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United States Patent Application |
20110172140
|
Kind Code
|
A1
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Ley; Arthur C.
;   et al.
|
July 14, 2011
|
Poly-Pegylated Protease Inhibitors
Abstract
Disclosed are compounds that comprise: (i) a Kunitz domain polypeptide
that comprises a Kunitz domain that binds to and inhibits a protease; and
(ii) a plurality of polyethylene glycol moieties attached to the Kunitz
domain polypeptide. Each accessible primary amine of the Kunitz domain
polypeptide can be attached to one of the moieties. Also disclosed are
related methods.
Inventors: |
Ley; Arthur C.; (Newton, MA)
; Sato; Aaron K.; (Somerville, MA)
; Ladner; Robert C.; (Ijamsville, MD)
; Stochl; Mark; (North Attleboro, MA)
|
Assignee: |
Dyax Corp.
Cambridge
MA
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Serial No.:
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471875 |
Series Code:
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12
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Filed:
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May 26, 2009 |
Current U.S. Class: |
514/1.1; 530/324; 530/350; 530/402 |
Class at Publication: |
514/1.1; 530/324; 530/350; 530/402 |
International Class: |
A61K 38/55 20060101 A61K038/55; C07K 2/00 20060101 C07K002/00; C07K 14/00 20060101 C07K014/00; A61P 29/00 20060101 A61P029/00; A61P 7/00 20060101 A61P007/00; C07K 1/00 20060101 C07K001/00 |
Claims
1. A compound comprising: (i) a Kunitz domain polypeptide that comprises
a Kunitz domain that binds to and inhibits a protease; and (ii) a
plurality of polyethylene glycol moieties attached to the Kunitz domain
polypeptide, wherein the average molecular weight of each of the moieties
is less than 12 kDa, and each accessible primary amine of the Kunitz
domain polypeptide is attached to one of the moieties.
2-22. (canceled)
23. A preparation that comprises Kunitz domain polypeptides that
specifically bind and inhibit a protease, wherein at least 80% of the
Kunitz domain polypeptides in the preparation (i) bind and inhibit the
protease, and (ii) have a polyethylene glycol moiety attached at a first
common site and a polyethylene glycol moiety attached at a second common
site and wherein the average molecular weight of each of the attached
polyethylene glycol moieties is less than 12 kDa or have a plurality of
polyethylene glycol moieties attached to said Kunitz domain and wherein
the average molecular weight of each of the attached polyethylene glycol
moieties is less than 12 kDa.
24-51. (canceled)
52. A preparation that comprises Kunitz domain polypeptides that comprise
(i) the amino acid sequence of DX-890, wherein at least 80% of the
DX-890-containing Kunitz domain polypeptides in the preparation have a
polyethylene glycol moiety attached to each of four lysine residues and
to the N-terminus of the polypeptide; (ii) the amino acid sequence of
DX-88, wherein at least 80% of the DX-88-containing Kunitz domain
polypeptides in the preparation have a polyethylene glycol moiety
attached to each of three lysine residues and to the N-terminus of the
polypeptide; or (iii) the amino acid sequence of DX-1000, wherein at
least 80% of the DX-1000-containing Kunitz domain polypeptides in the
preparation have a polyethylene glycol moiety attached to each of three
lysine residues and to the N-terminus of the polypeptide.
53-54. (canceled)
55. A method of providing a pegylated Kunitz domain, the method
comprising: providing a polypeptide that comprises a Kunitz domain that
has at least one lysine in the framework region of the Kunitz domain; and
contacting the polypeptide with activated polyethylene glycol, of average
molecular weight less than 12 kDa, under conditions in which a plurality
of polyethylene glycol moieties are attached to the polypeptide, at least
one of which is attached to the lysine and at least one is attached to
the N-terminal primary amine.
56. A method of providing a PEGylated Kunitz domain, the method
comprising: providing a polypeptide that comprises a Kunitz domain that
has at least two primary amine groups in the framework region of the
Kunitz domain; and contacting the polypeptide with activated polyethylene
glycol, of average molecular weight less than 12 kDa, under conditions in
which a plurality of polyethylene glycol moieties are attached to the
polypeptide.
57-67. (canceled)
68. A method of treating a disorder characterized by excessive or
undesired activity of a protease, the method comprising, administering to
a subject having the disorder or suspected of having the disorder to
pharmaceutical composition comprising the preparation of claim 1, wherein
the Kunitz domain polypeptide of the preparation inhibits the protease.
69-74. (canceled)
75. A preparation comprising molecules that comprise: (i) a Kunitz domain
polypeptide that comprises a Kunitz domain that binds to and inhibits a
protease, and (ii) a plurality of polyethylene glycol moieties attached
to the Kunitz domain polypeptide, wherein the average molecular weight of
each of the moieties is less than 12 kDa.
76-77. (canceled)
78. A method of treating a disorder characterized by excessive or
undesired activity of a protease, the method comprising, administering to
a subject having the disorder or suspected of having the disorder to
pharmaceutical composition comprising the preparation of claim 75,
wherein the Kunitz domain polypeptide of the preparation inhibits the
protease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No.
11/458,773, filed on Jul. 20, 2006, which is a continuation of U.S.
application Ser. No. 10/931,153, filed on Aug. 30, 2004, now abandoned,
which claims the benefit of U.S. Provisional Application Ser. No.
60/498,845, filed on Aug. 29, 2003, and of U.S. Provisional Application
Ser. No. 60/598,967, filed on Aug. 4, 2004.
BACKGROUND
[0002] The invention relates to modified protease inhibitors.
SUMMARY
[0003] In one aspect, the invention features a compound that include: a) a
polypeptide including a Kunitz domain that specifically binds and/or
inhibits a protease; and b) a plurality of non-protein moieties that are
physically associated with the polypeptide and increases the molecular
weight of the compound. The term "poly-PEGylated Kunitz domain" refers
herein to the afore-mentioned compound. Typically, the non-protein
moieties of a poly-PEGylated Kunitz domain include polyethylene glycol.
[0004] The compound (i.e., the polypeptide plus the plurality of
non-protein moieties) has a molecular weight of greater than 12, 14 or 16
kDa. In one embodiment, each non-protein moiety has an average molecular
weight of between 3 and 20, 3 and 12 kDa, 3 and 10 kDa, 3 and 8 kDa, 4
and 6 kDa, e.g., about 4, 5, 6, 7, or 8 kDa.
[0005] The protease that is bound and/or inhibited can be, for example, an
elastase (e.g., human neutrophil elastase (hNE)), a plasmin, a
kallikrein, or other protease, e.g., a protease described herein. For
example, the protease can be a serine protease.
[0006] In one embodiment, non-protein moieties are attached to each
available primary amine on the Kunitz domain, e.g., the N-terminal
primary amine and any solvent-accessible primary amines, e.g., accessible
primary amines of lysine side chains in the Kunitz domain. For example,
all possible primary amines are conjugated to one of the non-protein
moieties. The Kunitz domain may have at least one, two, three, or four
lysines. For example, the Kunitz domain may have only one, two, three,
four, or five lysines. In one embodiment, the polypeptide has an
N-terminal primary amine. In another embodiment, the polypeptide does not
include an N-terminal primary amine (e.g., the polypeptide can be
chemically modified, e.g., with a non-polymeric compound, at its
N-terminal primary amine so that the polypeptide does not include a
primary amine at that position).
[0007] A non-protein moiety can be attached at 2 or more of the primary
amines in the polypeptide. For example, all lysines or all lysines that
have a solvent accessible primary amine are attached to a non-protein
moiety. Preferably, the Kunitz domain does not include a lysine within
one of its binding loops, e.g., about residues corresponding to amino
acids 11-21 of BPTI and 31-42 of BPTI. Lysines within such binding loops
can be replaced, e.g., with arginine residues. For example, the
polypeptide is attached to at least three of molecules of the polymer.
Each lysine of the polypeptide, or one, two, three or more of the lysines
can be attached to a molecule of the polymer. Unless otherwise stated,
when it is said that a primary amine, e.g., that of a particular lysine
or at the N terminus, is modified or has a non-protein moiety attached
thereto, it is understood that the specified primary amine position on
every molecule in a preparation may not be so modified. The preparations
need not be perfectly homogeneous to be within the invention. Homogeneity
is desirable in some embodiments but it need not be absolute. In
preferred embodiments, at least 60, 70, 80, 90, 95, 97, 98, 99, or 100%
of a primary amine which is designated as modified will have a
non-protein moiety attached thereto. Other embodiments however, include
preparations that contains a mixture of species in which most of the
molecules, e.g., at least 60, 70, 80, 90, 95, 97, 98, 99, or 100% are
PEGylated at two or more sites but the sites (and in some cases the
number of sites modified) on molecules in the preparation will vary.
E.g., some molecules will have lysines A, B, and D modified while other
molecules will have the amino terminus and lysines A, B, C, and D
modified.
[0008] In one embodiment, the non-protein moiety includes a hydrophilic
polymer, e.g., a substantially homogeneous polymer. The polymer can be
branched or unbranched. For example, the moiety of polymer has a
molecular weight (e.g., an average molecular weight of the moieties added
to the compound) that is less than 20, 18, 15, 12, 10, 8, 7, or 6 or at
least 1.5, 2, 2.5, 3, 5, 6, 10 kDa, e.g., about 5 kDa. In one embodiment,
the sum of the molecular weight of the PEG moieties on the compound is at
least 15, 20, 25, 30, or 35, and/or less than 60, 50, 40, 35, 30, 25, or
23 kDa.
[0009] In one embodiment, the polymer is a polyalkylene oxide. For
example, at least 20, 30, 50, 70, 80, 90, or 95% of the copolymer blocks
of the polymer are ethylene glycol. In one embodiment, the polymer is
polyethylene glycol.
[0010] In one embodiment, the compound has the following structure:
P--X.sup.0-[(CR'R'').sub.n--X.sup.1].sub.a--(CH.sub.2).sub.m--X.sup.2--R-
.sup.t
[0011] wherein P is the polypeptide,
[0012] each of R' and R'' is, independently, H, or C.sub.1-C.sub.12 alkyl;
[0013] X.sup.0 is O, N--R.sup.1, S, or absent, wherein R.sup.1 is H,
C.sub.1-C.sub.12 alkyl or aryl,
[0014] X.sup.1 is O, N--R.sup.2, S, wherein R.sup.2 is H, alkyl or aryl,
[0015] X.sup.2 is O, N--R.sup.3, S, or absent, wherein R.sup.3 is H, alkyl
or aryl,
[0016] each n is between 1 and 5, e.g., 2,
[0017] a is at least 4,
[0018] m is between 0 and 5, and
[0019] R.sup.t is H, C.sub.1-C.sub.12 alkyl or aryl.
[0020] R' and R'' can be H. In one embodiment, R' or R'' is independently,
H, or C1-C4, C1-C6, or C1-C10 alkyl.
[0021] In one embodiment, the compound has the following structure:
P--X.sup.0-[CH.sub.2CH.sub.2O].sub.a--(CH.sub.2).sub.m--X.sup.2--R.sup.t
[0022] wherein P is the polypeptide,
[0023] a is at least 4,
[0024] m is between 0 and 5,
[0025] X.sup.2 is O, N--R.sup.1, S, or absent, wherein R.sup.1 is H, alkyl
or aryl,
[0026] X.sup.0 is O, N--R.sup.2, S, or absent, wherein R.sup.2 is H, alkyl
or aryl, and
[0027] R.sup.t is H, C.sub.1-C.sub.12 alkyl or aryl. For example, X.sup.2
is O, and R.sup.t is CH.sub.3. (The use of mPEG is preferred.)
[0028] In one embodiment, the Kunitz domain polypeptide is less than 14,
8, or 7 kDa in molecular weight. In one embodiment, the Kunitz domain
polypeptide includes only one Kunitz domain. Generally, the compound
includes only one Kunitz domain, but in some embodiments, may include
more than one.
[0029] In one embodiment, the Kunitz domain includes the amino acid
sequence of DX-890, DX-88, or DX-1000 or an amino acid sequence that
differs by at least one, but fewer than six, five, four, three, or two
amino acid differences (e.g., substitutions, insertions, or deletions)
from the amino acid sequence of DX-890, DX-88, or DX-1000. Typically, the
Kunitz domain does not naturally occur in humans. The Kunitz domain may
include an amino acid sequence that differs by fewer than ten, seven, or
four amino acids from a human Kunitz domain.
[0030] In one embodiment, the K.sub.i of the compound is within a factor
of 0.5 to 1.5, 0.8 to 1.2, 0.3 to 3.0, 0.1 to 10.0, or 0.02 to 50.0 of
the K.sub.i of the unmodified polypeptide for elastase. For example, the
K.sub.i for hNE can be less than 100, 50, 18, 12, 10, or 9 pM.
[0031] In one embodiment, the compound has a circulatory half life of the
longest-lived component ("longest phase circulatory half life") in a
rabbit or mouse model that is at least 1.5, 2, 4, or 8 fold greater than
a substantially identical compound that does not include the polymer. The
compound can have a longest phase circulatory half life in a rabbit or
mouse model that has an amplitude at least 1.5, 2, 2.5, or 4 fold greater
than a substantially identical compound that does not include the
non-protein moiety. The compound can have an alpha-phase circulatory half
life in a rabbit or mouse model that has an amplitude at least 20, 30,
40, or 50% smaller than a substantially identical compound that does not
include the non-protein moiety. For example, the compound has a longest
phase circulatory half life with an amplitude of at least 40, 45, 46, 50,
53, 54, 60, or 65%. In one embodiment, the compound has a beta phase
circulatory half life in a mouse or rabbit model of at least 2, 3, 4, 5,
6, or 7 hours. In one embodiment, the compound has a longest phase
circulatory half life in a 70 kg human of at least 6 hours, 12 hours, 24
hours, 2 days, 5 days, 7 days, or 10 days.
[0032] In one embodiment, the compound has a longest phase circulatory
half life in a rabbit model of at least 4200 minutes, 4700 minutes, or
4980 minutes (or about 83 hours). In one embodiment, the compound has a
longest phase circulatory half-life that is longer than a similarly sized
molecule with the same Kunitz domain, but only a single PEG moiety (i.e.,
a mono-PEGylated version of the same Kunitz domain). The longest phase
half-life can be at least 5, 10, 20, 30, or 50% longer. In one
embodiment, in a mouse, the longest phase circulatory half life has an
amplitude of greater than 50, 55, 60, or 65%. The longest phase half life
can be, e.g., greater than 550, 600, 700, 750, 900, 1000, 1100 minutes.
[0033] In one embodiment, the compound has increased solubility (e.g.,
1.5, 2, 4, or 8 fold greater) in an aqueous solution having a pH between
5 and 8 and an ionic strength less than the ionic strength of 0.5 M NaCl
than the polypeptide that does not include the non-protein moiety.
[0034] In one embodiment, the polyethylene glycol is attached by coupling
monomethoxy-PEG propionaldehyde or monomethoxy-PEG succinimidyl propionic
acid to the polypeptide. The compound can formed by coupling of mPEG
(CH.sub.3--(O--CH.sub.2--CH.sub.2).sub.n--) at a pH that enables
attachment to accessible amino groups on lysine side chains and to the
N-terminal amino group, e.g., a pH 6.8 to 8.8, e.g., between 7.4 and 8.8.
[0035] In another aspect, the invention features a compound that includes
(1) a polypeptide including the amino acid sequence of DX-890, DX-88, or
DX-1000 or an amino acid sequence that differs by at least one, but fewer
than six, five, four, three, or two amino acid differences (e.g.,
substitutions, insertions, or deletions) from the amino acid sequence of
DX-890, DX-88, or DX-1000, and (2) a plurality of polyethylene glycol
moieties. Each polyethylene glycol moiety can be less than 20, 19, 18,
15, 12, 11, 10, 9, 8, 7, or 6 kDa in molecular weight and is attached.
Each polyethylene moiety can be attached to the polypeptide by a single
covalent bond.
[0036] In one embodiment, a molecule of polyethylene glycol is attached to
each lysine side chain of the polypeptide, e.g., where the polypeptide
includes more than one lysine, e.g., two, three or four lysines. For
example, the polypeptide is identical to the amino acid sequence of
DX-890 and a molecule of polyethylene glycol is attached to each of the
four lysine side chains of DX-890, and optionally also to the N-terminus.
In another example, the polypeptide is identical to the amino acid
sequence of DX-88 or DX-1000 and a molecule of polyethylene glycol is
attached to each of the three lysine side chains of DX-88 or DX-1000,
and, optionally, also to the N-terminus. In one embodiment, the molecules
of polyethylene glycol are between 4 and 12 kDa in molecular weight. In
one embodiment, the polyethylene glycol is attached to the N-terminus and
to each accessible lysine side chain.
[0037] In one embodiment, the amino acid sequence differs by at least one
amino acid from the amino acid sequence of DX-890. The amino acid
sequence is identical to the amino acid sequence of DX-890 at one or more
positions (e.g., at least two, three, five, seven, ten, twelve, thirteen,
fourteen, or all) corresponding to positions 5, 13, 14, 16, 17, 18, 19,
30, 31, 32, 34, 38, 39, 51, and 55 according to the BPTI numbering.
[0038] In another aspect, the invention features a preparation that
comprises Kunitz domain polypeptides that specifically bind and inhibit a
protease. At least 40, 50, 70, 80, 85, 90, 92, 95, 97, 98, 99, or 99.5%
of the Kunitz domain polypeptides in the preparation (i) bind and inhibit
the protease, and (ii) have a polyethylene glycol moiety attached at a
first common site and a polyethylene glycol moiety attached at a second
common site. Typically, the average molecular weight of each attached
polyethylene glycol moiety is less than 12, 10, or 8 kDa. In one
embodiment, the designated population of Kunitz domain polypeptides
further have a polyethylene glycol moiety attached at the third common
site and a polyethylene glycol moiety attached at the fourth common site.
For example, the designated population of Kunitz domain polypeptides have
a polyethylene glycol moiety attached to each accessible primary amine
and/or an N-terminal primary amine.
[0039] In one embodiment, each of the Kunitz domain polypeptides in the
preparation binds and inhibits the protease. For example, Kunitz domain
polypeptides that are not members of the designated population also bind
and inhibit a protease, e.g., the same or a different protease.
[0040] The Kunitz domain polypeptides of the population can include, for
example, other features described herein.
[0041] The invention also features a preparation that includes a compound
described herein, e.g., above. For example, the compound is present at a
concentration of at least 0.1, 1, 2, or 5 mg of polypeptide per
milliliter, e.g., in a solution between pH 6-8. In one embodiment, the
compound produces a major peak by size exclusion chromatography that
includes at least 70% the compound relative to the injectate. In one
embodiment, the molecular weight of 95% of the species of the compound
are within 5, 4, 3, 2, or 1 kDa of the average molecular weight of the
compound.
[0042] In another aspect, the invention features a pharmaceutical
preparation that includes (1) a compound described herein, and (2) a
pharmaceutically acceptable carrier. In one embodiment, at least 60, 70,
80, 85, 90, 95, 97, 98, 99, or 100% of the compounds in the preparation
have an identical distribution of PEG molecules attached thereto. The use
of chemical reaction in which all available primary amines (e.g., all
solvent accessible primary amines, or all primary amines) are modified
can be used to provide a preparation in which the compounds have an
identical distribution of PEG molecules. Of course, some variation will
be present in the molecule weight of the moieties attached to different
primary amines on a given molecule or among molecules since there is
variation about an average molecular weight for the PEG reagent used in
the chemical reaction. The preparation can also be made using a process
that provides a greater than 25, 30, 40, 50, 60, 70, 75, 80, or 85% yield
for input protein.
[0043] In one embodiment, the preparation is aqueous and the compound is
present at a concentration of at least 0.1 mg of polypeptide per
milliliter. In one embodiment, injection of the preparation into a mouse
results in less than 50, 30, 25, 15, or 10% of the compound is an SEC
peak with higher mobility than the preparation after 12 hours.
[0044] The preparation can be suitable for pulmonary delivery or for
gastrointestinal delivery (e.g., ingestion, rectal, etc.).
[0045] In another aspect, the invention features a pharmaceutical
preparation that includes (1) a compound described herein, and (2) a
pharmaceutically acceptable carrier. In one embodiment, at least 60, 70,
80, 85, 90, 95, 97, 98, 99, or 100% of the polypeptides in the
preparation have at least 2, 3 or 4 primary amines modified with a
non-protein moiety. The preparation can contain a mixture of species in
which most of the molecules, e.g., at least 60, 70, 80, 90, or 95% are
PEGylated at least 2 (or 3 or 4) sites but the sites (and in some cases
the number of sites modified) on molecules in the preparation will vary.
E.g., some molecules will have lysines A, B, and D modified while other
molecules will have the amino terminus and lysines A, B, C, and D
modified. In some embodiments, the preparation can include a small number
of compounds that are inactive (e.g., less than 5, 2, 1, or 0.1%), but
generally, most of the compounds (e.g., at least 50%, 90, 95, 98, 99,
99.5, or 99.9%) in the preparation are active, e.g., can inhibit a
protease.
[0046] In some aspects of the invention, the non-protein moiety attached
to different sites will be the same, in terms of identity or size. In
other aspects, a first non-protein moiety is attached at a first primary
amine, and a second non-protein moiety which is different, e.g., by type
or size, is attached to a different primary amine. E.g., it may be
desirable to attach a PEG of a first size to the primary amine of the N
terminus but to attach a PEG of a different size to a lysine position.
[0047] The invention also features a medical device that includes a
dispenser and a compartment that includes a pharmaceutical preparation
described herein. For example, the dispenser is configured to generate an
inhalable form of the pharmaceutical preparation. The invention also
features an implantable medical device that includes a dispenser and a
compartment that includes a pharmaceutical preparation described herein
wherein the dispenser is configured to delivery the pharmaceutical
preparation into the circulatory system of a subject. The invention also
features a suppository that includes a pharmaceutical preparation
described herein.
[0048] In another aspect, the invention features a preparation that
includes a poly-pegylated Kunitz domain. The preparation can be
substantially (e.g., at least 70, 75, 80, 85, 90, 95, or 100%)
monodisperse. For example, the poly-PEGylated compound is present at a
concentration of at least 0.05, 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, or 2.5
milligrams of polypeptide per milliliter or between 0.05 and 10
milligrams of polypeptide per milliliter. In one embodiment, the
preparation is dry. For example, the preparation includes particles or is
in the form of a powder.
[0049] In another aspect, the invention features a compound that includes
a polypeptide including the amino acid sequence of DX-890, DX-88, or
DX-1000 or other Kunitz domain sequence described herein in which at
least one lysine is substituted with a non-lysine amino acid, e.g.,
arginine. The compound is useful for reducing the number of lysines to
which PEG is coupled, e.g., without a substantial change in activity, to
produce a substantially homogenous conjugate. In one embodiment, the
amino acid sequence (e.g., of DX-890) has three lysine substitutions and
a single remaining lysine. In another embodiment, the amino acid has one
or two lysine substitutions. In one embodiment, the compound further
includes a non-protein moiety, e.g., a hydrophilic polymer described
herein. The polymer can be coupled to the remaining lysine residues,
e.g., single remaining lysine (e.g., the first, second, third, or fourth
lysine).
[0050] In another aspect, the invention features a preparation (e.g., an
aqueous preparation) that includes: a compound that includes a Kunitz
domain conjugated to a plurality of moieties of a hydrophilic and
substantially homogeneous polymer. For example, the concentration of
Kunitz domain component alone is greater than 2 mg per ml, the pH of the
preparation is greater than 3, and the ionic strength of the preparation
is less than the ionic strength of 0.5 M NaCl. In one embodiment, the
Kunitz domain includes the amino acid sequence of DX-890, DX-88, or
DX-1000 or an amino acid sequence that differs by at least one, but fewer
than six, five, four, three, or two amino acid differences (e.g.,
substitutions, insertions, or deletions) from the amino acid sequence of
DX-890. The invention also provides a sealed container that includes the
preparation. The container can be opaque to light. The container can
include printed information on an external region of the container.
[0051] In another aspect, the invention features a method that includes:
providing a polypeptide that includes a Kunitz domain; contacting the
polypeptide to a hydrophilic polymer (e.g., a polyalkylene oxide) that
includes a single reactive group that can form a covalent bond with the
polypeptide under conditions suitable for bond formation at a plurality
of available sites (e.g., a plurality of primary amines, e.g., all
available primary amines), thereby providing a modified protease
inhibitor.
[0052] In one embodiment, the hydrophilic polymer is mono-activated. For
example, the hydrophilic polymer is alkoxy-terminated. In one embodiment,
the polymer includes a succinimidyl group.
[0053] In one embodiment, the polymer is a polyethylene glycol, e.g.,
monomethoxy-polyethylene glycol. For example, the polymer is mPEG
propionaldehyde or mPEG succinimidyl propionic acid.
[0054] In one embodiment, the conditions are between pH 6.5 and 9.0, e.g.,
between 7.5 and 8.5. In one embodiment, the hydrophilic polymer is
covalently attached to the N-terminus of the polypeptide. In another
embodiment, the hydrophilic polymer is covalently attached to a lysine
side chain of the polypeptide.
[0055] The method can further include separating polypeptides that have a
single attached polymer from other products and reactants. The method can
further include chromatographically separating products of the
contacting, e.g., using ion exchange chromatography or size exclusion
chromatography.
[0056] The invention also features a modified Kunitz domain prepared by a
method described herein, e.g., the above method.
[0057] In another aspect, the invention features a method of treating a
disorder characterized by excessive or undesired activity of a protease.
The method includes: administering to a subject having the disorder or
suspected of having the disorder to pharmaceutical composition comprising
a compound or preparation described herein. The compound or preparation
includes a Kunitz domain polypeptide that inhibits the protease. For
example, a preparation has at least a certain percentage of molecules of
the Kunitz domain polypeptide in which a hydrophilic polymer is attached
to a first common site and a second common site. For example, at least a
certain percentage of molecules of the Kunitz domain polypeptide further
include the hydrophilic polymer attached to a third, fourth, and
optionally a fifth common site.
[0058] In one embodiment, the protease is elastase. For example, the
Kunitz domain polypeptide comprises the amino acid sequence of DX-890 or
a sequence that differs by at least one, but fewer than six amino acid
alterations from DX-890. Exemplary disorders that can be treated using a
Kunitz domain that inhibits elastase (e.g., human neutrophil elastase)
include cystic fibrosis, COPD, and an inflammatory disorder.
[0059] In one embodiment, the protease is a kallikrein. For example, the
Kunitz domain polypeptide comprises the amino acid sequence of DX-88 or a
sequence that differs by at least one, but fewer than six amino acid
alterations from DX-88. Exemplary disorders that can be treated using a
Kunitz domain that inhibits a kallikrein include disorders of
coagulation, fibrinolysis, hypotensions, inflammation, hemophilia,
post-operative bleeding, peri-operative bleeding, and hereditary
angioedema.
[0060] In one embodiment, the protease is plasmin and the Kunitz domain
polypeptide comprises the amino acid sequence of DX-1000 or a sequence
that differs by at least one, but fewer than six amino acid alterations
from DX-1000. Exemplary disorders that can be treated using a Kunitz
domain that inhibits plasmin include fibrinolysis or fibrinogenolysis,
excessive bleeding associated with thrombolytics, post-operative
bleeding, peri-operative bleeding, and inappropriate androgenesis.
[0061] In another aspect, the invention features a method of treating or
preventing a pulmonary disorder. The method includes administering a
compound described herein to a subject, e.g., in an amount effective to
ameliorate at least one symptom of the disorder. For example, the
compound includes a) a polypeptide including a Kunitz domain that
specifically binds and inhibits an elastase (e.g., human neutrophil
elastase (hNE)); and b) a non-protein moiety that is physically
associated with the polypeptide and increases the molecular weight of the
compound. For example, the compound includes (1) a polypeptide including
the amino acid sequence of DX-890 or an amino acid sequence that differs
by at least one, but fewer than six, five, four, three, or two amino acid
differences (e.g., substitutions, insertions, or deletions) from the
amino acid sequence of DX-890, and (2) polyethylene glycol wherein the
sum of the polyethylene glycol moieties is at least 15, 18, 20, 25, 27,
or 30 kDa in molecular weight.
[0062] In one embodiment, the compound is administered no more than once
every 12, 24, 36, or 72 hours. In another embodiment, the compound is
administered no more than once every four, seven, ten, twelve, or
fourteen days. The compound can be administered once or at multiple times
(e.g., regularly).
[0063] In one embodiment, the administering includes pulmonary delivery.
For example, the administering includes actuation of an inhaler and/or
nebulization. In one embodiment, the administering includes delivery of
the composition directly or indirectly into the circulatory system. For
example, the administering includes injection or intravenous delivery.
[0064] In one embodiment, the subject has cystic fibrosis or a genetic
defect in the cystic fibrosis gene. In another embodiment, the subject
has chronic obstructive pulmonary disease.
[0065] The symptom can be lung tissue integrity or an index of tissue
destruction.
[0066] In another aspect, the invention features a method of treating or
preventing a inflammatory disorder. The method includes: administering a
compound described herein to a subject, e.g., in an amount effective to
ameliorate at least one symptom of the disorder. For example, the
compound includes a) a polypeptide including a Kunitz domain that
specifically binds and inhibits an elastase (e.g., human neutrophil
elastase (hNE)); and b) a plurality of non-protein moieties that are
physically associated with the polypeptide and increase the molecular
weight of the compound. For example, the compound includes (1) a
polypeptide including the amino acid sequence of DX-890 or an amino acid
sequence that differs by at least one, but fewer than six, five, four,
three, or two amino acid differences (e.g., substitutions, insertions, or
deletions) from the amino acid sequence of DX-890, and (2) a plurality of
polyethylene glycol moieties, e.g., wherein each polyethylene glycol
moiety is less than 20, 18, 16, 12, 10, 9, 8, or 7 kDa in molecular
weight.
[0067] In one embodiment, the disorder is an inflammatory bowel disorder,
e.g., Crohn's disease or ulcerative colitis. In one embodiment, the
compound is delivered by a suppository.
[0068] In one embodiment, the compound is administered no more than once
every 12, 24, 36, or 72 hours. In another embodiment, the compound is
administered no more than once every four, seven, ten, twelve, or
fourteen days. The compound can be administered once or at multiple times
(e.g., regularly).
[0069] In another aspect, the invention features a method of treating or
preventing a disorder characterized at least in part by inappropriate
elastase activity or neutrophil activity. The method includes
administering a compound described herein to a subject, e.g., in an
amount effective to ameliorate at least one symptom of the disorder or to
alter elastase or neutrophil activity, e.g., to reduce elastase-mediated
proteolysis. For example, the disorder is rheumatoid arthritis.
[0070] In one embodiment, the compound is administered no more than once
every 12, 24, 36, or 72 hours. In another embodiment, the compound is
administered no more than once every four, seven, ten, twelve, or
fourteen days. The compound can be administered once or at multiple times
(e.g., regularly).
[0071] Many of the examples provided herein describe methods and
compositions that relate to Kunitz domains and a particular protease
target--elastase. However, these methods and compositions can be modified
to provide corresponding methods and compositions that relate to other
targets, e.g., other proteases or other proteins, e.g., protease other
than Kunitz domains, particularly proteins that include one or more
lysine residues. For example, the lysines may be positioned at a site
where their modification does not interfere with function. Similarly the
described methods and compositions can be modified to corresponding
methods and compositions that relate to polypeptides that do not include
a Kunitz domain or that include a Kunitz domain and other types of
domains.
[0072] As used herein, "binding affinity" refers to the apparent
association constant or Ka. The Ka is the reciprocal of the dissociation
constant (Kd). A ligand may, for example, have a binding affinity of at
least 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.11, or 10.sup.12 M.sup.-1 for a particular target molecule. Higher
affinity binding of a ligand to a first target relative to a second
target can be indicated by a higher Ka (or a smaller numerical value Kd)
for binding the first target than the Ka (or numerical value Kd) for
binding the second target. In such cases the ligand has specificity for
the first target relative to the second target. Ka measurements for
binding to hNE are typically made under the following conditions: 50 mM
HEPES, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100 at 30.degree. C. using
100 pM of the hNE.
[0073] Binding affinity can be determined by a variety of methods
including equilibrium dialysis, equilibrium binding, gel filtration,
ELISA, surface plasmon resonance, or spectroscopy (e.g., using a
fluorescence assay). These techniques can be used to measure the
concentration of bound and free ligand as a function of ligand (or
target) concentration. The concentration of bound ligand ([Bound]) is
related to the concentration of free ligand ([Free]) and the
concentration of binding sites for the ligand on the target where (N) is
the number of binding sites per target molecule by the following
equation:
[Bound]=N[Free]/((1/Ka)+[Free])
[0074] It is not always necessary to make an exact determination of Ka,
though, since sometimes it is sufficient to obtain a quantitative
measurement of affinity, e.g., determined using a method such as ELISA or
FACS analysis, is proportional to Ka, and thus can be used for
comparisons, such as determining whether a higher affinity is, e.g., 2
fold higher.
[0075] An "isolated composition" refers to a composition that is removed
from at least 90% of at least one component of a natural sample from
which the isolated composition can be obtained. Compositions produced
artificially or naturally can be "compositions of at least" a certain
degree of purity if the species or population of species of interests is
at least 5, 10, 25, 50, 75, 80, 90, 95, 98, or 99% pure on a
weight-weight basis.
[0076] An "epitope" refers to the site on a target compound that is bound
by a ligand, e.g., a polypeptide ligand such as a Kunitz domain, small
peptide, or antibody. In the case where the target compound is a protein,
for example, an epitope may refer to the amino acids that are bound by
the ligand. Such amino acids may be contiguous or non-contiguous with
respect to the underlying polypeptide backbone. Overlapping epitopes
include at least one common amino acid residue.
[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 Kunitz
domains, the second domain has the same specificity and, for example, has
at least 0.5, 5, or 50% of the binding affinity of the first domain. A
sufficient degree of identity may be about 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or higher.
[0078] Sequences similar or homologous (e.g., at least about 85% sequence
identity) to the sequences disclosed herein are also part of this
application. In some embodiment, the sequence identity can be about 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
Alternatively, substantial identity exists when the nucleic acid segments
will hybridize under selective hybridization conditions (e.g., highly
stringent hybridization conditions), to the complement of the strand. The
nucleic acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form.
[0079] Calculations of "homology" or "sequence identity" between two
sequences (the terms are used interchangeably herein) are 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.
[0080] The comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical algorithm.
In a preferred embodiment, the percent identity 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 identity 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 sequence identity or homology limitation of the
invention) 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.
[0081] As used herein, the term "homologous" is synonymous with
"similarity" and means that a sequence of interest differs from a
reference sequence by the presence of one or more amino acid
substitutions (although modest amino acid insertions or deletions) may
also be present. Presently preferred means of calculating degrees of
homology or similarity to a reference sequence are through the use of
BLAST algorithms (available from the National Center of Biotechnology
Information (NCBI), National Institutes of Health, Bethesda Md.), in each
case, using the algorithm default or recommended parameters for
determining significance of calculated sequence relatedness. The percent
identity between two amino acid or nucleotide sequences can also be
determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS,
4:11-17) which has been incorporated into the ALIGN program (version
2.0), using a PAM120 weight residue table, a gap length penalty of 12 and
a gap penalty of 4.
[0082] 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 preferably 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. Very high
stringency conditions (4) are the preferred conditions and the ones that
should be used unless otherwise specified. Accordingly, nucleic acids
that hybridize with appropriate stringency to nucleic acids that encode a
polypeptide described herein are provided as are polypeptides that are
encode by such nucleic acids. Such polypeptides can be similarly modified
as described herein.
[0083] It is understood that a polypeptide described herein (e.g., a
polypeptide that includes a Kunitz domain) may have mutations relative to
a particular polypeptide described herein (e.g., a conservative or
non-essential amino acid substitutions), which do not have a substantial
effect on the polypeptide functions. Whether or not a particular
substitution will be tolerated, i.e., will not adversely affect desired
biological properties, such as binding activity can be determined as
described in Bowie, et al. (1990) Science 247:1306-1310. A "conservative
amino acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain. Families
of amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains (e.g.,
lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains
(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). It is possible for many framework
and CDR amino acid residues to include one or more conservative
substitutions.
[0084] A "non-essential" amino acid residue is a residue that can be
altered from the wild-type sequence of the binding agent, e.g., the
antibody, without abolishing or more preferably, without substantially
altering a biological activity, whereas an "essential" amino acid residue
results in such a change.
[0085] The terms "polypeptide" or "peptide" (which may be used
interchangeably) refer to a polymer of three or more amino acids linked
by a peptide bond, e.g., between 3 and 30, 12 and 60, or 30 and 300, or
over 300 amino acids in length. The polypeptide may include one or more
unnatural amino acids. Typically, the polypeptide includes only natural
amino acids. A "protein" can include one or more polypeptide chains.
Accordingly, the term "protein" encompasses polypeptides. A protein or
polypeptide can also include one or more modifications, e.g., a
glycosylation, amidation, phosphorylation, and so forth. The term "small
peptide" can be used to describe a polypeptide that is between 3 and 30
amino acids in length, e.g., between 8 and 24 amino acids in length.
[0086] The term "alkyl" refers to a hydrocarbon chain that may be a
straight chain or branched chain, containing the indicated number of
carbon atoms. For example, C.sub.1-C.sub.12 alkyl indicates that the
group may have from 1 to 12 (inclusive) carbon atoms in it.
[0087] The term "aryl" refers to an aromatic monocyclic, bicyclic, or
tricyclic hydrocarbon ring system, wherein any ring atom capable of
substitution can be substituted by a substituent. Examples of aryl
moieties include, but are not limited to, phenyl, naphthyl, and
anthracenyl.
[0088] 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. All published patent
applications, patents, and references cited herein are incorporated by
reference in their entirety. In particular, U.S. Pat. Nos. 5,663,143;
5,223,409, 6,010,080, 6,103,499 and 6,333,402 are incorporated by
reference in their entireties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIG. 1 depicts the structure of DX-890 (SEQ ID NO:23) and the
position of its four lysine residues.
[0090] FIG. 2 depicts the structure of DX-88 (SEQ ID NO:24) and the
position of its three lysine residues.
[0091] FIG. 3 depicts the structure of DX-1000 (SEQ ID NO:25) and the
position of its three lysine residues.
[0092] FIG. 4 is an exemplary PEGylation scheme. The reaction pH can be
run at a pH of between 7.8 and 8.5.
[0093] FIG. 5 shows results of an exemplary MALDI analysis.
[0094] FIG. 6 shows results of exemplary GP-HPLC runs.
[0095] FIG. 7 shows exemplary results of SDS-PAGE analysis.
[0096] FIG. 8a shows exemplary results of clearance studies in mice and 8b
shows clearance studies in rabbits. The data were plotted using a double
4-parameter exponential decay. FIG. 8c shows an allometric extrapolation
to humans. Extrapolated values for long half life clearance phase in a 70
Kg human were as follows: DX-890, 8.4 hours; 5-PEG5-DX-890, 330 hours, or
about 14 days; DX-1000, 1.7 hours; 4-PEG5-DX-1000, 210 hours, or about 9
days.
[0097] FIG. 9 shows exemplary results of DX-88 poly-PEGylation at various
rations by SDS-PAGE analysis.
DETAILED DESCRIPTION
[0098] The invention provides, in part, compounds that bind to and inhibit
a protease (e.g., an elastase, e.g., a neutrophil elastase). The
compounds include (i) a polypeptide that includes a Kunitz domain and
(ii) a plurality of moieties (such as polymer moieties) that increases
the molecular weight of the compounds relative to the polypeptide alone.
The addition of the moieties to the compound can increase the in vivo
circulating half life of the compound. In some embodiments, the compounds
can inhibit neutrophil elastase with high affinity and selectivity.
Polymers
[0099] A variety of moieties can be used to increase the molecular weight
of a polypeptide that includes a Kunitz domain or other protease
inhibitor. In one embodiment, the moiety is a polymer, e.g., a water
soluble and/or substantially non-antigenic polymer such as a homopolymer
or a non-biological polymer. Substantially non-antigenic polymers
include, e.g., polyalkylene oxides or polyethylene oxides. The moiety may
improve stabilization and/or retention of the Kunitz domain in
circulation, e.g., in blood, serum, lymph, or other tissues, e.g., by at
least 1.5, 2, 5, 10, 50, 75, or 100 fold. A plurality of moieties are
attached to a Kunitz domain. For example, the polypeptide is attached to
at least three moieties of the polymer. Each lysine of the polypeptide
can be attached to a moiety of the polymer.
[0100] Suitable polymers can vary substantially by weight. For example, it
is possible to use polymers having average molecular weights ranging from
about 200 Daltons to about 40 kDa, e.g., 1-20 kDa, 4-12 kDa or 3-8 kDa,
e.g., about 4, 5, 6, or 7 kDa. In one embodiment, the average molecular
weight of individual moieties of the polymer that are associated with the
compound are less than 20, 18, 17, 15, 12, 10, 8, or 7 kDa. The final
molecular weight can also depend upon the desired effective size of the
conjugate, the nature (e.g. structure, such as linear or branched) of the
polymer, and the degree of derivatization.
[0101] A non-limiting list of exemplary 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. The polymer can be a hydrophilic polyvinyl
polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. Additional
useful polymers include polyoxyalkylenes such as polyoxyethylene,
polyoxypropylene, and block copolymers of polyoxyethylene and
polyoxypropylene (Pluronics); polylactic acid; polyglycolic acid;
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, cellulose, 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.
In some embodiments, the polymer includes a variety of different
copolymer blocks.
[0102] The polypeptide that includes a Kunitz domain can be physically
associated with the polymer in a variety of ways. Typically, the
polypeptide is covalently linked to the polymer at a plurality of sites.
For example, the polypeptide is conjugated to the polymer at a plurality
of primary amines, e.g., all accessible primary amines or all primary
amines. Other compounds can also be attached to the same polymer, e.g., a
cytotoxin, a label, or another targeting agent, e.g., another ligand that
binds to the same target as the Kunitz domain or a ligand that binds to
another target, e.g., a an unrelated ligand. Other compounds may also be
attached to the polypeptide.
[0103] In one embodiment, the polymer is water soluble prior to
conjugation to the polypeptide (although need not be). Generally, after
conjugation to the polypeptide, the product is water soluble, e.g.,
exhibits a water solubility of at least about 0.01 mg/ml, and more
preferably at least about 0.1 mg/ml, and still more preferably at least
about 1 mg/ml. In addition, the polymer should not be highly immunogenic
in the conjugate form, nor should it possess viscosity that is
incompatible with intravenous infusion or injection if the conjugate is
intended to be administered by such routes.
[0104] In one embodiment, the polymer contains only a single group which
is reactive. This helps to avoid conjugation of one polymer to multiple
protein molecules. 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 conjugation to the polypeptide. See,
e.g., U.S. Pat. No. 5,951,974.
[0105] In its most common form, poly(ethylene glycol), PEG, is a linear or
branched polyether terminated with hydroxyl groups. Linear PEG can have
the following general structure:
HO--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
PEG can be synthesized by anionic ring opening polymerization of ethylene
oxide initiated by nucleophilic attack of a hydroxide ion on the epoxide
ring. Particularly useful for polypeptide modification is monomethoxy
PEG, mPEG, having the general structure:
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
[0106] For further descriptions, see, e.g., Roberts et al. (2002) Advanced
Drug Delivery Reviews 54:459-476. In one embodiment, the polymer units
used for conjugation are mono-disperse or otherwise highly homogenous,
e.g., present in a preparation in which 95% or all molecules are with 7,
5, 4, 3, 2, or 1 kDa of one another. In another embodiment, the polymer
units are poly-disperse.
[0107] It is possible to select reaction conditions that reduce
cross-linking between polymer units or conjugation to multiple
polypeptides and to purify the reaction products through gel filtration
or ion exchange chromatography to recover substantially homogenous
derivatives, e.g., derivatives that include only a single Kunitz domain
polypeptide. In other embodiments, the polymer contains two or more
reactive groups for the purpose of linking multiple polypeptides (e.g.,
multiple units of the Kunitz domain polypeptide) to the polymer. Again,
gel filtration or ion exchange chromatography can be used to recover the
desired derivative in substantially homogeneous form.
[0108] The polypeptide that includes a Kunitz domain is generally attached
to a plurality of PEG molecules. For example, to form a compound that is
larger than 20 or 30 kDa, a Kunitz domain (about 7 kDa) can be attached
to at least three 8 kDa molecules of PEG. Other combinations are
possible, e.g., at least two, four, or five molecules of PEG. The
molecular weight of the PEG molecules can be selected so that the final
molecular weight of the compound is equal to or larger than a desired
molecular weight (e.g., between 17-35, or 20-25, or 27-33 kDa). The
plurality of PEG molecules can be attached to any region of the Kunitz
domain, preferably at least 5, 10, or 15 Angstroms from a region that
interacts with a target, or at least 2, 3, or 4 residues from an amino
acid that interacts with a target. The PEG molecules can be attached,
e.g., to lysine residues or a combination of lysine residues and the
N-terminus.
[0109] A covalent bond can be used to attach a polypeptide (e.g., a
polypeptide that includes a Kunitz domain) to a polymer, for example,
conjugation to the N-terminal amino group and epsilon amino groups found
on lysine residues, as well as other amino, imino, carboxyl, sulfhydryl,
hydroxyl or other hydrophilic groups. The polymer may be covalently
bonded directly to the polypeptide without the use of a multifunctional
(ordinarily bifunctional) crosslinking agent. Covalent binding to amino
groups can be accomplished by known chemistries based upon cyanuric
chloride, carbonyl diimidazole, aldehyde reactive groups (PEG alkoxide
plus diethyl acetyl of bromoacetaldehyde; PEG plus DMSO and acetic
anhydride, or PEG chloride plus the phenoxide of 4-hydroxybenzaldehyde,
activated succinimidyl esters, activated dithiocarbonate PEG,
2,4,5-trichlorophenylcloroformate or P-nitrophenylcloroformate activated
PEG.) Carboxyl groups can be derivatized by coupling PEG-primary amine
using carbodiimide. Sulfhydryl groups can be derivatized by coupling to
maleimido-substituted PEG (see, e.g., WO 97/10847) or PEG-maleimide
(e.g., commercially available from Shearwater Polymers, Inc., Huntsville,
Ala.). Alternatively, free amino groups on the polypeptide (e.g. epsilon
amino groups on lysine residues) can be thiolated with 2-imino-thiolane
(Traut's reagent) and then coupled to maleimide-containing derivatives of
PEG, e.g., as described in Pedley et al., Br. J. Cancer, 70: 1126-1130
(1994).
[0110] Functionalized PEG polymers that can be attached to a polypeptide
that includes Kunitz domain include polymers that are commercially
available, e.g., from Shearwater Polymers, Inc. (Huntsville, Ala.). Such
PEG derivatives include, e.g., amino-PEG, PEG amino acid esters,
PEG-hydrazide, PEG-thiol, PEG-succinate, carboxymethylated PEG,
PEG-propionic acid, PEG amino acids, PEG succinimidyl succinate, PEG
succinimidyl propionate, succinimidyl ester of carboxymethylated PEG,
succinimidyl carbonate of PEG, succinimidyl esters of amino acid PEGs,
PEG-oxycarbonylimidazole, PEG-nitrophenyl carbonate, PEG tresylate,
PEG-glycidyl ether, PEG-aldehyde, PEG vinylsulfone, PEG-maleimide,
PEG-orthopyridyl-disulfide, heterofunctional PEGs, PEG vinyl derivatives,
PEG silanes, and PEG phospholides. The reaction conditions for coupling
these PEG derivatives may vary depending on the polypeptide, the desired
degree of PEGylation, and the PEG derivative utilized. Some factors
involved in the choice of PEG derivatives include: the desired point of
attachment (such as lysine or cysteine R-groups), hydrolytic stability
and reactivity of the derivatives, stability, toxicity and antigenicity
of the linkage, suitability for analysis, etc.
[0111] The conjugates of a polypeptide that includes a Kunitz domain and a
polymer can be separated from the unreacted starting materials using
chromatographic methods, e.g., by gel filtration or ion exchange
chromatography, e.g., HPLC. Heterologous species of the conjugates are
purified from one another in the same fashion. Resolution of different
species (e.g. containing one or two PEG residues) is also possible due to
the difference in the ionic properties of the unreacted amino acids. See,
e.g., WO 96/34015.
Kunitz Domains
[0112] 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 sequence 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 or 2 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 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.
[0113] 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 seventy
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, and three
Kunitz domains of TFPI-2 (Sprecher et al., (1994) PNAS USA,
91:3353-3357). 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-00001
TABLE 1
Exemplary Natural Kunitz Domains
LACI: 1 MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit dtelpplklM
(SEQ ID 51 HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC
NO. 1) 101 KKMCTRDnan riikttlqqe kpdfCfleed pgiCrgyitr yfynnqtkqC
151 erfkyggClg nmnnfetlee CkniCedgpn gfqvdnygtq lnavnnsltp
201 qstkvpslfe fhgpswCltp adrglCrane nrfyynsvig kCrpfkysgC
251 ggnennftsk geClraCkkg fiqriskggl iktkrkrkkq rvkiayeeif
301 vknm
BPTI 1 2 3 4 5
(SEQ ID 1234567890123456789012345678901234567890123456789012345678
NO: 2) RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA
The signal sequence (1-28) is uppercase and underscored
LACI-K1 is uppercase
LACI-K2 is underscored
LACI-K3 is bold
[0114] 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.
[0115] Proteins containing exemplary Kunitz domains include the following,
with SWISS-PROT Accession Numbers in parentheses:
TABLE-US-00002
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)
TABLE-US-00003
TABLE 2
The amino-acid sequences of 19 human Kunitz
domains. Amino-acid sequences of 19 Human
Kunitz Domains Binding loops are underscored.
Collagen A1 VII
(SEQ ID NO: 3)
SDDPCSLPLDEGSCTAYTLRWYHRAVTEACHPFVYGGCGGNANRFGTR
EACERRCPPR
TFPI2-K1
(SEQ ID NO: 4)
NAEICLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTW
EACDDACWRI
AppI
(SEQ ID NO: 5)
VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTE
EYCMAVCGSA
Hep GF AI T2, K2
(SEQ ID NO: 6)
YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSE
EACMLRCFRQ
ITI, K1
(SEQ ID NO: 7)
KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTE
KECLQTCRTV
Chrome20
(SEQ ID NO: 8)
FQEPCMLPVRHGNCNHEAQRWHFDFKNYRCTPFKYRGCEGNANNFLNED
ACRTACMLIR
Hep GF AI T1, K1
(SEQ ID NO: 9)
TEDYCLASNKVGRCRGSFPRWYYDPTEQICKSFVYGGCLGNKNNYLREE
ECILACRGV
Hep GF AI T1, K2
(SEQ ID NO: 10)
DKGHCVDLPDTGLCKESIPRWYYNPFSEHCARFTYGGCYGNKNNFEEEQ
QCLESCRGI
TFPI2-K3
(SEQ ID NO: 11)
IPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSRE
DCKRACAKA
ITI, K2
(SEQ ID NO: 12)
AACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKEC
REYCGVP
Hep GF AI T2, K1
(SEQ ID NO: 13)
IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNNYLTKE
ECLKKCATV
App2
(SEQ ID NO: 14)
VKAVCSQEAMTGPCRAVMPRWYFDLSKGKCVRFIYGGCGGNRNNFESED
YCMAVCKAM
TFPI1 K2 = LACI-D2
(SEQ ID NO: 15)
KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLE
ECKNICEDG
TFPI2-K2
(SEQ ID NO: 16)
VPKVCRLQVSVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNRIENRFP
DEATCMGFCAPK
HKI B9
(SEQ ID NO: 17)
LPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFLRKE
KCEKFCKFT
TFPI1 K1 = LACI-D1
(SEQ ID NO: 18)
MHSFCAFKADDGPCKAIMKRFFFNIFTRQCEEFIYGGCEGNQNRFESLE
ECKKMCTRD
TFPI1 K3 = LACI-D3
(SEQ ID NO: 19)
GPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQ
ECLRACKKG
Collagen A3
(SEQ ID NO: 20)
ETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQK
ECEKVCAPV
CAB37635
(SEQ ID NO: 21)
KQDVCEMPKETGPCLAYFLHWWYDKKDNTCSMFVYGGCQGNNNNFQSKA
NCLNTCKNK
End Table 2.
[0116] 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 Propom 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. Natl. 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).
[0117] Useful Kunitz domains for selecting protease inhibitors can include
Kunitz domains that have a framework region with a particular number of
lysine residues. In one implementation, frameworks with four lysine
residues are useful and can be modified, e.g., by attachment of PEG
moieties of average molecular weight between 3-8 kDa, e.g., about 5 kDa.
For example, the ITI framework has four lysines. In another
implementation, frameworks with three lysines are useful and can be
modified e.g., by attachment of PEG moieties of average molecular weight
between 4-10 kDa, e.g., about 5 kDa or 7 kDa. LACI is one such framework.
Frameworks can also be altered to include fewer or additional lysines,
for example, to reduce the number of lysines that are within five, four,
or three residues of a binding loop, or to introduce a sufficient number
of lysines that the protein can be modified with small PEG moieties
(e.g., between 3-8 kDa PEG moieties) to increase the size of the protein
and stability of the protein in vivo.
[0118] 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 11-21 of BPTI. The
second loop region is between about residues corresponding to amino acids
31-42 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 include: positions 13, 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.
[0119] 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 11-21 of BPTI and 31-42 of BPTI.
[0120] Conversely, residues that are not at these particular positions or
which are not in the loop regions may tolerate a wider range of amino
acid substitution (e.g., conservative and/or non-conservative
substitutions) than these amino acid positions.
Elastase-Inhibiting Kunitz Domains
[0121] One exemplary polypeptide that binds to and inhibits human
neutrophil elastase (hNE) is DX-890 (also known as "EPI-hNE4"). DX-890 is
a highly specific and potent (Ki=4.times.10.sup.-12 M) inhibitor of human
neutrophil elastase (hNE). DX-890 includes the following amino acid
sequence:
TABLE-US-00004
(SEQ ID NO: 23)
Glu Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys
Ile Ala Phe Phe Pro Arg Trp Ala Phe Asp Ala Val
Lys Gly Lys Cys Val Leu Phe Pro Tyr Gly Gly Cys
Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys Glu
Cys Arg Glu Tyr Cys Gly Val Pro
[0122] DX-890 is derived from the second Kunitz-type domain of
inter-.alpha.-inhibitor protein (ITI-D2) and can be produced by
fermentation in Pichia pastoris. It includes 56 amino acids, with a
predicted MW of 6,237 Daltons. DX-890 is resistant to oxidative and
proteolytic inactivation.
[0123] In vitro, ex vivo and in vivo pharmacological studies have
demonstrated hNE inhibitory capacity and the protective effect of DX-890
against lesions induced by hNE of sputum from cystic fibrosis children
(see ref. Delacourt et al. 2002). Acute and subchronic 4-week studies of
aerosolized DX-890 in cynomolgus monkeys showed no signs of clinical or
biological toxicity, nor of histopathological lesions induced by the
administration of DX-890.
[0124] In clinical studies using healthy human volunteers, DX-890 was
found to be safe for administration by inhalation at 8 increasing doses
(up to 120 minutes of DX-890 in saline resulting in an inhaled mass of
about 72 mg).
[0125] Some of the consequences of elastase activity include: cleavage of
complement receptors and C3bi; cleavage of immunoglobulins; degradation
of elastin (and consequently plugging of airways, structural damage,
bronchiectasis); secretion of macromolecules; increased interleukin-8;
increase in PMN (and consequently release of oxygen, hydrogen peroxide,
leukotriene B4 and interleukin-8); and persistence of bacteria.
Inhibitors of elastase can be used to reduce one or more of these
activities.
[0126] DX-890 can be used as an anti-inflammatory drug targeted against
neutrophil mediated inflammation, e.g., in pulmonary CF lesions.
Exemplary pulmonary indications include Cystic Fibrosis (CF), Acute
Respiratory Distress Syndrome (ARDS), and Chronic Obstructive Pulmonary
Disease (COPD). In CF patients, the balance between proteinases and their
inhibitors may become severely disturbed. Activated polymorphonuclear
leukocytes (PMN) produce human neutrophil elastase (hNE) and other
proteases. hNE is considered to be a key cause of lung tissue damage
associated with cystic fibrosis. Inhibition of hNE is therefore a logical
avenue for treatment of CF lung disease since it attacks the original
source of damage rather than ameliorating symptoms and consequences of
the damage.
[0127] It is possible, for example, to deliver DX-890 to the lung by
nebulization. DX-890 activity was detected in broncho-alveolar lavages of
volunteer inhaling nebulized DX-890. 12 healthy volunteers received
during 14 days a single daily dose of DX-890, by nebulization lasting 5
or 20 minutes, corresponding to estimated inhaled mass of 3.75 or 15 mg
respectively. Tolerability was excellent; no significant adverse event
was reported. No clinical or biological abnormalities were observed.
[0128] With respect to pulmonary indications, DX-890 can be used to treat,
for example, Cystic Fibrosis (CF), Acute Respiratory Distress Syndrome
(ARDS) and Chronic Obstructive Pulmonary Disease (COPD).
[0129] There are also known correlations between the structure of DX-890
and its ability to bind to hNE. See, e.g., U.S. Pat. No. 5,663,143. U.S.
Pat. No. 5,663,143 also describes other Kunitz domains that inhibit
elastase. These and related domains (e.g., domains at least 70, 75, 80,
85, 90, or 95% identical) can also be used.
[0130] Exemplary Kunitz domains that inhibit plasma kallikrein are
described, for example, in U.S. Pat. No. 6,057,287.
[0131] Exemplary Kunitz domains that inhibit plasmin are described, for
example, in U.S. Pat. No. 6,103,499.
TABLE-US-00005
TABLE 3
Exemplary Amino Acids for hNE inhibitors
Some preferred Amino acids in hNE-inhibiting
Kunitz domains Position Allowed amino acids
at amino acid positions corresponding to
respective positions in BPTI
5 C
10 YSVN
11 TARQP
12 G
13 PAV
14 C
15 IV
16 AG
17 FILVM
18 F
19 PSQKR
20 R
21 YWF
30 C
31 QEV
32 TLP
33 F
34 VQP
35 Y
36 G
37 G
38 C
39 MQ
40 GA
41 N
42 G
43 N
45 F
51 C
55 C
[0132] "Protection against acute lung injury by intravenous or
intratracheal pretreatment with EPI-HNE4, a new potent neutrophil
elastase inhibitor." Delacourt C, Herigault S, Delclaux C, et al. Am J
Respir Cell Mol Biol 2002; 26:290-7 and Grimbert et al. (2003)
"Characteristics of EPI-hNE4 aerosol: a new elastase inhibitor for
treatment of cystic fibrosis" J Aerosol Med. 16(2):121-9.
Identifying Kunitz Domains and Other Protease Inhibitors
[0133] A variety of methods can be used to identify a protein that binds
to and/or inhibits a protease. These methods can be used to identify
natural and non-naturally occurring Kunitz domains that can be used as
components of the compounds described herein.
[0134] For example, a Kunitz domain can be identified from a library of
proteins in which each of a plurality of library members includes a
varied Kunitz domain. A variety of amino acids can be varied in the
domain. See, e.g., U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,663,143, and
U.S. Pat. No. 6,333,402. Kunitz domains can varied, e.g., using DNA
mutagenesis, DNA shuffling, chemical synthesis of oligonucleotides (e.g.,
using codons as subunits), and cloning of natural genes. See, e.g., U.S.
Pat. No. 5,223,409 and U.S. 2003-0129659.
[0135] The library can be an expression library that is used to produce
proteins. The proteins can be arrayed, e.g., using a protein array. U.S.
Pat. No. 5,143,854; De Wildt et al. (2000) Nat. Biotechnol. 18:989-994;
Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge (2000) Nucleic Acids
Res. 28, e3, I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763;
WO 0/98534, WO01/83827, WO02/12893, WO 00/63701, WO 01/40803 and WO
99/51773.
[0136] The proteins can also be displayed on a replicable genetic package,
e.g., in the form of a phage library such as a phage display, yeast
display library, ribosome display, or nucleic acid-protein fusion
library. See, e.g., U.S. Pat. No. 5,223,409; Smith (1985) Science
228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO
93/01288; WO 92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999)
J. Biol. Chem. 274:18218-30; Hoogenboom et al. (1998) Immunotechnology
4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et al.
(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths
et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol
226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.
(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology
9:1373-1377; Rebar et al. (1996) Methods Enzymol. 267:129-49; Hoogenboom
et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS
88:7978-7982 for examples of phage display and other methods. See, e.g.,
Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557 and WO 03/029456 for
examples of yeast cell display and other methods. See, e.g., Mattheakis
et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000)
Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol.
328:404-30. and Schaffitzel et al. (1999) J Immunol Methods.
231(1-2):119-35 for examples of ribosome display and other methods. See,
e.g., Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA
94:12297-12302, and U.S. Pat. No. 6,207,446 for examples of nucleic
acid-protein fusions. Such libraries can be screened in a high throughput
format. See, e.g., U.S. 2003-0129659.
[0137] Libraries of Kunitz domains can be generated by varying one or more
binding site loop amino acid residues using a Kunitz domain described
herein, e.g., a Kunitz domain having a framework described herein, e.g.,
a modified or naturally occurring framework region. In one embodiment,
the residues that are varied are varied among a plurality of amino acids.
The plurality is chosen such that lysine is unavailable.
Screening Display Libraries
[0138] This section describes exemplary methods of screening a display
library to identify a polypeptide that interacts with an elastase. These
methods can be modified to identify other polypeptides that interact with
other targets, e.g., other proteases or other proteins. The methods can
also be modified and used in combination with other types of libraries,
e.g., an expression library or a protein array, and so forth.
[0139] In an exemplary display library screen, a phage library is
contacted with and allowed to bind to the target elastase protein (e.g.,
an active or an inactivated form (e.g., mutant or chemically inactivated
protein) or a fragment thereof). To facilitate separation of binders and
non-binders in the screening process, it is often convenient to
immobilize the elastase on a solid support, although it is also possible
to first permit binding to elastase in solution and then segregate
binders from non-binders by coupling the target compound to a support. By
way of illustration, when incubated in the presence of the elastase,
phage displaying a polypeptide that interacts with elastase form a
complex with the elastase immobilized on a solid support whereas
non-binding phage remain in solution and may be washed away with buffer.
Bound phage may then be liberated from the elastase by a number of means,
such as changing the buffer to a relatively high acidic or basic pH
(e.g., pH 2 or pH 10), changing the ionic strength of the buffer, adding
denaturants, adding a competitor, adding a host cell which can be
infected, or other known means.
[0140] For example, to identify elastase-binding peptides, elastase can be
adsorbed to a solid surface, such as the plastic surface of wells in a
multi-well assay plate. Subsequently, an aliquot of a phage display
library is added to a well under appropriate conditions that maintain the
structure of the immobilized elastase and the phage, such as pH 6-7.
Phage in the libraries that display polypeptides that bind the
immobilized elastase are bound to the elastase and are retained in the
well. Non-binding phage can be removed. It is also possible to include a
blocking agent or competing ligand during the binding of the phage
library to the immobilized elastase.
[0141] Phage bound to the immobilized elastase may then be eluted by
washing with a buffer solution having a relatively strong acid pH (e.g.,
pH 2) or an alkaline pH (e.g., pH 8-9). The solutions of recovered phage
that are eluted from the elastase are then neutralized and may, if
desired, be pooled as an enriched mixed library population of phage
displaying elastase binding peptides. Alternatively the eluted phage from
each library may be kept separate as a library-specific enriched
population of elastase binders. Enriched populations of phage displaying
elastase binding peptides may then be grown up by standard methods for
further rounds of screening and/or for analysis of peptide displayed on
the phage and/or for sequencing the DNA encoding the displayed binding
peptide.
[0142] One of many possible alternative screening protocols uses elastase
target molecules that are biotinylated and that can be captured by
binding to streptavidin, for example, coated on particles.
[0143] Recovered phage may then be amplified by infection of bacterial
cells, and the screening process may be repeated with the new pool of
phage that is now depleted in non-elastase binders and enriched in
elastase binders. The recovery of even a few binding phage may be
sufficient to carry the process to completion. After a few rounds of
selection, the gene sequences encoding the binding moieties derived from
selected phage clones in the binding pool are determined by conventional
methods, revealing the peptide sequence that imparts binding affinity of
the phage to the target. An increase in the number of phage recovered
after each round of selection and the recovery of closely related
sequences indicate that the screening is converging on sequences of the
library having a desired characteristic.
[0144] After a set of binding polypeptides is identified, the sequence
information may be used to design other, secondary libraries. For
example, the secondary libraries can explore a smaller segment of
sequence space in more detail than the initial library. In some
embodiments, the secondary library includes proteins that are biased for
members having additional desired properties, e.g., sequences that have a
high percentage identity to a human protein.
[0145] Display technology can also be used to obtain polypeptides that are
specific to particular epitopes of a target. This can be done, for
example, by using competing non-target molecules that lack the particular
epitope or are mutated within the epitope, e.g., with alanine. Such
non-target molecules can be used in a negative selection procedure as
described below, as competing molecules when binding a display library to
the target, or as a pre-elution agent, e.g., to capture in a wash
solution dissociating display library
[0146] Iterative Selection. In one preferred embodiment, display library
technology is used in an iterative mode. A first display library is used
to identify one or more proteins that interacts with a target. These
identified proteins are then varied using a mutagenesis method to form a
second display library. Higher affinity proteins are then selected from
the second library, e.g., by using higher stringency or more competitive
binding and washing conditions.
[0147] In some implementations, the mutagenesis is targeted to regions
known or likely to be at the binding interface. Some exemplary
mutagenesis techniques include: error-prone PCR (Leung et al. (1989)
Technique 1:11-15), recombination, DNA shuffling using random cleavage
(Stemmer (1994) Nature 389-391; termed "nucleic acid shuffling"),
RACHITT.TM. (Coco et al. (2001) Nature Biotech. 19:354), site-directed
mutagenesis (Zoller et al. (1987) Nucl Acids Res 10:6487-6504), cassette
mutagenesis (Reidhaar-Olson (1991) Methods Enzymol. 208:564-586) and
incorporation of degenerate oligonucleotides (Griffiths et al. (1994)
EMBO J 13:3245). For Kunitz domains, many positions near the binding
interface are known. Such positions include, for example, positions 13,
16, 17, 18, 19, 31, 32, 34, and 39 with respect to the sequence of BPTI.
(according to the BPTI numbering in U.S. Pat. No. 6,333,402). Such
positions can be held constant and other positions can be varied or these
positions themselves may be varied.
[0148] In one example of iterative selection, the methods described herein
are used to first identify proteins from a display library that bind an
elastase with at least a minimal binding specificity for a target or a
minimal activity, e.g., an equilibrium dissociation constant for binding
of greater than 1 nM, 10 nM, or 100 nM. The nucleic acid sequences
encoding the initial identified proteins are used as a template nucleic
acid for the introduction of variations, e.g., to identify a second
protein ligand that has enhanced properties (e.g., binding affinity,
kinetics, or stability) relative to the initial protein ligand.
[0149] Off-Rate Selection. Since a slow dissociation rate can be
predictive of high affinity, particularly with respect to interactions
between proteins and their targets, the methods described herein can be
used to isolate proteins with a desired kinetic dissociation rate (i.e.
reduced) for a binding interaction to a target.
[0150] To select for slow dissociating proteins from a display library,
the library is contacted to an immobilized target, e.g., immobilized
elastase. The immobilized target is then washed with a first solution
that removes non-specifically or weakly bound biomolecules. Then the
immobilized target is eluted with a second solution that includes a
saturation amount of free target, i.e., replicates of the target that are
not attached to the particle. The free target binds to biomolecules that
dissociate from the target. Rebinding is effectively prevented by the
saturating amount of free target relative to the much lower concentration
of immobilized target.
[0151] The second solution can have solution conditions that are
substantially physiological or that are stringent. Typically, the
solution conditions of the second solution are identical to the solution
conditions of the first solution. Fractions of the second solution are
collected in temporal order to distinguish early from late fractions.
Later fractions include biomolecules that dissociate at a slower rate
from the target than biomolecules in the early fractions.
[0152] Further, it is also possible to recover display library members
that remain bound to the target even after extended incubation. These can
either be dissociated using chaotropic conditions or can be amplified
while attached to the target. For example, phage bound to the target can
be contacted to bacterial cells.
[0153] Selecting or Screening for Specificity. The display library
screening methods described herein can include a selection or screening
process that discards display library members that bind to a non-target
molecule, e.g., a protease other than elastase, such as trypsin. In one
embodiment, the non-target molecule is elastase that has been activated
by treatment with an irreversibly bound inhibitor, e.g., a covalent
inhibitor.
[0154] In one implementation, a so-called "negative selection" step or
"depletion" is used to discriminate between the target and a related, but
distinct or an unrelated non-target molecules. The display library or a
pool thereof is contacted to the non-target molecule. Members of the
sample that do not bind the non-target are collected and used in
subsequent selections for binding to the target molecule or even for
subsequent negative selections. The negative selection step can be prior
to or after selecting library members that bind to the target molecule.
[0155] In another implementation, a screening step is used. After display
library members are isolated for binding to the target molecule, each
isolated library member is tested for its ability to bind to a non-target
molecule (e.g., a non-target listed above). For example, a
high-throughput ELISA screen can be used to obtain this data. The ELISA
screen can also be used to obtain quantitative data for binding of each
library member to the target. The non-target and target binding data are
compared (e.g., using a computer and software) to identify library
members that specifically bind to the target.
Modifying and Varying Polypeptides
[0156] It is also possible to vary a protein described herein to obtain
useful variant protein that has similar or improved or altered
properties. Typically, a number of variants are possible. A variant can
be prepared and then tested, e.g., using a binding assay described herein
(such as fluorescence anisotropy).
[0157] One type of variant is a truncation of a ligand described herein or
isolated by a method described herein. In this example, the variant is
prepared by removing one or more amino acid residues of the ligand from
the N or C terminus. In some cases, a series of such variants is prepared
and tested. Information from testing the series is used to determine a
region of the ligand that is essential for binding the elastase protein.
A series of internal deletions or insertions can be similarly constructed
and tested. For Kunitz domains, it can be possible to remove, e.g.,
between one and five residues or one and three residues that are
N-terminal to C.sub.5, the first cysteine, and between one and five
residues or one and three residues that are C-terminal to C.sub.55, the
final cysteine, wherein each of the cysteines corresponds to a
respectively numbered cysteine in BPTI.
[0158] Another type of variant is a substitution. In one example, the
ligand is subjected to alanine scanning to identify residues that
contribute to binding activity. In another example, a library of
substitutions at one or more positions is constructed. The library may be
unbiased or, particularly if multiple positions are varied, biased
towards an original residue. In some cases, the substitutions are all
conservative substitutions.
[0159] Another type of variant includes one or more non-naturally
occurring amino acids. Such variant ligands can be produced by chemical
synthesis or modification. One or more positions can be substituted with
a non-naturally occurring amino acid. In some cases, the substituted
amino acid may be chemically related to the original naturally occurring
residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic)
or an isostere of the original residue.
[0160] It may also be possible to include non-peptide linkages and other
chemical modifications. For example, part or all of the ligand may be
synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon et al.
(1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends
Biotechnol. 13:132-4). See also other modifications discussed below.
Characterization of Binding Interactions
[0161] The binding properties of a protein (e.g., a polypeptide that
includes a Kunitz domain) can be readily assessed using various assay
formats. For example, the binding property of a protein can be measured
in solution by fluorescence anisotropy, which provides a convenient and
accurate method of determining a dissociation constant (K.sub.D) or
association constant (Ka) of the protein for a particular target. In one
such procedure, the protein to be evaluated is labeled with fluorescein.
The fluorescein-labeled protein is mixed in wells of a multi-well assay
plate with various concentrations of the particular target (e.g.,
elastase). Fluorescence anisotropy measurements are carried out using a
fluorescence polarization plate reader.
[0162] ELISA. The binding interactions can also be analyzed using an ELISA
assay. For example, the protein to be evaluated is contacted to a
microtitre plate whose bottom surface has been coated with the target,
e.g., a limiting amount of the target. The molecule is contacted to the
plate. The plate is washed with buffer to remove non-specifically bound
molecules. Then the amount of the protein bound to the plate is
determined by probing the plate with an antibody that recognizes the
protein. For example, the protein can include an epitope tag. The
antibody can be linked to an enzyme such as alkaline phosphatase, which
produces a colorimetric product when appropriate substrates are provided.
In the case where a display library member includes the protein to be
tested, the antibody can recognize a region that is constant among all
display library members, e.g., for a phage display library member, a
major phage coat protein.
[0163] Homogeneous Assays. A binding interaction between a protein and a
particular target can be analyzed using a homogenous assay, i.e., after
all components of the assay are added, additional fluid manipulations are
not required. For example, fluorescence energy transfer (FET) can be used
as a homogenous assay (see, for example, Lakowicz et al., U.S. Pat. No.
5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A
fluorophore label on the first molecule (e.g., the molecule identified in
the fraction) is selected such that its emitted fluorescent energy can be
absorbed by a fluorescent label on a second molecule (e.g., the target)
if the second molecule is in proximity to the first molecule. The
fluorescent label on the second molecule fluoresces when it absorbs to
the transferred energy. Since the efficiency of energy transfer between
the labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in the assay should be maximal.
An FET binding event can be conveniently measured through standard
fluorometric detection means well known in the art (e.g., using a
fluorimeter). By titrating the amount of the first or second binding
molecule, a binding curve can be generated to estimate the equilibrium
binding constant.
[0164] Surface Plasmon Resonance (SPR). A binding interaction between a
protein and a particular target can be analyzed using SPR. For example,
after sequencing of a display library member present in a sample, and
optionally verified, e.g., by ELISA, the displayed protein can be
produced in quantity and assayed for binding the target using SPR. SPR or
real-time Biomolecular Interaction Analysis (BIA) detects biospecific
interactions in real time, without labeling any of the interactants
(e.g., BIAcore). Changes in the mass at the binding surface (indicative
of a binding event) of the BIA chip result in alterations of the
refractive index of light near the surface (the optical phenomenon of
surface plasmon resonance (SPR)). The changes in the refractivity
generate a detectable signal, which are measured as an indication of
real-time reactions between biological molecules. Methods for using SPR
are described, for example, in U.S. Pat. No. 5,641,640; Raether (1988)
Surface Plasmons Springer Verlag; Sjolander, S, and Urbaniczky, C. (1991)
Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705.
[0165] Information from SPR can be used to provide an accurate and
quantitative measure of the equilibrium dissociation constant (K.sub.d),
and kinetic parameters, including k.sub.on and k.sub.off, for the binding
of a biomolecule to a target. Such data can be used to compare different
biomolecules. For example, proteins selected from a display library can
be compared to identify individuals that have high affinity for the
target or that have a slow k.sub.off. This information can also be used
to develop structure-activity relationship (SAR) if the biomolecules are
related. For example, if the proteins are all mutated variants of a
single parental antibody or a set of known parental antibodies, variant
amino acids at given positions can be identified that correlate with
particular binding parameters, e.g., high affinity and slow k.sub.off.
[0166] Additional methods for measuring binding affinities include
fluorescence polarization (FP) (see, e.g., U.S. Pat. No. 5,800,989),
nuclear magnetic resonance (NMR), and binding titrations (e.g., using
fluorescence energy transfer).
[0167] Other solution measures for studying binding properties include
fluorescence resonance energy transfer (FRET) and NMR.
Characterization of Elastase Inhibition
[0168] With respect to embodiments in which the compound includes a
polypeptide that has a Kunitz domain specific for elastase, it may be
useful to characterize the ability of the polypeptide to inhibit
elastase.
[0169] Kunitz domains can be screened for binding to elastase and for
inhibition of elastase proteolytic activity. Kunitz domains can be
selected for their potency and selectivity of inhibition of elastase. In
one example, elastase and its substrate are combined under assay
conditions permitting reaction of the protease with its substrate. The
assay is performed in the absence of the Kunitz domain, and in the
presence of increasing concentrations of the Kunitz domain. The
concentration of test compound at which 50% of the elastase activity is
inhibited by the test compound is the IC.sub.50 value (Inhibitory
Concentration) or EC.sub.50 (Effective Concentration) value for that
compound. Within a series or group of Kunitz domain, those having lower
IC.sub.50 or EC.sub.50 values are considered more potent inhibitors of
the elastase than those compounds having higher IC.sub.50 or EC.sub.50
values. Preferred compounds according to this aspect have an IC.sub.50
value of 100 nM or less as measured in an in vitro assay for inhibition
of elastase activity.
[0170] Kunitz domain can also be evaluated for selectivity toward
elastase. A test compound is assayed for its potency toward a panel of
serine proteases and other enzymes and an IC.sub.50 value is determined
for each peptide. A Kunitz domain that demonstrates a low IC.sub.50 value
for the elastase enzyme, and a higher IC.sub.50 value for other enzymes
within the test panel (e.g., trypsin, plasmin, kallikrein), is considered
to be selective toward elastase. Generally, a compound is deemed
selective if its IC.sub.50 value is at least one order of magnitude less
than the next smallest IC.sub.50 value measured in the panel of enzymes.
[0171] Specific methods for evaluating inhibition of elastase are
described in the Example below.
[0172] It is also possible to evaluate Kunitz domain activity in vivo or
in samples (e.g., pulmonary lavages) of subjects to which a compound
described herein has been administered.
Protease Targets
[0173] Proteases are involved in a wide variety of biological processes,
including inflammation and tissue injury. Serine proteases produced by
inflammatory cells, including neutrophils, are implicated in various
disorders, such as pulmonary emphysema. Neutrophil elastase is a serine
protease produced by polymorphonuclear leukocytes with activity against
extracellular matrix components and pathogens. Pulmonary emphysema is
characterized by alveolar destruction leading to a major impairment in
lung function.
[0174] A deficiency of a serine protease inhibitor, .alpha.1-protease
inhibitor (API, or .alpha.1-PI, formerly known as .alpha.-1 antitrypsin)
is a risk factor for the development of pulmonary emphysema (Laurell, C.
B. and Eriksson, S. (1963) Scand. J. Clin. Lab. Invest. 15:132-140;
Brantly, M. L., et al. (1988) Am. Rev. Respir. Dis. 138:327-336). API
deficiency may lead to uncontrolled activity of neutrophil elastase and
contribute to the destruction of lung tissue in pulmonary emphysema.
Likewise, API inactivation and chronic inflammation can lead to excess
neutrophil elastase activity and pathologic destruction of pulmonary
tissue.
[0175] Human neutrophil elastase consists of approximately 218 amino acid
residues, contains 2 asparagine-linked carbohydrate side chains, and is
joined together by 2 disulfide bonds (Sinha, S., et al. Proc. Nat. Acad.
Sci. 84: 2228-2232, 1987). It is normally synthesized in the developing
neutrophil as a proenzyme but stored in the primary granules in its
active form, ready with full enzymatic activity when released from the
granules, normally at sites of inflammation (Gullberg U, et al. Eur J
Haematol. 1997; 58:137-153; Borregaard N, Cowland J B. Blood. 1997;
89:3503-3521).
[0176] Other exemplary protease targets include: plasmin, kallikrein,
Factor VIIa, Factor XIa, thrombin, urokinase, and Factor IIa. Classes of
relevant proteases include: proteases associated with blood coagulation,
proteases associated with complement, proteases that digest extracellular
matrix components, proteases that digest basement membranes, and
proteases associated with endothelial cells. For example, the protease is
a serine protease.
Protein Production
[0177] Recombinant production of polypeptides. Standard recombinant
nucleic acid methods can be used to express a polypeptide component of a
compound described herein (e.g., a polypeptide that includes a Kunitz
domain). Generally, a nucleic acid sequence encoding the polypeptide is
cloned into a nucleic acid expression vector. If the polypeptide is
sufficiently small, e.g., the protein is a peptide of less than 50 amino
acids, the protein can be synthesized using automated organic synthetic
methods.
[0178] The expression vector for expressing the polypeptide can include a
segment encoding the polypeptide and regulatory sequences, for example, a
promoter, operably linked to the coding segment. Suitable vectors and
promoters are known to those of skill in the art and are commercially
available for generating the recombinant constructs of the present
invention. See, for example, the techniques described in Sambrook &
Russell, Molecular Cloning: A Laboratory Manual, 3.sup.rd Edition, Cold
Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al., Current
Protocols in Molecular Biology (Greene Publishing Associates and Wiley
Interscience, N.Y. (1989).
[0179] Scopes (1994) Protein Purification: Principles and Practice, New
York:Springer-Verlag and other texts provide a number of general methods
for purifying recombinant (and non-recombinant) proteins.
[0180] Synthetic production of peptides. The polypeptide component of a
compound can also be produced by synthetic means. See, e.g., Merrifield
(1963) J. Am. Chem. Soc., 85: 2149. For example, the molecular weight of
synthetic peptides or peptide mimetics can be from about 250 to about
8,0000 Daltons. A peptide can be modified, e.g., by attachment to a
moiety that increases the effective molecular weight of the peptide. If
the peptide is oligomerized, dimerized and/or derivatized, e.g., with a
hydrophilic polymer (e.g., to increase the affinity and/or activity of
the peptides), its molecular weights can be greater and can range
anywhere from about 500 to about 50,000 Daltons.
Pharmaceutical Compositions
[0181] Also featured is a composition, e.g., a pharmaceutically acceptable
composition, that includes a poly-PEGylated Kunitz domain. In one
embodiment, the Kunitz domain binds to a protease such as elastase,
plasmin, or kallikrein. As used herein, "pharmaceutical compositions"
encompass compounds (e.g., labeled compounds) for diagnostic (e.g., in
vivo imaging) use as well as compounds for therapeutic or prophylactic
use.
[0182] As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like
that are physiologically compatible. In one embodiment, the carrier is
other than water. Preferably, the carrier is suitable for intravenous,
intramuscular, subcutaneous, parenteral, spinal or epidermal
administration (e.g., by injection or infusion). Depending on the route
of administration, the active compound may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0183] A "pharmaceutically acceptable salt" refers to a salt that retains
the desired biological activity of the parent compound and does not
impart any undesired toxicological effects (see e.g., Berge, S. M., et
al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include those
derived from nontoxic inorganic acids, such as hydrochloric, nitric,
phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like,
as well as from nontoxic organic acids such as aliphatic mono- and
dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic
acids, aromatic acids, aliphatic and aromatic sulfonic acids and the
like. Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like, as
well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, procaine and the like.
[0184] The compositions of this invention may be in a variety of forms.
These include, for example, liquid, semi-solid and solid dosage forms,
such as liquid solutions (e.g., injectable and infusible solutions),
dispersions or suspensions, tablets, pills, powders, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions, such
as compositions similar to those used for administration of humans with
antibodies. The preferred mode of administration is parenteral (e.g.,
intravenous, subcutaneous, intraperitoneal, intramuscular). In a
preferred embodiment, the compound is administered by intravenous
infusion or injection. In another preferred embodiment, the compound is
administered by intramuscular or subcutaneous injection.
[0185] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other than
enteral and topical administration, usually by injection, and includes,
without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural and
intrasternal injection and infusion.
[0186] Pharmaceutical compositions typically must be sterile and stable
under the conditions of manufacture and storage. A pharmaceutical
composition can also be tested to insure it meets regulatory and industry
standards for administration. For example, endotoxin levels in the
preparation can be tested using the Limulus amebocyte lysate assay (e.g.,
using the kit from Bio Whittaker lot # 7L3790, sensitivity 0.125 EU/mL)
according to the USP 24/NF 19 methods. Sterility of pharmaceutical
compositions can be determined using thioglycollate medium according to
the USP 24/NF 19 methods. For example, the preparation is used to
inoculate the thioglycollate medium and incubated at 35.degree. C. for 14
or more days. The medium is inspected periodically to detect growth of a
microorganism.
[0187] The composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high drug
concentration. Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients enumerated
above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into a
sterile vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and freeze-drying that
yields a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of surfactants.
Prolonged absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0188] The poly-PEGylated Kunitz domains described herein can be
administered by a variety of methods known in the art. For many
applications, the route/mode of administration is intravenous injection
or infusion. For example, for therapeutic applications, the compound can
be administered by intravenous infusion at a rate of less than 30, 20,
10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m.sup.2 or 7 to
25 mg/m.sup.2. The route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the active
compound may be prepared with a carrier that will protect the compound
against rapid release, such as a controlled release formulation,
including implants, and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
and polylactic acid. Many methods for the preparation of such
formulations are patented or generally known. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel
Dekker, Inc., New York, 1978. Pharmaceutical formulation is a
well-established art, and is further described in Gennaro (ed.),
Remington: The Science and Practice of Pharmacy, 20.sup.th ed.,
Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 7.sup.th Ed.,
Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and
Kibbe (ed.), Handbook of Pharmaceutical Excipients American
Pharmaceutical Association, 3.sup.rd ed. (2000) (ISBN: 091733096X).
[0189] In certain embodiments, the composition may be orally administered,
for example, with an inert diluent or an assimilable edible carrier. The
compound (and other ingredients, if desired) may also be enclosed in a
hard or soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compound may be incorporated with excipients and used
in the form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. To administer a
compound by other than parenteral administration, it may be necessary to
coat the compound with, or co-administer the compound with, a material to
prevent its inactivation.
[0190] Pharmaceutical compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
pharmaceutical composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices disclosed in
U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880,
4,790,824, or 4,596,556. Examples of well-known implants and modules
useful in the present invention include: U.S. Pat. No. 4,487,603, which
discloses an implantable micro-infusion pump for dispensing medication at
a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic
device for administering medicants through the skin; U.S. Pat. No.
4,447,233, which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which
discloses a variable flow implantable infusion apparatus for continuous
drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat. No.
4,475,196, which discloses an osmotic drug delivery system. Of course,
many other such implants, delivery systems, and modules are also known.
[0191] In certain embodiments, the compound can be formulated to ensure
proper distribution in vivo. For example, the blood-brain barrier (BBB)
excludes many highly hydrophilic compounds. To ensure that the
therapeutic compounds of the invention cross the BBB (if desired), they
can be formulated, for example, in liposomes. For methods of
manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;
and 5,399,331. The liposomes may comprise one or more moieties that are
selectively transported into specific cells or organs, thus enhance
targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.
Pharmacol. 29:685).
[0192] Also within the scope of the invention are kits comprising
poly-PEGylated Kunitz domain and instructions for use, e.g., treatment,
prophylactic, or diagnostic use. In one embodiment, the kit includes (a)
the compound, e.g., a composition that includes the compound, 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 compound for the
methods described herein. For example, in the case of a Kunitz domain
that inhibits elastase activity, the informational material describes
methods for administering the compound to reduce elastase activity or to
treat or prevent a pulmonary disorder (e.g., CF or COPD), an inflammatory
disorder (e.g., IBD), or a disorder characterized by excessive elastase
activity.
[0193] In one embodiment, the informational material can include
instructions to administer the compound in a suitable manner, e.g., in a
suitable dose, dosage form, or mode of administration (e.g., a dose,
dosage form, or mode of administration described herein). In another
embodiment, the informational material can include instructions for
identifying a suitable subject, e.g., a human, e.g., a human having, or
at risk for a disorder characterized by excessive elastase activity. 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. The
informational material of the kits is not limited in its form. In many
cases, the informational material, e.g., instructions, is provided in
printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a
label or printed sheet. However, the informational material can also be
provided in other formats, such as Braille, computer readable material,
video recording, or audio recording. In another embodiment, the
informational material of the kit is a link or contact information, e.g.,
a physical address, email address, hyperlink, website, or telephone
number, where a user of the kit can obtain substantive information about
the compound and/or its use in the methods described herein. Of course,
the informational material can also be provided in any combination of
formats.
[0194] In addition to the compound, the composition of the kit can include
other ingredients, such as a solvent or buffer, a stabilizer or a
preservative, and/or a second agent for treating a condition or disorder
described herein, e.g. a pulmonary (e.g., CF or COPD) or inflammatory
(e.g., IBD or RA) disorder. Alternatively, the other ingredients can be
included in the kit, but in different compositions or containers than the
compound. In such embodiments, the kit can include instructions for
admixing the compound and the other ingredients, or for using the
compound together with the other ingredients.
[0195] The compound can be provided in any form, e.g., liquid, dried or
lyophilized form. It is preferred that the compound be substantially pure
and/or sterile. When the compound is provided in a liquid solution, the
liquid solution preferably is an aqueous solution, with a sterile aqueous
solution being preferred. When the compound is 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.
[0196] The kit can include one or more containers for the composition
containing the compound. 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 compound. For
example, the kit includes a plurality of syringes, ampules, foil packets,
or blister packs, each containing a single unit dose of the compound. The
containers of the kits can be air tight, waterproof (e.g., impermeable to
changes in moisture or evaporation), and/or light-tight.
[0197] In one embodiment wherein the compound contains a polypeptide that
binds to an elastase, the instructions for diagnostic applications
include the use of the compound to detect elastase, in vitro, e.g., in a
sample, e.g., a biopsy or cells from a patient having a pulmonary
disorder, or in vivo. In another embodiment, the instructions for
therapeutic applications include suggested dosages and/or modes of
administration in a patient with a pulmonary disorder. The kit can
further contain a least one additional reagent, such as a diagnostic or
therapeutic agent, e.g., a diagnostic or therapeutic agent as described
herein, and/or one or more additional agents to treat the pulmonary
disorder (e.g., another elastase inhibitor), formulated as appropriate,
in one or more separate pharmaceutical preparations.
Treatments
[0198] A poly-PEGylated Kunitz domain has therapeutic and prophylactic
utilities.
[0199] In one embodiment, poly-PEGylated Kunitz domain inhibits an
elastase, e.g., a neutrophil elastase. The compound can be administered
to a subject to treat, prevent, and/or diagnose a variety of disorders,
such as diseases characterized by unwanted or aberrant elastase activity.
For example, the disease or disorder can be characterized by enhanced
elastolytic activity of neutrophils. The disease or disorder may result
from an increased neutrophil burden on a tissue, e.g., an epithelial
tissue such as the epithelial surface of the lung. For example, the
polypeptide that inhibits elastase can be used to treat or prevent
pulmonary diseases such as cystic fibrosis (CF) or chronic obstructive
pulmonary disorder (COPD), e.g., emphysema. The compound can also be
administered to cells, tissues, or organs in culture, e.g. in vitro or ex
vivo.
[0200] Poly-PEGylated Kunitz domains that inhibit other proteases can also
be used to treat or prevent disorders associated with the activity of
such other respective proteases.
[0201] As used herein, the term "treat" or "treatment" is defined as the
application or administration of poly-PEGylated Kunitz domain, alone or
in combination with, a second agent to a subject, e.g., a patient, or
application or administration of the agent to an isolated tissue or cell,
e.g., cell line, from a subject, e.g., a patient, who has a disorder
(e.g., a disorder as described herein), a symptom of a disorder or a
predisposition toward a disorder, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect the
disorder, the symptoms of the disorder or the predisposition toward the
disorder. Treating a cell refers to the inhibition, ablation, killing of
a cell in vitro or in vivo, or otherwise reducing capacity of a cell,
e.g., an aberrant cell, to mediate a disorder, e.g., a disorder as
described herein (e.g., a pulmonary disorder). In one embodiment,
"treating a cell" refers to a reduction in the activity and/or
proliferation of a cell, e.g., a leukocyte or neutrophil. Such reduction
does not necessarily indicate a total elimination of the cell, but a
reduction, e.g., a statistically significant reduction, in the activity
or the number of the cell.
[0202] As used herein, an amount of a poly-PEGylated Kunitz domain
effective to treat a disorder, or a "therapeutically effective amount"
refers to an amount of the compound which is effective, upon single or
multiple dose administration to a subject, in treating a subject, e.g.,
curing, alleviating, relieving or improving at least one symptom of a
disorder in a subject to a degree beyond that expected in the absence of
such treatment. For example, the disorder can be a pulmonary disorder,
e.g., a pulmonary disorder described herein.
[0203] A "locally effective amount" refers to the amount (e.g.,
concentration) of the compound which is effective at detectably
modulating activity of a target protein (e.g., elastase) in a tissue,
e.g., in a region of the lung exposed to elastase, or a
elastase-producing cell, such as a neutrophil. Evidence of modulation can
include, e.g., increased amount of substrate, e.g., reduced proteolysis
of the extracellular matrix.
[0204] As used herein, an amount of poly-PEGylated Kunitz domain effective
to prevent a disorder, or a "a prophylactically effective amount" of the
compound refers to an amount of an elastase-binding compound, e.g., a
polypeptide-polymer compound described herein, which is effective, upon
single- or multiple-dose administration to the subject, in preventing or
delaying the occurrence of the onset or recurrence of a disorder, e.g., a
pulmonary disorder.
[0205] The terms "induce," "inhibit," "potentiate," "elevate," "increase,"
"decrease" or the like, e.g., which denote quantitative differences
between two states, refer to a difference, e.g., a statistically
significant difference (e.g., P<0.05, 0.02, or 0.005), between the two
states.
[0206] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single bolus may
be administered, several divided doses may be administered over time or
the dose may be proportionally reduced or increased as indicated by the
exigencies of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used herein
refers to physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity of
active compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier.
[0207] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a compound described herein is
0.1-20 mg/kg, more preferably 1-10 mg/kg. The compound can be
administered by intravenous infusion at a rate of less than 20, 10, 5, or
1 mg/min to reach a dose of about 1 to 50 mg/m.sup.2 or about 5 to 20
mg/m.sup.2. It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage regimens
should be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising the
administration of the compositions, and that dosage ranges set forth
herein are only exemplary.
[0208] A pharmaceutical composition may include a "therapeutically
effective amount" or a "prophylactically effective amount" of a compound
described herein, e.g., a compound that includes a polypeptide that binds
and inhibits a protease (e.g., elastase). A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic result. A therapeutically
effective amount of the composition may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of the compound to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the composition is outweighed by the
therapeutically beneficial effects. A "therapeutically effective dosage"
preferably inhibits a measurable parameter, e.g., an increase in
pulmonary function, relative to untreated subjects. The ability of a
compound to inhibit a measurable parameter can be evaluated in an animal
model system predictive of efficacy in a human disorder. Alternatively,
this property of a composition can be evaluated by examining the ability
of the compound to inhibit, such inhibition in vitro by assays known to
the skilled practitioner, e.g., an assay described herein.
[0209] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve the
desired prophylactic result. Typically, since a prophylactic dose is used
in subjects prior to or at an earlier stage of disease, the
prophylactically effective amount may be less than the therapeutically
effective amount.
[0210] As used herein, the term "subject" is intended to include human and
non-human animals. The term "non-human animals" of the invention includes
all vertebrates, e.g., non-mammals (such as chickens, amphibians,
reptiles) and mammals, such as non-human primates, sheep, dog, cow, pig,
etc.
[0211] In one embodiment, the subject is a human subject. Alternatively,
the subject can be a non-human mammal expressing a human neutrophil
elastase or an endogenous non-human neutrophil elastase protein or an
elastase-like antigen to which an elastase-binding compound cross-reacts.
A compound of the invention can be administered to a human subject for
therapeutic purposes (discussed further below). Moreover, an
elastase-binding compound can be administered to a non-human mammal
expressing the elastase-like antigen to which the compound binds (e.g., a
primate, pig or mouse) for veterinary purposes or as an animal model of
human disease. Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of the compound (e.g., testing of
dosages and time courses of administration).
[0212] The subject method can be used on cells in culture, e.g. in vitro
or ex vivo. The method can be performed on cells present in a subject, as
part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in
vivo embodiments, the contacting step is effected in a subject and
includes administering the elastase-binding compound to the subject under
conditions effective to permit both binding of the compound to a target
(e.g., an elastase) in the subject.
[0213] The compounds which inhibit elastase can reduce elastase-mediated
degradation and its sequalae, such as persistent infection and
inflammation, leading to destruction of tissue (e.g., destruction of
airway epithelium).
[0214] Methods of administering compounds are described in "Pharmaceutical
Compositions". Suitable dosages of the compounds used will depend on the
age and weight of the subject and the particular drug used. The compounds
can be used as competitive agents to inhibit, reduce an undesirable
interaction, e.g., between a natural or pathological agent and the
elastase, e.g., between the extracellular matrix and elastase.
[0215] In one embodiment, the compounds are used to kill or ablate cells
that express elastase in vivo. The compounds can be used by themselves or
conjugated to an agent, e.g., a cytotoxic drug, radioisotope. This method
includes: administering the compound alone or attached to a cytotoxic
drug, to a subject requiring such treatment.
[0216] The terms "cytotoxic agent" and "cytostatic agent" refer to agents
that have the property of inhibiting the growth or proliferation (e.g., a
cytostatic agent), or inducing the killing of cells.
[0217] Poly-PEGylated Kunitz domain may also be used to deliver a variety
of drugs including therapeutic drugs, a compound emitting radiation,
molecules of plants, fungal, or bacterial origin, biological proteins,
and mixtures thereof. For example, the Kunitz domain can be used to
target the payload to a region of a subject which includes a protease
that specifically interacts with the Kunitz domain.
[0218] Enzymatically active toxins and fragments thereof are exemplified
by diphtheria toxin A fragment, nonbinding active fragments of diphtheria
toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, .alpha.-sacrin, certain Aleurites fordii
proteins, certain Dianthin proteins, Phytolacca americana proteins (PAP,
PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin, Saponaria
officinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin, and
enomycin. Procedures for preparing enzymatically active polypeptides of
the immunotoxins are described in WO 84/03508 and WO 85/03508. Examples
of cytotoxic moieties that can be conjugated to the antibodies include
adriamycin, chlorambucil, daunomycin, methotrexate, neocarzinostatin, and
platinum.
[0219] In the case of polypeptide toxins, recombinant nucleic acid
techniques can be used to construct a nucleic acid that encodes the
polypeptide including a Kunitz domain and the cytotoxin (or a polypeptide
component thereof) as translational fusions. The recombinant nucleic acid
is then expressed, e.g., in cells and the encoded fusion polypeptide
isolated. Then the fusion protein is physically associated with a moiety
that increases the molecular weight of the compound, e.g., to stabilize
half-life in vivo, and then attached to a moiety (e.g., a polymer).
[0220] Procedures for conjugating proteins with the cytotoxic agents have
been previously described. For conjugating chlorambucil with proteins,
see, e.g., Flechner (1973) European Journal of Cancer, 9:741-745; Ghose
et al. (1972) British Medical Journal, 3:495-499; and Szekerke, et al.
(1972) Neoplasma, 19:211-215. For conjugating daunomycin and adriamycin
to proteins, see, e.g., Hurwitz, E. et al. (1975) Cancer Research,
35:1175-1181 and Arnon et al. (1982) Cancer Surveys, 1:429-449. For
preparing protein-ricin conjugates, see, e.g., U.S. Pat. No. 4,414,148
and by Osawa, T., et al. (1982) Cancer Surveys, 1:373-388 and the
references cited therein. Coupling procedures as also described in EP 226
419.
[0221] Also encompassed by the present invention is a method of killing or
ablating which involves using the compound for prophylaxis. For example,
these materials can be used to prevent or delay development or
progression of a lung disease.
[0222] Use of the therapeutic methods of the present invention to treat
lung diseases has a number of benefits. Since the polypeptide portion of
the compound specifically recognizes elastase, other tissue is spared and
high levels of the agent are delivered directly to the site where therapy
is required. Treatment in accordance with the present invention can be
effectively monitored with clinical parameters. Alternatively, these
parameters can be used to indicate when such treatment should be
employed.
Pulmonary Disorders and Methods and Formulations
[0223] hNE inhibitor polypeptides that are physically associated with a
moiety (e.g., a polymer) can be used to treat pulmonary disorders such as
emphysema, cystic fibrosis, COPD, bronchitis, pulmonary hypertension,
acute respiratory distress syndrome, interstitial lung disease, asthma,
smoke intoxication, bronchopulmonary dysplasia, pneumonia, thermal
injury, and lung transplant rejection.
[0224] Cystic Fibrosis. Cystic fibrosis (CF) is a genetic disease
affecting approximately 30,000 children and adults in the United States.
A defect in the CF gene causes the body to produce an abnormally thick,
sticky mucus that clogs the lungs and leads to life-threatening lung
infections. A diagnostic for the genetic disorder includes a sweat test
which can include measuring chloride concentration in sweat collected on
gauze or filter paper, measuring sodium concentration in sweat collected
on gauze or filter paper, and pilocarpine delivery and current density in
sweat collection. The gene that causes CF has been identified and a
number of mutations in the gene are known.
[0225] In one embodiment, a hNE inhibitor polypeptide that is physically
associated with a moiety (e.g., a polymer) is used to ameliorate at least
one symptom of CF, e.g., to reduce pulmonary lesions in the lungs of a CF
patient.
[0226] This compound can also be used to ameliorate at least one symptom
of a chronic obstructive pulmonary disease (COPD). Emphysema, along with
chronic bronchitis, is part of chronic obstructive pulmonary disease
(COPD). It is a serious lung disease and is progressive, usually
occurring in elderly patients. COPD causes over-inflation of structures
in the lungs known as alveoli or air sacs. The walls of the alveoli break
down resulting in a decrease in the respiratory ability of the lungs.
Patients with this disease may first experience shortness of breath and
cough. One clinical index for evaluating COPD is the destructive index
which measures a measure of alveolar septal damage and emphysema, and has
been proposed as a sensitive index of lung destruction that closely
reflects functional abnormalities, especially loss of elastic recoil.
See, e.g., Am Rev Respir Dis 1991 July; 144(1):156-9. The compound can be
used to reduce the destructive index in a patient, e.g., a statistically
significant amount, e.g., at least 10, 20, 30, or 40% or at least to
within 50, 40, 30, or 20% of normal of a corresponding age and
gender-matched individual.
[0227] In one aspect, the invention provides a composition that
poly-PEGylated Kunitz domain that is an hNE inhibitor for treatment of a
pulmonary disorder (e.g., cystic fibrosis, COPD). The composition can be
formulated for inhalation or other mode of pulmonary delivery.
Accordingly, the compounds described herein can be administered by
inhalation to pulmonary tissue. The term "pulmonary tissue" as used
herein refers to any tissue of the respiratory tract and includes both
the upper and lower respiratory tract, except where otherwise indicated.
A hNE inhibitor polypeptide that is physically associated with a moiety
(e.g., a polymer) can be administered in combination with one or more of
the existing modalities for treating pulmonary diseases.
[0228] In one example the compound is formulated for a nebulizer. In one
embodiment, the compound can be stored in a lyophilized form (e.g., at
room temperature) and reconstituted in solution prior to inhalation. In
another embodiment, the compound is stored at an acidic pH (e.g., a pH
less than 5, 4, or 3) and then combined with a neutralizing buffer having
a basic pH prior to inhalation.
[0229] It is also possible to formulate the compound for inhalation using
a medical device, e.g., an inhaler. See, e.g., U.S. Pat. Nos. 6,102,035
(a powder inhaler) and 6,012,454 (a dry powder inhaler). The inhaler can
include separate compartments for the active compound at an acidic pH and
the neutralizing buffer and a mechanism for combining the compound with a
neutralizing buffer immediately prior to atomization. In one embodiment,
the inhaler is a metered dose inhaler.
[0230] The three common systems used to deliver drugs locally to the
pulmonary air passages include dry powder inhalers (DPIs), metered dose
inhalers (MDIs) and nebulizers. MDIs, the most popular method of
inhalation administration, may be used to deliver medicaments in a
solubilized form or as a dispersion. Typically MDIs comprise a Freon or
other relatively high vapor pressure propellant that forces aerosolized
medication into the respiratory tract upon activation of the device.
Unlike MDIs, DPIs generally rely entirely on the inspiratory efforts of
the patient to introduce a medicament in a dry powder form to the lungs.
Nebulizers form a medicament aerosol to be inhaled by imparting energy to
a liquid solution. Direct pulmonary delivery of drugs during liquid
ventilation or pulmonary lavage using a fluorochemical medium has also
been explored. These and other methods can be used to deliver a hNE
inhibitor polypeptide that is physically associated with a moiety (e.g.,
a polymer).
[0231] For example, for administration by inhalation, poly-PEGylated
Kunitz domain that inhibits hNE are delivered in the form of an aerosol
spray from pressured container or dispenser which contains a suitable
propellant or a nebulizer. The compound may be in the form of a dry
particle or as a liquid. Particles that include the compound can be
prepared, e.g., by spray drying, by drying an aqueous solution of the
poly-PEGylated Kunitz domain that inhibits hNE with a charge neutralizing
agent and then creating particles from the dried powder or by drying an
aqueous solution in an organic modifier and then creating particles from
the dried powder.
[0232] The compound may be conveniently delivered in the form of an
aerosol spray presentation from pressurized packs or a nebulizer, with
the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dielilorotetrafluoroctliane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol, the dosage unit
may be determined by providing a valve to deliver a metered amount.
Capsules and cartridges for use in an inhaler or insufflator may be
formulated containing a powder mix of the poly-PEGylated Kunitz domain
that inhibits hNE and a suitable powder base such as lactose or starch,
if the particle is a formulated particle. In addition to the formulated
or unformulated compound, other materials such as 100% DPPC or other
surfactants can be mixed with the poly-PEGylated Kunitz domain that
inhibit hNE to promote the delivery and dispersion of formulated or
unformulated compound. Methods of preparing dry particles are described,
for example, in PCT Publication WO 02/32406.
[0233] The poly-PEGylated Kunitz domain that inhibits hNE, e.g., as dry
aerosol particles, when administered can be rapidly absorbed and can
produce a rapid local or systemic therapeutic result. Administration can
be tailored to provide detectable activity within 2 minutes, 5 minutes, 1
hour, or 3 hours of administration. In some embodiments, the peak
activity can be achieved even more quickly, e.g., within one half hour or
even within ten minutes. Alternatively, a poly-PEGylated Kunitz domain
that inhibits hNE can be formulated for longer biological half-life can
be used as an alternative to other modes of administration, e.g., such
that the compound enters circulation from the lung and is distributed to
other organs or to a particular target organ.
[0234] In one embodiment, poly-PEGylated Kunitz domain that inhibits hNE
is delivered in an amount such that at least 5% of the mass of the
polypeptide is delivered to the lower respiratory tract or the deep lung.
Deep lung has an extremely rich capillary network. The respiratory
membrane separating capillary lumen from the alveolar air space is very
thin (.ltoreq.6 Tm) and extremely permeable. In addition, the liquid
layer lining the alveolar surface is rich in lung surfactants. In other
embodiments, at least 2%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or
80% of the composition of a poly-PEGylated Kunitz domain that inhibits
hNE is delivered to the lower respiratory tract or to the deep lung.
Delivery to either or both of these tissues results in efficient
absorption of the compound and high bioavailability. In one embodiment,
the compound is provided in a metered dose using, e.g., an inhaler or
nebulizer. For example, the compound is delivered in a dosage unit form
of at least about 0.02, 0.1, 0.5, 1, 1.5, 2, 5, 10, 20, 40, or 50 mg/puff
or more.
[0235] The percent bioavailability can be calculated as follows: the
percent bioavailability=(AUC.sub.non-invasive/AUC.sub.i.v. or
s.c.).times.(dose.sub.i.v. or s.c./dose.sub.non-invasive).times.100.
[0236] Although not necessary, delivery enhancers such as surfactants can
be used to further enhance pulmonary delivery. A "surfactant" as used
herein refers to a compound having a hydrophilic and lipophilic moiety,
which promotes absorption of a drug by interacting with an interface
between two immiscible phases. Surfactants are useful in the dry
particles for several reasons, e.g., reduction of particle agglomeration,
reduction of macrophage phagocytosis, etc. When coupled with lung
surfactant, a more efficient absorption of the compound can be achieved
because surfactants, such as DPPC, will greatly facilitate diffusion of
the compound. Surfactants are well known in the art and include but are
not limited to phosphoglycerides, e.g., phosphatidylcholines,
L-alpha-phosphatidylcholine dipalmitoyl (DPPC) and diphosphatidyl
glycerol (DPPG); hexadecanol; fatty acids; polyethylene glycol (PEG);
polyoxyethylene-9-; auryl ether; palmitic acid; oleic acid; sorbitan
trioleate (Span 85); glycocholate; surfactin; poloxomer; sorbitan fatty
acid ester; sorbitan trioleate; tyloxapol; and phospholipids.
IBD and Methods and Formulations Therefor
[0237] In one embodiment, a poly-PEGylated Kunitz domain that inhibits hNE
is used to ameliorate at least one symptom of an inflammatory bowel
disease, e.g., ulcerative colitis or Crohn's disease.
[0238] Inflammatory bowel diseases (IBD) are generally chronic, relapsing
intestinal inflammation. IBD refers to two distinct disorders, Crohn's
disease and ulcerative colitis (UC). Both diseases may involve either a
dysregulated immune response to GI tract antigens, a mucosal barrier
breach, and/or an adverse inflammatory reaction to a persistent
intestinal infection (see, e.g., MacDermott, R. P., J Gastroenterology,
31:907:-916 (1996)).
[0239] In patients with IBD, ulcers and inflammation of the inner lining
of the intestines lead to symptoms of abdominal pain, diarrhea, and
rectal bleeding. Ulcerative colitis occurs in the large intestine, while
in Crohn's, the disease can involve the entire GI tract as well as the
small and large intestines. For most patients, IBD is a chronic condition
with symptoms lasting for months to years. The clinical symptoms of IBD
are intermittent rectal bleeding, crampy abdominal pain, weight loss and
diarrhea. Diagnosis of IBD is based on the clinical symptoms, the use of
a barium enema, but direct visualization (sigmoidoscopy or colonoscopy)
is the most accurate test.
[0240] Symptoms of IBD include, for example, abdominal pain, diarrhea,
rectal bleeding, weight loss, fever, loss of appetite, and other more
serious complications, such as dehydration, anemia and malnutrition. A
number of such symptoms are subject to quantitative analysis (e.g. weight
loss, fever, anemia, etc.). Some symptoms are readily determined from a
blood test (e.g. anemia) or a test that detects the presence of blood
(e.g. rectal bleeding). A clinical index can also be used to monitor IBD
such as the Clinical Activity Index for Ulcerative Colitis. See also,
e.g., Walmsley et al. Gut. 1998 July; 43(1):29-32 and Jowett et al.
(2003) Scand J Gastroenterol. 38(2):164-71.
[0241] In one embodiment, administration of the compound to a subject
having or predisposed to having ulcerative colitis causes amelioration of
the index, e.g., a statistically significant change in the index. The
compound includes hNE inhibitor polypeptide that is physically associated
with a moiety (e.g., a hydrophilic polymer)
[0242] In one embodiment, administration of the compound to a subject
having or predisposed to having IBD causes amelioration of at least one
symptom of IBD.
[0243] Crohn's disease, an idiopathic inflammatory bowel disease, is
characterized by chronic inflammation at various sites in the
gastrointestinal tract. While Crohn's disease most commonly affects the
distal ileum and colon, it may manifest itself in any part of the
gastrointestinal tract from the mouth to the anus and perianal area. The
prognosis and diagnosis of Crohn's disease can be measured using a
clinical index, e.g., Crohn's Disease Activity Index. See, e.g., American
Journal of Natural Medicine, July/August 1997, and Best W R, et al.,
"Development of a Crohn's disease activity index." Gastroenterology
70:439-444, 1976. In one embodiment, administration of the compound to a
subject having or predisposed to having Crohn's disease causes
amelioration of the index, e.g., a statistically significant change in
the index, or amelioration of at least one symptom of Crohn's disease.
[0244] Accordingly, in one aspect, the invention provides a composition
that includes poly-PEGylated Kunitz domain that inhibits hNE for
treatment of a bowel disease (e.g., a colitis such as ulcerative colitis,
Crohn's disease or IBP) or other gastrointestinal or rectal disease. The
composition can be formulated as a suppository. Suppositories can be
formulated with base ingredients such as waxes, oils, and fatty alcohols
with characteristics of remaining in solid state at room temperatures and
melting at body temperatures. The active ingredients of this invention
with or without optional therapeutic ingredients, like hydrocortisone
(1.0%), topical anesthetics like benzocaine (1.0 to 6.0%) or others as
already listed may be prepared at appropriate pH values; for example pH 5
liquid fatty alcohols, such as oleyl alcohol (range 45% to 65%) or solid
higher fatty alcohols like cetyl or stearyl alcohol (30% to 50%). The
base ingredients are well known in the art of this industry. See, e.g.,
U.S. Pat. Nos. 4,945,084 and 5,196,405.
[0245] The composition may also be used as an active ingredient in creams,
lotions, ointments, sprays, pads, patches, enemas, foams and
suppositories and others or in delivery vehicles such as
micro-encapsulation in liposomes or glycospheres. Other delivery
technologies include microsponges or the substitute cell membrane
(Completech.TM.) which entrap the active ingredients for both protection
and for slower release. Rectal foams can be prepared as topical aerosol
compositions may also be used, e.g., to treat (ulcerative colitis, Crohns
colitis, and others).
Diagnostic Uses
[0246] A poly-PEGylated Kunitz domain has diagnostic utilities.
[0247] In one aspect, the present invention provides a diagnostic method
for detecting the presence of a elastase protein, in vitro (e.g., a
biological sample, such as tissue, biopsy or in vivo (e.g., in vivo
imaging in a subject). The method includes: (i) contacting a sample with
a poly-PEGylated Kunitz domain, e.g., a Kunitz domain that binds to a
target protease, e.g., elastase, plasmin, or kallikrein; and (ii)
detecting formation of a complex between the elastase ligand and the
sample. The method can also include contacting a reference sample (e.g.,
a control sample) with the ligand, and determining the extent of
formation of the complex between the ligand and the sample relative to
the same for the reference sample. A change, e.g., a statistically
significant change, in the formation of the complex in the sample or
subject relative to the control sample or subject can be indicative of
the presence of elastase in the sample.
[0248] Another method includes: (i) administering the compound to a
subject; and (iii) detecting formation of a complex between the compound,
and the target protease. The detecting can include determining location
or time of formation of the complex.
[0249] The compound can be directly or indirectly labeled with a
detectable substance to facilitate detection of the bound or unbound
antibody. Suitable detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials and
radioactive materials.
[0250] Complex formation between the compound and target protease can be
detected by measuring or visualizing either the ligand bound to the
target protease or unbound ligand. Conventional detection assays can be
used, e.g., an enzyme-linked immunosorbent assays (ELISA), a
radioimmunoassay (RIA) or tissue immunohistochemistry. Further to
labeling the compound, the presence of target protease can be assayed in
a sample by a competition immunoassay utilizing standards labeled with a
detectable substance and an unlabeled protease ligand. In one example of
this assay, the biological sample, the labeled standards and the compound
are combined and the amount of labeled standard bound to the unlabeled
ligand is determined. The amount of target protease in the sample is
inversely proportional to the amount of labeled standard bound to the
compound.
[0251] Fluorophore and chromophore labeled protein ligands can be
prepared. A variety of suitable fluorescers and chromophores are
described by Stryer (1968) Science, 162:526 and Brand, L. et al. (1972)
Annual Review of Biochemistry, 41:843-868. The protein ligands can be
labeled with fluorescent chromophore groups by conventional procedures
such as those disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and
4,376,110. One group of fluorescers having a number of the desirable
properties described above is the xanthene dyes, which include the
fluoresceins and rhodamines. Another group of fluorescent compounds are
the naphthylamines. Once labeled with a fluorophore or chromophore, the
protein ligand can be used to detect the presence or localization of the
a target protease in a sample, e.g., using fluorescent microscopy (such
as confocal or deconvolution microscopy).
[0252] Protein Arrays. The compound can also be immobilized on a protein
array. The protein array can be used as a diagnostic tool, e.g., to
screen medical samples (such as isolated cells, blood, sera, biopsies,
and the like). Methods of producing polypeptide arrays are described,
e.g., above.
[0253] In vivo Imaging. In still another embodiment, the invention
provides a method for detecting the presence of a target protease or a
target protease-expressing tissue in vivo. The method includes (i)
administering to a subject (e.g., a patient having a pulmonary or
respiratory disorder) a compound that includes a Kunitz domain and that
is polyPEGylated, conjugated to a detectable marker; (ii) exposing the
subject to a means for detecting said detectable marker to the target
protease-expressing tissues or cells. For example, the subject is imaged,
e.g., by NMR or other tomographic means.
[0254] Examples of labels useful for diagnostic imaging in accordance with
the present invention include radiolabels such as .sup.131I, .sup.111In,
.sup.123I, .sup.99mTc, .sup.32P, .sup.125I, .sup.3H, .sup.14C, and
.sup.188Rh, fluorescent labels such as fluorescein and rhodamine, nuclear
magnetic resonance active labels, positron emitting isotopes detectable
by a positron emission tomography ("PET") scanner, chemiluminescers such
as luciferin, and enzymatic markers such as peroxidase or phosphatase.
Short-range radiation emitters, such as isotopes detectable by
short-range detector probes can also be employed. The compound that
includes the Kunitz domain can be labeled with such reagents using known
techniques. For example, see Wensel and Meares (1983) Radioimmunoimaging
and Radioimmunotherapy, Elsevier, N.Y. for techniques relating to the
radiolabeling of proteins and D. Colcher et al. (1986) Meth. Enzymol.
121: 802-816.
[0255] A radiolabeled compound of this invention can also be used for in
vitro diagnostic tests. The specific activity of an isotopically-labeled
compound depends upon the half-life, the isotopic purity of the
radioactive label, and how the label is incorporated into the compound.
[0256] Procedures for labeling polypeptides (e.g., the polypeptide portion
of the compound) with the radioactive isotopes (such as .sup.14C,
.sup.3H, .sup.35S, .sup.125I, .sup.32P, .sup.131I) are generally known.
For example, tritium labeling procedures are described in U.S. Pat. No.
4,302,438. Iodinating, tritium labeling, and .sup.35S labeling
procedures, e.g., as adapted for murine monoclonal antibodies, are
described, e.g., by Goding, J. W. (Monoclonal antibodies: principles and
practice: production and application of monoclonal antibodies in cell
biology, biochemistry, and immunology 2nd ed. London; Orlando: Academic
Press, 1986. pp 124-126) and the references cited therein. Other
procedures for iodinating polypeptides, are described by Hunter and
Greenwood (1962) Nature 144:945, David et al. (1974) Biochemistry
13:1014-1021, and U.S. Pat. Nos. 3,867,517 and 4,376,110. Radiolabeling
elements which are useful in imaging include .sup.123I, .sup.131I,
.sup.111In, and .sup.99mTc, for example. Procedures for iodinating
polypeptides are described by Greenwood, F. et al. (1963) Biochem. J.
89:114-123; Marchalonis, J. (1969) Biochem. J. 113:299-305; and Morrison,
M. et al. (1971) Immunochemistry 289-297. Procedures for
.sup.99mTc-labeling are described by Rhodes, B. et al. in Burchiel, S. et
al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer,
New York: Masson 111-123 (1982) and the references cited therein.
Procedures suitable for .sup.111In-labeling antibodies are described by
Hnatowich, D. J. et al. (1983) J. Immul. Methods, 65:147-157, Hnatowich,
D. et al. (1984) J. Applied Radiation, 35:554-557, and Buckley, R. G. et
al. (1984) F.E.B.S. 166:202-204.
[0257] In the case of a radiolabeled compound, the compound is
administered to the patient, is localized to the tissue the antigen with
which the compound interacts, and is detected or "imaged" in vivo using
known techniques such as radionuclear scanning using e.g., a gamma camera
or emission tomography. See e.g., A. R. Bradwell et al., "Developments in
Antibody Imaging", Monoclonal Antibodies for Cancer Detection and
Therapy, R. W. Baldwin et al., (eds.), pp 65-85 (Academic Press 1985).
Alternatively, a positron emission transaxial tomography scanner, such as
designated Pet VI located at Brookhaven National Laboratory, can be used
where the radiolabel emits positrons (e.g., .sup.11C, .sup.18F, .sup.15O,
and .sup.13N).
[0258] MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses NMR to
visualize internal features of living subject, and is useful for
prognosis, diagnosis, treatment, and surgery. MRI can be used without
radioactive tracer compounds for obvious benefit. Some MRI techniques are
summarized in EP-A-0 502 814. Generally, the differences related to
relaxation time constants T1 and T2 of water protons in different
environments is used to generate an image. However, these differences can
be insufficient to provide sharp high resolution images.
[0259] The differences in these relaxation time constants can be enhanced
by contrast agents. Examples of such contrast agents include a number of
magnetic agents paramagnetic agents (which primarily alter T1) and
ferromagnetic or superparamagnetic (which primarily alter T2 response).
Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (and
reduce toxicity) of some paramagnetic substances (e.g., Fe.sup.+3,
Mn.sup.+2, Gd.sup.+3). Other agents can be in the form of particles,
e.g., less than 10 .mu.m to about 10 nM in diameter). Particles can have
ferromagnetic, antiferromagnetic or superparamagnetic properties.
Particles can include, e.g., magnetite (Fe.sub.3O.sub.4),
.gamma.--Fe.sub.2O.sub.3, ferrites, and other magnetic mineral compounds
of transition elements. Magnetic particles may include: one or more
magnetic crystals with and without nonmagnetic material. The nonmagnetic
material can include synthetic or natural polymers (such as sepharose,
dextran, dextrin, starch and the like.
[0260] The compounds can also be labeled with an indicating group
containing of the NMR-active .sup.19F atom, or a plurality of such atoms
inasmuch as (i) substantially all of naturally abundant fluorine atoms
are the .sup.19F isotope and, thus, substantially all fluorine-containing
compounds are NMR-active; (ii) many chemically active polyfluorinated
compounds such as trifluoracetic anhydride are commercially available at
relatively low cost, and (iii) many fluorinated compounds have been found
medically acceptable for use in humans such as the perfluorinated
polyethers utilized to carry oxygen as hemoglobin replacements. After
permitting such time for incubation, a whole body MRI is carried out
using an apparatus such as one of those described by Pykett (1982)
Scientific American, 246:78-88 to locate and image cancerous tissues.
[0261] Also within the scope of the invention are kits comprising the
compound that binds to a target protease and instructions for use, e.g.,
the use of the compound (e.g., poly-PEGylated Kunitz domain) to detect
the target protease, in vitro, e.g., in a sample, e.g., a biopsy or cells
from a patient having a pulmonary disorder, or in vivo, e.g., by imaging
a subject. The kit can further contain a least one additional reagent,
such as a label or additional diagnostic agent. For in vivo use the
compound can be formulated as a pharmaceutical composition.
An exemplary amino acid sequence of a human neutrophil elastase: (Also
listed in GenBank.RTM. under: gi|4503549|ref|NP.sub.--001963.1| elastase
2, neutrophil [Homo sapiens])
TABLE-US-00006
(SEQ ID NO: 22)
MTLGRRLACLFLACVLPALLLGGTALASEIVGGRRARPHAWPFMVSLQ
LRGGHFCGATLIAPNFVMSAAHCVANVNVRAVRVVLGAHNLSRREPTR
QVFAVQRIFENGYDPVNLLNDIVILQLNGSATINANVQVAQLPAQGRR
LGNGVQCLAMGWGLLGRNRGIASVLQELNVTVVTSLCRRSNVCTLVRG
RQAGVCFGDSGSPLVCNGLIHGIASFVRGGCASGLYPDAFAPVAQFVN
WIDSIIQRSEDNPCPHPRDPDPASRTH
The following non-limiting examples further illustrate aspects of the
invention:
Example
[0262] Peptides and small proteins are rapidly cleared from circulation in
vivo. The rapid clearance often greatly limits therapeutic potency. High
doses and frequent administration are needed to achieve therapeutic
effects.
[0263] DX-890 consists of 56 amino acids, contains three intramolecular
disulfide bonds, and has a molecular weight of 6,237 Da. For primary
amine-based coupling, there are five potential PEGylation sites on
DX-890, each of the four lysine residues and the N-terminus. Use of mPEG
succinimidyl propionic acid can be used to couple PEG to each of these
sites, e.g., at four lysine residues and the N-terminus. The PEG reagent
that can be used may be mPEG that has an average molecular weight of
about 5 kDa.
[0264] The reaction can be allowed to proceed to completion at a pH that
permits modification of the amino groups on the lysine side chains and to
the N-terminus. For example, the pH can be greater than 7.5, e.g.,
between 7.8 and 8.5. The reaction is quenched, e.g., with Tris. The
reaction can be loaded onto an ion exchange or size exclusion column and
fractions that contain PEGylated DX-890 are collected. These relevant
fractions can be dialyzed, further purified, and then stored or analyzed.
[0265] DX-1000, a human plasmin inhibitor, is a Kunitz domain with fewer
lysines than DX-890. It has a three available lysines and an N-terminus
for modification with mPEG. DX-1000 can be combined with an mPEG
succinimidyl propionic acid reagent having an average molecular weight of
about 5 kDa or 7 kDa. DX-1000 can be modified and purified, e.g., as
described for DX-890. U.S. Pat. No. 6,103,499 also describes other
plasmin inhibitors, including DX-1000 related inhibitors. Kunitz domains
having sequences or conforming to motifs described in U.S. Pat. No.
6,103,499 can be modified as described herein.
[0266] DX-88, a kallikrein inhibitor is a Kunitz domain with fewer lysines
than DX-890. It has a three available lysines and an N-terminus for
modification with mPEG. DX-88 can be combined with an mPEG succinimidyl
propionic acid reagent having an average molecular weight of about 5 kDa
or 7 kDa. DX-88 can be modified and purified, e.g., as described for
DX-890. U.S. Pat. No. 6,333,402 also describes other kallikrein
inhibitors, including DX-88 related inhibitors. See, e.g., Tables 6 and
103 described therein. Kunitz domains having sequences or conforming to
motifs described in U.S. Pat. No. 6,333,402 can be modified as described
herein.
[0267] The predicted or actual structures of DX-890, DX-88, and DX-1000
are shown with the lysine residues indicated in FIGS. 1, 2, and 3,
respectively.
Example
[0268] The Example above is further detailed by the following methods for
PEGylating a protein of interest at multiple or all possible reactive
sites, in the following implementations, the method is used to
poly-PEGylate Kunitz domains at multiple or all possible primary amines.
[0269] A 5 kDa amino-reactive monofunctional PEG (mPEG-SPA) from NEKTAR
Therapeutics (cat. no.: 2M4M0H01) was used as material of the PEGylation
reactions.
[0270] We found that it is possible to poly-PEGylate DX-88, DX-890 and
DX-1000 with four or five 5 kDa PEGs. Moreover, the poly-PEGylated
proteins maintained the desired therapeutic activity while having
increased circulating half-life. Additionally, reaction conditions were
very efficient in terms of the conversion of unmodified protein to the
desired PEGylated form. The reactions can be used, or scaled up, to
provide consistently homogenous preparations of poly-PEGylated product.
Because of this great efficiency and few reaction side products,
preparations of the poly-PEGylated products can be synthesized with
higher yield and lower cost than Kunitz domains that include a single PEG
moiety. This approach makes for easier manufacturability with more
controlled batch-to-batch consistency and a final product which is easier
to fully characterize.
Materials
[0271] mPEG-SPA, MW 5,000 Da, NEKTAR Therapeutics, cat. no.: 2M4M0H01
(succinimidyl ester of methoxy-capped polyethylene glycol propionic acid)
[0272] DX-88 API, MW 7,054 Da, .about.10 mg/ml in PBS, pH 7.0 [0273]
DX-890 API, MW 6,231 Da, .about.10 mg/ml in 10 mM NaAc, pH 3.0 [0274]
DX-1000 API, MW 7,167 Da, .about.10 mg/ml in PBS, pH 7.0 [0275] 0.2-0.3M
Hepes, pH 7.8-8.5 [0276] 1M Tris, pH 8.0 [0277] 1N HCl
PEGylation Reaction I:
[0277] [0278] 1) Calculate the amount of PEG needed for reacting the
Kunitz domain polypeptide at approximately 10:1 molar ratio of
PEG:reactive group. For example, DX-890 has 5 total reactive groups, so a
50:1 molar ratio of PEG:DX-890 is used. Depending upon the Kunitz domain
polypeptide and/or reaction conditions, a ratio of 25:1 to 50:1 is
typically used. For example, for PEGylating 10 mg of DX-890 (MW 6,231 Da)
at a 50:1 molar ratio of PEG:peptide, 401 mg of PEG (MW 5,000 Da) would
be used. [0279] 2) Just prior to reacting the Kunitz domain polypeptide
with the PEG, dilute the required volume of Kunitz domain polypeptide
stock 1:1 with 0.2M Hepes, pH 7.8-8.5 buffer. The peptide stock is
typically .about.10.0 mg/ml. Therefore upon dilution, the concentration
of the Kunitz domain polypeptide is .about.5.0 mg/ml in 0.1M Hepes, pH
7.8-8.5 buffer. Both DX-88 and DX-1000 are relatively stable in terms of
solubility upon dilution. DX-890, while initially soluble upon dilution,
however, may precipitate over time. Reaction times can be chosen to
minimize precipitation. [0280] 3) Immediately add the 1:1 diluted Kunitz
domain polypeptide solution directly to the PEG powder and quickly
dissolve the PEG by vortexing. Once completely dissolved, cap the tube,
wrap in foil and allow to react while slowly rocking/tumbling for 2.5-3
hours at 2-8.degree. C. to 25.degree. C. [0281] 4) Quench the reaction by
adding 1/9.sup.th. volume of 1 M Tris, pH 8.0 for 30-60 minutes at
2-8.degree. C. to 25.degree. C. while slowly rocking/tumbling. [0282] 5)
Carefully and slowly adjust the pH of the quench reaction mixture to
.about.pH 7 with small additions of 1N HCl while mixing. [0283] 6) The
neutralized reaction can be stored at 2-8.degree. C. or frozen at
-20.degree. C. to -80.degree. C. until purification. The direct addition
of the Kunitz domain polypeptide solution to PEG powder can help simplify
the number of steps in the reaction process and reduce hydrolysis prior
to reaction.
PEGylation Reaction II:
[0284] The following is another method for poly-PEGylating a polypeptide.
[0285] 1) PEG is weighed out, as described for Reaction I, and placed
aside for use just prior to reaction. [0286] 2) Dilute the Kunitz domain
polypeptide to 3-5 mg/ml in 0.3M Hepes, pH 7.8-8.5. [0287] 3) Just prior
to reaction, quickly prepare a 200-250 mg/ml solution of PEG (in slight
excess) in dH.sub.20 that has been previously degassed and
N.sub.2-saturated. Add the water to the PEG and quickly and completely
dissolve by vortexing. [0288] 4) Immediately add the required volume of
PEG solution to the Kunitz domain polypeptide solution while mixing. Cap
the tube, wrap in foil and allow to react while slowly rocking/tumbling
for 2.5-3 hours at 2-8 C to 25 C. [0289] 5) Continue with steps 4)
through 6) above.
Example
Analytical Methods
[0290] Modified Kunitz domains can be analyzed and characterized by a
variety of methods. Exemplary methods include the following:
[0291] The unpurified reaction mix may be analyzed for the extent of
PEGylation by both reducing/non-reducing SDS-PAGE analysis with both
Coomassie and iodine staining as described in a separate protocol and
size-exclusion high performance liquid chromatography (SEC-HPLC) by
monitoring both refractive index (RI) and absorbance at 280 nm (UV). The
SDS-PAGE analysis by Coomassie stain detects only the polypeptide
component of the reaction mix (free and coupled) whereas staining with
iodine preferentially detects the PEG (free and coupled). SEC-HPLC
analysis by UV (abs. 280 nm) detects the peptide (free and coupled) and
RI detects both peptide and PEG. Dynamic light scattering (LS) detection
allows for determination of absolute MW and MW distribution.
[0292] SDS-PAGE and SEC-HPLC can show the distribution of PEGylated
products, but the absolute molecular weights should be determined by
MALDI-TOF or other methods. The reason is that PEGylated proteins run
more slowly on gels and SEC-HPLC than do unPEGylated proteins, due to the
PEG moieties large hydrodynamic radius, leading to overestimation of
molecular weight. This could be overcome by using PEGylated Kunitz
domains of known absolute molecular weight as standards.
Iodine Staining
[0293] Gels are loaded with approximately 2-3 .mu.g of protein initially
(for DX-1000, DX-88, and DX-890) for PEGylated samples that will resolve
into one or two bands only. This loading is most often appropriate for
the 25:1 and 50:1 PEG:protein reactions if the coupling was successful.
However, for samples that were PEGylated at the lower reaction ratios
(1:1, 5:1, and 10:1) and are expected to exhibit multiple PEGylated
species, 10-15 .mu.g of protein per lane is more appropriate (since 4-5
bands may appear). Samples are mixed with the appropriate amount of
NuPAGE LDS Sample Buffer. Samples are vortexed and heated at 70.degree.
C. for 10 minutes prior to loading.
[0294] Gels can be prepared and resolved according to standard methods,
e.g. using the Invitrogen NuPAGE system with a 4-12% Bis-Tris gels. See,
e.g., NuPAGE Novex Bis-Tris Gels Quick Reference Card, Invitrogen Life
Technologies.
[0295] Gels are rinsed briefly in deionized water, then covered with a 5%
barium chloride solution for 10 minutes on the shaker. The gel is rinsed
again with deionized water and then immersed in a 0.1N Iodine solution
for at least 10 minutes on the shaker. Bands should be visible almost
immediately. Full staining will be complete after 10 minutes. The gel is
then photographed, for example, with UVP Epi Chem II Darkroom and the
Ethidium bromide filter.
[0296] After iodine staining, the protein can be stained for proteins with
Coomassie. The gel is first rinsed in water to destain then mixed with
Coomassie and then destained in 300 mL methanol, 100 mL glacial acetic
acid, and 600 mL water. UnPEGylated protein bands appear dark blue, and
PEGylated protein may appear very light blue, if at all.
Chromatography
[0297] The chromatography system (Waters Corporation) used here was the
600 system (pump/controller) running EMPOWER.TM. software with 717 plus
auto sampler, 996 photodiode array detector (PDA) and 2414 refractive
index detector. In addition, a PD2010+ dynamic light scattering (LS)
detector (Precision Detectors, Inc.) was also run in series.
[0298] SEC column chromatography can include the following features: SEC
column: TSK G3000SW.sub.x1 (7.8 mm ID.times.30 cm L) with guard (Tosoh
Bioscience, cat. no.: 08541 and 08543); Flowrate: 0.5 ml/minute; Run
time: 35 minutes; Mobile phase: PBS, pH 7.2 with 0.05% NaN.sub.3; Sample
injection volume: 25-100 .mu.L; Sample load: 50-100 .mu.g per injection;
Detection: UV (280 nm), RI and LS; SEC Standards: BioRad, cat. no.:
151-1901
MALDI-TOF
[0299] MALDI-TOF (matrix-assisted laser desorption ionization-time of
flight) Mass Spectrometry (ABI, Applied Biosystems Voyager-DE) can be
used to evaluate actual mass of reaction products and subjects. For
polypeptide analysis (e.g., prior to reaction),
alpha-cyano-4-hydroxycinnamic acid can be used as a matrix. For analysis
of reaction products or poly-PEGylated species, 2,5-dihydroxybenzoic acid
(DHB) can be used as a matrix. Chips can be spotted 1:1 (0.5 .mu.L:0.5
.mu.L) of sample:matrix, and air dried prior to analysis
Ki Measurement
[0300] The equilibrium inhibition constants (Ki) for a poly-PEGylated
protein (e.g., a poly-PEGylated DX-890) can be determined according to
the tight-binding inhibition model with formation of a reversible complex
(1:1 stoichiometry). Reactions are set up with 100 pM enzyme (e.g.,
elastase) and a range of inhibitor concentrations (0-4 nM) at 30.degree.
C. in 50 mM HEPES, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100. Following
a 24 h incubation, substrate is added (25 .mu.M) to the enzyme-inhibitor
solution and the rate of substrate hydrolysis is monitored at an
excitation of 360 nm and an emission of 460 nm. Plots of the percent
remaining activity versus active inhibitor concentration are fit by
nonlinear regression analysis to Equation 1 to determine equilibrium
dissociation constants. Unmodified protein and poly-PEGylated protein can
be analyzed for comparison.
% A = 100 - ( ( I + E + K i ) - ( I + E + K
i ) 2 - 4 E I 2 E ) 100 Equation 1
##EQU00001##
Where:
[0301] % A=percent activity I=Kunitz domain protein concentration (e.g.,
DX-890) E=enzyme (e.g., HNE) concentration K.sub.i=equilibrium inhibition
constant
Pharmacokinetics in Animals
[0302] The following methods can be used to evaluate the pharmacokinetics
(PK) of proteins such as poly-PEGylated proteins in animals, e.g., mice
and rabbits.
[0303] The protein to be tested is labeled with iodine (.sup.125I) using
the iodogen method (Pierce). The reaction tube is rinsed with reaction
buffer (25 mM Tris, 0.4 M NaCl, pH 7.5). The tube is emptied and then
replaced with 0.1 ml of reaction buffer and 12 .mu.l of carrier free
iodine-126, about 1.6 mCi. After six minutes, the activated iodine is
transferred to a tube containing the protein to be tested. After nine
minutes, the reaction is terminated with 25 .mu.l of saturated tyrosine
solution. The reaction can be purified on a 5 ml D-salt polyacrylamide
6000 column in Tris/NaCl. HSA can be used to minimize sticking to the
gel.
[0304] A sufficient number of mice (about 36) are obtained. The weight of
each animal is recorded. In the case of mice, the animals are injected in
the tail vein with about 5 .mu.g of the protein to be tested. Samples are
recovered at each time point per animal, with four animals per time
point, at approximately 0, 7, 15, 30, and 90 minutes, 4 hours, 8 hours,
16 hours, and 24 hours post injection. Samples (about 0.5 ml) are
collected into anti-coagulant (0.02 ml EDTA). Cells are spun down and
separated from plasma/serum. Samples can be analyzed by radiation
counting and SEC peptide column on HPLC with inline radiation detection.
[0305] For rabbits, the material is injected into the ear vein. Samples
can be collected at 0, 7, 15, 30, 90 minutes, 4, 8, 16, 24, 48, 72, 96,
120, and 144 hours post-injection. Samples can be collected and analyzed
as for mice.
[0306] Data can be fit to a bi-exponential (equation 2) or a
tri-exponential (equation 3) decay curve describing "fast", "slow", and
"slowest" phases of in vivo clearance:
y=Ae.sup.-.alpha.t+Be.sup.-.beta.t Equation 2
y=Ae.sup..alpha.t+Be.sup.-.beta.t+Ce.sup.-.gamma.t Equation 3
Where:
[0307] y=Amount of label remaining in plasma at time=t post-administration
A=Total label in "fast" clearance phase B=Total label in "slow" clearance
phase C=Total label in "slowest" clearance phase .alpha.="Fast" clearance
phase decay constant .beta.="Slow" clearance phase decay constant
.gamma.="Slowest" clearance phase decay constant t=Time post
administration
[0308] The .alpha., .beta., and .gamma. phase decay constants can be
converted to half-lives for their respective phases as:
.alpha. Phase Half-life=0.69(1/.alpha.)
.beta. Phase Half-life=0.69(1/.beta.)
.gamma. Phase Half-life=0.69(1/.gamma.)
[0309] In the case where the data are fit using the bi-exponential
equation, the percentages of the total label cleared from in vivo
circulation through the .alpha. and .beta. phases are calculated as:
% .alpha. Phase=[A/(A+B)].times.100
% .beta. Phase=[B/(A+B)].times.100
[0310] In the case where the data are fit using the tri-exponential
equation, the percentages of the total label cleared from in vivo
circulation through the .alpha. and 13 phases are calculated as:
% .alpha. Phase=[A/(A+B+C)].times.100
% .beta. Phase=[B/(A+B+C)].times.100
% .gamma. Phase=[C/(A+B+C)].times.100
TABLE-US-00007
TABLE 4
Plasma Clearance in Mice
T.sub.1/2 alpha Clearance T.sub.1/2 beta Clearance
(min.) (%) (min.) (%)
DX-890 1.3 79 59.2 21
DX-1000 1.5 87 26.9 13
T.sub.1/2 alpha Clearance T.sub.1/2 beta Clearance
(hrs.) (%) (hrs.) (%)
5xPEG5-DX-890 1.1 33 20.2 67
4xPEG5-DX-1000 0.3 38 12.5 62
TABLE-US-00008
TABLE 5
Plasma Clearance in Rabbit
T.sub.1/2 alpha Clearance T.sub.1/2 gamma Clearance
(min.) (%) T.sub.1/2 beta (hrs.) Clearance (%) (hrs.) (%)
DX-890 1.7 83 3.4 17
DX-1000 0.9 85 1 15
5xPEG5-DX-890 2.8 28 4.5 34 97.6 38
4xPEG5-DX-1000 1.9 34 3 32 69.3 34
[0311] In the case of both the mouse (Table 4) and rabbit (Table 5),
PEGylation of either DX-890 or of DX-1000 results in a decrease in the
fraction of clearance through the alpha pathway. At the same time the
fraction of clearance through the longer lived pathways (beta and gamma)
increases.
[0312] The poly-PEGylated proteins also showed good in vivo stability by
SEC analysis.
Purification:
[0313] One exemplary purification method is as follows: [0314] 1)
Purification of polyPEGylated-protein from excess/unreacted PEG and trace
amounts of both high molecular weight and lower molecular weight
PEGylated species may be accomplished by ion-exchange chromatography on
an AKTA Basic 10/100 chromatography system (Amersham). [0315] 2) For
example, a column of appropriate size and capacity may be packed with a
strong cation exchange resin (i.e.: Poros 50HS, Applied Biosystems, prod.
code: 1-3359-11) in the case of at least PEGylated DX-88 and DX-1000.
[0316] 3) Briefly, a volume of the PEGylation reaction mix is diluted
5-15 fold or as necessary, with water followed by pH adjustment to
pH.about.3.0 with 1 M acetic acid (100-200 mM final) and conductivity
<3 mS/cm. [0317] 4) The column is first equilibrated with 100 mM
acetic acid, pH 3.0. Linear flowrate of 100 cm/hr. [0318] 5) Loaded and
washed with same for .about.5 column volumes. Linear flowrate during
loading is 50 cm/hr. [0319] 6) The PEGylated protein is eluted from the
column in a series of step gradients. [0320] 7) The first step elution is
100 mM acetic acid, with 20 mM NaCl, pH 3.2 to help remove HMW components
(.about.20 CV at 100 cm/hr). [0321] 8) The second step elution is 100 mM
acetic acid with 50 mM NaCl, pH 3.8 (.about.10 CV at 100 cm/hr) elutes
the main product (i.e.: 4.times.5 kDa PEG/peptide for DX-88 and DX-1000).
[0322] 9) The third and final step elution is PBS, pH 7.2 (.about.5 CV
100 cm/hr) to help remove trace amounts of LMW PEGylated species. [0323]
10) Followed by 0.2M NaOH cleaning (.about.5 CV with contact time of 30
minutes). [0324] 11) Followed by column storage in 20% ethanol (.about.10
CV). [0325] 12) Fractions are collected across the profile and analyzed
by SDS-PAGE prior to pooling. [0326] 13) The final pool of purified
PEGylated protein is then UF/DF into PBS, pH 7.2 using conventional means
available. The final material is then 0.22 um filtered, quantitated by
abs. 280 nm (as previously described), aliquoted and frozen at
-20.degree. C. to -80.degree. C. until use.
[0327] Another exemplary purification method, and one that can be used to
purify poly-PEGylated DX-88, is as follows.
[0328] Reaction products are loaded on a cation exchange column. Poros
50HS was found to have a fair binding capacity (.about.3 mg DX88-PEG5K/ml
resin) at this small scale that would allow for separation of free PEG
and a fairly concentrated eluate that includes the poly-PEGylated
species. Conductivity can be maintained below 2 mS/cm. For example, a 9.5
cm AKTA Poros 50HS column (1.1 cm w..times.10 cm h.) can be used. The
column is washed and cleaned to remove endotoxin and other contaminants.
The column can be equilibrated and loaded in 10 mM sodium acetate pH 3.5.
UF/DF and Final DX-88-PEG5K Pool Analysis
[0329] Fractions containing poly-PEGylated DX-88 were pooled for a total
sample volume of .about.6 mL. The sample was buffer exchanged into
1.times.DPBS pH 7.2 (unmodified) from Invitrogen (endotoxin specification
<0.25 EU/ml) and concentrated using two Amicon Ultra-15 Centrifugal
Filter Devices with a molecular weight cut-off of 10,000 kDa and a
centrifugal force of 4500.times.g. The CENTRICONs.TM. were washed with
0.1 N NaOH (diluted from 1 N NaOH, Acros, for low endotoxin production)
for one hour followed by several rinses with HyClone water prior to use.
The final exchange factor was 300 fold into 1.times.DPBS. A total of 3 mL
of concentrated and purified DX-88-PEG5K were recovered from the
centricons. The final sample concentration, 4.97 mg/mL, was determined by
diluting the sample 1:10 and measuring the O.D. 280 nm against
1.times.DPBS using an extinction coefficient for DX-88 of 0.954. The
final sample pH was .about.2 measured using Whatman pH Indicator strips
(pH 0-14). All filtrates (33 mL total) were analyzed for protein content
and had an O.D. 280 nm of 0.003 or less measured against 1.times.DPBS.
The purified DX-88-PEG5K sample was aliquoted into 0.5 mL fractions (2.5
mg each) in sterile tubes and frozen at -80.degree. C. The 1:10 diluted
sample was analyzed by SDS-PAGE in a dilution series to estimate the
purity of the main product of interest, 4-PEG5K-DX-88. The purity of
4-PEG5K-DX-88 is approximately 90%.
[0330] DX-890. We prepared a poly-PEGylated DX-890. Gel electrophoresis
and chromatographic analysis indicated that a reaction with a 1:50 or
1:63 ratio of DX-890 to 5K PEG reagent produced a reaction product that
was predominantly (>85%) a modified DX-890 with five attached PEG
moieties. DX-890 pegylated under a variety of ratios maintained its
specific activity relative to a control (about 10 U/mg).
[0331] Poly-pegylated DX-890 is predicted to have five PEG moieties (each
having about 5,266 Daltons molecular weight) plus the mass of DX-890
(6,237 Daltons, theoretical; 6,229 Daltons, observed). The predicted
total mass is 34,682 Daltons. The mass of the species observed by
MALDI-TOF was about 34,219 Daltons, in agreement with the theoretical
prediction, as the mass of individual PEG moieties can vary.
[0332] DX-88. We prepared a poly-PEGylated DX-88. Gel electrophoresis and
chromatographic analysis indicated that a reaction with a 1:50 ratio of
DX-890 to 5K PEG reagent at pH 7.8 produced a reaction product that was
predominantly (>85%) a modified DX-88 with four attached PEG moieties.
[0333] Poly-pegylated DX-88 is predicted to have four PEG moieties (each
having about 5,266 Daltons molecular weight) plus the mass of DX-88
(7,054 Daltons). The predicted total mass is 28,126 Daltons. The mass of
the species observed by MALDI-TOF was about 29,680 Daltons, in agreement
with the theoretical prediction, as the mass of individual PEG moieties
can vary.
[0334] Other embodiments are within the following claims.
Sequence CWU
1
251304PRTHomo sapiens 1Met Ile Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala
Ser Val Cys 1 5 10 15Leu
Leu Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu 20
25 30Asp Glu Glu His Thr Ile Ile Thr
Asp Thr Glu Leu Pro Pro Leu Lys 35 40
45Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys
50 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 Ser 85 90 95Leu
Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile
100 105 110Ile Lys Thr Thr Leu Gln Gln
Glu Lys Pro Asp Phe Cys Phe Leu Glu 115 120
125Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr
Asn 130 135 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 Glu 165 170
175Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn
180 185 190Ala Val Asn Asn Ser Leu
Thr Pro Gln Ser Thr Lys Val Pro Ser Leu 195 200
205Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp
Arg Gly 210 215 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 Asn 245 250
255Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly Phe Ile
260 265 270Gln Arg Ile Ser Lys
Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys 275
280 285Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe
Val Lys Asn Met 290 295 300258PRTBos
taurus 2Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1
5 10 15Arg Ile Ile Arg
Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20
25 30Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn
Asn Phe Lys Ser Ala 35 40 45Glu
Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55358PRTHomo
sapiens 3Ser Asp Asp Pro Cys Ser Leu Pro Leu Asp Glu Gly Ser Cys Thr Ala
1 5 10 15Tyr Thr Leu Arg
Trp Tyr His Arg Ala Val Thr Glu Ala Cys His Pro 20
25 30Phe Val Tyr Gly Gly Cys Gly Gly Asn Ala Asn
Arg Phe Gly Thr Arg 35 40 45Glu
Ala Cys Glu Arg Arg Cys Pro Pro Arg 50 55458PRTHomo
sapiens 4Asn Ala Glu Ile Cys Leu Leu Pro Leu Asp Tyr Gly Pro Cys Arg Ala
1 5 10 15Leu Leu Leu Arg
Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln 20
25 30Phe Leu Tyr Gly Gly Cys Glu Gly Asn Ala Asn
Asn Phe Tyr Thr Trp 35 40 45Glu
Ala Cys Asp Asp Ala Cys Trp Arg Ile 50 55558PRTHomo
sapiens 5Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala
1 5 10 15Met Ile Ser Arg
Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro 20
25 30Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn
Asn Phe Asp Thr Glu 35 40 45Glu
Tyr Cys Met Ala Val Cys Gly Ser Ala 50 55658PRTHomo
sapiens 6Tyr Glu Glu Tyr Cys Thr Ala Asn Ala Val Thr Gly Pro Cys Arg Ala
1 5 10 15Ser Phe Pro Arg
Trp Tyr Phe Asp Val Glu Arg Asn Ser Cys Asn Asn 20
25 30Phe Ile Tyr Gly Gly Cys Arg Gly Asn Lys Asn
Ser Tyr Arg Ser Glu 35 40 45Glu
Ala Cys Met Leu Arg Cys Phe Arg Gln 50 55758PRTHomo
sapiens 7Lys Glu Asp Ser Cys Gln Leu Gly Tyr Ser Ala Gly Pro Cys Met Gly
1 5 10 15Met Thr Ser Arg
Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys Glu Thr 20
25 30Phe Gln Tyr Gly Gly Cys Met Gly Asn Gly Asn
Asn Phe Val Thr Glu 35 40 45Lys
Glu Cys Leu Gln Thr Cys Arg Thr Val 50 55859PRTHomo
sapiens 8Phe Gln Glu Pro Cys Met Leu Pro Val Arg His Gly Asn Cys Asn His
1 5 10 15Glu Ala Gln Arg
Trp His Phe Asp Phe Lys Asn Tyr Arg Cys Thr Pro 20
25 30Phe Lys Tyr Arg Gly Cys Glu Gly Asn Ala Asn
Asn Phe Leu Asn Glu 35 40 45Asp
Ala Cys Arg Thr Ala Cys Met Leu Ile Arg 50
55956PRTHomo sapiens 9Thr Glu Asp Tyr Cys Leu Asn Lys Val Gly Arg Cys Arg
Gly Ser Phe 1 5 10 15Pro
Arg Trp Tyr Tyr Asp Pro Thr Glu Gln Ile Cys Lys Ser Phe Val 20
25 30Tyr Gly Gly Cys Leu Gly Asn Lys
Asn Asn Tyr Leu Arg Glu Glu Glu 35 40
45Cys Ile Leu Ala Cys Arg Gly Val 50
551058PRTHomo sapiens 10Asp Lys Gly His Cys Val Asp Leu Pro Asp Thr Gly
Leu Cys Lys Glu 1 5 10
15Ser Ile Pro Arg Trp Tyr Tyr Asn Pro Phe Ser Glu His Cys Ala Arg
20 25 30Phe Thr Tyr Gly Gly Cys Tyr
Gly Asn Lys Asn Asn Phe Glu Glu Glu 35 40
45Gln Gln Cys Leu Glu Ser Cys Arg Gly Ile 50
551156PRTHomo sapiens 11Ile Pro Ser Phe Cys Pro Lys Asp Glu Gly Leu Cys
Ser Ala Asn Val 1 5 10
15Thr Arg Tyr Tyr Phe Asn Pro Arg Tyr Arg Thr Cys Asp Ala Phe Thr
20 25 30Tyr Thr Gly Cys Gly Gly Asn
Asp Asn Asn Phe Val Ser Arg Glu Asp 35 40
45Cys Lys Arg Ala Cys Ala Lys Ala 50
551256PRTHomo sapiens 12Ala Ala Cys Asn Leu Pro Ile Val Arg Gly Pro Cys
Arg Ala Phe Ile 1 5 10
15Gln Leu Trp Ala Phe Asp Ala Val Lys Gly Lys Cys Val Leu Phe Pro
20 25 30Tyr Gly Gly Cys Gln Gly Asn
Gly Asn Lys Phe Tyr Ser Glu Lys Glu 35 40
45Cys Arg Glu Tyr Cys Gly Val Pro 50
551358PRTHomo sapiens 13Ile His Asp Phe Cys Leu Val Ser Lys Val Val Gly
Arg Cys Arg Ala 1 5 10
15Ser Met Pro Arg Trp Trp Tyr Asn Val Thr Asp Gly Ser Cys Gln Leu
20 25 30Phe Val Tyr Gly Gly Cys Asp
Gly Asn Ser Asn Asn Tyr Leu Thr Lys 35 40
45Glu Glu Cys Leu Lys Lys Cys Ala Thr Val 50
551458PRTHomo sapiens 14Val Lys Ala Val Cys Ser Gln Glu Ala Met Thr Gly
Pro Cys Arg Ala 1 5 10
15Val Met Pro Arg Trp Tyr Phe Asp Leu Ser Lys Gly Lys Cys Val Arg
20 25 30Phe Ile Tyr Gly Gly Cys Gly
Gly Asn Arg Asn Asn Phe Glu Ser Glu 35 40
45Asp Tyr Cys Met Ala Val Cys Lys Ala Met 50
551558PRTHomo sapiens 15Lys Pro Asp Phe Cys Phe Leu Glu Glu Asp Pro Gly
Ile Cys Arg Gly 1 5 10
15Tyr Ile Thr Arg Tyr Phe Tyr Asn Asn Gln Thr Lys Gln Cys Glu Arg
20 25 30Phe Lys Tyr Gly Gly Cys Leu
Gly Asn Met Asn Asn Phe Glu Thr Leu 35 40
45Glu Glu Cys Lys Asn Ile Cys Glu Asp Gly 50
551661PRTHomo sapiens 16Val Pro Lys Val Cys Arg Leu Gln Val Ser Val Asp
Asp Gln Cys Glu 1 5 10
15Gly Ser Thr Glu Lys Tyr Phe Phe Asn Leu Ser Ser Met Thr Cys Glu
20 25 30Lys Phe Phe Ser Gly Gly Cys
His Arg Asn Arg Ile Glu Asn Arg Phe 35 40
45Pro Asp Glu Ala Thr Cys Met Gly Phe Cys Ala Pro Lys 50
55 601758PRTHomo sapiens 17Leu Pro Asn Val
Cys Ala Phe Pro Met Glu Lys Gly Pro Cys Gln Thr 1 5
10 15Tyr Met Thr Arg Trp Phe Phe Asn Phe Glu
Thr Gly Glu Cys Glu Leu 20 25
30Phe Ala Tyr Gly Gly Cys Gly Gly Asn Ser Asn Asn Phe Leu Arg Lys
35 40 45Glu Lys Cys Glu Lys Phe Cys Lys
Phe Thr 50 551858PRTHomo sapiens 18Met His Ser Phe
Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala 1 5
10 15Ile Met Lys Arg Phe Phe Phe Asn Ile Phe
Thr Arg Gln Cys Glu Glu 20 25
30Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu
35 40 45Glu Glu Cys Lys Lys Met Cys Thr
Arg Asp 50 551958PRTHomo sapiens 19Gly Pro Ser Trp
Cys Leu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala 1 5
10 15Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val
Ile Gly Lys Cys Arg Pro 20 25
30Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn Phe Thr Ser Lys
35 40 45Gln Glu Cys Leu Arg Ala Cys Lys
Lys Gly 50 552058PRTHomo sapiens 20Glu Thr Asp Ile
Cys Lys Leu Pro Lys Asp Glu Gly Thr Cys Arg Asp 1 5
10 15Phe Ile Leu Lys Trp Tyr Tyr Asp Pro Asn
Thr Lys Ser Cys Ala Arg 20 25
30Phe Trp Tyr Gly Gly Cys Gly Gly Asn Glu Asn Lys Phe Gly Ser Gln
35 40 45Lys Glu Cys Glu Lys Val Cys Ala
Pro Val 50 552158PRTHomo sapiens 21Lys Gln Asp Val
Cys Glu Met Pro Lys Glu Thr Gly Pro Cys Leu Ala 1 5
10 15Tyr Phe Leu His Trp Trp Tyr Asp Lys Lys
Asp Asn Thr Cys Ser Met 20 25
30Phe Val Tyr Gly Gly Cys Gln Gly Asn Asn Asn Asn Phe Gln Ser Lys
35 40 45Ala Asn Cys Leu Asn Thr Cys Lys
Asn Lys 50 5522267PRTHomo sapiens 22Met Thr Leu Gly
Arg Arg Leu Ala Cys Leu Phe Leu Ala Cys Val Leu 1 5
10 15Pro Ala Leu Leu Leu Gly Gly Thr Ala Leu
Ala Ser Glu Ile Val Gly 20 25
30Gly Arg Arg Ala Arg Pro His Ala Trp Pro Phe Met Val Ser Leu Gln
35 40 45Leu Arg Gly Gly His Phe Cys Gly
Ala Thr Leu Ile Ala Pro Asn Phe 50 55
60Val Met Ser Ala Ala His Cys Val Ala Asn Val Asn Val Arg Ala Val65
70 75 80Arg Val Val Leu Gly
Ala His Asn Leu Ser Arg Arg Glu Pro Thr Arg 85
90 95Gln Val Phe Ala Val Gln Arg Ile Phe Glu Asn
Gly Tyr Asp Pro Val 100 105
110Asn Leu Leu Asn Asp Ile Val Ile Leu Gln Leu Asn Gly Ser Ala Thr
115 120 125Ile Asn Ala Asn Val Gln Val
Ala Gln Leu Pro Ala Gln Gly Arg Arg 130 135
140Leu Gly Asn Gly Val Gln Cys Leu Ala Met Gly Trp Gly Leu Leu
Gly145 150 155 160Arg Asn
Arg Gly Ile Ala Ser Val Leu Gln Glu Leu Asn Val Thr Val
165 170 175Val Thr Ser Leu Cys Arg Arg
Ser Asn Val Cys Thr Leu Val Arg Gly 180 185
190Arg Gln Ala Gly Val Cys Phe Gly Asp Ser Gly Ser Pro Leu
Val Cys 195 200 205Asn Gly Leu Ile
His Gly Ile Ala Ser Phe Val Arg Gly Gly Cys Ala 210
215 220Ser Gly Leu Tyr Pro Asp Ala Phe Ala Pro Val Ala
Gln Phe Val Asn225 230 235
240Trp Ile Asp Ser Ile Ile Gln Arg Ser Glu Asp Asn Pro Cys Pro His
245 250 255Pro Arg Asp Pro Asp
Pro Ala Ser Arg Thr His 260
2652356PRTArtificial SequenceSynthetically generated peptide 23Glu Ala
Cys Asn Leu Pro Ile Val Arg Gly Pro Cys Ile Ala Phe Phe 1 5
10 15Pro Arg Trp Ala Phe Asp Ala Val
Lys Gly Lys Cys Val Leu Phe Pro 20 25
30Tyr Gly Gly Cys Gln Gly Asn Gly Asn Lys Phe Tyr Ser Glu Lys
Glu 35 40 45Cys Arg Glu Tyr Cys
Gly Val Pro 50 552460PRTArtificial
SequenceSynthetically generated peptide 24Glu Ala Met His Ser Phe Cys Ala
Phe Lys Ala Asp Asp Gly Pro Cys 1 5 10
15Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg
Gln Cys 20 25 30Glu Glu Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu 35
40 45Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg
Asp 50 55 602560PRTArtificial
SequenceSynthetically generated peptide 25Glu Ala Met His Ser Phe Cys Ala
Phe Lys Ala Glu Thr Gly Pro Cys 1 5 10
15Arg Ala Arg Phe Asp Arg Trp Phe Phe Asn Ile Phe Thr Arg
Gln Cys 20 25 30Glu Glu Phe
Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu 35
40 45Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg
Asp 50 55 60
* * * * *