U.S. patent application number 11/931941 was filed with the patent office on 2009-10-01 for kallikrein inhibitors and anti-thrombolytic agents and uses thereof.
This patent application is currently assigned to DYAX CORP.. Invention is credited to Thomas Beck, Henry Blair, Robert C. Ladner.
Application Number | 20090247453 11/931941 |
Document ID | / |
Family ID | 36119484 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090247453 |
Kind Code |
A1 |
Blair; Henry ; et
al. |
October 1, 2009 |
Kallikrein Inhibitors and Anti-thrombolytic Agents and Uses
Thereof
Abstract
Methods, kits and compositions are described that include a
non-naturally occurring kallikrein inhibitor and an
anti-thrombolytic agent, e.g., an anti-fibrinolytic agent, for
preventing or reducing blood loss and/or ischemia, e.g., ischemia
associated with perioperative blood loss and cerebral ischemia, the
onset of systemic inflammatory response, and/or reperfusion injury,
e.g., reperfusion injury associated with cerebral ischemia or a
focal brain ischemia, e.g., in patients subjected to invasive
surgical procedures, especially procedures requiring
cardiopulmonary bypass.
Inventors: |
Blair; Henry; (Boston,
MA) ; Beck; Thomas; (Concord, MA) ; Ladner;
Robert C.; (Ijamsville, MD) |
Correspondence
Address: |
LANDO & ANASTASI, LLP
ONE MAIN STREET, SUITE 1100
CAMBRIDGE
MA
02142
US
|
Assignee: |
DYAX CORP.
Cambridge
MA
|
Family ID: |
36119484 |
Appl. No.: |
11/931941 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11804388 |
May 18, 2007 |
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11931941 |
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11125639 |
May 9, 2005 |
7235530 |
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11804388 |
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10953902 |
Sep 27, 2004 |
7153829 |
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11125639 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 31/195 20130101;
A61P 9/00 20180101; A61P 43/00 20180101; A61K 38/55 20130101; A61K
45/06 20130101; A61P 29/00 20180101; A61P 7/04 20180101; A61K 38/57
20130101; A61P 9/10 20180101; A61P 7/00 20180101; A61P 7/02
20180101; A61K 31/195 20130101; A61K 2300/00 20130101; A61K 38/57
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/11 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61P 7/02 20060101 A61P007/02 |
Claims
1.-28. (canceled)
29. A kit comprising a non-naturally occurring kallikrein inhibitor
and Desmopressin (DDAVP).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/804,388, filed May 18, 2007, which is a continuation (and
claims the benefit of priority under 35 USC 120) of U.S.
application Ser. No. 11/125,639, filed May 9, 2005, issued as U.S.
Pat. No. 7,235,530, which is a continuation-in-part of U.S. patent
application Ser. No. 10/953,902, filed Sep. 27, 2004, issued as
U.S. Pat. No. 7,153,829, the contents of all of which are
incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention is generally in the field of invasive
surgical procedures associated with contact activation of
complement components and the coagulation/fibrinolysis systems.
More specifically, the invention provides methods, kits and
compositions utilizing a combination of a non-naturally occurring
kallikrein inhibitor and an anti-thrombolytic agent, e.g., an
anti-fibrinolytic agent, to reduce or prevent blood loss, e.g.,
perioperative blood loss, and/or injury associated with various
ischemias, e.g., in patients subjected to invasive surgical
procedures. For example, the invention provides methods, kits and
compositions for reducing blood loss associated with procedures
requiring cardiopulmonary bypass.
BACKGROUND
[0003] Owing to the many advances in medicine a number of highly
invasive surgical procedures are carried out each day that result
in blood loss, or place patients at a high risk for blood loss.
Such patients must be carefully monitored to restore and maintain
normal blood supply and hemostasis, and they may need blood
transfusions. Surgical procedures that involve blood loss include
those involving extra-corporeal circulation methods such as
cardiopulmonary bypass (CPB). In such methods, a patient's heart is
stopped and the circulation, oxygenation, and maintenance of blood
volume are carried out artificially using an extra-corporeal
circuit and a synthetic membrane oxygenator. These techniques are
commonly used during cardiac surgery. Additionally, it is apparent
that surgery involving extensive trauma to bone, such as the
sternal split necessary in coronary artery bypass grafting (CABG)
or hip replacement procedures, is also associated with activation
of the contact activation system (CAS), which can result in a
variety of disruptions in the blood and vasculature.
[0004] Atherosclerotic coronary artery disease (CAD) causes a
narrowing of the lumen of one or several of the coronary arteries;
this limits the flow of blood to the myocardium (i.e., the heart
muscle) and can cause angina, heart failure, and myocardial
infarcts. In the end stage of coronary artery atherosclerosis, the
coronary circulation can be almost completely occluded, causing
life threatening angina or heart failure, with a very high
mortality. CABG procedures may be required to bridge the occluded
blood vessel and restore blood to the heart; these are potentially
life saving. CABG procedures are among the most invasive of
surgeries in which one or more healthy veins or arteries are
implanted to provide a "bypass" around the occluded area of the
diseased vessel.
[0005] The number of CABG procedures performed in the United States
in 1998 was approximately 500,000. CABG procedures carry with them
a small but important perioperative risk, but they are very
successful in providing patients with immediate relief from the
mortality and morbidity of atherosclerotic cardiovascular disease.
Despite these very encouraging results, repeat CABG procedures are
not uncommon, as indicated by a clear increase in the number of
patients who eventually undergo second and even third procedures;
the perioperative mortality and morbidity seen in primary CABG
procedures is increased in these re-do procedures.
[0006] There have been improvements in minimally invasive surgical
techniques for uncomplicated CAD. However, nearly all CABG
procedures performed for valvular and/or congenital heart disease,
heart transplantation, and major aortic procedures, are still
carried out on patients supported by CPB. In CPB, large cannulae
are inserted into the great vessels of a patient to permit
mechanical pumping and oxygenation of the blood using a membrane
oxygenator. The blood is returned to the patient without flowing
through the lungs, which are hypoperfused during this procedure.
The heart is stopped using a cardioplegic solution, the patient
cooled to help prevent brain damage, and the peripheral circulating
volume increased by an extracorporeal circuit, i.e., the CPB
circuit, which requires "priming" with donor blood and saline
mixtures are used to fill the extracorporeal circuit. CPB has been
extensively used in a variety of procedures performed for nearly
half a century with successful outcomes. The interaction between
artificial surfaces, blood cells, blood proteins, damaged vascular
endothelium, and extravascular tissues, such as bone, disturbs
hemostasis and frequently activates the CAS, which, as noted above,
can result in a variety of disruptions in the blood and
vasculature. Such disruption leads to excess perioperative
bleeding, which then requires immediate blood transfusion. A
consequence of circulating whole blood through an extracorporeal
circuit in CPB may also include the systemic inflammatory response
(SIR), which is initiated by contact activation of the coagulation
and complement systems. Indeed, much of the morbidity and mortality
associated with seemingly mechanically successful CPB surgical
procedures is the result of the effects of activating coagulation,
fibrinolysis, or complement systems. Such activation may damage the
pulmonary system, leading to adult respiratory distress syndrome
(ARDS), impairment of kidney and splanchnic circulation, and
induction of a general coagulopathy leading to blood loss and the
need for transfusions. In addition to the dangers of perioperative
blood loss, additional pathologies associated with SIR include
neurocognitive deficits, stroke, renal failure, acute myocardial
infarct, and cardiac tissue damage.
[0007] Blood transfusions also present a significant risk of
infection and elevate the cost of CABG or other similar procedures
that require CPB. In the absence of any pharmacological
intervention, three to seven units of blood must typically be
expended on a patient, even with excellent surgical techniques.
Accordingly, there is considerable incentive for the development of
new and improved treatments to reduce or prevent perioperative
bleeding and SIR in patients subjected to CPB and CABG
procedures.
SUMMARY
[0008] The disclosure is based, at least in part, on the discovery
that a combination of a non-naturally occurring kallikrein
inhibitor, e.g., a Kunitz domain kallikrein inhibitor polypeptide,
and an anti-thrombolytic agent, e.g., an anti-fibrinolytic agent,
can be administered to a subject to eliminate or reduce blood loss,
e.g., perioperative blood loss, as well as injury associated with
ischemia (including ischemia associated with perioperative blood
loss and cerebral ischemia), the onset of systemic inflammatory
response, and/or reperfusion injury, e.g., reperfusion injury
associated with cerebral ischemia or a focal brain ischemia. In one
embodiment, the treatment can reduce or eliminate blood loss by one
or more of: reducing or eliminating bleeding, capillary leakage and
alterations in body fluid balance. The treatment can be in patients
subjected to invasive surgical procedures such as, e.g.,
cardiothoratic surgery (e.g., cardiopulmonary bypass), orthopedic
surgery (e.g., hip or knee replacement or fracture), hepatectomy,
nephrectomy. The invasive surgical procedure can involve the use of
extracorporeal circulation or dialysis. Preferably, the treatment
is more effective because of the combined administration. For
example, the anti-thrombolytic agent is more effective, e.g., an
equivalent effect is seen with less of the anti-thrombolytic agent,
the anti-thrombolytic treatment reduces symptoms to a greater
extent than would be seen if the anti-thrombolytic treatment were
administered in the absence of the non-naturally occurring
kallikrein inhibitor, and/or an unwanted side effect associated
with the anti-thrombolytic agent is seen less than would be seen if
the anti-thrombolytic agent was administered in the absence of the
non-naturally occurring kallikrein inhibitor, or the analogous
situation is seen with the non-naturally occurring kallikrein
inhibitor.
[0009] Accordingly, the disclosure features methods, compositions
and kits that include a non-naturally occurring kallikrein
inhibitor, e.g., a plasma kallikrein inhibitor, and an
anti-thrombolytic agent, to eliminate or reduce blood loss and/or
injury associated with ischemias. Preferably, the anti-thrombolytic
agent is an anti-fibrinolytic agent, e.g., an anti-fibrinolytic
agent described herein. Anti-fibrinolytic agents can be one or more
of: tranexamic acid (Cyklokapron.TM.), epsilon amino caproic acid
(Amicar.TM.), aprotinin (Trasyol.TM.), Desmopressin (DDAVP), and
pirfenidone.
[0010] In one aspect, the disclosure features a method for
preventing or reducing blood loss in a patient that includes
administering to the patient a non-naturally occurring inhibitor of
kallikrein, e.g., a plasma kallikrein, in combination with an
anti-thrombolytic agent, e.g., an anti-fibrinolytic agent.
Typically, the patient is a human patient. The combination of the
inhibitor of kallikrein and the anti-thrombolytic agent can be
administered in an amount effective to prevent or reduce blood loss
(e.g., prevent or reduce one or more of: bleeding, capillary
leakage and alterations in body fluid balance). In a particular
embodiment, the blood loss is perioperative blood loss is due to a
surgical procedure performed on the patient. The surgical procedure
can be, e.g., a cardiothoracic surgery, (e.g., cardiopulmonary
bypass or coronary artery bypass grafting); orthopedic surgery
(e.g., hip or knee replacement or bone fracture); hepatectomy;
nephrectomy; procedures that utilize extracorporeal circulation or
dialysis; and any other procedure which can result in perioperative
blood loss. The inhibitor and/or the anti-thrombolytic agent can be
administered before, during, or after the procedure. In one
embodiment, the method reduces the amount or need for a transfusion
before, during or after the procedure.
[0011] In another aspect, the disclosure features a kit for
preventing or reducing blood loss, e.g., perioperative blood loss
due to a surgical procedure performed on the patient. The kit can
include a non-naturally occurring inhibitor of kallikrein, e.g.,
plasma kallikrein, and instructions for administering the inhibitor
in combination with an anti-thrombolytic agent, e.g., an
anti-fibrinolytic agent. In one embodiment, the instructions
provide a dosing regimen, dosing schedule, and/or route of
administration of the inhibitor that differs from the dosing
regimen, dosing schedule and/or route of administration for the
inhibitor in the absence of the anti-thrombolytic agent. In one
embodiment, the kit further includes an anti-thrombolytic agent,
e.g., an anti-fibrinolytic agent.
[0012] In another aspect, the disclosure features a method for
preventing or reducing injury associated with ischemia in a patient
that includes administering to the patient a non-naturally
occurring inhibitor of kallikrein, e.g., a plasma kallikrein, in
combination with an anti-thrombolytic agent, e.g., an
anti-fibrinolytic agent. Typically, the patient is a human patient.
The combination of the inhibitor of kallikrein and the
anti-thrombolytic agent can be administered in an amount effective
to prevent or reduce an injury associated with ischemia in the
patient. In a particular embodiment, the ischemia is at least
partially due to blood loss, e.g., perioperative blood loss due to
a surgical procedure performed on the patient. The surgical
procedure can be, e.g., a cardiothoracic surgery, (e.g.,
cardiopulmonary bypass or coronary artery bypass grafting);
orthopedic surgery (e.g., hip or knee replacement or bone
fracture); hepatectomy; nephrectomy; procedures that utilize
extracorporeal circulation or dialysis; and any other procedure
which can result in perioperative blood loss. The inhibitor and/or
the anti-thrombolytic agent can be administered before, during, or
after the procedure.
[0013] In another aspect, the disclosure features a kit for
preventing or reducing injury associated with ischemia in a
patient, e.g., ischemia at least partially due to blood loss, e.g.,
perioperative blood loss due to a surgical procedure performed on
the patient. The kit can include a non-naturally occurring
inhibitor of kallikrein, e.g., plasma kallikrein, and instructions
for administering the inhibitor in combination with an
anti-thrombolytic agent, e.g., an anti-fibrinolytic agent. In one
embodiment, the instructions provide a dosing regimen, dosing
schedule, and/or route of administration of the inhibitor that
differs from the dosing regimen, dosing schedule and/or route of
administration for the inhibitor in the absence of the
anti-thrombolytic agent. In one embodiment, the kit further
includes an anti-thrombolytic agent, e.g., an anti-fibrinolytic
agent.
[0014] In another aspect, the disclosure features a method for
preventing or reducing a systemic inflammatory response, e.g., a
response associated with a surgical procedure in a patient or its
onset. The method includes: administering to the patient a
non-naturally occurring inhibitor of kallikrein, e.g., plasma
kallikrein, in combination with an anti-thrombolytic agent, e.g.,
an anti-fibrinolytic agent. Typically, the patient is a human
patient. The inhibitor and/or the anti-thrombolytic agent can be
administered before, during, or after surgery. In one embodiment,
the surgical procedure is a cardiothoracic surgery, (e.g.,
cardiopulmonary bypass or coronary artery bypass grafting);
orthopedic surgery (e.g., hip or knee replacement or bone
fracture); hepatectomy; nephrectomy; a procedure that utilize
extracorporeal circulation or dialysis; and any other procedure
which can result in perioperative blood loss.
[0015] In another aspect, the disclosure features a kit for
preventing or reducing systemic inflammatory response, e.g., a
response associated with a surgical procedure in a patient or its
onset. The kit can include a non-naturally occurring inhibitor of
kallikrein, e.g., plasma kallikrein, and instructions for
administering the inhibitor in combination with an
anti-thrombolytic agent, e.g., an anti-fibrinolytic agent. In one
embodiment, the instructions provide a dosing regimen, dosing
schedule, and/or route of administration of the inhibitor that
differs from the dosing regimen, dosing schedule and/or route of
administration for the inhibitor in the absence of the
anti-thrombolytic agent. In one embodiment, the kit further
includes an anti-thrombolytic agent, e.g., an anti-fibrinolytic
agent.
[0016] In another aspect, the disclosure features a method for
treating a brain or central nervous system (CNS) injury. The method
can be used to prevent or reduce adverse effects of cerebral
ischemia, e.g., stroke, and/or reperfusion injury, e.g.,
reperfusion injury associated with cerebral ischemia, in a patient
including administering to the patient a non-naturally occurring
inhibitor of kallikrein, e.g., a plasma kallikrein, in combination
with an anti-thrombolytic agent, e.g., an anti-fibrinolytic agent.
In one embodiment, the cerebral ischemia is stroke, e.g.,
embolism-, thrombus- or hemorrhage-associated stroke. The method
can include administering the inhibitor and/or the
anti-thrombolytic agent, before, during, or after the ischemia,
e.g., at the time of reperfusion or at a time between 1-12 hours
after an ischemic event, e.g., between 1-5 hours after such an
event.
[0017] In another aspect, the disclosure features a kit for
treating a brain or central nervous system (CNS) injury, e.g., to
prevent or reduce adverse effects of cerebral ischemia, e.g.,
stroke, and/or reperfusion injury, e.g., reperfusion injury
associated with cerebral ischemia. In one embodiment, the cerebral
ischemia is stroke, e.g., embolism-, thrombus- or
hemorrhage-associated stroke. The kit can include a non-naturally
occurring inhibitor of kallikrein, e.g., plasma kallikrein, and
instructions for administering the inhibitor in combination with an
anti-thrombolytic agent, e.g., an anti-fibrinolytic agent. In one
embodiment, the instructions provide a dosing regimen, dosing
schedule, and/or route of administration of the inhibitor that
differs from the dosing regimen, dosing schedule and/or route of
administration for the inhibitor in the absence of the
anti-thrombolytic agent. In one embodiment, the kit further
includes an anti-thrombolytic agent, e.g., an anti-fibrinolytic
agent.
[0018] The disclosure also features a composition that includes a
non-naturally occurring inhibitor of kallikrein, e.g., a plasma
kallikrein, and an anti-thrombolytic agent, e.g., an
anti-fibrinolytic agent. The composition can further include a
pharmaceutically acceptable carrier, stabilizer and/or
excipient.
[0019] The non-naturally occurring kallikrein inhibitor used in any
disclosed method, kit or composition can have one or more of the
characteristics described below.
[0020] The kallikrein inhibitor can have a Ki for kallikrein, e.g.,
plasma kallikrein, of less than 50 nM, 40 nM, 30 nM, 20 nM, 5 nM, 1
nM, 500 pM, 100 pM, 50 pM, e.g., about 44 pM. The kallikrein
inhibitor can preferentially inhibit plasma kallikrein at least
100, 200, 500, or 1000 more than another kallikrein, e.g., human
urine kallikrein, or another protease, e.g., plasmin or
thrombin.
[0021] In one embodiment, the kallikrein inhibitor is an agent that
can cross the blood-brain barrier.
[0022] In one embodiment, the kallikrein inhibitor includes a
polypeptide that includes a Kunitz domain such as the amino acid
sequence:
TABLE-US-00001 (SEQ ID NO: 1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7
Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19
Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys
Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42
Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53
Xaa54 Cys Xaa56 Xaa57 Xaa58.
[0023] The framework of the Kunitz domain can be human or can
differ from a human Kunitz domain framework by fewer than six,
five, four, three, or two amino acids. For example, the framework
of the Kunitz domain can be the framework of one of the Kunitz
domains of human lipoprotein-associated coagulation inhibitor
(LACI) protein, e.g., the first second or third Kunitz domain. LACI
is also known as "Tissue Factor Pathway Inhibitor" or "TFPI".
Typically, the polypeptide differs from BPTI and/or one or more of
the LACI Kunitz domains by at least one, two, three, or four amino
acids, e.g., at least one, two or three amino acids in the binding
loops and/or at least two, three, four, or six amino acids in the
framework region. For example, the polypeptide can include a
non-naturally occurring Kunitz domain that is derived from a
naturally occurring Kunitz domain, e.g., a human Kunitz domain. In
one embodiment, an inhibitor that includes a Kunitz domain binds to
plasma kallikrein with an affinity that is at least 10, 100, or 500
fold better than BPTI and/or LACI.
[0024] In one embodiment, the polypeptide that inhibits kallikrein
is not immunogenic on second use.
[0025] In one embodiment, the polypeptide that inhibits kallikrein
can have one or more of the following features: Xaa1, Xaa2, Xaa3,
Xaa4, Xaa56, Xaa57 or Xaa58 are each individually an amino acid or
absent; Xaa10 is an amino acid selected from the group consisting
of: Asp and Glu; Xaa11 is an amino acid selected from the group
consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 is
an amino acid selected from the group consisting of: Arg, His, Pro,
Asn, Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acid
selected from the group consisting of: Arg, Lys, Ala, Ser, Gly,
Met, Asn and Gln; Xaa16 is an amino acid selected from the group
consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an amino acid
selected from the group consisting of: Ala, Asn, Ser, Ile, Gly,
Val, Gln and Thr; Xaa18 is an amino acid selected from the group
consisting of: His, Leu, Gln and Ala; Xaa19 is an amino acid
selected from the group consisting of: Pro, Gln, Leu, Asn and Ile;
Xaa21 is an amino acid selected from the group consisting of: Trp,
Phe, Tyr, His and Ile; Xaa22 is an amino acid selected from the
group consisting of: Tyr and Phe; Xaa23 is an amino acid selected
from the group consisting of: Tyr and Phe; Xaa31 is an amino acid
selected from the group consisting of: Glu, Asp, Gln, Asn, Ser,
Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected from
the group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser,
Ala, Gly and Val; Xaa34 is an amino acid selected from the group
consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is
an amino acid selected from the group consisting of: Tyr, Trp and
Phe; Xaa39 is an amino acid selected from the group consisting of:
Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid selected from
the group consisting of: Gly and Ala; Xaa43 is an amino acid
selected from the group consisting of: Asn and Gly; Xaa45 is an
amino acid selected from the group consisting of: Phe and Tyr; and
wherein the polypeptide inhibits kallikrein.
[0026] In a particular embodiment, individual amino acid positions
of a kallikrein inhibitor that includes the amino acid sequence of
SEQ ID NO:1 has one or more of the following: Xaa10 is Asp, Xaa11
is Asp, Xaa13 is Pro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala,
Xaa18 is His, Xaa19 is Pro, Xaa21 is Trp, Xaa31 is Glu, Xaa32 is
Glu, Xaa34 is Ile, Xaa35 is Tyr, Xaa39 is Glu.
[0027] The polypeptide that inhibits kallikrein can include (or
consist of) the following amino acid sequence: Met His Ser Phe Cys
Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe
Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu
Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr
Arg Asp (amino acids 3-60 of SEQ ID NO:2), or a fragment thereof,
e.g., a fragment that binds and inhibits kallikrein. For example,
the polypeptide can have fewer than 80, 70, 65, 60, 58, 55 or 52
amino acids.
[0028] The polypeptide that inhibits kallikrein can include (or
consist of) a polypeptide described in U.S. Pat. No. 5,786,328, the
contents of which are incorporated by reference.
[0029] Methods, kits and compositions described herein can include
an inhibitor that comprises a non-naturally occurring, Kunitz
domain polypeptide having any of the amino acid sequences described
herein and an additional flanking sequence of one to six amino
acids at the amino and/or carboxy terminal end domains. Such
additional amino acids may be artifacts of expressing a particular
non-naturally occurring kallikrein inhibitor polypeptide or Kunitz
domain polypeptide in any of a variety of recombinant expression
vector systems, such as used in yeast, bacteria, mammalian cell
lines, insect cells, and the like. Preferably, such additional
amino acids at the amino and/or carboxy termini of a non-naturally
occurring Kunitz domain described herein do not diminish the
affinity for kallikrein or kallikrein inhibition activity of the
domain or a polypeptide comprising the domain.
[0030] The inhibitor polypeptide can include a non-naturally
occurring Kunitz domain polypeptide having an amino acid sequence
of SEQ ID NO:1 and an amino terminal flanking sequence as the
result of producing the polypeptide as a recombinant protein in
yeast. An example of a particularly preferred yeast recombinant
expression system comprises fusing a nucleotide coding sequence for
a non-naturally occurring Kunitz domain of SEQ ID NO:1 to a
nucleotide sequence encoding the mat.alpha. Prepro peptide leader
sequence of Saccharomyces cerevisiae and expressing the recombinant
coding sequence in the yeast Pichia pastoris. The resulting
expressed fusion protein comprises an amino acid sequence of SEQ ID
NO:1 and an amino terminal flanking dipeptide, Glu-Ala. A
particularly preferred species of an inhibitor polypeptide of the
invention produced in a yeast expression system has the amino acid
sequence of SEQ ID NO:2:
TABLE-US-00002 (SEQ ID NO: 2) Glu Ala Met His Ser Phe Cys Ala Phe
Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn
Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn
Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg
Asp.
[0031] In one embodiment, the polypeptide that inhibits kallikrein
is modified, e.g., to include one or more moieties, e.g., one or
more moieties that extend half life of the polypeptide, e.g., a
polymer moiety or a plurality of polymer moieties, e.g., as
described in U.S. Ser. No. 10/931,153, filed Aug. 30, 2004, bearing
attorney docket number 10280-119001. For example, the polypeptide
can include a plurality of polyethylene glycol moieties, e.g., one
on an N-terminal amine and one attached to each lysine of the
polypeptide. The polyethylene glycol moieties can be less than 10,
8, 7, or 6 kDa in average molecular weight. In other embodiments,
the moiety can be, e.g., serum albumin, e.g., human serum albumin.
Other exemplary modifications include a label, e.g., a radioactive
or MRI-detectable label. In some embodiments, the polypeptide is
part of a mixture that includes modified and unmodified
polypeptides that inhibit kallikrein. For example, the mixture can
include one or more modified polypeptides that inhibit kallikrein
and that include a polymer moiety such as a polyethylene glycol
moiety and one or more unmodified polypeptides that inhibit
kallikrein and do not include a polymer moiety. In one embodiment,
approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or all of
the polypeptides that inhibit kallikrein in the mixture are
modified.
[0032] The kallikrein inhibitor polypeptides useful in the methods,
compositions and kits may be any of the non-naturally occurring
Kunitz domain polypeptides described herein or larger polypeptides
comprising any such Kunitz domains, provided the kallikrein
inhibitor polypeptides bind and inhibit kallikrein as determined in
standard assays.
[0033] The anti-thrombolytic agent used in any disclosed method,
kit or composition can be an anti-fibrinolytic agent. Examples of
anti-fibrinolytic agents include: tranexamic acid
(Cyklokapron.TM.), epsilon amino caproic acid (Amicar.TM.),
aprotinin (Trasyol.TM.), Desmopressin (DDAVP), pirfenidone, and
combinations thereof. In one embodiment, the anti-thrombolytic
agent is an anti-fibrinolytic agent selected from epsilon amino
caproic acid (Amicar.TM.), aprotinin (Trasyol.TM.), and
combinations thereof.
[0034] The methods described herein can include administering an
effective amount of the combination treatment. Such an amount can
be an amount sufficient to produce an improvement detectable to one
skilled in the art, to ameliorate at least one symptom, or to
modulate (e.g., improve) at least one physiological parameter,
e.g., to a statistically significant degree.
[0035] Preferred compositions, e.g., used in any method or kit
described herein, may further comprise one or more pharmaceutically
acceptable buffers, carriers, and excipients, which may provide a
desirable feature to the composition including, but not limited to,
enhanced administration of the composition to a patient, enhanced
circulating half-life of the inhibitor and/or anti-thrombolytic
agent, enhanced compatibility of the composition with patient blood
chemistry, enhanced storage of the composition, and/or enhanced
efficacy of the composition upon administration to a patient.
[0036] Preferred methods described herein are useful for preventing
or reducing perioperative blood loss and/or SIR in a patient
subjected to a surgical procedure such as a surgical procedure that
requires extra-corporeal circulation (e.g., cardiopulmonary bypass
(CPB)) or dialysis. Particularly preferred are methods of the
invention for preventing or reducing perioperative blood loss
and/or SIR in a patient subjected to a surgical procedure
comprising administering to the patient of a kallikrein inhibitor
polypeptide described herein, in combination with an
anti-thrombolytic agent, e.g., an anti-fibrinolytic agent, wherein
the surgical procedure requires cardiopulmonary bypass (CPB) and
the surgical procedure is a coronary artery bypass graft (CABG)
procedure.
[0037] Methods described herein may be carried out on a patient
before, during, and/or after the surgical procedure. Particularly
preferred is the use of the methods before and during a surgical
procedure, especially in the case of CABG procedures to prevent
perioperative blood loss by activation of the contact activation
system and the onset of SIR.
[0038] In another embodiment, the invention provides nucleic acid
molecules comprising nucleotide sequences coding for a
non-naturally occurring Kunitz domain or kallikrein inhibitor
polypeptide described herein. Such nucleic acid molecules may be
any of a variety of nucleic acid molecules including, but not
limited to, a recombinant phage genome, a recombinant mammalian
viral vector, a recombinant insect viral vector, a yeast mini
chromosome, and a plasmid. Preferred plasmid molecules of the
invention include but are not limited to yeast expression plasmids,
bacterial expression plasmids, and mammalian expression plasmids.
Nucleic acid molecules useful in the invention may comprise a
specific nucleotide sequence described herein or a degenerate form
thereof.
[0039] Particularly preferred nucleic acid molecules of the
invention include a nucleic acid molecule comprising a nucleotide
sequence as shown in FIG. 2 encoding a fusion protein comprising a
mat.alpha. Prepro signal peptide fused to a heretofore undisclosed
kallikrein inhibitor polypeptide, nucleic acid molecules comprising
the nucleotide sequence encoding the kallikrein inhibitor
polypeptide having an amino acid of SEQ ID NO:2, and nucleic acid
molecules comprising a nucleotide sequence encoding a kallikrein
inhibitor polypeptide having an amino acid sequence of amino acids
3-60 of SEQ ID NO:2.
[0040] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 is a simplified diagram of major multiple pathways
and related events involved in the contact activation system and
systemic inflammatory response (SIR) that may arise in a patient
subjected to soft and bone tissue trauma such as that associated
with a coronary artery bypass grafting (CABG) procedure, especially
when the CABG procedure involves extra-corporeal blood circulation,
such as cardiopulmonary bypass (Bypass Apparatus). Arrows indicate
activation from one component or event to another component or
event in the cascade. Arrows in both directions indicate activating
effects of components or events in both directions. Broken arrows
indicate likely participation of one component or event in the
activation of another component or event. Abbreviations: "tPA",
tissue plasminogen activator; "C5a", a protein component of the
complement system; "fXIIa", activator protein of prekallikrein to
form active kallikrein; "Extrinsic", extrinsic coagulation system;
"Intrinsic", intrinsic coagulation system.
[0042] FIG. 2 shows a portion of a DNA and corresponding deduced
amino acid for an exemplary kallikrein inhibitor polypeptide in
plasmid pPIC-K503. The inserted DNA encodes the mat.alpha. Prepro
signal peptide of Saccharomyces cerevisiae (underlined) fused in
frame to the amino terminus of the PEP-1 polypeptide having the
amino acid sequence enclosed by the boxed area. The amino acid
sequence of the PEP-1 polypeptide shown in the boxed region is SEQ
ID NO:2, and the corresponding nucleotide coding sequence is SEQ ID
NO:3. The dashed arrows indicate the location and direction of two
PCR primer sequences in AOX regions that were used to produce
sequencing templates. DNA sequence for the entire nucleotide
sequence of the figure includes the structural coding sequence for
the fusion protein and is designated SEQ ID NO:27. The double
underlined portion of the sequence indicates a diagnostic probe
sequence. BstB I and EcoR I indicate locations of their respective
palindromic, hexameric, restriction endonuclease sites in the
sequence. Asterisks denote translational stop codons. See text for
details.
[0043] FIGS. 3A and 3B show an alignment of exemplary amino acid
sequences, the native LACI sequence from which these variants were
derived (SEQ ID NO:32), and other known Kunitz domains (SEQ ID
NOS:29-31 and 33-53). Cysteine residues are highlighted.
DETAILED DESCRIPTION
[0044] The invention is based on the discovery that the use of a
group of kallikrein inhibitor (KI) polypeptides that bind to and
inhibit plasma kallikrein with a specificity, in combination with
an anti-thrombolytic agent, e.g., an anti-fibrinolytic agent,
provides improved methods of preventing or reducing blood loss,
e.g., perioperative blood loss, e.g., in patients undergoing
surgical procedures. The methods can also reduce or prevent injury
associated with various ischemias and/or a systemic inflammatory
response (SIR) in patients undergoing surgical procedures. Surgical
procedures can include, e.g., a cardiothoracic surgery, (e.g.,
cardiopulmonary bypass (CPB) or coronary artery bypass grafting
(CABG)); orthopedic surgery (e.g., hip or knee replacement or bone
fracture); hepatectomy; nephrectomy; procedures that utilize
extracorporeal circulation or dialysis; and any other procedure
which can result in perioperative blood loss. "Blood loss" as used
herein refers restoring blood supply and/or hemostasis, maintaining
blood supply and/or hemostasis, and/or reducing the amount or need
for a transfusion. For example, the methods, kits and compositions
described herein can be used to reduce or prevent bleeding,
capillary leakage and/or alterations in body fluid balance.
Administered "in combination", as used herein, means that two (or
more) different treatments are delivered to the subject while the
subject is at risk for blood loss or during the course of the blood
loss, e.g., the two or more treatments are delivered after the
subject has been determined to be at risk for the disorder and
before the disorder has been prevented, cured or eliminated or
treatment has ceased for other reasons. In some embodiments, the
delivery of one treatment is still occurring when the delivery of
the second begins, so that there is overlap in terms of
administration. This is sometimes referred to herein as
"simultaneous" or "concurrent delivery." In other embodiments, the
delivery of one treatment ends before the delivery of the other
treatment begins. In some embodiments of either case, the treatment
is more effective because of combined administration. For example,
the anti-thrombolytic agent is more effective, e.g., an equivalent
effect is seen with less of the anti-thrombolytic agent, the
anti-thrombolytic agent reduces symptoms to a greater extent than
would be seen if the anti-thrombolytic agent were administered in
the absence of the kallikrein inhibitor, and/or a side effect
associated with the anti-thrombolytic agent is seen to a lesser
extent than if the anti-thrombolytic agent were administered in the
absence of the kallikrein inhibitor, or the analogous situation is
seen with the kallikrein inhibitor. In some embodiments, delivery
is such that the reduction in a symptom, or other parameter related
to the disorder is greater than what would be observed with one
treatment delivered in the absence of the other. The effect of the
two treatments can be partially additive, wholly additive, or
greater than additive. The delivery can be such that an effect of
the first treatment delivered is still detectable when the second
is delivered.
[0045] The combination treatment can be used to prevent or treat
disorders associated with blood loss or blood fluidity. For
example, the combination of a kallikrein inhibitor and an
anti-thrombolytic agent can be used to treat or prevent
perioperative blood loss, a systemic inflammatory response (SIR)
induced by kallikrein (especially, for example, in patients
undergoing surgical procedures and particularly surgical procedures
involving cardiothoracic surgery, e.g., cardiopulmonary bypass
(CPB), such as a coronary artery bypass graft (CABG) procedures as
well as in patients with other disorders), cerebral ischemia and/or
reperfusion injury associated with ischemia, e.g., cerebral
ischemia.
[0046] Further examples of applications of the combination
treatment include pediatric cardiac surgery, lung transplantation,
total hip replacement and orthotopic liver transplantation, and to
reduce or prevent perioperative stroke during CABG, extracorporeal
membrane oxygenation (ECMO) and cerebrovascular accidents (CVA)
during these procedures. The combination treatment can also be used
for stroke, e.g., embolism, thrombus and/or hemorrhage associated
stroke and for reperfusion injury associated with stroke.
[0047] Cardiothoracic surgery is surgery of the chest area, most
commonly the heart and lungs. Typical diseases treated by
cardiothoracic surgery include coronary artery disease, tumors and
cancers of the lung, esophagus and chest wall, heart vessel and
valve abnormalities, and birth defects involving the chest or
heart. Where cardiothoracic surgery is utilized for treatment, the
risk of blood loss (and, e.g., surgery-induced ischemia) and the
onset of a systemic inflammatory response (SIR) is incurred.
Surgery-induced SIR can result in severe organ dysfunction
(systemic inflammatory response syndrome; SIRS).
Kunitz Domains
[0048] A number of useful inhibitors of kallikrein include a Kunitz
domain.
[0049] As used herein, a "Kunitz domain" is a polypeptide domain
having at least 51 amino acids and containing at least two, and
preferably three, disulfides. The domain is folded such that the
first and sixth cysteines, the second and fourth, and the third and
fifth cysteines form disulfide bonds (e.g., in a Kunitz domain
having 58 amino acids, cysteines can be present at positions
corresponding to amino acids 5, 14, 30, 38, 51, and 55, according
to the number of the BPTI homologous sequences provided below, and
disulfides can form between the cysteines at position 5 and 55, 14
and 38, and 30 and 51), or, if two disulfides are present, they can
form between a corresponding subset of cysteines thereof. The
spacing between respective cysteines can be within 7, 5, 4, 3, 2, 1
or 0 amino acids of the following spacing between positions
corresponding to: 5 to 55, 14 to 38, and 30 to 51, according to the
numbering of the BPTI sequence provided below. The BPTI sequence
can be used as a reference to refer to specific positions in any
generic Kunitz domain. Comparison of a Kunitz domain of interest to
BPTI can be performed by identifying the best fit alignment in
which the number of aligned cysteines in maximized.
[0050] The 3D structure (at high resolution) of the Kunitz domain
of BPTI is known. One of the X-ray structures is deposited in the
Brookhaven Protein Data Bank as "6PTI". The 3D structure of some
BPTI homologues (Eigenbrot et al., (1990) Protein Engineering,
3(7):591-598; Hynes et al., (1990) Biochemistry, 29:10018-10022)
are known. At least eighty one Kunitz domain sequences are known.
Known human homologues include three Kunitz domains of LACI (Wun et
al., (1988) J. Biol. Chem. 263(13):6001-6004; Girard et al., (1989)
Nature, 338:518-20; Novotny et al, (1989) J. Biol. Chem.,
264(31):18832-18837) two Kunitz domains of Inter-.alpha.-Trypsin
Inhibitor, APP-I (Kido et al., (1988) J. Biol. Chem.,
263(34):18104-18107), a Kunitz domain from collagen, three Kunitz
domains of TFPI-2 (Sprecher et al., (1994) PNAS USA, 91:3353-3357),
the Kunitz domains of hepatocyte growth factor activator inhibitor
type 1, the Kunitz domains of Hepatocyte growth factor activator
inhibitor type 2, the Kunitz domains described in U.S. Patent
Publication No.: 20040152633. LACI is a human serum
phosphoglycoprotein with a molecular weight of 39 kDa (amino acid
sequence in Table 1) containing three Kunitz domains.
TABLE-US-00003 TABLE 1 Exemplary Natural Kunitz Domains LACI: 1
MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit dtelpplklM (SEQ ID 51
HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC NO. 54) 101
KKMCTRDnan riikttlqqe kpdfCfleed pgiCrgyitr yfynnqtkqC 151
erfkyggClg nmnnfetlee CkniCedgpn gfqvdnygtq lnavnnsltp 201
qstkvpslfe fhgpswCltp adrglCrane nrfyynsvig kCrpfkysgC 251
ggnennftsk qeClraCkkg fiqriskggl iktkrkrkkq rvkiayeeif 301 vknm The
signal sequence (1-28) is uppercase and underscored LACI-K1
(50-107) is uppercase LACI-K2 (121-178) is underscored LACI-K3
(211-270) is bold BPTI 1 2 3 4 5 (SEQ ID
1234567890123456789012345678901234567890123456789012345678 NO: 55)
RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA
[0051] The Kunitz domains above are referred to as LACI-K1
(residues 50 to 107), LACI-K2 (residues 121 to 178), and LACI-K3
(213 to 270). The cDNA sequence of LACI is reported in Wun et al.
(J. Biol. Chem., 1988, 263(13):6001-6004). Girard et al. (Nature,
1989, 338:518-20) reports mutational studies in which the P1
residues of each of the three Kunitz domains were altered. LACI-K1
inhibits Factor VIIa (F.VIIa) when F.VIIa is complexed to tissue
factor and LACI-K2 inhibits Factor Xa.
[0052] Proteins containing exemplary Kunitz domains include the
following, with SWISS-PROT Accession Numbers in parentheses:
TABLE-US-00004 A4_HUMAN (P05067), A4_MACFA (P53601), A4_MACMU
(P29216), A4_MOUSE (P12023), A4_RAT (P08592), A4_SAISC (Q95241),
AMBP_PLEPL (P36992), APP2_HUMAN (Q06481), APP2_RAT (P15943),
AXP1_ANTAF (P81547), AXP2_ANTAF (P81548), BPT1_BOVIN (P00974),
BPT2_BOVIN (P04815), CA17_HUMAN (Q02388), CA36_CHICK (P15989),
CA36_HUMAN (P12111), CRPT_BOOMI (P81162), ELAC_MACEU (O62845),
ELAC_TRIVU (Q29143), EPPI_HUMAN (O95925), EPPI_MOUSE (Q9DA01),
HTIB_MANSE (P26227), IBP_CARCR (P00993), IBPC_BOVIN (P00976),
IBPI_TACTR (P16044), IBPS_BOVIN (P00975), ICS3_BOMMO (P07481),
IMAP_DROFU (P11424), IP52_ANESU (P10280), ISC1_BOMMO (P10831),
ISC2_BOMMO (P10832), ISH1_STOHE (P31713), ISH2_STOHE (P81129),
ISIK_HELPO (P00994), ISP2_GALME (P81906), IVB1_BUNFA (P25660),
IVB1_BUNMU (P00987), IVB1_VIPAA (P00991), IVB2_BUNMU (P00989),
IVB2_DABRU (P00990), IVB2_HEMHA (P00985), IVB2_NAJNI (P00986),
IVB3_VIPAA (P00992), IVBB_DENPO (P00983), IVBC_NAJNA (P19859),
IVBC_OPHHA (P82966), IVBE_DENPO (P00984), IVBI_DENAN (P00980),
IVBI_DENPO (P00979), IVBK_DENAN (P00982), IVBK_DENPO (P00981),
IVBT_ERIMA (P24541), IVBT_NAJNA (P20229), MCPI_MELCP (P82968),
SBPI_SARBU (P26228), SPT3_HUMAN (P49223), TKD1_BOVIN (Q28201),
TKD1_SHEEP (Q29428), TXCA_DENAN (P81658), UPTI_PIG (Q29100),
AMBP_BOVIN (P00978), AMBP_HUMAN (P02760), AMBP_MERUN (Q62577),
AMBP_MESAU (Q60559), AMBP_MOUSE (Q07456), AMBP_PIG (P04366),
AMBP_RAT (Q64240), IATR_HORSE (P04365), IATR_SHEEP (P13371),
SPT1_HUMAN (O43278), SPT1_MOUSE (Q9R097), SPT2_HUMAN (O43291),
SPT2_MOUSE (Q9WU03), TFP2_HUMAN (P48307), TFP2_MOUSE (O35536),
TFPI_HUMAN (P10646), TFPI_MACMU (Q28864), TFPI_MOUSE (O54819),
TFPI_RABIT (P19761), TFPI_RAT (Q02445), YN81_CAEEL (Q03610)
[0053] A variety of methods can be used to identify a Kunitz domain
from a sequence database. For example, a known amino acid sequence
of a Kunitz domain, a consensus sequence, or a motif (e.g., the
ProSite Motif) can be searched against the GenBank sequence
databases (National Center for Biotechnology Information, National
Institutes of Health, Bethesda Md.), e.g., using BLAST; against
Pfam database of HMMs (Hidden Markov Models) (e.g., using default
parameters for Pfam searching; against the SMART database; or
against the ProDom database. For example, the Pfam Accession Number
PF00014 of Pfam Release 9 provides numerous Kunitz domains and an
HMM for identify Kunitz domains. A description of the Pfam database
can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and
a detailed description of HMMs can be found, for example, in
Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al.
(1987) Proc. Nat. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994)
J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci.
2:305-314. The SMART database (Simple Modular Architecture Research
Tool, EMBL, Heidelberg, D E) of HMMs as described in Schultz et al.
(1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al.
(2000) Nucl. Acids Res 28:231. The SMART database contains domains
identified by profiling with the hidden Markov models of the HMMer2
search program (R. Durbin et al. (1998) Biological sequence
analysis: probabilistic models of proteins and nucleic acids.
Cambridge University Press). The database also is annotated and
monitored. The Propom protein domain database consists of an
automatic compilation of homologous domains (Corpet et al. (1999),
Nucl. Acids Res. 27:263-267). Current versions of Propom are built
using recursive PSI-BLAST searches (Altschul et al. (1997) Nucleic
Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and
Chemistry 23:333-340.) of the SWISS-PROT 38 and TREMBL protein
databases. The database automatically generates a consensus
sequence for each domain. Prosite lists the Kunitz domain as a
motif and identifies proteins that include a Kunitz domain. See,
e.g., Falquet et al. Nucleic Acids Res. 30:235-238 (2002).
[0054] Kunitz domains interact with target protease using,
primarily, amino acids in two loop regions ("binding loops"). The
first loop region is between about residues corresponding to amino
acids 13-20 of BPTI. The second loop region is between about
residues corresponding to amino acids 31-39 of BPTI. An exemplary
library of Kunitz domains varies one or more amino acid positions
in the first and/or second loop regions. Particularly useful
positions to vary, when screening for Kunitz domains that interact
with kallikrein or when selecting for improved affinity variants,
include: positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 with
respect to the sequence of BPTI. At least some of these positions
are expected to be in close contact with the target protease. It is
also useful to vary other positions, e.g., positions that are
adjacent to the aforementioned positions in the three-dimensional
structure.
[0055] The "framework region" of a Kunitz domain is defined as
those residues that are a part of the Kunitz domain, but
specifically excluding residues in the first and second binding
loops regions, i.e., about residues corresponding to amino acids
13-20 of BPTI and 31-39 of BPTI. Conversely, residues that are not
in the binding loop may tolerate a wider range of amino acid
substitution (e.g., conservative and/or non-conservative
substitutions).
[0056] In one embodiment, these Kunitz domains are variant forms of
the looped structure including Kunitz domain 1 of human
lipoprotein-associated coagulation inhibitor (LACI) protein. LACI
contains three internal, well-defined, peptide loop structures that
are paradigm Kunitz domains (Girard, T. et al., 1989. Nature,
338:518-520). Variants of Kunitz domain 1 of LACI described herein
have been screened, isolated and bind kallikrein with enhanced
affinity and specificity (see, for example, U.S. Pat. Nos.
5,795,865 and 6,057,287, incorporated herein by reference). These
methods can also be applied to other Kunitz domain frameworks to
obtain other Kunitz domains that interact with kallikrein, e.g.,
plasma kallikrein. Useful modulators of kallikrein function
typically bind and/or inhibit kallikrein, as determined using
kallikrein binding and inhibition assays.
[0057] An exemplary polypeptide that includes a Kunitz domain that
inhibits kallikrein has the amino acid sequence defined by amino
acids 3-60 of SEQ ID NO:2.
[0058] An exemplary polypeptide includes the amino acid
sequence:
TABLE-US-00005 (SEQ ID NO: 1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7
Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19
Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys
Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42
Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53
Xaa54 Cys Xaa56 Xaa57 Xaa58
[0059] "Xaa" refers to a position in a peptide chain that can be
any of a number of different amino acids. In a first example, Xaa
can by any amino acid except cysteine. In another example, one or
more of the following apply: Xaa10 can be Asp or Glu; Xaa11 can be
Asp, Gly, Ser, Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg,
His, Asn, Ser, Thr, Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys,
Ala, Ser, Gly, Met, Asn or Gln; Xaa16 can be Ala, Gly, Ser, Asp or
Asn; Xaa17 can be Ala, Asn, Ser, Ile, Gly, Val, Gln or Thr; Xaa18
can be His, Leu, Gln or Ala; Xaa19 can be Pro, Gln, Leu, Asn or
Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile; Xaa31 can be Glu, Asp,
Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32 can be Glu, Gln,
Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 can be Ile,
Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp or Phe;
Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,
Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,
Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and
Xaa54 can be any amino acid.
[0060] Additionally, each of the first four and at last three amino
acids of SEQ ID NO:1 can optionally be present or absent and can be
any amino acid, if present, e.g., any non-cysteine amino acid.
[0061] In one embodiment, the polypeptide has a sequence with one
or more of the following properties: Xaa11 can be Asp, Gly, Ser or
Val; Xaa13 can be Pro, Arg, His or Asn; Xaa15 can be Arg or Lys;
Xaa16 can be Ala or Gly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18
can be His, Leu or Gln; Xaa19 can be Pro, Gln or Leu; Xaa21 can be
Trp or Phe; Xaa31 is Glu; Xaa32 can be Glu or Gln; Xaa34 can be
Ile, Thr or Ser; Xaa35 is Tyr; and Xaa39 can be Glu, Gly or
Ala.
[0062] An exemplary polypeptide can include the following amino
acids: Xaa10 is Asp; Xaa11 is Asp; Xaa13 can be Pro or Arg; Xaa15
is Arg; Xaa16 can be Ala or Gly; Xaa17 is Ala; Xaa18 is His; Xaa19
is Pro; Xaa21 is Trp; Xaa31 is Glu; Xaa32 is Glu; Xaa34 can be Ile
or Ser; Xaa35 is Tyr; and Xaa39 is Gly.
[0063] It is also possible to use portions of the polypeptides
described herein. For example, polypeptides could include binding
domains for specific kallikrein epitopes. For example, the binding
loops of Kunitz domains can by cyclized and used in isolation or
can be grafted onto another domain, e.g., a framework of another
Kunitz domain. It is also possible to remove one, two, three, or
four amino acids from the N-terminus of an amino acid sequence
described herein, and/or one, two, three, four, or five amino acids
from the C-terminus of an amino acid sequence described herein.
[0064] Examples of sequences encompassed by SEQ ID NO:1 are
described by the following (where not indicated, "Xaa" refers to
any amino acid, any non-cysteine amino acid or any amino acid from
the same set of amino acids that are allowed for SEQ ID NO:1):
TABLE-US-00006 (SEQ ID NO: 56) Met His Ser Phe Cys Ala Phe Lys Ala
Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21
Phe Phe Asn Ile Phe Thr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly
Gly Cys Xaa39 Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys
Lys Met Cys Thr Arg Asp. (amino acids 3-60 of SEQ ID NO: 2) Met His
Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys
Met Cys Thr Arg Asp, (SEQ ID NO: 4) Met His Ser Phe Cys Ala Phe Lys
Ala Asp Asp Gly Pro Cys Lys Ala Asn His Leu Arg Phe Phe Phe Asn Ile
Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln
Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,
(SEQ ID NO: 5) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His
Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys
Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 6) Met His
Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr Gly
Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys
Met Cys Thr Arg Asp, (SEQ ID NO: 7) Met His Ser Phe Cys Ala Phe Lys
Ala Asp Asp Gly His Cys Lys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile
Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln
Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,
(SEQ ID NO: 8) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His
Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys
Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser
Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 9) Met His
Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Gly Ala His Leu
Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys
Met Cys Thr Arg Asp, (SEQ ID NO: 10) Met His Ser Phe Cys Ala Phe
Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn
Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn
Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg
Asp, (SEQ ID NO: 11) Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly
Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg
Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe
Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:
12) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala
Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu
Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 13) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala His Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO: 14) Met His Ser Phe Cys Ala Phe Lys Ala
Asp Val Gly Arg Cys Arg Gly Ala Gln Pro Arg Phe Phe Phe Asn Ile Phe
Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn
Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ
ID NO: 15) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Ser Cys
Arg Ala Ala His Leu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu
Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu
Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 16) Met His
Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Ser Cys Arg Ala Ala His Gln
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys
Met Cys Thr Arg Asp, (SEQ ID NO: 17) Met His Ser Phe Cys Ala Phe
Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu Arg Phe Phe Phe Asn
Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn
Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg
Asp, (SEQ ID NO: 18) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp
Gly His Cys Arg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg
Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe
Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:
19) Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly
Asn Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe
Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu
Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 20) Met His Ser Phe
Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg Gly Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys
Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys
Thr Arg Asp, (SEQ ID NO: 21) Met His Ser Phe Cys Ala Phe Lys Ala
Asp Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe Asn Ile Phe
Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn
Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ
ID NO: 22) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys
Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu
Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu
Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.
[0065] Additional examples of sequence include those that differ by
at least one amino acid, but fewer than seven, six, five, four,
three, or two amino acids differences relative to an amino acid
sequence described herein, e.g., an amino acid sequence provided
above. In one embodiment, fewer than three, two, or one differences
are in one of the binding loops. For example, the first binding
loop may have no differences relative to an amino acid sequence
described herein, e.g., an amino acid sequence provided above. In
another example, neither the first nor the second binding loop
differs from an amino acid sequence described herein, e.g., an
amino acid sequence provided above.
[0066] FIGS. 3A and 3B provides an amino acid sequence alignment of
these sequences, the native LACI sequence from which these variants
were derived (SEQ ID NO:32), and other known Kunitz domains (SEQ ID
NOS: 29-31 and 33-53). Still others polypeptides that inhibit
kallikrein include an about 58-amino acid sequence of amino acids
3-60 of SEQ ID NO:2 or the PEP-1 polypeptide having the 60-amino
acid sequence of SEQ ID NO:2. The term "PEP-1" and "DX-88" as used
herein refer to the 60-amino acid sequence of SEQ ID NO:2. A
nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2
is provided in SEQ ID NO:3 (see, e.g., nucleotides 309-488 in FIG.
2). It is understood that based on the known genetic code,
degenerate forms of the nucleotide sequence of SEQ ID NO:3 can be
obtained by simply substituting one or more of the known degenerate
codons for each amino acid encoded by the nucleotide sequence.
Nucleotides 7-180 of SEQ ID NO:3, and degenerate forms thereof,
encode the non-naturally occurring Kunitz domain polypeptide that
includes the 58-amino acid sequence of amino acids 3-60 of SEQ ID
NO:2, a related sequence, or a functional fragment thereof.
[0067] In one embodiment, the polypeptide is other than aprotinin,
e.g., differs from aprotinin, by at least one, two, three, five,
ten, or fifteen amino acids.
[0068] Polypeptides described herein can be made synthetically
using any standard polypeptide synthesis protocol and equipment.
For example, the stepwise synthesis of a polypeptide can be carried
out by the removal of an amino (N) terminal-protecting group from
an initial (i.e., carboxy-terminal) amino acid, and coupling
thereto of the carboxyl end of the next amino acid in the sequence
of the polypeptide. This amino acid is also suitably protected. The
carboxyl group of the incoming amino acid can be activated to react
with the N-terminus of the bound amino acid by formation into a
reactive group such as formation into a carbodiimide, a symmetric
acid anhydride, or an "active ester" group such as
hydroxybenzotriazole or pentafluorophenyl esters. Preferred
solid-phase peptide synthesis methods include the BOC method, which
utilizes tert-butyloxycarbonyl as the I-amino protecting group, and
the FMOC method, which utilizes 9-fluorenylmethloxycarbonyl to
protect the alpha-amino of the amino acid residues. Both methods
are well known to those of skill in the art (Stewart, J. and Young,
J., Solid-Phase Peptide Synthesis (W. H. Freeman Co., San Francisco
1989); Merrifield, J., 1963. Am. Chem. Soc., 85:2149-2154;
Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis
(Springer-Verlag, New York 1984)). If desired, additional amino-
and/or carboxy-terminal amino acids can be designed into the amino
acid sequence and added during polypeptide synthesis.
[0069] Polypeptides can also be produced using recombinant
technology. Recombinant methods can employ any of a number of cells
and corresponding expression vectors, including but not limited to
bacterial expression vectors, yeast expression vectors, baculovirus
expression vectors, mammalian viral expression vectors, and the
like. A polypeptide described herein can be produced by a
transgenic animal, e.g., in the mammary gland of a transgenic
animal. In some cases, it could be necessary or advantageous to
fuse the coding sequence for a polypeptide that inhibits kallikrein
(e.g., a polypeptide that includes a Kunitz domain) to another
coding sequence in an expression vector to form a fusion
polypeptide that is readily expressed in a host cell. Part or all
of the additional sequence can be removed, e.g., by protease
digestion.
[0070] An exemplary recombinant expression system for producing a
polypeptide that inhibits kallikrein (e.g., a polypeptide that
includes a Kunitz domain) is a yeast expression vector, which
permits a nucleic acid sequence encoding the amino acid sequence
for the inhibitor polypeptide to be linked in the same reading
frame with a nucleotide sequence encoding the MAT.alpha. prepro
leader peptide sequence of Saccharomyces cerevisiae, which in turn
is under the control of an operable yeast promoter. The resulting
recombinant yeast expression plasmid can be transformed by standard
methods into the cells of an appropriate, compatible yeast host,
which cells are able to express the recombinant protein from the
recombinant yeast expression vector. Preferably, a host yeast cell
transformed with such a recombinant expression vector is also able
to process the fusion protein to provide an active inhibitor
polypeptide. An other exemplary yeast host for producing
recombinant polypeptides is Pichia pastoris.
[0071] As noted above, polypeptides that inhibit kallikrein can
include a Kunitz domain polypeptide described herein. Some
polypeptides can include an additional flanking sequence,
preferably of one to six amino acids in length, at the amino and/or
carboxy-terminal end, provided such additional amino acids do not
significantly diminish kallikrein binding affinity or kallikrein
inhibition activity so as to preclude use in the methods and
compositions described herein. Such additional amino acids can be
deliberately added to express a polypeptide in a particular
recombinant host cell or can be added to provide an additional
function, e.g., to provide a linker to another molecule or to
provide an affinity moiety that facilitates purification of the
polypeptide. Preferably, the additional amino acid(s) do not
include cysteine, which could interfere with the disulfide bonds of
the Kunitz domain.
[0072] An exemplary Kunitz domain polypeptide includes the amino
acid sequence of residues 3-60 of SEQ ID NO:2. When expressed and
processed in a yeast fusion protein expression system (e.g., based
on the integrating expression plasmid pHIL-D2), such a Kunitz
domain polypeptide retains an additional amino terminal Glu-Ala
dipeptide from the fusion with the MATalpha-prepro leader peptide
sequence of S. cerevisiae. When secreted from the yeast host cell,
most of the leader peptide is processed from the fusion protein to
yield a functional polypeptide (referred to herein as "PEP-1")
having the amino acid sequence of SEQ ID NO:2 (see boxed region in
FIG. 2).
[0073] In one embodiment, an inhibitor of kallikrein, e.g., a
polypeptide inhibitor, has a binding affinity for kallikrein that
is on the order of 1000 times higher than that of aprotinin, which
is currently approved for use in CABG procedures to reduce blood
loss. The surprisingly high binding affinities of such kallikrein
inhibitors combined with their high degree of specificity for
kallikrein to the exclusion of other molecular targets (see Table
1, below) provide for particularly useful inhibitors. However,
inhibitors with lesser affinity or specificity also have their
applications.
[0074] A typical Kunitz domain, e.g., that includes, SEQ ID NO:1,
contains a number of invariant positions, e.g., positions
corresponding to position 5, 14, 30, 33, 38, 45, 51 and 55 in the
BPTI numbering scheme are cysteine. The spacing between these
positions may vary to the extent allowable within the Kunitz domain
fold, e.g., such that three disulfide bonds are formed. Other
positions such as, for example, positions 6, 7, 8, 9, 20, 24, 25,
26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53 and 54, or
positions corresponding to those positions, can be any amino acid
(including non-genetically encoded occurring amino acids). In a
particularly preferred embodiment, one or more amino acids
correspond to that of a native sequence (e.g., SEQ ID NO:32, see
FIG. 3). In another embodiment, at least one variable position is
different from that of the native sequence. In yet another
preferred embodiment, the amino acids can each be individually or
collectively substituted by a conservative or non-conservative
amino acid substitution.
[0075] Conservative amino acid substitutions replace an amino acid
with another amino acid of similar chemical nature and may have no
affect on protein function. Non-conservative amino acid
substitutions replace an amino acid with another amino acid of
dissimilar chemical structure. Examples of conserved amino acid
substitutions include, for example, Asn->Gln, Arg->Lys and
Ser->Thr. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 of these amino
acids can be independently or collectively, in any combination,
selected to correspond to the corresponding position of SEQ ID
NO:2.
[0076] Other positions, for example, positions 10, 11, 13, 15, 16,
17, 18, 19, 21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, or
positions corresponding to those positions can be any of a selected
set of amino acids. For example, SEQ ID NO:1 defines a set of
possible sequences. Each member of this set contains, for example,
a cysteine at positions 5, 14, 30, 51 and 55, and any one of a
specific set of amino acids at positions 10, 11, 13, 15, 16, 17,
18, 19, 21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, or positions
corresponding to those positions. In a preferred embodiment, 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and/or 19
of these amino acids can be independently or collectively, in any
combination, selected to correspond to the corresponding position
of SEQ ID NO:2. The polypeptide preferably has at least 80%, 85%,
90%, 95, 97, 98, or 99% identity to SEQ ID NO:2.
[0077] As used herein, the term "substantially identical" (or
"substantially homologous") is used herein to refer to a first
amino acid or nucleotide sequence that contains a sufficient number
of identical or equivalent (e.g., with a similar side chain, e.g.,
conserved amino acid substitutions) amino acid residues or
nucleotides to a second amino acid or nucleotide sequence such that
the first and second amino acid or nucleotide sequences have
similar activities. In the case of antibodies, the second antibody
has the same specificity and has at least 50% of the affinity of
the same.
[0078] Calculations of "homology" between two sequences can be
performed as follows. The sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in one or both of
a first and a second amino acid or nucleic acid sequence for
optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). In a preferred embodiment, the length of
a reference sequence aligned for comparison purposes is at least
30%, preferably at least 40%, more preferably at least 50%, even
more preferably at least 60%, and even more preferably at least
70%, 80%, 90%, 100% of the length of the reference sequence. The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0079] The comparison of sequences and determination of percent
homology between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
homology between two amino acid sequences is determined using the
Needleman and Wunsch (1970), J. Mol. Biol. 48:444-453, algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent homology between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of parameters (and the one that should be used if the
practitioner is uncertain about what parameters should be applied
to determine if a molecule is within a homology limitation) are a
Blossum 62 scoring matrix with a gap penalty of 12, a gap extend
penalty of 4, and a frameshift gap penalty of 5.
[0080] Useful polypeptides can also be encoded by a nucleic acid
that hybridizes to a nucleic acid that encodes a polypeptide
described herein. The nucleic acids can hybridize under medium,
high, or very high stringency conditions. As used herein, the term
"hybridizes under low stringency, medium stringency, high
stringency, or very high stringency conditions" describes
conditions for hybridization and washing. Guidance for performing
hybridization reactions can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6,
which is incorporated by reference. Aqueous and nonaqueous methods
are described in that reference and either can be used. Specific
hybridization conditions referred to herein are as follows: (1) low
stringency hybridization conditions in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
two washes in 0.2.times.SSC, 0.1% SDS at least at 50.degree. C.
(the temperature of the washes can be increased to 55.degree. C.
for low stringency conditions); (2) medium stringency hybridization
conditions in 6.times.SSC at about 45.degree. C., followed by one
or more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; (3)
high stringency hybridization conditions in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C.; and (4) very high stringency
hybridization conditions are 0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C.
Modifications
[0081] It is possible to modify polypeptides that inhibit a Kunitz
domain in a variety of ways. For example, the polypeptides can be
attached to one or more polyethylene glycol moieties to stabilize
the compound or prolong retention times, e.g., by at least 2, 4, 5,
8, 10, 15, 20, 50, 100, 500 or 1000 fold.
[0082] A polypeptide that inhibits kallikrein can be associated
with (e.g., conjugated to) a polymer, e.g., a substantially
non-antigenic polymers, such as polyalkylene oxides or polyethylene
oxides. Suitable polymers will vary substantially by weight.
Polymers having molecular number average weights ranging from about
200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 to
about 12,500) can be used. A plurality of polymer moieties can be
attached to one polypeptide, e.g., at least two, three, or four
such moieties, e.g., having an average molecular weight of about
2,000 to 7,000 Daltons.
[0083] For example, the polypeptide can be conjugated to a water
soluble polymer, e.g., hydrophilic polyvinyl polymers, e.g.
polyvinylalcohol and polyvinylpyrrolidone. A non-limiting list of
such polymers include polyalkylene oxide homopolymers such as
polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers
thereof, provided that the water solubility of the block copolymers
is maintained. Additional useful polymers include polyoxyalkylenes
such as polyoxyethylene, polyoxypropylene, and block copolymers of
polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched
polysaccharides which comprise the saccharide monomers D-mannose,
D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,
D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g. polymannuronic acid, or alginic acid), D-glucosamine,
D-galactosamine, D-glucose and neuraminic acid including
homopolysaccharides and heteropolysaccharides such as lactose,
amylopectin, starch, hydroxyethyl starch, amylose, dextrane
sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit
of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of
sugar alcohols such as polysorbitol and polymannitol; heparin or
heparon.
[0084] Other compounds can also be attached to the same polymer,
e.g., a cytotoxin, a label, or another targeting agent or an
unrelated agent. Mono-activated, alkoxy-terminated polyalkylene
oxides (PAO's), e.g., monomethoxy-terminated polyethylene glycols
(mPEG's); C.sub.1-4 alkyl-terminated polymers; and bis-activated
polyethylene oxides (glycols) can be used for crosslinking. See,
e.g., U.S. Pat. No. 5,951,974.
Anti-Thrombolytic Agents
[0085] Anti-thrombolytic agents are agents that reduce or prevent
dissolution of a blood clot, stabilize a blood clot, increase
clotting and/or prevent abnormal amounts of blood loss such as
hemorrhaging by maintaining, stabilizing or increasing a blood
clot. Preferably, the anti-thrombolytic agent is an
anti-fibrinolytic agent. Anti-fibrinolytic agents are agents that
prevent or reduce the dissolution or breakdown of fibrin. Examples
of anti-fibrinolytic agents include tranexamic acid
(Cyklokapron.TM.), epsilon amino caproic acid (Amicar.TM.),
aprotinin (Trasyol.TM.), Desmopressin (DDAVP), pirfenidone, and
combinations thereof. The anti-fibrinolytic activity of an agent
may be determined by any in vitro clot lysis activity known in the
art, such as the purified clot lysis assay described by Carlson, et
al., Anal. Biochem. 168, 428-435 (1988) and its modified form
described by Bennett, W. F. et al., 1991, supra, the entire
contents of which are hereby incorporated by reference.
Methods and Compositions
[0086] The kallikrein inhibitors and the anti-thrombolytic agents
described herein can be used in methods for preventing or reducing
blood loss, e.g., perioperative blood loss; methods for preventing
or reducing injury associated with ischemia (e.g., reperfusion
injury associated with ischemia); and/or a systemic inflammatory
response (SIR) in a patient, especially associated with surgery.
The surgery can be, e.g., a cardiothoracic surgery, (e.g.,
cardiopulmonary bypass or coronary artery bypass grafting);
orthopedic surgery (e.g., hip or knee replacement or bone
fracture); liver surgery; kidney surgery; procedures that utilize
extracorporeal circulation or dialysis; and any other procedure
which can result in perioperative blood loss. The method includes
administering a non-naturally occurring inhibitor of kallikrein,
e.g., plasma kallikrein, in combination with an anti-thrombolytic
agent, e.g., an anti-fibrinolytic agent.
[0087] In one embodiment, a method for treatment includes
administration of a non-naturally occurring polypeptide comprising
a Kunitz domain as the inhibitor of kallikrein. One embodiment of
the method uses a polypeptide containing an amino acid sequence of
SEQ ID NO:1 that has an affinity for kallikrein that is
approximately 30-fold or more higher than that of a broad range
serine protease, e.g., aprotinin, which is isolated from bovine
lung and currently approved for use in CABG procedures
(TRASYLOL.TM., Bayer Corporation Pharmaceutical Division, West
Haven, Conn.).
[0088] Patients subjected to any of a number of surgical
procedures, especially those involving extra-corporeal circulation,
e.g., cardiothoracic surgery, such as, for example, CPB, and/or
bone trauma, such as sternal split or hip replacement, are at risk
for perioperative blood loss and inflammation. Contact of a
patient's blood with the cut surfaces of bone or of CPB equipment
is sufficient to activate one or several undesirable cascade
responses, including a contact activation system (CAS), which can
lead to extensive perioperative blood loss requiring immediate
blood transfusion, as well as a systemic inflammatory response
(SIR), which, in turn, can result in permanent damage to tissues
and organs. While not desiring to be limited to any particular
mechanism or theory, it appears that the blood loss that occurs
associated with cardiothoracic surgery, e.g., CPB, as in a CABG
procedure, probably results from extensive capillary leakage, which
can result in significant loss of blood that must be replaced by
immediate blood transfusion.
[0089] The combination treatment described herein can be used to
prevent or reduce perioperative blood loss as well as various
ischemias and SIR in a patient subjected to a surgical procedure,
and especially wherein the surgical procedure requires
extra-corporeal circulation, e.g., cardiothoracic surgery, such as,
for example, CPB. The combination treatment can be particularly
useful for preventing or reducing perioperative blood loss and/or
SIR in a patient subjected to a CABG procedure requiring CPB or
other cardiac surgery. Further, the combination treatment described
herein can be used to prevent or reduce cerebral ischemia (such as
stroke) and/or reperfusion injury associated with cerebral ischemia
(e.g., stroke).
[0090] Exemplary compositions for medical use comprise a kallikrein
inhibitor described herein, an anti-thrombolytic agent described
herein or both a kallikrein inhibitor described herein and an
anti-thrombolytic agent described herein. Such compositions can
further include one or more pharmaceutically acceptable buffers,
carriers, and excipients, which can provide a desirable feature to
the composition including, but not limited to, enhanced
administration of the composition to a patient, enhanced
circulating half-life of the inhibitor and/or anti-thrombolytic
agent, enhanced compatibility of the inhibitor and/or
anti-thrombolytic agent with patient blood chemistry, enhanced
storage of the composition, and/or enhanced delivery and/or
efficacy of the inhibitor and/or anti-thrombolytic agent upon
administration to a patient. In addition to a kallikrein inhibitor
and/or anti-thrombolytic agent described herein, compositions can
further include one or more other pharmaceutically active compounds
that provide an additional prophylactic or therapeutic benefit to a
patient, e.g., a patient of an invasive surgical procedure or a
patent otherwise at risk for, having or previously had cerebral
ischemia and/or reperfusion injury associated with cerebral
ischemia. For example, the compositions can include another
compound described herein.
[0091] Perioperative Blood Loss and Reduced Heart Bloodflow
[0092] Due to the many advances in medicine, a number of highly
invasive surgical procedures are carried out each day that result
in blood loss, or place patients at a high risk for blood loss.
Such patients are generally carefully monitored to restore and
maintain normal blood supply and hemostasis, and they may need
blood transfusions. Surgical procedures that involve blood loss
include those involving extra-corporeal circulation methods such as
cardiothoracic surgery, e.g., CPB. In such methods, a patient's
heart is stopped and the circulation, oxygenation, and maintenance
of blood volume are carried out artificially using an
extra-corporeal circuit and a synthetic membrane oxygenator. These
techniques are commonly used during cardiac surgery. Additionally,
it is apparent that surgery involving extensive trauma to bone,
such as the sternal split necessary in CABG or hip replacement
procedures, is also associated with activation of the CAS, which
can result in a variety of disruptions in the blood and
vasculature.
[0093] Atherosclerotic coronary artery disease (CAD) causes a
narrowing of the lumen of one or several of the coronary arteries;
this limits the flow of blood to the myocardium (i.e., the heart
muscle) and can cause angina, heart failure, and myocardial
infarcts. In the end stage of coronary artery atherosclerosis, the
coronary circulation can be almost completely occluded, causing
life threatening angina or heart failure, with a very high
mortality. CABG procedures may be required to bridge the occluded
blood vessel and restore blood to the heart; these are potentially
life saving. CABG procedures are among the most invasive of
surgeries in which one or more healthy veins or arteries are
implanted to provide a "bypass" around the occluded area of the
diseased vessel. CABG procedures carry with them a small but
important perioperative risk, but they are very successful in
providing patients with immediate relief from the mortality and
morbidity of atherosclerotic cardiovascular disease. Despite these
very encouraging results, repeat CABG procedures are frequently
necessary, as indicated by an increase in the number of patients
who eventually undergo second and even third procedures; the
perioperative mortality and morbidity seen in primary CABG
procedures is increased in these re-do procedures.
[0094] There have been improvements in minimally invasive surgical
techniques for uncomplicated CAD. However, nearly all CABG
procedures performed for valvular and/or congenital heart disease,
heart transplantation, and major aortic procedures, are still
carried out on patients supported by CPB. In CPB, large cannulae
are inserted into the great vessels of a patient to permit
mechanical pumping and oxygenation of the blood using a membrane
oxygenator. The blood is returned to the patient without flowing
through the lungs, which are hypoperfused during this procedure.
The heart is stopped using a cardioplegic solution, the patient
cooled to help prevent brain damage, and the peripheral circulating
volume increased by an extracorporeal circuit, i.e., the CPB
circuit, which requires "priming" with donor blood and saline
mixtures are used to fill the extracorporeal circuit. CPB has been
extensively used in a variety of procedures performed for nearly
half a century with successful outcomes. The interaction between
artificial surfaces, blood cells, blood proteins, damaged vascular
endothelium, and extravascular tissues, such as bone, disturbs
hemostasis and frequently activates the CAS, which, as noted above,
can result in a variety of disruptions in the blood and
vasculature. Such disruption leads to excess perioperative
bleeding, which then requires immediate blood transfusion. A
consequence of circulating whole blood through an extracorporeal
circuit in CPB can also include the systemic inflammatory response
(SIR), which is initiated by contact activation of the coagulation
and complement systems. Indeed, much of the morbidity and mortality
associated with seemingly mechanically successful CPB surgical
procedures is the result of the effects of activating coagulation,
fibrinolysis, or complement systems. Such activation can damage the
pulmonary system, leading to adult respiratory distress syndrome
(ARDS), impairment of kidney and splanchnic circulation, and
induction of a general coagulopathy leading to blood loss and the
need for transfusions. In addition to the dangers of perioperative
blood loss, additional pathologies associated with SIR include
neurocognitive deficits, stroke, renal failure, acute myocardial
infarct, and cardiac tissue damage.
[0095] Blood transfusions also present a significant risk of
infection and elevate the cost of CABG or other similar procedures
that require CPB. In the absence of any pharmacological
intervention, three to seven units of blood must typically be
expended on a patient, even with excellent surgical techniques.
Accordingly, there is considerable incentive for the development of
new and improved pharmacologically effective compounds and
treatment protocols to reduce or prevent perioperative bleeding and
SIR in patients subjected to CPB and CABG procedures. Use of the
inhibitors described herein in combination with various
anti-thrombolytic agents (e.g., anti-fibrinolytic agents) can
improve these various treatments and lead to reduction and/or
amelioration of the undesirable symptoms that can occur.
[0096] Cerebral Ischemia and Reperfusion Injury
[0097] The methods described herein are useful for reducing or
preventing cerebral ischemia as well as reperfusion injury
associated with cerebral ischemia. A "cerebral ischemic attack" or
"cerebral ischemia" is an ischemic condition in which blood supply
to the brain is blocked. This interruption in the blood supply to
the brain may result from a variety of causes including, but not
limited to, an intrinsic blockage or occlusion of the blood vessel
itself, a remotely originated source of occlusion, decreased
perfusion pressure or increased blood viscosity resulting in
decreased cerebral blood flow, or ruptured or leaky blood vessels
in the subarachnoid space or intracerebral tissue. Cerebral
ischemia may result in either transient or permanent deficits and
the seriousness of the neurological damage in a patient who has
experienced cerebral ischemia depends on the intensity and duration
of the ischemia event. A transient ischemia attack (TIA) is one in
which the blood flow to the brain is briefly interrupted and causes
temporary neurological deficits. Symptoms of TIA include numbness
of weakness of face or limbs, loss of ability to speak clearly
and/or understand the speech of others, a loss of vision or dimness
of vision and dizziness. Permanent cerebral ischemia attacks, also
called strokes, are caused by a longer interruption in blood flow
to the brain resulting from an embolism, a thrombus or bleeding in
the brain (e.g., a hemorrhage). The term "thromboembolic stroke" or
"thromboembolism" is used herein to refer to a stroke caused by
either a thrombosis or an embolism. A stroke causes a loss of
neurons typically resulting in a neurological deficit that may
improve but does not entirely resolve. The combination treatments
described herein are useful in preventing or reducing stroke
including embolic-, thrombolic-, thromboembolic- and
hemorrhage-associated strokes. Strokes can be caused by a variety
of causes. One category includes perioperative strokes that can be
associated with thrombus or embolism formation.
[0098] In stroke patients, there is a core of the neurological
deficit marked by total ischemia and/or tissue necrosis. This area
is normally surrounded by ischemic tissue, referred to as the
ischemic penumbra, that receives collateral circulation. Ischemia
in the penumbra does not always result in irreversible damage. In
some cases, restoration of blood flow (reperfusion) into the
penumbra may prevent total ischemia and necrosis in this area.
However, reperfusion has also been associated with injury to the
tissue surrounding the core. Once blood flow is returned, blood
cells such as neutrophils, attack the damaged tissue which can
cause additional inflammation and/or damage. Reperfusion injury is
associated with an influx of neutrophils into the affected tissue
and subsequent activation of the neutrophils. Neutrophils can
release lytic enzymes that directly induce tissue damage and
proinflammatory mediators such as cytokines that amplify local
inflammatory reaction. The influx of neutrophils to a site of
ischemic damage can also plug capillaries and cause
vasoconstriction. It has been found that kallikrein plays a role in
neutrophil chemotaxis, neutrophil activation and reperfusion
injury. Thus, the kallikrein inhibitors described herein can be
used to prevent or reduce reperfusion injury, e.g., by reducing or
preventing one or more of: 1) neutrophil infiltration, 2)
neutrophil activation; 3) cytokine release; 4) elastase release;
and 5) vasodilation. For example, a kallikrein inhibitor can be
used to inhibit bradykinin and Factor XII. The kallikrein
inhibitors can be used in combination with one or more
anti-thrombolytic agent, e.g., one or more anti-thrombolytic agent
described herein.
Administration
[0099] A kallikrein inhibitor and/or anti-thrombolytic agent can be
administered to a patient before, during, and/or after an event
that causes or is associated with blood loss, e.g., a surgical
procedure, or an ischemic event, e.g., a cerebral ischemic attack,
in a pharmaceutically acceptable composition or in connection with
another disorder or event described herein. The patient is
generally a human, but may also be a non-human mammal. Human
patients include adults, e.g., patients between ages 19-25, 26-40,
41-55, 56-75, and 76 and older, and pediatric patients, e.g.,
patients between ages 0-2, 3-6, 7-12, and 13-18.
[0100] The term "pharmaceutically acceptable" composition refers to
a non-toxic carrier or excipient that may be administered to a
patient, together with a kallikrein inhibitor and/or
anti-thrombolytic agent described herein. The carrier or excipient
is chosen to be compatible with the biological or pharmacological
activity of the composition. The inhibitors and/or
anti-thrombolytic agents described herein can be administered
locally or systemically by any suitable means for delivery of an
inhibitory amount of the inhibitor and/or anti-thrombolytic agent
to a patient including but not limited to systemic administrations
such as, for example, intravenous and inhalation. Parenteral
administration is particularly preferred.
[0101] For parenteral administration, the kallikrein inhibitor
and/or the anti-thrombolytic agent can be injected intravenously,
intramuscularly, intraperitoneally, or subcutaneously. Intravenous
administration is preferred. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Other pharmaceutically acceptable carriers include,
but are not limited to, sterile water, saline solution, and
buffered saline (including buffers like phosphate or acetate),
alcohol, vegetable oils, polyethylene glycols, gelatin, lactose,
amylose, magnesium stearate, talc, silicic acid, paraffin, etc.
Where necessary, the composition can also include a solubilizing
agent and a local anaesthetic such as lidocaine to ease pain at the
site of the injection, preservatives, stabilizers, wetting agents,
emulsifiers, salts, lubricants, etc. as long as they do not react
deleteriously with the active compounds. Similarly, the composition
can comprise conventional excipients, e.g., pharmaceutically
acceptable organic or inorganic carrier substances suitable for
parenteral, enteral or intranasal application which do not
deleteriously react with the active compounds. Generally, the
ingredients will be 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 in
activity units. Where the composition is to be administered by
infusion, it can be dispensed with an infusion bottle containing
sterile pharmaceutical grade "water for injection" or saline. Where
the composition is to be administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients can be mixed prior to administration.
[0102] In one embodiment, the kallikrein inhibitor and/or
anti-thrombolytic agent is administered to a patient as an
intravenous infusion according to any approved procedure. For
example, a non-naturally occurring kallikrein inhibitor described
herein and an anti-thrombolytic agent (e.g., an anti-fibrinolytic
agent) can be administered to a patient subjected to a CABG
procedure at the times similar to those currently used in approved
protocols for administering aprotinin and in an amount necessary to
provide a patient with a required number or concentration of
kallikrein inhibitory units (KIU). In another embodiment, each of
the non-naturally occurring kallikrein inhibitor and the
anti-thrombolytic agent, e.g., anti-fibrinolytic agent, is
administered in an amount necessary to provide a patient with a
required number or concentration of kallikrein inhibitory units
(KIU).
[0103] A kallikrein inhibitor and/or anti-thrombolytic agent
described herein can also be administered to a patient in the
immediate postoperative period, when bleeding abnormalities can
occur as a consequence of downstream effects of SIR. For example,
in a procedure involving CPB, an inhibitor and/or anti-thrombolytic
agent described herein can be administered to a patient as an
initial loading dose, e.g., an effective amount over the course of
a convenient time, such as 10 minutes, prior to induction of
anesthesia. Then, at induction of anesthesia, a second dose of the
inhibitor and/or anti-thrombolytic agent can be injected into the
CPB priming fluid ("pump prime volume"). The patient can then be
placed on a continuous and controlled intravenous infusion dose for
the duration of the surgical procedure, and after the procedure if
indicated.
[0104] In other embodiments, a kallikrein inhibitor and/or
anti-thrombolytic agent can be administered after an ischemic
event, e.g., after a stroke, e.g., 5, 10, 15, 30, 45 minutes, 1, 2,
3, 5, 10, 15, 20 hours or more after a stroke. Preferably, the
inhibitor and/or anti-thrombolytic agent is administered within 12
to 60 hours, e.g., within 24 to 48 hours, after a stroke. In some
embodiments, a kallikrein inhibitor and/or anti-thrombolytic agent
is administered after an ischemic event, e.g., after a stroke, but
prior to reperfusion of the damaged tissue. In other embodiments, a
kallikrein inhibitor and/or anti-thrombolytic agent is administered
during reperfusion or after reperfusion has begun. In yet another
embodiment, a kallikrein inhibitor and/or anti-thrombolytic agent
is administered after reperfusion has occurred. An "effective"
amount in this context is an amount sufficient to reduce one or
more symptoms associated with cerebral ischemia and/or reperfusion
injury associated with cerebral ischemia which otherwise would have
occurred in a subject experiencing a cerebral ischemia and/or
reperfusion injury associated with cerebral ischemia absent the
treatment. Several physiological parameters may be used to assess
stroke and reperfusion injury associated with stroke including
infarct size, regional cerebral blood flow, intracranial pressure,
anterograde amnesia, retrograde amnesia, dementia, cognitive
function and/or emotion, and cerebral edema, for example, as
compared to pretreatment patient parameters, untreated stroke
patients or stroke patients treated with the other therapeutic
agent but not the combination with the inhibitor (e.g., the Kunitz
domain polypeptide or other compound described herein) or visa
versa.
[0105] Parameters that can be evaluated for determining a dose of
the kallikrein inhibitor, the anti-thrombolytic agent, or both are
described below with regards to DX-88 (a non-naturally occurring
kallikrein inhibitor) and aprotinin (an anti-fibrinolytic agent).
By determining information regarding, for example, the KIU and
binding specificity, an appropriate dose of each of the kallikrein
inhibitor and the anti-thrombolytic agent can be determined for the
desired therapeutic or prophylactic effect.
[0106] With respect to an implementation in which DX-88 or a
DX-88-related inhibitor is used, the affinity constant (Ki) of
DX-88 is at least about 1000 times greater than aprotinin for
kallikrein inhibition. Accordingly, a dose of DX-88 or an inhibitor
of similar affinity can be, e.g., at least about 5, 10, 15, 20, 30,
50, 100, 500 or 1000 times lower than aprotinin on a mole per mole
basis. The dose could also be modulated as a function of the amount
of kallikrein activated during an event (e.g., CPB), the
specificity of the DX-88-kallikrein interaction in vivo, the
concentration of kallikrein eliciting SIRS, and pharmacological
distribution. In one aspect, the dose of DX-88 or an inhibitor of
similar affinity administered in combination with an
anti-fibrinolytic agent such as aprotinin can be adjusted such that
the KIU for the combination is the same as it would be if only
DX-88 or only aprotinin were administered. In other embodiments,
each of DX-88 (or an inhibitor of similar affinity) and the
anti-fibrinolytic agent, e.g., aprotinin, is administered at a dose
the same or similar to that given in the absence of the other
agent. Similar adjustments can be made for other anti-thrombolytic
agents, e.g., anti-fibrinolytic agents, described herein.
[0107] The total amount of circulating prekallikrein in plasma is
reported to be approximately 500 nM to 600 nM. Silverberg, M. et
al., "The Contact System and Its Disorders," in Blood: Principles
and Practice of Hematology, Handin, R. et al., eds, J B Lippincott
Co., Philadelphia, 1995). If all prekallikrein is activated, about
520 nmoles/L of DX-88 can be used to inhibit kallikrein in a
stoichiometric manner. An individual having 5 L of plasma would
require a dose of 2.6 micromoles DX-88, or approximately 18 mg
based on the molecular weight of DX-88 of 7,054 Daltons. This was
calculated as follows: the K.sub.i of DX88 is 0.044 nM. When it is
desired to have a concentration of plasma kallikrein (PK) of, e.g.,
1 nM, the formula K.sub.i=0.044
nM=[DX88].times.[PK]/[DX88-PK]=[DX88].times.1 nm/499 nM, indicates
that the concentration of free DX-88 is 22.0 nM. Thus, the total
amount of DX-88 needed would be 499+22 or 521 nM. The dose can be
reduced proportionally if not all of the prekallikrein is activated
or if a portion of the kallikrein is deactivated by an endogenous
inhibitor, e.g., C1 esterase inhibitor (C1INH). Thus, in certain
embodiments, about 5, 10, 15, 20, 30, 40, 60, 80, 120, 250, 500,
600, 700, 800, 1000 mg of DX-88 can be administered to a subject,
e.g., over a twenty-four hour period. In other embodiments, less
than 5, 10, 15, 20, 30, 40, 60, 80, 120, 250, 500, 600, 700, 80,
1000 mg of DX-88 can be administered, e.g., over a twenty-four hour
period, such that the combination of DX-88 with an
anti-thrombolytic agent has a similar (or better) effect on one or
more symptom of the disorder than if DX-88 were administered
alone.
[0108] As the concentration of active kallikrein may have to rise
above a certain level to contribute to increased fluid and blood
loss post-operatively, in many cases, it is not necessary to
inactivate all active kallikrein. DX-88 would be expected to be
effective at a significantly lower dose compared to aprotinin on
the basis of its higher affinity for kallikrein. Using the same
calculations described above, if all prekallikrein is activated,
about 15,469 nmoles/L of aprotinin can be used to inhibit
kallikrein in a stoichiometric manner. Therefore, an individual
having 5 L of plasma would require a dose of 77.5 micromoles
aprotinin, or approximately 542 mg.
[0109] DX-88 also has greater specificity for kallikrein inhibition
compared to aprotinin in vitro. Therefore, proteases other than
kallikrein that are inhibited by aprotinin may lower the effective
concentration of the inhibitor, thereby increasing the amount of
aprotinin needed for a therapeutic effect and leading to unwanted
side effects.
[0110] Currently there are two regimens approved in the United
States for administering aprotinin to a patient undergoing a CABG
procedure (see, product label and insert for TRASYLOL.TM., Bayer
Corporation Pharmaceutical Division, West Haven, Conn., the
contents of which are incorporated herein).
[0111] Several considerations regarding dosing with a polypeptide
inhibitor of kallikrein can be illustrated by way of example with
the representative DX-88 polypeptide.
[0112] Table 1, below, provides a comparison of the affinity
(Ki,app) of the DX-88 polypeptide for kallikrein and eleven other
known plasma proteases.
TABLE-US-00007 TABLE 1 Protease Substrate DX-88 K.sub.i, app (pM)
Aprotinin K.sub.i, app (pM) human plasma kallikrein 44 3.0 .times.
10.sup.4 human urine kallikrein >1 .times. 10.sup.8 4.0 .times.
10.sup.3 porcine pancreatic 2.7 .times. 10.sup.7 550 kallikrein
human C1r, activated >2.0 .times. 10.sup.8 >1.0 .times.
10.sup.7 human C1s, activated >2.0 .times. 10.sup.7 >1.0
.times. 10.sup.8 human plasma factor XIa 1.0 .times. 10.sup.4 ND
human plasma factor XIIa >2.0 .times. 10.sup.7 >1.0 .times.
10.sup.8 human plasmin 1.4 .times. 10.sup.5 894 human pancreatic
trypsin >2 .times. 10.sup.7 ND human pancreatic >2.0 .times.
10.sup.7 7.3 .times. 10.sup.5 chymotrypsin human neutrophil
elastase >2.0 .times. 10.sup.7 1.7 .times. 10.sup.6 human plasma
thrombin >2.0 .times. 10.sup.7 >1.0 .times. 10.sup.8 ND = not
determined
[0113] Clearly, the DX-88 polypeptide is highly specific for human
plasma kallikrein. Furthermore, the affinity (K.sub.i,app) of DX-88
for kallikrein is 700 times higher than the affinity of aprotinin
for kallikrein: the K.sub.i,app of DX-88 for kallikrein is about 44
pM (Table 1), whereas the K.sub.i,app of aprotinin for kallikrein
is 30,000 pM. Thus, a dose of DX-88 could be lower than that used
for aprotinin on a per mole basis. Using this information, the dose
of each of DX-88 and aprotinin can be determined.
[0114] Consideration of several other factors may provide a more
accurate estimation of the dose of DX-88 required in practice. Such
factors include the amount of kallikrein activated during CPB in a
particular patient, the concentration of kallikrein required to
elicit an SIR, the bioavailability and pharmacological distribution
of DX-88 in a patient and the effect of C-1 esterase inhibitor on
endogenous plasma kallikrein inhibition. Nevertheless, use of a
polypeptide that includes a Kunitz domain that inhibits kallikrein
in doses currently approved for the use of aprotinin is still
expected to provide significant improvements over the current use
of the less specific, lower affinity, bovine aprotinin.
Accordingly, lower doses, e.g., at least half, or a tenth of the
approved aprotinin dose may be used for a kallikrein inhibitor
which inhibits kallikrein at least 2, 5, 10, 20, 30, 50 or 100 fold
better than aprotinin.
[0115] Another factor to consider is the threshold concentration of
kallikrein required to induce a SIR in a patient. If the
concentration of active kallikrein must be maintained below, e.g.,
1 nM, then owing to its high affinity for kallikrein, DX-88 offers
a significant advantage over aprotinin in the amount of protein
that would be required to inhibit SIR.
[0116] In some embodiment, the kallikrein inhibitor polypeptide is
administered in a dose of about 1-500 mg/m.sup.2, preferably about
1-250 mg/m.sup.2, 1-100 mg/m.sup.2. For example, a kallikrein
inhibitor polypeptide, e.g., a kallikrein inhibitor polypeptide
described herein, can be administered to a subject at risk for
cerebral ischemia, suffering from cerebral ischemia, or who has
suffered a cerebral ischemic attack at a dose of 1-100 mg/m.sup.2.
In other embodiments, the dose of the kallikrein inhibitor
polypeptide can be less than the doses provided above. For example,
the dose of the kallikrein inhibitor can be reduced, such that the
combination of the kallikrein inhibitor polypeptide and the
anti-thrombolytic agent, give the same or a similar effect as the
kallikrein inhibitor polypeptide given at a higher dose.
[0117] Suggested dosage regimens for other anti-thrombolytic
agents, e.g., anti-fibrinolytic agents, are known. In some
embodiments, the suggested dose of the anti-thrombolytic agent,
e.g., anti-fibrinolytic agent, can be adjusted such that the
combination treatment has, e.g., the same (or better) therapeutic
effect than the anti-thrombolytic agent, e.g., anti-fibrinolytic
agent given at its suggested dose. Current dosing regimens for
epsilon amino caproic acid (Amicar.TM.) are as follows: in adult
patients, epsilon amino caproic acid is administered is given at 4
to 5 grams in the first hour and then at 1-1.25 grams/hour, three
to four times a day. The current dosing regimen for tranexamic acid
(Cyklokapron.TM.) is 10 mg/kg every 6-8 hours for 7 to 10 days.
[0118] The kallikrein inhibitor can be administered before,
concurrently with, or after the administration of the
anti-thrombolytic agent.
[0119] The methods described herein can further include
administration of another agent or agents other than the kallikrein
inhibitor and the anti-thrombolytic agent. For example, an
anti-coagulation agent or anti-platelet agent can also be
administered to the patient.
[0120] Anticoagulation agents prevent the coagulation of blood
components and thus prevent clot formation. Anticoagulants include,
but are not limited to, heparin, warfarin, coumadin, dicumarol,
phenprocoumon, acenocoumarol, ethyl biscoumacetate, hirudin,
bivalarutin, and other direct thrombin inhibitors, and indandione
derivatives.
[0121] Anti-platelet agents inhibit platelet aggregation and are
often used to prevent thromboembolic stroke in patients who have
experienced a transient ischemic attack or stroke. Anti-platelet
agents include, but are not limited to, aspirin, thienopyridine
derivatives such as ticlopodine and clopidogrel, dipyridamole and
sulfinpyrazone, as well as RGD mimetics.
[0122] The kallikrein inhibitor polypeptides are non-naturally
occurring, and can be, e.g., produced synthetically or
recombinantly, as noted above, thereby avoiding potential
contamination of transmissible diseases that can arise during
isolation of a protein from a natural animal source, such as in the
case of aprotinin, which is isolated from bovine lung. Increasingly
important to administrative and public acceptance of a treatment or
pharmaceutical composition comprising a polypeptide is the
avoidance of possible contamination with and transmission to human
patients of various pathological agents. Of particular interest for
the safety of proteins isolated from a bovine tissue is the
elimination of the possible risk of exposure to viral mediated
diseases, bacterial mediated diseases, and, especially,
transmissible bovine spongiform encephalopathies.
[0123] As variants of the Kunitz domain 1 of the human LACI
protein, fewer side effects are expected from administering the
kallikrein inhibitor polypeptides to patients than for aprotinin,
which is a bovine protein that is documented to cause anaphylactic
and a naphylactoid responses in patients, especially in repeat
administrations, such as second time CABG procedures. Additionally,
the highly specific binding of the kallikrein inhibitor
polypeptides described herein to kallikrein can limit or eliminate
the thrombotic tendencies observed with aprotinin, and/or reduce
the problems observed with graft patency following CABG
procedures.
[0124] In some embodiments, the kallikrein inhibitor polypeptide is
administered in combination with aprotinin, and one or more side
effect associated with aprotinin is reduced or eliminated. One or
more side effect of aprotinin that can be reduced or prevented by a
combination treatment with a non-naturally occurring kallikrein
inhibitor include, but are not limited to: hypersensitivity and
pseudo-allergic reactions; itching; rash; sweating; urticaria; skin
eruptions; pallor or cyanosis; dyspnea; nausea; drop in blood
pressure; tachycardia or bradycardia; airway obstruction; severe
hypotension and anaphylactic shock; renal dysfunction; kidney
failure; increase risk of graft closure; increased risk of
myocardial infarction.
[0125] In other embodiments, the kallikrein inhibitor polypeptide
is administered in combination with one or more of epsilon amino
caproic acid and/or tranexamic acid and, e.g., one or more side
effect associated with administration epsilon amino caproic acid
and/or tranexamic acid is reduced or eliminated. One or more side
effect of epsilon amino caproic acid and/or tranexamic acid that
can be reduced or prevented by a combination treatment with a
non-naturally occurring kallikrein inhibitor include, but are not
limited to: blood clots; headache; loss of coordination; pains in
chest, groin, or legs, especially the calves; shortness of breath;
slurred speech; vision changes; weakness or numbness in arm or leg;
ringing or buzzing in ears; skin rash; slow or irregular heart
beat; stomach cramps; swelling of face, feet or lower legs; unusual
tiredness; weight gain; decrease in amount of urine in patient;
diarrhea, nausea or vomiting; seizures; and hallucination.
Devices and Kits
[0126] Pharmaceutical compositions that include the kallikrein
inhibitor and/or the anti-thrombolytic agent (e.g., the
anti-fibrinolytic agent) can be administered with a medical device.
The device can designed with features such as portability, room
temperature storage, and ease of use so that it can be used in
emergency situations, e.g., by an untrained subject or by emergency
personnel in the field, removed to medical facilities and other
medical equipment. The device can include, e.g., one or more
housings for storing pharmaceutical preparations that include a
non-naturally occurring kallikrein inhibitor and/or an
anti-thrombolytic agent, and can be configured to deliver one or
more unit doses of the agent or agents.
[0127] For example, the pharmaceutical composition can be
administered with a device disclosed in U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; and U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery. Many other devices, implants, delivery
systems, and modules are also known.
[0128] A non-naturally occurring kallikrein inhibitor and/or
anti-thrombolytic agent can be provided in a kit. In one
embodiment, the kit includes (a) a container that contains a
composition that includes a non-naturally occurring kallikrein
inhibitor, and optionally (b) informational material. The
informational material can be descriptive, instructional, marketing
or other material that relates to the methods described herein
and/or the use of the agents for therapeutic benefit. In an
embodiment, the kit includes also includes an anti-thrombolytic
agent. For example, the kit includes a first container that
contains a composition that includes the non-naturally occurring
kallikrein inhibitor, and a second container that includes the
anti-thrombolytic agent.
[0129] The informational material of the kits is not limited in its
form. In one embodiment, the informational material can include
information about production of the compound, molecular weight of
the compound, concentration, date of expiration, batch or
production site information, and so forth. In one embodiment, the
informational material relates to methods of administering the
non-naturally occurring kallikrein inhibitor, e.g., in a suitable
dose, dosage form, or mode of administration (e.g., a dose, dosage
form, or mode of administration described herein), to treat a
subject who has or is at risk for blood loss, injury associated
with ischemia (e.g., ischemia associated with perioperative blood
loss, cerebral ischemia, reperfusion injury, e.g., reperfusion
injury associated with cerebral ischemia or a focal brain
ischemia), and/or the onset of systemic inflammatory response,
e.g., in patients subjected to invasive surgical procedures,
especially procedures requiring cardiopulmonary bypass. In one
embodiment, the instructions provide a dosing regimen, dosing
schedule, and/or route of administration of the kallikrein
inhibitor that differs from the dosing regimen, dosing schedule
and/or route of administration for the kallikrein inhibitor in the
absence of the anti-thrombolytic agent, e.g., a dosing regimen
described herein. The information can be provided in a variety of
formats, include printed text, computer readable material, video
recording, or audio recording, or a information that provides a
link or address to substantive material.
[0130] In addition to the non-naturally occurring kallikrein
inhibitor and/or anti-thrombolytic agent, the composition in the
kit can include other ingredients, such as a solvent or buffer, a
stabilizer, or a preservative. The non-naturally occurring
kallikrein inhibitor and/or anti-thrombolytic agent can be provided
in any form, e.g., liquid, dried or lyophilized form, preferably
substantially pure and/or sterile. When the agents are provided in
a liquid solution, the liquid solution preferably is an aqueous
solution. When the agents are provided as a dried form,
reconstitution generally is by the addition of a suitable solvent.
The solvent, e.g., sterile water or buffer, can optionally be
provided in the kit.
[0131] The kit can include one or more containers for the
composition or compositions containing the agents. In some
embodiments, the kit contains separate containers, dividers or
compartments for the composition and informational material. For
example, the composition can be contained in a bottle, vial, or
syringe, and the informational material can be contained in a
plastic sleeve or packet. In other embodiments, the separate
elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle,
vial or syringe that has attached thereto the informational
material in the form of a label. In some embodiments, the kit
includes a plurality (e.g., a pack) of individual containers, each
containing one or more unit dosage forms (e.g., a dosage form
described herein) of the agents. The containers can include a
combination unit dosage, e.g., a unit that includes both the
non-naturally occurring kallikrein inhibitor and the
anti-thrombolytic agent, e.g., in a desired ratio. For example, the
kit includes a plurality of syringes, ampules, foil packets,
blister packs, or medical devices, e.g., each containing a single
combination unit dose. The containers of the kits can be air tight,
waterproof (e.g., impermeable to changes in moisture or
evaporation), and/or light-tight.
[0132] The kit optionally includes a device suitable for
administration of the composition, e.g., a syringe or other
suitable delivery device. The device can be provided pre-loaded
with one or both of the agents or can be empty, but suitable for
loading.
[0133] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
56158PRTArtificial SequencePolypeptide Inhibiting Kallikrein 1Xaa
Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Cys Xaa Xaa1 5 10
15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa20
25 30Phe Xaa Xaa Gly Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa35 40 45Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa50
55260PRTArtificialArtificial Kunitz Domain 2Glu Ala Met His Ser Phe
Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys1 5 10 15Arg Ala Ala His Pro
Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys20 25 30Glu Glu Phe Ile
Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu35 40 45Ser Leu Glu
Glu Cys Lys Lys Met Cys Thr Arg Asp50 55
603180DNAArtificialArtificial Kunitz Domain coding sequence
3gaggctatgc actctttctg tgctttcaag gctgacgacg gtccgtgcag agctgctcac
60ccaagatggt tcttcaacat cttcacgcgt caatgcgagg agttcatcta cggtggttgt
120gagggtaacc aaaacagatt cgagtctcta gaggagtgta agaagatgtg
tactagagac 180458PRTArtificial SequenceSynthetically generated
peptide 4Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys
Lys Ala1 5 10 15Asn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln
Cys Glu Glu20 25 30Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg
Phe Glu Ser Leu35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp50
55558PRTArtificial SequenceSynthetically generated peptide 5Met His
Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala1 5 10 15Asn
His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25
30Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35
40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp50 55658PRTArtificial
SequenceSynthetically generated peptide 6Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly His Cys Lys Ala1 5 10 15Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln20 25 30Phe Thr Tyr Gly
Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 55758PRTArtificial
SequenceSynthetically generated peptide 7Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly His Cys Lys Ala1 5 10 15Ser Leu Pro Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ile Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 55858PRTArtificial
SequenceSynthetically generated peptide 8Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly His Cys Lys Ala1 5 10 15Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 55958PRTArtificial
SequenceSynthetically generated peptide 9Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly His Cys Lys Gly1 5 10 15Ala His Leu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551058PRTArtificial
SequenceSynthetically generated peptide 10Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly1 5 10 15Ala His Leu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ile Tyr Gly
Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551158PRTArtificial
SequenceSynthetically generated peptide 11Met His Ser Phe Cys Ala
Phe Lys Ala Asp Gly Gly Arg Cys Arg Gly1 5 10 15Ala His Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551258PRTArtificial
SequenceSynthetically generated peptide 12Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala1 5 10 15Ala His Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551358PRTArtificial
SequenceSynthetically generated peptide 13Met His Ser Phe Cys Ala
Phe Lys Ala Asp Val Gly Arg Cys Arg Gly1 5 10 15Ala His Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551458PRTArtificial
SequenceSynthetically generated peptide 14Met His Ser Phe Cys Ala
Phe Lys Ala Asp Val Gly Arg Cys Arg Gly1 5 10 15Ala Gln Pro Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551558PRTArtificial
SequenceSynthetically generated peptide 15Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Ser Cys Arg Ala1 5 10 15Ala His Leu Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551658PRTArtificial
SequenceSynthetically generated peptide 16Met His Ser Phe Cys Ala
Phe Lys Ala Glu Gly Gly Ser Cys Arg Ala1 5 10 15Ala His Gln Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551758PRTArtificial
SequenceSynthetically generated peptide 17Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly1 5 10 15Ala His Leu Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551858PRTArtificial
SequenceSynthetically generated peptide 18Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly His Cys Arg Gly1 5 10 15Ala Leu Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 551958PRTArtificial
SequenceSynthetically generated peptide 19Met His Ser Phe Cys Ala
Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly1 5 10 15Asn Leu Pro Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 552058PRTArtificial
SequenceSynthetically generated peptide 20Met His Ser Phe Cys Ala
Phe Lys Ala Asp Ser Gly Arg Cys Arg Gly1 5 10 15Asn His Gln Arg Phe
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 552158PRTArtificial
SequenceSynthetically generated peptide 21Met His Ser Phe Cys Ala
Phe Lys Ala Asp Gly Gly Arg Cys Arg Ala1 5 10 15Ile Gln Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 552258PRTArtificial
SequenceSynthetically generated peptide 22Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Arg Cys Arg Gly1 5 10 15Ala His Pro Arg Trp
Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe Ser Tyr Gly
Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40 45Glu Glu Cys
Lys Lys Met Cys Thr Arg Asp50 55236DNAArtificial SequenceModified
Cloning Site 23ttcgaa 6248DNAArtificial SequenceModified Cloning
Site 24ttcgcgaa 8256DNAArtificial SequenceModified Cloning Site
25gacgtc 62610DNAArtificial SequenceModified Cloning Site
26gacgtacgtc 1027548DNAArtificial SequenceNucleotide Sequence of
Fusion Protein 27cgacttttaa cgacaacttg agaagatcaa aaaacaacta
attattcgaa acgatgagat 60tcccatctat cttcactgct gttttgttcg ctgcttcctc
tgctttggct gctccagtta 120acaccactac tgaagacgag actgctcaaa
ttcctgctga ggctgtcatc ggttactctg 180acttggaagg tgacttcgac
gtcgctgttt tgccattctc taactctact aacaacggtt 240tgttgttcat
caacactacc atcgcttcta tcgctgctaa ggaggaaggt gtttccctcg
300agaagagaga ggctatgcac tctttctgtg ctttcaaggc tgacgacggt
ccgtgcagag 360ctgctcaccc aagatggttc ttcaacatct tcacgcgtca
atgcgaggag ttcatctacg 420gtggttgtga gggtaaccaa aacagattcg
agtctctaga ggagtgtaag aagatgtgta 480ctagagacta gtaagaattc
gccttagaca tgactgttcc tcagttcaag ttgggcactt 540acgagaag
54828145PRTArtificial SequenceFusion Protein 28Met Arg Phe Pro Ser
Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Ala
Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln20 25 30Ile Pro Ala
Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe35 40 45Asp Val
Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu50 55 60Phe
Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val65 70 75
80Ser Leu Glu Lys Arg Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala85
90 95Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn
Ile100 105 110Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys
Glu Gly Asn115 120 125Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys
Lys Met Cys Thr Arg130 135 140Asp1452958PRTBos taurus 29Arg Pro Asp
Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala1 5 10 15Arg Ile
Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr20 25 30Phe
Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala35 40
45Glu Asp Cys Met Arg Thr Cys Gly Gly Ala50 553058PRTArtificial
SequenceSynthetically generated peptide 30Lys Glu Asp Ser Cys Gln
Leu Gly Tyr Ser Ala Gly Pro Cys Met Gly1 5 10 15Met Thr Ser Arg Tyr
Phe Tyr Asn Gly Thr Ser Met Ala Cys Glu Thr20 25 30Phe Gln Tyr Gly
Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr Glu35 40 45Lys Glu Cys
Leu Gln Thr Cys Arg Thr Val50 553158PRTArtificial
SequenceSynthetically generated peptide 31Thr Val Ala Ala Cys Asn
Leu Pro Ile Val Arg Gly Pro Cys Arg Ala1 5 10 15Phe Ile Gln Leu Trp
Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu20 25 30Phe Pro Tyr Gly
Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu35 40 45Lys Glu Cys
Arg Glu Tyr Cys Gly Val Pro50 553258PRTHomo sapiens 32Met His Ser
Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala1 5 10 15Ile Met
Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu20 25 30Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu35 40
45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp50 553358PRTArtificial
SequenceSynthetically generated peptide 33Lys Pro Asp Phe Cys Phe
Leu Glu Glu Asp Pro Gly Ile Cys Arg Gly1 5 10 15Tyr Ile Thr Arg Tyr
Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg20 25 30Phe Lys Tyr Gly
Gly Cys Leu Gly Asn Met Asn Asn Phe Glu Thr Leu35 40 45Glu Glu Cys
Lys Asn Ile Cys Glu Asp Gly50 553458PRTArtificial
SequenceSynthetically generated peptide 34Gly Pro Ser Trp Cys Leu
Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala1 5 10 15Asn Glu Asn Arg Phe
Tyr Tyr Asn Ser Val Ile Gly Lys Cys Arg Pro20 25 30Phe Lys Tyr Ser
Gly Cys Gly Gly Asn Glu Asn Asn Phe Thr Ser Lys35 40 45Gln Glu Cys
Leu Arg Ala Cys Lys Lys Gly50 553558PRTArtificial
SequenceSynthetically generated peptide 35Leu Pro Asn Val Cys Ala
Phe Pro Met Glu Lys Gly Pro Cys Gln Thr1 5 10 15Tyr Met Thr Arg Trp
Phe Phe Asn Phe Glu Thr Gly Glu Cys Glu Leu20 25 30Phe Ala Tyr Gly
Gly Cys Gly Gly Asn Ser Asn Asn Phe Leu Arg Lys35 40 45Glu Lys Cys
Glu Lys Phe Cys Lys Phe Thr50 553658PRTArtificial
SequenceSynthetically generated peptide 36Glu Thr Asp Ile Cys Lys
Leu Pro Lys Asp Glu Gly Thr Cys Arg Asp1 5 10 15Phe Ile Leu Lys Trp
Tyr Tyr Asp Pro Asn Thr Lys Ser Cys Ala Arg20 25 30Phe Trp Tyr Gly
Gly Cys Gly Gly Asn Glu Asn Lys Phe Gly Ser Gln35 40 45Lys Glu Cys
Glu Lys Val Cys Ala Pro Val50 553758PRTArtificial
SequenceSynthetically generated peptide 37Asn Ala Glu Ile Cys Leu
Leu Pro Leu Asp Tyr Gly Pro Cys Arg Ala1 5 10 15Leu Leu Leu Arg Tyr
Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln20 25 30Phe Leu Tyr Gly
Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr Thr Trp35 40 45Glu Ala Cys
Asp Asp Ala Cys Trp Arg Ile50 553861PRTArtificial
SequenceSynthetically generated peptide 38Val Pro Lys Val Cys Arg
Leu Gln Val Ser Val Asp Asp Gln Cys Glu1 5 10 15Gly Ser Thr Glu Lys
Tyr Phe Phe Asn Leu Ser Ser Met Thr Cys Glu20 25 30Lys Phe Phe Ser
Gly Gly Cys His Arg Asn Arg Ile Glu Asn Arg Phe35 40 45Pro Asp Glu
Ala Thr Cys Met Gly Phe Cys Ala Pro Lys50 55 603958PRTArtificial
SequenceSynthetically generated peptide 39Ile Pro Ser Phe Cys Tyr
Ser Pro Lys Asp Glu Gly Leu Cys Ser Ala1 5 10 15Asn Val Thr Arg Tyr
Tyr Phe Asn Pro Arg Tyr Arg Thr Cys Asp Ala20 25 30Phe Thr Tyr Thr
Gly Cys Gly Gly Asn Asp Asn Asn Phe Val Ser Arg35 40 45Glu Asp Cys
Lys Arg Ala Cys Ala Lys Ala50 554059PRTArtificial
SequenceSynthetically generated peptide 40Arg Asn Arg Glu Val Cys
Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg1 5 10 15Ala Met Ile Ser Arg
Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala20 25 30Pro Phe Phe Tyr
Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr35 40 45Glu Glu Tyr
Cys Met Ala Val Cys Gly Ser Ala50 554158PRTArtificial
SequenceSynthetically generated peptide 41Arg Pro Asp Phe Cys Leu
Glu Pro Pro Tyr Thr Gly Pro Cys Val Ala1 5 10 15Met Phe Pro Arg Tyr
Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr20 25 30Phe Val Tyr Gly
Gly Cys Met Gly Asn Gly Asn Asn Phe Lys Ser Ala35 40 45Glu Asp Cys
Met Arg Thr Cys Gly Gly Ala50 554258PRTArtificial
SequenceSynthetically generated peptide 42Arg Pro Asp Phe Cys Gln
Leu Gly Tyr Ser Ala Gly Pro Cys Val Ala1 5 10 15Met Phe Pro Arg Tyr
Phe Tyr Asn Gly Thr Ser Met Ala Cys Gln Thr20
25 30Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr
Glu35 40 45Lys Asp Cys Leu Gln Thr Cys Arg Gly Ala50
554358PRTArtificial SequenceSynthetically generated peptide 43Arg
Pro Asp Phe Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Val Ala1 5 10
15Met Phe Pro Arg Tyr Phe Tyr Asn Gly Ala Ser Met Ala Cys Gln Thr20
25 30Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr
Glu35 40 45Lys Asp Cys Leu Gln Thr Cys Arg Gly Ala50
554458PRTArtificial SequenceSynthetically generated peptide 44Arg
Pro Asp Phe Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Val Ala1 5 10
15Met Phe Pro Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys Glu Thr20
25 30Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr
Glu35 40 45Lys Asp Cys Leu Gln Thr Cys Arg Gly Ala50
554558PRTArtificial SequenceSynthetically generated peptide 45Arg
Pro Asp Phe Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Val Gly1 5 10
15Met Phe Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys Gln Thr20
25 30Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val Thr
Glu35 40 45Lys Asp Cys Leu Gln Thr Cys Arg Gly Ala50
554662PRTArtificial SequenceSynthetically generated peptide 46Glu
Ala Glu Ala Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly1 5 10
15Pro Cys Ile Ala Phe Phe Pro Arg Tyr Phe Tyr Asn Ala Lys Ala Gly20
25 30Leu Cys Gln Thr Phe Val Tyr Gly Gly Cys Met Gly Asn Gly Asn
Asn35 40 45Phe Lys Ser Ala Glu Asp Cys Met Arg Thr Cys Gly Gly
Ala50 55 604756PRTArtificial SequenceSynthetically generated
peptide 47Ala Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Ile Ala
Phe Phe1 5 10 15Pro Arg Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val
Leu Phe Pro20 25 30Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr
Ser Glu Lys Glu35 40 45Cys Arg Glu Tyr Cys Gly Val Pro50
554856PRTArtificial SequenceSynthetically generated peptide 48Ala
Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Ile Ala Phe Phe1 5 10
15Pro Arg Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro20
25 30Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys
Glu35 40 45Cys Arg Glu Tyr Cys Gly Val Pro50 554956PRTArtificial
SequenceSynthetically generated peptide 49Glu Ala Cys Asn Leu Pro
Ile Val Arg Gly Pro Cys Ile Ala Phe Phe1 5 10 15Pro Arg Trp Ala Phe
Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro20 25 30Tyr Gly Gly Cys
Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu35 40 45Cys Arg Glu
Tyr Cys Gly Val Pro50 555060PRTArtificial SequenceSynthetically
generated peptide 50Glu Ala Val Arg Glu Val Cys Ser Glu Gln Ala Glu
Thr Gly Pro Cys1 5 10 15Ile Ala Phe Phe Pro Arg Trp Tyr Phe Asp Val
Thr Glu Gly Lys Cys20 25 30Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly
Asn Arg Asn Asn Phe Asp35 40 45Thr Glu Glu Tyr Cys Met Ala Val Cys
Gly Ser Ala50 55 605160PRTArtificial SequenceSynthetically
generated peptide 51Glu Ala Asn Ala Glu Ile Cys Leu Leu Pro Leu Asp
Tyr Gly Pro Cys1 5 10 15Ile Ala Phe Phe Pro Arg Tyr Tyr Tyr Asp Arg
Tyr Thr Gln Ser Cys20 25 30Arg Gln Phe Leu Tyr Gly Gly Cys Glu Gly
Asn Ala Asn Asn Phe Tyr35 40 45Thr Trp Glu Ala Cys Asp Asp Ala Cys
Trp Arg Ile50 55 605260PRTArtificial SequenceSynthetically
generated peptide 52Glu Ala Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp
Pro Gly Ile Cys1 5 10 15Ile Gly Phe Phe Pro Arg Tyr Phe Tyr Asn Asn
Gln Ala Lys Gln Cys20 25 30Glu Arg Phe Val Tyr Gly Gly Cys Leu Gly
Asn Met Asn Asn Phe Glu35 40 45Thr Leu Glu Glu Cys Lys Asn Ile Cys
Glu Asp Gly50 55 605360PRTArtificial SequenceSynthetically
generated peptide 53Glu Ala Glu Thr Asp Ile Cys Lys Leu Pro Lys Asp
Glu Gly Thr Cys1 5 10 15Ile Ala Phe Phe Pro Arg Trp Tyr Tyr Asp Pro
Asn Thr Lys Ser Cys20 25 30Ala Arg Phe Val Tyr Gly Gly Cys Gly Gly
Asn Glu Asn Lys Phe Gly35 40 45Ser Gln Lys Glu Cys Glu Lys Val Cys
Ala Pro Val50 55 6054304PRTHomo sapiens 54Met Ile Tyr Thr Met Lys
Lys Val His Ala Leu Trp Ala Ser Val Cys1 5 10 15Leu Leu Leu Asn Leu
Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu20 25 30Asp Glu Glu His
Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys35 40 45Leu Met His
Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys50 55 60Ala Ile
Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu65 70 75
80Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser85
90 95Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg
Ile100 105 110Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys
Phe Leu Glu115 120 125Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr
Arg Tyr Phe Tyr Asn130 135 140Asn Gln Thr Lys Gln Cys Glu Arg Phe
Lys Tyr Gly Gly Cys Leu Gly145 150 155 160Asn Met Asn Asn Phe Glu
Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu165 170 175Asp Gly Pro Asn
Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn180 185 190Ala Val
Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu195 200
205Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg
Gly210 215 220Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser
Val Ile Gly225 230 235 240Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys
Gly Gly Asn Glu Asn Asn245 250 255Phe Thr Ser Lys Gln Glu Cys Leu
Arg Ala Cys Lys Lys Gly Phe Ile260 265 270Gln Arg Ile Ser Lys Gly
Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys275 280 285Lys Gln Arg Val
Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met290 295
3005558PRTBos taurus 55Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr
Gly Pro Cys Lys Ala1 5 10 15Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys
Ala Gly Leu Cys Gln Thr20 25 30Phe Val Tyr Gly Gly Cys Arg Ala Lys
Arg Asn Asn Phe Lys Ser Ala35 40 45Glu Asp Cys Met Arg Thr Cys Gly
Gly Ala50 555658PRTArtificial SequenceSynthetically generated
peptide 56Met His Ser Phe Cys Ala Phe Lys Ala Xaa Xaa Gly Xaa Cys
Xaa Xaa1 5 10 15Xaa Xaa Xaa Arg Xaa Phe Phe Asn Ile Phe Thr Arg Gln
Cys Xaa Xaa20 25 30Phe Xaa Xaa Gly Gly Cys Xaa Gly Asn Gln Asn Arg
Phe Glu Ser Leu35 40 45Glu Glu Cys Lys Lys Met Cys Thr Arg Asp50
55
* * * * *