U.S. patent application number 12/471875 was filed with the patent office on 2011-07-14 for poly-pegylated protease inhibitors.
This patent application is currently assigned to Dyax Corp.. Invention is credited to Robert C. Ladner, Arthur C. Ley, Aaron K. Sato, Mark Stochl.
Application Number | 20110172140 12/471875 |
Document ID | / |
Family ID | 34278618 |
Filed Date | 2011-07-14 |
United States Patent
Application |
20110172140 |
Kind Code |
A1 |
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
|
Family ID: |
34278618 |
Appl. No.: |
12/471875 |
Filed: |
May 26, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11458773 |
Jul 20, 2006 |
7550427 |
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12471875 |
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10931153 |
Aug 30, 2004 |
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11458773 |
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60498845 |
Aug 29, 2003 |
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60598967 |
Aug 4, 2004 |
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Current U.S.
Class: |
514/1.1 ;
530/324; 530/350; 530/402 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
9/14 20180101; A61P 7/04 20180101; A61K 49/0002 20130101; C07K
14/8114 20130101; A61P 7/06 20180101; A61P 1/12 20180101; A61P 9/00
20180101; A61P 43/00 20180101; A61P 7/00 20180101; A61P 11/00
20180101; A61K 47/60 20170801; A61P 1/06 20180101; A61P 29/00
20180101 |
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
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
* * * * *