. 7A). As
expected, LDLR mRNA levels were not affected by expression of the
PCSK9 mRNAs in these cells (FIG. 7B), consistent with previous
studies using known PCSK9 variants and mutants (Maxwell K. N.,
Breslow J. L., Proc. Nat. Amer. Sci., 101:7100-7105 (2004);
Benjannet S., et al., J. Bio. Chem. 279:48865-48875 (2004)).
Western blot results using PCSK9 antibody demonstrated that both
variant b and variant c were also expressed as protein in the
transfected cells. Variant b was expressed as an approximately
33-kDa protein and variant c was expressed as an approximately
57-kDa protein (FIG. 8A). The apparent molecular weights of the
variant proteins as judged by SDS-PAGE mobility corresponded to
their predicted molecular weight from sequence analysis. Compared
with wild type PCSK9 protein, the protein expression levels of the
two variants were lower yet readily detectable by antibody.
[0708] In the conditioned media of the cell culture, both PCSK9-b
and -c protein expression were observed by Western blot (FIG. 8B),
indicating that these two variants may be secreted by the
cells.
[0709] PCSK9 Variants Exhibit Functional Activity in DiI-LDL Uptake
Assay.
[0710] DiI-LDL uptake is a standardized functional assay for LDLR
receptor, while PCSK9 activity reduces LDLR. Therefore DiI-LDL
uptake by cells can be used as a functional assay for PCSK9
activity. Transient expression of PCSK9 variant c decreased the
uptake of DiI-LDL in HepG2 cells, compared to vector control,
indicating that variant c is competent to express PCSK9 functional
activity on LDLR (FIG. 9A). In this assay, variant c showed greater
activity than wild-type PCSK9. Variant b showed greater activity
than wildtype PCSK9, but was less than the PCSK9b variant. Based on
sequence deduction and the molecular weights seen in Western blots,
variant PCSK9c appears to form a nearly complete PCSK9 protein
(compared to wild type), while variant PCSK9c lacks part of the
C-terminal domain, consistent with the activity assay findings.
[0711] Consistent with the DiI-LDL uptake assay, expression of
PCSK9 variant c decreased LDLR protein level in CHO cells assayed
by Western blot, while expression of variant b did not appear to
affect LDLR protein level under these conditions (FIG. 9B).
Conclusions
[0712] The two PCSK9 variants, b and c, were examined in a series
of experiments to detect expression and function These two variants
differ from the wild type PCSK9 by the lack of the signal peptide
and prodomain as well as 22 amino acid in the N-terminus of the
catalytic domain. In variant b, a portion of the C-terminal domain
is also absent.
[0713] Both variants were expressed at the mRNA level in basal
(sterol suppressed) HepG2 cells, with mRNA approximately 1% of the
wild-type PCSK9 mRNA level in the presence of medium containing 10%
fetal bovine serum (data not shown). Media containing 10% fetal
bovine serum (FBS) is known to suppress sterol-sensitive genes,
including PCSK9. However, under conditions including 5%
lipoprotein-deficient serum (LPDS) in the medium, which is known to
induce sterol-sensitive genes including PCSK9, the PCSK9c variant
mRNA was highly induced, with levels reaching approximately 10% of
the wild-type PCSK9 mRNA level (data not shown). Transient
expression of the two variants using standard plasmid expression
vectors in mammalian cell lines resulted in the PCSK9b variant
being expressed as a 33 kDa protein, and the PCSK9c variant being
expressed as a 57 kDa protein. Both of the PCSK9 variant proteins
were detectable in conditioned medium as well as in cell lysates,
indicating that both of them may also be secreted from cells. Based
on the DiI-LDL uptake assay and western blot data, transient
expression of variant c decreased LDLR levels in cells, while
variant b exhibited lesser effects.
[0714] These data suggest that the novel PCSK9 variants, especially
variant c, are not only expressed but are functionally competent
and exhibit PCSK9 activity by interacting with the LDLR and
decreasing LDLR levels, the main function identified for wild-type
PCSK9 to date. The data support a biological role for the novel
variants in modulating the LDLR and suggest they are useful in
biotechnology and discovery applications involving PCSK9
expression, function, and screening.
Example 5
Method of Measuring the Proteinase Activity of the PCSK9b or PCSK9c
Polypeptides of the Present Invention
[0715] A number of assays known in the art may be utilized to
demonstrate the proteinase activity of the PCSK9b and PCSK9c
polypeptides of the present invention. Specifically, the method
outlined in Naureckiene et al. (Arch. Biochem. Biophys., 420:55-67
(2003); which is hereby incorporated by reference in its entirety
herein) may be utilized. Briefly:
Expression of PCSK9b and PCSK9c
[0716] Escherichia coli BL21 (DE3) cells may be transformed with
prN1 (.DELTA.SP; .DELTA.C@Q453)/6.times.His. Transformants are
grown in LB medium (supplemented to 100 .mu.g/ml with
carbenicillin) at 30.degree. C. to an OD.sub.600 of 0.6. Expression
is induced with 0.1 mM IPTG and the culture grown for an additional
3.5 h. Cells are harvested by centrifugation 30 min. at 14,000 g,
and stored at -80 C.
Purification of PCSK9b and PCSK9c
[0717] Procedures may be performed at 4.degree. C. Cells from 2 L
of culture are resuspended in 25 ml lysis buffer (50 mM Tris-HCl,
pH 8.0; 150 mM NaCl; 10% glycerol; and 1 M NDSB) in the presence or
absence of an EDTA-free proteinase inhibitor cocktail (Roche).
After cell lysis by sonication, the cell debris is removed by
centrifugation (30 min, 14,000 g). The supernatant containing
soluble proteins is loaded onto a pre-equilibrated 1 ml
Ni-nitrilotriacetic acid (NTA) Agarose column (Qiagen). The column
is washed with 20 ml lysis buffer containing 20 mM imidazole to
remove weakly bound contaminating proteins. The purified protein is
eluted with 5 ml lysis buffer containing 200 mM imidazole. Eluted
protein is dialyzed overnight against 1 L Buffer A: 50 mM Tris-HCl,
pH 8.0, 50 mM NaCl, and 10% glycerol. Dialyzed protein is loaded
onto a 1 ml MONO Q.RTM. column (Amersham Biosciences) equilibrated
with the same buffer. The column is washed with 10 ml of the same
buffer and proteins are eluted with 12 ml NaCl gradient (0.05-1.0
M) in the same buffer. One milliliter fractions are collected and
analyzed for purity on SDS-PAGE. Fractions containing purified
protein are pooled and dialyzed against 1000 ml storage buffer (50
mM Tris-HCl, pH 8.0, 50 mM NaCl, and 10% glycerol), aliquoted, and
stored at -80 C. In parallel, a purification from cells transformed
with empty vector pET21a is performed to generate material for use
as a negative control in enzymatic assays.
Proteinase Activity Assay
[0718] Subtilisin-, furin-, and TPP-specific fluorogenic substrates
may be purchased from Bachem. Custom-made proteinase substrates can
be synthesized by New England Peptide, Inc. Sequences of peptide
substrates are as follows:
TABLE-US-00010 Peptide Sequence A Dnp-FAQSIPK-AMC (SEQ ID NO: 18) B
Dnp-DSLVFAK-AMC (SEQ ID NO: 19) C Dnp-FANAIPK-AMC (SEQ ID NO:
20)
[0719] Proteolytic activity may be assayed at 37.degree. C. using
100 .mu.M substrate and 0.12 .mu.M enzyme in various buffers,
including 50 mM Tris-HCL, pH 7.5 to pH 11, 1 mM to 20 mM CaCl2, and
0.5% TX-100 at 37.degree. C. Protein derived from an empty vector
transformation is included as a negative control for enzymatic
activity. Continuous and end-point fluorimetric measurements may be
performed on spectrophotometer (.lamda..sub.ex=350 nm,
.lamda..sub.em=450 nm).
Example 6
Method of Screening for Compounds that Interact with the PCSK9b or
PCSK9c Polypeptide
[0720] The following assays are designed to identify compounds that
bind to the PCSK9b or PCSK9c polypeptide, bind to other cellular
proteins that interact with the PCSK9b or PCSK9c polypeptide, and
to compounds that interfere with the interaction of the PCSK9b or
PCSK9c polypeptide with other cellular proteins.
[0721] Such compounds can include, but are not limited to, other
cellular proteins. Specifically, such compounds can include, but
are not limited to, peptides, such as, for example, soluble
peptides, including, but not limited to Ig-tailed fusion peptides,
comprising extracellular portions of PCSK9b or PCSK9c polypeptide
transmembrane receptors, and members of random peptide libraries
(see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghton, R.
et al., 1991, Nature 354:84-86), made of D- and/or L-configuration
amino acids, phosphopeptides (including, but not limited to,
members of random or partially degenerate phosphopeptide libraries;
see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778), antibodies
(including, but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab').sub.2 and FAb expression libary fragments, and
epitope-binding fragments thereof), and small organic or inorganic
molecules.
[0722] Compounds identified via assays such as those described
herein can be useful, for example, in elaborating the biological
function of the PCSK9b or PCSK9c polypeptide, and for ameliorating
symptoms of tumor progression, for example. In instances, for
example, whereby a tumor progression state or disorder results from
a lower overall level of PCSK9b or PCSK9c expression, PCSK9b or
PCSK9c polypeptide, and/or PCSK9b or PCSK9c polypeptide activity in
a cell involved in the tumor progression state or disorder,
compounds that interact with the PCSK9b or PCSK9c polypeptide can
include ones which accentuate or amplify the activity of the bound
PCSK9b or PCSK9c polypeptide. Such compounds would bring about an
effective increase in the level of PCSK9b or PCSK9c polypeptide
activity, thus ameliorating symptoms of the tumor progression
disorder or state. In instances whereby mutations within the PCSK9b
or PCSK9c polypeptide cause aberrant PCSK9b or PCSK9c polypeptides
to be made which have a deleterious effect that leads to tumor
progression, compounds that bind PCSK9b or PCSK9c polypeptide can
be identified that inhibit the activity of the bound PCSK9b or
PCSK9c polypeptide. Assays for testing the effectiveness of such
compounds are known in the art and discussed, elsewhere herein.
Example 7
Method of Identifying Compounds that Interfere with PCSK9b or
PCSK9c Polypeptide/Cellular Product Interaction
[0723] The PCSK9b or PCSK9c polypeptide of the invention can, in
vivo, interact with one or more cellular or extracellular
macromolecules, such as proteins. Such macromolecules include, but
are not limited to, polypeptides, particularly PCSK9 ligands, and
those products identified via screening methods described,
elsewhere herein. For the purposes of this discussion, such
cellular and extracellular macromolecules are referred to herein as
"binding partner(s)". For the purpose of the present invention,
"binding partner" may also encompass polypeptides, small molecule
compounds, polysaccarides, lipids, and any other molecule or
molecule type referenced herein. Compounds that disrupt such
interactions can be useful in regulating the activity of the PCSK9b
or PCSK9c polypeptide, especially mutant PCSK9b or PCSK9c
polypeptide. Such compounds can include, but are not limited to
molecules such as antibodies, peptides, and the like described in
elsewhere herein.
[0724] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between the PCSK9b or
PCSK9c polypeptide and its cellular or extracellular binding
partner or partners involves preparing a reaction mixture
containing the PCSK9b or PCSK9c polypeptide, and the binding
partner under conditions and for a time sufficient to allow the two
products to interact and bind, thus forming a complex. In order to
test a compound for inhibitory activity, the reaction mixture is
prepared in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of PCSK9b or PCSK9c
polypeptide and its cellular or extracellular binding partner.
Control reaction mixtures are incubated without the test compound
or with a placebo. The formation of any complexes between the
PCSK9b or PCSK9c polypeptide and the cellular or extracellular
binding partner is then detected. The formation of a complex in the
control reaction, but not in the reaction mixture containing the
test compound, indicates that the compound interferes with the
interaction of the PCSK9b or PCSK9c polypeptide and the interactive
binding partner. Additionally, complex formation within reaction
mixtures containing the test compound and normal PCSK9b or PCSK9c
polypeptide can also be compared to complex formation within
reaction mixtures containing the test compound and mutant PCSK9b or
PCSK9c polypeptide. This comparison can be important in those cases
wherein it is desirable to identify compounds that disrupt
interactions of mutant but not normal PCSK9b or PCSK9c
polypeptide.
[0725] The assay for compounds that interfere with the interaction
of the PCSK9b or PCSK9c polypeptide and binding partners can be
conducted in a heterogeneous or homogeneous format. Heterogeneous
assays involve anchoring either the PCSK9b or PCSK9c polypeptide or
the binding partner onto a solid phase and detecting complexes
anchored on the solid phase at the end of the reaction. In
homogeneous assays, the entire reaction is carried out in a liquid
phase. In either approach, the order of addition of reactants can
be varied to obtain different information about the compounds being
tested. For example, test compounds that interfere with the
interaction between the PCSK9b or PCSK9c polypeptide and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance;
i.e., by adding the test substance to the reaction mixture prior to
or simultaneously with the PCSK9b or PCSK9c polypeptide and
interactive cellular or extracellular binding partner.
Alternatively, test compounds that disrupt preformed complexes,
e.g. compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are described briefly below.
[0726] In a heterogeneous assay system, either the PCSK9b or PCSK9c
polypeptide or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface, while the non-anchored
species is labeled, either directly or indirectly. In practice,
microtitre plates are conveniently utilized. The anchored species
can be immobilized by non-covalent or covalent attachments.
Non-covalent attachment can be accomplished simply by coating the
solid surface with a solution of the PCSK9b or PCSK9c polypeptide
or binding partner and drying. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface. The surfaces can be
prepared in advance and stored.
[0727] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized species
is pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the non-immobilized
species is not pre-labeled, an indirect label can be used to detect
complexes anchored on the surface; e.g., using a labeled antibody
specific for the initially non-immobilized species (the antibody,
in turn, can be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds which inhibit complex formation
or which disrupt preformed complexes can be detected.
[0728] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds which inhibit complex
or which disrupt preformed complexes can be identified.
[0729] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of the
PCSK9b or PCSK9c polypeptide and the interactive cellular or
extracellular binding partner product is prepared in which either
the PCSK9b or PCSK9c polypeptide or their binding partners are
labeled, but the signal generated by the label is quenched due to
complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein
which utilizes this approach for immunoassays). The addition of a
test substance that competes with and displaces one of the species
from the preformed complex will result in the generation of a
signal above background. In this way, test substances which disrupt
PCSK9b or PCSK9c polypeptide-cellular or extracellular binding
partner interaction can be identified.
[0730] In a particular embodiment, the PCSK9b or PCSK9c polypeptide
can be prepared for immobilization using recombinant DNA techniques
known in the art. For example, the PCSK9b or PCSK9c polypeptide
coding region can be fused to a glutathione-S-transferase (GST)
gene using a fusion vector such as pGEX-5X-1, in such a manner that
its binding activity is maintained in the resulting fusion product.
The interactive cellular or extracellular product can be purified
and used to raise a monoclonal antibody, using methods routinely
practiced in the art and described above. This antibody can be
labeled with the radioactive isotope .sup.125 I, for example, by
methods routinely practiced in the art. In a heterogeneous assay,
e.g., the GST-PCSK9b or PCSK9c polypeptide fusion product can be
anchored to glutathione-agarose beads. The interactive cellular or
extracellular binding partner product can then be added in the
presence or absence of the test compound in a manner that allows
interaction and binding to occur. At the end of the reaction
period, unbound material can be washed away, and the labeled
monoclonal antibody can be added to the system and allowed to bind
to the complexed components. The interaction between the PCSK9b or
PCSK9c polypeptide and the interactive cellular or extracellular
binding partner can be detected by measuring the amount of
radioactivity that remains associated with the glutathione-agarose
beads. A successful inhibition of the interaction by the test
compound will result in a decrease in measured radioactivity.
[0731] Alternatively, the GST-PCSK9b or PCSK9c polypeptide fusion
product and the interactive cellular or extracellular binding
partner product can be mixed together in liquid in the absence of
the solid glutathione-agarose beads. The test compound can be added
either during or after the binding partners are allowed to
interact. This mixture can then be added to the glutathione-agarose
beads and unbound material is washed away. Again the extent of
inhibition of the binding partner interaction can be detected by
adding the labeled antibody and measuring the radioactivity
associated with the beads.
[0732] In another embodiment of the invention, these same
techniques can be employed using peptide fragments that correspond
to the binding domains of the PCSK9b or PCSK9c polypeptide product
and the interactive cellular or extracellular binding partner (in
case where the binding partner is a product), in place of one or
both of the full length products.
[0733] Any number of methods routinely practiced in the art can be
used to identify and isolate the protein's binding site. These
methods include, but are not limited to, mutagenesis of one of the
genes encoding one of the products and screening for disruption of
binding in a co-immunoprecipitation assay. Compensating mutations
in the gene encoding the second species in the complex can be
selected. Sequence analysis of the genes encoding the respective
products will reveal the mutations that correspond to the region of
the product involved in interactive binding. Alternatively, one
product can be anchored to a solid surface using methods described
in this Section above, and allowed to interact with and bind to its
labeled binding partner, which has been treated with a proteolytic
enzyme, such as trypsin. After washing, a short, labeled peptide
comprising the binding domain can remain associated with the solid
material, which can be isolated and identified by amino acid
sequencing. Also, once the gene coding for the cellular or
extracellular binding partner product is obtained, short gene
segments can be engineered to express peptide fragments of the
product, which can then be tested for binding activity and purified
or synthesized.
Example 8
Isolation of a Specific Clone from the Deposited Sample
[0734] The deposited material in the sample assigned the ATCC.RTM.
Deposit Number cited in Table I for any given cDNA clone also may
contain one or more additional plasmids, each comprising a cDNA
clone different from that given clone. Thus, deposits sharing the
same ATCC.RTM. Deposit Number contain at least a plasmid for each
cDNA clone identified in Table I. Typically, each ATCC.RTM. deposit
sample cited in Table I comprises a mixture of approximately equal
amounts (by weight) of about 1-10 plasmid DNAs, each containing a
different cDNA clone and/or partial cDNA clone; but such a deposit
sample may include plasmids for more or less than 2 cDNA
clones.
[0735] Two approaches can be used to isolate a particular clone
from the deposited sample of plasmid DNA(s) cited for that clone in
Table I. First, a plasmid is directly isolated by screening the
clones using a polynucleotide probe corresponding to SEQ ID
NO:1.
[0736] Particularly, a specific polynucleotide with 30-40
nucleotides is synthesized using an Applied Biosystems DNA
synthesizer according to the sequence reported. The oligonucleotide
is labeled, for instance, with 32P-(-ATP using T4 polynucleotide
kinase and purified according to routine methods. (E.g., Maniatis
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press, Cold Spring, N.Y. (1982).) The plasmid mixture is
transformed into a suitable host, as indicated above (such as XL-1
Blue (Stratagene)) using techniques known to those of skill in the
art, such as those provided by the vector supplier or in related
publications or patents cited above. The transformants are plated
on 1.5% agar plates (containing the appropriate selection agent,
e.g., ampicillin) to a density of about 150 transformants
(colonies) per plate. These plates are screened using Nylon
membranes according to routine methods for bacterial colony
screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press,
pages 1.93 to 1.104), or other techniques known to those of skill
in the art.
[0737] Alternatively, two primers of 17-20 nucleotides derived from
both ends of the SEQ ID NO:1 or SEQ ID NO:3 (i.e., within the
region of SEQ ID NO:1 or SEQ ID NO:3 bounded by the 5' NT and the
3' NT of the clone defined in Table I) are synthesized and used to
amplify the desired cDNA using the deposited cDNA plasmid as a
template. The polymerase chain reaction is carried out under
routine conditions, for instance, in 25 ul of reaction mixture with
0.5 ug of the above cDNA template. A convenient reaction mixture is
1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP,
dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min;
annealing at 55 degree C. for 1 min; elongation at 72 degree C. for
1 min) are performed with a Perkin-Elmer Cetus automated thermal
cycler. The amplified product is analyzed by agarose gel
electrophoresis and the DNA band with expected molecular weight is
excised and purified. The PCR product is verified to be the
selected sequence by subcloning and sequencing the DNA product.
[0738] The polynucleotide(s) of the present invention, the
polynucleotide encoding the polypeptide of the present invention,
or the polypeptide encoded by the deposited clone may represent
partial, or incomplete versions of the complete coding region
(i.e., full-length gene). Several methods are known in the art for
the identification of the 5' or 3' non-coding and/or coding
portions of a gene which may not be present in the deposited clone.
The methods that follow are exemplary and should not be construed
as limiting the scope of the invention. These methods include but
are not limited to, filter probing, clone enrichment using specific
probes, and protocols similar or identical to 5' and 3' "RACE"
protocols that are well known in the art. For instance, a method
similar to 5' RACE is available for generating the missing 5' end
of a desired full-length transcript. (Fromont-Racine et al.,
Nucleic Acids Res. 21(7):1683-1684 (1993)).
[0739] Briefly, a specific RNA oligonucleotide is ligated to the 5'
ends of a population of RNA presumably containing full-length gene
RNA transcripts. A primer set containing a primer specific to the
ligated RNA oligonucleotide and a primer specific to a known
sequence of the gene of interest is used to PCR amplify the 5'
portion of the desired full-length gene. This amplified product may
then be sequenced and used to generate the full-length gene.
[0740] This above method starts with total RNA isolated from the
desired source, although poly-A+ RNA can be used. The RNA
preparation can then be treated with phosphatase if necessary to
eliminate 5' phosphate groups on degraded or damaged RNA that may
interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid
pyrophosphatase in order to remove the cap structure present at the
5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the 5' end of the cap cleaved RNA which can then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
[0741] This modified RNA preparation is used as a template for
first strand cDNA synthesis using a gene specific oligonucleotide.
The first strand synthesis reaction is used as a template for PCR
amplification of the desired 5' end using a primer specific to the
ligated RNA oligonucleotide and a primer specific to the known
sequence of the gene of interest. The resultant product is then
sequenced and analyzed to confirm that the 5' end sequence belongs
to the desired gene. Moreover, it may be advantageous to optimize
the RACE protocol to increase the probability of isolating
additional 5' or 3' coding or non-coding sequences. Various methods
of optimizing a RACE protocol are known in the art, though a
detailed description summarizing these methods can be found in B.
C. Schaefer, Anal. Biochem., 227:255-273, (1995).
[0742] An alternative method for carrying out 5' or 3' RACE for the
identification of coding or non-coding sequences is provided by
Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002
(1988). Briefly, a cDNA clone missing either the 5' or 3' end can
be reconstructed to include the absent base pairs extending to the
translational start or stop codon, respectively. In some cases,
cDNAs are missing the start of translation, therefor. The following
briefly describes a modification of this original 5' RACE
procedure. Poly A+ or total RNAs reverse transcribed with
SUPERSCRIPT.RTM. II (Gibco/BRL) and an antisense or I complementary
primer specific to the cDNA sequence. The primer is removed from
the reaction with a MICROCON.RTM. Concentrator (Amicon). The
first-strand cDNA is then tailed with dATP and terminal
deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence
is produced which is needed for PCR amplification. The second
strand is synthesized from the dA-tail in PCR buffer, Taq DNA
polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing
three adjacent restriction sites (XhoIJ Sail and ClaI) at the 5'
end and a primer containing just these restriction sites. This
double-stranded cDNA is PCR amplified for 40 cycles with the same
primers as well as a nested cDNA-specific antisense primer. The PCR
products are size-separated on an ethidium bromide-agarose gel and
the region of gel containing cDNA products the predicted size of
missing protein-coding DNA is removed. cDNA is purified from the
agarose with the Magic PCR Prep kit (Promega), restriction digested
with XhoI or SalI, and ligated to a plasmid such as
PBLUESCRIPT.RTM. SKII (Stratagene) at XhoI and EcoRV sites. This
DNA is transformed into bacteria and the plasmid clones sequenced
to identify the correct protein-coding inserts. Correct 5' ends are
confirmed by comparing this sequence with the putatively identified
homologue and overlap with the partial cDNA clone. Similar methods
known in the art and/or commercial kits are used to amplify and
recover 3' ends.
[0743] Several quality-controlled kits are commercially available
for purchase. Similar reagents and methods to those above are
supplied in kit form from Gibco/BRL for both 5' and 3' RACE for
recovery of full length genes. A second kit is available from
Clontech which is a modification of a related technique, SLIC
(single-stranded ligation to single-stranded cDNA), developed by
Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major
differences in procedure are that the RNA is alkaline hydrolyzed
after reverse transcription and RNA ligase is used to join a
restriction site-containing anchor primer to the first-strand cDNA.
This obviates the necessity for the dA-tailing reaction which
results in a polyT stretch that is difficult to sequence past.
[0744] An alternative to generating 5' or 3' cDNA from RNA is to
use cDNA library double-stranded DNA. An asymmetric PCR-amplified
antisense cDNA strand is synthesized with an antisense
cDNA-specific primer and a plasmid-anchored primer. These primers
are removed and a symmetric PCR reaction is performed with a nested
cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase Protocol for Generating the 5' or 3' End Sequences to
Obtain Full Length Genes
[0745] Once a gene of interest is identified, several methods are
available for the identification of the 5' or 3' portions of the
gene which may not be present in the original cDNA plasmid. These
methods include, but are not limited to, filter probing, clone
enrichment using specific probes and protocols similar and
identical to 5' and 3'RACE. While the full-length gene may be
present in the library and can be identified by probing, a useful
method for generating the 5' or 3' end is to use the existing
sequence information from the original cDNA to generate the missing
information. A method similar to 5'RACE is available for generating
the missing 5' end of a desired full-length gene. (This method was
published by Fromont-Racine et al., Nucleic Acids Res., 21(7):
1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is
ligated to the 5' ends of a population of RNA presumably 30
containing full-length gene RNA transcript and a primer set
containing a primer specific to the ligated RNA oligonucleotide and
a primer specific to a known sequence of the gene of interest, is
used to PCR amplify the 5' portion of the desired full length gene
which may then be sequenced and used to generate the full length
gene. This method starts with total RNA isolated from the desired
source, poly A RNA may be used but is not a prerequisite for this
procedure. The RNA preparation may then be treated with phosphatase
if necessary to eliminate 5' phosphate groups on degraded or
damaged RNA which may interfere with the later RNA ligase step. The
phosphatase if used is then inactivated and the RNA is treated with
tobacco acid pyrophosphatase in order to remove the cap structure
present at the 5' ends of messenger RNAs. This reaction leaves a 5'
phosphate group at the 5' end of the cap cleaved RNA which can then
be ligated to an RNA oligonucleotide using T4 RNA ligase. This
modified RNA preparation can then be used as a template for first
strand cDNA synthesis using a gene specific oligonucleotide. The
first strand synthesis reaction can then be used as a template for
PCR amplification of the desired 5' end using a primer specific to
the ligated RNA oligonucleotide and a primer specific to the known
sequence of the apoptosis related of interest. The resultant
product is then sequenced and analyzed to confirm that the 5' end
sequence belongs to the relevant apoptosis related.
Example 9
Bacterial Expression of a Polypeptide
[0746] A polynucleotide encoding a polypeptide of the present
invention is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' ends of the DNA sequence, as
outlined herein, to synthesize insertion fragments. The primers
used to amplify the cDNA insert should preferably contain
restriction sites, such as BamHI and XbaI, at the 5' end of the
primers in order to clone the amplified product into the expression
vector. For example, BamHI and XbaI correspond to the restriction
enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic
resistance (Ampr), a bacterial origin of replication (ori), an
IPTG-regulatable promoter/operator (P/O), a ribosome binding site
(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning
sites.
[0747] The pQE-9 vector is digested with BamHI and XbaI and the
amplified fragment is ligated into the pQE-9 vector maintaining the
reading frame initiated at the bacterial RBS. The ligation mixture
is then used to transform the E. coli strain M15/rep4 (Qiagen,
Inc.) which contains multiple copies of the plasmid pREP4, that
expresses the lad repressor and also confers kanamycin resistance
(Kanr). Transformants are identified by their ability to grow on LB
plates and ampicillin/kanamycin resistant colonies are selected.
Plasmid DNA is isolated and confirmed by restriction analysis.
[0748] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells are grown to
an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lad
repressor, clearing the P/O leading to increased gene
expression.
[0749] Cells are grown for an extra 3 to 4 hours. Cells are then
harvested by centrifugation (20 mins at 6000.times.g). The cell
pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl
by stirring for 3-4 hours at 4 degree C. The cell debris is removed
by centrifugation, and the supernatant containing the polypeptide
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).
[0750] 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 polypeptide is eluted with 6 M
guanidine-HCl, pH 5.
[0751] 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 are eluted by the addition of 250 mM imidazole.
Imidazole 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.
Example 10
Purification of a Polypeptide from an Inclusion Body
[0752] The following alternative method can be used to purify a
polypeptide 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.
[0753] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10 degree C. and the
cells 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.
[0754] The cells are 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.
[0755] 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 polypeptide containing supernatant is incubated at 4 degree
C. overnight to allow further GuHCl extraction.
[0756] 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.
[0757] To clarify the refolded polypeptide solution, a previously
prepared tangential filtration unit equipped with 0.16 um 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.RTM. HS-50,
Perceptive 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 nm of the effluent is continuously monitored. Fractions are
collected and further analyzed by SDS-PAGE.
[0758] Fractions containing the 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.RTM. HQ-50, Perceptive Biosystems) and weak anion
(POROS.RTM. CM-20, Perceptive 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 A280 monitoring of the effluent. Fractions
containing the polypeptide (determined, for instance, by 16%
SDS-PAGE) are then pooled.
[0759] The resultant polypeptide should exhibit greater than 95%
purity after the above refolding and purification steps. No major
contaminant bands should be observed from Coomassie blue stained
16% SDS-PAGE gel when 5 ug of purified protein is loaded. The
purified protein can also be tested for endotoxin/LPS
contamination, and typically the LPS content is less than 0.1 ng/ml
according to LAL assays.
Example 11
Cloning and Expression of a Polypeptide in a Baculovirus Expression
System
[0760] In this example, the plasmid shuttle vector pAc373 is used
to insert a polynucleotide into a baculovirus to express a
polypeptide. A typical baculovirus expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction
sites, which may include, for example BamHI, Xba I and Asp718. The
polyadenylation site of the simian virus 40 ("SV40") is often 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.
[0761] Many other baculovirus vectors can be used in place of the
vector above, such as 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).
[0762] A polynucleotide encoding a polypeptide of the present
invention is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' ends of the DNA sequence, as
outlined in herein, to synthesize insertion fragments. The primers
used to amplify the cDNA insert should preferably contain
restriction sites at the 5' end of the primers in order to clone
the amplified product into the expression vector. Specifically, the
cDNA sequence contained in the deposited clone, including the AUG
initiation codon and the naturally associated leader sequence
identified elsewhere herein (if applicable), is amplified using the
PCR protocol described herein. If the naturally occurring signal
sequence is used to produce the protein, the vector used does not
need a second signal peptide. Alternatively, the vector can be
modified to include a baculovirus leader sequence, using the
standard methods 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).
[0763] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit (GENECLEAN.RTM., BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[0764] The plasmid is digested with the corresponding restriction
enzymes 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.RTM., BIO 101 Inc., La Jolla, Calif.).
[0765] The 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 containing the plasmid are identified
by digesting DNA from individual colonies and analyzing the
digestion product by gel electrophoresis. The sequence of the
cloned fragment is confirmed by DNA sequencing.
[0766] Five ug of a plasmid containing the polynucleotide is
co-transformed with 1.0 ug of a commercially available linearized
baculovirus DNA ("BACULOGOLD.RTM. baculovirus DNA", Pharmingen, San
Diego, Calif.), using the lipofection method described by Feigner
et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of
BACULOGOLD.RTM. virus DNA and 5 ug of the plasmid are mixed in a
sterile well of a microtiter plate containing 50 ul of serum-free
Grace's medium (Life Technologies Inc., Gaithersburg, Md.).
Afterwards, 10 ul LIPOFECTIN.RTM. plus 90 ul 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 degrees 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
degrees C. for four days.
[0767] 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 ul 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.
[0768] To verify the expression of the polypeptide, Sf9 cells are
grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells are infected with the recombinant baculovirus containing
the polynucleotide 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 uCi of 35S-methionine and 5 uCi
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).
[0769] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the produced protein.
Example 12
Expression of a Polypeptide in Mammalian Cells
[0770] The polypeptide of the present invention can be expressed in
a mammalian cell. A typical mammalian expression vector contains a
promoter element, which mediates the initiation of transcription of
mRNA, a 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 is 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).
[0771] 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), pBC12MI (ATCC.RTM. 67109), pCMVSport
2.0, and pCMVSport 3.0. 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.
[0772] Alternatively, the polypeptide can be expressed in stable
cell lines containing the polynucleotide integrated into a
chromosome. The co-transformation with a selectable marker such as
dhfr, gpt, neomycin, hygromycin allows the identification and
isolation of the transformed cells.
[0773] The transformed gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful in developing cell lines that carry several
hundred or even several thousand copies of the gene of interest.
(See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370
(1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta,
1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology
9:64-68 (1991).) 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.
[0774] A polynucleotide of the present invention is amplified
according to the protocol outlined in herein. If the naturally
occurring signal sequence is used to produce the protein, the
vector does not need a second signal peptide. Alternatively, if the
naturally occurring signal sequence is not used, the vector can be
modified to include a heterologous signal sequence. (See, e.g., WO
96/34891.) The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit (GENECLEAN.RTM., BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[0775] The amplified fragment is then digested with the same
restriction enzyme and purified 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 pC6 using, for instance, restriction enzyme analysis.
[0776] Chinese hamster ovary cells lacking an active DHFR gene is
used for transformation. Five .mu.g of an expression plasmid is
cotransformed with 0.5 ug of the plasmid pSVneo using
LIPOFECTIN.RTM. (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 methotrexate 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 (1 uM,
2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until
clones are obtained which grow at a concentration of 100-200 uM.
Expression of the desired gene product is analyzed, for instance,
by SDS-PAGE and Western blot or by reversed phase HPLC
analysis.
Example 13
Method of Creating N- and C-Terminal Deletion Mutants Corresponding
to PCSK9, PCSK9b or PCSK9c Polypeptides of the Present
Invention
[0777] As described elsewhere herein, the present invention
encompasses the creation of N- and C-terminal deletion mutants, in
addition to any combination of N- and C-terminal deletions thereof,
corresponding to the PCSK9, PCSK9b or PCSK9c polypeptide of the
present invention. A number of methods are available to one skilled
in the art for creating such mutants. Such methods may include a
combination of PCR amplification and gene cloning methodology.
Although one of skill in the art of molecular biology, through the
use of the teachings provided or referenced herein, and/or
otherwise known in the art as standard methods, could readily
create each deletion mutant of the present invention, exemplary
methods are described below.
[0778] Briefly, using the isolated cDNA clone encoding the
full-length PCSK9b or PCSK9c polypeptide sequence (as described
herein, for example), appropriate primers of about 15-25
nucleotides derived from the desired 5' and 3' positions of SEQ ID
NO:1 or SEQ ID NO:3 or SEQ ID NO:38 may be designed to PCR amplify,
and subsequently clone, the intended N- and/or C-terminal deletion
mutant. Such primers could comprise, for example, an inititation
and stop codon for the 5' and 3' primer, respectively. Such primers
may also comprise restriction sites to facilitate cloning of the
deletion mutant post amplification. Moreover, the primers may
comprise additional sequences, such as, for example, flag-tag
sequences, kozac sequences, or other sequences discussed and/or
referenced herein.
[0779] For example, in the case of the PCSK9b L16 to R315
N-terminal deletion mutant, the following primers could be used to
amplify a cDNA fragment corresponding to this deletion mutant:
TABLE-US-00011 5' Primer 5'-GCAGCA GCGGCCGC
CTAGACACCAGCATACAGAGTGACC-3' (SEQ ID NO: 22) NotI 3' Primer
5'-GCAGCA GTCGAC TCTGGGGCGCAGCGGGCGATGGCTG -3' (SEQ ID NO: 23)
SalI
[0780] For example, in the case of the PCSK9b M1 to P284 C-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant:
TABLE-US-00012 5' Primer 5'-GCAGCA GCGGCCGC
ATGTCGCCTTGGAAAGACGGAGGCA-3' (SEQ ID NO: 24) NotI 3' Primer
5'-GCAGCA GTCGAC AGGGCCTGCCCCATGGGTGCTGGGG-3' (SEQ ID NO: 25)
SalI
[0781] For example, in the case of the PCSK9c L16 to Q523
N-terminal deletion mutant, the following primers could be used to
amplify a cDNA fragment corresponding to this deletion mutant:
TABLE-US-00013 5' Primer 5'-GCAGCA GCGGCCGC
CTAGACACCAGCATACAGAGTGACC-3' (SEQ ID NO: 26) NotI 3' Primer
5'-GCAGCA GTCGAC CTGGAGCTCCTGGGAGGCCTGCGCC-3' (SEQ ID NO: 27)
SalI
[0782] For example, in the case of the PCSK9c M1 to A306 C-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant:
TABLE-US-00014 5' Primer 5'- GCAGCA GCGGCCGC
ATGTCGCCTTGGAAAGACGGAGGCA-3' (SEQ ID NO: 28) NotI 3' Primer 5'-
GCAGCA GTCGAC GGCGATGGCTGTGGCCATCCGTGTA-3' (SEQ ID NO: 29) SalI
[0783] For example, in the case of the PCSK9 L16 to Q692 N-terminal
deletion mutant, the following primers could be used to amplify a
cDNA fragment corresponding to this deletion mutant:
TABLE-US-00015 5' Primer 5'-GCAGCA GCGGCCGC
CTGCTGCTGCTGCTGCTGCTGCTCC-3' (SEQ ID NO: 39) NotI 3' Primer
5'-GCAGCA GTCGAC CTGGAGCTCCTGGGAGGCCTGCGCC-3' (SEQ ID NO: 40)
SalI
[0784] Representative PCR amplification conditions are provided
below, although the skilled artisan would appreciate that other
conditions may be required for efficient amplification. A 100 ul
PCR reaction mixture may be prepared using 10 ng of the template
DNA (cDNA clone of PCSK9b or PCSK9c), 200 uM 4dNTPs, 1 uM primers,
0.25 U Taq DNA polymerase (PE), and standard Taq DNA polymerase
buffer. Typical PCR cycling condition are as follows:
[0785] 20-25 cycles: 45 sec, 93 degrees [0786] 2 min, 50 degrees
[0787] 2 min, 72 degrees
[0788] 1 cycle: 10 min, 72 degrees
[0789] After the final extension step of PCR, 5 U Klenow Fragment
may be added and incubated for 15 min at 30 degrees.
[0790] Upon digestion of the fragment with the NotI and SalI
restriction enzymes, the fragment could be cloned into an
appropriate expression and/or cloning vector which has been
similarly digested (e.g., pSport1, among others). The skilled
artisan would appreciate that other plasmids could be equally
substituted, and may be desirable in certain circumstances. The
digested fragment and vector are then ligated using a DNA ligase,
and then used to transform competent E. coli cells using methods
provided herein and/or otherwise known in the art.
[0791] The 5' primer sequence for amplifying any additional
N-terminal deletion mutants may be determined by reference to the
following formula: (S+(X*3)) to ((S+(X*3))+25), wherein `S` is
equal to the nucleotide position of the initiating start codon of
PCSK9b (SEQ ID NO:1) or PCSK9c (SEQ ID NO:3) or PCSK9 (SEQ ID
NO:38), and `X` is equal to the most N-terminal amino acid of the
intended N-terminal deletion mutant. The first term will provide
the start 5' nucleotide position of the 5' primer, while the second
term will provide the end 3' nucleotide position of the 5' primer
corresponding to sense strand of SEQ ID NO:1 or SEQ ID NO:3 or SEQ
ID NO:38. Once the corresponding nucleotide positions of the primer
are determined, the final nucleotide sequence may be created by the
addition of applicable restriction site sequences to the 5' end of
the sequence, for example. As referenced herein, the addition of
other sequences to the 5' primer may be desired in certain
circumstances (e.g., kozac sequences, etc.).
[0792] The 3' primer sequence for amplifying any additional
N-terminal deletion mutants may be determined by reference to the
following formula: (S+(X*3)) to ((S+(X*3))-25), wherein `S` is
equal to the nucleotide position of the initiating start codon of
PCSK9b (SEQ ID NO:1) or PCSK9c (SEQ ID NO:3) or PCSK9 (SEQ ID
NO:38), and `X` is equal to the most C-terminal amino acid of the
intended N-terminal deletion mutant. The first term will provide
the start 5' nucleotide position of the 3' primer, while the second
term will provide the end 3' nucleotide position of the 3' primer
corresponding to the anti-sense strand of SEQ ID NO:1 or SEQ ID
NO:3 or SEQ ID NO:38. Once the corresponding nucleotide positions
of the primer are determined, the final nucleotide sequence may be
created by the addition of applicable restriction site sequences to
the 5' end of the sequence, for example. As referenced herein, the
addition of other sequences to the 3' primer may be desired in
certain circumstances (e.g., stop codon sequences, etc.). The
skilled artisan would appreciate that modifications of the above
nucleotide positions may be necessary for optimizing PCR
amplification.
[0793] The same general formulas provided above may be used in
identifying the 5' and 3' primer sequences for amplifying any
C-terminal deletion mutant of the present invention. Moreover, the
same general formulas provided above may be used in identifying the
5' and 3' primer sequences for amplifying any combination of
N-terminal and C-terminal deletion mutant of the present invention.
The skilled artisan would appreciate that modifications of the
above nucleotide positions may be necessary for optimizing PCR
amplification.
Example 14
Protein Fusions
[0794] The polypeptides of the present invention are preferably
fused to other proteins. These fusion proteins can be used for a
variety of applications. For example, fusion of the present
polypeptides to His-tag, HA-tag, protein A, IgG domains, and
maltose binding protein facilitates purification. (as described
herein; see also EP A 394,827; Traunecker, et al., Nature 331:84-86
(1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases
the half-life time in vivo. Nuclear localization signals fused to
the polypeptides of the present invention can target the protein to
a specific subcellular localization, while covalent heterodimer or
homodimers can increase or decrease the activity of a fusion
protein. Fusion proteins can also create chimeric molecules having
more than one function. Finally, fusion proteins can increase
solubility and/or stability of the fused protein compared to the
non-fused protein. All of the types of fusion proteins described
above can be made by modifying the following protocol, which
outlines the fusion of a polypeptide to an IgG molecule.
[0795] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian expression vector. Note
that the polynucleotide is cloned without a stop codon, otherwise a
fusion protein will not be produced.
[0796] The naturally occurring signal sequence may be used to
produce the protein (if applicable). Alternatively, if the
naturally occurring signal sequence is not used, the vector can be
modified to include a heterologous signal sequence. (See, e.g., WO
96/34891 and/or U.S. Pat. No. 6,066,781, supra.)
Human IgG Fc Region
TABLE-US-00016 [0797] (SEQ ID NO: 21)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 15
Production of an Antibody from a Polypeptide
[0798] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing a polypeptide of the
present invention are administered to an animal to induce the
production of sera containing polyclonal antibodies. In a preferred
method, a preparation of the 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.
[0799] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or 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., pp. 563-681 (1981).) In
general, such procedures involve immunizing an animal (preferably a
mouse) with polypeptide or, more preferably, with a
polypeptide-expressing cell. 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 degrees C.), and
supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
[0800] 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 (SP20), available
from the ATCC.RTM.. 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 polypeptide.
[0801] Alternatively, additional antibodies capable of binding to
the polypeptide can be produced in a two-step procedure using
anti-idiotypic antibodies. Such a method makes use of the fact that
antibodies are themselves antigens, and therefore, it is possible
to obtain an antibody that binds to a second antibody. In
accordance with this method, 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 that produce an
antibody whose ability to bind to the protein-specific antibody can
be blocked by the polypeptide. Such antibodies comprise
anti-idiotypic antibodies to the protein-specific antibody and can
be used to immunize an animal to induce formation of further
protein-specific antibodies.
[0802] 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, protein-binding fragments can be
produced through the application of recombinant DNA technology or
through synthetic chemistry.
[0803] For in vivo use of antibodies 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).)
[0804] Moreover, in another preferred method, the antibodies
directed against the polypeptides of the present invention may be
produced in plants. Specific methods are disclosed in U.S. Pat.
Nos. 5,959,177, and 6,080,560, which are hereby incorporated in
their entirety herein. The methods not only describe methods of
expressing antibodies, but also the means of assembling foreign
multimeric proteins in plants (i.e., antibodies, etc,), and the
subsequent secretion of such antibodies from the plant.
Example 16
Regulation of Protein Expression Via Controlled Aggregation in the
Endoplasmic Reticulum
[0805] As described more particularly herein, proteins regulate
diverse cellular processes in higher organisms, ranging from rapid
metabolic changes to growth and differentiation. Increased
production of specific proteins could be used to prevent certain
diseases and/or disease states. Thus, the ability to modulate the
expression of specific proteins in an organism would provide
significant benefits.
[0806] Numerous methods have been developed to date for introducing
foreign genes, either under the control of an inducible,
constitutively active, or endogenous promoter, into organisms. Of
particular interest are the inducible promoters (see, M. Gossen, et
al., Proc. Natl. Acad. Sci. USA., 89:5547 (1992); Y. Wang, et al.,
Proc. Natl. Acad. Sci. USA, 91:8180 (1994), D. No., et al., Proc.
Natl. Acad. Sci. USA, 93:3346 (1996); and V. M. Rivera, et al.,
Nature Med, 2:1028 (1996); in addition to additional examples
disclosed elsewhere herein). In one example, the gene for
erthropoietin (Epo) was transferred into mice and primates under
the control of a small molecule inducer for expression (e.g.,
tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,
(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.
Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X.
Ye et al., Science, 283:88 (1999). Although such systems enable
efficient induction of the gene of interest in the organism upon
addition of the inducing agent (i.e., tetracycline, rapamycin,
etc.), the levels of expression tend to peak at 24 hours and trail
off to background levels after 4 to 14 days. Thus, controlled
transient expression is virtually impossible using these systems,
though such control would be desirable.
[0807] A new alternative method of controlling gene expression
levels of a protein from a transgene (i.e., includes stable and
transient transformants) has recently been elucidated (V. M.
Rivera., et al., Science, 287:826-830 (2000)). This method does not
control gene expression at the level of the mRNA like the
aforementioned systems. Rather, the system controls the level of
protein in an active secreted form. In the absence of the inducing
agent, the protein aggregates in the ER and is not secreted.
However, addition of the inducing agent results in dis-aggregation
of the protein and the subsequent secretion from the ER. Such a
system affords low basal secretion, rapid, high level secretion in
the presence of the inducing agent, and rapid cessation of
secretion upon removal of the inducing agent. In fact, protein
secretion reached a maximum level within 30 minutes of induction,
and a rapid cessation of secretion within 1 hour of removing the
inducing agent. The method is also applicable for controlling the
level of production for membrane proteins.
[0808] Detailed methods are presented in V. M. Rivera., et al.,
Science, 287:826-830, (2000)), briefly:
[0809] Fusion protein constructs are created using polynucleotide
sequences of the present invention with one or more copies
(preferably at least 2, 3, 4, or more) of a conditional aggregation
domain (CAD) a domain that interacts with itself in a
ligand-reversible manner (i.e., in the presence of an inducing
agent) using molecular biology methods known in the art and
discussed elsewhere herein. The CAD domain may be the mutant domain
isolated from the human FKBP12 (Phe.sup.36 to Met) protein (as
disclosed in V. M. Rivera., et al., Science, 287:826-830, (2000),
or alternatively other proteins having domains with similar
ligand-reversible, self-aggregation properties. As a principle of
design the fusion protein vector would contain a furin cleavage
sequence operably linked between the polynucleotides of the present
invention and the CAD domains. Such a cleavage site would enable
the proteolytic cleavage of the CAD domains from the polypeptide of
the present invention subsequent to secretion from the ER and upon
entry into the trans-Golgi (J. B. Denault, et al., FEBS Lett.,
379:113, (1996)). Alternatively, the skilled artisan would
recognize that any proteolytic cleavage sequence could be
substituted for the furin sequence provided the substituted
sequence is cleavable either endogenously (e.g., the furin
sequence) or exogenously (e.g., post secretion, post purification,
post production, etc.). The preferred sequence of each feature of
the fusion protein construct, from the 5' to 3' direction with each
feature being operably linked to the other, would be a promoter,
signal sequence, "X" number of (CAD)x domains, the furin sequence
(or other proteolytic sequence), and the coding sequence of the
polypeptide of the present invention. The artisan would appreciate
that the promotor and signal sequence, independent from the other,
could be either the endogenous promotor or signal sequence of a
polypeptide of the present invention, or alternatively, could be a
heterologous signal sequence and promotor.
[0810] The specific methods described herein for controlling
protein secretion levels through controlled ER aggregation are not
meant to be limiting are would be generally applicable to any of
the polynucleotides and polypeptides of the present invention,
including variants, homologues, orthologs, and fragments
therein.
Example 17
Alteration of Protein Glycosylation Sites to Enhance
Characteristics of Polypeptides of the Invention
[0811] Many eukaryotic cell surface and proteins are
post-translationally processed to incorporate N-linked and O-linked
carbohydrates (Kornfeld and Kornfeld (1985) Annu. Rev. Biochem.
54:631-64; Rademacher et al., (1988) Annu. Rev. Biochem.
57:785-838). Protein glycosylation is thought to serve a variety of
functions including: augmentation of protein folding, inhibition of
protein aggregation, regulation of intracellular trafficking to
organelles, increasing resistance to proteolysis, modulation of
protein antigenicity, and mediation of intercellular adhesion
(Fieidler and Simons (1995) Cell, 81:309-312; Helenius (1994) Mol.
Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473;
Caton et al., (1982) Cell, 37:417-427; Alexamnder and Elder (1984),
Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem.,
269:14015-14020). In higher organisms, the nature and extent of
glycosylation can markedly affect the circulating half-life and
bio-availability of proteins by mechanisms involving receptor
mediated uptake and clearance (Ashwell and Morrell, (1974), Adv.
Enzymol., 41:99-128; Ashwell and Harford (1982), Ann. Rev.
Biochem., 51:531-54). Receptor systems have been identified that
are thought to play a major role in the clearance of serum proteins
through recognition of various carbohydrate structures on the
glycoproteins (Stockert (1995), Physiol. Rev., 75:591-609; Kery et
al., (1992), Arch. Biochem. Biophys., 298:49-55). Thus, production
strategies resulting in incomplete attachment of terminal sialic
acid residues might provide a means of shortening the
bioavailability and half-life of glycoproteins. Conversely,
expression strategies resulting in saturation of terminal sialic
acid attachment sites might lengthen protein bioavailability and
half-life.
[0812] In the development of recombinant glycoproteins for use as
pharmaceutical products, for example, it has been speculated that
the pharmacodynamics of recombinant proteins can be modulated by
the addition or deletion of glycosylation sites from a
glycoproteins primary structure (Berman and Lasky (1985a) Trends in
Biotechnol., 3:51-53). However, studies have reported that the
deletion of N-linked glycosylation sites often impairs
intracellular transport and results in the intracellular
accumulation of glycosylation site variants (Machamer and Rose
(1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992), J.
Virology., 66:7136-7145; Collier et al., (1993), Biochem.,
32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,
1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521).
While glycosylation site variants of proteins can be expressed
intracellularly, it has proved difficult to recover useful
quantities from growth conditioned cell culture medium.
[0813] Moreover, it is unclear to what extent a glycosylation site
in one species will be recognized by another species glycosylation
machinery. Due to the importance of glycosylation in protein
metabolism, particularly the secretion and/or expression of the
protein, whether a glycosylation signal is recognized may
profoundly determine a proteins ability to be expressed, either
endogenously or recombinately, in another organism (i.e.,
expressing a human protein in E. coli, yeast, or viral organisms;
or an E. coli, yeast, or viral protein in human, etc.). Thus, it
may be desirable to add, delete, or modify a glycosylation site,
and possibly add a glycosylation site of one species to a protein
of another species to improve the proteins functional, bioprocess
purification, and/or structural characteristics (e.g., a
polypeptide of the present invention).
[0814] A number of methods may be employed to identify the location
of glycosylation sites within a protein. One preferred method is to
run the translated protein sequence through the PROSITE computer
program (Swiss Institute of Bioinformatics). Once identified, the
sites could be systematically deleted, or impaired, at the level of
the DNA using mutagenesis methodology known in the art and
available to the skilled artisan, Preferably using PCR-directed
mutagenesis (See Maniatis, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). Similarly,
glycosylation sites could be added, or modified at the level of the
DNA using similar methods, preferably PCR methods (See, Maniatis,
supra). The results of modifying the glycosylation sites for a
particular protein (e.g., solubility, secretion potential,
activity, aggregation, proteolytic resistance, etc.) could then be
analyzed using methods know in the art.
[0815] The skilled artisan would acknowledge the existence of other
computer algorithms capable of predicting the location of
glycosylation sites within a protein. For example, the Motif
computer program (Genetics Computer Group suite of programs)
provides this function, as well.
Example 18
Method of Enhancing the Biological Activity/Functional
Characteristics of Invention Through Molecular Evolution
[0816] Although many of the most biologically active proteins known
are highly effective for their specified function in an organism,
they often possess characteristics that make them undesirable for
transgenic, therapeutic, and/or industrial applications. Among
these traits, a short physiological half-life is the most prominent
problem, and is present either at the level of the protein, or the
level of the proteins mRNA. The ability to extend the half-life,
for example, would be particularly important for a proteins use in
gene therapy, transgenic animal production, the bioprocess
production and purification of the protein, and use of the protein
as a chemical modulator among others. Therefore, there is a need to
identify novel variants of isolated proteins possessing
characteristics which enhance their application as a therapeutic
for treating diseases of animal origin, in addition to the proteins
applicability to common industrial and pharmaceutical
applications.
[0817] Thus, one aspect of the present invention relates to the
ability to enhance specific characteristics of invention through
directed molecular evolution. Such an enhancement may, in a
non-limiting example, benefit the inventions utility as an
essential component in a kit, the inventions physical attributes
such as its solubility, structure, or codon optimization, the
inventions specific biological activity, including any associated
enzymatic activity, the proteins enzyme kinetics, the proteins Ki,
Kcat, Km, Vmax, Kd, protein-protein activity, protein-DNA binding
activity, antagonist/inhibitory activity (including direct or
indirect interaction), agonist activity (including direct or
indirect interaction), the proteins antigenicity (e.g., where it
would be desirable to either increase or decrease the antigenic
potential of the protein), the immunogenicity of the protein, the
ability of the protein to form dimers, trimers, or multimers with
either itself or other proteins, the antigenic efficacy of the
invention, including its subsequent use a preventative treatment
for disease or disease states, or as an effector for targeting
diseased genes. Moreover, the ability to enhance specific
characteristics of a protein may also be applicable to changing the
characterized activity of an enzyme to an activity completely
unrelated to its initially characterized activity. Other desirable
enhancements of the invention would be specific to each individual
protein, and would thus be well known in the art and contemplated
by the present invention.
[0818] Directed evolution is comprised of several steps. The first
step is to establish a library of variants for the gene or protein
of interest. The most important step is to then select for those
variants that entail the activity you wish to identify. The design
of the screen is essential since your screen should be selective
enough to eliminate non-useful variants, but not so stringent as to
eliminate all variants. The last step is then to repeat the above
steps using the best variant from the previous screen. Each
successive cycle, can then be tailored as necessary, such as
increasing the stringency of the screen, for example.
[0819] Over the years, there have been a number of methods
developed to introduce mutations into macromolecules. Some of these
methods include, random mutagenesis, "error-prone" PCR, chemical
mutagenesis, site-directed mutagenesis, and other methods well
known in the art (for a comprehensive listing of current
mutagenesis methods, see Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)).
Typically, such methods have been used, for example, as tools for
identifying the core functional region(s) of a protein or the
function of specific domains of a protein (if a multi-domain
protein). However, such methods have more recently been applied to
the identification of macromolecule variants with specific or
enhanced characteristics.
[0820] Random mutagenesis has been the most widely recognized
method to date. Typically, this has been carried out either through
the use of "error-prone" PCR (as described in Moore, J., et al,
Nature Biotechnology 14:458, (1996), or through the application of
randomized synthetic oligonucleotides corresponding to specific
regions of interest (as described by Derbyshire, K. M. et al, Gene,
46:145-152, (1986), and Hill, D E, et al, Methods Enzymol.,
55:559-568, (1987). Both approaches have limits to the level of
mutagenesis that can be obtained. However, either approach enables
the investigator to effectively control the rate of mutagenesis.
This is particularly important considering the fact that mutations
beneficial to the activity of the enzyme are fairly rare. In fact,
using too high a level of mutagenesis may counter or inhibit the
desired benefit of a useful mutation.
[0821] While both of the aforementioned methods are effective for
creating randomized pools of macromolecule variants, a third
method, termed "DNA Shuffling", or "sexual PCR" (WPC, Stemmer,
PNAS, 91:10747, (1994)) has recently been elucidated. DNA shuffling
has also been referred to as "directed molecular evolution",
"exon-shuffling", "directed enzyme evolution", "in vitro
evolution", and "artificial evolution". Such reference terms are
known in the art and are encompassed by the invention. This new,
preferred, method apparently overcomes the limitations of the
previous methods in that it not only propagates positive traits,
but simultaneously eliminates negative traits in the resulting
progeny.
[0822] DNA shuffling accomplishes this task by combining the
principal of in vitro recombination, along with the method of
"error-prone" PCR. In effect, you begin with a randomly digested
pool of small fragments of your gene, created by Dnase I digestion,
and then introduce said random fragments into an "error-prone" PCR
assembly reaction. During the PCR reaction, the randomly sized DNA
fragments not only hybridize to their cognate strand, but also may
hybridize to other DNA fragments corresponding to different regions
of the polynucleotide of interest--regions not typically accessible
via hybridization of the entire polynucleotide. Moreover, since the
PCR assembly reaction utilizes "error-prone" PCR reaction
conditions, random mutations are introduced during the DNA
synthesis step of the PCR reaction for all of the
fragments--further diversifying the potential hybridization sites
during the annealing step of the reaction.
[0823] A variety of reaction conditions could be utilized to
carry-out the DNA shuffling reaction. However, specific reaction
conditions for DNA shuffling are provided, for example, in PNAS,
91:10747, (1994). Briefly:
[0824] Prepare the DNA substrate to be subjected to the DNA
shuffling reaction. Preparation may be in the form of simply
purifying the DNA from contaminating cellular material, chemicals,
buffers, oligonucleotide primers, deoxynucleotides, RNAs, etc., and
may entail the use of DNA purification kits as those provided by
Qiagen, Inc., or by the Promega, Corp., for example.
[0825] Once the DNA substrate has been purified, it would be
subjected to Dnase I digestion. About 2-4 ug of the DNA
substrate(s) would be digested with 0.0015 units of Dnase I (Sigma)
per ul in 100 ul of 50 mM Tris-HCL, pH 7.4/1 mM MgCl2 for 10-20
min. at room temperature. The resulting fragments of 10-50 bp could
then be purified by running them through a 2% low-melting point
agarose gel by electrophoresis onto DE81 ion-exchange paper
(Whatmann) or could be purified using MICROCON.RTM. concentrators
(Amicon) of the appropriate molecular weight cutoff, or could use
oligonucleotide purification columns (Qiagen), in addition to other
methods known in the art. If using DE81 ion-exchange paper, the
10-50 bp fragments could be eluted from said paper using 1M NaCl,
followed by ethanol precipitation.
[0826] The resulting purified fragments would then be subjected to
a PCR assembly reaction by re-suspension in a PCR mixture
containing: 2 mM of each dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM
Tris.HCL, pH 9.0, and 0.1% Triton X-100, at a final fragment
concentration of 10-30 ng/ul. No primers are added at this point.
Taq DNA polymerase (Promega) would be used at 2.5 units per 100 ul
of reaction mixture. A PCR program of 94 C for 60 s; 94 C for 30 s,
50-55 C for 30 s, and 72 C for 30 s using 30-45 cycles, followed by
72 C for 5 min using an MJ RESEARCH.RTM. (Cambridge, Mass.) PTC-150
thermocycler. After the assembly reaction is completed, a 1:40
dilution of the resulting primerless product would then be
introduced into a PCR mixture (using the same buffer mixture used
for the assembly reaction) containing 0.8 um of each primer and
subjecting this mixture to 15 cycles of PCR (using 94 C for 30 s,
50 C for 30 s, and 72 C for 30 s). The referred primers would be
primers corresponding to the nucleic acid sequences of the
polynucleotide(s) utilized in the shuffling reaction. Said primers
could consist of modified nucleic acid base pairs using methods
known in the art and referred to else where herein, or could
contain additional sequences (i.e., for adding restriction sites,
mutating specific base-pairs, etc.).
[0827] The resulting shuffled, assembled, and amplified product can
be purified using methods well known in the art (e.g., Qiagen PCR
purification kits) and then subsequently cloned using appropriate
restriction enzymes.
[0828] Although a number of variations of DNA shuffling have been
published to date, such variations would be obvious to the skilled
artisan and are encompassed by the invention. The DNA shuffling
method can also be tailored to the desired level of mutagenesis
using the methods described by Zhao, et al. (Nucl Acid Res.,
25(6):1307-1308, (1997).
[0829] As described above, once the randomized pool has been
created, it can then be subjected to a specific screen to identify
the variant possessing the desired characteristic(s). Once the
variant has been identified, DNA corresponding to the variant could
then be used as the DNA substrate for initiating another round of
DNA shuffling. This cycle of shuffling, selecting the optimized
variant of interest, and then re-shuffling, can be repeated until
the ultimate variant is obtained. Examples of model screens applied
to identify variants created using DNA shuffling technology may be
found in the following publications: J. C., Moore, et al., J. Mol.
Biol., 272:336-347, (1997), F. R., Cross, et al., Mol. Cell. Biol.,
18:2923-2931, (1998), and A. Crameri., et al., Nat. Biotech.,
15:436-438, (1997).
[0830] DNA shuffling has several advantages. First, it makes use of
beneficial mutations. When combined with screening, DNA shuffling
allows the discovery of the best mutational combinations and does
not assume that the best combination contains all the mutations in
a population. Secondly, recombination occurs simultaneously with
point mutagenesis. An effect of forcing DNA polymerase to
synthesize full-length genes from the small fragment DNA pool is a
background mutagenesis rate. In combination with a stringent
selection method, enzymatic activity has been evolved up to 16000
fold increase over the wild-type form of the enzyme. In essence,
the background mutagenesis yielded the genetic variability on which
recombination acted to enhance the activity.
[0831] A third feature of recombination is that it can be used to
remove deleterious mutations. As discussed above, during the
process of the randomization, for every one beneficial mutation,
there may be at least one or more neutral or inhibitory mutations.
Such mutations can be removed by including in the assembly reaction
an excess of the wild-type random-size fragments, in addition to
the random-size fragments of the selected mutant from the previous
selection. During the next selection, some of the most active
variants of the polynucleotide/polypeptide/enzyme, should have lost
the inhibitory mutations.
[0832] Finally, recombination enables parallel processing. This
represents a significant advantage since there are likely multiple
characteristics that would make a protein more desirable (e.g.
solubility, activity, etc.). Since it is increasingly difficult to
screen for more than one desirable trait at a time, other methods
of molecular evolution tend to be inhibitory. However, using
recombination, it would be possible to combine the randomized
fragments of the best representative variants for the various
traits, and then select for multiple properties at once.
[0833] DNA shuffling can also be applied to the polynucleotides and
polypeptides of the present invention to decrease their
immunogenicity in a specified host. For example, a particular
variant of the present invention may be created and isolated using
DNA shuffling technology. Such a variant may have all of the
desired characteristics, though may be highly immunogenic in a host
due to its novel intrinsic structure. Specifically, the desired
characteristic may cause the polypeptide to have a non-native
structure which could no longer be recognized as a "self" molecule,
but rather as a "foreign", and thus activate a host immune response
directed against the novel variant. Such a limitation can be
overcome, for example, by including a copy of the gene sequence for
a xenobiotic ortholog of the native protein in with the gene
sequence of the novel variant gene in one or more cycles of DNA
shuffling. The molar ratio of the ortholog and novel variant DNAs
could be varied accordingly. Ideally, the resulting hybrid variant
identified would contain at least some of the coding sequence which
enabled the xenobiotic protein to evade the host immune system, and
additionally, the coding sequence of the original novel variant
that provided the desired characteristics.
[0834] Likewise, the invention encompasses the application of DNA
shuffling technology to the evolution of polynucleotides and
polypeptides of the invention, wherein one or more cycles of DNA
shuffling include, in addition to the gene template DNA,
oligonucleotides coding for known allelic sequences, optimized
codon sequences, known variant sequences, known polynucleotide
polymorphism sequences, known ortholog sequences, known homologue
sequences, additional homologous sequences, additional
non-homologous sequences, sequences from another species, and any
number and combination of the above.
[0835] In addition to the described methods above, there are a
number of related methods that may also be applicable, or desirable
in certain cases. Representative among these are the methods
discussed in PCT applications WO 98/31700, and WO 98/32845, which
are hereby incorporated by reference. Furthermore, related methods
can also be applied to the polynucleotide sequences of the present
invention in order to evolve invention for creating ideal variants
for use in gene therapy, protein engineering, evolution of whole
cells containing the variant, or in the evolution of entire enzyme
pathways containing polynucleotides of the invention as described
in PCT applications WO 98/13485, WO 98/13487, WO 98/27230, WO
98/31837, and Crameri, A., et al., Nat. Biotech., 15:436-438,
(1997), respectively.
[0836] Additional methods of applying "DNA Shuffling" technology to
the polynucleotides and polypeptides of the present invention,
including their proposed applications, may be found in U.S. Pat.
No. 5,605,793; PCT Application No. WO 95/22625; PCT Application No.
WO 97/20078; PCT Application No. WO 97/35966; and PCT Application
No. WO 98/42832; PCT Application No. WO 00/09727 specifically
provides methods for applying DNA shuffling to the identification
of herbicide selective crops which could be applied to the
polynucleotides and polypeptides of the present invention;
additionally, PCT Application No. WO 00/12680 provides methods and
compositions for generating, modifying, adapting, and optimizing
polynucleotide sequences that confer detectable phenotypic
properties on plant species; each of the above are hereby
incorporated in their entirety herein for all purposes.
Example 19
Method of Determining Alterations in a Gene Corresponding to a
Polynucleotide
[0837] RNA isolated from entire families or individual patients
presenting with a phenotype of interest (such as a disease) is be
isolated. cDNA is then generated from these RNA samples using
protocols known in the art. (See, Sambrook.) The cDNA is then used
as a template for PCR, employing primers surrounding regions of
interest in SEQ ID NO: 1. Suggested PCR conditions consist of 35
cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58
degrees C.; and 60-120 seconds at 70 degrees C., using buffer
solutions described in Sidransky et al., Science 252:706
(1991).
[0838] PCR products are then sequenced using primers labeled at
their 5' end with T4 polynucleotide kinase, employing SequiTherm
Polymerase. (Epicentre Technologies). The intron-exon borders of
selected exons is also determined and genomic PCR products analyzed
to confirm the results. PCR products harboring suspected mutations
is then cloned and sequenced to validate the results of the direct
sequencing.
[0839] PCR products are cloned into T-tailed vectors as described
in Holton et al., Nucleic Acids Research, 19:1156 (1991) and
sequenced with T7 polymerase (United States Biochemical). Affected
individuals are identified by mutations not present in unaffected
individuals.
[0840] Genomic rearrangements are also observed as a method of
determining alterations in a gene corresponding to a
polynucleotide. Genomic clones isolated according to the methods
described herein are nick-translated with digoxigenindeoxy-uridine
5'-triphosphate (Boehringer Manheim), and FISH performed as
described in Johnson et al., Methods Cell Biol. 35:73-99 (1991).
Hybridization with the labeled probe is carried out using a vast
excess of human cot-1 DNA for specific hybridization to the
corresponding genomic locus.
[0841] Chromosomes are counterstained with
4,6-diamino-2-phenylidole and propidium iodide, producing a
combination of C- and R-bands. Aligned images for precise mapping
are obtained using a triple-band filter set (Chroma Technology,
Brattleboro, Vt.) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, Ariz.) and variable excitation
wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75
(1991).) Image collection, analysis and chromosomal fractional
length measurements are performed using the ISEE.RTM. Graphical
Program System. (Inovision Corporation, Durham, N.C.) Chromosome
alterations of the genomic region hybridized by the probe are
identified as insertions, deletions, and translocations. These
alterations are used as a diagnostic marker for an associated
disease.
Example 20
Method of Detecting Abnormal Levels of a Polypeptide in a
Biological Sample
[0842] A polypeptide of the present invention can be detected in a
biological sample, and if an increased or decreased level of the
polypeptide is detected, this polypeptide is a marker for a
particular phenotype. Methods of detection are numerous, and thus,
it is understood that one skilled in the art can modify the
following assay to fit their particular needs.
[0843] For example, antibody-sandwich ELISAs are used to detect
polypeptides in a sample, preferably a biological sample. Wells of
a microtiter plate are coated with specific antibodies, at a final
concentration of 0.2 to 10 ug/ml. The antibodies are either
monoclonal or polyclonal and are produced by the method described
elsewhere herein. The wells are blocked so that non-specific
binding of the polypeptide to the well is reduced.
[0844] The coated wells are then incubated for >2 hours at RT
with a sample containing the polypeptide. Preferably, serial
dilutions of the sample should be used to validate results. The
plates are then washed three times with deionized or distilled
water to remove unbounded polypeptide.
[0845] Next, 50 ul of specific antibody-alkaline phosphatase
conjugate, at a concentration of 25-400 ng, is added and incubated
for 2 hours at room temperature. The plates are again washed three
times with deionized or distilled water to remove unbounded
conjugate.
[0846] Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or
p-nitrophenyl phosphate (NPP) substrate solution to each well and
incubate 1 hour at room temperature. Measure the reaction by a
microtiter plate reader. Prepare a standard curve, using serial
dilutions of a control sample, and plot polypeptide concentration
on the X-axis (log scale) and fluorescence or absorbance of the
Y-axis (linear scale). Interpolate the concentration of the
polypeptide in the sample using the standard curve.
Example 21
Method of Isolating Antibody Fragments Directed Against PCSK9b or
PCSK9c from a Library of scFvs
[0847] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against PCSK9b or PCSK9c to which the donor may or may
not have been exposed (see e.g., U.S. Pat. No. 5,885,793
incorporated herein by reference in its entirety).
[0848] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100
.mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of
0.8 with shaking. Five ml of this culture is used to inoculate 50
ml of 2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper
(M13 delta gene III, see PCT publication WO 92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 .mu.g/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[0849] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phage particles are
purified and concentrated from the culture medium by two
PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS
and passed through a 0.45 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[0850] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of
a polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 .mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[0851] Characterization of Binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing. These ELISA positive clones may
also be further characterized by techniques known in the art, such
as, for example, epitope mapping, binding affinity, receptor signal
transduction, ability to block or competitively inhibit
antibody/antigen binding, and competitive agonistic or antagonistic
activity.
[0852] Moreover, in another preferred method, the antibodies
directed against the polypeptides of the present invention may be
produced in plants. Specific methods are disclosed in U.S. Pat.
Nos. 5,959,177, and 6,080,560, which are hereby incorporated in
their entirety herein. The methods not only describe methods of
expressing antibodies, but also the means of assembling foreign
multimeric proteins in plants (i.e., antibodies, etc,), and the
subsequent secretion of such antibodies from the plant.
Sequence CWU 1
1
4013175DNAHomo sapiensCDS(250)..(1194) 1gatgacttgg gtccttcttg
gcagtagcat tgccagctga tggccttgga cagttacctg 60ccctctctag gcctcccttt
ccttgtctat gaaatacatt atagaatagg atgtagtgtg 120tgaggatttt
ttggaggtta aacgagtgaa tatatttaag gcgctttcac cagtgcctgg
180gatgtgctct gtagtttctg tgtgttaact ataaggttga ctttatgctc
attccctcct 240ctcccacaa atg tcg cct tgg aaa gac gga ggc agc ctg gtg
gag gtg tat 291 Met Ser Pro Trp Lys Asp Gly Gly Ser Leu Val Glu Val
Tyr 1 5 10 ctc cta gac acc agc ata cag agt gac cac cgg gaa atc gag
ggc agg 339Leu Leu Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu
Gly Arg 15 20 25 30 gtc atg gtc acc gac ttc gag aat gtg ccc gag gag
gac ggg acc cgc 387Val Met Val Thr Asp Phe Glu Asn Val Pro Glu Glu
Asp Gly Thr Arg 35 40 45 ttc cac aga cag gcc agc aag tgt gac agt
cat ggc acc cac ctg gca 435Phe His Arg Gln Ala Ser Lys Cys Asp Ser
His Gly Thr His Leu Ala 50 55 60 gga gtg gtc agc ggc cgg gat gcc
ggc gtg gcc aag ggt gcc agc atg 483Gly Val Val Ser Gly Arg Asp Ala
Gly Val Ala Lys Gly Ala Ser Met 65 70 75 cgc agc ctg cgc gtg ctc
aac tgc caa ggg aag ggc acg gtt agc ggc 531Arg Ser Leu Arg Val Leu
Asn Cys Gln Gly Lys Gly Thr Val Ser Gly 80 85 90 acc ctc ata ggc
ctg gag ttt att cgg aaa agc cag ctg gtc cag cct 579Thr Leu Ile Gly
Leu Glu Phe Ile Arg Lys Ser Gln Leu Val Gln Pro 95 100 105 110 gtg
ggg cca ctg gtg gtg ctg ctg ccc ctg gcg ggt ggg tac agc cgc 627Val
Gly Pro Leu Val Val Leu Leu Pro Leu Ala Gly Gly Tyr Ser Arg 115 120
125 gtc ctc aac gcc gcc tgc cag cgc ctg gcg agg gct ggg gtc gtg ctg
675Val Leu Asn Ala Ala Cys Gln Arg Leu Ala Arg Ala Gly Val Val Leu
130 135 140 gtc acc gct gcc ggc aac ttc cgg gac gat gcc tgc ctc tac
tcc cca 723Val Thr Ala Ala Gly Asn Phe Arg Asp Asp Ala Cys Leu Tyr
Ser Pro 145 150 155 gcc tca gct ccc gag gtc atc aca gtt ggg gcc acc
aat gcc cag gac 771Ala Ser Ala Pro Glu Val Ile Thr Val Gly Ala Thr
Asn Ala Gln Asp 160 165 170 cag ccg gtg acc ctg ggg act ttg ggg acc
aac ttt ggc cgc tgt gtg 819Gln Pro Val Thr Leu Gly Thr Leu Gly Thr
Asn Phe Gly Arg Cys Val 175 180 185 190 gac ctc ttt gcc cca ggg gag
gac atc att ggt gcc tcc agc gac tgc 867Asp Leu Phe Ala Pro Gly Glu
Asp Ile Ile Gly Ala Ser Ser Asp Cys 195 200 205 agc acc tgc ttt gtg
tca cag agt ggg aca tca cag gct gct gcc cac 915Ser Thr Cys Phe Val
Ser Gln Ser Gly Thr Ser Gln Ala Ala Ala His 210 215 220 gtg gct ggc
att gca gcc atg atg ctg tct gcc gag ccg gag ctc acc 963Val Ala Gly
Ile Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr 225 230 235 ctg
gcc gag ttg agg cag aga ctg atc cac ttc tct gcc aaa gat gtc 1011Leu
Ala Glu Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp Val 240 245
250 atc aat gag gcc tgg ttc cct gag gac cag cgg gta ctg acc ccc aac
1059Ile Asn Glu Ala Trp Phe Pro Glu Asp Gln Arg Val Leu Thr Pro Asn
255 260 265 270 ctg gtg gcc gcc ctg ccc ccc agc acc cat ggg gca ggc
cct ttt tgc 1107Leu Val Ala Ala Leu Pro Pro Ser Thr His Gly Ala Gly
Pro Phe Cys 275 280 285 agg ttg gca gct gtt ttg cag gac tgt gtg gtc
agc aca ctc ggg gcc 1155Arg Leu Ala Ala Val Leu Gln Asp Cys Val Val
Ser Thr Leu Gly Ala 290 295 300 tac acg gat ggc cac agc cat cgc ccg
ctg cgc ccc aga tgaggagctg 1204Tyr Thr Asp Gly His Ser His Arg Pro
Leu Arg Pro Arg 305 310 315 ctgagctgct ccagtttctc caggagtggg
aagcggcggg gcgagcgcat ggaggcccaa 1264gggggcaagc tggtctgccg
ggcccacaac gcttttgggg gtgagggtgt ctacgccatt 1324gccaggtgct
gcctgctacc ccaggccaac tgcagcgtcc acacagctcc accagctgag
1384gccagcatgg ggacccgtgt ccactgccac caacagggcc acgtcctcac
aggctgcagc 1444tcccactggg aggtggagga ccttggcacc cacaagccgc
ctgtgctgag gccacgaggt 1504cagcccaacc agtgcgtggg ccacagggag
gccagcatcc acgcttcctg ctgccatgcc 1564ccaggtctgg aatgcaaagt
caaggagcat ggaatcccgg cccctcagga gcaggtgacc 1624gtggcctgcg
aggagggctg gaccctgact ggctgcagtg ccctccctgg gacctcccac
1684gtcctggggg cctacgccgt agacaacacg tgtgtagtca ggagccggga
cgtcagcact 1744acaggcagca ccagcgaaga ggccgtgaca gccgttgcca
tctgctgccg gagccggcac 1804ctggcgcagg cctcccagga gctccagtga
cagccccatc ccaggatggg tgtctgggga 1864gggtcaaggg ctggggctga
gctttaaaat ggttccgact tgtccctctc tcagccctcc 1924atggcctggc
acgaggggat ggggatgctt ccgcctttcc ggggctgctg gcctggccct
1984tgagtggggc agcctccttg cctggaactc actcactctg ggtgcctcct
ccccaggtgg 2044aggtgccagg aagctccctc cctcactgtg gggcatttca
ccattcaaac aggtcgagct 2104gtgctcgggt gctgccagct gctcccaatg
tgccgatgtc cgtgggcaga atgactttta 2164ttgagctctt gttccgtgcc
aggcattcaa tcctcaggtc tccaccaagg aggcaggatt 2224cttcccatgg
ataggggagg gggcggtagg ggctgcaggg acaaacatcg ttggggggtg
2284agtgtgaaag gtgctgatgg ccctcatctc cagctaactg tggagaagcc
cctgggggct 2344ccctgattaa tggaggctta gctttctgga tggcatctag
ccagaggctg gagacaggtg 2404tgcccctggt ggtcacaggc tgtgccttgg
tttcctgagc cacctttact ctgctctatg 2464ccaggctgtg ctagcaacac
ccaaaggtgg cctgcgggga gccatcacct aggactgact 2524cggcagtgtg
cagtggtgca tgcactgtct cagccaaccc gctccactac ccggcagggt
2584acacattcgc acccctactt cacagaggaa gaaacctgga accagagggg
gcgtgcctgc 2644caagctcaca cagcaggaac tgagccagaa acgcagattg
ggctggctct gaagccaagc 2704ctcttcttac ttcacccggc tgggctcctc
atttttacgg gtaacagtga ggctgggaag 2764gggaacacag accaggaagc
tcggtgagtg atggcagaac gatgcctgca ggcatggaac 2824tttttccgtt
atcacccagg cctgattcac tggcctggcg gagatgcttc taaggcatgg
2884tcgggggaga gggccaacaa ctgtccctcc ttgagcacca gccccaccca
agcaagcaga 2944catttatctt ttgggtctgt cctctctgtt gcctttttac
agccaacttt tctagacctg 3004ttttgctttt gtaacttgaa gatatttatt
ctgggttttg tagcattttt attaatatgg 3064tgacttttta aaataaaaac
aaacaaacgt tgtcctaaaa aaaaaaaaaa aaaaaaaaaa 3124aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 31752315PRTHomo
sapiens 2Met Ser Pro Trp Lys Asp Gly Gly Ser Leu Val Glu Val Tyr
Leu Leu 1 5 10 15 Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile Glu
Gly Arg Val Met 20 25 30 Val Thr Asp Phe Glu Asn Val Pro Glu Glu
Asp Gly Thr Arg Phe His 35 40 45 Arg Gln Ala Ser Lys Cys Asp Ser
His Gly Thr His Leu Ala Gly Val 50 55 60 Val Ser Gly Arg Asp Ala
Gly Val Ala Lys Gly Ala Ser Met Arg Ser 65 70 75 80 Leu Arg Val Leu
Asn Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu 85 90 95 Ile Gly
Leu Glu Phe Ile Arg Lys Ser Gln Leu Val Gln Pro Val Gly 100 105 110
Pro Leu Val Val Leu Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val Leu 115
120 125 Asn Ala Ala Cys Gln Arg Leu Ala Arg Ala Gly Val Val Leu Val
Thr 130 135 140 Ala Ala Gly Asn Phe Arg Asp Asp Ala Cys Leu Tyr Ser
Pro Ala Ser 145 150 155 160 Ala Pro Glu Val Ile Thr Val Gly Ala Thr
Asn Ala Gln Asp Gln Pro 165 170 175 Val Thr Leu Gly Thr Leu Gly Thr
Asn Phe Gly Arg Cys Val Asp Leu 180 185 190 Phe Ala Pro Gly Glu Asp
Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr 195 200 205 Cys Phe Val Ser
Gln Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala 210 215 220 Gly Ile
Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala 225 230 235
240 Glu Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys Asp Val Ile Asn
245 250 255 Glu Ala Trp Phe Pro Glu Asp Gln Arg Val Leu Thr Pro Asn
Leu Val 260 265 270 Ala Ala Leu Pro Pro Ser Thr His Gly Ala Gly Pro
Phe Cys Arg Leu 275 280 285 Ala Ala Val Leu Gln Asp Cys Val Val Ser
Thr Leu Gly Ala Tyr Thr 290 295 300 Asp Gly His Ser His Arg Pro Leu
Arg Pro Arg 305 310 315 33756DNAHomo sapiensCDS(881)..(2449)
3tgctagcagc aacctgcctg aagtcttcct ttggcctggc tgagagtttc tgagacctgc
60gctggagcgg aggtgcttcc ttccttgctt cctttcttcc tctctccctt ctccatccag
120caggctggac ctgcctggca tctgtgagct ctccctactt tctcctatac
cctaaccttt 180gtcctgcatg ggcgactccc ccagtgagtc tcttgcagct
tttaccccag tgcctgcttc 240ttggagaatc caaactgatc cagttaggga
tgataaagtg tagggtaggc gctcggtgac 300tgttttctct gaggttgtga
ctcgtgtgag gcagaagcag tccccgtgag ccctcctggt 360atcttgtgga
gtggagaacg cttggacctg gagccaggag gcccagacat acatcctgtc
420cgagctgcag cttcctgtct ctaaaatgag ccggccagcg caggtggcca
gacatcactg 480ttattctcct ttgagtcttt aaatcttgtt gtctttcttg
cagactcggt gagctgtgaa 540aggctataat aggggcttta ttttacactt
tgatactatt ttttgaacat tcatattatt 600gttagatatt gatattcata
tgaaggagca ggatgacttg ggtccttctt ggcagtagca 660ttgccagctg
atggccttgg acagttacct gccctctcta ggcctccctt tccttgtcta
720tgaaatacat tatagaatag gatgtagtgt gtgaggattt tttggaggtt
aaacgagtga 780atatatttaa ggcgctttca ccagtgcctg ggatgtgctc
tgtagtttct gtgtgttaac 840tataaggttg actttatgct cattccctcc
tctcccacaa atg tcg cct tgg aaa 895 Met Ser Pro Trp Lys 1 5 gac gga
ggc agc ctg gtg gag gtg tat ctc cta gac acc agc ata cag 943Asp Gly
Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln 10 15 20
agt gac cac cgg gaa atc gag ggc agg gtc atg gtc acc gac ttc gag
991Ser Asp His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu
25 30 35 aat gtg ccc gag gag gac ggg acc cgc ttc cac aga cag gcc
agc aag 1039Asn Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala
Ser Lys 40 45 50 tgt gac agt cat ggc acc cac ctg gca ggg gtg gtc
agc ggc cgg gat 1087Cys Asp Ser His Gly Thr His Leu Ala Gly Val Val
Ser Gly Arg Asp 55 60 65 gcc ggc gtg gcc aag ggt gcc agc atg cgc
agc ctg cgc gtg ctc aac 1135Ala Gly Val Ala Lys Gly Ala Ser Met Arg
Ser Leu Arg Val Leu Asn 70 75 80 85 tgc caa ggg aag ggc acg gtt agc
ggc acc ctc ata ggc ctg gag ttt 1183Cys Gln Gly Lys Gly Thr Val Ser
Gly Thr Leu Ile Gly Leu Glu Phe 90 95 100 att cgg aaa agc cag ctg
gtc cag cct gtg ggg cca ctg gtg gtg ctg 1231Ile Arg Lys Ser Gln Leu
Val Gln Pro Val Gly Pro Leu Val Val Leu 105 110 115 ctg ccc ctg gcg
ggt ggg tac agc cgc gtc ctc aac gcc gcc tgc cag 1279Leu Pro Leu Ala
Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln 120 125 130 cgc ctg
gcg agg gct ggg gtc gtg ctg gtc acc gct gcc ggc aac ttc 1327Arg Leu
Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe 135 140 145
cgg gac gat gcc tgc ctc tac tcc cca gcc tca gct ccc gag gtc atc
1375Arg Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile
150 155 160 165 aca gtt ggg gcc acc aat gcc cag gac cag ccg gtg acc
ctg ggg act 1423Thr Val Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr
Leu Gly Thr 170 175 180 ttg ggg acc aac ttt ggc cgc tgt gtg gac ctc
ttt gcc cca ggg gag 1471Leu Gly Thr Asn Phe Gly Arg Cys Val Asp Leu
Phe Ala Pro Gly Glu 185 190 195 gac atc att ggt gcc tcc agc gac tgc
agc acc tgc ttt gtg tca cag 1519Asp Ile Ile Gly Ala Ser Ser Asp Cys
Ser Thr Cys Phe Val Ser Gln 200 205 210 agt ggg aca tca cag gct gct
gcc cac gtg gct ggc att gca gcc atg 1567Ser Gly Thr Ser Gln Ala Ala
Ala His Val Ala Gly Ile Ala Ala Met 215 220 225 atg ctg tct gcc gag
ccg gag ctc acc ctg gcc gag ttg agg cag aga 1615Met Leu Ser Ala Glu
Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg 230 235 240 245 ctg atc
cac ttc tct gcc aaa gat gtc atc aat gag gcc tgg ttc cct 1663Leu Ile
His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro 250 255 260
gag gac cag cgg gta ctg acc ccc aac ctg gtg gcc gcc ctg ccc ccc
1711Glu Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro
265 270 275 agc acc cat ggg gca ggt tgg cag ctg ttt tgc agg act gtg
tgg tca 1759Ser Thr His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val
Trp Ser 280 285 290 gca cac tcg ggg cct aca cgg atg gcc aca gcc atc
gcc cgc tgc gcc 1807Ala His Ser Gly Pro Thr Arg Met Ala Thr Ala Ile
Ala Arg Cys Ala 295 300 305 cca gat gag gag ctg ctg agc tgc tcc agt
ttc tcc agg agt ggg aag 1855Pro Asp Glu Glu Leu Leu Ser Cys Ser Ser
Phe Ser Arg Ser Gly Lys 310 315 320 325 cgg cgg ggc gag cgc atg gag
gcc caa ggg ggc aag ctg gtc tgc cgg 1903Arg Arg Gly Glu Arg Met Glu
Ala Gln Gly Gly Lys Leu Val Cys Arg 330 335 340 gcc cac aac gct ttt
ggg ggt gag ggt gtc tac gcc att gcc agg tgc 1951Ala His Asn Ala Phe
Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys 345 350 355 tgc ctg cta
ccc cag gcc aac tgc agc gtc cac aca gct cca cca gct 1999Cys Leu Leu
Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala 360 365 370 gag
gcc agc atg ggg acc cgt gtc cac tgc cac caa cag ggc cac gtc 2047Glu
Ala Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val 375 380
385 ctc aca ggc tgc agc tcc cac tgg gag gtg gag gac ctt ggc acc cac
2095Leu Thr Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His
390 395 400 405 aag ccg cct gtg ctg agg cca cga ggt cag ccc aac cag
tgc gtg ggc 2143Lys Pro Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln
Cys Val Gly 410 415 420 cac agg gag gcc agc atc cac gct tcc tgc tgc
cat gcc cca ggt ctg 2191His Arg Glu Ala Ser Ile His Ala Ser Cys Cys
His Ala Pro Gly Leu 425 430 435 gaa tgc aaa gtc aag gag cat gga atc
ccg gcc cct cag gag cag gtg 2239Glu Cys Lys Val Lys Glu His Gly Ile
Pro Ala Pro Gln Glu Gln Val 440 445 450 acc gtg gcc tgc gag gag ggc
tgg acc ctg act ggc tgc agt gcc ctc 2287Thr Val Ala Cys Glu Glu Gly
Trp Thr Leu Thr Gly Cys Ser Ala Leu 455 460 465 cct ggg acc tcc cac
gtc ctg ggg gcc tac gcc gta gac aac acg tgt 2335Pro Gly Thr Ser His
Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys 470 475 480 485 gta gtc
agg agc cgg gac gtc agc act aca ggc agc acc agc gaa ggg 2383Val Val
Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly 490 495 500
gcc gtg aca gcc gtt gcc atc tgc tgc cgg agc cgg cac ctg gcg cag
2431Ala Val Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln
505 510 515 gcc tcc cag gag ctc cag tgacagcccc atcccaggat
gggtgtctgg 2479Ala Ser Gln Glu Leu Gln 520 ggagggtcaa gggctggggc
tgagctttaa aatggttccg acttgtccct ctctcagccc 2539tccatggcct
ggcacgaggg gatggggatg cttccgcctt tccggggctg ctggcctggc
2599ccttgagtgg
ggcagcctcc ttgcctggaa ctcactcact ctgggtgcct cctccccagg
2659tggaggtgcc aggaagctcc ctccctcact gtggggcatt tcaccattca
aacaggtcga 2719gctgtgctcg ggtgctgcca gctgctccca atgtgccgat
gtccgtgggc agaatgactt 2779ttattgagct cttgttccgt gccaggcatt
caatcctcag gtctccacca aggaggcagg 2839attcttccca tggatagggg
agggggcggt aggggctgca gggacaaaca tcgttggggg 2899gtgagtgtga
aaggtgctga tggccctcat ctccagctaa ctgtggagaa gcccctgggg
2959gctccctgat taatggaggc ttagctttct ggatggcatc tagccagagg
ctggagacag 3019gtgcgcccct ggtggtcaca ggctgtgcct tggtttcctg
agccaccttt actctgctct 3079atgccaggct gtgctagcaa cacccaaagg
tggcctgcgg ggagccatca cctaggactg 3139actcggcagt gtgcagtggt
gcatgcactg tctcagccaa cccgctccac tacccggcag 3199ggtacacatt
cgcaccccta cttcacagag gaagaaacct ggaaccagag ggggcgtgcc
3259tgccaagctc acacagcagg aactgagcca gaaacgcaga ttgggctggc
tctgaagcca 3319agcctcttct tacttcaccc ggctgggctc ctcattttta
cgggtaacag tgaggctggg 3379aaggggaaca cagaccagga agctcggtga
gtgatggcag aacgatgcct gcaggcatgg 3439aactttttcc gttatcaccc
aggcctgatt cactggcctg gcggagatgc ttctaaggca 3499tggtcggggg
agagggccaa caactgtccc tccttgagca ccagccccac ccaagcaagc
3559agacatttat cttttgggtc tgtcctctct gttgcctttt tacagccaac
ttttctagac 3619ctgttttgct tttgtaactt gaagatattt attctgggtt
ttgtagcatt tttattaata 3679tggtgacttt ttaaaataaa aacaaacaaa
cgttgtccta aaaaaaaaaa aaaaaaaaaa 3739aaaaaaaaaa aaaaaaa
37564523PRTHomo sapiens 4Met Ser Pro Trp Lys Asp Gly Gly Ser Leu
Val Glu Val Tyr Leu Leu 1 5 10 15 Asp Thr Ser Ile Gln Ser Asp His
Arg Glu Ile Glu Gly Arg Val Met 20 25 30 Val Thr Asp Phe Glu Asn
Val Pro Glu Glu Asp Gly Thr Arg Phe His 35 40 45 Arg Gln Ala Ser
Lys Cys Asp Ser His Gly Thr His Leu Ala Gly Val 50 55 60 Val Ser
Gly Arg Asp Ala Gly Val Ala Lys Gly Ala Ser Met Arg Ser 65 70 75 80
Leu Arg Val Leu Asn Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu 85
90 95 Ile Gly Leu Glu Phe Ile Arg Lys Ser Gln Leu Val Gln Pro Val
Gly 100 105 110 Pro Leu Val Val Leu Leu Pro Leu Ala Gly Gly Tyr Ser
Arg Val Leu 115 120 125 Asn Ala Ala Cys Gln Arg Leu Ala Arg Ala Gly
Val Val Leu Val Thr 130 135 140 Ala Ala Gly Asn Phe Arg Asp Asp Ala
Cys Leu Tyr Ser Pro Ala Ser 145 150 155 160 Ala Pro Glu Val Ile Thr
Val Gly Ala Thr Asn Ala Gln Asp Gln Pro 165 170 175 Val Thr Leu Gly
Thr Leu Gly Thr Asn Phe Gly Arg Cys Val Asp Leu 180 185 190 Phe Ala
Pro Gly Glu Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr 195 200 205
Cys Phe Val Ser Gln Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala 210
215 220 Gly Ile Ala Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu
Ala 225 230 235 240 Glu Leu Arg Gln Arg Leu Ile His Phe Ser Ala Lys
Asp Val Ile Asn 245 250 255 Glu Ala Trp Phe Pro Glu Asp Gln Arg Val
Leu Thr Pro Asn Leu Val 260 265 270 Ala Ala Leu Pro Pro Ser Thr His
Gly Ala Gly Trp Gln Leu Phe Cys 275 280 285 Arg Thr Val Trp Ser Ala
His Ser Gly Pro Thr Arg Met Ala Thr Ala 290 295 300 Ile Ala Arg Cys
Ala Pro Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe 305 310 315 320 Ser
Arg Ser Gly Lys Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly 325 330
335 Lys Leu Val Cys Arg Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr
340 345 350 Ala Ile Ala Arg Cys Cys Leu Leu Pro Gln Ala Asn Cys Ser
Val His 355 360 365 Thr Ala Pro Pro Ala Glu Ala Ser Met Gly Thr Arg
Val His Cys His 370 375 380 Gln Gln Gly His Val Leu Thr Gly Cys Ser
Ser His Trp Glu Val Glu 385 390 395 400 Asp Leu Gly Thr His Lys Pro
Pro Val Leu Arg Pro Arg Gly Gln Pro 405 410 415 Asn Gln Cys Val Gly
His Arg Glu Ala Ser Ile His Ala Ser Cys Cys 420 425 430 His Ala Pro
Gly Leu Glu Cys Lys Val Lys Glu His Gly Ile Pro Ala 435 440 445 Pro
Gln Glu Gln Val Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr 450 455
460 Gly Cys Ser Ala Leu Pro Gly Thr Ser His Val Leu Gly Ala Tyr Ala
465 470 475 480 Val Asp Asn Thr Cys Val Val Arg Ser Arg Asp Val Ser
Thr Thr Gly 485 490 495 Ser Thr Ser Glu Gly Ala Val Thr Ala Val Ala
Ile Cys Cys Arg Ser 500 505 510 Arg His Leu Ala Gln Ala Ser Gln Glu
Leu Gln 515 520 5692PRTHomo sapiens 5Met Gly Thr Val Ser Ser Arg
Arg Ser Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu
Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25 30 Asp Glu Asp
Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu 35 40 45 Glu
Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 50 55
60 His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val
65 70 75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr
Ala Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu
Thr Lys Ile Leu 100 105 110 His Val Phe His Gly Leu Leu Pro Gly Phe
Leu Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu Leu Ala Leu Lys
Leu Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp Ser Ser Val Phe
Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg 145 150 155 160 Ile Thr Pro
Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly 165 170 175 Gly
Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp 180 185
190 His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val
195 200 205 Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys
Cys Asp 210 215 220 Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly
Arg Asp Ala Gly 225 230 235 240 Val Ala Lys Gly Ala Ser Met Arg Ser
Leu Arg Val Leu Asn Cys Gln 245 250 255 Gly Lys Gly Thr Val Ser Gly
Thr Leu Ile Gly Leu Glu Phe Ile Arg 260 265 270 Lys Ser Gln Leu Val
Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro 275 280 285 Leu Ala Gly
Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu 290 295 300 Ala
Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp 305 310
315 320 Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr
Val 325 330 335 Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly
Thr Leu Gly 340 345 350 Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala
Pro Gly Glu Asp Ile 355 360 365 Ile Gly Ala Ser Ser Asp Cys Ser Thr
Cys Phe Val Ser Gln Ser Gly 370 375 380 Thr Ser Gln Ala Ala Ala His
Val Ala Gly Ile Ala Ala Met Met Leu 385 390 395 400 Ser Ala Glu Pro
Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 405 410 415 His Phe
Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp 420 425 430
Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr 435
440 445 His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala
His 450 455 460 Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys
Ala Pro Asp 465 470 475 480 Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser
Arg Ser Gly Lys Arg Arg 485 490 495 Gly Glu Arg Met Glu Ala Gln Gly
Gly Lys Leu Val Cys Arg Ala His 500 505 510 Asn Ala Phe Gly Gly Glu
Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu 515 520 525 Leu Pro Gln Ala
Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 530 535 540 Ser Met
Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr 545 550 555
560 Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro
565 570 575 Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly
His Arg 580 585 590 Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro
Gly Leu Glu Cys 595 600 605 Lys Val Lys Glu His Gly Ile Pro Ala Pro
Gln Glu Gln Val Thr Val 610 615 620 Ala Cys Glu Glu Gly Trp Thr Leu
Thr Gly Cys Ser Ala Leu Pro Gly 625 630 635 640 Thr Ser His Val Leu
Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val 645 650 655 Arg Ser Arg
Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val 660 665 670 Thr
Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser 675 680
685 Gln Glu Leu Gln 690 6196PRTHomo sapiens 6Met Ser Pro Trp Lys
Asp Gly Gly Ser Leu Val Glu Val Tyr Leu Leu 1 5 10 15 Asp Thr Ser
Ile Gln Ser Asp His Arg Glu Ile Glu Gly Arg Val Met 20 25 30 Val
Thr Asp Phe Glu Asn Val Pro Glu Glu Asp Gly Thr Arg Phe His 35 40
45 Arg Gln Ala Ser Lys Cys Asp Ser His Gly Thr His Leu Ala Gly Val
50 55 60 Val Ser Gly Arg Asp Ala Gly Val Ala Lys Gly Ala Ser Met
Arg Ser 65 70 75 80 Leu Arg Val Leu Asn Cys Gln Gly Lys Gly Thr Val
Ser Gly Thr Leu 85 90 95 Ile Gly Leu Glu Phe Ile Arg Lys Ser Gln
Leu Val Gln Pro Val Gly 100 105 110 Pro Leu Val Val Leu Leu Pro Leu
Ala Gly Gly Tyr Ser Arg Val Leu 115 120 125 Asn Ala Ala Cys Gln Arg
Leu Ala Arg Ala Gly Val Val Leu Val Thr 130 135 140 Ala Ala Gly Asn
Phe Arg Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser 145 150 155 160 Ala
Pro Glu Gly Arg Thr Ser Leu Val Pro Pro Ala Thr Ala Ala Pro 165 170
175 Ala Leu Cys His Arg Val Gly His His Arg Leu Leu Pro Thr Trp Leu
180 185 190 Ala Leu Gln Pro 195 780DNAHomo sapiens 7ggcgctttca
ccagtggctg ggatgtgctc tgtagtttct gtgtgttaac tataaggttg 60actttatgct
cattccctcc 80820DNAHomo sapiens 8ctaggcctcc ctttccttgt 20920DNAHomo
sapiens 9ttccaaggtg acatttgtgg 201017DNAHomo sapiens 10cctgcgcgtg
ctcaact 171122DNAHomo sapiens 11ccgaataaac tccaggccta tg
221223DNAHomo sapiens 12ccgctaaccg tgcccttccc ttg 231320DNAHomo
sapiens 13ctaggcctcc ctttccttgt 201479DNAHomo sapiens 14tggcaggcgg
cgttgaggac gcggctgtac ccacccgcca ggggcagcag caccaccagt 60ggccccacag
gctggacca 791520DNAHomo sapiens 15gcctggagtt tattcggaaa
201620DNAHomo sapiens 16gagtagaggc aggcatcgtc 201720DNAHomo sapiens
17gagtagaggc aggcatcgtc 20187PRTArtificialSynthesized Polypeptide.
18Phe Ala Gln Ser Ile Pro Lys 1 5 197PRTArtificialSynthesized
Polypeptide. 19Asp Ser Leu Val Phe Ala Lys 1 5
207PRTArtificialSynthesized Polypeptide. 20Phe Ala Asn Ala Ile Pro
Lys 1 5 21733DNAHomo sapiens 21gggatccgga gcccaaatct tctgacaaaa
ctcacacatg cccaccgtgc ccagcacctg 60aattcgaggg tgcaccgtca gtcttcctct
tccccccaaa acccaaggac accctcatga 120tctcccggac tcctgaggtc
acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180tcaagttcaa
ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg
240aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
caccaggact 300ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca acccccatcg 360agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac accctgcccc 420catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480atccaagcga
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga
540ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag
ctcaccgtgg 600acaagagcag gtggcagcag gggaacgtct tctcatgctc
cgtgatgcat gaggctctgc 660acaaccacta cacgcagaag agcctctccc
tgtctccggg taaatgagtg cgacggccgc 720gactctagag gat 7332239DNAHomo
sapiens 22gcagcagcgg ccgcctagac accagcatac agagtgacc 392337DNAHomo
sapiens 23gcagcagtcg actctggggc gcagcgggcg atggctg 372439DNAHomo
sapiens 24gcagcagcgg ccgcatgtcg ccttggaaag acggaggca 392537DNAHomo
sapiens 25gcagcagtcg acagggcctg ccccatgggt gctgggg 372639DNAHomo
sapiens 26gcagcagcgg ccgcctagac accagcatac agagtgacc 392725DNAHomo
sapiens 27ctggagctcc tgggaggcct gcgcc 252839DNAHomo sapiens
28gcagcagcgg ccgcatgtcg ccttggaaag acggaggca 392937DNAHomo sapiens
29gcagcagtcg acggcgatgg ctgtggccat ccgtgta 373020DNAHomo sapiens
30tgtctttgcc cagagcatcc 203119DNAHomo sapiens 31tattcatccg
cccggtacc 193220DNAHomo sapiens 32agatgtcatc aatgaggcct
203320DNAHomo sapiens 33agctgccaac ctgcaaaaac 203423DNAHomo sapiens
34ctctgaggtt gtgactcgtg tga 233521DNAHomo sapiens 35agcgttctcc
actccacaag a 213621DNAHomo sapiens 36gagaatgatc tgcagcaccc a
213721DNAHomo sapiens 37tgctgatgac ggtgtcatag g 21383636DNAHomo
sapiens 38cagcgacgtc gaggcgctca tggttgcagg cgggcgccgc cgttcagttc
agggtctgag 60cctggaggag tgagccaggc agtgagactg gctcgggcgg gccgggacgc
gtcgttgcag 120cagcggctcc cagctcccag ccaggattcc gcgcgcccct
tcacgcgccc tgctcctgaa 180cttcagctcc tgcacagtcc tccccaccgc
aaggctcaag gcgccgccgg cgtggaccgc 240gcacggcctc taggtctcct
cgccaggaca gcaacctctc ccctggccct catgggcacc 300gtcagctcca
ggcggtcctg gtggccgctg ccactgctgc tgctgctgct gctgctcctg
360ggtcccgcgg gcgcccgtgc gcaggaggac gaggacggcg actacgagga
gctggtgcta 420gccttgcgtt ccgaggagga cggcctggcc gaagcacccg
agcacggaac cacagccacc 480ttccaccgct gcgccaagga tccgtggagg
ttgcctggca cctacgtggt ggtgctgaag 540gaggagaccc acctctcgca
gtcagagcgc actgcccgcc gcctgcaggc ccaggctgcc 600cgccggggat
acctcaccaa gatcctgcat gtcttccatg gccttcttcc tggcttcctg
660gtgaagatga gtggcgacct gctggagctg gccttgaagt tgccccatgt
cgactacatc 720gaggaggact cctctgtctt tgcccagagc atcccgtgga
acctggagcg gattacccct 780ccacggtacc gggcggatga ataccagccc
cccgacggag gcagcctggt ggaggtgtat 840ctcctagaca ccagcataca
gagtgaccac cgggaaatcg agggcagggt catggtcacc 900gacttcgaga
atgtgcccga ggaggacggg acccgcttcc acagacaggc cagcaagtgt
960gacagtcatg gcacccacct ggcaggggtg gtcagcggcc gggatgccgg
cgtggccaag 1020ggtgccagca tgcgcagcct gcgcgtgctc aactgccaag
ggaagggcac ggttagcggc 1080accctcatag gcctggagtt tattcggaaa
agccagctgg tccagcctgt ggggccactg 1140gtggtgctgc tgcccctggc
gggtgggtac agccgcgtcc tcaacgccgc ctgccagcgc 1200ctggcgaggg
ctggggtcgt gctggtcacc gctgccggca acttccggga cgatgcctgc
1260ctctactccc cagcctcagc
tcccgaggtc atcacagttg gggccaccaa tgcccaagac 1320cagccggtga
ccctggggac tttggggacc aactttggcc gctgtgtgga cctctttgcc
1380ccaggggagg acatcattgg tgcctccagc gactgcagca cctgctttgt
gtcacagagt 1440gggacatcac aggctgctgc ccacgtggct ggcattgcag
ccatgatgct gtctgccgag 1500ccggagctca ccctggccga gttgaggcag
agactgatcc acttctctgc caaagatgtc 1560atcaatgagg cctggttccc
tgaggaccag cgggtactga cccccaacct ggtggccgcc 1620ctgcccccca
gcacccatgg ggcaggttgg cagctgtttt gcaggactgt atggtcagca
1680cactcggggc ctacacggat ggccacagcc gtcgcccgct gcgccccaga
tgaggagctg 1740ctgagctgct ccagtttctc caggagtggg aagcggcggg
gcgagcgcat ggaggcccaa 1800gggggcaagc tggtctgccg ggcccacaac
gcttttgggg gtgagggtgt ctacgccatt 1860gccaggtgct gcctgctacc
ccaggccaac tgcagcgtcc acacagctcc accagctgag 1920gccagcatgg
ggacccgtgt ccactgccac caacagggcc acgtcctcac aggctgcagc
1980tcccactggg aggtggagga ccttggcacc cacaagccgc ctgtgctgag
gccacgaggt 2040cagcccaacc agtgcgtggg ccacagggag gccagcatcc
acgcttcctg ctgccatgcc 2100ccaggtctgg aatgcaaagt caaggagcat
ggaatcccgg cccctcagga gcaggtgacc 2160gtggcctgcg aggagggctg
gaccctgact ggctgcagtg ccctccctgg gacctcccac 2220gtcctggggg
cctacgccgt agacaacacg tgtgtagtca ggagccggga cgtcagcact
2280acaggcagca ccagcgaagg ggccgtgaca gccgttgcca tctgctgccg
gagccggcac 2340ctggcgcagg cctcccagga gctccagtga cagccccatc
ccaggatggg tgtctgggga 2400gggtcaaggg ctggggctga gctttaaaat
ggttccgact tgtccctctc tcagccctcc 2460atggcctggc acgaggggat
ggggatgctt ccgcctttcc ggggctgctg gcctggccct 2520tgagtggggc
agcctccttg cctggaactc actcactctg ggtgcctcct ccccaggtgg
2580aggtgccagg aagctccctc cctcactgtg gggcatttca ccattcaaac
aggtcgagct 2640gtgctcgggt gctgccagct gctcccaatg tgccgatgtc
cgtgggcaga atgactttta 2700ttgagctctt gttccgtgcc aggcattcaa
tcctcaggtc tccaccaagg aggcaggatt 2760cttcccatgg ataggggagg
gggcggtagg ggctgcaggg acaaacatcg ttggggggtg 2820agtgtgaaag
gtgctgatgg ccctcatctc cagctaactg tggagaagcc cctgggggct
2880ccctgattaa tggaggctta gctttctgga tggcatctag ccagaggctg
gagacaggtg 2940cgcccctggt ggtcacaggc tgtgccttgg tttcctgagc
cacctttact ctgctctatg 3000ccaggctgtg ctagcaacac ccaaaggtgg
cctgcgggga gccatcacct aggactgact 3060cggcagtgtg cagtggtgca
tgcactgtct cagccaaccc gctccactac ccggcagggt 3120acacattcgc
acccctactt cacagaggaa gaaacctgga accagagggg gcgtgcctgc
3180caagctcaca cagcaggaac tgagccagaa acgcagattg ggctggctct
gaagccaagc 3240ctcttcttac ttcacccggc tgggctcctc atttttacgg
gtaacagtga ggctgggaag 3300gggaacacag accaggaagc tcggtgagtg
atggcagaac gatgcctgca ggcatggaac 3360tttttccgtt atcacccagg
cctgattcac tggcctggcg gagatgcttc taaggcatgg 3420tcgggggaga
gggccaacaa ctgtccctcc ttgagcacca gccccaccca agcaagcaga
3480catttatctt ttgggtctgt cctctctgtt gcctttttac agccaacttt
tctagacctg 3540ttttgctttt gtaacttgaa gatatttatt ctgggttttg
tagcattttt attaatatgg 3600tgacttttta aaataaaaac aaacaaacgt tgtcct
36363939DNAHomo sapiens 39gcagcagcgg ccgcctgctg ctgctgctgc
tgctgctcc 394037DNAHomo sapiens 40gcagcagtcg acctggagct cctgggaggc
ctgcgcc 37
* * * * *