Note: Descriptions are shown in the official language in which they were submitted.
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Protein C Derivatives
This application claims priority of Provisional
Application Serial No. 60/131,801 filed April 30, 1999.
This invention relates to novel polynucleotides,
polypeptides encoded by them and to the use of such
polynucleotides and polypeptides. More specifically, the
invention relates to protein C derivatives resistant to
serpin inactivation, to their production, and to
pharmaceutical compositions comprising these protein C
derivatives.
Protein C is a serine protease and naturally occurring
anticoagulant that plays a role in the regulation of
homeostasis by inactivating Factors Va and VIIIa in the
coagulation cascade. Human protein C is made in vivo as a
single polypeptide of 461 amino acids. This polypeptide
undergoes multiple post-translational modifications
including 1) cleavage of a 42 amino acid signal sequence;
2) cleavage of lysine and arginine residues (positions 156
and 157) to make a 2-chain inactive precursor or zymogen (a
155 amino acid residue light chain attached via a disulfide
bridge to a 262 amino acid residue heavy chain); 3) vitamin
K-dependent carboxylation of nine glutamic acid residues of
the light chain, resulting in nine gamma-carboxyglutamic
acid residues; and 4) carbohydrate attachment at four sites
(one in the light chain and three in the heavy chain).
Finally, the 2-chain zymogen may be activated by removal of
a dodecapeptide at the N-terminus of the heavy chain,
producing activated protein C (aPC) possessing greater
enzymatic activity than the 2-chain zymogen.
In conjunction with other polypeptides, aPC functions
as an anti-coagulant important in protecting against
thrombosis, has anti-inflammatory effects through its
inhibition of cytokine generation (e.g., TNF and IL-1), and
exerts profibrinolytic properties that facilitate clot
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lysis. Thus, aPC provides a mechanism for anti-coagulation,
anti-inflammation, and fibrinolysis.
The critical role of aPC in controlling hemostasis is
exemplified by the increased rate of thrombosis in
heterozygous deficiency, protein C resistance (e.g., due to
the common Factor V Leiden mutation) and the fatal outcome
of untreated homozygous protein C deficiency. Plasma-
derived and recombinantly produced aPC have been shown to be
effective and safe antithrombotic agents in a variety of
animal models of both venous and arterial thrombosis.
Protein C levels have also been shown to be abnormally
low in the following diseases and conditions: disseminated
intravascular coagulation (DIC)[Fourrier, et al., Chest
101:816-823, 1992], sepsis [Gerson, et al., Pediatrics
91:418-422, 1993], major trauma/major surgery [Thomas, et
al., Am J Surg. 158:491-494, 1989], burns [Lo, et al., Burns
20:186-187 (1994)], adult respiratory distress syndrome
CARDS)[Hasegawa, et al., Chest 105(1):268-277, 1994], and
transplantations [Gordon, et al., Bone Marrow Trans. 11:61-
65 (1993)]. In addition, there are numerous diseases with
thrombotic abnormalities or complications that aPC may be
useful in treating, such as: heparin-induced
thrombocytopenia (HIT) [Phillips, et al., Annals of
Pharmacotherapy 28: 43-45, 1994], sickle cell disease or
thalassemia [Karayalcin, et al., The American Journal of
Pediatric Hematology/Oncology 11(3):320-323, 1989], viral
hemorrhagic fever [Lacy, et al., Advances in Pediatric
Infectious Diseases 12:21-53, 1997], thrombotic
thrombocytopenic purpura (TTP)and hemolytic uremic syndrome
(HUS)[Moake, Seminars in Hematology :34(2):83-89, 1997]. In
addition, aPC in combination with Bactericidal Permeability
Increasing Protein (BPI) may be useful in the treatment of
sepsis [Fisher, et al., Crit. Care Med. 22(4):553-558,
1994] .
Finally, platelet inhibition is efficacious in both
prevention and treatment of thrombotic disease. However,
the use of antiplatelet agents, such as aspirin, increase
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the risk of bleeding, which limits the dose of the agent and
duration of treatment. The combination of aPC and
antiplatelet agents results in a synergy that allows the
reduction of the dosages of both aPC and the antiplatelet
agent(s). The reduction of the dosages of the agents in
combination therapy in turn results in reduced side effects
such as increased bleeding often observed in combination
anti-coagulant/anti-platelet therapy.
Various methods of obtaining protein C from plasma and
producing protein C, aPC and protein C/aPC polypeptides
through recombinant DNA technology are known in the art and
have been described. See e.g., U.S. Patent Nos. 4,775,624
and 5,358,932. Despite improvements in methods to produce
aPC through recombinant DNA technology, aPC and polypeptides
thereof are difficult and costly to produce and have a
relatively short half-life in vivo.
A reason for the short half-life is that blood levels
of aPC are regulated by molecules known as serpins (Serine
Protease Inhibitors), which covalently bind to aPC forming
an inactive serpin/aPC complex. The serpin/aPC complexes
are formed when aPC binds and proteolytically cleaves a
reactive site loop within the serpin; upon cleavage, the
serpin undergoes a conformational change irreversibly
inactivating aPC. The serpin/aPC complex is then eliminated
from the bloodstream via hepatic receptors for the
serpin/aPC complex. As a result, aPC has a relatively short
half-life compared to the zymogen; approximately 20 minutes
for aPC versus approximately 10 hours for human protein C
zymogen (Okajima, et al., Thromb Haemost 63(1):48-53, 1990).
It has been shown that changes to serine protease amino
acid sequences at residues which interact directly with the
substrate (generally within or near the active site) can
alter the specificity of the serine protease, potentially
providing increased specific activity towards appropriate
coagulation factors, as well as increased resistance to
serpins (Rezaie, J Biol Chem 271(39):23807-23814, 1996;
Rezaie and Esmon, Eur. J. Biochem 242:477-484, 1996).
*rB
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Therefore, an aPC polypeptide exhibiting increased
resistance to serpin inactivation, while maintaining the
desirable biological activities of aPC (e. g., anti-
coagulant, fibrinolytic, and anti-inflammatory activities),
provides a compound that has an increased plasma half-life
and, therefore, is effectively more potent than the parent
compound, requiring substantially reduced dosage levels or
less frequent administrations for therapeutic applications.
The potency advantages are especially important in disease
states in which serpin levels are elevated.
Physiologically, the two serpins that serve as the
primary inactivators of aPC are protein C inhibitor (PCI)
and al-antitrypsin (al-AT) [Heeb, et al., J Biol Chem
263(24):11613-6, 1988). Both PCI and al-AT have been
demonstrated to be the primary physiological inactivators of
aPC in disease states such as disseminated intravascular
coagulation (Scully, et al., Thromb Haemost 69(5):448-53,
1993), and elevated levels of al-AT have been observed in a
number of disease states involving an inflammatory response
(Somayajulu, et al., J Pathol Microbiol 39(4):271-5, 1996;
Morgan, et al., Int J Biochem Cell Biol 29(12):1501-11,
1997). The elevated serpin levels inactivate aPC resulting
in an increased susceptibility of coagulapathies associated
with decreased protein C levels. Attempts have been made to
increase the plasma half-life of aPC by increasing the
resistance to serpins by modifying the human protein C
molecule (e.g., U.S. Patent No. 5,358,932). An increase in
immunogenicity is often observed when a natural protein is
significantly modified and then administered to a patient.
Through scientific experiment and analysis, we
identified serpin and protein C binding sites essential to
formation of serpin/aPC complexes. We modified targeted
amino acid residues in the aPC molecule and surprisingly
found that we were able to inhibit farmation of the
serpin/aPC complex (the complex which irreversibly
inactivates aPC) while at the same time retaining the
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specificity of the aPC polypeptide for aPC's natural
substrates (e. g. factor Va and VIIIa). In particular, three
sites of recognition within the aPC active site show
distinctive differences between substrate recognition
sequences and inhibitor recognition sequences: S2, S3', and
S4'. We found inhibition of serpin/human aPC polypeptide
binding by substituting one or more of the following amino
acids: 194 (Leu), 195 (Ala), 228 (Leu), 249 (Tyr), 254
(Thr), 302 (Tyr), and 316 (Phe) of SEQ ID NO: 7 with an
amino acids) selected from Ser, Ala, Thr, His, Lys, Arg,
Asn, Asp, Glu, Gly, and Gln, provided that 195 is not
substituted with Ala and 254 is not substituted with Thr.
Accordingly, the present invention describes novel
protein C derivatives. These protein C derivatives retain
the important biological activity of the wild-type protein C
(SEQ ID NO: 7) and have substantially longer half-lives in
human blood. Therefore, these compounds provide various
advantages, eg. less frequent administration and/or smaller
dosages and thus a reduction in the overall cost of
production of the therapy. Additionally, these compounds
exhibit an advantage in disease states with significantly
elevated al-AT levels such as sepsis. Importantly, the
increases in protein C derivative plasma half-lives may be
achieved via single amino acid substitutions, which are less
likely to be immunogenic in comparison to molecules which
contain multiple amino acid substitutions (U.S. Patent No.
5,358,932; Holly, et al., Biochemistry 33:1876-1880, 1994).
The present invention provides a protein C derivative
comprising SEQ ID NO: 1 and the corresponding amino acids in
SEQ ID NO: 2, wherein one or more of amino acids 194, 195,
228, 249, 254, 302, or 316 is substituted with an amino acid
selected from Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu,
Gly, and Gln, provided that amino acid 195 is not Ala and
amino acid 254 is not Thr. The invention further provides
the activated form of the above-identified protein C
derivatives.
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The present invention also provides recombinant DNA
molecules encoding the protein C derivatives in the
preceding paragraph, in particular those comprising SEQ ID
NOS: 8, 9, and 10.
Another aspect of the present invention provides
protein sequences of these same protein C derivatives,
particularly those comprising SEQ ID NOS: 3, 4, and 5 and
the activated forms of these protein C derivatives.
The present invention comprises methods of treating
vascular occlusive disorders and hypercoagulable states
including: sepsis, disseminated intravascular coagulation,
purpura fulminans, major trauma, major surgery, burns, adult
respiratory distress syndrome, transplantations, deep vein
thrombosis, heparin-induced thrombocytopenia, sickle cell
disease, thalassemia, viral hemorrhagic fever, thrombotic
thrombocytopenic purpura, and hemolytic uremic syndrome.
The invention further provides treating these same diseases
and conditions employing the activated form of the above-
identified protein C derivatives.
Another embodiment of the present invention is a method
of treating sepsis comprising the administration to a
patient in need thereof a pharmaceutically effective amount
of a protein C derivative of this invention in combination
with bacterial permeability increasing protein.
The present invention comprises methods of treating
acute coronary syndromes such as myocardial infarction and
unstable angina.
The present invention further comprises methods of
treating thrombotic disorders. Such disorders include, but
are not limited to, stroke, abrupt closure following
angioplasty or stent placement, and thrombosis as a result
of peripheral vascular surgery.
The present invention also provides a pharmaceutical
composition comprising a protein C derivative of this
invention.
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Human protein C derivatives for the above-mentioned
indications and pharmaceutical compositions are preferably
selected from L194S, L194S:T254S, and L194A:T254S.
Methods and aspects of producing the novel isolated
human protein polypeptides are also an aspect of this
invention.
Finally, an aspect of the invention comprises treating
the diseases and conditions caused or resulting from protein
C deficiency as defined herein, by inhibiting binding to
inhibitor recognition sequences S2, S3', and S4' of the
serpins, PCI and al-AT. This final aspect of the invention
contemplates any and all modifications to any aPC molecule
resulting in inhibition of the binding to said inhibitor
recognition sequences of the serpins PCI and al-AT. The
inhibition of binding to the specific inhibitor recognition
sequences of the serpins (S2, S3', and S4') being an
important contribution to this aspect of the invention.
Figure 1. Inactivation of human aPC polypeptides
during incubation with normal human plasma. Remaining
activity is measured as amidolytic activity normalized to
activity at the start of the experiment (time=0); error bars
indicate the standard error of triplicate experiments. Data
are shown for the wild-type protein C (WT, circles), T254S
(squares), L194S (triangles), and A195G (diamonds).
Figure 2. Inactivation of human aPC polypeptides
during incubation with normal human plasma. Remaining
activity is measured as amidolytic activity normalized to
activity at the start of the experiment (time=0); error bars
indicate the standard error of triplicate experiments. Data
are shown for the wild-type protein C (WT, circles),
L194A/T254S (squares), and L194S/T254S (triangles).
Figure 3. Inactivation of human aPC polypeptides by
normal human plasma containing Heparin (10 U/mL). Remaining
activity is measured as amidolytic activity normalized to
activity at the start of the experiment (time=0); error bars
indicate the standard error of triplicate experiments. Data
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are shown for the wild-type protein C(WT, circles), T254S
(squares), L194S (triangles), and A195G (diamonds).
Figure 4. Inactivation of human aPC polypeptides by
purified al-antitrypsin. Remaining activity is measured as
amidolytic activity normalized to activity at the start of
the experiment (time=0); error bars indicate the standard
error of triplicate experiments. Data are shown for the
wild-type protein (WT, circles), T254S (squares), L194S
(triangles), and A195G (diamonds).
Figure 5. Plasma aPC levels following a bolus IV dose
of 0.1 mg/kg in normal, conscious rabbits (N=3). Activated
protein C levels were determined using immunocapture assay,
and compared to a standard curve generated from dilutions of
the purified protein in rabbit plasma; the standard curve
ranged from 1 to 250 ng/mL, with the calculated values
within 10~ of the standard samples. Data are shown for the
wild-type protein (WT, circles), T254S (squares), L194S
(triangles), and L194S/T254S (diamonds). The values plotted
are the mean and standard error for the three animals.
For purposes of the present invention, as disclosed and
claimed herein, the following terms are as defined below.
Antiplatelet agent - one or more agents alone or in
combination which reduces the ability of platelets to
aggregate. Agents understood and appreciated in the art
include those cited in, for example, Remington, The Science
and Practice of Pharmacy, Nineteenth Edition, Vol II, pages
924-25, Mack Publishing Co., herein incorporated by
reference. Such agents include but are not limited to
aspirin (ASA), clopidogrel, ReoProG (abciximab),
dipyridamole, ticlopidine and IIb/IIIa antagonists.
aPC or activated protein C refers to recombinant aPC.
aPC includes and is preferably recombinant human aPC
although aPC may also include other species having protein C
proteolytic, amidolytic, esterolytic, and biological (anti-
coagulant, anti-inflammatory, or pro-fibrinolytic)
activities.
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Protein C derivatives) refers to the recombinantly
produced polypeptides of this invention that differ from
wild-type human protein C but when activated retain the
essential properties i.e., proteolytic, amidolytic,
esterolytic, and biological (anti-coagulant, anti-
inflammatory, pro-fibrinolytic activities). The definition
of protein C derivatives as used herein also includes the
activated form of the above identified protein C
derivatives.
Treating - describes the management and care of a
patient for the purpose of combating a disease, condition,
or disorder whether to eliminate the disease, condition, or
disorder, or prophylactically to prevent the onset of the
symptoms or complications of the disease, condition, or
disorder.
Continuous infusion - continuing substantially
uninterrupted the introduction of a solution or suspension
into a vein for a specified period of time.
Bolus injection - the injection of a drug in a defined
quantity (called a bolus) over a period of time up to about
120 minutes.
Suitable for administration - a lyophilized formulation
or solution that is appropriate to be given as a therapeutic
agent.
Unit dosage form - refers to physically discrete units
suitable as unitary dosages for human subjects, each unit
containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient.
Hypercoagulable states - excessive coagulability
associated with disseminated intravascular coagulation, pre-
thrombotic conditions, activation of coagulation, or
congenital or acquired deficiency of clotting factors such
as aPC.
Zymogen - protein C zymogen, as used herein, refers to
secreted, inactive forms, whether one chain or two chains,
of protein C.
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Pharmaceutically effective amount - a therapeutically
efficacious amount of a pharmaceutical compound. The
particular dose of the compound administered according to
this invention will, of course, be determined by the
attending physician evaluating the particular circumstances
surrounding the case, including the compound administered,
the particular condition being treated, the patient
characteristics and similar considerations.
Acute coronary syndromes - clinical manifestations of
coronary atherosclerosis complicated by coronary plaque
rupture, superimposed coronary thrombosis, and jeopardized
coronary blood flow resulting in coronary ischemia and/or
myocardial infarction. The spectrum of acute coronary
syndromes includes unstable angina, non-Q-wave (i.e., non-
ST-segment elevation) myocardial infarction, and Q-wave
(i.e., ST-segment elevation) myocardial infarction.
Thrombotic disorders - a disorder relating to, or
affected with the formation or presence of a blood clot
within a blood vessel. Such disorders include, but are not
limited to, stroke, abrupt closure following angioplasty or
stent placement, and thrombosis as a result of peripheral
vascular surgery.
Purpura fulminans - ecchymotic skin lesions, fever,
hypotension associated with bacterial sepsis, viral,
bacterial or protozoan infections. Disseminated
intravascular coagulation is usually present.
Serpin - any of a group of structurally related
proteins that typically are serine protease inhibitors whose
inhibiting activity is conferred by an active site in a
highly variable and mobile peptide loop and that include but
are not limited to protein C inhibitor (PCI) and al-
antitrypsin (al-AT).
Inhibitor recognition sequence S2: the 2nd residue
N-terminal to the cleavage site of PCI or al-AT.
Inhibitor recognition sequence S3': the 3rd residue
C-terminal to the cleavage site of PCI or al-AT.
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Inhibitor recognition sequence S4': the 4"' residue
C-terminal to the cleavage site of PCI or al-AT.
Wild-type protein C - the type of protein C that
predominates in a natural population of humans in contrast
to that of natural or laboratory mutant or polypeptide forms
of protein C.
Bactericidal permeability increasing protein - includes
naturally and recombinantly produced bactericidal
permeability increasing (BPI) protein; natural, synthetic,
and recombinant biologically active polypeptide fragments of
BPI protein; biologically active polypeptide variants of BPI
protein or fragments thereof, including hybrid fusion
proteins and dimers; biologically active variant analogs of
BPI protein or fragments or variants thereof, including
cysteine-substituted analogs; and BPI-derived peptides. The
complete amino acid sequence of human BPI, as well as the
nucleotide sequence of DNA encoding BPI have been elucidated
by Gray, et al., 1989, J. Biol. Chem 264:9505. Recombinant
genes encoding and methods for expression of BPI proteins,
including BPI holoprotein and fragments of BPI are disclosed
in U.S. Patent No. 5,198,541, herein incorporated by
reference.
The amino acid abbreviations are accepted by the United
States Patent and Trademark Office as set forth in 37 C.F.R.
1.822 (d)(1) (1998).
The activated form of aPC or isolated human aPC
polypeptides may be produced by activating recombinant human
protein C zymogen or recombinant protein C derivative
zymogen in vitro or by direct secretion of the activated
form of protein C. The means by which the activation occurs
is not critical and the process aspects of this invention
include any and all means of activation. Protein C
derivatives may be produced in eukaryotic cells, transgenic
animals, or transgenic plants, including, for example,
secretion from human kidney 293 cells as a zymogen then
purified and activated by techniques known to the skilled
artisan.
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The present invention provides protein C derivatives,
including activated forms thereof, which have increased
resistance to serpins, and consequently result in extended
plasma half-lives. Specific protein C derivatives include
L194S, L194S:T254S, and L194A:T254S and activated forms
thereof.
Protein C derivative L194S preferably contains a serine
residue at position 194 rather than a leucine residue
normally found at this position. One with skill in the art
would realize that other amino acid substitutions at residue
194 in addition to Ser may impart increased resistance to
serpins in the resulting polypeptide molecule. Examples of
such amino acid substitutions include, Ala, Thr, His, Lys,
Arg, Asn, Asp, Glu, and Gln. The activated form of protein
C derivative L194S demonstrates prolonged half-life in
plasma (Figure 1) and increased resistance to serpins, for
example, al-antitrypsin (al-AT}, Figure 4.
Protein C derivative L194S:T254S preferably contains a
serine residue at position 194 rather than a leucine residue
normally found at this position and a serine residue at
position 254 rather than a threonine residue normally found
at this position. It is apparent to one with skill in the
art that other amino acid substitutions at residues 194 and
254 in addition to Ser may impart increased resistance to
serpins in the resulting polypeptide molecule. Examples of
such amino acid substitutions include Ala, Thr, His, Lys,
Arg, Asn, Asp, Glu, Gln, and Gly, provided that amino acid
254 is not substituted with Thr. The activated form of
human protein C derivative L194S:T254S demonstrates a
prolonged half-life in normal human plasma compared to wild-
type protein C, Figure 2.
Protein C derivative L194A:T254S preferably contains an
alanine residue at position 194 rather than a leucine
residue normally found at this position and a serine residue
at position 254 rather than a threonine residue normally
found at this position. It is apparent to one with skill in
the art that other amino acid substitutions at residues 194
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and 254 in addition to Ser may impart increased resistance
to serpins in the resulting polypeptide molecule. Examples
of such amino acid substitutions include Ala, Thr, His, Lys,
Arg, Asn, Asp, Glu, Gln, and Gly, provided that amino acid
254 is not substituted with Thr. The activated form of human
protein C derivative L194A:T254S demonstrates a prolonged
half-life in normal human plasma compared to wild-type
protein C, Figure 2.
Further embodiments of the present invention include
protein C derivatives: L194T, L194A, A195G, L228Q, T254S,
F316N, Y249E, and Y302Q, and activated forms thereof which
have increased resistance to serpins.
Protein C derivatives L194T or L194A preferably contain
a threonine residue or an alanine residue at position 194
rather than a leucine residue normally found at this
position. One with skill in the art would realize that
other amino acid substitutions at residue 194 in addition to
Ser may impart increased resistance to serpins in the
resulting polypeptide molecule. Examples of such amino acid
substitutions include, His, Lys, Arg, Asn, Asp, Glu, and
Gln.
Protein C derivative A195G preferably contains a
glycine residue at position 195 rather than an alanine
residue normally found at this position. One with skill in
the art would realize that other amino acid substitutions at
residue 195 in addition to Gly may impart increased
resistance to serpins in the resulting polypeptide molecule.
Examples of such amino acid substitutions include Ser, Ala,
Thr, His, Lys, Arg, Asn, Asp, Glu, and Gln. The activated
form of protein C derivative A195G demonstrates prolonged
half-life in plasma (Figure 1) and increased resistance to
serpins, for example, a1-antitrypsin (al-AT), Figure 4.
Protein C derivative L228Q preferably contains a
glutamine residue at position 228 rather than a leucine
residue normally found at this position. One with skill in
the art would realize that other amino acid substitutions at
residue 228 in addition to Gln may impart increased
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resistance to serpins in the resulting polypeptide molecule.
Examples of such amino acid substitutions include, Ser, Ala,
Thr, His, Lys, Arg, Asn, Asp, Glu, and Gly.
Protein C derivative T254S preferably contains a serine
residue at position 254 rather than a threonine residue
normally found at this position. It is apparent to one with
skill in the art that other amino acid substitutions at
residue 254 in addition to Ser may impart increased
resistance to serpins in the resulting polypeptide molecule.
Examples of such amino acid substitutions include Ala, Thr,
His, Lys, Arg, Asn, Asp, Glu, Gln, and Gly, provided that
amino acid 254 is not substituted with Thr. The activated
form of protein C derivative T254S demonstrates prolonged
half-life in plasma (Figure 1) and increased resistance to
serpins, for example, al-antitrypsin (al-AT), Figure 4.
Protein C derivative F316N preferably contains an
asparagine residue at position 316 rather than a
phenylalanine residue normally found at this position. One
with skill in the art would realize that other amino acid
substitutions at residue 316 in addition to Asn may impart
increased resistance to serpins in the resulting polypeptide
molecule. Examples of such amino acid substitutions
include, Ser, Ala, Thr, His, Lys, Arg, Asp, Glu, Gln, and
Gly.
Protein C derivative Y249E preferably contains a
glutamic acid residue at position 249 rather than a tyrosine
residue normally found at this position. An additional
polypeptide contains an Asp at position 249 rather than a
tyrosine residue. One with skill in the art would realize
that other amino acid substitutions at residue 249 in
addition to Glu and Asp may impart increased resistance to
serpins in the resulting polypeptide molecule. Examples of
such amino acid substitutions include, Ser, Ala, Thr, His,
Lys, Arg, Asn, Gln, and Gly.
Protein C derivative Y302Q preferably contains a
glutamine residue at position 302 rather than a tyrosine
residue normally found at this position. An additional
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polypeptide contains a Glu at position 302 rather than a
tyrosine residue. One with skill in the art would realize
that other amino acid substitutions at residue 302 in
addition to Glu or Gln may impart increased resistance to
serpins in the resulting polypeptide molecule. Examples of
such amino acid substitutions include, Ser, Ala, Thr, His,
Lys, Arg, Asn, Asp, and Gly.
In addition, protein C derivatives may include proteins
that represent functionally equivalent gene products. Such
an equivalent protein C derivative may contain deletions,
additions, or substitutions of amino acid residues within
the~amino acid sequence encoded by the protein C polypeptide
gene sequences described above, but which result in a silent
change, thus producing a functionally equivalent protein C
derivative gene product. Amino acid substitutions may be
made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues involved.
Thus, the polypeptides of the present invention include
polypeptides having an amino acid sequence at least
identical to that of SEQ ID NOS: 3, 4, or 5, or fragments
thereof with at least 90% identity to the corresponding
fragment of SEQ ID NOS: 3, 4, or 5. Preferably, all of
these polypeptides retain the biological activity of human
aPC. Preferred polypeptides are those that vary from SEQ ID
NOS: 3, 4, or 5 by conservative substitutions i.e., those
that substitute a residue with another of like
characteristics. Typical substitutions are among Ala, Val,
Leu and Ile; among Ser and Thr; among the acidic residues
Asp and Glu; among Asn and Gln; and among the basic residues
Lys and Arg; or aromatic residues Phe and Tyr. Particularly
preferred are polypeptides in which several, 5-10, 1-5, or
1-2 amino acids are substituted, deleted, or added in any
combination.
The invention also provides DNA compounds for use in
making the protein C derivatives. These DNA compounds
comprise the coding sequence for the light chain of human
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protein C zymogen or protein C derivative zymogen positioned
immediately adjacent to, downstream of, and in translational
reading frame with the prepropeptide sequence of human
protein C zymogen or protein C derivative zymogen. The DNA
sequences preferably encode the Lys-Arg dipeptide which is
processed during maturation of the protein C molecule, the
activation peptide and the heavy chain of the protein C
derivative.
Those skilled in the art will recognize that, due to
l0 the degeneracy of the genetic code, a variety of DNA
compounds can encode the polypeptides described above. U.S.
Patent No. 4,775,624, the entire teaching of which is herein
incorporated by reference, discloses the wild-type form of
the human protein C molecule. The skilled artisan could
readily determine which changes in the DNA sequences which
could encode the exact polypeptides as disclosed herein.
The invention is not limited to the specific DNA sequences
disclosed. Consequently, the construction described below
and in the accompanying Examples for the preferred DNA
compounds are merely illustrative and do not limit the scope
of the invention.
All of the DNA compounds of the present invention were
prepared by the use of site-directed mutagenesis to change
particular positions within human protein C zymogen. The
methods used for the identification of residues which form
critical contacts in these particular positions are
described in Example 1.
The protein C derivatives can be made by techniques
well known in the art utilizing eukaryotic cell lines,
transgenic animals, or transgenic plants. Skilled artisans
will readily understand that appropriate host eukaryotic
cell lines include but are not limited to HepG2, LLC-MK2,
CHO-K1, 293, or AV12 cells, examples of which are described
in U.S. Patent No. 5,681,932, herein incorporated by
reference. Furthermore, examples of transgenic production
of recombinant proteins are described in U.S. Patent Nos.
5,589,604 and 5,650,503, herein incorporated by reference.
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Skilled artisans recognize that a variety of vectors
are useful in the expression of a DNA sequence of interest
in a eukaryotic host cell. Vectors that are suitable for
expression in mammalian cells include, but are not limited
to: pGT-h, pGT-d; pCDNA 3.0, pCDNA 3.1, pCDNA 3.1+Zeo, and
pCDNA 3.1+Hygro (Invitrogen); and, pIRES/Hygro, and
pIRES/neo (Clonetech). The preferred vector of the present
invention is pIG3 as described in Example 2.
To be fully active and operable under the present
methods, the protein C derivatives made by any of these
methods must undergo post-translational modifications such
as the addition of nine gamma-carboxy-glutamates, the
addition of one erythro-beta-hydroxy-Asp (beta-
hydroxylation), the addition of four Asn-linked
oligosaccharides (glycosylation) and, the removal of the
leader sequence (42 amino acid residues). Without such
post-translational modifications, the protein C polypeptides
are not fully functional or are non-functional.
Methods for the activation of zymogen forms of human
protein C and protein C derivatives to activated human
protein C and activated protein C derivatives are old and
well known in the art. Human protein C may be activated by
thrombin alone, by a thrombin/thrombomodulin complex, by
RW-X, a protease from Russell~s Viper venom, by pancreatic
trypsin or by other proteolytic enzymes.
The recombinant protein C derivatives of the present
invention are useful for the treatment of vascular occlusive
disorders or hypercoagulable states associated with sepsis,
disseminated intravascular coagulation, major trauma, major
surgery, burns, adult respiratory distress syndrome,
transplantations, deep vein thrombosis, heparin-induced
thrombocytopenia, sickle cell disease, thalassemia, viral
hemorrhagic fever, thrombotic thrombocytopenic purpura, and
hemolytic uremic syndrome. In another embodiment, the
recombinant protein C derivatives of the present invention
are useful for the treatment of sepsis in combination with
bacterial permeability increasing protein. In yet another
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aspect of this invention the activated protein C derivatives
of the present invention are combined with an antiplatelet
agents? to treat or prevent various disorders, such as,
thrombotic disease.
The present invention further provides for the
treatment of acute coronary syndromes comprising myocardial
infarction, and unstable angina with human protein C
derivatives with resistance to serpin inactivation as
compared to wild-type aPC.
The recombinant human protein C derivatives of the
present invention are also useful for the treatment of
thrombotic disorders such as stroke, abrupt closure
following angioplasty or stent placement, and thrombosis as
a result of peripheral vascular surgery.
The protein C derivatives can be formulated according
to known methods to prepare a pharmaceutical composition
comprising as the active agent an aPC polypeptide and a
pharmaceutically acceptable solid or carrier. For example,
a desired formulation would be one that is a stable
lyophilized product of high purity comprising a bulking
agent such as sucrose, a salt such as sodium chloride, a
buffer such as sodium citrate and an activated protein C
derivative. A preferred stable lyophilized formulation
comprises: 2.5 mg/ml activated protein C polypeptide, 15
mg/ml sucrose, 20 mg/ml NaCl and a citrate buffer, said
formulation having a pH of 6Ø An additional stable
lyophilized formulation comprises: 5.0 mg/ml activated
protein C polypeptide, 30 mg/ml sucrose, 38 mg/ml NaCl and a
citrate buffer, said formulation having a pH of 6Ø
Preferably, the human aPC polypeptides will be
administered parenterally to ensure delivery into the
bloodstream in an effective form by injecting the
appropriate dose as a continuous infusion for 1 to 240
hours. More preferably, the human aPC polypeptides will be
administered as a continuous infusion for 1 to 192 hours.
Even more preferably, the human aPC polypeptides will be
administered as a continuous infusion for 1 to 144 hours.
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Yet even more preferably, the aPC polypeptides will be
administered as a continuous infusion for 1 to 96 hours.
The amount of human aPC polypeptide administered will
be from about 0.01 ~.g/kg/hr to about 50 ~.g/kg/hr. More
preferably, the amount of human aPC polypeptide administered
will be about 0.1 ~.g/kg/hr to about 25 ~.g/kg/hr. Even more
preferably the amount of human aPC polypeptide administered
will be about 1 ~.g/kg/hr to about 15 ~.g/kg/hr. The most
preferable amounts of human aPC polypeptide administered
will be about 5 ~.g/kg/hr or about 10 ~Cg/kg/hr.
Alternatively, the human aPC polypeptide will be
administered by injecting a portion (1/3 to 1/2) of the
appropriate dose per hour as a bolus injection over a time
from about 5 minutes to about 120 minutes, followed by
continuous infusion of the appropriate dose for up to 240
hours.
In another alternative the human aPC derivatives will
be administered by injecting a dose of 0.01 mg/kg/day to
about 1.0 mg/kg/day, B.I.D. (2 times a day), for one to ten
days. More preferably, the human aPC derivatives will be
administered B.I.D. for three days.
In yet another alternative, the human aPC polypeptides
will be administered subcutaneously to ensure a slower
release into the bloodstream. Formulation for subcutaneous
preparations will be done using known methods to prepare
such pharmaceutical compositions.
An additional aspect of the invention comprises
treating the diseases and conditions caused or resulting
from protein C deficiency as defined herein, by inhibiting
binding to inhibitor recognition sequences S2, S3', and S4'
of the serpins, PCI and al-AT, as described in Example 1.
This final aspect of the invention contemplates any and all
modifications to any aPC molecule resulting in inhibition of
the binding to said inhibitor recognition sequences of the
serpins PCI and al-AT.
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The human aPC polypeptides described in this invention
have essentially the same type of biological activity as the
wild-type human aPC, with substantially longer half-lives in
human blood. Therefore., these compounds will require either
less frequent administration and/or smaller dosage.
Additionally, these compounds will exhibit an advantage in
disease states with significantly elevated al-AT levels such
as sepsis. Finally, superior increases in human aPC
polypeptide plasma half-lives may be achieved via one or two
amino acid substitutions, which are less likely to be
immunogenic compared to greater numbers of substitutions.
The following Examples are provided merely to further
illustrate the present invention. The scope of the
invention shall not be construed as merely consisting of the
following Examples.
Example 1
Site-Directed Mutagenesis
The use of site-directed mutagenesis to change
particular positions within human protein C molecule that
decrease inactivation by serpins, and consequently result in
extended plasma half-lives is described. The recognition
sequences in the two primary aPC inhibitors al-AT and PCI
reveal some differences that can be exploited by altering
the residues in aPC that interact with these sequences.
Table I depicts the sequences recognized by aPC. The
cleavage site occurs between the two residues shown in
italics. Residues occupying the specific subsites, S2, S3',
and S4', are underlined.
In general, the recognized sites in factor Va are
different from the sites in either factor VIIIa or the
inhibitors, therefore, it is possible to engineer the active
site of aPC to preferentially cleave the more critical
coagulant factor Va, while at the same time decrease aPC's
likelihood of being inhibited by serpins.
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Table I.
~agulation Factors S2 S3~ S4
Factor Va 300-313 N C P K K _TR N L K KI T
R
Factor Va 500-513 S R S L D _RR G I _ _A A
_Q_R A
Factor Va 673-685 S T V M A _TR K M _H_DR L
E
Factor VIIIa 330-341 P E E P Q _LR M K _N_NE E
A
Factor VIIIa 560-571 K E S V D _QR G N _Q_IM S
D
Serpins
PCI G T I F T _FR S A _R_LN S
Q
al-AT F L E A I P M S I P PE V
K
In particular, three sites of recognition within the
active site show distinctive differences between substrate
recognition sequences and inhibitor recognition sequences:
S2 (the 2nd residue N-terminal to the cleavage site), S3'
site, and S4'. The S2 site is primarily occupied by polar
residues in the factor Va sequences; unlike PCI and al-AT,
which have hydrophobic residues at this position. The S3'
site occupied by polar side chains in all of the substrate
sequences, but notably, a hydrophobic side chain in the al-
AT sequence. The S4' site is occupied by charged residues
in all three factor Va sequences, but is occupied by
hydrophobic residues in the factor VIIIa and inhibitor
sequences.
Based upon the crystal structures of the PPACK-
inhibited aPC (blather, et al., EMBO J. 15(24):6822-6831,
1996) and Hirulog 3-inhibited thrombin (Qiu, et al.,
Biochemistry 31(47):11689-97, 1992), two aPC-substrate model
structures were created and energy minimized using a CHARMm
protocol:
(1) The sequence representing the factor Va R506
cleavage sequence.
(2) The recognition sequence of al-AT, with the Met
substituted with Arg (corresponding to a polypeptide of al-
AT which exhibits extremely high affinity for aPC).
These models allowed for the identification of residues
which form critical contacts in these three specific sites.
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A summary of residues which may form specific contacts
within the active site, and replacements that are expected
to provide enhanced specificity and/or activity are
summarized in Table II. In general, mutations of residues
that form contacts within the specific subsites of the
active site are designed to reflect changes in the
environment to drive the specificity of human aPC
polypeptides away from the recognition of the two primary
physiological inhibitors, and potentially enhance human aPC
polypeptide's proteolytic activity.
Table II. Mutations constructed for alteration of
specificity
Site ~ aPC ;Constructed ;Substrate Coatact
Residue ;re lacemeats
________________________________;____~?
____________________,_________________________________________
S2 Thr254 ;Ser ;Aliphatic part of
;sidechain
___________________.________________________________________
; _.
1
____________;.___________________________ ,
S3' Tyr302 ;Glu, Gln ;End of
sidechain
S4' Leu194 ' ~Ser, Thr, Ala ;Aliphatic part of
;sidechain
S4' A1a195 ;Gly Aliphatic part of
;sidechain
____________t____________________;__________________________
_;_________________________________________
S4' Leu228 ;Gln ;End of
sidechain
S4' Phe316 'Asn Aliphatic part of
~sidechain
Example 2
Protein C Polypeptide Construction and Production.
Protein C derivatives were constructed using the
polymerase chain reaction (PCR) following standard methods.
The source of the wild-type coding sequence was plasmid pLPC
(Bio/Technology 5:1189-1192, 1987). The universal PCR
primers used include: PCOOlb; 5'-
GCGATGTCTAGAccaccATGTGGCAGCTCACAAGCCTCCTGC -3', which
encodes for an XbaI restriction site (underlined) used for
subcloning, a Kozak consensus sequence (lowercase) (Kozak, J
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Cell Biol 108(2):229-41, 1989), and the 5' end of the coding
region for protein C: PC002E; 5'-
CAGGGATGATCACTAAGGTGCCCAGCTCTTCTGG-3', which encodes for the
3' end of the coding region for human protein C, and
includes a BclI restriction site (underlined) for
subcloning. Mutagenic PCR primers (sense and anti-sense
directions, respectively) include: PC194SF, 5'-
CTCAAAGAAGAAGTCCGCCTGCGGGGCAGTGC-3' and PC194SR, 5'-
GCACTGCCCCGCAGGCGGACTTCTTCTTTGAG-3' which encode for a Leu
(CTG) to Ser (TCC) mutation at position 194 (boldfaced
type); PCA195GF, 5'-GAAGAAGCTGGGGTGCGGGGCAGTGC-3', and
PCA195GR, 5'-GCACTGCCCCGCACCCCAGCTTCTTC-3', which encode for
a Ala(GCC) to Gly(GGG) mutation; PCT254SF, 5'-
GCAAGAGCACCAGCGACAATGAC-ATCGC-3' and PCT254SR, 5'-
GCGATGTCATTGTCGCTGGTGCTCTTGC-3', which encode for a Thr
(ACC) to Ser (AGC) mutation at position 254 (boldfaced
type). The first round of PCR was used to amplify two
fragments of the protein C gene; the 5' fragment was
generated using PCOOlb and the antisense mutagenic primer,
and the 3' fragment was generated using PC002e and the sense
mutagenic primer. The resulting amplified products were
purified by standard procedures. These fragments were
combined and then used as a template for a second round of
PCR using primers PCOOlb and PC002e. The final PCR product
was digested with XbaI and BclI and subcloned into similarly
digested expression vector pIG3. A wild-type construct was
similarly generated by PCR using the two universal primers
and the plasmid pLPC as the template, followed by subcloning
into pIG3. The mutations were confirmed by DNA sequencing
of both the coding and non-coding strands. The completed
expression plasmids were designated pIG3-HPC (wild-type
protein C), pGH41 (T254S), pGH51(A195G), and pGH94 (L194S).
The pIG3 vector was generated by the insertion of an
"internal ribosome entry site" (IRES) (Jackson, et al.,
Trends Biochem Sci 15(12):447-83, 1990) and green
fluorescent protein (GFP) (Cormack, et al., Gene 173:33-38,
1996) gene into the mammalian expression vector pGTD
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(Gerlitz,et al., Biochem J 295(Pt 1):131-40, 1993). When a
cDNA of interest is cloned into the multiple cloning site of
pIG3, the GBMT promoter (Berg, Nucleic Acids Res
20(20):5485-6, 1992} drives expression of a bicistronic mRNA
(5'- cDNA - IRES - GFP - 3'). Efficient translation of the
first cistron is initiated by classical assembly of ribosome
subunits on the 5'-methylated cap structure of the mRNA;
while the normally inefficient translation of a second
cistron is overcome by the IRES sequence which allows for
internal ribosome assembly on the mRNA. The coupling of the
cDNA and reporter on a single mRNA, translated as separate
proteins, allows one to screen for the highest-producing
clones on the basis of fluorescence intensity. The
expression vector also contains an ampicillin resistance
cassette for maintenance of the plasmid in E. coli, and a
murine DHFR gene with appropriate expression sequences for
selection and amplification purposes in mammalian tissue
expression.
The adenovirus-transformed Syrian hamster AV12-664 cell
line was grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 50 ~,g/mL
gentamicin, 200 ~g /mL Geneticin (G418), and 10 ~g /mL
vitamin K1. One day prior to transfection, cells were
plated at a density of about 105 cells/ 25 cm2. FspI-
linearized plasmids were transfected using either the
calcium phosphate method (ProFection, Gibco BRL-Life
Technologies) or FuGene-6 (Boehringer Mannheim), following
the manufacturer's instructions. Approximately 48 hours
after transfection, the medium was replaced with medium
containing 250 nM methotrexate for selection. Colonies
resistant to methotrexate were pooled 2-3 weeks after
applying drug selection and expanded. The pools were
subjected to fluorescence activated cell sorting based upon
GFP fluorescence intensity (Cormack, 1996), with the most
intense 5% of fluorescent cells being retained and expanded.
To obtain material for purification, recombinant cells were
*rB
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grown in a modified mixture of Dulbecco's modified Eagle's
and Ham's F-12 media (1:3) containing 1 ~g/mL human insulin,
1 ~g/mL human transferrin, and 10 ~g/mL vitamin K1.
Conditioned media were collected, adjusted to a final
concentration of 5 mM benzamidine and 5 mM EDTA, pH 8.0, and
protein C was purified via anion-exchange chromatography as
described (Yan, et al., Bio/Technology 8:655-661, 1990).
Purified protein was desalted/concentrated in Ultrafree-CL
30,000 NMWL filtration units (Millipore) using Buffer A (150
mM NaCl, 20 mM Tris-HC1, pH 7.4), and quantitated by Pierce
BCA assay using bovine serum albumin (BSA) as the standard.
Example 3
Activation of Recombinant Protein C
Complete activation of the zymogen forms of protein C
and polypeptides was accomplished by incubation with
thrombin-sepharose. Thrombin-sepharose was washed
extensively with Buffer A. 200 ~L of packed thrombin-
sepharose was mixed with 250 ~g of protein C in 1 mL of the
same buffer and incubated at 37°C for 4 hours with gentle
shaking on a rotating platform. During the course of the
incubation, the degree of protein C activation was monitored
by briefly pelleting the thrombin-sepharose, and assaying a
small aliquot of the supernatant for aPC activity using the
chromogenic substrate S-2366 (DiaPharma). Following
complete activation, the thrombin-sepharose was pelleted,
and the supernatant collected. aPC concentration was
verified by Pierce BCA assay, and the aPC was either assayed
directly, or frozen in aliquots at -80°C. All polypeptides
were analyzed by SDS-PAGE with either Coomassie-blue
staining or Western Blot analysis to confirm complete
activation (Laemmli, Nature 227:680-685, 1970).
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Example 4
Functional Characterization
The amidolytic activity of recombinant human aPC
polypeptides were determined by hydrolysis of the tri-
peptide substrates S-2366 (Glu-Pro-Arg-p-nitroanilide), S-
2238 (Pip-Pro-Arg-p-nitroanilide), and S-2288 (Ile-Pro-Arg-
p-nitroanilide), Table III. The anticoagulant activity is
shown as measured clotting time in an aPTT at 500 ng mL-1
aPC. Amidolytic activites were measured using the
ZO chromogenic substrate S-2366.
Assays were performed at 25°C, in Buffer A containing
1 mg mL-1 BSA, 3 mM CaCl2, and 0.5 nM aPC. Reactions (200
~L/well) were performed in a 96-well microtiter plate, and
amidolytic activity was measured as the change in
absorbance units/min at 405 nm as monitored in a ThermoMax
kinetic micrometer plate reader. Kinetic constants were
derived by fitting velocity data at varying substrate
concentrations (16 ~M to 2 mM) to the Michaelis-Menten
equation. Changes in A405 were converted to mmol product
using a path length of 0.53 cm (Molecular Devices Technical
Applications Bulletin 4-1), and an extinction coefficient
for the released p-nitroanilide of 9620 M-1 cm-1 (Pfleiderer,
Methods Enzymol 19:514-521, 1970). Anti-coagulant activity
was assessed by measuring the prolongation of clotting time
in the activated partial thromboplastin time clotting assay
(Helena Laboratories). Clotting reactions were monitored
in a ThermoMax kinetic microtiter plate reader, measuring
the time to Vmax in the change in turbidity.
Table III. Functional characterization of protein C
polypeptides
Protein ;Anticoagulant activity Amidolytic
APTT Clotting Time activity i
______________ _________________________________.________ __
xcat/Itm__(mM_s_1)_____'
Control J 36 seconds N/A
WT-aPC ' 114 seconds 98
________________;_________.__________________________________~_________________
____________________
Leu194S 108 seconds 84
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A1a195G 120 seconds 66
Thr254S 108 seconds 63
Example 5
Inactivation of aPC Polypeptides
The rates of inactivation of aPC polypeptides were
determined by incubating normal human plasma (Helena Labs}
with 20 nM aPC (or either polypeptide) at 37°C (Figure 1.).
Plasma concentration was 90% (v/v) in the final reaction
buffer containing 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg
mL-1 BSA. Aliquots were removed at selected times, arid
activity was measured as amidolytic activity using S-2366 at
a final concentration of lmM. The measured half-lives are
summarized in Table IV. To assess the impact of activated
protein C polypeptide inactivation by PCI, heparin (10 U mL-
1), which is known to cause about 100-fold stimulation in
the inactivation of aPC by PCI (Heeb, et al., J Biol Chem
263(24):11613-11616, 1988; Espana, et al., Thromb Res
55(3):369-84, 1989; Aznar, et al., Thromb Haemost
76(6):983-988, 1996), was added to a similar reaction
(Figure 3). Inactivation by al-antitrypsin (al-AT) was
determined by incubation of aPC or derivatives at 20 nM with
40 mM al-AT (Sigma) in a reaction buffer consisting of 3 mM
CaCl2, 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL-1 BSA.
Aliquots were removed at selected times, and activity was
measured as amidolytic activity using S-2366 at a final
concentration of lmM.
Table IV. Half-lives for inactivation of activated protein
C polypeptides in normal human plasma.
ro ein -- tl/2 -r~ola increase
(min) relative to wild-type
Wild-Type 28 1
Leu194Ser 180 6.5
._~~~_________________________~~____....J____________________J___._.___________
._____________________._..__
Leu194A1a 88 3.1
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A1a195G1y 50 1.8
Thr254Ser 50 1.8
___________;____ ___________________________________________________
;__________
___________._________________253 9.1
Leu194A1a/Thr254Ser
J
_ 280 10.1
Leu194A1a/Thr254Ser
Example 6
In vivo Pharmacokinetics
In vivo pharmacokinetic experiments were performed in
normal rabbits to verify the observed in vitro effects in
half-life as a result of the mutations. A marginal ear vein
and a central ear artery was cannulated i.n the conscious
rabbit. Activated protein C polypeptides in buffer A (300
~g/ml) were used to administer a dose of 100 ~g/Kg or 0.1
mg/kg bolus through the marginal ear vein catheter. Blood
was sampled (0.45 ml) into a syringe containing 0.05 ml of
3.8% citrate containing benzamidine - adjustments were made
to compensate for the syringe/needle dead space to yield the
final concentration of 1 part citrate/benzamidine: 9 parts
blood. Samples were collected 0, 5, 10, 15, 30, 45, 60, 90,
120, 180, 240, 300 and 360 minutes post treatment, spun as
soon as convenient after collection, and 200 ~1 of plasma
was aliquoted into 96-well plates. The level of activated
protein C polypeptides were determined using an enzyme
capture assay (ECA), as described previously (Gruber, et
al., Blood 79(9):2340-2348, 1992), compared to standards
ranging from 1 to 250 ng/mL diluted in pooled rabbit plasma.
The results for wild-type and Leu194Ser are shown in Figure
5.
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SEQUENCE LISTING
<110> Gerlitz, Bruce E.
Jones, Bryan E.
<120> ACTIVATED PROTEIN C POLYPEPTIDES
<130> X-11901
<140> X-11901
<141> 2000-04-28
<150> P-11901
<151> 1999-04-30
<160> 10
<170> PatentIn Ver. 2.0
<210> 1
<211> 419
<212> PRT
<213> Homo sapiens
<400> 1
Ala Asn Ser Phe Leu Glu Glu Leu Arg His Ser Ser Leu Glu Arg Glu
1 5 10 15
Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln
20 25 30
Asn Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His Val Asp Gly Asp
35 40 45
Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala Ser Leu Cys Cys
50 55 60
Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys
65 70 75 80
Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg Glu Val Ser Phe Leu
85 90 95
Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu
i00 105 110
Val Gly Trp Arg Arg Cys Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp
115 120 125
1
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Asp Leu Leu Gln Cys His Pro Ala Val Lys Phe Pro Cys Gly Arg Pro
130 135 140
Trp Lys Arg Met Glu Lys Lys Arg Ser His Leu Lys Arg Asp Thr Glu
145 150 I55 160
Asp Gln Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Met Thr
165 170 175
Arg Arg Gly Asp Ser Pro Trp Gln Val Val Leu Leu Asp Ser Lys Lys
180 185 190
Lys Leu Ala Cys Gly Ala Val Leu Ile His Pro Ser Trp Val Leu Thr
195 200 205
Ala Ala His Cys Met Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly
210 215 220
Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile
225 230 235 240
Lys Glu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn
245 250 255
Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr
260 265 270
Ile Val Pro Ile Gds Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu
275 280 285
Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser
290 295 300
Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu Asn Phe
305 310 315 320
Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser Glu Val Met Ser
325 330 335
Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly Ile Leu Gly Asp Arg
340 345 350
Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Met Val Ala Ser Phe
355 360 365
His Gly Thr Trp Phe Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cars
370 375 380
2
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Gly Leu Leu His Asn Tyr Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu
385 390 395 400
Asp Trp Ile His Gly His Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser
405 410 415
Trp Ala Pro
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Met Trp Gln Leu Thr Ser Leu Leu Leu Phe Val Ala Thr Trp Gly Ile
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Ser Gly Thr Pro Ala Pro Leu Asp Ser Val Phe Ser Ser Ser Glu Arg
20 25 30
Ala His Gln Val Leu Arg Ile Arg Lys Arg Ala Asn Ser Phe Leu Glu
35 40 45
Glu Leu Arg His Ser Ser Leu Glu Arg Glu Cys Ile Glu Glu Ile Cys
50 55 60
Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln Asn Val Asp Asp Thr Leu
65 70 75 80
Ala Phe Trp Ser Lys His Val Asp Gly Asp Gln Cys Leu Val Leu Pro
85 90 95
Leu Glu His Pro Cys Ala Ser Leu Cys Cys Gly His Gly Thr Cys Ile
100 105 110
Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys Arg Ser Gly Trp Glu Gly
115 120 125
Arg Phe Cys Gln Arg Glu Val Ser Phe Leu Asn Cys Ser Leu Asp Asn
130 135 140
Gly Gly Cys Thr His Tyr Cys Leu Glu Glu Val Gly Trp Arg Arg Cys
145 150 155 160
Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp Asp Leu Leu Gln Cys His
3
His Gly Thr Trp Phe Leu Val
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
165 170 175
Pro Ala Val Lys Phe Pro Cys Gly Arg Pro Trp Lys Arg Met Glu Lys
180 185 190
Lys Arg Ser His Leu Lys Arg Asp Thr Glu Asp Gln Glu Asp Gln Val
195 200 205
Asp Pro Arg Leu Ile Asp Gly Lys Met Thr Arg Arg Gly Asp Ser Pro
210 215 220
Trp Gln Val Val Leu Leu Asp Ser Lys Lys Lys Leu Ala Cys Gly Ala
225 230 235 240
Val Leu Ile His Pro Ser Trp Val Leu Thr Ala Ala His Cys Met Asp
245 250 255
Glu Ser Lys Lys Leu Leu Val Arg Leu Gly Glu Tyr Asp Leu Arg Arg
260 265 270
Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile Lys Glu Val Phe Val His
275 280 285
Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn Asp Ile Ala Leu Leu His
290 295 300
Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr Ile Val Pro Ile Cys Leu
305 310 315 320
Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu Asn Gln Ala Gly Gln Glu
325 330 335
Thr Leu Val Thr Gly Trp Gly Tyr His Ser Ser Arg Glu Lys Glu Ala
340 345 350
Lys Arg Asn Arg Thr Phe Val Leu Asn Phe Ile Lys Ile Pro Val Val
355 360 365
Pro His Asn Glu Cys Ser Glu Val Met Ser Asn Met Val Ser Glu Asn
370 375 380
Met Leu Cys Ala Gly Ile Leu Gly Asp Arg Gln Asp Ala Cys Glu Gly
385 390 395 400
Asp Ser Gly Gly Pro Met Val Ala Ser Phe His Gly Thr Trp Phe Leu
405 4I0 415
Val Gly Leu Val Ser Trp Gly Glu Gly Cys Gly Leu Leu His Asn Tyr
4
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
420 425 430
Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu Asp Trp Ile His Gly His
435 440 445
Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser Trp Ala Pro
450 455 460
<210> 3
<211> 419
<212> PRT
<213> Homo sapiens
<400> 3
Ala Asn Ser Phe Leu Glu Glu Leu Arg His Ser Ser Leu Glu Arg Glu
1 5 10 15
Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln
20 25 30
Asn Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His Val Asp Gly Asp
35 40 45
Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala Ser Leu Cys Cys
50 55 60
Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys
65 70 75 80
Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg Glu Val Ser Phe Leu
85 90 95
Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu
100 105 110
Val Gly Trp Arg Arg Cys Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp
115 120 125
Asp Leu Leu Gln Cys His Pro Ala Val Lys Phe Pro Cys Gly Arg Pro
130 135 140
Trp Lys Arg Met Glu Lys Lys Arg Ser His Leu Lys Arg Asp Thr Glu
145 150 155 160
Asp Gln Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Met Thr
165 170 175
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
Arg Arg Gly Asp Ser Pro Trp Gln Val Val Leu Leu Asp Ser Lys Lys
180 185 190
Lys Ser Ala Cys Gly Ala Val Leu Ile His Pro Ser Trp Val Leu Thr
195 200 205
Ala Ala His Cys Met Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly
210 215 220
Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile
225 230 235 240
Lys Glu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Thr Asp Asn
245 250 255
Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr
260 265 270
Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu
275 280 285
Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser
290 295 300
Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu Asn Phe
305 310 315 320
Ile Lys Ile Pro Val Val Pro His Aen Glu Cys Ser Glu Val Met Ser
325 330 335
Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly Ile Leu Gly Asp Arg
340 345 350
Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Met Val Ala Ser Phe
355 360 365
His Gly Thr Trp Phe Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cys
370 375 380
Gly Leu Leu His Asn Tyr Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu
385 390 395 400
Asp Trp Ile His Gly His Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser
405 410 415
Trp Ala Pro
6
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
<210> 4
<211> 419
<212> PRT
<213> Homo sapiens
<400> 4
Ala Asn Ser Phe Leu Glu Glu Leu Arg His Ser Ser Leu Glu Arg Glu
1 5 10 15
Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln
20 25 30
Asn Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His Val Asp Gly Asp
35 40 45
Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala Ser Leu Cys Cys
50 55 60
Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys
65 70 75 80
Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg Glu Val Ser Phe Leu
85 90 95
Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu
100 105 110
Val Gly Trp Arg Arg Cys Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp
115 120 125
Asp Leu Leu Gln Cys His Pro Ala Val Lys Phe Pro Cys Gly Arg Pro
130 135 140
Trp Lys Arg Met Glu Lys Lys Arg Ser His Leu Lys Arg Asp Thr Glu
145 150 155 160
Asp Gln Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Met Thr
165 170 175
Arg Arg Gly Asp Ser Pro Trp Gln Val Val Leu Leu Asp Ser Lys Lys
180 185 190
Lys Ser Ala Cys Gly Ala Val Leu Ile His Pro Ser Trp Val Leu Thr
195 200 205
Ala Ala His Cys Met Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly
210 215 220
7
CA 02338799 2001-O1-26
WO 00166754 PCT/US00/08722
Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asg Ile
225 230 235 240
Lys Glu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Ser Asp Asn
245 250 255
Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr
260 265 270
Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu
275 280 285
Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser
290 295 300
Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu Asn Phe
305 310 315 320
Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser ~Glu Val Met Ser
325 330 335
Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly Ile Leu Gly Asp Arg
340 345 350
Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Met Val Ala Ser Phe
355 360 365
His Gly Thr Trp Phe Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cys
370 375 380
Gly Leu Leu His Asn Tyr Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu
385 390 395 400
Asp Trp Ile His Gly His Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser
405 410 415
Trp Ala Pro
<210>5
<211>419
<212>PRT
<213>Homo sapiens
<400> 5
Ala Asn Ser Phe Leu Glu Glu Leu Arg His Ser Ser Leu Glu Arg Glu
8
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
1 5 10 15
Cys Ile Glu Glu Ile Cys Asp Phe Glu Glu Ala Lys Glu Ile Phe Gln
20 25 30
Asn Val Asp Asp Thr Leu Ala Phe Trp Ser Lys His Val Asp Gly Asp
35 40 45
Gln Cys Leu Val Leu Pro Leu Glu His Pro Cys Ala Ser Leu Cys Cps
50 55 60
Gly His Gly Thr Cys Ile Asp Gly Ile Gly Ser Phe Ser Cys Asp Cys
65 70 75 80
Arg Ser Gly Trp Glu Gly Arg Phe Cys Gln Arg Glu Val Ser Phe Leu
85 90 95
Asn Cys Ser Leu Asp Asn Gly Gly Cys Thr His Tyr Cys Leu Glu Glu
100 105 110
Val Gly Trp Arg Arg Cys Ser Cys Ala Pro Gly Tyr Lys Leu Gly Asp
115 120 125
Asp Leu Leu Gln Cys His Pro Ala Val Lys Phe Pro Cys Gly Arg Pro
130 135 140
Trp Lys Arg Met Glu Lys Lys Arg Ser His Leu Lys Arg Asp Thr Glu
145 150 155 160
Asp Gln Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys Met Thr
165 170 175
Arg Arg Gly Asp Ser Pro Trp Gln Val Val Leu Leu Asp Ser Lys Lys
180 185 190
Lys Ala Ala Cys Gly Ala Val Leu Ile His Pro Ser Trp Val Leu Thr
195 200 205
Ala Ala His Cys Met Asp Glu Ser Lys Lys Leu Leu Val Arg Leu Gly
210 215 220
Glu Tyr Asp Leu Arg Arg Trp Glu Lys Trp Glu Leu Asp Leu Asp Ile
225 230 235 240
Lys Glu Val Phe Val His Pro Asn Tyr Ser Lys Ser Thr Ser Asp Asn
245 250 255
Asp Ile Ala Leu Leu His Leu Ala Gln Pro Ala Thr Leu Ser Gln Thr
9
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00108722
260 265 270
Ile Val Pro Ile Cys Leu Pro Asp Ser Gly Leu Ala Glu Arg Glu Leu
275 280 285
Asn Gln Ala Gly Gln Glu Thr Leu Val Thr Gly Trp Gly Tyr His Ser
290 295 300
Ser Arg Glu Lys Glu Ala Lys Arg Asn Arg Thr Phe Val Leu Asn Phe
305 310 315 320
Ile Lys Ile Pro Val Val Pro His Asn Glu Cys Ser Glu Val Met Ser
325 330 335
Asn Met Val Ser Glu Asn Met Leu Cys Ala Gly Ile Leu Gly Asp Arg
340 345 350
Gln Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Met Val Ala Ser Phe
355 360 365
His Gly Thr Trp Phe Leu Val Gly Leu Val Ser Trp Gly Glu Gly Cys
370 375 380
Gly Leu Leu His Asn Tyr Gly Val Tyr Thr Lys Val Ser Arg Tyr Leu
385 390 395 400
Asp Trp Ile His Gly His Ile Arg Asp Lys Glu Ala Pro Gln Lys Ser
405 410 415
Trp Ala Pro
<210>6
<2I1>1260
<212>DNA
<213>Homo sapiens
<400> 6
gccaactcct tcctggagga gctccgtcac agcagcctgg agcgggagtg catagaggag 60
atctgtgact tcgaggaggc caaggaaatt ttccaaaatg tggatgacac actggccttc 120
tggtccaagc acgtcgacgg tgaccagtgc ttggtcttgc ccttggagca cccgtgcgcc 180
agcctgtgct gcgggcacgg cacgtgcatc gacggcatcg gcagcttcag ctgcgactgc 240
cgcagcggct gggagggccg cttctgccag cgcgaggtga gcttcctcaa ttgctcgctg 300
gacaacggcg gctgcacgca ttactgccta gaggaggtgg gctggcggcg ctgtagctgt 360
gcgcctggct acaagctggg ggacgacctc ctgcagtgtc accccgcagt gaagttccct 420
tgtgggaggc cctggaagcg gatggagaag aagcgcagtc acctgaaacg agacacagaa 480
gaccaagaag accaagtaga tccgcggctc attgatggga agatgaccag gcggggagac 540
l0
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
agcccctggc aggtggtcct gctggactca aagaagaagc tggcctgcgg ggcagtgctc 600
atccacccct cctgggtgct gacagcggcc cactgcatgg atgagtccaa gaagctcctt 660
gtcaggcttg gagagtatga cctgcggcgc tgggagaagt gggagctgga cctggacatc 720
aaggaggtct tcgtccaccc caactacagc aagagcacca ccgacaatga catcgcactg 780
ctgcacctgg cccagcccgc caccctctcg cagaccatag tgcccatctg cctcccggac 840
agcggccttg cagagcgcga gctcaatcag gccggccagg agaccctcgt gacgggctgg 900
ggctaccaca gcagccgaga gaaggaggcc aagagaaacc gcaccttcgt cctcaacttc 960
atcaagattc ccgtggtccc gcacaatgag tgcagcgagg tcatgagcaa catggtgtct 1020
gagaacatgc tgtgtgcggg catcctcggg gaccggcagg atgcctgcga gggcgacagt 1080
ggggggccca tggtcgcctc cttccacggc acctggttcc tggtgggcct ggtgagctgg 1140
ggtgagggct gtgggctcct tcacaactac ggcgtttaca ccaaagtcag ccgctacctc 1200
gactggatcc atgggcacat cagagacaag gaagcccccc agaagagctg ggcaccttag 1260
<210> 7
<211> 1386
<212> DNA
<213> Homa Sapiens
<400> 7
atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60
gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120
aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180
gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240
gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300
tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360
gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420
tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480
agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540
ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600
acagaagacc aagaagacca agtagatccg cggctcattg atgggaagat gaccaggcgg 660
ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagctggc ctgcggggca 720
gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780
ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840
gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccaccga caatgacatc 900
gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960
ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020
ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080
aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140
gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200
gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260
agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320
tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380
ccttag 1386
<210> 8
<211> 1386
<212> DNA
<213> Homo Sapiens
11
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
<400> 8
atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60
gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120
aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180
gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240
gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300
tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360
gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420
tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480
agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540
ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600
acagaagacc aagaagacca agtagatccg cggctcattg atgggaagat gaccaggcgg 660
ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagtccgc ctgcggggca 720
gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780
ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840
gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccaccga caatgacatc 900
gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960
ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020
ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080
aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140
gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200
gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260
agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320
tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380
ccttag 1386
<210> 9
<211> 1386
<212> DNA
<213> Homo sapiens
<400> 9
atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60
gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120
aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180
gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240
gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300
tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360
gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420
tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480
agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540
ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600
acagaagacc aagaagacca agtagatccg cggctcattg atgggaagat gaccaggcgg 660
ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaagtccgc ctgcggggca 720
gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780
ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840
gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccagcga caatgacatc 900
12
CA 02338799 2001-O1-26
WO 00/66754 PCT/US00/08722
gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960
ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020
ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080
aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140
gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200
gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260
agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320
tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380
ccttag 1386
<210> 10
<211> 1386
<212> DNA
<213> Homo Sapiens
<400> 10
atgtggcagc tcacaagcct cctgctgttc gtggccacct ggggaatttc cggcacacca 60
gctcctcttg actcagtgtt ctccagcagc gagcgtgccc accaggtgct gcggatccgc 120
aaacgtgcca actccttcct ggaggagctc cgtcacagca gcctggagcg ggagtgcata 180
gaggagatct gtgacttcga ggaggccaag gaaattttcc aaaatgtgga tgacacactg 240
gccttctggt ccaagcacgt cgacggtgac cagtgcttgg tcttgccctt ggagcacccg 300
tgcgccagcc tgtgctgcgg gcacggcacg tgcatcgacg gcatcggcag cttcagctgc 360
gactgccgca gcggctggga gggccgcttc tgccagcgcg aggtgagctt cctcaattgc 420
tcgctggaca acggcggctg cacgcattac tgcctagagg aggtgggctg gcggcgctgt 480
agctgtgcgc ctggctacaa gctgggggac gacctcctgc agtgtcaccc cgcagtgaag 540
ttcccttgtg ggaggccctg gaagcggatg gagaagaagc gcagtcacct gaaacgagac 600
acagaagacc aagaagacca agtagatccg cggctcattg atgggaagat gaccaggegg 660
ggagacagcc cctggcaggt ggtcctgctg gactcaaaga agaaggccgc ctgcggggca 720
gtgctcatcc acccctcctg ggtgctgaca gcggcccact gcatggatga gtccaagaag 780
ctccttgtca ggcttggaga gtatgacctg cggcgctggg agaagtggga gctggacctg 840
gacatcaagg aggtcttcgt ccaccccaac tacagcaaga gcaccagcga caatgacatc 900
gcactgctgc acctggccca gcccgccacc ctctcgcaga ccatagtgcc catctgcctc 960
ccggacagcg gccttgcaga gcgcgagctc aatcaggccg gccaggagac cctcgtgacg 1020
ggctggggct accacagcag ccgagagaag gaggccaaga gaaaccgcac cttcgtcctc 1080
aacttcatca agattcccgt ggtcccgcac aatgagtgca gcgaggtcat gagcaacatg 1140
gtgtctgaga acatgctgtg tgcgggcatc ctcggggacc ggcaggatgc ctgcgagggc 1200
gacagtgggg ggcccatggt cgcctccttc cacggcacct ggttcctggt gggcctggtg 1260
agctggggtg agggctgtgg gctccttcac aactacggcg tttacaccaa agtcagccgc 1320
tacctcgact ggatccatgg gcacatcaga gacaaggaag ccccccagaa gagctgggca 1380
ccttag 1386
13
