Note: Descriptions are shown in the official language in which they were submitted.
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PROTEASE SCREENING METHODS AND PROTEASES IDENTIFIED THEREBY
RELATED APPLICATIONS
This application is a division of application 2,656,531 filed July 5, 2007.
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FIELD OF THE INVENTION
Methods for identifying modified proteases with modified substrate specificity
or other properties are provided. The methods screen candidate and modified
proteases by contacting them with a substrate that traps them upon cleavage of
the
substrate.
BACKGROUND
Proteases are protein-degrading enzymes. Because proteases can specifically
interact with and inactivate or activate a target protein, they have been
employed as
therapeutics. Naturally-occurring proteases often are not optimal therapeutics
since
they do not exhibit the specificity, stability and/or catalytic activity that
renders them
suitable as biotherapeutics`(see, e.g., Fernandez-Gacio et al. (2003) Trends
in Biotech.
21: 408-414). Among properties of therapeutics that are important are lack of
immunogenicity or reduced immunogenicity; specificity for a target molecule,
and
limited side-effects. Naturally-occurring proteases generally are deficient in
one or =
more of these properties.
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Attempts have been made to engineer proteases with improved properties.
Among these approaches include 1) rational design, which requires information
about
the structure, catalytic mechanisms, and molecular modeling of a protease; and
2)
directed evolution, which is a process that involves the generation of a
diverse mutant
repertoire for a protease, and selection of those mutants that exhibit a
desired
characteristic (Bertschinger et al. (2005) in Phage display in Biotech. and
Drug
Discovery (Sidhu s, ed), pp. 461-491). For the former approach, a lack of
information
regarding the structure-function relationship of proteases limits the ability
to
rationally design mutations for most proteases. Directed evolution
methodologies
have been employed with limited success.
Screening for improved protease activity often leads to a loss of substrate
selectivity and vice versa. An optimal therapeutic protease should exhibit a
high
specificity for a target substrate and a high catalytic efficiency. Because of
the
limited effectiveness of available methods to select for proteases with
optimized
specificity and optimized activity, there remains a need to develop alternate
methods
of protease selection. Accordingly, it is among the objects herein to provide
methods .
for selection of proteases or mutant proteases with desired substrate
specificities and
activities.
SUMMARY
Provided are methods for selection or identification of proteases or mutant
proteases or catalytically active portions thereof with desired or
predetermined
substrate specificities and activities. In particular, provided herein are
protease
screening methods that identify proteases that have an altered, improved, or
optimized
or otherwise altered substrate specificity and/or activity for a target
substrate or
substrates. The methods can be used, for example, to screen for proteases that
have
an altered substrate specificity and/or activity for a target substrate
involved in the
etiology of a disease or disorder. By virtue of the altered, typically
increased,
specificity and/or activity for a target substrate, the proteases identified
or selected in
the methods provided herein are candidates for use as reagents or therapeutics
in the
treatment of diseases or conditions for which the target substrate is
involved. In
practicing the methods provided herein, a collection of proteases or
catalytically
active portion thereof is contacted with a protease trap polypeptide resulting
in the
formation of stable complexes of the protease trap polypeptide with proteases
or
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catalytically active portion thereof in the collection. In some examples, the
protease
trap polypeptide is modified to be cleaved by a protease having a
predetermined
substrate specificity and/or activity for a target substrate, for example, a
target
substrate involved in a disease or disorder. The method can further comprise
screening the complexes for substrate specificity for the cleavage sequence of
the
target substrate. In such examples, the identified or selected protease has an
altered
activity ancUor specificity towards the target substrate. In one example, the
stable
complex is formed by covalent linkage of a selected protease with a protease
trap
polypeptide. The selected proteases or catalytically active portion thereof
are
identified or selected from the complex in the methods provided herein. The
methods
provided herein can further include the step of separating the complexed
proteases
from the uncomplexed protease members of the collection. In one example, the
protease trap polypeptide is labeled for detection or separation and
separation is
effected by capture of complexes containing the detectable protease trap
polypeptide
and the protease or catalytically active portion thereof. Capture can be
effected in
suspension, solution or on a solid support. In instances where capture is by a
solid
support, the protease trap polypeptide is attached to the solid support, which
can be
effected before, during or subsequent to contact of the protease trap
polypeptide with
the collection of proteases or catalytically active portions thereof. The
solid support
can include, for example, a well of a 96-well plate. In some examples, the
protease
trap polypeptide is labeled with biotin. In other examples, the protease trap
polypeptide can be labeled with a His tag and separation can be 'effected by
capture
with a metal chelating agent such as, but not limited to, nickel sulphate
(NiSO4),
cobalt chloride (CoC12), copper sulphate (CuSO4) and zinc chloride (ZnC12).
The
metal chelating agent can be conjugated to a solid support, such as for
example, on
beads such as sepharose beads or magnetic beads.
In the methods provided herein, the method can further include a step of
amplifying the protease or catalytically active portion thereof in the
separated
complexes. In some examples, the protease or catalytically active portion
thereof in
the separated complex is displayed on a phage, and amplification is effected
by
infecting a host cell with the phage. The host cells can include a bacteria,
for
example, E.coli. The amplified protease, either from bacterial cell medium,
bacterial
periplasm, phage supernatant or purified protein, can be screened for
specificity
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and/or activity towards a target substrate. Typically, the target substrate is
a
polypeptide or cleavage sequence in a polypeptide involved in the etiology of
a
disease or disorder.
Also provided herein is a multiplexing method whereby the collection of
proteases are contacted with a plurality of different protease trap
polypeptides,
including modified forms thereof, where each of the protease trap polypeptides
are
labeled such that they can be identifiably detected. In such methods, at least
two
protease trap polypeptides are identifiable labeled such that more than one
stable
complex can form and more than one protease is identified.
In the methods provided herein, the methods also include successive rounds of
screening to optimize protease selection where proteases are amplified
following their
identification or selection in a first round of the screening methods herein,
to thereby
produce a second collection of proteases or catalytically active portions
thereof. The
second collection of proteases are contacted with a protease trap polypeptide,
that is
the same or different than the first protease trap polypeptide, to produce a
second set
of stable complexes. The proteases in the second set of stable complexes are
identified or selected.
In the methods provided herein, the protease trap polypeptide is a serpin, a
= member of the alpha macroglobulin family, or a member of the p35 family.
Such a
polypeptide molecule used in the methods provided herein forms a stable
complex by
covalent linkage of a protease or catalytically active portion thereof with
the protease
trap polypeptide.
In one aspect of the method provided herein, proteases are identified that
have
a desired substrate specificity by contacting a collection of protease and/or
proteolytically active portions of proteases with a protease trap polypeptide
to form
stable complexes of the protease trap polypeptide with a protease upon
cleavage of
the protease trap polypeptide. The Protease trap polypeptide, or modified form
thereof, is selected for use in the methods for purposes of being cleaved by a
protease
having the desired substrate specificity. In the methods, the protease or =
proteolytically active portion thereof is identified to select for a protease
having a
desired substrate specificity.
The collection of proteases used in the methods provided herein are any
collection of proteases or catalytically active portions thereof and include
members
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with at least, about, or equal to 5, 10, 50, 100, 103, 104, i05, 106 or more
different
members. In some aspects, the proteases are serine and/or cysteine proteases.
In the
methods provided herein, the collection of proteases or catalytically active
portions
thereof are displayed for contact with a protease trap polypeptide. In one
example,
.. the protease or proteolytically active portion thereof are displayed on a
solid support,
cell surface, or on a surface of a microorganism. The protease can be
displayed on
yeast, bacterium, a virus, a phage, a nucleic acid, an mRNA molecule, or on
ribosomes. Where the protease or proteolytically active portion thereof is
displayed
on a microorganism the microorganism includes, but is not limited to, E. coli,
S.cerevisiae, or a virus such as an M13, fd, or T7 phage, or a baculovirus. In
the
methods provided herein, the proteases or proteolytically active portions
thereof are
displayed on a phage display library and the protease collection is a protease
phage
display library. In some embodiments, the proteases are provided in the
collections,
such as by display, as proteolytically active portions of a full-length
protease. In
some examples, contact of a protease collection with a protease trap
polypeptide is in
a homogenous mixture.
Provided herein is a method of protease selection where at least two different
protease trap polypeptides are contacted with the collection, but where only
one of the
protease trap polypeptides is detectably labeled. The protease trap
polypeptide that is
detectably labeled permits the capture of stable complexes containing the
detectable
protease trap polypeptide and a protease or catalytically active portion
thereof. In
some examples of this method, the one or more other protease trap polypeptides
that
are not detectably labeled are present in excess in the reaction compared to
the
detectably labeled protease trap polypeptide. In the methods, the label is any
label for
detection thereof, such as a fluorescent label or an epitope label such as a
His tag. In
other examples, the detectable label is biotin.
The collection of proteases for which selection is made in the methods
provided herein include any collection of proteases. In some examples, the
proteases
are serine or cysteine proteases. The collection of proteases include those
that are
members of the chymotrypsin and subtilisin family of serine proteases or from
the
caspases of the papain family of cysteine proteases. The proteases include any
proteases, or catalytically active portion thereof, set forth in Table 7. In
some
examples, the protease or catalytically active portion thereof are collections
of
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urokinase plasminogen activator (u-PA) proteases, tissue plasminogen activator
(t-
PA) proteases, or MT-SP1 proteases.
In one aspect, the protease trap polypeptides used in the methods provided
herein are serpins, p35 family members, alpha-macroglobulin family members, or
any
modified forms thereof. A protease trap polypeptide used in the methods
provided
herein include, but is not limited to, plasminogen inhibitor-1 (PAT-1),
antithrombin
(AT3), or alpha 2-macroglobulin, or modified forms thereof. Modified forms of
a
protease trap polypeptide used in the methods provided herein included those
containing amino acid replacement, deletions, or substitutions in the reactive
site of
.. the protease trap polypeptide. In some examples, the modification is any
one or more
amino acid replacements corresponding to a cleavage sequence of a target
substrate.
The target substrate can be any protein involved in an etiology of a disease
or
disorder. Examples of target substrates include, but are not limited to, a
VEGFR, at-
PA cleavage sequence, or a complement protein. For example, target substrates
include, but are not limited to, VEGFR2 or complement protein C2. The cleavage
sequence of a target substrate includes, but is not limited to, any set forth
in any of
SEQ ID NOS: 389, 479 and 498. In some aspects, the protease trap polypeptide
is a
serpin and the one or more amino acid replacements is/are in the reactive site
loop of
the serpin polypeptide. The one or more replacements in the reactive site loop
(RSL)
.. include those in any one or more of the P4-P2' positions. Exemplary of such
serpins
used in the methods provided herein are any set forth in any of SEQ ID NOS:
497,
499, 610 and 611. In another example, the protease trap polypeptide is an
alpha 2
macroglobulin and the one or more amino acid replacements are in the bait
region of
the polypeptide. The .proteases identified or selected in the methods herein
against. a
modified protease trap polypeptide can be screened or selected for altered
substrate
specificity for the target substrate as compared to a non-target substrate. In
such
examples, the non-target substrate includes a substrate of the corresponding
template
protease. Typically, the substrate specificity of the identified or selected
protease is
increased by 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold,
300-fold,
400-fold, 500-fold or more.
The methods provided herein include those that are iterative where the method
of identifying and/or selecting for proteases with a desired substrate
specificity is
repeated or performed a plurality of times. In such methods, a plurality of
different
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proteases can be identified in the first iteration, or the first round, of the
method. In
other examples, a plurality of proteases are generated and prepared after the
first
iteration based on the identified proteases selected in the first round of
iteration.
Additionally, in some examples, the amino acid sequences of selected proteases
identified in the first round or iteration and/or in successive rounds are
compared to
identify hot spots. Hot spots are those positions that are recognized as a
modified
locus in multiple rounds, such as occur in at least 2, 3, 4, 5 or more
identified
proteases, such as compared to a wild-type or template protease, from a
collection of
modified proteases used in the method.
Also provided in the method herein is a further step of, after identifying a
protease or proteases, preparing a second collection of proteases, where an
identified
protease is used a template to make further mutations in the protease sequence
or
catalytically active portion thereof such that members of the second
collection (are
based on) contain polypeptides having mutations of the identified proteases
and
additional mutations; then contacting the second collection with a protease
trap
polypeptide that is either identical or different from the protease trap used
to isolate
the first protease or proteases, where the protease trap is modified to be
cleaved by a
protease having the desired substrate specificity; and identifying a
protease(s) or
proteolytically active portion(s) of a protease from the collection in a
complex,
whereby the identified protease(s) has greater activity or specificity towards
the
desired substrate than the first identified protease. The second collection
can contain
random or focused mutations compared to the sequence of amino acids of the
identified template protease(s) or proteolytically active portion of a
protease. Focused
mutations, include, for example, hot spot positions, such as positions 30, 73,
89 and
155, based on chymotrypsin numbering, in a serine protease, such as u-PA.
In practicing the methods, the reaction for forming stable complexes can be
modulated by controlling one or more parameters. Such parameters are any that
alter
the rate or extent of reaction or efficiency of the reaction, such as, but are
not limited
to, reaction time, temperature, pH, ionic strength, library concentration and
protease
trap polypeptide concentration.
The reactions can be performed in the presence of a competitor of the reaction
between a protease trap polypeptide and a protease or proteolytically active
portion
thereof to thereby enhance selectivity of identified protease(s) or
proteolytically
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active portion(s). Competitors include, for example, serum or plasma. Such as
human
serum or human plasma, a cell or tissue extract, a biological fluid, such as
urine or
blood, a purified or partially purified wild type form (or other modified
form) of the
protease trap. Exemplary of such competitors is a purified or partially
purified wild-
type form of a protease trap polypeptide or one or more specific variants of a
protease
trap polypeptide.
Iterative methods for evolving or selecting or identifying a protease or
proteolytically active portion thereof with specificity/selectivity and/or
activity for at
least two cleavage sequences are provided. The methods include the steps of:
a) con-
tacting a collection of proteases and/or proteolytically active portions of
proteases
with a first protease trap polypeptide to form, upon cleavage of the protease
trap
polypeptide by the protease or proteolytically active portion thereof; stable
complexes containing the protease trap polypeptide with a protease or
catalytically
active portion thereof in the collection, wherein contacting is effected in
the presence
.. of a competitor; b) identifying or selecting proteases or proteolytically
active portions
thereof that form complexes with the first protease trap polypeptide; c)
contacting
proteases or proteolytically active portion thereof that form complexes with
the first
protease trap polypeptide with a second protease trap polypeptide in the
presence of a
competitor; and d) identifying or selecting proteases or proteolytically
active portions
thereof that form complexes with the first protease trap polypeptide. The two
cleavage sequences can be in one target substrate or can be in two different
target
substrates. The identified, selected or evolved protease or proteolytically
active ,
portion thereof has substrate specificity and/or cleavage activity for at
least two
different cleavage sequences in one or two different target substrates. The
first and
second protease trap polypeptide can be the same or different. Typically, the
first and
second protease trap polypeptide used in the method are different and each are
modified to be cleaved by a protease having the predetermined substrate
specificity
for different target substrates. The method can further include repeating
steps a) and
b) or a)-d) at least once more until a protease with a desired or
predetermined
substrate specificity and cleavage activity to at least two recognition
sequences is
isolated. Substrate specificity and cleavage activity typically are increased
compared
with a template protease.
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Competitors for use in the methods include anything with which the protease
trap polypeptide can interact, typically with lesser stability than a target
protease.
Competitors include, but are not limited to, serum, plasma, human serum or
human
plasma, a cell or tissue extract, a biological fluid such as urine or blood, a
purified or
partially purified wild-type form of the protease trap, and one or more
specific
variants of a protease trap polypeptide.
Also provided are methods of protease selection that include the steps of:
a) contacting a collection of proteases or proteolytically active portions
thereof with a
first protease trap polypeptide to form, upon cleavage of the protease trap
poly-
peptide, covalent complexes of the protease trap polypeptide with any protease
or
catalytically active portion thereof in the collection; b) separating the
complexed
proteases from uncomplexed protease trap polypeptide(s); c) isolating or
selecting or
identifying the complexed proteases; d) generating a second collection of
proteases or
proteolytically active portions of proteases based on the selected proteases;
and
e) repeating steps a) - c) by contacting the second collection of proteases or
proteo-
lytically active portions thereof with a second protease trap polypeptide that
is
different from the first protease trap polypeptide to form complexes;
separating the
complexes; and isolating, selecting or identifying complexed proteases. The
first and
second protease trap polypeptides can be modified to contain two different
target
substrate recognition sequences, whereby the identified or selected protease
has
specificity and high cleavage activity to at least two recognition sequences.
These
methods can be repeated a plurality of times. In these methods, the collection
of
proteases or proteolytically active portions thereof can be contacted with the
first
and/or second protease trap polypeptide in the presence of a competitor (see
above).
In any of the methods of protease selection provided herein, the collections
can contain modified proteases. The modifications in the proteases can be
random or
focused or in a target region of the polypeptide.
Also provided are combinations that contain a collection of proteases and/or
proteolytically active portions thereof; and at least one protease trap
polypeptide. The
components can be provided separately or as a mixture. The protease trap
polypeptide include, among serpins, p35 family members, alpha-macroglobulin
family members, modified forms of each, and mixtures thereof.
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The collection of proteases and/or catalytically active portions thereof can
be
provided in solution or suspension or in a solid phase or otherwise displayed,
such as
on a solid support (matrix material) or in a display library, such as, but not
limited
to, a phage display library, where members display at least a proteolytically
active
portion of a protease.
Kits containing the combinations are provided. Typically the kits contain the
packaged components and, optionally, additional reagents and instructions for
performing the methods.
Also provided are methods for modifying the substrate specificity of a serine
protease, such as u-PA, by modifying one or more of residues selected from 30,
73,
89 and 155 based on chymotrypsin numbering.
Also provided are modified proteases identified by the methods herein. The
modified proteases provided herein exhibit altered substrate specificity
and/or activity
by virtue of the identified modification. Any of the modifications provided
herein
identified using the selection method can be made in a wild-type protease, any
allelic
or species variant thereof, or in any other variant of the protease. In
addition, also
provided herein are modified proteases containing 80%, 85%, 90%, 91%, 92%,
93%, =
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type
protease,
or allelic or species variant thereof, so long as the modification identified
in the
methods herein is present.
Among such proteases are modified proteases, including modified serine
proteases in the chymotrypsin family, such as urinary plasminogen activator (u-
PA)
polypeptides, or catalytically active fragments thereof containing one or more
mutations in hot spot positions selected from among positions 30, 73, 89, and
155,
based on chymotrypsin numbering, whereby substrate specificity is altered.
Provided herein also include modified urinary plasminogen activator (u-PA)
proteases identified in the method herein that exhibit increased specificity
and/or
activity towards a target substrate involved in the etiology of a disease or
disorder.
Such target substrates include, but are not limited to, a VEGFR or a tissue
plasminogen activator (t-PA) substrate. Hence, also provided are modified
serine
proteases or catalytically active portions thereof that cleave a t-PA
substrate. In
particular, among such proteases are modified urinary plasminogen activator (u-
PA)
polypeptides in which the u-PA polypeptide or catalytically active portion
thereof
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positions 21, 24,
30, 39, 61(A), 80, 82, 84, 89, 92, 156, 158, 159, and 187, based on
chymotrypsin =
numbering. Also provided are such modified u-PA polypeptides or catalytically
active portions thereof containing one or more mutations selected from among
F21V,
I24L, F30V, F3OL, T39A, Y61(A)H, E80G, K82E, E84IC, 189V, K92E, K156T,
Ti 58A, V159A, and K187E. Also provided are these modified u-PA polypeptides
where the u-PA polypeptide or catalytically active portions thereof contain
two or=
more mutations selected from among F30V/Y61(a)H; F30V/K82E; F30V/K156T;
F30V/K82E1VI 59A; F30V/K82E/T39A1VI 59A; F30V/K82E/T158AN159A;
F30V/Y61(a)H/K92E; F30V/K82E/V159A/E80G/189V/K187E; and
F30V/K82EN I 59A/E80G/E841089W(187E.
Also provided are modified serine proteases or catalytically active portions
thereof that cleave VEGFR, particularly VEGFR-2. In particular, provided are
modified urinary plasminogen activator (u-PA) polypeptides and catalytically
active
portions thereof, wherein the u-PA polypeptide or catalytically active portion
thereof
contains one or more modifications in positions selected from among positions
38, 72,
73, 75, 132, 133, 137, 138, 155, 160, and 217, based on chymotrypsin
numbering,
whereby substrate specificity to a VEGFR-2, or sequence thereof, is altered.
Also
provided are such polypeptides that contain one or more mutations selected
from
among V38D, R72G, L73A, L73P, 875P, F132L, 01331), E1370, n38T, LI55P, =
L155V, L155M, V160A, and R217C. These polypeptides can further include a
modification at position 30 and/or position 89, based on chymotrypsin
numbering,
such as modifications.selected from among F301, F30T, F3OL, F30V, F300, F30M,
and 189V. Also provided are these u-PA polypeptides or catalytically active
portions
thereof that contain one or more mutations, and in some instances two or more
mutations selected from among L73A/I89V; L73P; R217C; L155P;
S75P1189V/1138T; E1370; R72G/L155P; G133D; V160A; V38D; F132L1V160A;
L73A/189V/F30T; L73A/189V/F3OL; L73A/189V/F30V; L73A/189V/F30G;
L73A/I89V/L155V; L73A/189V/F30M; L73A/I89V/L155M;
L73A/189V/F30L/L155M; and L73A/189V/F30G/L155M.
Also provided are modified u-PA polypeptide, wherein the u-PA polypeptide
or catalytically active portion thereof contains one or more mutations
selected from
among F301, F30T, F300, and F30M.
RECTIFIED SHEET (RULE 91) ISA/EP
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Also provided herein are modified MT-SP1 polypeptides identified by the
methods herein. Such modified polypeptides include any having one or more
amino
acid modifications selected from among D23E, 14 IF, I4 1T, L52M, Y60(g)s,
T65K,
H71R, F93L, N95K, F97Y, F97L, T98P, F99L, A126T, V129D, P13 1S, I136T,
1136V, H143R, T1441, 1154V, N164D, T166A, L171F, P173S, F184(a)L, Q192H,
S201I, Q209L, Q221(a)L, R230W, F234L, and V2440, based on chymotrypsin
numbering, in an MT-SP1 polypeptide set forth in SEQ ID NO:253. In some
examples, the modifications are in a catalytically active portion of an MT-SPI
having
a sequence of amino acids set forth in SEQ ID NO:505. In other examples, the
modifications are man MT-SP1 polypeptide further comprising a modification
corresponding to modification of C122S in an MT-SP1 polypeptide set forth in
SEQ
ID NO:253, based on chymotrypsin numbering, for example, an MT-SP1 set forth
in
SEQ ID NO: 507 or 517. In particular of modifications provided herein are any
selected from among I4 1F, F97Y, L171F and V244G. The modified MT-SP1
polypeptides provided herein can further include one or more modifications
corresponding to Q175R or D217V in an MT-SP1 polypeptide set forth in SEQ ID
NO:253. Such modifications include any selected from among
1136T/N164D/T166A/F184(A)L/D217V; I41F; I41F/ A12611V244G;
D23E/I41F/T98P /T1441; I41F/ L171F/V244G; H143R/Q175R; 141F/ L171F;
R230W; I41F /I154VN244G; I41F/L52M/ V129D/Q221(A)L; F99L; F97Y/
I136V/Q192H/S2011; H71R/ P131S/D217V; D217V; T65K/F93L/F97Y/ D217V;
I41T/ P1735/Q209L; F97L/ F234L; Q175R; N95K; and Y60(G)S. Any of the above
modified MT-SP1 polypeptides exhibit modifications that increase one or both
of
specificity for a C2 complement protein or activity towards C2 complement
protein.
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- 13a -
Also provided herein is use of a pharmaceutical composition, comprising a
modified MT-SP1 polypeptide or catalytically active fragment thereof for
inhibition of
complement activation for the treatment of a disease or condition that is
mediated by a
complement protein, wherein: the modified MT-SP1 polypeptide or catalytically
active fragment
thereof, comprises the amino acid replacement F94L, whereby one or both of
substrate
specificity and activity towards a complement protein is greater than the
activity of the MT-SP1
polypeptide not containing the replacement(s); the complement protein is
selected from among
Clr, Cis, C2, C3, C3a, C4, C5a, C5b-9, and C9; numbering of replacements is
with reference to
amino acid positions of the protease domain whose sequence is set forth in SEQ
ID NO:505; and
the amino acid replacements are in a polypeptide whose sequence comprises the
sequence of
amino acids set forth in any one of SEQ ID NOS:253, 515, 505 and 507.
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Nucleic acid molecules encoding the modified proteases, including the u-PA
proteases and MT-SP1 proteases and catalytically active portions thereof are
provided. Also provided are vectors containing the nucleic acid molecules and
cells
containing the nucleic acid molecules or vectors.
Methods of treatment of subjects having a disease or condition, such as, but
not limited to, a disease or condition selected from among arterial
thrombosis, venous
thrombosis and thromboembolism, ischemic stroke, acquired coagulation
disorders,
disseminated intravascular coagulation, bacterial infection and periodontitis,
and
neurological conditions, that is treated by administration oft-PA, by
administering the
pharmaceutical compositions containing the modified u-PA proteases or
proteolytically active portions thereof or encoding nucleic acid molecules or
the cells
are provided. The methods are effected by administering a nucleic acid
molecule, a
cell or a pharmaceutical composition to the subject.
Also provided are methods of treating a subject having a disease or condition
that is mediated by a VEGFR, particularly a VEGFR-2. Such diseases and
conditions
include, but are not limited to, cancer, angiogenic diseases, ophthalmic
diseases, such
as macular degeneration, inflammatory diseases, and diabetes, particularly
complications therefrom, such as diabetic retinopathies. The methods are
effected by
administering a nucleic acid molecule or cell or composition containing or
encoding
the modified u-PA proteases that exhibit substrate specificity, particularly
increased
compared to the unmodified form, for VEGFR-2. The methods optionally include
administering another agent for treatment of the disease or condition, such as
administering an anti-tumor agent where the disease is cancer.
Also provided are methods of treating a subject having a disease or condition
.. that is mediated by a complement protein, particularly C2. Such diseases
and
conditions include, but are not limited to sepsis, Rheumatoid arthritis (RA),
membranoproliferative glomerulonephritis (MPGN), Multiple Sclerosis (MS),
Myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex
(IC)-mediated acute inflammatory tissue injury, Alzheimer's Disease (AD),
Ischemia-
reperfusion injury, and Guillan-Barre syndrome. In some examples, the ischemia-
reperfusion injury is caused by an event or treatment, such as, but not
limited to,
= myocardial infarct (MI), stroke, angioplasty, coronary artery bypass
graft,
cardiopulmonary bypass (CPB), and hemodialysis. The methods are effected by
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administering a nucleic acid molecule or cell or composition containing or
encoding
the modified MT-SP1 proteases that exhibit substrate specificity, particularly
increased compared to the unmodified form, for C2. In other examples, the
disease or
conditions results from treatment of a subject. For example, the treatment can
result
in complement-mediated ischemia-reperfusion injury. Such treatments include,
but
are not limited to, angioplasty or coronary artery bypass graft. In such
examples, a
modified MT-SP1 protease is administered prior to treatment of a subject. The
modified MT-SP1 polypeptides can be administered by contacting a body fluid or
tissue sample in vitro, ex vivo or in vivo.
Also provided herein are modified serpin polypeptides used in the methods
provided herein. Such modified serpin polypeptides are modified in its
reactive site
loop at positions corresponding to positions P4-P2' with a cleavage sequence
for a
target substrate. Typically, the target substrate is involved in the etiology
of a disease
or disorder. Such target substrates include, but are not limited to, a VEGFR,
a
complement protein or a t-PA substrates. For example, target substrates
include
VEGFR2 and complement protein C2. Exemplary of modified serpins provided
herein are modified plasminogen-activator inhibitor-1 (PAI-1) and antithrombin-
3
(AT3). Exemplary modified serpins are set forth in any of SEQ ID NOS: 497,
499,
610 and 611.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 depicts the mechanism of inhibition of a protease by a serpin and the
generation of a stable inhibited complex. Following contact, a serpin (I) and
protease
(E) initially form a noncovalent, Michaelis-like complex (El). This is
followed by
cleavage of the P I-P1' scissile bond and nucleophilic attack by the catalytic
serine of
a protease on a reactive site loop (RSL) carbonyl of a serpin and the
formation of a
covalent acyl-enzyme intermediate (EI ). The kinetically trapped covalent
inhibitory
product (Er) is the result of RSL insertion, protease translocation, protease
active site
distortion, and deformation of the overall protease structure. The minor non-
inhibitory pathway releases normal cleavage product, serpin (I*), and reactive
protease (E).
DETAILED DESCRIPTION
Outline
A. Definitions
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B. Method for Screening Proteases
C. Protease Trap Polypeptides
1. Serpins: Structure, Function, and Expression
2. Protease Catalysis, Inhibitory Mechanism of Serpins, and
Formation of Acyl Enzyme Intermediate
a. Exemplary Serpins
i. PAT-1
Antithrombin (AT3)
3. Other Protease Trap Polypeptides
a. p35
b. Alpha Macroglobulins (aM)
4. Protease Trap Polypeptide Competitors
5. Variant Protease Trap Polypeptides
D. Proteases
1. Candidate Proteases
a. Classes of Proteases
i. Serine Proteases
(a) Urokinase-type Plasminogen Activator
(u-PA)
(b) Tissue Plasminogen Activator (t-PA)
(c) MT-SP1
Cysteine Proteases
E. Modified Proteases and Collections for Screening
1. Generation of Variant Proteases
a. Random Mutagenesis
b. Focused Mutagenesis
2. Chimeric Forms of Variant Proteases
3. Combinatorial Libraries and Other Libraries
a. Phage Display Libraries
b. Cell Surface Display Libraries
c. Other Display Libraries
F. Methods of Contacting, Isolating, and Identifying Selected
Proteases
1. Iterative Screening
= 2. Exemplary Selected Proteases
G. Methods of Assessing Protease Activity and Specificity
H. Methods of Producing Nucleic Acids Encoding Protease Trap
Polypeptides (i.e. Serpins) or Variants Thereof or Proteases/Modified
Proteases
1. Vectors and Cells
2. Expression
a. Prokaryotic Cells
b. Yeast Cells
c. Insect Cells
d. Mammalian Cells
e. Plants
3. Purification Techniques
4. Fusion Proteins
5. Nucleotide Sequences
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I.
Preparation, Formulation and Administration of Selected Protease
Polypeptides
1. Compositions and Delivery
2. In vivo Expression of Selected Proteases and Gene Therapy
a. Delivery of Nucleic Acids
i. Vectors ¨ Episomal and Integrating
Artificial Chromosomes and Other Non-viral
Vector Delivery Methods
iii. Liposomes and Other Encapsulated Forms and
Administration of Cells Containing Nucleic
Acids
b. In vitro and Ex vivo Delivery
c.. Systemic, Local and Topical Delivery
2. Combination Therapies
3. Articles of Manufacture and Kits
J. Exemplary Methods of Treatment with Selected Protease
Polypeptides
1. Exemplary Methods of Treatment for Selected uPA
Polypeptides That Cleave tPA Targets
a. Thrombotic Diseases and Conditions
i. Arterial Thrombosis
Venous Thrombosis and Thromboembolism
(a) Ischemic stroke
iii. Acquired Coagulation Disorders
(a) Disseminated Intravascular Coagulation
(DIC)
(b) Bacterial Infection and Periodontitis
b. Other tPA Target-associated Conditions
c. Diagnostic Methods
2. Exemplary Methods of Treatment for Selected Protease
Polypeptides That Cleave VEGF or VEGFR Targets
a. Angiogenesis, Cancer, and Other Cell Cycle Dependent
Diseases or Conditions
b. Combination Therapies with Selected Proteases That
Cleave VEGF or VEGFR
3, Exemplary Methods of Treatment for Selected MT-SP1
Polypeptides that cleave complement protein targets
a. Immune-mediated Inflammatory Diseases
b. Neurodegenerative Disease
c. Cardiovascular Disease
K. Examples
A. DEFINITIONS
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=
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as is commonly understood by one of skill in the art to which
the
invention(s) belong. In the event that there are a plurality of
definitions for terms herein, those in this section prevail. Where reference
is made to
a URL or other such identifier or address, it understood that such identifiers
can
change and particular information on the intemet can come and go, but
equivalent
information can be found by searching the intemet. Reference thereto evidences
the =
availability and public dissemination of such information.
As used herein, a "protease trap" or "protease rap polypeptide", refers to a
substrate that is cleaved by a protease and that, upon cleavage, forms a
stable complex
with the protease to thereby trap the proteases as the protease goes through
an actual
transition state to form an enzyme complex, thereby inhibiting activity of the
proteases and capturing it. Thus, protease traps are inhibitors of proteases.
Protease
traps are polypeptides or molecules that include amino acid residues that are
cleaved
by a protease such that upon cleavage a stable complex is formed. The complex
is
sufficiently stable to permit separation of complexes from unreacted trap or
from trap
that has less stable interactions with the proteases. Protease traps include
any
molecule, synthetic, modified or naturally-occurring that is cleaved by the
protease
and, upon cleavage, forms a complex with the protease to permit separation of
the
reacted protease or complex from unreacted trap. Exemplary of such protease
traps
are serpins, modified serpins, molecules that exhibit a mechanism similar to
serpins,
such as for example, p35, and any other molecule that is cleaved by a protease
and
traps the protease as a stable complex, such as for example, alpha 2
macroglobulin.
Also included as protease traps are synthetic polypeptides that are cleaved by
a
protease (or proteolytically active portion thereof) and, upon cleavage, form
a stable
complex with the protease or proteolytically active portion thereof.
As used herein, serpins refer to a group of structurally related proteins that
inhibit proteases following cleavage of their reactive site by a protease
resulting in the
formation of a stable acyl-enzyme intermediate and the trapping of the
protease in a
stable covalent complex. Serpins include allelic and species variants and
other
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variants so long as the serpin molecule inhibits a protease by forming a
stable
covalent complex. Serpins also include truncated or contiguous fragments of
amino
acids of a full-length serpin polypeptide that minimally includes at least a
sufficient
portion of the reactive site loop (RSL) to facilitate protease inhibition and
the
formation of a stable covalent complex with the protease. Exemplary serpins
are set
forth in Table 2 and/or have a sequence of amino acids set forth in any one of
SEQ ID
NOS: 1-38, allelic variants, or truncated portions thereof.
As used herein a "mutant" or "variant" serpin refers to a serpin that contains
'
amino acid modifications, particularly modifications in the reactive site loop
of the
serpin. The modifications can be replacement, deletion, or substitution of one
or more
amino acids corresponding to Pn-P15-P14-P13....P4-P3-P2-P1-P1 '-P2'-P3' ..
positions. Typically, the serpin contains amino acid replacements in 1, 2, 3,
4, 5, 6, 7,
8, 9, 10 or more amino acid positions in the reactive site loop as compared to
a wild-
type serpin. Most usually, the replacements are in one or more amino acids
corresponding to positions P4-P2'. For example, for the exemplary PAT-1 serpin
set
forth in SEQ ID NO:11, the P4-P1' positions (VSARM) corresponding to amino
acid
positions 366-370 in SEQ ID NO:11 can be modified. Example 1 describes
modification of the VSARM amino acid residues to RRVRM or PFGRS.
As used herein, a "scissile bond" refers to the bond in a polypeptide cleaved
by a protease and is denoted by the bond formed between the Pl-P1' position in
the
cleavage sequence of a substrate.
As used herein, reactive site refers to the portion of the sequence of a
target
substrate that is cleaved by a protease. Typically, a reactive site includes
the PI-P1'
scissile bond sequence.
As used herein, reactive site loop (RSL; also called reactive center loop,
RCL)
refers to a sequence of amino acids in a serpin molecule (typically 17 to 22
contiguous amino acids) that serve as the protease recognition site and
generally
contain the sole or primary determinants of protease specificity. Cleavage of
the RSL
sequence and conformational changes thereof are responsible for the trapping
of the
protease by the serpin molecule in a stable covalent complex. For purposes
herein,
any one or more amino acids in the RSL loop of a serpin can be modified to
correspond to cleavage sequences in a desired target protein. Such modified
serpins,
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or portions thereof containing the variant RSL sequence, can be used to select
for
proteases with altered substrate specificity.
As used herein, partitioning refers to the process by which serpins partition
between a stable serpin-protease complex versus cleaved serpins. The reason
for
partitioning in serpins pertains to the nature of the inhibitory pathway,
which results
from a large translocation of the cleaved reactive-site loop across the serpin
surface.
If the protease has time to dissociate (i.e. deacylate the enzyme-serpin
complex)
before adopting the inhibited location, then partitioning occurs. The outcome
of a
given serpin¨protease interaction, therefore, depends on the partitioning
ratio between
the inhibitory (k4) and substrate (k5) pathways (such as is depicted in Figure
1), which
is represented by the stoichiometry of inhibition (SI = 1 + k5/k4); good
inhibitors have
the value of I because most of the serpin molecules partition into complex
formation
and k5/k4 is close to 0. If the RSL loop, however, is not inserted fast enough
into the
protease, partitioning occurs and the reaction proceeds directly to the
cleaved product.
As used herein, catalytic efficiency or keat/km is a measure of the efficiency
with which a protease cleaves a substrate and is measured under steady state
conditions as is well known to those skilled in the art.
As used herein, second order rate constant of inhibition refers to the rate
constant for the interaction of a protease with an inhibitor. Generally the
interaction
of a protease with an inhibitor, such as a protease trap, such as a serpin, is
a second
order reaction proportional to the product of the concentration of each
reactant, the
inhibitor and the protease. The second order rate constant for inhibition of a
protease
by a tight binding or irreversible inhibitor or a protease trap is a constant,
which when
multiplied by the enzyme concentration and the inhibitor concentration yields
the rate
of enzyme inactivation by a particular inhibitor. The rate constant for each
protease
trap and enzyme pair uniquely reflects their interaction. As a second order
reaction,
an increase in the second order rate constant means that the interaction
between a
modified selected protease and inhibitor is faster compared to the interaction
of an
unmodified protease and the inhibitor. Thus, a change in the second order rate
constant reflects a change in the interaction between the components, the
protease
and/or inhibitor, of the reaction. An increased second order rate constant
when
screening for proteases can reflect a desired selected modification in the
protease.
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As used herein, acyl enzyme intermediate refers to the covalent intermediate
formed during the first step in the catalysis between a substrate and an
essential serine
in the catalytic center of a serine protease (typically Ser195, based on
chymotrypsin
numbering). The reaction proceeds as follows: the serine ¨OH attacks the
carbonyl
carbon at the P1 position of the substrate, the nitrogen of the histidine
accepts the
hydrogen from the ¨OH of the serine, and a pair of electrons from the double
bond of
the carbonyl oxygen moves to the oxygen. This results in the generation of a
negatively charged tetrahedral intermediate. The bond joining the nitrogen and
the
carbon in the peptide bond of the substrate is now broken. The covalent
electrons
creating this bond move to attack the hydrogen of the histidine thereby
breaking the
connection. The electrons that previously moved from the carbonyl oxygen
double
bond move back from the negative oxygen to recreate the bond resulting in the
formation of a covalent acyl enzyme intermediate. The acyl enzyme intermediate
is
hydrolyzed by water, resulting in deacylation and the formation of a cleaved
substrate
and free enzyme.
As used herein, a collection of proteases refers to a collection containing at
least 10 different proteases and/or proteolytically active portions thereof,
and
generally containing at least 50, 100, 500, 1000, 104, 105 or more members.
The
collections typically contain proteases (or proteolytically active portions
thereof) to be
screened for substrate specificity. Included in the collections are naturally
occurring
proteases (or proteolytically active portions thereof) and/or modified
proteases (or
proteolytically active portions thereof). The modifications include random
mutations
along the length of the proteases and/or modifications in targeted or selected
regions
(i.e focused mutations). The modifications can be combinatorial and can
include all
permutations, by substitution of all amino acids at a particular locus or at
all loci or
subsets thereof The collections can include proteases of full length or
shorter,
including only the protease domain. The proteases can include any proteases,
such
as serine proteases and cysteine proteases. The size of the collection and
particular
collection is determined by the user. In other embodiments, the collection can
contain
as few as 2 proteases.
As used herein, "combinatorial collections" or "combinatorial libraries"
refers to a collection of protease polypeptides having distinct and diverse
amino acid
mutations in its sequence with respect to the sequence of a starting template
protease
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polypeptide sequence. The mutations represented in the collection can be
across the
sequence of the polypeptide or can be in a specified region or regions of the
polypeptide sequence. The mutations can be made randomly or can be targeted
mutations designed empirically or rationally based on structural or functional
information.
As used herein, a "teinplate protease" refers to a protease having a sequence
of
amino acids that is used for mutagenesis thereof. A template protease can be
the
sequence of a wild-type protease, or a catalytically active portion thereof,
or it can be
the sequence of a variant protease, or catalytically active portion thereof,
for which
additional mutations are made. For example, a mutant protease identified in
the
selection methods herein, can be used as a starting template for further
mutagenesis to
be used in subsequent rounds of selection.
As used herein, random mutation refers to the introduction of one or more
amino acid changes across the sequence of a polypeptide without regard or bias
as to
the mutation. Random mutagenesis can be facilitated by ,a variety of
techniques
known to one of skill in the art including, for example, UV irradiation,
chemical
methods, and PCR methods (e.g., error-prone PCR).
As used herein, a focused mutation refers to one or more amino acid changes
in a specified region (or regions) or a specified position (or positions) of a
polypeptide. For example, targeted mutation of the amino acids in the
specificity
binding pocket of a protease can be made. Focused mutagenesis can be
performed,
for example, by site directed mutagenesis or multi-site directed mutagenesis
using
standard recombinant techniques known in the art.
As used herein, a stable complex between a protease trap and a protease or a
proteolytically active portion thereof refers to a complex that is
sufficiently stable to
be separated from proteases that did not form complexes with the protease trap
(i.e.
uncomplexed proteases). Such complexes can be formed via any stable
interaction,
including covalent, ionic and hydrophobic interactions, but are sufficiently
stable
under the reaction conditions to remain complexed for sufficient time to
separate
complexes for isolation. Typically such interactions, such as between serpins
and
cleaved proteases, are covalent bonds.
As used herein, a "hot spot" refers to a position that is mutated in multiple
variants resulting from the protease selection that exhibit improved activity
and/or
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selectivity for the desired new substrate sequence. One or more "hot spots"
can be
identified during protease selection. Hence, such positions are specificity
and/or
selectivity determinants for the protease and thereby contribute to substrate
specificity
and also can occur as broad specificity and/or selectivity determinants across
the
corresponding locus in more than one member of a protease family, such as a
serine
protease family or a particular protease family, such as based on chymotrypsin
numbering.
As used herein, desired specificity with reference to substrate specificity
refers
to cleavage specificity for a predetermined or preselected or otherwise
targeted
substrate.
As used herein, "select" or grammatical variations thereof refers to picking
or
choosing a protease that is in complex with a protease trap polypeptide.
Hence, for
purposes herein, select refers to pulling out the protease based on its
association in
stable complexes with a protease trap polypeptide. Generally, selection can be
facilitated by capture of the covalent complexes, and if desired, the protease
can be
isolated. For example, selection can be facilitated by labeling the protease
trap
polypeptide, for example, with a predetermined marker, tag or other detectable
moiety, to thereby identify the protease based on its association in the
stable complex.
As used herein, "identify" and grammatical variations thereof refers to the
recognition of or knowledge of a protease in a stable complex. Typically, in
the
methods herein, the protease is identified by its association in a stable
complex with a
protease trap polypeptide, which can be accomplished*, for example, by
amplification
(i.e. growth in an appropriate host cell) of the bound proteases in the
complex
followed by DNA sequencing.
As used herein, labeled for detection or separation means that that the
molecule, such as a protease trap polypeptide, is associated with a detectable
label,
such as a fluorophore, or is associated with a tag or other moiety, such as
for
purification or isolation or separation. Detectably labeled refers to a
molecule, such
as a protease trap polypeptide, that is labeled for detection or separation.
As used herein, reference to amplification of a protease or proteolytically
active portion of a protease, means that the amount of the protease or
proteolytically
active portion is increased, such as through isolation and cloning and
expression, or,
where the protease or proteolytically active portion is displayed on a
microorganism,
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the microorganism is introduced into an appropriate host and grown or cultured
so
that more displayed protease or proteolytically active portion is produced.
As used herein, homogeneous with reference to a reaction mixture means that
the reactants are in the liquid phase as a mixture, including as a solution or
suspension.
As used herein, recitation that a collection of proteases or proteolytically
active portions of proteases is "based on" a particular protease means that
the
collection is derived from the particular protease, such as by random or
directed
mutagenesis or rational design or other modification scheme or protocol, to
produce a
collection.
As used herein, a disease or condition that is treated by administration oft-
PA
refers to a disease or condition for which one of skill in the art would
administer t-PA.
Such conditions include, but are not limited to, fibrinolytic conditions, such
as arterial
thrombosis, venous thrombosis and thromboembolism, ischemic stroke, acquired
coagulation disorders, disseminated intravascular coagulation, and precursors
thereto,
such as bacterial or viral infections, periodontitis, and neurological
conditions.
As used herein, a disease or condition that is mediated by VEGFR-2 is
involved in the pathology or etiology. Such conditions include, but are not
limited to,
inflammatory and angiogenic conditions, such as cancers, diabetic
retinopathies, and
ophthalmic disorders, including macular degeneration.
As used herein, "proteases," "proteinases" and "peptidases" are
interchangeably used to refer to enzymes that catalyze the hydrolysis of
covalent
peptidic bonds. These designations include zymogen forms and activated single-
,
two- and multiple-chain forms thereof. For clarity, reference to protease
refers to all
forms. Proteases include, for example, serine proteases, cysteine proteases,
aspartic
proteases, threonine and metallo-proteases depending on the catalytic activity
of their
active site and mechanism of cleaving peptide bonds of a target substrate.
As used herein, a zymogen refers to a protease that is activated by
proteolytic
cleavage, including maturation cleavage, such as activation cleavage, and/or
complex
formation with other protein(s) and/or cofactor(s). A zymogen is an inactive
precursor
of a proteolytic enzyme. Such precursors are generally larger, although not
necessarily larger than the active form. With reference to serine proteases,
zymogens
are converted to active enzymes by specific cleavage, including catalytic and
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autocatalytic cleavage, or by binding of an activating co-factor, which
generates an
active enzyme. A zymogen, thus, is an enzymatically inactive protein that is
converted to a proteolytic enzyme by the action of an activator. Cleavage can
be
effected autocatalytically Zymogens, generally, are inactive and can be
converted to
mature active polypeptides by catalytic or autocatalytic cleavage of the
proregion
from the zymogen.
As used herein, a "proregion," "propeptide," or "pro sequence," refers to a
region or a segment that is cleaved to produce a mature protein. This can
include
segments that function to suppress enzymatic activity by masking the catalytic
machinery and thus preventing formation of the catalytic intermediate (i.e.,
by
sterically occluding the substrate binding site). A proregion is a sequence of
amino
acids positioned at the amino terminus of a mature biologically active
polypeptide and
can be as little as a few amino acids or can be a multidomain structure.
As used herein, an activation sequence refers to a sequence of amino acids in
a
zymogen that are the site required for activation cleavage or maturation
cleavage to
form an active protease. Cleavage of an activation sequence can be catalyzed
autocatalytically or by activating partners.
Activation cleavage is a type of maturation cleavage in which a
conformational change required for activity occurs. This is a classical
activation
pathway, for example, for serine proteases in which a cleavage generates a new
N-
terminus which interacts with the conserved regions of catalytic machinery,
such as
catalytic residues, to induce conformational changes required for activity.
Activation
can result in production of multi-chain forms of the proteases. In some
instances,
single chain forms of the protease can exhibit proteolytic activity as a
single chain.
As used herein, domain refers to a portion of a molecule, such as proteins or
the encoding nucleic acids, that is structurally and/or functionally distinct
from other
portions of the molecule and is identifiable.
As used herein, a protease domain is the catalytically active portion of a
protease. Reference to a protease domain of a protease includes the single,
two- and
multi-chain forms of any of these proteins. A protease domain of a protein
contains
all of the requisite properties of that protein required for its proteolytic
activity, such
as for example, its catalytic center.
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As used herein, a catalytically active portion or proteolytically active
portion
of a protease refers to the protease domain, or any fragment or portion
thereof that
retains protease activity. Significantly, at least in vitro, the single chain
forms of the
proteases and catalytic domains or proteolytically active portions thereof
(typically C-
terminal truncations) exhibit protease activity.
As used herein, a "nucleic acid encoding a protease domain or catalytically
active portion of a protease" refers to a nucleic acid encoding only the
recited single
chain protease domain or active portion thereof, and not the other contiguous
portions
of the protease as a continuous sequence.
As used herein, recitation that a polypeptide consists essentially of the
protease domain means that the only portion of the polypeptide is a protease
domain
or a catalytically active portion thereof. The polypeptide can optionally, and
generally will, include additional non-protease-derived sequences of amino
acids.
As used herein, "Sl-S4" refers to amino acid residues that form the binding
sites for P1-P4 residues of a substrate (see, e.g., Schecter and Berger (1967)
Biochem
Biophys Res Commun 27:157-162). Each of Sl-S4 contains one, two or more
residues, which can be non-contiguous. These sites are numbered sequentially
from
the recognition site N-terminal to the site of proteolysis, referred to as the
scissile
bond.
As used herein, the terms "Pl-P4" and "P l'-P4" refer to the residues in a
substrate peptide that specifically interact with the S1-S4 residues and S I '-
S4'
residues, respectively, and are cleaved by the protease. PI-P4 refer to the
residue
positions on the N-terminal side of the cleavage site; P1 '-P4' refer to the
residue
positions to the C-terminal side of the cleavage site. Amino acid residues are
labeled
from N to C termini of a polypeptide substrate (Pi, ..., P3, P2, P1, P1', P2',
P3', Pj).
The respective binding sub-sites are labeled (Si,..., S3, S2, SI, S1', S2',
S3',..., Sj).
The cleavage is catalyzed between P1 and Pl.'
As used herein, a "binding pocket" refers to the residue or residues that
interact with a specific amino acid or amino acids on a substrate. A
"specificity
pocket" is a binding pocket that contributes more energy than the others (the
most
important or dominant binding pocket). Typically, the binding step precedes
the
formation of the transition state that is necessary for the catalytic process
to occur.
S1-S4 and Sl'- S4' amino acids make up the substrate sequence binding pocket
and
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facilitate substrate recognition by interaction with PI-P4 and Pl'-P4'amino
acids of a
peptide, polypeptide or protein substrate, respectively. Whether a protease
interacts
with a substrate is a function of the amino acids in the Sl-S4 and S I '-S4'
positions.
If the amino acids in any one or more of the Si, S2, S3, S4, Si', S2', S3' and
S4' sub-
sites interact with or recognize any one or more of the amino acids in the P1,
P2, P3,
P4, P I ', P2', P3' and P4' sites in a substrate, then the protease can cleave
the
substrate. A binding pocket positions a target amino acid with a protease so
that
catalysis of a peptide bond and cleavage of a substrate is achieved. For
example,
serine proteases typically recognize P4-P2' sites in a substrate; other
proteases can
have extended recognition beyond P4-P2'.
As used herein, amino acids that "contribute to extended substrate
specificity"
refers to those residues in the active site cleft in addition to the
specificity pocket.
These amino acids include the Sl-S4, Sl'-S4' residues in a protease.
As used herein, secondary sites of interaction are outside the active site
cleft.
These can contribute to substrate recognition and catalysis. These amino acids
include amino acids that can contribute second and third shell interactions
with a
substrate. For example, loops in the structure of a protease surrounding the
Sl-S4.
S1'-S4' amino acids play a role in positioning P1-P4, P1'-P4' amino acids in
the
substrate thereby registering the scissile bond in the active site of a
protease.
As used herein, active site of a protease refers to the substrate binding site
where catalysis of the substrate occurs. The structure and chemical properties
of the
active site allow the recognition and binding of the substrate and subsequent
hydrolysis and cleavage of the scissile bond in the substrate. The active site
of a
protease contains amino acids that contribute to the catalytic mechanism of
peptide
cleavage as well as amino acids that contribute to substrate sequence
recognition,
such as amino acids that contribute to extended substrate binding specificity.
As used herein, a "catalytic triad" or "active site residues" of a serine or
cysteine protease refers to a combination of amino acids, typically three
amino acids,
that are in the active site of a serine or cysteine protease and contribute to
the catalytic
mechanism of peptide cleavage. Generally, a catalytic triad is found in serine
proteases and provides an active nucelophile and acid/base catalysis. The
catalytic
triad of serine proteases contains three amino acids, which in chymotrypsin
are
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Aspi 2, His57, and Ser195. These residues are critical for the catalytic
efficiency of a
serine protease.
As used herein, the "substrate recognition site" or "cleavage sequence" refers
to the sequence recognized by the active site of a protease that is cleaved by
a
protease. Typically, for example, for a serine protease, a cleavage sequence
is made
up of the PI-P4 and P I '-P4' amino acids in a substrate, where cleavage
occurs after
the PI position. Typically, a cleavage sequence for a serine protease is six
residues in
length to match the extended substrate specificity of many proteases, but can
be
longer or shorter depending upon the protease. For example, the substrate
recognition
site or cleavage sequence of MT-SP1 required for autocatalysis is RQARVV,
where
R is at the P4 position, Q is at the P3 position, A is at the P2 position and
R is at the
P1 position. Cleavage in MT-SP1 occurs after position R followed by the
sequence
VVGG.
As used herein, target substrate refers to a substrate that is specifically
cleaved
at its substrate recognition site by a protease. Minimally, a target substrate
includes
the amino acids that make up the cleavage sequence. Optionally, a target
substrate
includes a peptide containing the cleavage sequence and any other amino acids.
A
full-length protein, allelic variant, isoform, or any portion thereof,
containing a
cleavage sequence recognized by a protease, is a target substrate for that
protease.
Additionally, a target substrate includes a peptide or protein containing an
additional
moiety that does not affect cleavage of the substrate by a protease. For
example, a
target substrate can include a four amino acid peptide or a full-length
protein .
chemically linked to a fluorogenic moiety.
As used herein, cleavage refers to the breaking of peptide bonds by a
protease.
The cleavage site motif for a protease involves residues N- and C-terminal to
the
scissile bond (the unprimed and primed sides, respectively, with the cleavage
site for
a protease defined as ... P3-P2-PI-P1'-P2'-P3' ..., and cleavage occurs
between the PI
and P1 residues). Typically, cleavage of a substrate is an activating cleavage
or an
inhibitory cleavage. An activating cleavage refers to cleavage of a
polypeptide from
an inactive form to an active form. This includes, for example, cleavage of a
zymogen to an active enzyme, and/or cleavage of a progrowth factor into an
active
growth factor. For example, MT-SP1 can auto-activate by cleaving a target
substrate
at the PI-P4 sequence of RQAR. An activating cleavage also is cleavage whereby
a
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protein is cleaved into one or more proteins that themselves have activity.
For
example, activating cleavage occurs in the complement system, which is an
irreversible cascade of proteolytic cleavage events whose termination results
in the
formation of multiple effector molecules that stimulate inflammation,
facilitate
antigen phagocytosis, and lyse some cells directly.
As used herein, an inhibitory cleavage is cleavage of a protein into one or
more degradation products that are not functional. Inhibitory cleavage results
in the
diminishment or reduction of an activity of a protein. Typically, a reduction
of an
activity of a protein reduces the pathway or process for which the protein is
involved.
In one example, the cleavage of any one ore more target proteins, such as for
example
a VEGFR, that is an inhibitory cleavage results in the concomitant reduction
or
inhibition of any one or more functions or activities of the target substrate.
For
example, for cleavage of a VEGFR, activities that can be inhibited include,
but are not
limited to, ligand binding, kinase activity, or angiogenic activity such as
angiogenic
activity in vivo or in vitro. To be inhibitory, the cleavage reduces activity
by at least
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99.9% or more
compared to a native form of the protein. The percent cleavage of a protein
that is
required for the cleavage to be inhibitory varies among proteins but can be
determined
by assaying for an activity of the protein.
As used herein, a protease polypeptide is a polypeptide having an amino acid
sequence corresponding to any one of the candidate proteases, or variant
proteases
thereof described herein.
As used herein, a "modified protease," or "mutein protease" refers to a
protease polypeptide (protein) that has one or more modifications in primary
sequence
compared to a wild-type or template protease. The one or more mutations can be
one
or more amino acid replacements (substitutions), insertions, deletions and any
combination thereof A modified protease polypeptide includes those with 1, 2,
3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modified
positions. A
modified protease can be a full-length protease, or can be a catalytically
active portion
thereof of a modified full length protease as long as the modified protease is
proteolytically active. Generally, these mutations change the specificity and
activity
of the wild-type or template proteases for cleavage of any one or more desired
or
predetermined target substrates. In addition to containing modifications in
regions
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,
that alter the substrate specificity of a protease, a modified protease also
can tolerate
other modifications in regions that are non-essential to the substrate
specificity of a
protease. Hence, a modified protease typically has 60%, 70%, 80 %, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a
corresponding sequence of amino acids of a wildtype or scaffold protease. A
modified full-length protease or a catalytically active portion thereof of a
modified
protease can include proteases that are fusion proteins as long as the fusion
itself does
not alter substrate specificity of a protease.
As used herein, chymotrypsin numbering refers to the amino acid numbering
of a mature chymotrypsin polypeptide of SEQ ID NO:391. Alignment of a protease
of the chymotrypsin family (i.e. u-PA, t-PA, MT-SP I, and others), including
the
protease domain, can be made with chymotrypsin. In such an instance, the amino
acids of the protease that correspond to amino acids of chymotrypsin are given
the
numbering of the chymotrypsin amino acids. Corresponding positions can be
determined by such alignment by one of skill in the art using manual
alignments or by
using the numerous alignment programs available (for example, BLASTP).
Corresponding positions also can be based on structural alignments, for
example by
using computer simulated alignments of protein structure. Recitation that
amino acids
of a polypeptide correspond to amino acids in a disclosed sequence refers to
amino
acids identified upon alignment of the polypeptide with the disclosed sequence
to
maximize identity or homology (where conservedsainino acids are aligned) using
a
standard alignment algorithm, such as the GAP algorithm. For example, upon
alignment of u-PA with the mature chymotrypsin polypeptide amino acid C168 in
the
precursor sequence of u-PA set forth in SEQ ID NO:191 aligns with amino acid
Cl of
the mature chymotrypsin polypeptide. Thus, amino acid C168 in u-PA also is Cl
based on chymotrypsin numbering. Using such a chymotrypsin numbering standard,
amino acid L244 in the precursor tiTA sequence set forth in SEQ ID NO:191 is
the
same as L73 based on chymotrypsin numbering and amino acid and 1260 is the
same
as 189 based on chymotrypsin numbering. In another example, upon alignment of
the
serine protease domain of MT-SP1 (corresponding to amino acids 615 to 855 in
SEQ
ID NO:253) with mature chymotrypsin, V at position 615 in MT-SP1 is given the
chymotrypsin numbering of V16. Subsequent amino acids are numbered
accordingly.
Thus, an F at amino acid position 708 of full-length MT-SP I (SEQ ID NO:253),
*Trademark
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corresponds to F99 based on chymotrypsin numbering. Where a residue exists in
a
protease, but is not present in chymotrypsin, the amino acid residue is given
a letter
notation. For example, residues in chymotrypsin that are part of a loop with
amino
acid 60 based on chymotrypsin numbering, but are inserted in the MT-SP1
sequence
compared to chymotrypsin, are referred to for example as Asp6Ob or Arg60c.
As used herein, specificity for a target substrate refers to a preference for
cleavage of a target substrate by a protease compared to another substrate,
referred to
as a non-target substrate. Specificity is reflected in the second order rate
constant or
specificity constant (km/Km), which is a measure of the affinity of a protease
for its
substrate and the efficiency of the enzyme.
As used herein, a specificity constant for cleavage is (kcm/Km), wherein Km is
the Michaelis-Menton constant ([S] at one half Vmõ) and Icat is the Vma,J[E-
r], where
ET is the final enzyme concentration. The parameters kcat, Km and kcat/Km can
be
calculated by graphing the inverse of the substrate concentration versus the
inverse of
the velocity of substrate cleavage, and fitting to the Lineweaver-Burk
equation
(1/velocity=(Km/Vmax)(10]) 1Nmõ; where Vmõ=[Er]kcat). Any method to
determine the rate of increase of cleavage over time in the presence of
various
concentrations of substrate can be used to calculate the specificity constant.
For
example, a substrate is linked to a fluorogenic moiety, which is released upon
cleavage by a protease. By determining the rate of cleavage at different
enzyme
concentrations, kw can be determined for a particular protease. The
specificity
constant can be used to determine the site specific preferences of an amino
acid in any
one or more of the S I-S4 pockets of a protease for a concomitant PI-P4 amino
acid in
a substrate using standard methods in the art, such as a positional scanning
combinatorial library (PS-SCL). Additionally, the specificity constant also
can be
used to determine the preference of a protease for one target substrate over
another
substrate. =
As used herein, a substrate specificity ratio is the ratio of specificity
constants
and can be used to compare specificities of two or more proteases or a
protease for
two more substrates. For example, substrate specificity of a protease for
competing
substrates or of competing proteases for a substrate can be compared by
comparing
kem/Km For example, a protease that has a specificity constant of 2 X 106 M-
Iseel for
a target substrate and 2 X 104 M'isec-1 for a non-target substrate is more
specific for
=
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the target substrate. Using the specificity constants from above, the protease
has a
substrate specificity ratio of 100 for the target protease.
As used herein, preference for a target substrate can be expressed as a
substrate specificity ratio. The particular value of the ratio that reflects a
preference
is a function of the substrates and proteases at issue. A substrate
specificity ratio that
is greater than 1 signifies a preference for a target substrate and a
substrate specificity
less than 1 signifies a preference for a non-target substrate. Generally, a
ratio of at
least or about 1 reflects a sufficient difference for a protease to be
considered a
candidate therapeutic.
As used herein, altered specificity refers to a change in substrate
specificity of
a modified or selected protease compared to a starting wild-type or template
protease.
Generally, the change in specificity is a reflection of the change in
preference of a
modified protease for a target substrate compared to a wildtype substrate of
the
template protease (herein referred to as a non-target substrate). Typically,
modified
proteases or selected proteases provided herein exhibits increased substrate
specificity
for any one or more predetermined or desired cleavage sequences of a target
protein
compared to the substrate specificity of a template protease. For example, a
modified
protease or selected protease that has a substrate specificity ratio of 100
for a target
substrate versus a non-target substrate exhibits a 10-fold increased
specificity
compared to a scaffold protease with a substrate specificity ratio of 10. In
another
example, a modified protease that has a substrate specificity ratio of 1
compared to a
ratio of 0.1, exhibits a 10-fold increase in substrate specificity. To exhibit
increased
specificity compared to a template protease, a modified protease has a 1.5-
fold, 2-
fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-
fold or more
greater substrate specificity for any.one of more of the predetermined target
substrates.
, As used herein, "selectivity" can be used interchangeably with specificity
when referring to the ability of a protease to choose and cleave one target
substrate
from among a mixture of competing substrates. Increased selectivity of a
protease for
a target substrate compared to any other one or more target substrates can be
determined, for example, by comparing the specificity constants of cleavage of
the
target substrates by a protease. For example, if a protease has a specificity
constant of
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:
cleavage of 2 X 106 M' sec for a target substrate and 2 X 104 sec-1
for any other
one of more substrates, the protease is more selective for the former target
substrate.
As used herein, activity refers to a functional activity or activities of a
polypeptide or portion thereof associated with a full-length (complete)
protein.
Functional activities include, but are not limited to, biological activity,
catalytic or
enzymatic activity, antigenicity (ability to bind to or compete with a
polypeptide for
binding to an anti-polypeptide antibody), immunogenicity, ability to form
multimers,
and the ability to specifically bind to a receptor or ligand for the
polypeptide.
As used herein, catalytic activity or cleavage activity refers to the activity
of a
protease as assessed in in vitro proteolytic assays that detect proteolysis of
a selected
substrate. Cleavage activity can be measured by assessing catalytic efficiency
of a
protease.
As used herein, activity towards a target substrate refers to cleavage
activity
and/or functional activity, or other measurement that reflects the activity of
a protease
on or towards a target substrate. Cleavage activity can be measured by
assessing
catalytic efficiency of a protease. For purposes herein, an activity is
increased if a
protease exhibits greater protcolysis or cleavage of a target substrate and/or
modulates
(i.e. activates or inhibits) a functional activity of a target substrate
protein as
compared to the absence of the protease.
As used herein, serine protease or serine endopeptidases refers to a class of
peptidases, which are characterized by the presence of a serine residue in the
active
center of the enzyme. Serine proteases participate in a wide range of
functions in the
body, including blood clotting, inflammation as well as digestive enzymes in
prokaryotes and eukaryotes. The mechanism of cleavage by "serine proteases,"
is
based on nucleophilic attack of a targeted peptidic bond by a serine.
Cysteine,
threonine or water molecules associated with aspartate or metals also can play
this
role. Aligned side chains of serine, histidine and aspartate form a catalytic
triad
common to most serine proteases. The active site of serine proteases is shaped
as a
cleft where the polypeptide substrate binds. Exemplary serine proteases
include
urinary plasminogen activator (u-PA) set forth in SEQ ID NO: 433 and MT-SP I
set
forth in SEQ ID NO:253, and catalytically active portions thereof, for example
the
MT-SP1 protease domain (also called the B-chain) set forth in SEQ ID NO:505.
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As used herein, a human protein is one encoded by a nucleic acid molecule,
such as DNA, present in the genome of a human, including all allelic variants
and
conservative variations thereof. A variant or modification of a protein is a
human
protein if the modification is based on the wildtype or prominent sequence of
a human
protein.
As used herein, the residues of naturally occurring a-amino acids are the
residues of those 20 a-amino acids found in nature which are incorporated into
protein
by the specific recognition of the charged tRNA molecule with its cognate mRNA
codon in humans.
As used herein, non-naturally occurring amino acids refer to amino acids that
are not genetically encoded.
As used herein, nucleic acids include DNA, RNA and analogs thereof,
including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can
be
single or double-stranded. When referring to probes or primers, which are
optionally
labeled, such as with a detectable label, such as a fluorescent or radiolabel,
single-
stranded molecules are contemplated. Such molecules are typically of a length
such
that their target is statistically unique or of low copy number (typically
less than 5,
generally less than 3) for probing or priming a library. Generally a probe or
primer
contains at least 14, 16 or 30 contiguous nucleotides of sequence
complementary to or
identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100
or more
nucleic acids long.
As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino
acids in length.
As used herein, the amino acids which occur in the various sequences of
amino acids provided herein are identified according to their known, three-
letter or
one-letter abbreviations (Table 1). The nucleotides which occur in the various
nucleic
acid fragments are designated with the standard single-letter designations
used
routinely in the art.
As used herein, an "amino acid" is an organic compound containing an amino
group and a carboxylic acid group. A polypeptide contains two or more amino
acids.
For purposes herein, amino acids include the twenty naturally-occurring amino
acids,
non-natural amino acids and amino acid analogs (i.e., amino acids wherein the
a-
carbon has a side chain).
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¨ As used herein, "amino acid residue" refers to an amino acid
formed upon
chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The
amino
acid residues described herein are presumed to be in the "L" isomeric form.
Residues
in the "D" isomeric form, which are so designated, can be substituted for any
L-amino
acid residue as long as the desired functional property is retained by the
polypeptide.
NH2 refers to the free amino group present at the amino terminus of a
polypeptide.
COOH refers to¨the free carboxy group present at the carboxyl terminus of a
polypeptide. In keeping with standard polypeptide nomenclature described in J.
BioL
= Chem., 243: 3552-3559 (1969), abbreviations
for amino acid residues are shown in Table I:
Table 1 ¨ Table of Correspondence
SYMBOL
=
1-Letter 3-Letter AMINO ACID
. Tyr Tyrosine
Gly Glycine
Phe Phenylalanine
Met Methionine
A Ala Alanine
Ser Serine
Ile Isoleucine
Leu Leucine
Thr Threonine
V Val Valine
Pro proline
Lys Lysine
His Histidine
Gin Glutamine
Glu glutamic acid
Glx Glu and/or Gin
Trp Tryptophan
=
Arg Arginine
Asp aspartic acid
=N Asn asparagines
Asx Asn and/or Asp
Cys Cysteine
X Xaa Unknown or other
It should be noted that all amino acid residue sequences represented herein by
formulae have a left to right orientation in the conventional direction of
amino-
terminus to carboxyl-terminus. In addition, the phrase "amino acid residue" is
broadly defined to include the amino acids listed in the Table of
Correspondence
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(Table 1) and modified and unusual amino acids. Furthermore, it should
be noted that a dash at the beginning or end of .an amino acid residue
sequence
indicates a peptide bond to a further sequence of one or more amino acid
residues, to
an amino-terminal group such as NI-12 or to a carboxyl-terminal group such as
COOH.
As used herein, "naturally occurring amino acids" refer to the 20 L-amino
acids that occur in polypeptides.
As used herein, "non-natural amino acid" refers to an organic compound that
has a structure_similar to a natural amino acid but has been modified
structurally to
mimic the structure and reactivity of a natural amino acid. Non-naturally
occurring
amino acids thus include, for example, amino acids or analogs of amino acids
other
than the 20 naturally-occurring amino acids and include, but are not limited
to, the D-
isostereomers of amino acids. Exemplary non-natural amino acids are described
herein and are known to those of skill in the art.
As used herein, an isokinetic mixture is one in which the molar ratios of
amino acids has been adjusted based on their reported reaction rates (see,
e.g.,
Ostresh et al., (1994) Biopolymers 34:1681).
As used herein, a DNA construct is a single or double stranded, linear or
circular DNA molecule that contains segments of DNA combined and juxtaposed in
a
manner not found in nature. DNA constructs exist as a result of human
manipulation,
and include clones and other copies of manipulated molecules.
As used herein, a DNA segment is a portion of a larger DNA molecule having
specified attributes. For example, a DNA segment encoding a specified
polypeptide
is .a portion of a longer DNA molecule, such as a plasmid or plasmid fragment,
which,
when read from the 5' to 3' direction ,encodes the sequence of amino acids of
the
specified polypeptide.
As used herein, the term ortholog means a polypeptide or protein obtained
from one species that is the functional counterpart or a polypeptide or
protein from a
different species. Sequence differences among orthologs are the result of
speciation.
As used herein, the term polynucleotide means a single- or double-stranded
polymer of deoxyribonucleotides or ribonucleotide bases read from the 5' to
the 3'
end. Polynucleotides include RNA and DNA, and can be isolated from natural
sources, synthesized in vitro, or prepared from a combination of natural and
synthetic
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molecules. The length of a polynucleotide molecule is given herein in terms of
nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term
nucleotides
is used for single- and double-stranded molecules where the context permits.
When
the term is applied to double-stranded molecules it is used to denote overall
length
and will be understood to be equivalent to the term base pairs. It will be
recognized
by those skilled in the art that the two strands of a double-stranded
polynucleotide can
differ slightly in length and that the ends thereof can be staggered; thus all
nucleotides
within a double-stranded polynucleotide molecule can not be paired. Such
unpaired
ends will, in general, not exceed 20 nucleotides in length.
As used herein, ''similarity" between two proteins or nucleic acids refers to
the
relatedness between the sequence of amino acids of the proteins or the
nucleotide
sequences of the nucleic acids. Similarity can be based on the degree of
identity
and/or homology of sequences of residues and the residues contained therein.
Methods for assessing the degree of similarity between proteins or nucleic
acids are
known to those of skill in the art. For example, in one method of assessing
sequence
similarity, two amino acid or nucleotide sequences are aligned in a manner
that yields
a maximal level of identity between the sequences. "Identity" refers to the
extent to
which the amino acid or nucleotide sequences are invariant. Alignment of amino
acid
sequences, and to some extent nucleotide sequences, also can take into account
conservative differences and/or frequent substitutions in amino acids (or
nucleotides).
Conservative differences are those that preserve the physico-chemical
properties of
the residues involved. Alignments can be global (alignment of the compared
sequences over the entire length of the sequences and including all residues)
or local
(the alignment of a portion of the sequences that includes only the most
similar region
or regions).
"Identity" per se has an art-recognized meaning and can be calculated using
published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A.M.,
ed.,
Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome
Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis
of
Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press,
New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M
Stockton Press, New York, 1991). While there exists a number of methods to
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measure identity between two polynucleotide or polypeptides, the term
"identity" is
well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math
48:1073 (1988)).
As used herein, homologous (with respect to nucleic acid and/or amino acid
sequences) means about greater than or equal to 25% sequence homology,
typically
greater than or equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95%
sequence homology; the precise percentage can be specified if necessary. For
purposes herein the terms "homology" and "identity" are often used
interchangeably,
unless otherwise indicated. In general, for determination of the percentage
homology
or identity, sequences are aligned so that the highest order match is obtained
(see, e.g.:
Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New
York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part!,
Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994;
Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and
Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York,
1991; Carillo etal. (1988) SIAM J Applied Math 48:1073). By sequence homology,
the number of conserved amino acids is determined by standard alignment
algorithms
programs, and can he used with default gap penalties established by each
supplier.
Substantially homologous nucleic acid molecules would hybridize typically at
moderate stringency. or at high stringency all along the length of the nucleic
acid of
interest. Also contemplated are nucleic acid molecules that contain degenerate
codons in place of codons in the hybridizing nucleic acid molecule.
Whether any two molecules have nucleotide sequences or amino acid
sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
"identical" or "homologous" can be determined using known computer algorithms
such as the "FASTA" program, using for example, the default parameters as in
Pearson et al. (1988) Proc. Natl. Acad. Set. USA 85:2444 (other programs
include the
GCG program package (Devereux, J, et al., Nucleic Acids Research 12(0:387
*
(1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., etal., J Molec Biol 215:403
(1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San
Diego, 1994, and Carillo etal. (1988) SIAM J Applied Math 48:1073). For
example,
*.
the BLAST function of the National Center for Biotechnology Information
database
*Trademark
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=
can be used to determine identity. Other commercially or publicly available
programs,.
include, DNAStar "MegAlign" program (Madison, WI) and the University of
Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison WI).
Percent homology or identity of proteins and/or nucleic acid molecules can be
determined, for example, by comparing sequence information using a GAP
computer
program (e.g., Needleman et al. (1970)J. Mol. Biol. 48:443, as revised by
Smith and
Waterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP program defines
simi-
larity as the number of aligned symbols (i.e., nucleotides or amino acids),
which are
similar, divided by the total number of symbols in the shorter of the two
sequences.
Default parameters for the GAP program can include: (1) a unary comparison
matrix
(containing a value of 1 for identities and 0 for non-identities) and the
weighted com-
parison matrix of Gribskov etal. (1986) Nucl. Acids Res. 14:6745, as described
by
Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE;
National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of
3.0
for each gap and an additional 0.10 penalty for each symbol in each gap; and
(3) no
penalty for end gaps.
Therefore, as used herein, the term "identity" or "homology" represents a
comparison between a test and a reference polypeptide or polynucleotide. As
used
herein, the term at least "90% identical to" refers to percent identities from
90 to 99.99
relative to the reference nucleic acid or amino acid sequence of the
polypeptide.
Identity at a level of 90% or more is indicative of the fact that, assuming
for
exemplification purposes a test and reference polypeptide length of 100 amino
acids
are compared. No more than 10% (i.e., 10 out of 100) of the amino acids in the
test
polypeptide differs from that of the reference polypeptide. Similar
comparisons can
be made between test and reference polynucleotides. Such differences can be
represented as point mutations randomly distributed over the entire length of
a
polypeptide or they can be clustered in one or more locations of varying
length up to
the maximum allowable, e.g. 10/100 amino acid difference (approximately 90%
identity). Differences are defined as nucleic acid or amino acid
substitutions,
insertions .or deletions. At the level of homologies or identities above about
85-90%,
the result should be independent of the program and gap parameters set; such
high
levels of identity can be assessed readily, often by manual alignment without
relying
on software.
*Trademark
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As used herein, an aligned sequence refers to the use of homology (similarity
and/or identity) to align corresponding positions in a sequence of nucleotides
or
amino acids. Typically, two or more sequences that are related by 50% or more
identity are aligned. An aligned set of sequences refers to 2 or more
sequences that
.. are aligned at corresponding positions and can include aligning sequences
derived
from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.
As used herein, "primer" refers to a nucleic acid molecule that can act as a
point of initiation of template-directed DNA synthesis under appropriate
conditions
(e.g., in the presence of four different nucleoside triphosphates and a
polymerization
agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an
appropriate buffer and at a suitable temperature. It will be appreciated that
a certain
nucleic acid molecules can serve as a "probe" and as a "primer." A primer,
however,
has a 3' hydroxyl group for extension. A primer can be used in a variety of
methods,
including, for example, polymerase chain reaction (PCR), reverse-transcriptase
(RT)-
.. PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression
PCR, 3 and 5' RACE, in situ PCR, ligation-mediated PCR and other amplification
protocols.
As used herein, "primer pair" refers to a set of primers that includes a 5'
(upstream) primer that hybridizes with the 5' end of a sequence to be
amplified (e.g.
.. by PCR) and a 3' (downstream) primer that hybridizes with the complement of
the 3'
end of the sequence to be amplified.
As used herein, "specifically hybridizes" refers to annealing, by
complementary base-pairing, of a nucleic acid molecule (e.g. an
oligonucleotide) to a
target nucleic acid molecule. Those of skill in the art are familiar with in
vitro and in
vivo parameters that affect specific hybridization, such as length and
composition of
the particular molecule. Parameters particularly relevant to in vitro
hybridization
further include annealing and washing temperature, buffer composition and salt
concentration. Exemplary washing conditions for removing non-specifically
bound
nucleic acid molecules at high stringency are 0.1 x SSPE, 0.1% SDS, 65 C, and
at
medium stringency are 0.2 x SSPE, 0.1% SDS, 50 C. Equivalent stringency
conditions are known in the art. The skilled person can readily adjust these
parameters to achieve specific hybridization of a nucleic acid molecule to a
target
nucleic acid molecule appropriate for a particular application.
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As used herein, substantially identical to a product means sufficiently
similar
so that the property of interest is sufficiently unchanged so that the
substantially
identical product can be used in place of the product.
As used herein, it also is understood that the terms "substantially identical"
or
.. "similar" varies with the context as understood by those skilled in the
relevant art.
As used herein, an allelic variant or allelic variation references any of two
or
more alternative forms of a gene occupying the same chromosomal locus. Allelic
variation arises naturally through mutation, and can result in phenotypic
polymorphism within populations. Gene mutations can be silent (no change in
the
encoded polypeptide) or can encode polypeptides having altered amino acid
sequence.
The term "allelic variant" also is used herein to denote a protein encoded by
an allelic
variant of a gene. Typically the reference form of the gene encodes a wildtype
form
and/or predominant form of a polypeptide from a population or single reference
member of a species. Typically, allelic variants, which include variants
between and
among species typically have at least 80%, 90% or greater amino acid identity
with a
wildtype and/or predominant form from the same species; the degree of identity
depends upon the gene and whether comparison is interspecies or intraspecies.
Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or
95%
identity or greater with a wildtype and/or predominant form, including 96%,
97%,
98%, 99% or greater identity with a wildtype and/or predominant form of a
polypeptide. Reference to an allelic variant herein generally refers to
variations n
proteins among members of the same species.
As used herein, "allele," which is used interchangeably herein with "allelic
variant" refers to alternative forms of a gene or portions thereof. Alleles
occupy the
same locus or position on homologous chromosomes. When a subject has two
identical alleles of a gene, the subject is said to be homozygous for that
gene or allele.
When a subject has two different alleles of a gene, the subject is said to be
heterozygous for the gene. Alleles of a specific gene can differ from each
other in a
single nucleotide or several nucleotides, and can include substitutions,
deletions and
insertions of nucleotides. An allele of a gene also can be a form of a gene
containing
a mutation.
As used herein, species variants refer to variants in polypeptides among
different species, including different mammalian species, such as mouse and
human.
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As used herein, a splice variant refers to a variant produced by differential
processing of a primary transcript of genomic DNA that results in more than
one type
of mRNA.
As used herein, modification is in reference to modification of a sequence of
amino acids of a polypeptide or a sequence of nucleotides in a nucleic acid
molecule
and includes deletions, insertions, and replacements of amino acids and
nucleotides,
respectively. Methods of modifying a polypeptide are routine to those of skill
in the
art, such as by using recombinant DNA methodologies.
As used herein, a peptidomimetic is a compound that mimics the conformation
and certain stereochemical features of the biologically active form of a
particular
peptide. In general, peptidomimetics are designed to mimic certain desirable
properties of a compound, but not the undesirable properties, such as
flexibility, that
lead to a loss of a biologically active conformation and bond breakdown.
Peptidomimetics can be prepared from biologically active compounds by
replacing
certain groups or bonds that contribute to the undesirable properties with
bioisosteres.
Bioisosteres are known to those of skill in the art. For example the methylene
bioisostere CH2S has been used as an amide replacement in enlcephalin analogs
(see,
e.g., Spatola (1983) pp. 267-357 in Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins, Weinstein, Ed. volume 7, Marcel Dekker, New York).
Mbrphine, which can be administered orally, is a compound that is a
peptidomimetic
of the peptide endorphin. For purposes herein, cyclic peptides are included
among
peptidomimetics as are polypeptides in which one or more peptide bonds is/are
replaced by a mimic.
As used herein, a polypeptide comprising a specified percentage of amino
acids set forth in a reference polypeptide refers to the proportion of
contiguous
identical amino acids shared between a polypeptide and a reference
polypeptide. For
example, an isoform that comprises 70% of the amino acids set forth in a
reference
polypeptide having a sequence of amino acids set forth in SEQ ID NO:XX, which
recites 147 amino acids, means that the reference polypeptide contains at
least 103
contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:XX.
As used herein, the term promoter means a portion of a gene containing DNA
sequences that provide for the binding of RNA polymerase and initiation of
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transcription. Promoter sequences are commonly, but not always, found in the
5'
non-coding region of genes.
As used herein, isolated or purified polypeptide or protein or biologically-
active portion thereof is substantially free of cellular material or other
contaminating
proteins from the cell or tissue from which the protein is derived, or
substantially free
from chemical precursors or other chemicals when chemically synthesized.
Preparations can be determined to be substantially free if they appear free of
readily
detectable impurities as determined by standard methods of analysis, such as
thin
layer chromatography (TLC), gel electrophoresis and high performance liquid
chromatography (HPLC), used by those of skill in the art to assess such
purity, or
sufficiently pure such that further purification would not detectably alter
the physical
and chemical properties, such as enzymatic and biological activities, of the
substance.
Methods for purification of the compounds to produce substantially chemically
pure
compounds are known to those of skill in the art. A substantially chemically
pure
compound, however, can be a mixture of stereoisomers. In such instances,
further
purification might increase the specific activity of the compound.
The term substantially free of cellular material includes preparations of
proteins in which the protein is separated from cellular components of the
cells from
which it is isolated or recombinantly-produced. In one embodiment, the term
substantially free of cellular material includes preparations of protease
proteins having
less that about 30% (by dry weight) of non-protease proteins (also referred to
herein
as a contaminating protein), generally less than about 20% of non-protease
proteins or
10% of non-protease proteins or less that about 5% of non-protease proteins.
When
the protease protein or active portion thereof is recombinantly produced, it
also is
substantially free of culture medium, i.e., culture medium represents less
than about or
at 20%, 10% or 5% of the volume of the protease protein preparation.
As used herein, the term substantially free of chemical precursors or other
chemicals includes preparations of protease proteins in which the protein is
separated
from chemical precursors or other chemicals that are involved in the synthesis
of the
protein. The term includes preparations of protease proteins having less than
about
30% (by dry weight) 20%, 10%, 5% or less of chemical precursors or non-
protease
chemicals or components.
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As used herein, synthetic, with reference to, for example, a synthetic nucleic
acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic
acid
molecule or polypeptide molecule that is produced by recombinant methods
and/or by
chemical synthesis methods.
As used herein, production by recombinant means by using recombinant DNA
methods means the use of the well known methods of molecular biology for
expressing proteins encoded by cloned DNA.
As used herein, vector (or plasmid) refers to discrete elements that are used
to
introduce a heterologous nucleic acid into cells for either expression or
replication
thereof. The vectors typically remain episomal, but can be designed to effect
integration of a gene or portion thereof into a chromosome of the genome. Also
contemplated are vectors that are artificial chromosomes, such as yeast
artificial
chromosomes and mammalian artificial chromosomes. Selection and use of such
vehicles are well known to those of skill in the art.
As used herein, an expression vector includes vectors capable of expressing
DNA that is operatively linked with regulatory sequences, such as promoter
regions,
that are capable of effecting expression of such DNA fragments. Such
additional
segments can include promoter and terminator sequences, and optionally can
include
one or more origins of replication, one or more selectable markers, an
enhancer, a
.. polyadenylation signal, and the like. Expression vectors are generally
derived from
plasmid or viral DNA, or can contain elements of both. Thus, an expression
vector
refers to a recombinant DNA or RNA construct, such as a plasmid, a phage,
recombinant virus or other vector that, upon introduction into an appropriate
host cell,
results in expression of the cloned DNA. Appropriate expression vectors are
well
known to those of skill in the art and include those that are replicable in
eukaryotic
cells and/or prokaryotic cells and those that remain episomal or those which
integrate
into the host cell genome.
As used herein, vector also includes "virus vectors" or "viral vectors." Viral
vectors are engineered viruses that are operatively linked to exogenous genes
to
transfer (as vehicles or shuttles) the exogenous genes into cells.
As used herein, an adenovirus refers to any of a group of DNA-containing
viruses that cause conjunctivitis and upper respiratory tract infections in
humans. As
used herein, naked DNA refers to histone-free DNA that can be used for
vaccines and
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gene therapy. Naked DNA is the genetic material that is passed from cell to
cell
during a gene transfer processed called transformation. In transformation,
purified or
naked DNA is taken up by the recipient cell which will give the recipient cell
a new
characteristic or phenotype.
As used herein, operably or operatively linked when referring to DNA
segments means that the segments are arranged so that they function in concert
for
their intended purposes, e.g., transcription initiates in the promoter and
proceeds
through the coding segment to the terminator.
As used herein, protein binding sequence refers to a protein or peptide
sequence that is capable of specific binding to other protein or peptide
sequences
generally, to a set of protein or peptide sequences or to a particular protein
or peptide
sequence.
As used herein, epitope tag refers to a short stretch of amino acid residues
corresponding to an epitope to facilitate subsequent biochemical and
immunological
analysis of the epitope tagged protein or peptide. Epitope tagging is achieved
by
adding the sequence of the epitope tag to a protein-encoding sequence in an
appropriate expression vector. Epitope tagged proteins can be affinity
purified using
highly specific antibodies raised against the tags.
As used herein, metal binding sequence refers to a protein or peptide sequence
that is capable of specific binding to metal ions generally, to a set of metal
ions or to a
particular metal ion.
As used herein the term assessing is intended to include quantitative and
qualitative determination in the sense of obtaining an absolute value for the
activity of
a protease, or a domain thereof, present in the sample, and also of obtaining
an index,
ratio, percentage, visual or other value indicative of the level of the
activity.
Assessment can be direct or indirect and the chemical species actually
detected need
not of course be the proteolysis product itself but can for example be a
derivative
thereof or some further substance. For example, detection of a cleavage
product of a
complement protein, such as by SDS-PAGE and protein staining with Coomasie
blue.
As used herein, biological activity refers to the in vivo activities of a
compound or physiological responses that result upon in vivo administration of
a
compound, composition or other mixture. Biological activity, thus, encompasses
therapeutic effects and pharmaceutical activity of such compounds,
compositions and
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mixtures. Biological activities can be observed in in vitro systems designed
to test or
use such activities. Thus, for purposes herein a biological activity of a
protease is its
=
catalytic activity in which a polypeptide is hydrolyzed.
As used herein equivalent, when referring to two sequences of nucleic acids,
means that the two sequences in question encode the same sequence of amino
acids or
equivalent proteins. When equivalent is used in referring to two proteins or
peptides,
it means that the two proteins or peptides have substantially the same amino
acid
sequence with only amino acid substitutions that do not substantially alter
the activity
or function of the protein or peptide. When equivalent refers to a property,
the
property does not need to be present to the same extent (e.g., two peptides
can exhibit
different rates of the same type of enzymatic activity), but the activities
are usually
substantially the same. Complementary, when referring to two nucleotide
sequences,
means that the two sequences of nucleotides are capable of hybridizing,
typically with
less than 25%, 15% or 5% mismatches between opposed nucleotides. If necessary,
the percentage of complementarity will be specified. Typically the two
molecules are
selected such that they will hybridize under conditions of high stringency.
As used herein, an agent that modulates the activity of a protein or
expression
of a gene or nucleic acid either decreases or increases or otherwise alters
the activity
of the protein or, in some manner, up- or down-regulates or otherwise alters
expression of the nucleic acid in a cell.
As used herein, a pharmaceutical effect or therapeutic effect refers to an
effect
observed upon administration of an agent intended for treatment of a disease
or
disorder or for amelioration of the symptoms thereof.
As used herein, "modulate" and "modulation" or "alter" refer to a change of
an activity of a molecule, such as a protein. Exemplary activities include,
but are not
limited to, biological activities, such as signal transduction. Modulation can
include
an increase in the activity (i.e., up-regulation or agonist activity) a
decrease in
activity (i.e., down-regulation or inhibition) or any other alteration in an
activity (such
as a change in periodicity, frequency, duration, kinetics or other parameter)
.
Modulation can be context dependent and typically modulation is compared to a
designated state, for example, the wildtype protein, the protein in a
constitutive state,
or the protein as expressed in a designated cell type or condition.
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=
As used herein, inhibit and inhibition refer to a reduction in an activity
relative
to the uninhibited activity.
As used herein, a composition refers to any mixture. It can be a solution,
suspension, liquid, powder, paste, aqueous, non-aqueous or any combination
thereof.
As used herein, a combination refers to any association between or among two
or more items. The combination can be two or more separate items, such as two
compositions or two collections, can be a mixture thereof, such as a single
mixture of
the two or more items, or any variation thereof. The elements of a combination
are
generally functionally associated or related. A kit is a packaged combination
that
optionally includes instructions for use of the combination or elements
thereof.
As used herein, "disease or disorder" refers to a pathological condition in an
organism resulting from cause or condition including, but not limited to,
infections,
acquired conditions, genetic conditions, and characterized by identifiable
symptoms.
Diseases and disorders of interest herein are those involving complement
activation,
including those mediated by complement activation and those in which
complement
activation plays a role in the etiology or pathology. Diseases and disorders
also
include those that are caused by the absence of a protein such as an immune
deficiency, and of interest herein are those disorders where complement
activation
does not occur due to a deficiency in a complement protein.
As used herein, "treating" a subject with a disease or condition means that
the
subject's symptoms are partially or totally alleviated, or remain static
following
treatment. Hence treatment encompasses prophylaxis, therapy and/or cure.
Prophylaxis refers to prevention of a potential disease and/or a prevention of
worsening of symptoms or progression of a disease. Treatment also encompasses
any
pharmaceutical use of a modified interferon and compositions provided herein.
As used herein, a therapeutic agent, therapeutic regimen, radioprotectant, or
chemotherapeutic mean conventional drugs and drug therapies, including
vaccines,
which are known to those skilled in the art. Radiotherapeutic agents are well
known
in the art.
As used herein, treatment means any manner in which the symptoms of a
condition, disorder or disease or other indication, are ameliorated or
otherwise
beneficially altered.
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As used herein therapeutic effect means an effect resulting from treatment of
a
subject that alters, typically improves or ameliorates the symptoms of a
disease or
condition or that cures a disease or condition. A therapeutically effective
amount
refers to the amount of a composition, molecule or compound which results in a
therapeutic effect following administration to a subject.
As used herein, the term "subject" refers to an animal, including a mammal,
such as a human being.
As used herein, a patient refers to a human subject.
As used herein, amelioration of the symptoms of a particular disease or
.. disorder by a treatment, such as by administration of a pharmaceutical
composition or
other therapeutic, refers to any lessening, whether permanent or temporary,
lasting or
transient, of the symptoms that can be attributed to or associated with
administration
of the composition or therapeutic.
As used herein, prevention or prophylaxis refers to methods in which the risk
of developing disease or condition is reduced.
As used herein, an effective amount is the quantity of a therapeutic agent
necessary for preventing, curing, ameliorating, arresting or partially
arresting a
symptom of a disease or disorder.
As used herein, administration of a protease, such as a modified protease,
refers to any method in which the protease is contacted with its substrate.
Adminstration can be effected in vivo or ex vivo or in vitro. For example, for
ex vivo
administration a body fluid, such as blood, is removed from a subject and
contacted
outside the body with the modified non-complement protease. For in vivo
administration, the modified protease can be introduced into the body, such as
by
local, topical, systemic and/or other route of introduction. In vitro
administration
encompasses methods, such as cell culture methods.
As used herein, unit dose form refers to physically discrete units suitable
for
human and animal subjects and packaged individually as is known in the art.
As used herein, a single dosage formulation refers to a formulation for direct
administration.
As used herein, an "article of manufacture" is a product that is made and
sold.
As used throughout this application, the term is intended to encompass
modified
protease polypeptides and nucleic acids contained in articles of packaging.
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As used herein, fluid refers to any composition that can flow. Fluids thus
encompass compositions that are in the form of semi-solids, pastes, solutions,
aqueous
mixtures, gels, lotions, creams and other such compositions.
As used herein, a "kit" refers to a combination of a modified protease
polypeptide or nucleic acid molecule provided herein and another item for a
purpose
including, but not limited to, administration, diagnosis, and assessment of a
biological
activity or property. Kits optionally include instructions for use.
As used herein, a cellular extract or lysate refers to a preparation or
fraction
. which is made from a lysed or disrupted cell.
As used herein, animal includes any animal, such as, but are not limited to
primates including humans, gorillas and monkeys; rodents, such as mice and
rats;
fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine,
such as
pigs and other animals. Non-human animals exclude humans as the contemplated
animal. The proteases provided herein are from any source, animal, plant,
prokaryotic
and fungal. Most proteases are of animal origin, including mammalian origin.
As used herein, a control refers to a sample that is substantially identical
to the
test sample, except that it is not treated with a test parameter, or, if it is
a sample
plasma sample, it can be from a normal volunteer not affected with the
condition of
interest. A control also can be an internal control.
As used herein, the singular forms "a," "an" and "the" include plural
referents
unless the context clearly dictates otherwise. Thus, for example, reference to
compound, comprising "an extracellular domain' includes compounds with one or
a
plurality of extracellular domains.
As used herein, ranges and amounts can be expressed as "about" a particular
= 25 value or range. About also includes the exact amount. Hence
"about 5 bases" means
"about 5 bases" and also "5 bases."
As used herein, "optional" or "optionally" means that the subsequently
described event or circumstance does or does not occur, and that the
description
includes instances where said event or circumstance occurs and instances where
it
does not. For example, an optionally substituted group means that the group is
=
unsubstituted or is substituted.
As used herein, the abbreviations for any protective groups, amino acids and
other compounds, are, unless indicated otherwise, in accord with their common
usage,
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recognized abbreviations, or the IUPAC-IUB Commission on Biochemical
Nomenclature (see, (1972) Biochem. 11:1726).
B. METHOD FOR SCREENING PROTEASES
Provided are methods for screening for proteases with altered properties,
particularly substrate specificity and selectivity. The methods also provide
such
altered proteases that exhibit substantially unchanged or with sufficient
activity for a
therapeutic use. The methods provided herein can be employed with any method
for
=
protease modification and design of modified proteases. Such methods include
random methods for producing libraries, use of existing libraries, and also
directed
evolution methods
A variety of selection schemes to identify proteases having altered substrate
specificity/selectivity have been employed, but each has limitations. The
methods =
provided herein overcome such limitations. Generally, selection schemes
include
those that 1) select for protease binding or 2) select for protease catalysis.
Examples
of strategies that take advantage of protease binding include, for example,
the use of
transition state analogues (TSAs) and those that employ small molecule suicide
substrates. A TSA is a stable compound that mimics the electronic and
structural
features of the transition state of a protease: substrate reaction. The
strongest
interaction between a protease and the substrate typically occurs at the
transition state
of a reaction. A TSA is employed as a model substrate to select for proteases
with
high binding affinity. A TSA is never a perfect mimic of a true transition
state and
their syntheses are difficult (Bertschinger et al. (2005) in Phage display in
Biotech.
and Drug Discovery (Sidhu S, ed), pp. 461-491). Such a strategy has identified
protease variants with altered substrate specificity, but such proteases
generally
exhibit reduced activity because a requirement for protease catalysis is not
part of the
selection scheme.
In an alternate strategy, small molecule suicide substrates (also called
mechanism-based inhibitors) have been used to select for proteases based on
binding.
Such suicide substrates typically are small molecule inhibitors that bind
covalently to
the active site of an enzyme. These suicide substrates contain a reactive
electrophile
that reacts with an enzymes nucleophile to form a covalent bond. Cleavage of a
natural peptide bond by the protease is not required for this reaction.
Typically, such
inhibitors produce a reactive nucleophile only upon binding to the correct
enzyme and
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undergoing normal catalytic steps (see, e.g., Bertschinger et al. (2005) in
Phage
display in Biotech. and Drug Discovery (Sidhu S, ed), pp. 461-491). In many
cases,
the substrate inhibitor mimics the conformation of the first transition state
involved in
catalysis, but do not allow completion of the catalytic cycle. As a result,
the use of
such inhibitors effectively selects for strong binding instead of catalysis
and results in
the selection of inactive enzymes with impaired dissociation of the substrate
(Droge et
al. (2006) ChemBioChem, 7:149-157). Also, due to their size and the lack of
requirement for cleavage of the substrate, they do not recapitulate the
interaction of a
protease with a natural protein substrate.
A protease selection strategy that selects for catalysis instead of binding
also
has been attempted (see, e.g., Heinis et al. (2001), Protein Engineering, 14:
1043-
1052). One of the major limitations in assaying for catalysis is that reaction
products
diffuse away quickly after the reaction is complete making it difficult to
isolate the
catalytically active enzyme. Consequently, strategies that select for
catalysis rely on
anchoring the substrate and the enzyme to phage such that they are in close
proximity.
For example, the protein calmodulin has been used as an immobilization agent
(Demartis (1999) J Mol. Biol., 286:617-633). Reaction substrates are non-
covalently
anchored on calmodulin-tagged phage enzymes using calmodulin-binding peptide
derivatives. Following catalysis, phage displaying the reaction product are
isolated
from non-catalytically active phage using anti-product affinity reagents.
Since the
substrate is attached to the phage particle, however, the catalysis reaction
can be
hindered. Therefore, these and other methods for protease selection, suffer
limitations and do not identify proteases with altered specificity and
substantially
unchanged with sufficient activity for therapeutic applications. The methods
provided herein address these limitations.
Provided herein are method of protease selection to identify proteases and/or
protease variants with altered, optimized or improved substrate specificity.
Such
proteases are identified for optimization and use as therapeutic proteases
that can
cleave and inactivate (or activate) desired protein targets such as, for
example, protein
targets involved in the etiology of a disease or disorder. In the methods for
screening
proteases provided herein, candidate proteases are trapped as stable
intermediate
complexes of the protease enzymatic reaction, and then identified. The stable
intermediate complexes typically are covalent complexes or other complexes
that
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permit separation thereof from non-complexed molecules. Such intermediates,
include, for example, an acyl enzyme intermediate, that permits capture and
ultimately identification of the proteases that have a selected or
predetermined
substrate specificity. Capture (trapping) of the protease is effected by
contacting a
collection of proteases with a protease trap polypeptide that is cleaved by
the protease,
and, upon cleavage, forms the stable complex. Exemplary of such protease trap
polypeptides are serpins, alpha 2 macroglobulin, and other such molecules. The
protease trap polypeptide can be naturally-occurring and/or can be modified to
select
for a particular target substrate.
In practicing the methods, collections of proteases, typically modified or
mutant proteases and/or collections of natural proteases, are contacted with a
protease
trap polypeptide that reacts with the protease following substrate cleavage to
form the
complex containing the trapped intermediate. These methods can be used to
identify
proteases having a desired substrate specificity/selectivity. To achieve
identification
of proteases having a desired substrate specificity/selectivity, the amino
acid sequence
of the scissile bond, and/or surrounding sequences in the reactive site, such
as the
reactive loop sequence or analogous sequence, can be modified in the protease
trap
polypeptide to mimic the substrate cleavage sequence of a desired target
substrate.
The screening reaction is performed by contacting a collection of proteases
with the protease trap polypeptide under conditions whereby stable complexes,
typically covalent complexes form. The complexes are of sufficient stability
to
permit their separation from other less stable complexes and tuu-eacted
protease trap
polypeptides.
The protease trap polypeptides can be identifiable labeled or affinity-tagged
to
facilitate identification of complexes. For example, labeling of the protease
trap
polypeptides, such as by a fluorescent moiety, affinity tag or other such
labeling/tagging agent facilitates the isolation of the protease-inhibitor
complex and
identification of the selected protease. Selected proteases can be analyzed
for activity
to assess proteolytic efficiency and substrate specificity. The identified or
selected
proteases also can be identified, such as by sequencing or other
identification
protocol, including mass spectrometric methods, or by other labeling methods,
to
identify selected proteases in the complexes.
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The methods provided herein also include optional iterative screening steps,
such that the method can be performed once, or can be performed in multiple
rounds
hone in on proteases of a desired or predetermined specificity/selectivity
and/or
cleavage activity. For example, proteases selection can include randomly or
empirically or systematically modifying the selected protease (in targeted
regions
and/or along the length), and repeating (in one, two, three, four or more
rounds) the
method of contacting the proteases collection with one or more protease trap
polypeptide.
The methods provided herein can be multiplexed, such as by including two or
more differentially labeled or differentially identifiable protease trap
polypeptides.
In the methods provided herein, it is not necessary that the protease trap
polypeptide exhibit 100% or even very high efficiency in the complexing
reaction as
long as at least a detectable percentage, typically at least 1%, 2%, 5%, 10%,
20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or more, can form a stable complex that can
be separated or otherwise identified from among less stable complexes or
unreacted
protease trap polypeptides. Thus, proteases can be selected where partitioning
occurs
in the reaction in which there is than 100% inhibition by the protease trap
polypeptide, such as for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% 90%, 95%, 99% or more inhibition of protease catalyzed reaction. In the
methods provided herein, the stringency of the selection and other parameters
can be
modulated, such as by controlling reaction time, temperature, pH, ionic
strength,
and/or library and substrate concentrations. Specificity constraints also can
be
modulated during selection by including competitors such as, for example,
specific
competitors containing an undesired substrate cleavage sequence or broader
classes of
competitors, such as for example, human plasma.
The method provided herein also can be performed by contacting a collection
of proteases with one protease trap polypeptide or mixtures of different
protease trap
polypeptides such as by multiplexing. Where a plurality of different protease
trap
polypeptides are used, each protease trap polypeptide can be individually and
distinctly labeled so that they can be identifiably detected. Such a method
enables the
isolation and identification of multiple proteases from a collection of
proteases in a
single reaction.
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The methods provided herein permit collections of proteases to be screened at
once to identify those having a desired or predetermined substrate
specificity. The
collections of proteases include, any collection of proteases, including
collections of
various wild-type proteases, modified proteases, mixtures thereof, and also
proteolytically active portions thereof. Any collection can be employed. The
collections also can be made as a set of mutant proteases, or proteolytically
active
portions thereof that contain the mutation. Such collections include,
combinatorial
collections in which members in the collection contain diverse mutations. The
mutation can be random along the length of a protease (or catalytically active
portion
thereof) or can be targeted to a particular position or region, such as for
example, the
specificity binding pocket of the protease. The methods provided herein can
identify
and discover non-contact residues not previously appreciated to be involved as
specificity determinants (i.e. buried residues). Hence, the protease selection
technology method provided herein can be used to create proteases with
entirely new
specificities and activities andfor to optimize the specificity or activity of
an existing
protease lead.
C. PROTEASE TRAP POLYPEPTIDES
A protease trap polypeptide used in the methods provided herein is a
polypeptide, or a polypeptide portion containing a reactive site, that serves
as a
substrate for a protease that upon cleavage results in the formation of a
protease-
substrate intermediate complex, that is stable. Generally, such a protease
trap
polypeptide is one that requires cleavage of a scissile bond (Pl-P1') by the
protease to
yield the generation of a trapped substrate-protease complex. The stable
complex is
typically an irreversible complex formed through the tight interactions
between the
protease and the protease trap polypeptide, such as due to covalent, ionic,
hydrophobic, or other tight linkages. As such the complex is generally stable
for
hours, days, weeks, or more thereby permitting isolation of the complex. In
one
example, the stable intermediate complex can be an acyl enzyme intermediate
that is
formed upon reaction of a serine or cysteine protease with a protease trap
polypeptide.
.. Most usually, following protease trap polypeptide cleavage a rapid
conformational
change in the complex distorts the protease and prevents deacylation of the
acyl-
enzyme complex. Thus, panning proteases with protease trap polypeptides allows
selection' for the rate limiting step of catalysis (i.e. cleavage of the P1-
P1' bond and
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acylation of the enzyme) while at the same time forming very tight (i.e.
covalent)
complexes that are easily isolated from collection mixtures.
Typically, such protease trap polypeptides are large (greater than 100 amino
acids), single domain proteins containing a reactive site sequence recognized
by a
protease. Generally, the reactive site cleavage sequence is part of a larger
reactive
loop that is flexible, exposed, and long to make it a target substrate
(Otlewski et al.
(2005) The EMBO J. 24: 1303-1310), however, so long as the protease trap
contains a
reactive site sequence that can be cleaved by a protease, thereby mimicking
substrate
cleavage, it can be used in the methods provided herein. Thus, any large
polypeptide
or synthetically produced polypeptide that contains a scissile bond cleaved by
a
protease resulting in the trapping of a protease in a long-lasting, stable
complex can
be used in the methods provided herein. Exemplary of such protease trap
polypeptides are serpins, such as any described herein. Other protease trap
polypeptides also can be used in the methods provided herein, such as any
whose
mechanism of action is similar to those of serpin molecules. These include,
for
example, synthetic or recombinantly generated serpin-like molecules, or
polypeptides
containing contiguous fragments or sequences of a serpin molecule including a
sufficient portion of a reactive site loop of a serpin molecule. In addition,
other
protease inhibitors whose mechanism of inhibition is similar to that of
serpins can be
used, such as for example, the baculovirus p35 protein that inhibits caspases
(Xu et al.
(2001) Nature, 410:494-497; Otlewski et al. (2005) The EMBO J. 24: 1303-1310).
Other protease trap polypeptides include any that trap a protease in a stable
complex
that can be easily isolated, such as, but not limited to, alpha 2
macroglobulin.
1. SERPINS: Structure, Function, and Expression
Serpins (serine protease inhibitors) are protease inhibitors that are large
protein molecules (about 330-500 amino acids) compared to other serine
protease
inhibitors that are normally about less than 60 amino acids. The serpin
superfamily is
the largest and most broadly distributed of protease inhibitors. Over 1,500
serpin
family members have been identified to date in a variety of different animals,
poxviruses, plants, bacteria, and archaea (Law etal. (2006) Genome Biology,
7:216),
with over thirty different human serpins studied thus far. Most human serpins
are
found in the blood where they function in a wide range of regulatory roles
including,
for example, inflammatory, complement, coagulation, and fibrinolytic cascades.
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Serpins also function intracellularly to perform cytoprotective roles, such as
for
example, regulating the inappropriate release of cytotoxic proteases. Although
most
serpins have an inhibitory role on protease activity, some serpins perform
other non-
inhibitory roles such as but not limited to, hormone transport, corticosteroid
binding
globulin, and blood pressure regulation (Silverman etal. (2001) JBC, 276:
33293-
33296). Among non-inhibitory serpins are steroid binding globulins and
ovalbumin.
Typically, serpins inhibit the action of serine proteases, although several
serpins have
been identified that are inhibitors of papain-like cysteine proteases or
caspases
(Whisstock et al. (2005) FEBS Journal, 272: 4868-4873).
The sequence identity among serpin family members is weak, however, their
structures are highly conserved. For example, members of the serpin family
share
about 30% amino acid sequence homology with the serpin alphal -antitrypsin and
have a conserved tertiary structure. Structurally, serpins are made up of
three p sheets
(A, B, and C) and 8-9 a-helices (A-I), which are organized into an upper 3-
barrel
domain and a lower helical domain. The two domains are bridged by the five
stranded B-sheet A, which is the main structural feature of serpins
(Huntington etal.
(2003), J. Thrombosis and Haemostasis, 1: 1535-1549). Serpins are metastable
proteins such that they are only partially stable in their active form; they
require
protease to adopt a completely stable conformation. A loop, termed the
reactive site
loop (RSL), is responsible for the altered conformation of the serpin
molecule. The
RSL is an exposed stretch of about 17 amino acid residues that protrudes out
from the
top of the molecule in a region between the A and C p-sheets. The RSL serves
as the
protease recognition site, and generally contains the sole determinants of
protease
specificity. The most stable form of the serpin structure is the RSL-cleaved
form.
Following protease cleavage, the amino terminal portion of the RSL inserts
into the
center of 3-sheet A to become strand four of the six-stranded 3-sheet. This
conformational change is termed the "stressed" to "relaxed" (or S to R)
transition.
This transformation is characterized by an increase in thermal stability of
the
molecule owing to the reorganization of the five-stranded 3-sheet A to a six-
stranded
anti-parallel form (Lawrence et al. (2000), J Biol. Chem., 275: 5839-5844). In
other
words, the native structure of serpins is equivalent to a latent intermediate,
which is
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only converted to a more stable structure following protease cleavage (Law et
al.
(2006) Ge,nome Biology, 7:216).
Typically, serpins target serine proteases, although some serpins inhibit
cysteine proteases using a similar mechanism. The RSL loop determines which
proteases are targeted for inhibition as it provides a pseudo-substrate for
the target
protease. In effect, the inhibitory specificity of a particular serpin is
mediated by the
RSL sequence, which is the most variable region among serpins (Travis et al.
(1990)
Biol. Chem. Hoppe Seyler, 371: 3-11). The RSL mimics the substrate recognition
sequence of a protease and thereby contains a reactive site numbered as ...Pn-
F3-P2-
where the reactive site is the scissile bond between P1 and 131'.
For mature al-antitrypsin, cleavage at the PI-PI' bond occurs at the Met358-
Ser359
bond (corresponding to amino acids Met382 and Ser389 of the sequence of amino
acids
set forth in SEQ ID NO:1). The corresponding binding site for the residues on
the
protease are ...Sn-S3-S2-Si-S1', S2', S3', Se'-... In the method provided
herein,
.. modification of the RSL sequence is made to select for proteases from a
display
library exhibiting altered substrate specificity, as discussed in detail
below.
2. Protease Catalysis, Inhibitory Mechanism of Serpins, and
Formation of Acyl Enzyme Intermediate
The protease selection method provided herein exploits the ability of
polypeptides to trap proteases, such as is exemplified by serpins, to identify
proteases
with altered substrate specificity. Mechanisms of protease catalysis differ
slightly
between classes of proteolytic enzymes: serine, cysteine, aspartic, threonine,
or
metallo-proteases. For example, serine peptidases have a serine residue
involved in
the active center, the aspartic have two aspartic acids in the catalytic
center, cysteine -
.. type peptidases have a cysteine residue, threonine-type peptidases have a
threonine
residue, and metallo-peptidases use a metal ion in the catalytic mechanism.
Generally, those proteases families that form covalent intermediates are the
target of
the protease selection method provided herein. These include, for example,
members
of the serine and cysteine protease family. As an example, for serine
proteases, the
.. first step in catalysis is the formation of an acyl enzyme intermediate
between the
substrate and the serine in the catalytic center of the protease. Formation of
this
covalent intermediate proceeds through a negatively charged tetrahedral
transition
state intermediate and then the PI-P1' peptide bond of the substrate is
cleaved.
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During the second step or deacylation, the acyl-enzyme intermediate is
hydrolyzed by
a water molecule to release the peptide and to restore the Ser-hydroxyl of the
enzyme.
The deacylation, which also involves the formation of a tetrahedral transition
state
intermediate, proceeds through the reverse reaction pathway of acylation. For
deacylation, a water molecule is the attacking nucleophile instead of the Ser
residue.
The His residue in the catalytic center of a serine protease provides a
general base and
accepts the OH group of the reactive Ser.
Serpins inhibit the catalysis reaction of both serine and cysteine target
proteases using the S to R transition as mentioned above. Their mechanism of
action
is unique among protease inhibitors by destroying the active site of the
protease
before deacylation progresses, thereby irreversibly impeding proteolysis
following the
formation of the acyl-enzyme intermediate (Otlewski etal. (2005) The EMBO
Journal, 24: 1303). The kinetic model of the reaction of a serpin with a
protease is
identical to that of proteolysis of a substrate (see e.g., Figure 1; Zhou
etal. (2001)1
Biol. Chem., 276: 27541-27547). Following interaction with a target protease,
the
serpin initially forms a non-covalent Michaelis-like complex through
interactions of
residues in the RSL flanking the P1-P1' scissile bond (Silverman etal. (2001),
J. Biol.
Chem., 276: 33293-33296). The serine residue (for serine proteases), in the
active site
of the protease attacks the P1-P I' bond, facilitating cleavage of the peptide
bond and
formation of a covalent ester linkage between the serine residue and the
backbone
carbonyl of the PI residue. After the RSL is cleaved, the RSL inserts into 13-
sheet A
of the serpin molecule. The first residue to insert is P14 (i.e. amino acid
345 in
mature al-antitrypsin, which corresponds to amino acid position 1369 in the
sequence of amino acids set forth in SEQ ID NO:1), and is followed by the
flexible
hinge region (P15-P9) of the RSL (Buck etal. (2005) Mot Biol. Evol., 22: 1627-
1634). Insertion of the RSL transports the covalently bound protease with it,
resulting in a conformational change of the protease characterized by a
distorted
active site (see Figure 1) as well as a transition of the serpin into a
"relaxed" state.
The conformational change of the protease alters the catalytic triad of the
active site
such that the PI side chain is removed from the S I pocket. The net result of
the
conformational rearrangements is trapping of the acyl enzyme intermediate
(Silverman et al. (2001), 1 Biol. Chem., 276: 33293-33296).
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The formation of an acyl enzyme is important to the serpin interaction, and
therefore, serpins are typically specific for classes of proteases that have
acyl enzyme
intermediates in catalysis. Among these classes of proteases are predominantly
members of the serine protease family including those in the chymotrypsin
superfamily and those in the subtilisin superfamily of proteases, which are
described
in more detail below. Additionally, serpins also are reactive against cysteine
proteases including, for example, those in the papain family and the caspases
family
of serine proteases. Typically, serpins do not inhibit proteases of the
metallo-,
threonine, or aspartic families. For example, interactions of serpins with
metalloproteases do not result in a covalent trapped intermediate, but instead
the
metalloprotease cleaves the inhibitor without the formation of any complex (Li
et al.
(2004) Cancer Res. 64: 8657-8665).
Thus, although most serpins inhibit serine proteases of the chymotrypsin
family, cross-class inhibitors do exist that inhibit cysteine proteases. Among
cross-
class inhibitors are the viral serpin CrmA and PI9 (SEPRINB9) that both
inhibit
caspases 1, and SCCA I (SERPINB3) that inhibits papain-like cysteine proteases
including cathepsins L, K, and S. The mechanism of serpin-mediated inhibition
of
serine proteases appears to be adapted to cysteine proteases as well. The
difference,
however, is that the kinetically trapped intermediate is a thiol ester rather
than an oxy
ester as is the case for serine proteases (Silverman etal. (2001) J. Biol.
Chem.,
276:33293-33296). The existence of a stable, covalent thiol ester-type linkage
is
supported by the detection of an SDS-stable complex between SCCA1 and
cathepsin
S (Silverman etal. (2001)J. Biol. Chem., 276:33293-33296; Schick et a/. (1998)
Biochemistry, 37:5258-5266).
The serpin-protease pair is highly stable for weeks up to years depending on
the serpin-protease pair, however, dissociation eventually will occur to yield
the
products of normal proteolysis (i.e. the cleaved serpin and the active
protease; see
e.g., Zhou et al. (2001) J. Biol. Chem., 276: 27541-27547). Further, if the
RSL loop
is not inserted fast enough into the protease, the reaction proceeds directly
to the
cleaved product. This phenomenon is termed partitioning and reflects the
existence of
a branched pathway that can occur leading to either a stable inhibitory
complex or
turnover of the serpin into a substrate such as is depicted in Figure 1 as the
formation
of an inhibited complex versus the non-inhibitory pathway (Lawrence et a/.
(2000), J.
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Biol. Chem., 275: 5839-5844). Partitioning of a serpin can be modulated by
changing
residues in the RSL loop, particularly in the hinge region of the RSL which
initiates
loop insertion (i.e. P14), or by altering the protease for which the serpin
optimally
interacts. For example, the inhibitory activity of the serpin plasminogen
activator
inhbitor-1 (PAI-1) differs between the proteases uPA, tPA, and thrombin, with
a
targeted preference for uPA and tPA. Further, variation of the RSL loop at,
for
example, the P14 position of the hinge region alters the targeted preference
of PAI-1:
mutation to charged amino acids (i.e. Arg, Lys, Asp, Glu) reduces the
inhibitory
activity of PA!-1 to each of uPA, tPA, and thrombin; mutation to neutral amino
acids
(i.e. His, Tyr, Gin, Asn) or to Gly which lacks a side chain results in a 10-
100-fold
reduced inhibitory activity of PAI-1 to tPA and thrombin as compared to uPA;
and
mutation to hydrophobic amino acids does not change the inhibitory activity of
PAI-1
as compared to wildtype PAI-1 (Lawrence etal. (2000), 1 Biol. Chem., 275: 5839-
5844).
An important factor in the success of the serpin-mediated inhibition of
protease catalysis is the length of the RSL loop, which must be of a precise
length to
ensure that the serpin and protease interact in a way that provides leverage
between
the body of the serpin and protease to allow for displacement of the catalytic
serine
from the active site and deformation of the protease (Zhou etal. (2001)1 Biol.
Chem., 276: 27541-27547; Huntington et al. (2000) Nature, 407:923-926). In
effect,
the protease is crushed against the body of the serpin. Most serpins have an
RSL that
is 17 residues in length, while only a few have been identified with loops of
16
residues (i.e. a2-antiplasmin, Cl-inhibitor, and CrmA). An a2-antiplasmin
variant
serpin having an 18 residue loop also has been identified from a patient with
a
bleeding disorder, although this variant is not a functional inhibitory serpin
(Zhou et
al. (2001)J. Biol. Chem., 276: 27541-27547). Thus, the serpin inhibitory
mechanism
can accommodate a shortening, but not a lengthening, of the RSL (Zhou eta!,
(2001)
J. Biol. Chem., 276: 27541-27547). In addition to a conservation of loop
length
among serpin family members, the RSLs of serpins also generally retain a
conserved
hinge region (P15-P9) composition and do not typically contain charged or
bulky P
residues.
a. Exemplary Serpins
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Serpins used in the method provided herein can be any serpin polypeptide, =
including but not limited to, recombinantly produced polypeptides,
synthetically
produced polypeptides and serpins extracted from cells, tissues, and blood.
Serpins
also include allelic variants and polypeptides from different species
including, but not
limited to, animals of human and non-human origin, poxviruses, plants,
bacteria, and
archaea. Typically, an allelic or species variant of a serpin differs from a
native or
wildtype serpin by about or at least 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99%. Human serpins include any serpin
provided herein (e.g., in Table 2 below), allelic variant isoforms, synthetic
molecules
from nucleic acids, proteins isolated from human tissues, cells, or blood, and
modified
forms of any human serpin polypeptide. Serpins also include truncated
polypeptide
. fragments so long as a sufficient portion of the RSL loop is present to
mediate
interaction with a protease and formation of a covalent acyl enzyme
intermediate. In the
Table 2 below, accession numbers (Acc.#) are provided from the UniProtKB/Swiss-
Prot
database, which is a protein sequence database.
TABLE 2: Exemplary Serpins
Serpin Protein name Nick- Function Ace. IS
Mature SEQ
name Poly- ID
peptide NO
(aa)
Extra- SERPINA I Alpha-I- AIAT Inhibits
elastase P01009 25-418 j
cellular antitrypsin
Inhibitory
Serpins
SERPINA2 Alpha-I- AIA May be a P20848 22-420 2
antitrypsin- 1.1 pscudogene
related protein
= SERPINF2 Alpha-2- A2AP Inhibits
plasmin P08697 40-491 3
antiplasmin and trypsin
SERPINA3 Alpha-I- AAG Inhibits P01011 24-
423 4
antichymotrypsi T neutrophil
cathepsin G and =
mast cell
cymase
SERPINCI Antithrombin- ANT3 Regulates
blood P01008 33-464 5
= Ill coagulation
cascade;
thrombin and
factor Xa
, inhibitor
SERPINDI Heparin cofactor HEP2 Regulates blood P05546
20-499 6
Ii coagulation
cascade;
thrombin
Inhibitor
SERPING1 Plasma protease ICI Regulation of
P05155 23-500 - 7
CI inhibitor complement ,
activation,
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blood
coagulation,
fibrinolysis and
the generation
of kinins; CI
esterase
inhibitor
SERPINA5 Plasma serine IPSP, Inhibits P05154 20-406
8
protease PAI-3 activated
inhibitor, Protein protein C and
C inhibitor plasminogen
activators
SERPINA4 Kallistatin KAIN Inhibits P29622 21-427 9
, PI4 amidolytic and
kininogenase
activities of
human tissue
kallikrein
SERPINII Neuroserpin NEUS Formation or Q99574 17-410
10
, PII2 reorganization
of synaptic
connections and
synaptic
plasticity;
inhibitor of tPA,
uPA, and
plasmin
SERPINE1 Plasminogen PAII Regulation of P05121 24-
402 11
activator fibrinolysis;
inhibitor-1 inhibitor of
thrombin, uPA,
= tPA, and
plasmin
SERPIN12 Myoepithelium- PII4 Inhibition of 075830 19-
405 12
derived serine cancer
proteinase metastasis
inhibitor
SERPINA I Protein Z- ZP1 Inhibits factor Z Q9UK55 22-444 13
0 dependent and XI
protease
inhibitor
SERPINE2 Protease nexin 1 PI", Inhibition of P07093 -- 20-
398 -- 14
, glia-derived GDN, uPA and tPA
nexin precursor PN-1
Infra- SERPIN B1 Leukocyte ILEU Inhibition of
P30740 1-379 15
cellular elastase neutrophil
inhibitory inhibitor, protease
Serpins monocytes
neutrophil
elastase inhibitor
SERPINB2 Plasminogen PAI2 Tissue-type P05120 1-415 16
activator plasminogen
inhibitor-2 activator,
intracellular
signaling;
inhibition of
uPA
SERP1NB6 Placental P116 Inhibits P35237 1-376 17
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=
thrombin thrombin
inhibitor
SERPINB1 Bomapin SB I 0, Haematopoiesis P48595 1-397 18
0 P110 , inhibition of
thrombin and
trypsin
SERPINB1 epipin SB I I Q96P15 1-392 19
1
SERPINB1 Yukopin SB12 Inhibits trypsin Q96P63 -- 1-405 --
20
2 and plasmin
SERPINB1 Headpin SB13, Proliferation or Q9UIV8 1-391 21
3 P113 differentiation
of
keratinocytes,
inhibition of
cathepsins L
and K
SERP1NB3 Squamous cell SCC1 Modulates P29508 1-390 22
= carcinoma immune
antigen 1 response
towards tumors,
inhibition of
cathepsins L, K,
S and V, and
papain
SERPINB4 Squamous cell SCC2 Modulates P48594 1-390 23
carcinoma immune
antigen 2 response
towards tumors,
inhibition of
Cathepsin G
and chymase
SERPINB7 Megsin SPB7 Maturation of 075635 1-390 24
megakaryocytes
SERPINB8 Cytoplasmic SPB8, Inhibition of P50452 1-374 25
antiproteinase 8 PI8 Furin
SERPINB9 Cytoplasmic SPB9, Granzyme B P50453 1-376 26
antiproteinase 9 P19 inhibitor
SERPINB6 Proteinase PI6, Inhibition of P35237 1-376
27
inhibitor-6, PTI cathepsin G,
placental inhibits
thrombin thrombin
inhibitor
Non- SERPINA8 Angiotensinogen ANG Blood pressure P01019 34-
485 28
inhibitory T regulation,
serpins hormone
precursor
SERPINA6 Corticosteroid- CBG Hormone P08185 23-405 29
binding globulin carrier
(glucocorticoids
and progestins),
cortisol binding
SERPINH1 47 kDa heat HS47 Molecular P29043 18-417 30
shock protein chaperone for
collagen
SERPINF1 Pigment PEDF Induces P36955 20-418 31
epithelium- neuronal
derived factor differentiation
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in
retinoblastoma
cells; inhibitor
of angiogenesis
SERPINB5 Maspin MAS Tumor P36952 1-375 32
suppressor,
prevents
metastasis
SERPINH2 Collagen- SIH2 Molecular P50454 19-418
33
binding protein chaperone for
2 collagen
SERPINA7 Thyroxine- THB Thyroid P05543 21-415
34
binding protein G hormone
transport,
thyroxine
binding
SERPINA9 Germinal center GCET Maintenance of Q86WD7 24-417 35
B-cell expressed I naive B cells
transcript 1
protein
SERPINA I Vaspin Insulin- Q8IW75 21-414 36
2 sensitizing
adipocytokine
SERPINA I Q86U17 20-422
37
SERPINA I Q6UXR4 22-307
38
3
Typically, a serpin used in the method provided herein is an inhibitory
serpin,
or fragment thereof, capable of forming a covalent acyl enzyme intermediate
between
the serpin and protease. Generally, such a serpin is used to select for
proteases
normally targeted by the serpin where close to complete inhibition of the
protease
occurs and partitioning is minimized between the inhibitory complex and
cleaved
serpin substrate. Table 3 depicts examples of serine proteases and their
cognate
serpin inhibitors. Such serpin/protease pairs are expected to have a high
association
constant or second ordered rate constant of inhibition and low or no
partitioning into a
non-inhibitory complex. For example, the major physiological inhibitor oft-PA
is the
serpin PAI-1, a glycoprotein of approximately 50 kD (Pannekoek et al. (1986)
EMBO
J., 5:2539-2544; Ginsberg etal., (1980)J. Clin. Invest., 78:1673-1680; and
Carrell et
al. In: Proteinase Inhibitors, Ed. Barrett, A.J. et al., Elsevier, Amsterdam,
pages 403-
420 (1986). Other serpin/protease pairs also can be used in the methods
provided
herein, however, even where association constants are lower and partitioning
is
higher. For example, although the association constants of other serpins, such
as Cl
esterase inhibitor and alpha-2-antiplasmin with tPA are orders of magnitude
lower
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than that of PAI-I (Ranby et al. (1982) Throm. Res., 27:175-183; Hekman etal.
(1988) Arch. Biochem. Biophys., 262:199-210), these serpins nevertheless
inhibit tPA
(see e.g., Lucore et al. (1988) Circ. 77:660-669).
TABLE 3:
Serine Protease Cognate Serpin Inhibitor
Activated protein C Protein C inhibitor
PAI-1
Cl esterase Cl esterase inhibitor
Cathepsin G Alpha-l-antitrypsin
Alpa-l-antichymotrypsin
Chymase Alpha-l-antichymotrypsin
Chymotrypsin Alpha-l-antichymotrypsin
Alpha-2-antiplasmin
Contrapsin
Coagulation Factors (VIIa, Xa, XIa, XIIa) Antithrombin III
Cl esterase inhibitor
Elastase Alpha-l-antitrypsin
Kallikrein Cl esterase inhibitor
Alpha-l-antitrypsin
Plasmin Alpha-2-antiplasmin
Thrombin Antithrombin III
Heparin cofactor II
tPA PAT-1, PAI-2, PAI-3
Trypsin Alpha-l-antitrypsin
Growth hormone regulated protein
Trypsin-like protease Protease nexin I
u-PA PAT-1, PAI-2, PAI-3
Thus, generally a serpin used for selection of a protease in the methods
provided herein yields a reaction product where 80%, 90%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% of the reaction product is the formation of the
inhibitory
complex. In some cases, however, increased partitioning between a serpin and
protease can occur in the methods provided herein, such as if the serpin used
in the
method does not optimally target the protease. Thus, in the method provided
herein a
serpin can be used to select a protease where the resulting reaction leads to
at or about
20%, 30%, 40%, 50%, 60%, 70%, 75%, or more of a stable inhibitory complex and
the remaining product is a cleaved serpin substrate. Factors that can be
altered to
optimize for protease selection where partitioning occurs include, for
example,
increased serpin concentration and increased reaction time. In some instances,
other
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non-inhibitory serpins, or mutants thereof as discussed below, can be used in
the
methods provided herein so long as the target protease for selection is able
to interact
with the serpin substrate to yield a covalent inhibitory complex that can be
captured.
i. PA!-!
Exemplary of serpins used in the protease selection methods is plasminogen
activator inhibitior-1 (PA!-!), or variants thereof. PAI-1 is the main
inhibitor of
tissue plasminogen activator (t-PA) and urokinase or urinary-plasminogen
activator
(u-PA), which are proteases involved in fibrinolyis due to the activation of
plasminogen. PAI-1 has a second order rate constant for t-PA and u-PA of about
2 X
107 PAI-1 is involved in tumor invasion, fibrinolysis, cell migration,
tissue
remodeling, tissue involution, ovulation, inflammation, trophoblast invasion,
and
malignant transformation (Salonen et al. (1988)J Biol. Chem., 264: 6339-6343).
PAI-1 is mainly produced by the endothelium, but also is secreted by other
tissue
types, such as for example, adipose tissue. Other related plasminogen
activator
inhibitors include PAI-2 and PAI-3. PAI-2, for example, also is an inhibitor
of u-PA
and t-PA, but is secreted by the placenta and typically is only present in
high amounts
during pregnancy.
PAI-1 is a single chain glycoprotein having a precursor sequence set forth in
SEQ ID NO:11, including a 23 amino acid signal sequence, which when cleaved
results in a 379 amino acid mature sequence. Like other serpins, PAI-1
transitions
from a latent form into an active form following cleavage by a protease at its
Pi-Pi'
reactive site located at Arg346-Met347 (i.e. corresponding to amino acids
Arg369 and
Met37 of a precursor sequence set forth in SEQ ID NO:11), thereby resulting
in the
formation of a stable covalent complex and the inactivation of the bound
protease.
Unlike other serpins, however, PAI-1 adopts a latent transition spontaneously
resulting in an inactive, highly stable but covalently intact form whereby
residues P15
to P4 of the RSL insert into the 3-sheet A to form strand four of the 3-sheet
(i.e. s4A),
and residues P3 to P10' form an extended loop at the surface of the molecule
(De
Taeye etal. (2003)J Biol. Chem., 278: 23899-23905). Thus, active PAT-1 is
relatively unstable at 37 C exhibiting a half-life of only 2.5 hours before
spontaneous
conversion to a latent conformation. This latent form, however, can be re-
activated
by denaturation, such as by denaturation with sodium dodecyl sulfate,
guanidiniurn
chloride, and urea (Declerek etal. (1992)J Biol. Chem., 267: 11693-11696) and
heat
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(Katagiri et al. (1988) Eur J. Biochem., 176: 81-87). The active form of PAI-1
also is
stabilized by interaction with vitronectin. Mutant PAI-1 have been identified
that are
unable to undergo conversion to a latent conformation and are therefore more
stable at
elevated temperature and pH for extended times periods (see e.g., Berkenpas et
al.
(1995) The EMBO J., 14:2969-2977).
Modifications of serine proteases (i.e. t-PA or u-PA) and/or of the inhibitory
serpin (i.e. PAI-1) have been made to modulate or alter the secondary rate
constants
of inhibition so as to make proteases resistant to inhibition by their cognate
serpin
inhibitor, or variant thereof, such as for use in therapeutic applications
where activity
of the wild-type protease is desired (see e.g, U.S. Patent Serial Nos.
5,866,413;
5,728,564; 5,550,042; 5,486602; 5,304,482).
Antithrombin (AT3)
Another exemplary serpin, or variant thereof, for use in the methods herein is
antithrombin (AT3). AT3 also is a member of the serpin family and inactivates
a
number of enzymes, including for example, those from the coagulation system
such
as, but not limited to, Factor X, Factor IX, Factor II (thrombin), Factor VII,
Factor XI,
and Factor XII. Typically, antithrombin is predominantly found in the blood
where it,
for example, prevents or inhibits coagulation by blocking the function of
thrombin.
The activity of AT3 is increased by the presence of one or more cofactors,
typically
heparin. Upon interaction with heparin, AT3 undergoes a conformational
rearrangement involving loop expulsion away from serpin structure and P1
exposure
resulting in an AT3 structure having an exposed protease-accessible
conformation. In
addition, heparin can bind to both the protease and inhibitor thereby
accelerating the
inhibitory mechanism (Law etal. (2006) Genome Biology, 7(216): 1-11).
The gene sequence for AT3 codes for a seven exon spanning DNA, encoding a
precursor protein set forth in SEQ ID NO:5. Cleavage of the signal sequence
corresponding to amino acids 1-32 of the sequence set forth in SEQ ID NO:5
results
in a mature protein of 432 amino acids that has a molecular weight of about
58,000
daltons. Six of the amino acids are cysteines, which results in the formation
of three
intramolecular disulfide bonds. The P4-P2' positions in the RSL of AT3 contain
the
amino acid residues IAGRSL (SEQ ID NO:478), which correspond to amino acids
422-427 in the sequence of amino acids set forth in SEQ ID NO:5, where
cleavage at
thp reartive citp PI-P1' nrriirs between am inn acids A ra425-ser426
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3.
Other Protease Trap Polypeptides
Additional protease trap polypeptides are known in the art or can be
identified
that exhibit a mechanism of inhibition similar to serpins (e.g. cleavage of
the target
substrate by a protease that produces a stable intermediate and a
conformational
change in the structure of the protease). Such protease trap polypeptides are
contemplated for use in the method provided herein. Exemplary of such a
protease
trap polypeptide is p35. In addition, any other molecule that is cleaved by a
protease
resulting in the trapping of a protease in a long-lasting, stable complex can
be used in
the methods provided herein.
a. p35
For example, the baculovirus p35 protein (SEQ ID NO: 473), which is a broad
spectrum caspase inhibitor, can inhibit caspases in this manner (Xu et al.
(2001)
Nature 410:494-497; Xu etal. (2003)J. Biol. Chem. 278(7):5455-5461). Cleavage
of
the PI-Pi' bond of p35 (at the caspase cleavage site DQMD") by caspases
produces a
covalent thioester intermediate between the amino segment of p35 loop (Asp87)
and
the cysteine residue of the caspase catalytic triad (Cys350 in caspase-8).
Upon
formation of the thioester linkage, the protease undergoes a conformational
change
allowing the amino segment of the cleaved loop to bury into the caspase, while
the N-
terminus of p35 containing a Cys residue at position 2 inserts into the
caspase active
site, thus blocking solvent accessibility of His 317 residue in caspase-8.
Inaccessibility to the hydrolytic water molecule thus prevents subsequent
hydrolysis
of thioester bond.
Similar viral caspase inhibitors in addition to p35 include, but are not
limited
to, p49 (SEQ ID NO: 491) and the serpin CrmA cowpox gene (SEQ ID NO: 492).
The p49 inhibitor exhibits a caspase inhibition mechanism similar to that of
p35 in
that a stable thioester linkage is formed with the active site of the caspase
upon
cleavage of the p49 caspase recognition sequence TVTD94.
Target substrates for the screening using the methods provided herein can
include a viral caspase inhibitor polypeptide, such as a p35, p49 or CrmA
polypeptide.
Methods of modification of the RSL loop of serpins provided herein can be
easily
adapted to modification of viral caspase inhibitor polypeptides. For example,
the
target site for cleavage in the p35 RSL can be modified to so as to select for
proteases
that have an altered reactivity or specificity for a target substrate. In wild-
type p35,
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caspase recognition is found at amino acid positions 84-87 (DQMD87).
Modifications to viral caspase inhibitor polypeptides can thus include
modifications
that alter the cleavage sequence and/or surrounding amino acid residues. For
example, such modified caspase inhibitor polypeptides, such as for example a
p35,
p49 or CrmA polypeptide, can be designed to mimic the cleavage sequence of a
desired target substrate, such as for example, a target substrate involved in
the
etiology of a disease or disorder. Any modification in the RSL loop sequence
of a
viral caspase inhibitor polypeptide can be made in the methods provided
herein.
Viral caspase inhibitor polypeptides such as a p35, p49 or CrmA polypeptide,
used in the methods provided herein can be any viral caspase inhibitor
polypeptide,
including but not limited to, recombinantly produced polypeptides,
synthetically
produced polypeptides and p35 pr p49 polypeptide produced by baculovirus
purification methods. Viral caspase inhibitor polypeptides also include
allelic
variants of polypeptides, such as p35, p49 or CrmA polypeptide variants.
b. Alpha Macroglobulins (aM)
The alpha macroglobulin (aM) family of proteases include protease inhibitors
such as the exemplary protease inhibitor alpha-2-macroglobulin (a2M; SEQ ID
NO:490), and are contemplated for use as protease traps in the methods
provided
herein, aM molecules inhibit all classes of proteases. aM protease traps are
characterized by a similar inhibition mechanism involving cleavage of a baif
region of
the inhibitor by a protease. The bait region is a segment that is susceptible
to
proteolytic cleavage, and which, upon cleavage, initiates a conformational
change in
the aM molecule resulting in the collapse of the structure around the
protease. For the
exemplary a2M sequence set forth in SEQ ID NO:490, the bait region corresponds
to
amino acids 690-728. In the resulting aM-protease stable complex, the active
site of
the protease is sterically shielded, thereby decreasing access to normal
protease
substrates. Typically, the trapped protease remains active against small
peptide
substrates, but loses its ability to interact with large protein substrates or
inhibitors.
In addition, aM molecules are characterized by the presence of a reactive
thiol ester,
which inactivates the inhibitory capacity by reaction of the thiol ester with
amines.
Further, the conformational change that occurs upon cleavage of the bait
region
exposes a conserved COOH-terminal receptor binding domain (RBD). Exposure of
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the RBD sequence facilitates the removal of the aM-protease complex from
circulation.
4. Protease Trap Competitors
Competitors can be used in the methods provided herein to modulate the
specificity and selectivity constraints of a selected protease for a target
substrate. The
competitors can be contacted with the protease, or collections thereof, at any
time,
such as before or after contact of the protease with the desired protease trap
polypeptide or the competitor and desired protease trap polypeptide can be
contacted
with the protease simultaneously. Competitors can be specific competitors or
broad
competitors.
Specific competitors are designed that mimic a predetermined non-target
substrate and thereby act as predetermined potential off-targets. Typically,
such
competitors are not labeled, so that stable protease complexes that form are
not
selected for. In addition, such competitors are added in large excess,
typically molar
excess, over the designed protease trap polypeptide used in the selection
scheme, such
that the competitors bind up the undesired proteases in the collection. In one
example
of specific competition, two different protease trap polypeptides, each
designed to
mimic different substrate recognition, are contacted with a collection of
proteases
where only one of the protease trap polypeptides is detectably labeled. For
example, a
competitor can include a polypeptide protease trap that is designed to have
its reactive
site mimic the cleavage sequence of a non-target substrate. Thus, a
competitor, such
as a serpin, can be designed to have its P4-P1' RSL residues replaced by the
cleavage
sequence of a predetermined non-target substrate. The competitor can be used
in
methods in combination with a protease trap polypeptide, such as for example
another
serpin polypeptide, whose RSL sequence has been modified to contain amino
acids in
the P4-P 1' positions that mimic the cleavage sequence of a desired or
predetermined
target substrate, and that is labeled for isolation thereof. Thus, both
protease trap
polypeptides select for proteases exhibiting selectivity for the target or non-
target
cleavage sequence, but only those stable protease complexes that exhibit the
desired
target substrate specificity and that are detectably labeled can be isolated
from the
reaction. Other examples of specific competitors include, for example, the
native
protease trap polypeptide for which the reactive site has been modified in the
methods
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provided herein. Example 6 exemplifies such a strategy where a plasma purified
AT3
serpin is used as a competitor against the modified serpin AT3SLGR-KI.
Broad competitors also can be used in the methods provided herein to
constrain the specificity and selectivity of selected proteases. Examples of
broad
competitors include, for example, human plasma or human serum which contains a
variety of natural protease inhibitors. Alternatively, a broad small molecule
library of
protease trap polypeptides can be generated where every position of P2, P3, or
P4 is
made to be different, such as for example an Acxxx-Thiaphine library.
5. Variant Protease Trap Polypeptides
Protease trap polypeptides that have been modified in their reactive site to
have an altered cleavage sequence can be used in the methods provided herein
to
select for proteases with a desired or predetermined target substrate. Thus,
protease
traps are modified in the region of their sequence that serves as the
recognized
cleavage site of a protease so as to select for proteases that have an altered
reactivity
or specificity for a target substrate. For example, serpins can be modified to
have an
altered cleavage sequence at or around the scissile bond in the RSL loop. In
another
example, a2M can be modified in its bait region to have an altered cleavage
sequence.
Such modified protease traps can be designed to mimic the cleavage sequence of
a
desired target substrate, such as for example, a target substrate involved in
the
etiology of a disease or disorder.
Any modification in the RSL loop sequence of a serpin molecule can be made
in the methods provided herein. Alignments of RSL sequences of exemplary wild-
type serpins are set forth in Table 4 below. In the Table below, the numbers
designating the P15 to P5' positions are with respect to a mature al-
antitrypsin
molecule (corresponding to amino acids 367- 387 of the sequence of amino acids
set
forth in SEQ ID NO:1). The identity of the RSL loop sequences are known to
those
of skill in the art and/or can be determined by alignments such as by
alignment with
serpins as set forth in Table 4 below.
TABLE 4: RSL LOOP SEQUENCE ALIGNMENT*
SERPIN RSL loop sequence SEQ
ID
NO
343 P15 PIO P4 2IP1' P5 363
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Manduca sexta serpin 1B EGAEAAAANAFGIVPKSLILY 397
Manduca sexta serpin 1K EGAEAAAANAFKITTYSFHEV 398
al-antichymotrypsin EGTEASAATAVKITLLSALVE 399
Antithrombin-III
EGSEAAASTAVVIAGRSLNPN. 400
PAL-II EGTEAAAGTGGVMTGRTGHGG 401
al-antitrypsin KGTEAAGAMFLEAIPMSIPPE 402
PAL-I SGTVASSSTAVIVSARMAPEE 403
PAI-III SGTRAAAATGTIFTFRSARLN 404
Ovalbumin AGREVVGSAEAGVDAASVSEE 405
* adapted from Ye et al. (2001) Nature Structural Biology 8: 979
Thus, amino acid sequences within the RSL loop of a serpin corresponding to
any one or more of amino acids in the reactive site of a serpin (i.e. any one
or more of
amino acids corresponding to P15 to P5' positions such as set forth, for
example, in
Table 4 above) can be modified. Typically, amino acids that are part of the
hinge
region of the RSL loop sequence are not modified (i.e. amino acids
corresponding to
P15-P9 positions). In one example, one or more amino acid in the P1 and/or P1'
position are modified corresponding to those amino acids that flank the
scissile bond.
In another example, any one or more amino acids corresponding to reactive site
positions P4-P2' are modified. For example, the P4-P1' of PAI-1 is VSARM (SEQ
ID NO:378), where cleavage occurs between the R (P1) and M (P1') amino acids.
Modification of any or more of amino acids of the VSARM sequence can be made
to
modify the cleavage sequence of PAI-I to select for proteases with altered
specificity.
Example 1 exemplifies modification of PAI-I where the VSARM sequence in the
reactive site loop is modified to be RRARM (SEQ ID NO:379). In another
example,
the reactive site loop the VSARM sequence can be modified to the known
efficient
peptide substrate PFGRS (SEQ ID NO:389). Exemplary of such mutant PAI-1 are
set
forth in SEQ ID NOS:610 and 611.
In another example, modifications can be made in the RSL of antithrombin III
(AT3). For example, the P4-P1' of AT3 is IAGRSL (SEQ ID NO:478), where
cleavage occurs between the R (P1) and S (P1') amino acids. Modification of
any one
or more of amino acids of the IAGRSL sequence can be made to modify the
cleavage
sequence of AT3 to select for proteases with altered specificity. Examples 6
and 7
exemplify modification of AT3 where the IAGRSL sequence in the reactive site
loop
is modified to be RRVRKE (SEQ ID NO:498). In another example, the IAGRSL
amino acid sequence in the reactive site loop can be modified to SLGRKI (SEQ
ID
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,
NO:479). Other modified AT3 polypeptides were made containing replacement of
the IAGRSL amino acid sequence with the amino acid sequence SKGRSL (SEQ ID
NO:501) or the amino acid sequence PRFKII (SEQ ID NO: 503). Exemplary of such
mutant AT3 molecules are set forth in any of SEQ ID NOS:497, 499, 500, and
502.
Alternatively, and if necessary, the modification in any one or more amino
acid positions P4-P2' can be made one at a time, two at a time, three at a
time, etc.,
and the resulting modified serpin can be separately tested in successive
rounds of
selection so as to optimize for proteases that exhibit substrate specificity
and/or
selectivity at each of the modified positions.
In most cases, amino acid residues that replace amino acid residues in the
reactive site loop of a wild-type serpin, or analogous sequence in another
protease
trap, are chosen based on cleavage sequences in a desired target substrate. A
target
substrate protein is one that is normally involved in a pathology, where
cleaving the
target protein at a given substrate sequence serves as a treatment for the
pathology
(see e.g. U.S. patent publication No. US 2004/0146938, US2006/0024289,
US2006/0002916, and provisional application serial No. 60/729,817). For
example,
the target protein can be one involved in rheumatoid arthritis e TNFR), sepsis
(i.e.
protein C), tumorigenicity (i.e. a growth factor receptor, such as a VEGFR),
or
inflammation (i.e. a complement protein). A target substrate also can be a
viral
20. protein such that upon cleavage of the viral protein the viruses would
be unable to
infect cells. Table 5 below sets forth exemplary target substrates.
TABLE 5: Exemplary Target Substrates
Target Indication Molecule Class
IL-5/IL-5R Asthma Cytokine
IL-1/IL-1R Asthma, inflammation, Cytokine
Rheumatic disorders
IL-13/IL-13R Asthma Cytokine
IL-12/1L-12R Immunological disorders Cytokine
IL-4/IL-4R Asthma Cytokine
TNF/TNFR Asthma, Crohres disease, HIV Cytokine
infection, inflammation,
psoriasis, rheumatoid arthritis,
inflammatory bowel disease
CCR5/CXCR4 HIV infection GPCR
gp120/gp41 HIV infection Fusion protein
CD4 HIV infection Immune Receptor
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Hemaglutinin Influenza infection Fusion Protein
RSV fusion protein RSV infection Fusion Protein
B7/CD28 Graft-v-host disorder, Immune Receptor
rheumatoid arthritis, transplant
rejection, diabetes mellitus
IgE/IgER Graft-v-host disorder, transplant Antibody receptor
rejection
CD2, CD3, CD4, Graft-v-host disorder, transplant Immune Receptor
CD40 rejection, psoriasis
IL-2/ IL-2R Autoimmune disorders, graft-v- Cytokine
host disorders, rheumatoid
arthritis
VEGF, FGF, EGF, Cancer Growth Factor
TGF
HER2/Neu Cancer (i.e. breast cancer) Growth Factor
Receptor
CCR1 Multiple sclerosis GPCR
CXCR3 Multiple sclerosis, rheumatoid GPCR
arthritis
CCR2 Atherosclerosis, rheumatoid GPCR
arthritis
Src Cancer, osteoporosis Kinase
Akt Cancer Kinase
Bc1-2 Cancer Protein-protein
BCR-Abl Cancer Kinase
GSK-3 Diabetes Kinase
Cdk-2/cdk-4 Cancer Kinase
EGFR Lung, breast, bladder, prostate,
colorectal, kidney, head & neck
cancer
VEGFR-1, neck cancer Growth Factor
Receptor
VEGFR-2
Complement Inflammatory diseases Immune molecules
Cleavage sites within target proteins are known or can be easily identified.
Cleavage sites within target proteins are identified by the following
criteria: 1) they
are located on the exposed surface of the protein; 2) they are located in
regions that
are devoid of secondary structure (i.e. not in P sheets of helices), as
determined by
atomic structure of structure prediction algorithms (these regions tend to be
loops on
the surface of proteins or stalk on cell surface receptors); 3) they are
located at sites
that are likely to inactive (or activate) the protein, based on its known
function.
Cleavage sequences are e.g., four residues in length (i.e. P I -P4 positions)
to match the
extended substrate specificity of proteases, but can be longer or shorter. For
example,
the P4-P 1 amino acid residues for a cleavage sequence in complement factor C2
is
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..
SLGR (SEQ ID NO:431), but also can be represented as the P4-P2' sequence of
SLGRKI (SEQ ID NO:479), where cleavage occurs between the P1 and P I '
position
(i.e. between R/K). Hence, any one or more residues within a cleavage
sequence,
including any one or more of residues P4-P2', including P4-P1, can be
introduced into
a protease trap polypeptide, such as in the RSL of a serpin to generate a
mutant
protease trap polypeptide.
Cleavage sequences can be identified in a target substrate by any method
known in the art (see e.g., published U.S. Application No. US 2004/0146938).
In one
example, cleavage of a target substrate is determined by incubating the target
substrate with any protease known to cleave the substrate. Following
incubation with
the protease, the target protein can be separated by SDS-PAGE and degradative
products can be identified by staining with a protein dye such as Coomassie
Brilliant
Blue. Proteolytic fragments can be sequenced to determine the identity of the
cleavage sequences, for example, the 6 amino acid P4-P2' cleavage sequence,
and in
particular, the four amino acid P4-P1 cleavage sequence residues. Table 6
identifies
cleavage sequences corresponding to positions P4-P1 for exemplary target
substrates.
TABLE 6: Cleavage Sequence for Exemplary Target Substrates
(P4-P1 residues)
Target Cleavage sequence
AEAK (406)
INF-RI. ENVK (407); GTED (408)
TNF-R2 SPTR (409); VSTR (410); STSF (411)
HER-2 KFPD (412); AEQR (413)
EGFR KYAD (414); NGPK (415)
VEGFR-1 SSAY (416); GTSD (417)
VEGFR-2 AQEK (418); RIDY (419); VLKD (480);
LVED (481); WFKD (482); RIYD (483);
KVGR (484); RVRK (485); RKTK (486);
KTKK. (487); TKKR (488); RRVR (489)
C3 REFK (420); GLAR (421); RLGR (422);
AEGK (423); QHAR (424); LPSR (425);
SLLR (426); LGLA (427); LSVV (428)
C4 HRGR (429)
C2 GATR (430); SLGR (431); VFAK (432)
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Hence, modification of an RSL of a serpin, or analogous sequence in other
protease traps, can be modified to any desired or predetermined cleavage
sequence of
a target substrate. In one example, the selected cleavage sequence can be one
that is a
particularly efficient cleavage sequence oft-PA. Such a cleavage sequence is,
for
example, PFGRS (SEQ ID NO:389; see e.g., Ding et al. (1995) PNAS, 92:7627-
7631). Thus, for example, a protease can be selected for that has an altered
substrate
specificity that is made to replicate the substrate specificity of t-PA. Since
t-PA is an
often used therapeutic for the treatment of fibrinolytic disorders, such a
selected
protease can be optimized to be an alternative t-PA therapeutic, while
minimizing
undesirable side effects often associated with t-PA therapies (i.e. excessive
bleeding).
In another example, a cleavage sequence for a complement protein can be
targeted as a predetermined or desired cleavage sequence for selection of a
protease
using the methods provided herein. A protease selected to have increased
substrate
specificity against any one or more complement proteins would be a therapeutic
candidate for treatment of disorders and diseases associated with inflammation
such
as, but not limited to, autoimmune diseases, such as rheumatoid arthritis and
lupus,
cardiac disorders, and other inflammatory disorders such as sepsis and
ischemia-
reperfusion injury (see e.g., United States provisional application serial No.
60/729,817).
Example 6 to Examples 15 exemplifiy selection of an MT-SP1 protease against an
AT3 serpin
molecule modified by replacements of its native P4-P2' residues IAGRSL (SEQ ID
NO:478) with a cleavage sequence of the C2 complement proteins (i.e. SLGRKI,
SEQ ID NO:479). Modification or replacement of amino acid residues by the
SLGRKI cleavage sequence, or intermediates thereof such as are described
below,
can be made in any protease trap polypeptide, such as any serpin polypeptide,
for
selection of any candidate protease as so desired.
In an additional example, a cleavage sequence can be selected in a VEGFR,
such as in the stalk region of a VEGFR, such that the VEGFR is inactivated
upon
cleavage by a protease having specificity for the cleavage sequence. Examples
of
cleavage sequences in a VEGFR are described herein and set forth in related
published U.S. application serial Nos. US20060024289 and US20060002916. For
= example, the RSL of a serpin, or analogous sequence in other protease
traps such as
the "bait" region in alpha-2 macroglobulin, can be modified to have any one or
more
of amino acid positions P4-P2' replaced with the cleavage sequence of a VEGFR.
In
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one example, amino acid residues in a native serpin can be modified to contain
the
P4-P1 positions corresponding to the RRVR (SEQ ID NO:489) cleavage sequence,
or
the entire P4-P2' sequence RRVRKE (SEQ ID NO:498). A protease selected against
such a modified serpin would be a candidate to treat VEGFR-mediated disorders,
such as for example, angiogenic disorders.
In some cases, in the methods provided herein, the modifications in any one or
more of the P4-P2' positions of a serpin RSL, or analogous sequence in other
protease
traps, can be made in successive rounds to optimize for selection of proteases
with a
desired or predetermined substrate specificity. For example, both u-PA and t-
PA
proteases prefer small amino acids at the P2 position and very different amino
acids in
the P3 and P4 positions. Thus, modified serpins can be generated that are
intermediates for the final target cleavage sequence, where a first
intermediate is
generated by modification of only the P3 and P4 positions to select for
proteases that
exhibit specificity at the P3 and P4 positions. The selected protease or
proteases can
then be used as a template for the generation of a new combinatorial library
against a
new serpin molecule modified to additionally have the P2 position changed.
Thus, in selection, for example, of a u-PA protease, or variant thereof, that
exhibits increased substrate specificity for the VEGFR cleavage sequence RRVR,
the
first round of selection can be made against an intermediate modified protease
trap
polypeptide, such as a serpin, where only the P3 and P4 positions are changed
as
compared to the native sequence at those positions. For example, where the
native
P4-P1' amino acids in the RSL loop of the serpin PAI-1 are VSARM, a modified
intermediate PAI-1 can be made by replacement of only the P4 and P3 VEGFR
cleavage sequence, to yield the intermediate serpin molecule containing RRARM
(SEQ ID NO:379) in the P4-P1' positions. Subsequent rounds of protease
selection
can be made against a PAI-1 serpin that has additionally been modified at the
P2
position.
Protease traps, including serpins, can be modified using any method known in
the art for modification of proteins. Such methods include site-directed
mutagenesis,
including single or multi-sited directed mutagenesis. Likewise, expression and
purification of protease-trap polypeptides, including variant protease-trap
polypeptides can be performed using methods standard in the art for expression
and
purification of polypeptides. Any host cell system can be used for expression,
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including, but not limited to, mammalian cells, bacterial cells or insect
cells. Further,
the protease trap polypeptides can be modified further to include additional
sequences
that aid in the identification and purification of the protease trap
polypeptide. For
example, epitope tags, such as but not limited to, His tags or Flag tags, can
be added
to aid in the affinity purification of the polypeptide. In some examples,
protease trap
polypeptides are directly biotinylated to aid in capture and/or purification.
An
exemplary method for biotinylating a protease-trap polypeptide is described in
Example 16.
Assays, such as assays for biological function of a serpin molecule or other
protease trap are known in the art and can be used to assess the activity of a
modified
protease trap as an inhibitor in the methods provided herein. Such assays are
dependent on the protease trap polypeptide modified for use in the methods
herein.
Exemplary of such assays for PAI-1 include, for example, active site titration
against
standard trypsin or titration of standard trypsin such as are exemplified in
Example 1.
Also exemplary of such assays are protease inhibition assays, which are known
in the
art, whereby the ability of the protease trap to inhibit the cleavage of a
fluorogenic
substrate by an active protease is used as a readout for protease trap
activity.
Exemplary of a protease inhibition assay is a matriptase (MT-SP1) inhibition
assay.
In one example of such an assay, the protease trap is a serpin. In a specific
example,
the serpin is AT3 or a variant AT3 protein made according to the methods
provided
herein, the fluorogenic substrate is RQAR-ACC. Cleavage of the substrate is
measured, for example, as exemplified in Example 14A. Thrombin inhibition
assays
also can be used to assess the activity of AT3, or modified AT3. Similar
assays can
be designed or are known to one of skill in the art depending on the cognate
protease
for which a protease trap polypeptide, or variant thereof, normally interacts.
Further,
it is expected and often is the case that a modified protease trap polypeptide
will have
reduced activity as compared to a wild-type protease trap polypeptide in
normal
assays of protease trap activity or function.
D. PROTEASES
In the methods provided herein, candidate proteases are selected for that
exhibit an altered substrate specificity, typically for a predetermined or
desired
substrate. Collections of proteases, mutant protease, or catalytically active
portions
thereof are contacted with a protease trap polypeptide, such as any provided
herein
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including, for example, serpins or modified serpins, to select for proteases
with
altered substrate specificity. The protease collections can be provided on a
solid
=
support or in a homogenous mixture such as in solution or suspension. The
selected
proteases can be isolated as stable complexes with the protease trap
polypeptide, and
can be identified. Selected proteases display increased catalytic efficiency
and
reactivity against the desired or predetermined target substrate, and are
thereby
candidates for use as therapeutics, such as in any disease or disorder for
which the
target substrate is involved.
1. Candidate Proteases
In the method provided herein, proteases are selected for that have an altered
and/or increased specificity for a desired substrate that is involved in a
disease or
disorder. Generally, proteases are highly specific proteins that hydrolyze
target
substrates while leaving others intact. For the cleavage of natural
substrates,
proteases exhibit a high degree of selectivity such that substrate cleavage is
favored,
whereas non-substrate cleavage is disfavored (Coombs etal. (1996).T Biol.
Chem.,
271: 4461-4467). Selecting for proteases with an altered specificity and
selectivity
for a desired target substrate would enable the use of proteases as
therapeutics t0
selectively activate or inactivate proteins to reduce, ameliorate, or prevent
a disease or
disorder. Target proteases used in the protease trap selection method provided
herein
can be any known class of protease capable of peptide bond hydrolysis for
which the
protease trap interacts. Typically, for serpins, such proteases are generally
serine or
cysteine proteases for which serpins react with to form a covalent
intermediate
complex. Exemplary of serine and cysteine proteases are any protease set forth
in
Table 7 below. Typically, a library of modified proteases are used in the
methods
provided herein to select for a protease variant that exhibits an increased
specificity
or selectivity for a target protease trap, or variant thereof, such as a
serpin, or variant
thereof.
Exemplary proteases that can be used, and/or modified to be used, in the
selection method provided herein are described, and include truncated
pdlypeptides
thereof that include a catalytically active portion. Exemplary candidate
proteases are
listed in Table 7 and described herein (see also e.g., Rawlings et al. (2004),
"MEROPS:
the peptidase database", Nucleic Acids Research, 32: D160-D164). The sequence
identifiers (SEQ ID NO) for the nucleotide sequence and encoded amino acid
precursor
sequence for each of the exemplary candidate proteases is depicted in
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the Table. The Table also sets forth the b.Ccession number (Ace. No.) in the
UniProtKB/Swiss-Prot
database or the GenBate National Institute of Health (NIH) genetic sequence
database
corresponding to each protease. The encoded amino acids corresponding to the
signal peptide or
propeptide sequence to yield a mature protein also are noted in the Table. In
addition, amino
acids designating the protease domain (i.e. peptidase unit) also are noted, as
are the
active site residues that make up, for example, the catalytic triad of the
respective
protease. Since interactions are dynamic, amino acid positions noted are for
reference
and exemplification. The noted positions reflects a range of loci that vary by
2, 3,4,
5 or more amino acids. Variations also exist among allelic variants and
species
variants. Those of skill in the art can identify corresponding sequences by
visual
comparison or other comparisons including readily available algorithms and
software.
Candidate proteases for selection typically are wild-type or modified or
variant
forms of a wildtype candidate protease, or catalytically active portion
thereof,
including' allelic variant and isoforms of anyone protein. A candidate
protease can be
produced or isolated by any method known in the art including isolation from
natural
sources, isolation of recombinantly produced.proteins in cells, tissues and
organisms,
and by recombinant methods and by methods including in silica steps, synthetic
methods and any methods known to those of skill in the art. Modification of a
candidate protease for selection can be by any method known to one of skill in
the art,
such as any method described herein below.
Table 7: Exemplary Candidate Proteases
Protease TvIerops Name Nt. ACC. Ni. A.A. A.A.
Signal/ Peptidas
Type Code NO: SEQ ACC. SEQ propeptld
e unit
ID NO: ID e sequence
(active
NO: NO: site
residues
Striae S01.010 gramme B, M17016 PI0144 40
1.18/19-20 21-247
Protease: human-type 39 (64,108,
Chymo- 203)
trypsin S01.011 Testisin NM 006799 41 NP_00679 42
1-19 /20-41 42-288
family (vi) 0 (82, 137;
NM 144956 43 44 238)
-(v2) = NP 65920
NM 144957 45 5 46
= -(v3)
NP_65920
6
S01.015 trypstase beta 1 NM 003294 47 NP
00328 48 1-18 /19-30 31-274
(Homo sapiens) ' 5 (74, 121,
(III) 224)
S01.017 kallikrein hk5 NM_012427 49 NP_03655
50 1-22/ 67-292
9 (108, 153,
245)
S01.019 Corin NM 006587 51 NP 00651
52 802-1037
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_
Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid
e unit
ID NO: ID e sequence (active
NO: NO: site
residues
8 (843,
892,
985)
S01.020 kallikrein 12 NM 019598 53 NP_06254 54 1-17/
22-248
(vi) 4 (843,
892,
NM_145894 55 56 985) '
(v2) NP 66590
NM_I45895 57 1 58
(v3)
NP_66590
2
S01.021 DESC1 oritease AF064819 59 AAF04328 60 191-422
(231, 276,
372)
S01.028 tryptase gamma NM_012467 61 NP_03659 62 1-19/
38-272
1 9 (78,
125,
= 222)
S01.029 kallikrein hK14 NM_022046 63 Q9P0G3 64 1-18/
19-24 25-249
(67, III,
204)
S01.033 hyaluronan- NM_004132 65 NP_00412 66 1-23/
314-557
binding serine 3 (362,
411,
protease (HGF 509)
activator-like
protein)
S01.034 transmembrean NM_019894 67 NP 06394 68 205-436
e protease, NM_183247 69 7 70
(245, 290,
serine 4 NP 89907 387)
0
S01.054 tryptase delta I NM_012217 71 Q98233 72
1.18/19-30 31-235
(Homo sapiens) (74,
121,
, 224)
S01.074 Marapsin NM_031948 73 NP_11415 74 1-22 / 23-34 35-
279
4 (75,
124,
229)
S01.075 Tryptase BC036846 75 AAN0405 76 37-281
homologue 2 5 (77,
126,
(Homo sapiens) 231)
S01.076 Tryptase Putative 77 78 ' 67-304
homologue 3 Only (107,
213,
(Homo sapiens) AC005570 259)
(Cosmid
407D8)
S01.079 transmembrane NM_024022 79 NP_07692 80 217-451
protease, serine (VA) 7 (257,
304,
3 NM 032401 81 82 401)
(vB) NP 11577
NM 032404 83 7 84
(vC)
NM 032405 85 NP_11578 86
(vD) 0
NP_11578
1
S01.081 kallikrein hK15 NM 023006 87 NP 07538 88 1-16
/ 17-21 22-256
(Homo sapiens) (v1) 2 (62,
106,
NM 138563 89 90 209)
(v2) NP 61263
NM_138564 91 0 92 =
(v3)
NM 017509 93 NP_61263 94
(v4) 1
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid e unit
ID NO: ID e sequence (active
NO: NO: site
residues
NP 05997
9
S01.085 Memame- BC035384 95 AAH3538 96 1-241
AA03 I 4 (56, 101,
peptidase 195)
(deduced from
ESTs by
MEROPS)
S01.087 membrane-type AB048796 97 8AB39741 98 321-556
mosaic serine (361, 409,
protease 506)
S01.088 memame- Putative CAC I 2709 99 10-142
AA038 Only (50,101)
peptidase ALI 36097
(RP11-62C3
clone)
SO 1.098 memame- Putative 100 AAH4160 101 -- 33-202
AA 128 Only 9 (50,152)
peptidase BC041609
(deduced from
ESTs by
MEROPS)
S01.127 cationic trypsin NM_002769 102 NP_00276 103 1-
15/ 16-23 24-246
(Homo sapiens- 0 (63, 107,
type I) 200)
(cationic)
S01.131 Neutrophils NM_001972 104 NP_00196 105 1-27 /
28-29 30-249
elastasc 3 (70, 117,
202)
S01.132 mannan-binding AF28442 I 106 AAK8407 107 1-
19/ 449-710
lectin- 1 (497, 553,
associated 664)
sense protease-
3
S01.133 cathepsin G NM_001911 108 NP_00190 109 1-18 /
19-20 21-245
2 (64, 108,
201)
S01.134 myeloblastin NM 002777 110 NP_00276 111 1-25 /
26-27 28-250
(proteinase 3) 8 (71, 118,
203)
S01.135 granzymeA NM J106144 112 NP_00613 113 1-26 /
27-28 29-261
(CTLA3) 5 (69, 114,
212)
S01.139 granzyme M NM_005317 114 NP_00530 115 1-23 /24-
25 26-256
8 (66,111,
207)
S01.140 chymase NM_001836 116 NP 00182 117 1-19 / 21-21
22-247
(human-type) 7 (66, 110,
203)
S01.143 tryptase alpha NM_003294 118 NP 00328 119 1-18/
19-30 31-274
(I) 5 (74, 121,
224)
S01.146 granzyme K NM_002104 120 NP_00209 121 1-24 /
26-26 27-261
(67, 116,
214)
S01.147 granzyme H NM_033423 122 NP_2 1949 123 1-18 /
19-20 21-246
(CTLA I) I (64, 108,
202)
S01.152 chymotrypsin B M24400 124 P17538 125 1-18 34-
263
(75, 120,
213)
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid -- e
unit
ID NO: ID e sequence (active
NO: NO: site
residues
S01.153 pancreatic NM 001971 126 NP 00196 127 1-8 /
9-18 19-256
elastase 2 (63, 111, .
206)
S01.154 pancreatic NM_005747 128 NP_00573 129 1-15
/16-28 29-270
endopeptidase E 8 (73, 123,
(A) 217)
S01.155 pancreatic M16652 130 AAA5238 131 1-16 / 7-
28 29-269
elastase II (HA) 0 (73, 121,
216)
S01.156 Enteropeptidase NM_002772 132 NP_00276 133 785-1019
3 (825, 876,
971)
S01.157 chymotrypsin C NM_007272 134 NP_00920
135 1-16/ 17-29 30-268
3 (74, 121,
216)
S01.159 Prostasin NM_002773 136 NP_00276 137 1-29/
30-32 45-288
4 (85, 134,
238)
S01.160 kallikrein 1 NM_002257 138 NP_00224 139 1-
18 / 19-24 25-261
(65, 120,
214)
S01.161 kallikrein hK2 NM 005551 140 NP_00554 141
1-18 / 19-24 25-260
(Homo sapiens) ,1) 2 (65, 120,
NM 001002 142 143 213)
23-1 (v2) NP 00100
NM 001002 144 2-231 145
23-2 (v3) NP 00100
/232
S01.162 kallikrein 3 NM 001648 146 NP_00163 147 1-
17 / 18-24 25-260
(vi) 9 (65, 120,
NM 001030 148 (v1) 149 213)
04-7 (v3) NP 00102
NM 001030 150 5218(v3) 151
048(v4) NP 00102
NM 001030 152 5219(v4) 153
04-9 (v5) NP 00102
NM 001030 154 5220(v5) 155
050(v6) NP 00102
5211 (v6)
S01.174 Mesotrypsin NM_002771 156 NP_00276 157 1-24/
24-246
2 (63, 107,
200)
S01.205 pancreatic NM_007352 158 NP_03137 159 1-15/
16-28 29-270
endopeptidase E 8 (73, 123,
form B (B) 217)
S01.206 pancreatic NM_015849 160 NP_05693 161 1-16
/ 17-28 29-269
elastase 11 form 3 (73, 121,
13 (Homos 216)
sapiens) (1113)
S01.211 coagulation NM_000505 162 NP_00049 163 1-
19/ 373-615
factor XI la 6 (412, 461,
563)
S01.212 plasma NM_000892 164 NP_00088 165 1-19/ 391-
628
kallikrein 3 (434, 483,
(KLK3) 578)
S01.213 coagulation NM 000128 166 NP_00011 167 1-
18/ 388-625
factor Xla (v1) 9 (431, 480,
(HAF) NM 019559 168 (v1) 169 575)
-62) NP_06250
(v2)
S01.214 coagulation = NM 000133 170 NP 00012 171 1-
28 / 29-46 227-461
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid -- e
unit
ID NO: ID e sequence (active
NO: NO: site
residues
factor IXa 4 (267, 315,
411)
S01.215 coagulation NM_000131 172 NP 00012 173 1-
20 / 21-60 213-454
factor Vila (v1) 2 (253, 302,
NM_019616 174 (v1) 175 404)
(v2) NP 06256
2
(v2)
S01.216 coagulation NM_000504 176 NP_00049 177 1-
31 /32-40 235-469
factor Xa 5 (276, 322,
419)
S01.217 Thrombin NM 000506 178 NP_00049 179 1-24 / 25-
43 364-620
7 (406, 462,
568)
S01.218 protein C NM_000312 180 NP_00030 181 1-
32 / 33-42 212-452
(activated) 3 (253, 299,
402)
S01.223 Acrosin NM 001097 182 NP 00108 183 1-19 43-
292
8 (88, 142,
240)
S01.224 Hepsin NM 182983 184 NP 89202 185 -- 163-407
(vi) 8 (203, 257,
NM 002151 186 187 353)
NP 00214
2
S01.228 hepatocyle NM_001528 188 NP_00151 189 1-
35 / 36- 408-648
growth factor. 9 372 (447, 497,
activator 598)
(HGFA)
S01.231 u-plasminogen NM_002658 190 NP_00264 191 1-20/ 179-426
activator (uPA) 9 (224, 275,
376)
S01.232 t-plasminogcn NM_000930 192 NP_00092
193 1-23 /24-32 311-562
activator (tPA) (v1) 1 and 33-35 (357, 406,
NM_000931 194 (v1) 195 513)
(v2) NP 00092
NM 033011 196 2 197
(v3) (v2)
NP 12750
9
(v3)
S01.233 Plasmin NM_000301 198 NP 00029 199 1-19 / 20-
97 581-810
2 (622, 665,
760)
S01.236 Neurosin NM 002774 200 NP 00276 201 1-16/ 17-
21 22-244
(VA) ¨5 (62, 106,
NM 001012 202 203 197)
964 (vB) NP 00101
NM 001012 204 1982 205
96-5 (vC) NP 00101
NM 001012 206 /983 207
966 (vD) NP 00101
1984
S01.237 Neurotrypsin NM_003619 208 NP_00361 209 1-20/ 631-875
0 (676, 726,
825)
S01.242 tryptase beta 2 NM_024164 210 NP_07707
211 1-30/ 31-268
(Homo sapiens) 8
(1)
S01.244 Neuropsin NM_007196 212 NP_00912 213 1-28 / 29-32
33-258
(v1) 7 (73, 120,
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid a unit
ID NO: ID e sequence (active
NO: NO: site
residues
NM 144505 2(4 (v1) 215 212)
(v2) NP_65308
NM 144506 216 8 217
(v3) (v2)
NM 144507 218 NP_65308 219
(v4) 9
(v3)
NP_65309
(v4)
S01.246 kallikrein hK10 NM 002776 220 NP_00276
221 . 1-30 / 35-276
(Homo sapiens) (vi) 7 (86, 137,
NM 145888 222 223 229)
(v2) NP 66589
S01.247 Epitheliasin NM_005656 224 NP_00564 225 256-491
7 (296, 345,
441)
S01.251 Prostase NM_004917 226 NP_00490 227 1-26 / 27-
30 31-254
8 (71, 116,
207)
S01.252 Brain serine NM_022119 228 NP_07140 229 1-32
50-292
proteinase 2 2 (90, 141,
242)
S01.256 Chymopasin NM_001907 230 NP_00189 231 1-18 / 19-
33 34-264
8 (75, 121,
214)
S01.257 kaIlikrein 11 NM 006853 232 NP_00684 -- 233 -- 22-250
(vi) 4 (62, 110,
NM 144947 234 (vi) 235 1-50/51-53 203)
(v2) NP 65919
6
(v2)
S01.258 anionic trypsin NM_002770 236 NP_00276 237 1-15 /
16-23 24-246
(Homo sapiens) 1 (63, 107,
(II) 200)
(TRY2, TRY8,
TRYP2)
S01.291 L0C144757 Putative 238 AAH4811 239 78-319
peptidase BC048112 2 (122, 171,
(Homo sapiens) 268)
S01.292 Memame- BN000133 240 CAD6798 241. 1-19 175-
406
AAI69 5 (215, 260,
peptidase 356)
SOI .294 Mername- Putative 242
AA171 No DNA
peptidase
S01.298 Memame- Putative AAC8020 243 24-246
AA174 no DNA seq 8 (63, 107,
peptidase 200)
(TRY6)
S01.299 Memame- NM_198464 244 NP 94086 245 68-302
AA175 6 (108, 156,
peptidase 250)
S01.300 stratum NM 005046 246 NP_00503 247 1-22123-29
30-250
corneum -(v1) 7 (70, 112,
chymotryptic NM_I39277 248 249 205)
enzyme (SCCE) (v2) NP 64480
6
S01.301 trypsin-like NM_004262 250 NP 00425 251 187-
471
enzyme, 3 (227, 272,
respiratory 368)
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid e
unit
ID NO: ID e sequence
(active
NO: NO: site
residues
(Transmem bran
e protease,
serine H D)
S01.302 Matripase AF 118224 252 AAD4276 253
615-855
(MTSP1) 5 (656, 711,
805)
S01.306 kallikrein hK13 NM 015596 254 NP_05641 255 1-16 /
36-263
1 (76, 124,
218)
S01.307 kallikrein hK9 NM 012315 256 NP_03644 257 1-15 /
23-250
(human 7 (63, 111,
numbering) 204)
S01.308 Mername- NM_153609 258 NP 70583 259 49-
283
AA035 7 (89, 140,
peptidase 234)
S01.309 umbilical vein NM_007173 260 NP 00910 261 1-23/
95-383
proteinase 4 (175, 246,
316)
S01.311 LCLP Peptide P34168 262 1-26
proteinase fragment (0)
(LCLP (N- No DNA
terminus))
S01.313 Spinesin NM_030770 263 NP_11039 264 218-455
7 (258, 308,
405)
S01.318 Mername- NM 183062 265 NP_89888 266 1-33
53-288
AA178 5 (93, 143,
peptidase 238)
S01.320 Mername- BN000120 267 CA06645 268 1-23/
52-301
AA180 2 (92, 142,
peptidase 240)
S01.322 Mername- BN000128 269 CAD6757 270 1-17 8-
298
AAI82 9 (87, 139,
peptidase 237)
S01.414 Mername- Putative 271 BAC11431 272 1-
177
AA122 AK075142 (12,64,
peptidase 168)
(deduced from
ESTs by =
MEROPS)
Cysteine C01.032 Cathepsin L X12451 273 P07711 274 1-17 /
18- 113-333
protease: 113 (132, 138,
= Papain = 276, 300)
family C01.009 Cathepsin V U13665 275 060911 276 1-
17/ 18- 114-334
113 (132, 138,
277, 301)
C01.036 Cathepsin K S93414 277 P43235 278 1-15 / 16-
115-329
114 (133, 139,
276, 296)
=
C01.034 Cathepsin S AJ007331 279 P25774 280 1-16 /
17- 115-331
114 (133, 139,
278, 298)
C01.018 Cathepsin F M14221 281 Q9UBX1 282 1-19 / 20-
271-484
270 (289, 295,
431, 451)
C01.060 Cathepsin B M15203 283 P07858 284 1-
17/ 18-79 80-331
(102, 108,
278, 298)
C01.001 Papain M84342 285 P00784 286 1-18/ 19-
135-342
133 (158,
292, 308)
C01.075 Cruzain Y14734 287 P25779 288 123-467/
124-334
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..
Protease Merops Name Nt. ACC. Nt. A.A. A.A.
Signal/ Peptides
Type Code NO: SEQ ACC. SEQ
propeptid e unit
ID NO: ID e sequence
(active
NO: NO: site
residues
(Cruzapain) (147, 284,
304,
Serine S08.001 Subtilisin X03341 290 P00780
289 1-29 130- 111-370
Protease: Carlsberg 105 (137,
subtilisin precursor ,
168,325)
Family S08.002 Subtilisin P07518 291 6-266
(Alkaline (32, 64,
mesentericopept 221)
idase)
S08.003 Subtilisin P29600 292 6-260
Savinase (32, 62,
(Alkaline 215)
protease)
S08.007 Therm itase P04072 293 13-264
(38,71,
225)
S08.009 Thermophilic 429506 295 Q45670
294 1-24 / 25- 134-391
serine 121 (160,193,
protcinasc 347)
precursor
(Ak. I protease)
S08.020 C5a peptidase J05229 297 P15926
296 1-31 / 120-339,
precursor 458-560
(130,
193, 512)
S08.021 fervidolysin AY035311 299
AAK6155 298 164-457
, 2
S08.035 Subtilisini M64743 301 P29142
300 1-29 / 30- 112-372
precursor 106 (138,
170, 327)
S08.036 Subtilisin E K01988 303 P04189
302 1-23 /24- 112-372
precursor 106 (138, 170,
327)
S08.037 Subtilisin DY P00781 304 6-
257
(32, 63,
220)
S08.054 Proteinase K X14689 306 P06873
305 1-15 (16- 134-373
precursor 105 (144,
(Endopeptidase 174,
329)
K)
S08.050 Alkaline serine M25499 308 P16588 307
1-21 /22- 155-412
exoprotease A 141 (180, 213,
precursor 363)
S08.060 Epidermin X052386 310 P30199
309 1-23 / 24 -? 123-451
leader peptide- (149,
processing 194, 402)
serine protease
epiP precursor
S08.063 Membrane- AF078105 312 Q922A8
311 1-14 / 18- 179-473
bound 186 (218, 249,
transcription 338, 414)
factor site 1
protease
precursor (Site-
! protease,
Subtilisiakexin
-isozyme 1,
SKI-I)
S08.066 Alkaline M87516 314 Q03420 313
1-20 / 21- 121-409
proteinase 120 (161, 192,
precursor (ALP) 353)
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptidas
Type - Code NO: SEQ ACC. SEQ propeptid e
unit
ID NO: ID e sequence (active
NO: NO: site
residues
S08.094 Extracellular M13469 316 P09489 315 1-27/
50-389
serine protease (76, 112,
precursor 341)
S08.090 Tripeptidy I- AF035251 318 Q9 V6K1 317 5-509
peruidase II (131, 359,
(TPP-11) 549)
S08.114 Minor M76590 320 P29141 319 1-28 / 29- 163-365,
extracellular 160 487-586
protease vpr (189, 233,
precursor 534)
S08.116 PIII-type .104962 322 P15292 321 1-33134- 190-
379,
proteinase 187 584-628
precursor (217, HI,
(Lactocepin) 620)
S08.048 Furin-like M81431 324 P30430 323 I-? /?-309
376-646
protease 1, (372, 413,
isoforrn 1-CRR 587)
precursor
508.070 Kexin precursor M22870 326 P13134 325 1-19/20-
149-445
(KEX2 109 and (175,
protease) 110-113 213, 385)
(Kex2-like
endoprotease I,
dKLIP-1)
S08.071 Furin precursor X17094 328 P09958 327 1-24 /
25- 131-421
(PACE) 107 (153, 194,
295, 368)
S08.075 Subtilisin/kexin M80482 330 P29122 329 1-63 /64-
182-473
-like protease 149 (205, 246,
PACE4 347, 420)
(Proprotein
convertase
subtilisindtexin
type 6
precursor)
S08.079 Calcium. X56955 332 P23916 331 254-521
dependent (233, 270,
protease 466)
precursor
(Trypsin)
Cysteine C14.001 Caspase-1 014647 334 P43527 333 /
1-118 120-404
protease: precursor (236, 284)
easpase (Interleukin-1
family beta convertase,
IL- IBC)
C14.002 Cell death L29052 336 P42573 335 235 ¨
495
protein 3 (315, 358)
precursor
C14.003 Caspase-3 013737 338 P42574 337 / 1-9 10-277
precursor (121, 163)
(Apopain,
Cys lei ne
protease)
C14.004 Caspase-7 039613 340 P55210 339 / 1-23 29.303
precursor (144, 186)
(ICE-like
apoptotic
protease 3,
Apoptotic
protease Mch-3)
C14,005 Caspase-6 020536 342 P55212 341 / 1-23 24-292
precursor (121, 163)
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Protease Merops Name Nt. ACC. Nt. AA. -- A.A. -- Signal/ --
Peptidas
Type Code NO: SEQ ACC. SEQ propeptid e unit
ID NO: ID e sequence (active
NO: NO: site
residues
(Apoptotic
protease Mch-2)
C14.006 Caspase-2 D28492 344 P29594 343 / 1-169 170-
432
precursor (277, 320)
(1CH-1
protease,
NEDD2
protein)
C14.007 Caspase-4 Z48810 346 P49662 345 / 1-80 93-377
precursor (210, 258)
(ICH-2
protease, TX
protease)
C14.008 Caspase-5 U280 I 5 348 P51879 347 /1-120 134-
418
precursor (251, 299)
(ICH-3
protease, TY
protease,
ICE(rel)-111)
C14.009 Caspase-8 X98172 350 Q14790 -- 349 -- /1-216 -- 193-
479
precursor (317, 360)
(FADD-like
ICE, ICE-like
apoptotic
protease 5)
C14.010 Caspase-9 U56390 352 P55211 351 117-416
precursor (237, 287)
(ICE-LA P6,
Apoptotic
protease Mch-6
C14.011 Caspase-10 U60519 354 Q92851 353 /1-219 243-
514
precursor (358, 401)
C14.012 Caspase-11 1159463 356 P70343 355 /1-80 89-373
(Caspase-4 (206, 254)
precursor)
C14.013 Caspase-12 Y13090 358 008736 357 133-419
precursor (250, 298)
C14.015 Caspase Y12261 360 001382 359 /1-28 (169,
211)
precursor
(insect)
(driCE)
C14.016 Caspase-1 AF001464 362 002002 361 /1-33 (154,
196)
precursor
(insect)
C14.017 Caspase- 13 AF078533 364 075601 363 (210,
258)
precursor
C14.018 Caspase-14 AF097874 366 P31944 365 1-242
precursor (89, 132)
CI4.019 Caspase Nc AF104357 368 Q9XYF4 367 /1-134 (271,
318)
precursor
(NEDD2-like
caspase
DRONC)
C14.026 MALT AFI 30356 370 Q9UDY8 369 337-523
lymphoma (415, 464)
translocation
protein 1
paracaspase
(paracaspase)
C14.971 CASP8 and 1385059 372 015519 371 260-433
FADD-like (315, 363)
apoptos is
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Protease Merops Name Nt. ACC. Nt. A.A. A.A. Signal/
Peptides
Type Code NO: SEQ ACC. SEQ propeptid e unit
ID NO: ID e sequence (active
NO: NO: site
residues
regulator
precursor (c-
FLIP)
a. Classes of Proteases
Proteases (also referred to as proteinases or peptidases) are protein-
degrading
enzymes that recognize sequences of amino acids or a polypeptide substrate
within a
target protein. Upon recognition of the substrate sequence of amino acids,
proteases
catalyze the hydrolysis or cleavage of a peptide bond within a target protein.
Such
hydrolysis of a target protein, depending on the location of the peptide bond
within
the context of the full-length sequence of the target sequence, can
inactivate, or in
some instances activate, a target.
Proteases are classified based on the way they attack the protein, either exo-
or
endo- proteases. Proteinases or endopeptidases attack inside the protein to
produce
large peptides. Peptidases or exopeptidases attack ends or fragments of
protein to
produce small peptides and amino acids. The peptidases are classified on their
action
pattern: aminopeptidase cleaves amino acids from the amino end:
carboxypeptidase
cleaves amino acids from the carboxyl end, dipeptidyl peptidase cleaves two
amino
acids; dipeptidase splits a dipeptide, and tripeptidase cleaves an amino acid
from a
tripeptide. Most proteases are small from 21,000 to 45,000 Daltons. Many
proteases
are synthesized and secreted as inactive forms called zymogens and
subsequently
activated by proteolysis. This changes the architecture of the active site of
the
enzyme.
Several distinct types of catalytic mechanisms are used by proteases ( Barret
et
al. (1994) Meth. Enzymol. 244:18-61; Barret et al. (1994) Meth. Enzymol
244:461-
486; Barret et al. (1994) Meth. Enzymol. 248:105-120; Barret etal. (1994)
Meth.
Enzymol. 248:183-228). Based on their catalytic mechanism, the
carboxypeptidases
are subdivided into serine-, metallo.and cysteine- type carboxypeptidases and
the
endopeptidases are the serine-, cysteine-, aspartic-, threonine- and
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metalloendopeptidases. Serine peptidases have a serine residue involved in the
active
center, the aspartic have two aspartic acids in the catalytic center, cysteine
-type
peptidases have a cysteine residue, threonine-type peptidases have a threonine
residue, and metallo-peptidases use a metal ion in the catalytic mechanism,
Generally, proteases can be divided into classes based on their catalytic
activity such
that classes of proteases can include serine, cysteine, aspartic, threonine,
or metallo-
proteases. The catalytic activity of the proteases is required to cleave a
target
substrate. Hence, modification of a protease to alter the catalytic activity
of a protease
can affect (i.e. modify specificity/ selectivity) the ability of a protease to
cleave a
particular substrate.
Each protease has a series of amino acids that lines the active site pocket
and
makes direct contact with the substrate. Crystallographic structures of
peptidases
show that the active site is commonly located in a groove on the surface of
the
molecule between adjacent structural domains, and the substrate specificity is
dictated
by the properties of binding sites arranged along the groove on one or both
sides of
the catalytic site that is responsible for hydrolysis of the scissile bond.
Accordingly,
the specificity of a peptidase is described by the ability of each subsite to
accommodate a sidechain of a single amino acid residue. The sites are numbered
from
the catalytic site, Si, S2...Sn towards the N-terminus of the substrate, and
Sr,
S2'...Sn' towards the C-terminus. The residues they accommodate are numbered
Pl,
P2...Pn, and P1', P2'...Pn', respectively. The cleavage of a target protein is
catalyzed
between PI and 131' where the amino acid residues from the N to C terminus of
the
polypeptide substrate are labeled (Pi, ..., P3, P2, PI, Pr, P2', P3', Pj)
and their
corresponding binding recognition pockets on the protease are labeled (Si,...,
S3, S2,
Si, Sr, S2', S3',..., Sj) (Schecter and Berger (1967) Biochem Biophys Res
Commun
27:157-162). Thus, P2 interacts with S2, P1 with Si, PI' with Si', etc.
Consequently, the substrate specificity of a protease comes from the Si -S4
positions
in the active site, where the protease is in contact with the P1 -P4 residues
of the
peptide substrate sequences. In some cases, there is little (if any)
interactions betweer
the S1-S4 pockets of the active site, such that each pocket appears to
recognize and
bind the corresponding residue on the peptide substrate sequence independent
of the
other pockets. Thus, the specificity determinants can be changed in one pocket
without affecting the specificity of the other pocket. Based upon numerous
structures
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and modeling of family members, surface residues that contribute to extended
substrate specificity and other secondary interactions with a substrate have
been
defined for many proteases including proteases of the serine, cysteine,
aspartic,
metallo-, and threonine families (see e.g. Wang et al., (2001) Biochemistry
40(34):
10038-46; Hopfner et al., (1999) Structure Fold Des. 7(8):989-96; Friedrich et
al.
(2002) J Biol Chem. 277(3):2160-8; Waugh etal., (2000) Nat Struct Biol.
7(9):762-5;
Cameron etal., (1993) J Biol Chem. 268:11711; Cameron et al., (1994) J Biol
Chem.
269: 11170).
i. Serine Proteas es
Serine proteases (SPs), which include secreted enzymes and enzymes
sequestered in cytoplasmic storage organelles, have a variety of physiological
roles,
including in blood coagulation, wound healing, digestion, immune responses and
tumor invasion and metastasis. For example, chymotrypsin, trypsin, and
elastase
function in the digestive tract; Factor 10, Factor 11, Thrombin, and Plasmin
are
involved in clotting and wound healing; and Clr, Cis, and the C3 convertases
play a
role in complement activation.
A class of cell surface proteins designated type II transmembrane serine
proteases are proteases which are membrane-anchored proteins with
extracellular
domains. As cell surface proteins, they play a role in intracellular signal
transduction
and in mediating cell surface proteolytic events. Other serine proteases are
membrane
bound and function in a similar manner. Others are secreted. Many serine
proteases
exert their activity upon binding to cell surface receptors, and, hence act at
cell
surfaces. Cell surface proteolysis is a mechanism for the generation of
biologically
active proteins that mediate a variety of cellular functions.
Serine proteases, including secreted and transmembrane serine proteases, are
involved in processes that include neoplastic development and progression.
While the
precise role of these proteases has not been fully elaborated, serine
proteases and
inhibitors thereof are involved in the control of many intra- and
extracellular
physiological processes, including degradative actions in cancer cell invasion
and
metastatic spread, and neovascularization of tumors that are involved in tumor
progression. Proteases are involved in the degradation and remodeling of
extracellular matrix (ECM) and contribute to tissue remodeling, and are
necessary for
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cancer invasion and metastasis. The activity and/or expression of some
proteases
have been shown to correlate with tumor progression and development.
Over 20 families (denoted SI-S27) of serine protease have been identified,
these being grouped into 6 clans (SA, SB, SC, SE, SF and SG) on the basis of
structural similarity and other functional evidence (Rawlings ND etal. (1994)
Meth.
Enzymol. 244: 19-61). There are similarities in the reaction mechanisms of
several
serine peptidases. Chymotrypsin, subtilisin and carboxypeptidase C clans have
a
catalytic triad of serine, aspartate and histidine in common: serine acts as a
nucleophile, aspartate as an electrophile, and histidine as a base. The
geometric
orientations of the catalytic residues are similar between families, despite
different
protein folds. The linear arrangements of the catalytic residues commonly
reflect clan
= relationships. For example the catalytic triad in the chymotrypsin clan
(SA) is ordered
HDS, but is ordered DHS in the subtilisin clan (SB) and SDH in the
carboxypeptidase
clan (SC).
Examples of serine proteases of the chymotrypsin superfamily include tissue-
type plasminogen activator (tPA), trypsin, trypsin-like protease,
chymotrypsin,
plasmin, elastase, urokinase (or urinary-type plasminogen activator, u-PA),
acrosin,
activated protein C, Cl esterase, cathepsin G, chymase, and proteases of the
blood
coagulation cascade including kallikrein, thrombin, and Factors Vila, IXa, Xa,
XIa,
and XIIa (Barret, A.J., In: Proteinase Inhibitors, Ed. Barrett, A.J., Et al.,
Elsevier,
Amsterdam, Pages 3-22 (1986); Strassburger, W. etal., (1983) FEBS Lett., 157
:219-
223; Dayhoff, M.O., Atlas of Protein Sequence and Structure, Vol 5, National
Biomedical Research Foundation, Silver Spring, Md. (1972); and Rosenberg, R.D.
et
at. (1986) Hosp. Prac., 21: 131-137).
The activity of proteases in the serine protease family is dependent on a set
of
amino acid residues that form their active site. One of the residues is always
a serine;
hence their designation as serine proteases. For example, chymotrypsin,
trypsin, and
elastase share a similar structure and their active serine residue is at the
same position
(Ser-195) in all three. Despite their similarities, they have different
substrate
specificities; they cleave different peptide bonds during protein digestion.
For
example, chymotrypsin prefers an aromatic side chain on the residue whose
carbonyl
carbon is part of the peptide bond to be cleaved. Trypsin prefers a positively
charged
Lys or Arg residue at this position. Serine proteases differ markedly in their
substrate
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recognition properties: some are highly specific (i.e. the proteases involved
in blood
coagulation and the immune complement system); some are only partially
specific
(i.e. the mammalian digestive proteases trypsin and chymotrypsin); and others,
like
subtilisin, a bacterial protease, are completely non-specific. Despite these
differences
in specificity, the catalytic mechanism of serine proteases is well conserved.
The mechanism of cleavage of a target protein by a serine protease is based on
nucleophilic attack of the targeted peptidic bond by a serine. Cysteine,
threonine or
water molecules associated with aspartate or metals also can play this role.
In many
cases the nucleophilic property of the group is improved by the presence of a
histidine, held in a "proton acceptor state" by an aspartate. Aligned side
chains of
serine, histidine and aspartate build the catalytic triad common to most
serine
proteases. For example, the active site residues of chymotrypsin, and serine
proteases
that are members of the same family as chymotrypsin, such as for example MTSP-
1,
are Asp102, His57, and Ser195.
The catalytic domains of all serine proteases of the chymotrypsin superfamily
have both sequence homology and structural homology. The sequence homology
includes the conservation of: 1) the characteristic active site residues
(e.g., Ser195,
His57, and Asp102 in the case of trypsin); 2) the oxyanion hole (e.g., Gly193,
Asp194
in the case of trypsin); and 3) the cysteine residues that form disulfide
bridges in the
structure (Hartley, B.S., (1974) Symp. Soc. Gen. Microbiol., 24: 152-182). The
structural homology includes 1) a common fold characterized by two Greek key
structures (Richardson, J. (1981) Adv. Prot. Chem., 34:167-339); 2) a common
disposition of catalytic residues; and 3) detailed preservation of the
structure within
the core of the molecule (Stroud, R.M. (1974) Sci. Am., 231: 24-88).
Throughout the chymotrypsin family of serine proteases, the backbone
interaction between the substrate and enzyme is completely conserved, but the
side
chain interactions vary considerably. The identity of the amino acids that
contain the
S1-S4 pockets of the active site determines the substrate specificity of that
particular
pocket. Grafting the amino acids of one serine protease to another of the same
fold
modifies the specificity of one to the other. Typically, the amino acids of
the protease
that contain the S1-S4 pockets are those that have side chains within 4 to 5
angstroms
of the substrate. The interactions these amino acids have with the protease
substrate
are generally called "first shell" interactions because they directly contact
the
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substrate. There, however, can be "second shell" and "third shell"
interactions that
ultimately position the first shell amino acids. First shell and second shell
substrate
binding effects are determined primarily by loops between beta-barrel domains.
Because these loops are not core elements of the protein, the integrity of the
fold is
maintained while loop variants with novel substrate specificities can be
selected
during the course of evolution to fulfill necessary metabolic or regulatory
niches at
the molecular level. Typically for serine proteases, the following amino acids
in the
primary sequence are determinants of specificity: 195, 102, 57 (the catalytic
triad);
189, 190, 191, 192, and 226 (S1); 57, the loop between 58 and 64, and 99 (S2);
192,
217, 218 (S3); the loop between Cys168 and Cys180, 215, and 97 to 100 (S4);
and 41
and 151 (S2'), based on chymotrypsin numbering, where an amino acid in an Si
position affects P1 specificity, an amino acid in an S2 position affects P2
specificity,
an amino acid in the S3 position affects P3 specificity, and an amino acid in
the S4
position affects P4 specificity. Position 189 in a serine protease is a
residue buried at
the bottom of the pocket that determines the Si specificity. Structural
determinants
for various serine proteases are listed in Table 8 with numbering based on the
numbering of mature chymotrypsin, with protease domains for each of the
designated
proteases aligned with that of the protease domain of chymotrypsin. The number
underneath the Cys168-Cys182 and 60's loop column headings indicate the number
of amino acids in the loop between the two amino acids and in the loop. The
yes/no
designation under the Cys191-Cys220 column headings indicates whether the
disulfide bridge is present in the protease. These regions are variable within
the
family of chymotrypsin-like serine proteases and represent structural
determinants in
themselves. Modification of a protease to alter any one or more of the amino
acids in
the S I-S4 pocket affect the specificity or selectivity of a protease for a
target
substrate.
Table 8: The structural determinants for various serine proteases
Residues that Determine Specificity
S4 S3 S2 Si
171 174 180 215 Cys168 192 218 99 57 60's 189 190 226 CysI91
Cys182 loop Cys220
Granzymc Leu Tyr Glu Tyr 14 Arg Asn Ile His 6 Gly Ser Arg No
Granzyme Asn Val Met Phe 17 Asn Leu Arg His 7 Asp Sec Gly Yes
A
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Granzyme Arg Ser Met Phe 15 Lys Arg Leu His 8 Ala Pro Pro
Yes
Cathepsin Phe Scr Gin Tyr 13 Lys Ser Ile His 6 Ala Ala Glu No
MT-SPI Leu Gin Met Trp 13 Gin Asp Phe His 16 Asp Ser Gly Yes
Neutrophil - Tyr S Phe Gly Leu His - 10 Gly
Val Asp Yes
alas Last
Chymase Phe Arg Gin Tyr 12 Lys Ser Phe His 6 Ser Ala Ala No
Alpha- Tyr Ile Met Trp 22 - Lys Glu Ile His ¨ 9
Asp Ser Gly Yes
tryptase
Beta- Tyr Ile Met Trp 22 Gin Glu Val His - 9
Asp Set- Gly Yes
tryptase(f)
Beta- Tyr Ile Met Trp 22 Lys Glu Thr His - 9
Asp Scr Gly Yes
tryptase
(II)
Chymo- Trp Arg Met T= rp 13 Met Ser Val His - 7 Ser
Ser Gly Yes
trypsin
Easter Tyr Ser Gin Phe 16 Arg Thr Gin His 14 Asp Ser Gly Yes
Collage. Tyr Ile - Phe 12 Asn Ala Ile ¨ His - 8
Gly Thr Asp Yes
nasc
Factor Xa Ser Phe Met Trp 13 Gln Glu Tyr His 8
Asp Ala Gly Yes
_
Protein C Met asn Met Trp 13 Glu Glu Thr His 8
Asp Ala Gly Yes
Plasma Tyr Gin Met Tyr 13 Arg Pro Phe His - 11
Asp Ala Ala - Yes
kallikrein
piasmin Glu Arg Glu Trp 15 Girt Leu Thr His - 11
Asp Ser Gly Yes
_
-bypsin Tyr Lys Met Trp 13 Gln Tyr Leu His 6 Asp Ser Gly Yes
Thrombin Thr Ile Met T= rp 13 Gin Glu Leu His '4-
16 Asp Ala Gly Yes
_
IPA Leu Thr Met Trp 15 Girt Leu Tyr His 11 Asp Ala Gly Yes
uPA His Ser Met T= rp 15 Gln Arg His - His ¨ ii
Asp Scr Gly yes
(a) Urokinase-type Plasminogen Activator (u-PA)
Urokinase-type plasminogen activator (u-PA, also called urinary plasminogen
activator) is an exemplary protease used as a candidate for selection in the
methods
herein, u-PA is set forth in SEQ ID NO:190 and encodes a precursor amino acid
sequence set forth in SEQ ID NO:191. u-PA is found in urine, blood, seminal
fluids,
and in many cancer tissues. It is involved in a variety of biological process,
which are
linked to its conversion of plasminogen to plasmin, which itself is a serine
protease.
Plasmin has roles in a variety of normal and pathological processes including,
for
example, cell migration and tissue destruction through its cleavage of a
variety of
molecules including fibrin, fibronectin, proteoglycans, and laminin. u-PA is
involved
in tissue remodeling during wound healing, inflammatory cell migration,
neovascularization and tumor cell invasion, u-PA also cleaves and activates
other
substrates, including, but not limited to, hepatocyte growth factor/scatter
factor
(HGF/SF), the latent form of membrane type 1 matrix metalloprotease (MT-SP1),
and
others.
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..
The mature form of u-PA is a 411 residue protein (corresponding to amino
acid residues 21 to 431 in the sequence of amino acids set forth in SEQ ID
NO:191,
which is the precursor form containing a 20 amino acid signal peptide). u-PA
contains three domains: the serine protease domain, the kringle domain and the
growth factor domain. In the mature form of human u-PA, amino acids 1-158
represent the N-terminal A chain including a growth factor domain (amino acids
I-
49), a kringle domain (amino acids 50-131), and an interdomain linker region
(amino
acids 132-158). Amino acids 159-411 represent the C-terminal serine protease
domain or B chain, u-PA is synthesized and secreted as a single-chain zymogen
molecule, which is converted into an active two-chain u-PA by a variety of
proteases
including, for example, plasmin, kallikrein, cathepsin B, and nerve growth
factor-
gamma. Cleavage into the two chain form occurs between residues 158 and 159 in
a
mature u-PA sequence (corresponding to amino acid residues 178 and 179 in SEQ
ID
NO:191). The two resulting chains are kept together by a disulfide bond,
thereby
forming the two-chain form of u-PA.
u-PA is regulated by the binding to a high affinity cell surface receptor,
uPAR.
Binding of u-PA to uPAR increases the rate of plasminogen activation and
enhances
extracellular matrix degradation and cell invasion. The binary complex formed
between uPAR and u-PA interact with membrane-associated plasminogen to form
higher order activation complexes that reduce the Km (i.e. kinetic rate
constant of the
approximate affinity for a substrate) for plasminogen activation (Bass et al.
(2002)
Biochem. Soc, Trans., 30: 189-194). In addition, binding of u-PA to uPAR
protects
the protease from inhibition by the cognate inhibitor, i.e. PAI-1. This is
because
single chain u-PA normally present in plasma is not susceptible to inhibition
by PA!-
1, and any active u-PA in the plasma will be inhibited by PAI-1. Active u-PA
that is
receptor bound is fully available for inhibition by PAI-1, however, PAI-1 is
unable to
access the bound active molecule (Bass et al. (2002) Biochem. Soc, Trans., 30:
189-
194). As a result, u-PA primarily functions on the cell surface and its
functions are
correlated with the activation of plasmin-dependent pericellular proteolysis.
The extended substrate specificity of u-PA and t-PA (discussed below) are
similar, owing to the fact that both are responsible for cleaving plasminogen
into
active plasmin. Both u-PA and t-PA have high specificity for cleavage after P1
Arg,
and they similarly show a preference for small amino acids at the P2 position.
Both
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4
of the P3 and P4 positions are specificity determinants for substrates of u-PA
and t-
PA, with a particularly prominent role of the P3 position (Ke et al. (1997) J.
Biol.
Chem., 272: 16603-16609). The preference for amino acids at the P3 position
are
distinct, and is the main determinant for altered substrate discrimination
between the
two proteases. t-PA has a preference for aromatic amino acids (Phe and Tyr) at
the
P3 position, while u-PA has a preference for small polar amino acids (Thr and
Ser)
(see e.g., Ke et al. (1997) J. Biol. Chem., 272: 16603-16609; Harris et al.
(2000)
PNAS, 97: 7754-7759).
(b) Tissue Plasminogen Activator (t-PA)
A candidate protease for selection against a protease trap in the methods
herein also includes the exemplary serine protease tissue plasminogen
activator (t-
PA), and variants thereof. t-PA is a serine protease that converts plasminogen
to
plasmin, which is involved in fibrinolysis or the formation of blood clots.
Recombinant t-PA is used as a therapeutic in diseases characterized by blood
clots,
such as for example, stroke. Alternative splicing of the t-PA gene produces
three
transcripts. The predominant transcript is set forth in SEQ ID NO:192 and
encodes a
precursor protein set forth in SEQ ID NO:193 containing a 20-23 amino acid
signal
sequence and a 12-15 amino acid pro-sequence. The other transcripts are set
forth in
SEQ ID NO:194 and 196, encoding precursor proteins having a sequence of amino
acids set forth in SEQ ID NOS: 195 and 197, respectively. The mature sequence
of t-
PA, lacking the signal sequence and propeptide sequence is 527 amino acids.
t-PA is secreted by the endothelium of blood vessels and circulates in the
blood as a single-chain form. Unlike many other serine proteases, the single-
chain or
"proenzyme" form of t-PA has high catalytic efficiency. The activity of t-PA
is
increased in the presence of fibrin. In the absence of fibrin, single-chain t-
PA is about
8% as active as compared to two-chain t-PA, however in the presence of fibrin
the
single- and two-chain forms oft-PA display similar activity. (Strandberg etal.
(1995)
J. Biol. Chem., 270: 23444-23449). Thus, activation of single-chain t-PA can
be
accomplished either by activation cleavage (i.e. zymogen cleavage), resulting
in a
two-chain form, or by binding to the co-factor fibrin. Activation cleavage
occurs
following cleavage by plasmin, tissue kallikrein, and activated Factor X at
amino acid
positions Arg275.11c276 (corresponding to Arg31 -I1e311 in the sequence of
amino acids
set forth in SEQ ID NO:193) resulting in the generation of the active two-
chain form
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of t-PA. The two-chain polypeptide contains an A and a B chain that are
connected
by an interchain disulfide bond.
The mature t-PA contains 16 disulfide bridges and is organized into five
distinct domains (Gething etal. (1988), The EMBO J, 7: 2731-2740). Residues 4-
50
of the mature protein form a finger domain, residues 51-87 form an EGF-like
domain,
residues 88-175 and 176-263 form two kringle domains that contain three
intradomain
disulfide bonds each, and residues 277-527 of the mature molecule
(corresponding to
amino acid residues 311-562 of the precursor sequence set forth in SEQ ID
NO:193)
make up the serine protease domain.
In contrast to u-PA, which acts as a cellular receptor-bound activator, t-PA
functions as a fibrin-dependent circulatory activation enzyme. Likewise, both
single-
and two-chain forms oft-PA are susceptible to inhibition by their cognate
inhibitors,
for example, PAI-1, although two-chain t-PA is inhibited by PAI-1
approximately 1.4
times more rapidly than single-chain t-PA (Tachias etal. (1997)J Biol. Chem.,
272:
14580-5). t-PA can become protected from inhibition by binding to its cellular
binding site Annexin-II on endothelial cells. Thus, although both t-PA and u-
PA both
cleave and activate plasminogen, the action oft-PA in the blood supports t-PA
as the
primary fibrinolytic activator of plasminogen, while u-PA is the primary
cellular
activator of plasminogen.
(c) MT-SP1
Membrane-type serine protease MT-SP1 (also called matriptase, TADG-15,
suppressor of tumorigenicity 14, ST14) is an exemplary protease for selection
in the
methods provided herein to select for variants with an altered substrate
specificity
against a desired or predetermined substrate cleavage sequence. The sequence
of
MT-SP1 is set forth in SEQ ID NO:252 and encodes an 855 amino acid polypeptide
having a sequence of amino acids set forth in SEQ ID NO:253. It is a
multidomain
proteinase with a C-terminal serine proteinase domain (Friedrich et al.
(2002)J Biol
Chem 277(3):2160). A 683 amino acid variant of the protease has been isolated,
but
this protein appears to be a truncated form or an ectodomain form.
MT-SP1 is highly expressed or active in prostate, breast, and colorectal
cancers and it can play a role in the metastasis of breast and prostate
cancer. MT-SP1
also is expressed in a variety of epithelial tissues with high levels of
activity and/or
expression in the human gastrointestinal tract and the prostate. Other species
of MT-
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SP1 are known. For example, a mouse homolog of MT-SP1 has been identified and
is called epithin.
MT-SP1 contains a transmembrane domain, two CUB domains, four LDLR
repeats, and a serine protease domain (or peptidase Si domain; also called the
B-
chain) between amino acids 615-854 (or 615-855 depending on variations in the
literature) in the sequence set forth in SEQ ID NO:253. The amino acid
sequence of
the protease domain is set forth in SEQ ID NO:505 and encoded by a sequence of
nucleic acids set forth in SEQ ID NO:504. MT-SP1 is synthesized as a zymogen,
and
activated to double chain form by cleavage. In addition, the single chain
proteolytic
domain alone is catalytically active and functional.
An MT-SPI variant, termed CB469, having a mutation of C122S
corresponding to the wild-type sequence of MT-SP1 set forth in either SEQ ID
NO:
253 or 505, based on chymotrypisn numbering, exhibits improved display on
phagemid vectors. Such a variant MT-SP1 is set forth in SEQ ID NO:515 (full
length
MT-SP1) or SEQ ID NO:507 (protease domain) and can be used in the methods
described herein below.
MT-SP1 belongs to the peptidase Si family of serine proteases (also referred
to as the chymotrypsin family), which also includes chymotrypsin and trypsin.
Generally, chymotrypsin family members share sequence and structural homology
with chymotrypsin. MT-SP1 is numbered herein according to the numbering of
mature chymotrypsin, with its protease domain aligned with that of the
protease
domain of chymotrypsin and its residues numbered accordingly. Based on
chymotrypsin numbering, active site residues are Asp102, His57, and Ser195
(corresponding to Asp711, His656, and Ser805 in SEQ ID NO:253). The linear
amino acid sequence can be aligned with that of chymotrypsin and numbered
according to the 13 sheets of chymotrypsin. Insertions and deletions occur in
the loops
between the beta sheets, but throughout the structural family, the core sheets
are
conserved. The serine protease interacts with a substrate in a conserved beta
sheet
manner. Up to 6 conserved hydrogen bonds can occur between the substrate and
enzyme. All serine proteases of the chymotrypsin family have a conserved
region at
their N-terminus of the protease domain that is necessary for catalytic
activity (i.e.
IIGG, VVGG, or IVGG, where the first amino acid in this quartet is numbered
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according to the chymotrypsin numbering and given the designation Ile16. This
numbering does not reflect the length of the precursor sequence).
The substrate specificity of MT-SP1 in the protease domain has been mapped
using a positional scanning synthetic combinatorial library and substrate
phage
display (Takeuchi et al. (2000)J Biol Chem 275: 26333). Cleavage residues in
substrates recognized by MT-SP1 contain Arg/Lys at P4 and basic residues or
Gin at
P3, small residues at P2, Arg or Lys at P1, and Ala at P1'. Effective
substrates
contain Lys-Arg-Ser-Arg in the P4 to PI sites, respectively. Generally, the
substrate
specificity for MT-SP1 reveals a trend whereby if P3 is basic, then P4 tends
to be
non-basic; and if P4 is basic, then P3 tends to be non-basic. Known substrates
for
MT-SP1, including, for example, proteinase-activated receptor-2 (PAR-2),
single-
chain uPA (se-uPA), the proform of MT-SP1, and hepatocyte growth factor (HGF),
conform to the cleavage sequence for MT-SP1 specific substrates.
MT-SP1 can cleave selected synthetic substrates as efficiently as trypsin, but
exhibit a more restricted specificity for substrates than trypsin. The
catalytic domain
of MT-SP1 has the overall structural fold of a (chymo)trypsin-like serine
protease, but
displays unique properties such as a hydrophobic/acidic S2/S4 sub-sites and an
exposed 60 loop. Similarly, MT-SP1 does not indiscriminately cleave peptide
substrates at accessible Lys or Arg residues, but requires recognition of
additional
residues surrounding the scissile peptide bond. This requirement for an
extended
primary sequence highlights the specificity of MT-SP1 for its substrates. For
example, although MT-SP1 cleaves proteinase activated receptor-2 (PAR-2)
(displaying a P4 to P1 target sequence of Ser-Lys-Gly-Arg), the enzyme does
not
activate proteins closely related to this substrate such as PAR-1, PAR-3, and
PAR-4
that do not display target sequences matching the extended MT-SP1 specificity
near
the scissile bond (see Friedrich et al. (2002)J Biol Chem 277: 2160).
The protease domain of MT-SP1 is composed of a pro-region and a catalytic
domain. The catalytically active portion of the polypeptide begins after the
autoactivation site at amino acid residue 611 of the mature protein (see, e.g,
SEQ ID
NO: 253 at RQAR followed by the residues VVGG). The S1 pocket of MT-SP1 and
trypsin are similar with good complementarity for Lys as well as Arg P1
residues,
thereby accounting for some similarities in substrate cleavage with trypsin.
The
accommodation of the PI-Lys residues is mediated by Ser19 whose side chain
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provides an additional hydrogen bond acceptor to stabilize the buried a-
ammonium
group (see Friedrich et al. (2002) J Biol Chem 277: 2160). The S2 pocket is
shaped to
accommodate small to medium-sized hydrophobic side chains of P2 amino acids
and
generally accepts a broad range of amino acids at the P2 position. Upon
substrate
binding, the S2 sub-site is not rigid as evidenced by the rotation of the
Phe99 benzyl
group. The substrate amino acids at positions P3 (for either Gin or basic
residues) and
P4 (for Arg or Lys residues) appears to be mediated by electrostatic
interactions in the
S3. and S4 pockets with the acidic side chains of Asp-217 and/or Asp-96 which
could
favorably pre-orient specific basic peptide substrates as they approach the
enzyme
active site cleft. The side chain of a P3 residue also is able to hydrogen
bond the
carboxamide group of Gln192 or alternatively, the P3 side chain can extend
into the S4
sub-site to form a hydrogen bond with Phe97 thereby weakening the inter-main
chain
hydrogen bonds with Gly216. In either conformation, a basic P3 side chain is
able to
interact favorably with the negative potential of the MT-SP1 S4 pocket. The
mutual
charge compensation and exclusion from the same S4 site explains the low
probability
of the simultaneous occurrence of Arg/Lys residues at P3 and P4 in good MT-SP1
substrates. Generally, the amino acid positions of MT-SP I (based on
chymotrypsin
numbering) that contribute to extended specificity for substrate binding
include: 146
and 151 (S1'); 189, 190, 191, 192, 216, 226 (Si); 57, 58, 59, 60, 61, 62, 63,
64,99
(S2); 192, 217, 218, 146 (S3); 96, 97, 98, 99, 100, 168, 169, 170, 170A, 171,
172,
173, 174, 175, 176, 178, 179, 180, 215, 217, 224 (S4).
Cysteine Proteases
Cysteine proteases have a catalytic mechanism that involves a cysteine
sulfhydryl group. Deprotonation of the cysteine sulfhydryl by an adjacent
histidine
residue is followed by nucleophilic attack of the cysteine on the peptide
carbonyl
carbon. A thioester linking the new carboxy-terminus to the cysteine thiol is
an
intermediate of the reaction (comparable to the acyl-enzyme intermediate of a
serine
protease). Cysteine proteases include papain, cathepsin, caspases, and
calpains.
Papain-like cysteine proteases are a family of thiol dependent endo-peptidases
related by structural similarity to papain. They form a two-domain protein
with the
domains labeled R and L (for right and left) and loops from both domains form
a
substrate recognition cleft. They have a catalytic triad made up of the amino
acids
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Cys25, His159, and Asn175. Unlike serine proteases which recognize and
proteolyze
a target peptide based on a beta-sheet conformation of the substrate, this
family of
proteases does not have well-defined pockets for substrate recognition. The
main
substrate recognition occurs at the P2 amino acid (compared to the PI residue
in
serine proteases).
The substrate specificity of a number of cysteine proteases (human cathepsin
L, V, K, S, F, B, papain, and cruzain) has been determined using a complete
diverse
positional scanning synthetic combinatorial library (PS-SCL). The complete
library
contains P1, P2, P3, and P4 tetrapeptide substrates in which one position is
held fixed
while the other three positions are randomized with equal molar mixtures of
the 20
possible amino acids, giving a total diversity of --160,000 tetrapeptide
sequences.
Overall, PI specificity is almost identical between the cathepsins, with Arg
and Lys being strongly favored while small aliphatic amino acids are
tolerated. Much
of the selectivity is found in the P2 position, where the human cathepsins are
strictly
selective for hydrophobic amino acids. Interestingly, P2 specificity for
hydrophobic
residues is divided between aromatic amino acids such as Phe, Tyr, and Trp
(cathepsin L, V), and bulky aliphatic amino acids such as Val or Leu
(cathepsin K, S,
F). Compared to the P2 position, selectivity at the P3 position is
significantly less
stringent. Several of the proteases, however, have a distinct preference for
proline
(cathepsin V, S, and papain), leucine (cathepsin B), or arginine (cathepsin S,
cruzain).
The proteases show broad specificity at the P4 position, as no one amino acid
is
selected over others.
The S2 pocket is the most selective and best characterized of the protease
substrate recognition sites. It is defined by the amino acids at the following
spatial
positions (papain numbering): 66, 67, 68, 133, 157, 160, and 205. Position 205
plays
a role similar to position 189 in the serine proteases - a residue buried at
the bottom of
the pocket that determines the specificity. The other specificity determinants
include
the following amino acids (numbering according to papain): 61 and 66 (S3); 19,
20,
and 158 (S1). The structural determinant for various cysteine proteases are
listed in
Table 9. Typically, modification of a cysteine protease, such as for example a
papain
protease, to alter any one or more of the amino acids in the extended
specificity
binding pocket or other secondary sites of interaction affect the specificity
or
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selectivity of a protease for a target substrate including a complement
protein target
substrate.
Table 9: The structural determinants for various cysteine proteases
Residues that Determine Specificity
Active Site
S3 52 Si
Residues
25 159 175 61 66 66 133 157 160 205 19 20 158
Cathepsin Cys His Asn Glu Gly Gly Ala Met Gly Ala Gin Gly Asp
Cathepsin Cys His Ass Gin Gly Gly Ala Leu Gly Ala Gin Lys Asp
V
Cathepsin Cys His Asn Asp Gly Gly Ala Leu Ala Leu Gin Gly Asn
Cathepsin Cys His Asn Lys Gly Gly Gly Val Gly Phe Gin Gly Asn
Cathepsin Cys His Asn Lys Gly Gly Ala Ile Ala Met Gin Gly Asp
Cathepsin Cys His Asn Asp Gly Gly Ala Gly Ala Glu Gin Gly Gly
papain Cys His Asn Tyr Gly Gly Val Val Ala Ser Gin Gly Asp
Cruzain Cys His Asn Ser Gly Gly Ala Leu Gly Glu Gin Gly Asp
E. MODIFIED PROTEASES AND COLLECTIONS FOR SCREENING
Proteases or variants thereof can be used in the methods herein to identify
proteases with a desired substrate specificity, most often a substrate
specificity that is
altered, improved, or optimized. Modified proteases to be used in the method
provided herein can be generated by mutating any one or more amino acid
residues of
a protease using any method commonly known in the art (see also published U.S.
Appin. No. 2004/0146938). Proteases for modification and the methods provided
herein include, for example, full-length wild-type proteases, known variant
forms of
proteases, or fragments of proteases that are sufficient for catalytic
activity, e.g.
proteolysis of a substrate. Such modified proteases can be screened
individually
against a target protease trap, such as a serpin or modified serpin, or they
can be
screened as a collection, such as for example by using a display library,
including a
combinatorial library where display of the protease is by, for example, phage
display,
cell-surface display, bead display, ribosome display, or others. Selection of
a protease
that exhibits specificity and/or selectivity for a protease trap or modified
form thereof,
due to the formation of a stable covalent inhibitory complex, can be
facilitated by any
detection scheme known to one of skill in the art including, but not limited
to, affinity
labeling and/or purification, ELISA, chromogenic assays, fluorescence-based
assays
(e.g. fluorescence quenching or FRET), among others.
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- 1. Generation of Variant Proteases
Examples of methods to mutate protease sequences include methods that result
in random mutagenesis across the entire sequence or methods that result in
focused
mutagenesis of a select region or domain of the protease sequence. In one
example,
-5 the number of mutations made to the protease is 1,2, 3, 4; 5, 6, 7,
8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20. In a preferred embodiment, the mutation(s)
confer
increased substrate specificity. In some examples, the activity of the
protease variant
is increased by at least 10-fold, 100-fold, or 1000-fold over the activity of
the wild-
type protease. In related aspects, the increase in activity is in substrate
specificity.
a, Random Mutagenesis
Random mutagenesis methods include, for example, use of E.coli XLIred, UV
irradiation, chemical modification such as by deamination, alkylation, or base
analog
mutagens, or PCR methods such as DNA shuffling, cassette mutagenesis, site-
directed random mutagenesis, or error prone PCR (see e.g. U.S. Application
No.:
2006-0115874). Such examples include, but are not limited to, chemical
modification
by hydroxylamine (Ruan, H., etal. (1997) Gene 188:35-39), the use of dNTP
analogs
(Zaccolo, M., et at. (1996)1 Mol. Biol. 255:589-603), or the use
of...commercially
available random mutagenesis kits such as, for example, GeneMorph PCR-based
random mutagenesis kits (Stratagene) or Diversify random mutagenesis kits
(Clontech). The Diversify random mutagenesis kit allows the selection of a
desired
mutation rate for a given DNA sequence (from 2 to 8 mutations/1000 base pairs)
by
varying the amounts of manganese (Mn2+) and dGTP in the reaction mixture.
Raising manganese levels initially increases the mutation rate, with a further
mutation
rate increase provided by increased concentration of dGTP. Even higher rates
of
mutation can be achieved by performing additional rounds of PCR.
b. Focused Mutagenesis =
Focused mutation can be achieved by making one or more mutations in a pre-
determined region of a gene sequence, for example, in regions of the protease
domain
that mediate catalytic activity. In one example, any one or more amino acids
of a
protease are mutated using any standard single or multiple site-directed
mutagenesis
kit such as for example QuikChange (Stratagene). In another example, any one
or
more amino acids of a protease are mutated by saturation mutagenesis (Zheng et
al.
(2004) Nucl. Acids. Res., 32:115), such as for example, mutagenesis of active
site
= *Trademark
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residues. In this example, residues that form the Si-S4 pocket of aprotease
(where
the protease is in contact with the PI-P4 residues of the peptide substrate)
and/or that
have been shown to be important determinants of specificity are mutated to
every
possible amino acid, either alone or in combination. In some cases, there is
little (if
any) interaction between the Si -S4 pockets of the active site, such that each
pocket
appears to recognize and bind the corresponding residue on the peptide
substrate
sequence independent of the other pockets. Thus, the specificity determinants
generally can be changed in one pocket without affecting the specificity of
the other
pockets. In one exemplary embodiment, a saturation mutagenesis technique is
used in
which the residue(s) lining the pocket are mutated to each of the 20 possible
amino
acids (see for example, Kunkel TA (2001) "Oligonucleotide-directed mutagenesis
without
phenotypic selection", Curr Protocol Mot Blot, 8:8.1). In such a technique, a
degenerate mutagenic
oligonucleotide primer can be synthesized which contains randomization of
nucleotides at the desired codon(s) encoding the selected amino acid(s).
Exemplary
randomization schemes include NNS- or NNK-randomization, where N represents
any nucleotide, S represents guanine or cytosine and K represents guanine or
thymine.
The degenerate mutagenic primer is annealed to the single stranded DNA
template
and DNA polymerase is added to synthesize the complementary strand of the
template. After ligation, the double stranded DNA template is transformed into
E.coli
for amplification.
Amino acids that form the extended substrate binding pocket of exemplary
proteases are described herein. Generally, the substrate specificity of a
protease is
known such as for example by molecular modeling based on three-dimensional
structures of the complex of a protease and substrate (see for example, Wang
etal.,
(2001) Biochemistry 40(34):10038; Hopfner et al., Structure Fold Des. 1999
7(8):989; Friedrich etal., (2002)J Blot Chem 277(3):2160; Waugh et al, (2000)
Nat
Struct Biol. 7(9):762). For example, focused mutations of MT-SP I can be in
any one
or more residues (based on chymotrypsin numbering) that contribute to
substrate
specificity including 195, 102, 157 (the catalytic triad); 189, 190, 191, 192,
216 and
226 (Si); 57, 58, 59, 60, 61, 62, 63, 64,99 (S2); 146, 192, 217, 218 (S3); 96,
97, 98,
99, 100, 168, 169, 170, 170A, 171, 172, 173, 174, 175, 176, 178, 179, 180,
215, 217,
224 (S4). In another example, mutation of amino acid residues in a papain
family
protease can be in any one or more residues that affect P2 specificity
(standard papain
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numbering) including 66-68, 133, 157, 160, and/or 215. In addition, residues
that do
not directly contact the protease substrate, but do affect the position and/or
conformation of contact residues (such as for example those listed above) also
can be
mutated to alter the specificity of a protease scaffold.
In another example, focused amino acids for mutagenesis can be selected by
sequence comparison of homologous proteases with similar substrate
specificities.
Consensus amino acid residues can be identified by alignment of the amino
sequences
of the homologous proteins, for example, alignment of regions of the protease
that are
involved in substrate binding. Typically, proteases with similar substrate
specificities
share consensus amino acids, for example, amino acids in the substrate binding
pocket
can be identical or similar between the compared proteases. Additionally, the
amino
acid sequences of proteases with differing substrate specificities can be
compared to
identify amino acids that can be involved in substrate recognition. These
methods can
be combined with methods, such as three-dimensional modeling, to identify
target
residues for mutagenesis.
In an additional example, focused mutagenesis can be restricted to amino acids
that are identified as hot spots in the initial rounds of protease screening.
For
example, following selection of proteases from randomly mutagenized
combinatorial
libraries, several "hot spot" positions are typically observed and selected
over and
over again in the screening methods. Most often, since random mutagenesis
broadly
mutates a polypeptide sequence but with only a few mutations at each site,
focused
mutagenesis is used as a second strategy to specifically target hot spot
positions for
further mutagenesis. Focused mutagenesis of hot spot positions allows for a
more
diverse and deep mutagenesis at particular specified positions, as opposed to
the more
shallow mutagenesis that occurs following random mutagenesis of a polypeptide
sequence. For example, saturation mutagenesis can be used to mutate "hot
spots"
such as by using oligos containing NNt/g or NNt/c at these positions. In one
example,
using the methods provided herein, the following hot spots have been
identified in u-
PA as contributing to increased substrate specificity: 73, 80, 30, and 155,
based on
chymotrypsin numbering. Mutation of these positions can be achieved, such as
for
example, by using saturation mutagenesis of a wild-type or template protease
sequence at one or more of these sites to create collections of protease
mutants to be
used in subsequent screenings.
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2. Chimeric Forms of Variant Proteases
Variant proteases provided herein can include chimeric or fusion proteins. In
one example, a protease fusion protein comprises at least one catalytically-
active
portion of a protease protein. In another example, a protease fusion protein
comprises
at least two or more catalytically-active portions of a protease. Within the
fusion
protein, the non-protease polypeptide can be fused to the N-terminus or C-
terminus of
the protease polypeptide. In one embodiment, the fusion protein can include a
flexible peptide linker or spacer, that separates the protease from a non-
protease
polypeptide. In another embodiment, the fusion protein can include a tag or
detectable polypeptide. Exemplary tags and detectable proteins are known in
the art
and include for example, but are not limited to, a histidine tag, a
hemagglutinin tag, a
myc tag or a fluorescent protein. In yet another embodiment, the fusion
protein is a
GST-protease fusion protein in which the protease sequences are fused to the N-
terminus of the GST (glutathione S-transferase) sequences. Such fusion
proteins can
facilitate the purification of recombinant protease polypeptides. In another
embodiment, the fusion protein is a Fc fusion in which the protease sequences
are
fused to the N-terminus of the Fc domain from immunoglobulin G. Such fusion
proteins can have better pharmacodynamic properties in vivo. In another
embodiment, the fusion protein is a protease protein containing a heterologous
signal
sequence at its N-terminus. In certain host cells (e.g., mammalian host
cells),
expression and/or secretion of protease can be increased through use of a
heterologous
signal sequence.
A protease chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation,
restriction enzyme digestion to provide for appropriate termini, filling-in of
cohesive
ends as appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and
enzymatic ligation. In another embodiment, the fusion gene can be synthesized
by
conventional techniques including automated DNA synthesizers. Alternatively,
PCR
amplification of gene fragments can be carried out using anchor primers that
give rise
to complementary overhangs between two consecutive gene fragments that can
subsequently be annealed and reamplified to generate a chimeric gene sequence
(see,
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e.g., Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, 1992). Moreover, many expression vectors are commercially
available that already encode a fusion moiety (e.g., a GST polypeptide). A
protease-
encoding nucleic acid can be cloned into such an expression vector such that
the
fusion moiety is linked in-frame to the protease protein.
3. Combinatorial Libraries and Other Libraries
The source of compounds for the screening assays, can be collections such as
libraries, including, but not limited to, combinatorial libraries. Methods for
synthesizing combinatorial libraries and characteristics of such combinatorial
libraries
are known in the art (See generally, Combinatorial Libraries: Synthesis,
Screening
and Application Potential (Cortese Ed.) Walter de Gruyter, Inc., 1995; Tietze
and
Lieb, Cur r. Opin. Chem. Biol., 2(3):363-71 (1998); Lam, Anticancer Drug Des.,
I2(3):145-67 (1997); Blaney and Martin, Curr. Op/n. Chem. Biol., 1(1):54-9
(1997);
and Schultz and Schultz, Biotechnol. Prog., 12(6):729-43 (1996)).
Methods and strategies for generating diverse libraries, including protease or
enzyme libraries, including positional scanning synthetic combinatorial
libraries
(PSSCL), have been developed using molecular biology methods and/or
simultaneous
chemical synthesis methodologies (see, e.g. Georgiou, et al. (1997) Nat.
Biotechnol.
15:29-34; Kim et a). (2000) App! Environ Microbiol. 66: 788 793; MacBeath,
G.P. et
al. (1998) Science 279:1958-1961; Soumillion, P.L. etal. (1994) Appl. Biochem.
Biotechnol. 47:175-189, Wang, C. I. et (1996). Methods
Enzymol. 267:52-68, U.S.
Patents 6,867,010, 6,168,919, U.S. Patent Application No. 2006-0024289). The
resulting combinatorial libraries potentially contain millions of compounds
that can be
screened to identify compounds that exhibit a selected activity.
In one example, the components of the collection or library of proteases can
be
displayed on a genetic package, including, but not limited to any replicable
vector,
such as a phage, virus, or bacterium, that can display a polypeptide moiety.
The
plurality of displayed polypeptides is displayed by a genetic package in such
a way as
to allow the polypeptide, such as a protease or catalytically active portion
thereof, to
bind and/or interact with a target polypeptide. Exemplary genetic packages
include,
but are not limited to, bacteriophages (see, e.g., Clackson et 25 a/. (1991)
Making
Antibody Fragments Using Phage Display Libraries, Nature, 352:624-628; Glaser
et
al. (1992) Antibody Engineering by Condon-Based Mutagenesis in a Filamentous
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Phage Vector System,J. Immunol., 149:3903 3913; Hoogenboom et al. (1991) Multi-
Subunit Proteins on the Surface of Filamentous Phage: Methodologies for
Displaying
Antibody (Fate) Heavy and 30 Light Chains, Nucleic Acids Res., 19:4133-41370),
baculoviruses (see, e.g., Boublik et a/. (1995) Eukaryotic Virus Display:
Engineering
.. the Major Surface Glycoproteins of the Autographa California Nuclear
Polyhedrosis
Virus (ACNPV) for the Presentation of Foreign Proteins on the Virus Surface,
Bio/Technology, 13:1079-1084), bacteria and other suitable vectors for
displaying a
protein, such as a phage-displayed protease. For example bacteriophages of
interest
include, but are not limited to, T4 phage, M13 phage and HI phage. Genetic
packages
are optionally amplified such as in a bacterial host. Any of these genetic
packages as
well as any others known to those of skill in the art, are used in the methods
provided
herein to display a protease or catalytically active portion thereof.
a. Pbage Display Libraries
Libraries of variant proteases, or catalytically active portions thereof, for
screening can be expressed on the surfaces bacteriophages, such as, but not
limited to,
M13, fd, fl, T7, and k phages (see, e.g., Santini (1998)J. Mol. Biol. 282:125-
135;
Rosenberg et al. (1996) Innovations 6:1-6; Houshmand et al. (1999)Anal Biochem
268:363-370, Zanghi et al. (2005) Nuc. Acid Res. 33(18)e160:1-8). The variant
proteases can be fused to a bacteriophage coat protein with covalent, non-
covalent, or
non-peptide bonds. (See, e.g., U.S. Pat. No. 5,223,409, Crameri et al. (1993)
Gene
137:69 and WO 01/05950). Nucleic acids encoding the variant proteases can be
fused
to nucleic acids encoding the coat protein to produce a protease-coat protein
fusion
protein, where the variant protein is expressed on the surface of the
bacteriophage.
For example, nucleic acid encoding the variant protease can be fused to
nucleic acids
encoding the C-terminal domain of filamentous phase M13 Gene III (gulp; SEQ ID
NO:512). In some examples, a mutant protease exhibiting improved display on
the
phage is used as a template to generate mutant phage display libraries as
described
herein. For example, as described in Example 8, a mutant MT-SP1 having the
mutation of serine to cysteine at position corresponding to position 122 of
wild-type
MT-SP1, based on chymotrypsin numbering exhibits improved phage display.
Hence, such a mutant can be used as the template from which to generate
diversity in
= the library.
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Additionally, the fusion protein can include a flexible peptide linker or
spacer,
a tag or detectable polypeptide, a protease site, or additional amino acid
modifications
to improve the expression and/or utility of the fusion protein. For example,
addition
of a protease site can allow for efficient recovery of desired bacteriophages
following
a selection procedure. Exemplary tags and detectable proteins are known in the
art
and include for example, but not limited to, a histidine tag, a hemagglutinin
tag, a myc
tag or a fluorescent protein. In another example, the nucleic acid encoding
the
protease-coat protein fusion can be fused to a leader sequence in order to
improve the
expression of the polypeptide. Exemplary of leader sequences include, but are
not
limited to, STII or OmpA. Phage display is described, for example, in Ladner
etal.,
U.S. Pat. No. 5,223,409; Rodi et al. (2002) Curr. Opin. Chem, Biol. 6:92-96;
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 etal. (1998)
Immunotechnology 4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8;
.Fuchs etal. (1991) Bio/Technoloa 9:1370-1372; Hay etal. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths etal.
(1993) EMBO J12:725-734; Hawkins etal. (1992)J Mol Biol 45:889-896; Clackson
etal. (1991) Nature 352:624-628; Gram etal. (1992) PNAS 89:3576-3580; Garrard
et
al. (1991) Bio/Technology 9:1373-1377; Rebar etal. (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.
Nucleic acids suitable for phage display, e.g., phage vectors, are known in
the
art (see, e.g., Andris-Widhopf et al. (2000)J Immunol Methods, 28: 159-81;
= 25 Armstrong et al. (1996) Academic Press, Kay et al., Ed. pp.35-53;
Corey etal. (1993)
Gene 128(1):129-34; Cwirla et al. (1990) Proc Nat! Acad Sci USA 87(16):6378-
82;
Fowlkes etal. (1992) Biotechniques 13(3):422-8; Hoogenboom etal. (1991) Nuc
Acid
Res 19(15):4133-7; McCafferty etal. (1990) Nature 348(6301):552-4; McConnell
et
al. (1994) Gene 151(1-2):115-8; Scott and Smith (1990) Science 249(4967):386-
90).
A library of nucleic acids encoding the protease-coat protein fusion proteins,
typically protease variants generated as described above, can be incorporated
into the
genome of the bacteriophage, or alternatively inserted into in a phagemid
vector. In a
phagemid system, the nucleic acid encoding the display protein is provided on
a
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phagemid vector, typically of length less than 6000 nucleotides. The phagemid
vector
includes a phage origin of replication so that the plasmid is incorporated
into
bacteriophage particles when bacterial cells bearing the plasmid are infected
with
helper phage, e.g. M13K01 or M13VCS. Phagemids, however, lack a sufficient set
of
phage genes in order to produce stable phage particles after infection. These
phage
genes can be provided by a helper phage. Typically, the helper phage provides
an
intact copy of the gene III coat protein and other phage genes required for
phage
replication and assembly. Because the helper phage has a defective origin of
replication, the helper phage genome is not efficiently incorporated into
phase
particles relative to the plasmid that has a wild type origin. See, e.g., U.S.
Pat. No.
5,821,047. The phagemid genome contains a selectable marker gene, e.g.
AmpR
or KanR (for ampicillin or kanamycin resistance, respectively) for the
selection
of cells that are infected by a member of the library.
In another example of phage display, vectors can be used that carry nucleic
acids encoding a set of phage genes sufficient to produce an infectious phage
particle
when expressed, a phage packaging signal, and an autonomous replication
sequence.
For example, the vector can be a phage genome that has been modified to
include a
sequence encoding the display protein. Phage display vectors can further
include a
site into which a foreign nucleic acid sequence can be inserted, such as a
multiple
cloning site containing restriction enzyme digestion sites. Foreign nucleic
acid
sequences, e.g., that encode display proteins in phage vectors, can be linked
to a
ribosomal binding site, a signal sequence (e.g., a M13 signal sequence), and a
transcriptional terminator sequence.
Vectors can be constructed by standard cloning techniques to contain sequence
encoding a polypeptide that includes a protease and a portion of a phage coat
protein,
and which is operably linked to a regulatable promoter. In some examples, a
phage
display vector includes two nucleic acid sequences that encode the same region
of a
phage coat protein. For example, the vector includes one sequence that encodes
such
a region in a position operably linked to the sequence encoding the display
protein,
and another sequence which encodes such a region in the context of the
functional
phage gene (e.g., a wild-type phage gene) that encodes the coat protein.
Expression
of both the wild-type and fusion coat proteins can aid in the production of
mature
phage by lowering the amount of fusion protein made. per phage particle. Such
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methods are particularly useful in situations where the fusion protein is less
tolerated
by the phage.
Phage display systems typically utilize filamentous phage, such as M13, fd,
and fl. In some examples using filamentous phage, the display protein is fused
to a
phage coat protein anchor domain. The fusion protein can be co-expressed with
another polypeptide having the same anchor domain, e.g., a wild-type or
endogenous
copy of the coat protein. Phage coat proteins that can be used for protein
display
include (i) minor coat proteins of filamentous phage, such as gene III protein
(gulp),
and (ii) major coat proteins of filamentous phage such as gene VIII protein
(gVIIIp).
Fusions to other phage coat proteins such as gene VI protein, gene VII
protein, or
gene IX protein also can be used (see, e.g., WO 00/71694).
Portions (e.g., domains or fragments) of these proteins also can be used.
Useful portions include domains that are stably incorporated into the phage
particle,
e.g., so that the fusion protein remains in the particle throughout a
selection
procedure. In one example, the anchor domain of gIIIp is used (see, e.g., U.S.
Pat.
No. 5,658,727 and Examples below). In another example, gVIIIp is used (see,
e.g.,
U.S. Pat. No. 5,223,409), which can be a mature, full-length gVIIIp fused to
the
display protein. The filamentous phage display systems typically use protein
fusions
to attach the heterologous amino acid sequence to a phage coat protein or
anchor
domain. For example, the phage can include a gene that encodes a signal
sequence,
the heterologous amino acid sequence, and the anchor domain, e.g., a gIIIp
anchor
domain.
Valency' of the expressed fusion protein can be controlled by choice of phage
coat protein. For example, gIIIp proteins typically are incorporated into the
phage
coat at three to five copies per virion. Fusion of gIIIp to variant proteases
thus
produces a low-valency. In comparison, gVIII proteins typically are
incorporated into
the phage coat at 2700 copies per virion (Marvin (1998) Curr. Opin. Struct.
Biol.
8:150-158). Due to the high-valency of gVIIIp, peptides greater than ten
residues are
generally not well tolerated by the phage. Phagemid systems can be used to
increase
the tolerance of the phage to larger peptides, by providing wild-type copies
of the coat
proteins to decrease the valency of the fusion protein. Additionally, mutants
of
gVIIIp can be used which are optimized for expression of larger peptides. In
one such
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example, a mutant gVIIp was obtained in a mutagenesis screen for gVIIIp with
improved surface display properties (Sidhu et al. (2000)J. Mol. Biol. 296:487-
495).
Regulatable promoters also can be used to control the valency of the display
protein. Regulated expression can be used to produce phage that have a low
valency
of the display protein. Many regulatable (e.g., inducible and/or repressible)
promoter
sequences are known. Such sequences include regulatable promoters whose
activity
can be altered or regulated by the intervention of user, e.g., by manipulation
of an
environmental parameter, such as, for example, temperature or by addition of
stimulatory molecule or removal of a repressor molecule. For example, an
exogenous
chemical compound can be added to regulate transcription of some promoters.
Regulatable promoters can contain binding sites for one or more
transcriptional
activator or repressor protein. Synthetic promoters that include transcription
factor
binding sites can be constructed and also can be used as regulatable
promoters.
Exemplary regulatable promoters include promoters responsive to an
environmental
parameter, e.g., thermal changes, hormones, metals, metabolites, antibiotics,
or
chemical agents. Regulatable promoters appropriate for use in E. coli include
promoters which contain transcription factor binding sites from the lac, tac,
trp, trc,
and let operator sequences, or operons, the alkaline phosphatase promoter
(pho), an
arabinose promoter such as an araBAD promoter, the rhamnose promoter, the
promoters themselves, or functional fragments thereof (see, e.g., Elvin etal.
(1990)
Gene 37: 123-126; Tabor and Richardson, (1998) Proc. Natl. Acad. Sci. U.S.A.
1074-
1078; Chang et al. (1986) Gene 44: 121-125; Lutz and Bujard, (1997) Nucl.
Acids.
Res. 25: 1203-1210; D. V Goeddel etal. (1979) Proc. Nat. Acad. Sci. U.S.A.,
76:106-
110; J. D. Windass et at (1982) Nucl. Acids. Res., 10:6639757; R. Crowl et al.
(1985)
Gene, 38:31-38; Brosius (1984) Gene 27: 161-172; Amanna and Brosius, (1985)
Gene 40: 183-190; Guzman etal. (1992) J. Bacteriol., 174: 7716-7728; Haldimann
et
al. (1998) J. Bacteriol., 180: 1277-1286).
The lac promoter, for example, can be induced by lactose or structurally
related molecules such as isopropyl-beta-D-thiogalactoside (IPTG) and is
repressed
by glucose. Some inducible promoters are induced by a process of derepression,
e.g.,
inactivation of a repressor molecule.
A regulatable promoter sequence also can be indirectly regulated. Examples
of promoters that can be engineered for indirect regulation include: the phage
lambda
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PR, PL, phage T7, SP6, and 15 promoters. For example, the regulatory sequence
is
repressed or activated by a factor whose expression is regulated, e.g., by an
environmental parameter. One example of such a promoter is a 17 promoter. The
expression of the T7 RNA polymerase can be regulated by an environmentally-
responsive promoter such as the lac promoter. For example, the cell can
include a
heterologous nucleic acid that includes a sequence encoding the 17 RNA
polymerase
and a regulatory sequence (e.g., the lac promoter) that is regulated by an
environmental parameter. The activity of the Ti RNA polymerase also can be
regulated by the presence of a natural inhibitor of RNA polymerase, such as T7
lysozyme.
In another configuration, the lambda PL can be engineered to be regulated by
an environmental parameter. For example, the cell can include a nucleic acid
sequence that encodes a temperature sensitive variant of the lambda repressor.
Raising
cells to the non-permissive temperature releases the PL promoter from
repression.
The regulatory properties of a promoter or transcriptional regulatory sequence
can be easily tested by operably linking the promoter or sequence to a
sequence
encoding a reporter protein (or any detectable protein). This promoter-report
fusion
sequence is introduced into a bacterial cell, typically in a plasmid or
vector, and the
abundance of the reporter protein is evaluated under a variety of
environmental
.. conditions. A useful promoter or sequence is one that is selectively
activated or
repressed in certain conditions.
In some embodiments, non-regulatable promoters are used. For example, a
promoter can be selected that produces an appropriate amount of transcription
under
the relevant conditions. An example of a non-regulatable promoter is the gIII
promoter.
b. Cell Surface Display Libraries
Libraries of variant proteases for screening can be expressed on the surfaces
of
cells, for example, prokaryotic or eukaryotic cells. Exemplary cells for cell
surface
expression include, but are not limited to, bacteria, yeast, insect cells,
avian cells,
plant cells, and mammalian cells (Chen and Georgiou (2002) Biotechnol Bioeng
79:
496-503). In one example, the bacterial cells for expression are Escherichia
coll.
Variant proteases can be expressed as a fusion protein with a protein that is
expressed on the surface of the cell, such as a membrane protein or cell
surface-
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associated protein. For example, a variant protease can be expressed in E.
coli as a
fusion protein with an E. coli outer membrane protein (e.g. OmpA), a
genetically
engineered hybrid molecule of the major E. coli lipoprotein (Lpp) and the
outer
membrane protein OmpA or a cell surface-associated protein (e.g. pili and
flagellar
subunits). Generally, when bacterial outer membrane proteins are used for
display of
heterologous peptides or proteins, it is achieved through genetic insertion
into
permissive sites of the carrier proteins. Expression of a heterologous peptide
or
protein is dependent on the structural properties of the inserted protein
domain, since
the peptide or protein is more constrained when inserted into a permissive
site as
compared to fusion at the N- or C-terminus of a protein. Modifications to the
fusion
protein can be done to improve the expression of the fusion protein, such as
the
insertion of flexible peptide linker or spacer sequences or modification of
the bacterial
protein (e.g by mutation, insertion, or deletion, in the amino acid sequence).
Enzymes, such as 13-lacatamase and the Cex exoglucanase of Cellulomonas fimi,
have
been successfully expressed as Lpp-OmpA fusion proteins on the surface of E.
coli
(Francisco J.A. and Georgiou G. Ann N Y Acad Sc!. 745:372-382 (1994) and
Georgiou G. etal. Protein Eng, 9:239-247 (1996)). Other peptides of 15-514
amino
acids have been displayed in the second, third, and fourth outer loops on the
surface
of OmpA (Samuelson et al. J. Biotechnol, 96: 129-154 (2002)). Thus, outer
membrane proteins can carry and display heterologous gene products on the
outer
surface of bacteria.
In another example, variant proteases can be fused to autotransporter domains
of proteins such as the N. gonorrhoeae IgAl protease, Serratia marcescens
serine
protease, the Shigella flexneri VirG protein, and the E. coli adhesin AIDA-I
(Klauser
et al. EMBO 1. 1991-1999 (1990); Shikata S, et al. J Biochem.114:723-731
(1993);
Suzuki T et at J Biol Chem. 270:30874-30880 (1995); and Maurer J et al. J
Bacteriol. 179:794-804 (1997)). Other autotransporter proteins include those
present
in gram-negative species (e.g. E. coli, Salmonella serovar Typhimurium, and S.
flexneri). Enzymes, such as 13-lactamase, have been successful expressed on
the
surface of E. coli using this system (Lattemann CT et al. J Bacterial.
182(13): 3726-
3733 (2000)).
Bacteria can be recombinantly engineered to express a fusion protein, such a
membrane fusion protein. Nucleic acids encoding the variant proteases can be
fused
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=
to nucleic acids encoding a cell surface protein, such as, but not limited to,
a bacterial
OmpA protein. The nucleic acids encoding the variant proteases can be inserted
into
a permissible site in the membrane protein, such as an extracellular loop of
the
membrane protein. Additionally, a nucleic acid encoding the fusion protein can
be
fused to a nucleic acid encoding a tag or detectable protein. Such tags and
detectable
proteins are known in the art and include for example, but are not limited to,
a
histidine tag, a hemagglutinin tag, a myc tag or a fluorescent protein. The
nucleic
acids encoding the fusion proteins can be operably linked to a promoter for
expression
in the bacteria. For example a nucleic acid can be inserted in a vector or
plasmid,
which can carry a promoter for expression of the fusion protein and
optionally,
additional genes for selection, such as for antibiotic resistance. The
bacteria can be
transformed with such plasmids, such as by electroporation or chemical
transformation. Such techniques are known to one of ordinary skill in the art.
Proteins in the outer membrane or periplasmic space are usually synthesized in
the cytoplasm as premature proteins, which are cleaved at a signal sequence to
produce the mature protein that is exported outside the cytoplasm. Exemplary
signal
sequences used for secretory production of recombinant proteins for E. coli
are
known. The N-terminal amino acid sequence, without the Met extension, can be
obtained after cleavage by the signal peptidase when a gene of interest is
correctly
fused to a signal sequence. Thus, a mature protein can be produced without
changing
the amino acid sequence of the protein of interest (Choi and Lee. App!.
Microbiol.
Biotechnol. 64: 625-635 (2004)).
Other cell surface display systems are known in the art and include, but are
not
limited to ice nucleation protein (Inp)-based bacterial surface display system
(Lebeault J M (1998) Nat Biotechnol. 16: 576 80), yeast display (e.g. fusions
with the
yeast Aga2p cell wall protein; see U.S. Pat. No. 6,423,538), insect cell
display (e.g.
baculovirus display; see Ernst et al. (1998) Nucleic Acids Research, Vol 26,
Issue 7
1718-1723), mammalian cell display, and other eukaryotic display systems (see
e.g.
5,789,208 and WO 03/029456).
. 30 c. Other Display Libraries
It also is possible to use other display formats to screen libraries of
variant
proteases, e.g., libraries whose variation is designed as described herein.
Exemplary
other display formats include nucleic acid-protein fusions, ribozyme display
(see e.g.
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Hanes and Pluckthun (1997) Proc. Natl. Acad. Sci. U.S.A. 13:4937-4942), bead
display (Lam, K. S. et al. Nature (1991) 354, 82-84; , K. S. etal. (1991)
Nature, 354,
82-84; Houghten, R. A. et al. (1991)Nature, 354, 84-86; Furka, A. et al.
(1991) Int.
J. Peptide Protein Res. 37, 487-493; Lam, K. S., et al. (1997) Chem. Rev., 97,
411-
448; U.S. Published Patent Application 2004-0235054) and protein arrays (see
e.g.
Cahill (2001) J. Immunol. Meth. 250:81-91, WO 01/40803, WO 99/51773, and
US2002-0192673-A1)
In specific other cases, it can be advantageous to instead attach the
proteases,
variant proteases, or catalytically active portions or phage libraries or
cells expressing
variant proteases to a solid support. For example, in some examples, cells
expressing
variant proteases can be naturally adsorbed to a bead, such that a population
of beads
contains a single cell per bead (Freeman et al. Biotechnol. Bioeng. (2004)
86:196-
200). Following immobilization to a glass support, microcolonies can be grown
and
screened with a chromogenic or fluorogenic substrate. In another example,
variant
proteases or phage libraries or cells expressing variant proteases can be
arrayed into
titer plates and immobilized.
F. METHODS OF CONTACTING, ISOLATING, AND IDENTIFYING
SELECTED PROTEASES
After a plurality of collections or libraries displaying proteases or
catalytically
active portions thereof have been chosen and prepared, the libraries are used
to
contact a target protease trap polypeptide with the protease components. The
target
substrates, including, for example, a protease trap polypeptide such as a
serpin
mutated in its RSL loop to have a desired cleavage sequence, are contacted
with the
displayed protease libraries for selection of a protease with altered
substrate
specificity. The protease and protease trap polypeptide can be contacted in
suspension, solution, or via a solid support. The components are contacted for
a
sufficient time, temperature, or concentration for interaction to occur and
for the
subsequent cleavage reaction and formation of a stable intermediate complex of
the
selected protease and protease trap polypeptide. The stringency by which the
reaction
is maintained can be modulated by changing one or more parameters from among
the
temperature of the reaction, concentration of the protease trap polypeptide
inhibitor,
concentration of a competitor (if included), concentration of the collection
of
proteases in the mixture, and length of time of the incubation.
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The selected proteases that form covalent complexes with the protease trap
polypeptide are captured and isolated. To facilitate capture, protease trap
polypeptides for screening against can be provided in solution, in suspension,
or
attached to a solid support, as appropriate for the assay method. For example,
the
protease trap polypeptide can be attached to a solid support, such as for
example, one
or more beads or particles, microspheres, a surface of a tube or plate, a
filter
membrane, and other solid supports known in the art. Exemplary solid support
systems include, but are not limited to, a flat surface constructed, for
example, of
glass, silicon, metal, nylon, cellulose, plastic or a composite, including
multiwell
plates or membranes; or can be in the form of a bead such as a silica gel, a
controlled
= pore glass, a magnetic (Dynabead) or cellulose bead. Such methods can be
adapted
for use in suspension or in the form of a column. Target protease trap
polypeptides
can be attached directly or indirectly to a solid support, such as a
polyacrylamide
bead. Covalent or non-covalent methods can be used for attachment. Covalent
methods of attachment of target compounds include chemical crosslinking
methods.
Reactive reagents can create covalent bonds between functional groups on the
target
molecule and the support. Examples of functional groups that can be chemically
reacted are amino, thiol, and carboxyl groups. N-ethylmaleimide,
iodoacetamide, N-
hydrosuccinimide, and glutaraldehyde are examples of reagents that react with
functional groups. In other examples, target substrates can be indirectly
attached to a
solid support by methods such as, but not limited to, immunoaffinity or ligand-
receptor interactions (e.g. biotin-streptavidin or glutathione S-transferase-
glutathione).
For example, a protease-trap polypeptide can be coated to an ELISA plate, or
other
similar addressable array. In one example, the wells of the plate can be
coated with
an affinity capture agent, which binds to and captures the protease-trap
polypeptide.
Example 9 exemplifies a method whereby biotinylated anti-His antibody is
coated
onto a streptavidin containing plate to facilitate capture of a protease-trap
polypeptide
containing a His-tag,
Attachment of the protease trap polypeptide to a solid support can be
performed either before, during, or subsequent to their contact with variant
proteases
or phage libraries or cells expressing variant proteases. For example, target
substrates
can be pre-absorbed to a solid support, such as a chromatography column, prior
to
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incubation with the variant protease. In other examples, the attachment of a
solid
support is performed after the target substrate is bound to the variant
protease.
In such an example, the solid support containing the complexed substrate-
protease pair can be washed to remove any unbound protease. The complex can be
recovered from the solid support by any Method known to one of skill in the
art, such
as for example, by treatment with dilute acid, followed by neutralization (Fu
et al.
(1997)s / Biol. Chem. 272:25678-25684) or with triethylamine (Chiswell et al.
(1992)
Trends Biotechnol. 10:80-84). This step can be optimized to ensure
reproducible and
quantitative recovery of the display source from the solid substrate. For
example, the
binding of the display source to the target substrate attached to the solid
support can
be monitored independently using methods well known to those of skill in the
art,
such as by using an antibody directed against the phage, such as against MI3
phage
(e.g., New England Biolabs, MA) and a standard ELISA (see e.g., Ausubel etal.
(1987) Current Protocols in Molecular Biology, John Wiley & Sons, New York).
Another method of capturing and isolating a substrate-protease complex is
from solution. Typically, in such a method, a protease trap polypeptide or
variant
thereof is contacted with a collection of proteases such as, for example, in a
small
volume of an appropriate binding buffer (i.e. 20, 30, 40, 50, 60, 70, 80, 90,
100, 200,
300, 400, 500 or more microliters) where each protease trap polypeptide is
associated
with a predetermined marker, tag, or other detectable moiety for
identification and
isolation thereof. The detectable moiety can be any moiety that facilitates
the
detection and isolation of substrate-protease complex. For example, the moiety
can
be an epitope tag for which an antibody specific for the tag exists (i.e. myc-
tag, His-
tag, or others). The antibody can be bound to a solid support, such as a bead,
to
facilitate capture of the stable complex. Other similar strategies can be used
and
include, for example, labeling of the target substrate with biotin and capture
using
streptavidin attached to a solid support such as magnetic beads or a
microtiter plate or
labeling with polyhistidinc (e.g., His 6-tag) and capture using a metal
chelating agent
such as, but not limited to, nickel sulphate (NiSO4), cobalt chloride (CoCl2),
copper
sulphate (CuSO4), or zinc chloride (ZnC12). The capturing agents can be
coupled to
large beads, such as for example, sepharose beads, whereby isolation of the
bound
beads can be easily achieved by centrifugation. Alternatively, capturing
agents can be
coupled to smaller beads, such as for example, magnetic beads (i.e. Miltenyi
Biotec),
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that can be easily isolated using a magnetic column. In addition, the moiety
can be a
fluorescent moiety. For example, in some display systems, such as for example,
cell
surface display systems, a fluorescent label can facilitate isolation of the
selected
complex by fluorescence activated cell sorting (FACS; see e.g., Levin et al.
(2006)
Molecular BioSystems, 2: 49-57).
In some instances, one or more distinct protease trap polypeptides are
contacted with a collection of proteases, where each of the protease trap
polypeptides
are associated with different detection moieties so as to individually isolate
one or
more than one protease trap polypeptide-protease complex. The ability to
include in a
single reaction 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct protease trap
polypeptides,
each with a different desired RSL cleavage sequence, permits the detection and
isolation of tens of hundreds or thousands of covalent complexes
simultaneously.
The selected proteases, captured as covalent complexes with the protease trap
polypeptide, can be separated from uncomplexed proteases from the collection
of
proteases. The selected proteasescan then be amplified to facilitate
identification of
the selected protease. After removal of any uncomplexed proteases to the
protease
trap polypeptide, the source of material to which the protease is displayed
(i.e. phage,
cells, beads, etc...) is amplified and expressed in an appropriate host cell.
For
example, where the protease is displayed on phage, generally, the protease-
phage in
complex with a protease trap polypeptide is incubated with a host cell to
allow phage
adsorption, followed by addition of a small volume of nutrient broth and
agitation of
the culture to facilitate phage probe DNA replication in the multiplying host.
In some
examples, this is done in the presence of helper phage in order to ensure that
the host
cells are infected by the phage. After this incubation, the media is
supplemented with
an antibiotic and/or an inducer. The phage protease genome also can contain a
gene
encoding resistance to the antibiotic to allow for selective growth of those
bacterial
cells that maintain the phage protease DNA. Typically, for amplification of
phage as
a source of phage supernatant containing selected proteases, rescue of the
phage is
required by the use of helper phage. In some examples, it is possible to assay
for the
presence of a selected protease without a rescue step. For example, following
incubation of the captured complex containing the selected or identified
protease with
a host cell, for example, bacteria, and growth in the presence of a selective
agent, the
periplasm or cell culture medium can be directly sampled as a source of the
selected
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protease, for example, to measure protease activity. Such a procedure is
described in
Example 17.
Additionally, the amplification of the display source, such as in a bacterial
host, can be optimized in a variety of ways. For example, the amount of
bacteria
added to the assay material, such as in microwells, can be in vast excess of
the phage
source recovered from the binding step thereby ensuring quantitative
transduction of
the phage genome. The efficiency of transduction optionally can be measured
when
phage are selected. The amplification step amplifies the genome of the display
source, such as phage genomes, allowing over-expression of the associated
signature
polypeptide and identification thereof, such as by DNA sequencing.
A panning approach can be used whereby proteases or catalytically active
portions thereof that interact with a target protein, such as a protease trap
polypeptide
or RSL variant thereof, are quickly selected. Panning is carried out, for
example, by
incubating a library of phage-displayed polypeptides, such as phage-displayed
proteases, with a surface-bound or soluble target protein, washing away the
unbound
phage, and eluting the specifically and covalently-bound phage. The eluted
phage is
then amplified, such as via infection of a host, and taken through additional
cycles of
panning and amplification to successively enrich the pool of phage for those
with the
highest affinities for the target polypeptide. After several rounds,
individual clones
are identified, such as by DNA sequencing, and their activity can be measured,
such
as by any method set forth in Section G below.
Once the selected protease is identified, it can be purified from the display
source and tested for activity. Generally, such methods include general
biochemical
and recombinant DNA techniques and are routine to those of skill in the art.
In one
method, polyethylene glycol (PEG) precipitation can be used to remove
potentially
contaminating protease activity in the purified selected phage supernatants.
In such
an example, following phage rescue in the presence of helper phage, phage
supernatant containing the selected protease can be precipated in the presence
of PEG.
One of skill in the art is able to determine the percentage of PEG required
for the
particular precipitation application. Generally, for precipitation of protease
supernatants, 20% PEG is used.
In some examples, the supernatant, either from the rescued phage supernatant,
or from the bacterial cell periplasm or cell medium (without phage rescue) can
be
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assayed for protease activity as described herein. Alternatively or
additionally, the
selected protease can be purified from the supernatant or other source. For
example,
DNA encoding the selected protease domain can be isolated from the display
source
to enable purification of the selected protein. For example, following
infection of
E.coli host cells with selected phage as set forth above, the individual
clones can be
picked and grown up for plasmid purification using any method known to one of
skill
in the art, and if necessary can be prepared in large quantities, such as for
example,
using the Midi Plasmid Purification Kit (Qiagen). The purified plasmid can
used for
DNA sequencing to identify the sequence of the variant protease, or can be
used to
transfect into any cell for expression, such as but not limited to, a
mammalian
expression system. If necessary, one or two-step PCR can be performed to
amplify
the selected sequence, which can be subcloned into an expression vector of
choice.
The PCR primers can be designed to facilitate subcloning, such as by including
the
addition of restriction enzyme sites. Example 4.exemplifies a two step PCR
procedure to accomplish amplification and purification of the full-length u-PA
gene,
where the selected protease phage contained only the protease domain of the u-
PA
gene. Following transfection into the appropriate cells for expression such as
is
described in detail below, conditioned medium containing the protease
polypeptide, or
catalytically active portion thereof can be tested in activity assays or can
be used for
further purification. In addition, if necessary, the protease can be processed
accordingly to yield an active protease, such as by cleavage of a single chain
form,
into a two chain form. Such manipulations are known to one of skill in the
art. For
example, single chain u-PA can be made active the cleavage of plasmin such as
is
described herein.
1. Iterative Screening
In the methods provided herein, iterative screening is employed to optimize
the modification of the proteases. thus, in methods of iterative screening, a
protease
can be evolved by performing the panning reactions a plurality of times under
various
parameters, Such as for example, by using different protease trap polypeptides
or
competitors. In such methods of iterative screening, the protease collection
can be
kept constant in successive rounds of screening. Alternatively, a new protease
collection can be generated containing only the selected proteases identified
in the
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preceding rounds and/or by creating a new collection of mutant proteases that
have
been further mutated as compared to a template protease identified in the
first round.
In one example, a first round screening of the protease library can identify
variant proteases containing one or more mutations which alter the specificity
of the
protease. A second round library synthesis can then be performed in which the
amino
acid positions of the one or mutations are held constant, and focused or
random
mutagenesis is carried out on the remainder of the protein or desired region
or residue.
After an additional round of screening, the selected protease can be subjected
to
additional rounds of library synthesis and screening. For example, 2, 3, 4, 5,
or more
rounds of library synthesis and screening can be performed. In some examples,
the
specificity of the variant protease toward the altered substrate is further
optimized
with each round of selection.
In another method of iterative screening, a first round screening of a
protease
collection can be against an intermediate protease trap polypeptide to
identify variant
proteases containing one or more mutations which alter the specificity of the
protease
to the intermediate substrate. The selected protease complexes can be
isolated, grown
up, and amplified in the appropriate host cells and used as the protease
collection in a
second round of screening against a protease trap polypeptide containing the
complete
cleavage sequence of a target polypeptide. For example, such an approach can
be
used to select for proteases having substrate specificity for a VEGFR cleavage
sequence where the one or more rounds of panning are against a RRARM
intermediate cleavage sequence, and subsequent rounds of panning are performed
against a protease trap polypeptide containing the VEGFR2 cleavage sequence
RRVR.
In an additional example of iterative screening, two or more protease trap
polypeptides containing different substrate recognition or cleavage sequences
for two
or more different polypeptides are used in the methods in alternative rounds
of
panning. Such a method is useful to select for proteases that are optimized to
have
selectivity for two different substrates. The selected variants typically have
narrow
specificity, but high activity towards two or more substrate recognition
sequences. In
such methods, a first round screening of a protease collection against a first
protease
trap polypeptide, that has been modified to select for a protease with a first
predetermined substrate specificity, can identify variant proteases containing
one or
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more mutations which alter the specificity of the protease. The selected
proteases can
be isolated, grown up, and amplified in the appropriate host cells and used as
the
protease collection in a second round of screening against a second protease
trap
polypeptide that has been modified to select for a protease with a second
predetermined substrate specificity. The first and second protease trap
polypeptide
used in the methods can be the same or different, but each is differently
modified in
its reactive site to mimic a substrate recognition site (i.e. cleavage
sequence) of
different target substrates. In some examples, the stringency in the selection
can be
enhanced in the presence of competitors, such as for example, narrow or broad
competitors as described herein.
2. Exemplary Selected Proteases
Provided herein are variant u-PA and MT-SP1 polypeptides identified in the
methods provided herein as having an altered and/or improved substrate
specificity.
Such variant u-PA and MT-SP1 polypeptides were identified as having an
increased
specificity for a selected or desired cleavage sequence of a target protein.
Exemplary
of such target proteins include, but are not limited to, a cleavage sequence
in a
VEGFR or a complement protein, for example, complement protein C2. Any
modified serpin can be used in the selection methods herein to identify
variant
proteases. Exemplary of such modified serpins are PAI-1 or AT3 modified in
their
RSL to contain cleavage sequences for a target protein, for example, a VEGFR
or C2,
as described herein above. The resulting selected modified proteases exhibit
altered,
typically improved, substrate specificity for the cleavage sequence in the
target
protein as compared to the template or starting protease, which does not
contain the
selected modifications. As described below, specificity is typically increased
and is
generally at least 2-fold, at least 4-fold, at least 5-fold, at least 6-fold,
at least 7-fold,
8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,
800, 900,
1000 times or more when compared to the specificity of a wild-type or template
protease for the target substrate selected against versus a non-target
substrate.
a. Variant u-PA polypeptides
For example, variant u-PA polypeptides provided herein were selected for to
have an increased reactivity for a mutant serpin polypeptide modified in its
RSL
sequence by replacement of the native P4-P1' reactive site amino acids with
those of a
desired or selected target protein. In one example, variant u-PA polypeptides
were
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identified against selection of a modified PAI-1 polypeptide. Examples of
modified
PAI-1 polypeptide molecules used in the u-PA selection methods provided herein
include, for example, PAI-1 modified in its native P4-P1' residues VSARM (SEQ
ID
NO:378) with amino acid residues for an intermediate VEGFR-2 cleavage sequence
RRARM (SEQ ID NO:379), where the desired cleavage sequence in the P4-P1
positions is the VEGFR-2 cleavage sequence RRVR (SEQ ID NO:489), or with
amino acid residues for the optimal t-PA cleavage sequence PFGRS (SEQ ID
NO:389).
Using the methods provided herein, the following positions were identified as
contributing to substrate specificity of a u-PA polypeptide: 21, 24, 30, 38,
39, 61(A),
72, 73, 75, 80, 82, 84, 89, 92, 132, 133, 137, 138, 155, 156, 158, 159, 160,
187, and
217, based on chymotrypsin numbering. Amino acid replacement or replacements
can be at any one or more positions corresponding to any of the following
positions
F21, 124, F30, V38, T39, Y61(A), R72, L73, S75, E80, K82, E84, 189, K92, F132,
.. G133, E137, 1138, L155, K156, T158, V159, V160, K187, and R217 of a u-PA
polypeptide, such as a u-PA polypeptide set forth in SEQ ID NO:433 or
catalytically
active portion thereof, based on chymotrypsin numbering. A modified u-PA
polypeptide provided herein that exhibits increased substrate specificity can
contain
one or more amino acid modifications corresponding to any one or more
modification
of F21V, I24L, F30I, F30V, F3OL, F30T, F30G, F30M, V38D, 139A, Y61(A)H,
R720, L73A, L73P, S75P, E80G, K82E, E84K, I89V, K92E, F132L, G133D, E137G,
I1381, L155P, L155V, L155M, K156Y, 1158A, V159A, V160A, K187E, and R217C
of a u-PA polypeptide, such as a u-PA polypeptide set forth in SEQ ID NO:433
or
catalytically active portion thereof, based on chymotrypsin numbering.
In one example, a modified u-PA polypeptide provided herein having
increased substrate specificity for a VEGFR-2 cleavage sequence contains one
or
more amino acid modifications corresponding to any one or more modifications
of
V38D, F30I, F30T, F3OL, F30V, F30G, F30M, R72G, L73A, L73P, S75P, 189V,
F132L, G133D, E137G, I138T, L155P, L155V, L155M, V160A, and R217C, based
on chymotrypsin numbering. Exemplary of such polypeptides are those u-PA
polypeptides containing one or more amino acid modifications corresponding to
any
of F30I; L73A/I89V; L73P; R217C; L155P; S75P/189V/I138T; E1370;
R72G/L155P; Gl 33D; VI 60A; V38D; F132LN160A; L73A/189V/F301;
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L73A/189V/F3OL; L73A/189V/F30V; L73A/189V/F30G; L73A/I89V/L155V;
L73A/189V/F30M; L73A/I89V/L155M; L73A/189V/F3OL/L155M; and
L73A/189V/F30G/L155M in a u-PA polypeptide, such as a u-PA polypeptide having
an amino acid sequence set forth in SEQ ID NO:433 or a catalytically active
fragment
thereof. Exemplary of such sequences are those set forth in any of SEQ ID NOS:
434-459, or fragments thereof of contiguous amino acids containing the
mutation and
having catalytic activity. In particular, modified u-PA polypeptides having
the
following amino acid modifications are provided: L73A/I89V; L155P; R720/L155P;
F132LN160A; L73A/189V/F30T; L73A/I89V/L155V; L73A/I89V/L155M; and
L73A/189V/F30L/L155M, based on chymotrypsin numbering.
In another example, a modified u-PA polypeptide provided herein having
increased specificity for a cleavage sequence recognized by t-PA contains one
or
more amino acid modifications corresponding to any one or more modifications
of
F21V, I24L, F30V, F3OL, T39A, Y61(A)H, E80G, K82E, E84K, I89V, K92E,
K1561, T158A, V159A, and K187E, based on chymotrypsin numbering. Exemplary
of such polypeptides are those u-PA polypeptides containing one or more amino
acid
modifications corresponding to any of F21V; I24L; F30V; F3OL; F30V/Y61(A)H;
F30V/K82E; F30V/K156T; F30V/K82EN159A; F30V/K82E/T39AN159A;
F30V/K82E/T158AN159A; F30V/Y61(A)H/K92E;
F30V/K82EN159A/E80G/189V/K187E; and
F30V/K82E/V159A/E80G/E84K/189V/K187E, in a u-PA polypeptide, such as au-PA
polypeptide having an amino acid sequence set forth in SEQ ID NO:433 or a
catalytically active fragment thereof. Exemplary of such sequences are those
set forth
in any of SEQ ID NOS:460-472, or fragments thereof of contiguous amino acids
=
containing the mutation and having catalytic activity.
Also provided herein are variant proteases of the chymotrypsin family having
the corresponding mutation as compared to the variant u-PA polypeptides
provided
herein, based on chymotrypsin numbering. For example, based on chymotrypsin
numbering, modification of position F30 in u-PA corresponds to modification of
position Q30 in t-PA, Q30 in trypsin, and Q30 in chymotrypsin (Bode etal.
(1997)
Current Opinion in Structural Biology, 7: 865-872). One of skill in the art
could
determine corresponding mutations in any other chymotrypsin family member,
including but not limited to modification of any protease set forth in Table 7
and
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having a sequence of amino acids set forth in any of SEQ ID NOS: 40, 42, 44,
46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94,
96, 98, 99, 101, 103, 105, 107; 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 129,
131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,
161, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 193, 195,
197, 199,
201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,
231, 233,
235, 237, 239, 241, 242, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,
262, 264,
266, 268, 270, 272, or catalytically active fragments thereof.
b. Variant MT-SP1 polypeptides
Irr another example, variant MT-SP1 polypeptides provided herein were
selected for to have an increased reactivity for a mutant serpin polypeptide
modified
in its RSL sequence by replacement of the native P4-P2' reactive site amino
acids
with those of a desired or selected target protein. In one example, variant MT-
SP1
polypeptides were identified against selection of a modified AT3 polypeptide.
Examples of modified AT3 polypeptide molecules used in the MT-SP1 selection
methods provided herein include, for example, AT3 modified in its native P4-
P2'
residues IAGRSL (SEQ ED NO:478) with amino acid residues for a complement
protein C2 cleavage sequence SLGRKI (SEQ ID NO :479).
Using the methods provided herein, the following positions were identified as
contributing to substrate specificity of an MT-SP1 polypeptide: 23, 41,
52,60(g), 65, .
71, 93, 95, 97, 98, 99, 126, 129, 131, 136, 143, 144, 154, 164, 166, 171, 173,
175, "
184(a), 192, 201, 209, 217, 221(a), 230, 234, and 244 ,based on chymotrypsin
numbering. Amino acid replacement or replacements can be at any one or more
positions corresponding to any of the following positions D23,141, L52,
Y60(g), T65,
H71, F93, N95, F97, 198, F99, A126, V129, P131, 1136, H143, 1144, 1154, N164,
T166, L171, P173, Q175, F184(a), Q192, S201, Q2op, D217, Q221(a), R230, F234,
and V244 of an MT-SP1 polypeptide, such as full-length MT-SP1 polypeptide set
forth in SEQ ID NO:253 or 515 or catalytically active portion thereof set
forth in SEQ
ID NO:505 or 507, based on chymotrypsin numbering. A modified MT-SP1
polypeptide provided herein that exhibits increased substrate specificity can
contain =
one or more amino acid modifications corresponding to any one or more
modification
of D23E, I41F,I4IT, L52M, Y60(g)s, T65K, H71R, F93L, N95K, F97Y, F97L,
198P, F99L, A1261, V129D, P131S, I1361, I136V, H143R, T1441, I154V, N164D,
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T166A, L171F, P173S, Q175R, F184(a)L, Q192H, S2011, Q209L, D217V, Q221(a)L,
R230W, F234L, and V244G of an MT-SP1 polypeptide, such as full-length MT-SP1
polypeptide set forth in SEQ ID NO:253 or 515 or catalytically active portion
thereof
set forth in SEQ ID NO:505 or 507, based on chymotrypsin numbering. In
particular,
.. a modified MT-SP1 polypeptide contains one or more amino acid modifications
corresponding to any one or more modification of141F, F97Y, L171F, Q175R,
D2I7V and V244G, for example, any one or more of I41F, F97Y, L171F and V244G.
Typically, such a modified MT-SP1 polypeptide exhibits increased substrate
specificity for complement protein C2. Exemplary of such polypeptides are
those
MT-SP1 polypeptides containing one or more amino acid modifications
corresponding to any of 1136T/N164D/T166A/F184(A)L/D217V; I41F; I41F/
A126T1V244G; D23E/I41F/T98P /T1441; I41F/ L171F/V244G; 11143R/Q175R; I41F/
L17IF; R230W; I41F /I154V/V244G; I41F/L52M/ VI29D/Q221(A)L; F99L; F97Y/
1136V/Q192H/S2011; H71R/ P131S/D217V; D217V; T65K/F93L/F97Y/ D217V;
I41T/ P173S/Q209L; F97L/ F234L; Q175R; N95K; and Y60(G)S in an MT-SP1
polypeptide, such as an MT-SP1 polypeptide having an amino acid sequence set
forth
in SEQ ID NO:253 or a catalytically active fragment thereof set forth in SEQ
ID
NO:505. Exemplary of such sequences are those set forth in any of SEQ ID NOS:
589-609, or fragments thereof of contiguous amino acids containing the
mutation and
having catalytic activity such as, for example, any set forth in any of SEQ ID
NOS: .
568-588. In some examples, the variant MT-SPI polypeptides provided herein
additionally contain a modification corresponding to C122S in an MT-SP1
polypeptide such as an MT-SP1 polypeptide having an amino acid sequence set
forth
in SEQ ID NO:253 or a catalytically active fragment thereof set forth in SEQ
ID
NO:505. Exemplary of such variant MT-SPI polypeptides are set forth in any of
SEQ=
ID NOS: 537-557, or fragments thereof of contiguous amino acids containing the
mutation and having catalytic activity such as, for example, any set forth in
any of
SEQ ID NOS: 516-536.
In particular, modified u-PA polypeptides having the following amino acid
modifications are provided: L73A/I89V; L155P; R72G/L155P; F132L1V160A;
L73A/189V/F30T; L73A/189V/L155V; L73A/189V/L155M; and
L73A/189V/F30L/L155M, based on chymotrypsin numbering.
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G. METHODS OF ASSESSING PROTEASE ACTIVITY AND
SPECIFICITY
Proteases selected in the methods provided herein can be tested to determine
if, following selection, the proteases retain catalytic efficiency and exhibit
the desired
substrate specificity. Activity assessment can be performed using supernatant
from
the amplified display source or from purified protein. For example, as
discussed
above, phage supernatant can be assayed following rescue of phage with helper
phage
and phage amplification. Alternatively, protease activity can be assayed
directly from
the cell medium or periplasm of infected bacteria. Protease activity of the
purified
selected protease also can be determined.
Catalytic efficiency and/or substrate specificity can be assessed by assaying
for substrate cleavage using known substrates of the protease. For example,
cleavage
of plasminogen can be asseesed in the case where t-PA or u-Pa are used in the
selection method herein. In another example, a peptide substrate recognized by
the
protease can be used. For example, RQAR (SEQ ID NO:513), which is the auto-
activation site of MT-SP1, can be used to assess the activity of selected MT-
SP1
proteases. In one embodiment, a fluorogenically tagged tetrapeptide of the
peptide
substrate can be used, for example, an ACC- or AMC- tetrapeptide. In addition,
a
fluorogenic peptide substrates designed based on the cleavage sequence of a
desired
target substrate for which the protease was selected against can be used to
assess
activity.
In some examples, the selected protease can be assessed for its activity
against
a known peptide substrate in the presence or absence of the variant protease
trap
polypeptide used in the selection method. Typically, such an activity
assessment is
performed in order to further select for those proteases that are inhibited in
the
presence of protease trap polypeptide containing the desired cleavage sequence
of the
target substrate, and thereby optimize for selected proteases having improved
selectivity for the target substrate. Comparisons of inhibition can be made
against the
wild-type or template protease and/or with all other proteases identified in
the
selection method.
Kinetic analysis of cleavage of native substrates of a selected protease can
be
compared to analysis of cleavage of desired target substrates to assess
specificity of
the selected protease for the target sequence. In addition, second order rate
constants
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of inhibition (ki) can be assessed to monitor the efficiency and reactivity of
a selected
protease for a substrate, such as for example, the protease trap polypeptide,
or variant
thereof, used in the selection method. Example 5 exemplifies various assays
used to
assess the catalytic efficiency and reactivity of mutant u-PA polypeptides
identified in
the methods provided herein. Example 10 and Example 12 exemplify various
assays
used to assess the catalytic efficiency of selected MT-SP1 phage supernatants.
Example 14 exemplifies various assays used to assess the catalytic efficiency
and
reactivity of selected purified variant MT-SP1 proteases.
In one example, selected proteases, such as for example selected u-PA or MT-
.. SP1 proteases, that are selected to match the desired specificity profile
of the mutated
protease trap polypepide, can be assayed using individual fluorogenic peptide
substrates corresponding to the desired cleavage sequence. For example, a
method of
assaying for a modified protease that can cleave any one or more of the
desired
cleavage sequences of a target substrate includes: (a) contacting a peptide
fluorogenic
.. sample (containing a desired target cleavage sequence) with a protease, in
such a
manner whereby a fluorogenic moiety is released from a peptide substrate
sequence
upon action of the protease, thereby producing a fluorescent moiety; and (b)
observing whether the sample undergoes a detectable change in fluorescence,
the
detectable change being an indication of the presence of the enzymatically
active
protease in the sample. In such an example, the desired cleavage sequence for
which
the protease was selected against is made into a fluorogenic peptide by
methods
known in the art. In one embodiment, the individual peptide cleavage sequences
can
be attached to a fluorogenically tagged substrate, such as for example an ACC
or
AMC fluorogenic leaving group, and the release of the fluorogenic moiety can
be
=
determined as a measure of specificity of a protease for a peptide cleavage
sequence.
The rate of increase in fluorescence of the target cleavage sequence can be
measured
such as by using a fluorescence spectrophotometer. The rate of increase in
fluorescence can be measured over time. Michaelis-Menton kinetic constants can
be
determined by the standard kinetic methods. The kinetic constants kcat, Km and
.. kcat/K,õ can be calculated by graphing the inverse of the substrate
concentration versus
the inverse of the velocity of substrate cleavage, and fitting to the
Lineweaver-Burk
equation (1 ivelocity=(KmNmax)(1/[5]) + 1Nmax; where Vmax=[ET]kcat). The
second
order rate constant or specificity constant (kcat/Km) is a measure of how well
a
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substrate is cut by a particular protease. For example, an ACC- or AMC-
tetrapeptide
such as Ac-RRAR-AMC, Ac-SLGR-AMC, Ac-SLGR-ACC, Ac-RQAR-ACC, can be
made and incubated with a protease selected in the methods provided herein and
activity of the protease can be assessed by assaying for release of the
fluorogenic
moiety. The choice of the tetrapeptide depends on the desired cleavage
sequence to
by assayed for and can be empirically determined. "
Assaying for a protease in a solution simply requires adding a quantity of the
stock solution to a protease to a fluorogenic protease indicator peptide and
measuring
the subsequent increase in fluorescence or decrease in excitation band in the
absorption spectrum. The solution and the fluorogenic indicator also can be
combined and assayed in a "digestion buffer" that optimizes activity of the
protease.
Buffers suitable for assaying protease activity are well known to those of
skill in the
art. In general, a buffer is selected with a PH which corresponds to the PH
optimum
of the particular protease. For example, a buffer particularly suitable for
assaying
elastase activity contains 50mM sodium phosphate, 1 mM EDTA at pH 8.9. The
measurement is most easily made in a fluorometer, an instrument that provides
an
"excitation" light source for the fluorophore and then measures the light
subsequently
emitted at a particular wavelength. Comparison with a control indicator
solution
lacking the protease provides a measure of the protease activity. The activity
level
can be precisely qtintified by generating a standard curve for the
protease/indicator
combination in which the rate of change in fluorescence produced by protease
solutions of known activity is determined.
While detection of fluorogenic compounds can be accomplished using a
fluorometer, detection can be accomplished by a variety of other methods well
known
to those of skill in the art. Thus, for example, when the fluorophores emit in
the
visible wavelengths, detection can be simply by visual inspection of
fluorescence in
response to excitation by a light source. Detection also can be by means of an
image
analysis system utilizing a video camera interfaced to a digitizer or other
image
acquisition system. Detection also can be by visualization through a filter,
as under a
fluorescence microscope. The microscope can provide a signal that is simply
visualized by the operator. Alternatively, the signal can be recorded on
photographic
= film or using a video analysis system. The signal also can simply be
quantified in
real time using either an image analysis system or a photometer.
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Thus, for example, a basic assay for protease activity of a sample involves
suspending or dissolving the sample in a buffer (at the pH optima of the
particular
protease being assayed) adding to the buffer a fluorogenic protease peptide
indicator,
and monitoring the resulting change in fluorescence using a spectrofluorometer
as
shown in e.g., Harris et al., (1998)J Biol Chem 273:27364. The
spectrofluorometer is
set to excite the fluorophore at the excitation wavelength of the fluorophore.
The
fluorogenic protease indicator is a substrate sequence of a protease that
changes in
fluorescence due to a protease cleaving the indicator.
Selected proteases also can be assayed to ascertain that they will cleave the
desired sequence when presented in the context of the full-length protein. In
one
example, a purified target protein, i.e. VEGFR2 or complement protein C2, can
be
incubated in the presence or absence of a selected protease and the cleavage
event can
be monitored by SDS-PAGE followed by Coomassie Brilliant Blue staining for
protein and analysis of cleavage products using densitometry. The specificity
constant of cleavage of a full length protein by a protease can be determined
by using
gel densitometry to assess changes in densitometry over time of a full-length
target
substrate band incubated in the presence of a protease. In addition, the
activity of the
target protein also can be assayed using methods well known in the art for
assaying
= the activity of a desired target protein, to verify that its function has
been destroyed by
the cleavage event.
In specific embodiments, comparisqn of the specificities of a selected
protease, typically a modified protease, can be used to determine if the
selected
protease exhibits altered, for example, increased, specificity compared to the
wild-
type or template protease. The specificity of a protease for a target
substrate can be
measured by observing how many disparate sequences a modified protease cleaves
at
a given activity compared to a wild-type or template protease. If the modified
protease cleaves fewer target substrates than the wildtype protease, the
modified
protease has greater specificity than the wild-type protease for those target
substrates.
The specificity of a protease for a target substrate can be determined from
the
specificity constant of cleavage of a target substrate compared to a non-
target
substrate (i.e. a native wildtype substrate sequence of a protease). A ratio
of the
specificity constants of a modified protease for a target substrate versus a
non-target
substrate can be made to determine a ratio of the efficiency of cleavage of
the
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protease. Comparison of the ratio of the efficiency of cleavage between a
modified
protease and a wild-type or template protease can be used to assess the fold
change in
specificity for a target substrate. Specificity can be at least 2-fold, at
least 4-fold, at
least 5-fold, at least 6-fold, at least 7-fold, 8, 9, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100,
200, 300, 400, 500, 600, 700, 800, 900, 1000 times or more when compared to
the
specificity of a wild-type or template protease for a target substrate versus
a non-
target substrate.
H. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING
PROTEASE TRAP POLYPEPTIDES (i.e. SERPINS) OR VARIANTS
THEREOF OR PROTEASES/MODIFIED PROTEASES
Polypeptides set forth herein, including protease trap polypeptides or
protease
polypeptides or catalytically active portions thereof, including modified u-PA
polypeptides or modified MT-SP1 polypeptides, can be obtained by methods well
known in the art for protein purification and recombinant protein expression.
Any
method known to those of skill in the art for identification of nucleic acids
that encode
desired genes can be used. Any method available in the art can be used to
obtain a
full length (i.e., encompassing the entire coding region) cDNA or genomic DNA
clone encoding a desired protease trap polypeptide or protease protein, such
as from a
cell or tissue source. Modified polypeptides, such as variant protease trap
polypeptides or selected variant proteases, can be engineered as described
herein from
a wildtype polypeptide, such as by site-directed mutagenesis.
Polypeptides can be cloned or isolated using any available methods known in
the art for cloning and isolating nucleic acid molecules. Such methods include
PCR
amplification of nucleic acids and screening of libraries, including nucleic
acid
hybridization screening, antibody-based screening and activity-based
screening.
Methods for amplification of nucleic acids can be used to isolate nucleic acid
molecules encoding a desired polypeptide, including for example, polymerase
chain
reaction (PCR) methods. A nucleic acid containing material can be used as a
starting
material from which a desired polypeptide-encoding nucleic acid molecule can
be
isolated. For example, DNA and mRNA preparations, cell extracts, tissue
extracts,
fluid samples (e.g. blood, serum, saliva), samples from healthy and/or
diseased
subjects can be used in amplification methods. Nucleic acid libraries also can
be used
as a source of starting material. Primers can be designed to amplify a desired
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polypeptide. For example, primers can be designed based on expressed sequences
from which a desired polypeptide is generated. Primers can be designed based
on
back-translation of a polypeptide amino acid sequence. Nucleic acid molecules
generated by amplification can be sequenced and confirmed to encode a desired
polypeptide.
Additional nucleotide sequences can be joined to a polypeptide-encoding
nucleic acid molecule, including linker sequences containing restriction
endonuclease
sites for the purpose of cloning the synthetic gene into a vector, for
example, a protein
expression vector or a vector designed for the amplification of the core
protein coding
DNA sequences. Furthermore, additional nucleotide sequences specifying
functional
DNA elements can be operatively linked to a polypeptide-encoding nucleic acid
molecule. Examples of such sequences include, but are not limited to, promoter
sequences designed to facilitate intracellular protein expression, and
secretion
sequences designed to facilitate protein secretion. Additional nucleotide
residues
sequences such as sequences of bases specifying protein binding regions also
can be
linked to protease-encoding nucleic acid molecules. Such regions include, but
are not
limited to, sequences of residues that facilitate or encode proteins that
facilitate
- uptake of a protease into specific target cells, or otherwise alter
pharrnacokinetics of
a product of a synthetic gene.
In addition, tags or other moieties can be added, for example, to aid in
detection or affinity purification of the polypeptide. For example, additional
nucleotide residues sequences such as sequences of bases specifying an epitope
tag or
other detectable marker also can be linked to protease-encoding nucleic acid
molecules or to a serpin-encoding nucleic acid molecule, or variants thereof
Exemplary of such sequences and nucleic acid sequences encoding a His tag
(e.g.,
6xHis, HHHHH; SEQ ID NO:496) or Flag Tag (DYKDDDDK; SEQ ID NO:495).
The identified and isolated nucleic acids can then be inserted into an
appropriate cloning vector. A large number of vector-host systems known in the
art
can be used. Possible vectors include, but are not limited to, plasmids or
modified
viruses, but the vector system must be compatible with the host cell used.
Such
, vectors include, but are not limited to, bacteriophages such as lambda
derivatives, or
plasmids such as pCM1/4, pBR322 or pUC plasmid derivatives or the Bluescript
vector (Stratagene, La Jolla, CA). The insertion into a cloning vector can,
for
*Trademark
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example, be accomplished by ligating the DNA fragment into a cloning vector-
which
has complementary cohesive termini. Insertion can be effected using TOPO
cloning
vectors (INVITROGEN, Carlsbad, CA). If the complementary restriction sites
used to
fragment the DNA are not present in the cloning vector, the ends of the DNA
molecules can be enzymatically modified. Alternatively, any site desired can
be
produced by ligating nucleotide sequences (linkers) onto the DNA termini;
these
ligated linkers can contain specific chemically synthesized oligonucleotides
encoding
restriction endonuclease recognition sequences. In an alternative method, the
cleaved
vector and protein gene can be modified by homopolymeric tailing. Recombinant
molecules can be introduced into host cells via, for example, transformation,
transfection, infection, electroporation and sonoporation, so that many copies
of the
gene sequence are generated.
In specific embodiments, transformation of host cells with recombinant DNA
molecules that incorporate the isolated protein gene, cDNA, or synthesized DNA
sequence enables generation of multiple copies of the gene. Thus, the gene can
be
obtained in large quantities by growing transformants, isolating the
recombinant DNA
molecules from the transformants and, when necessary, retrieving the inserted
gene
from the isolated recombinant DNA.
1. Vectors and cells
For recombinant expression of one or more of the desired proteins, such as any
described herein, the nucleic acid containing all or a portion of the
nucleotide
sequence encoding the protein can be inserted into an appropriate expression
vector,
i.e., a vector that contains the necessary elements for the transcription and
translation
of the inserted protein coding sequence. The necessary transcriptional and
translational signals also can be supplied by the native promoter for protease
genes,
and/or their flanking regions.
Also provided are vectors that contain a nucleic acid encoding the protease or
modified protease. Cells containing the vectors also are provided. The cells
include
= eukaryotic and prokaryotic cells, and the vectors are any suitable for
use therein.
Prokaryotic and eukaryotic cells, including endothelial cells, containing the
vectors are provided. Such cells include bacterial cells, yeast cells, fungal
cells,
Archea, plant cells, insect cells and animal cells. The cells are used to
produce a
protein thereof by growing the above-described cells under conditions whereby
the
*Trademark
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encoded protein is expressed by the cell, and recovering the expressed
protein. For
purposes herein, for example, the protease can be secreted into the medium.
In one embodiment, vectors containing a sequence of nucleotides that encodes
a polypeptide that has protease activity, such as encoding any of the u-PA
variant
polypeptide provided herein, and contains all or a portion of the protease
domain, or
multiple copies thereof, are provided. Also provided are vectors that contain
a
sequence of nucleotides that encodes the protease domain and additional
portions of a
protease protein up to and including a full length protease protein, as well
as multiple
copies thereof. The vectors can be selected for expression of the modified
protease
protein or protease domain thereof in the cell or such that the protease
protein is
expressed as a secreted protein. When the protease domain is expressed, the
nucleic
acid is linked to a nucleic acid encoding a secretion signal, such as the
Saccharomyces
= cerevisiae " mating factor signal sequence or a portion thereof, or the
native signal
sequence.
A variety of host-vector systems can be used to express the protein coding
sequence. These include but are not limited to mammalian cell systems infected
with
virus (e.g. vaccinia virus, adenovirus and other viruses); insect cell systems
infected
with virus (e.g. baculovirus); microorganisms such as yeast containing yeast
vectors;
or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
The expression elements of vectors vary in their strengths and specificities.
Depending on the host-vector system used, any one of a number of suitable
transcription and translation elements can be used.
Any methods known to those of skill in the art for the insertion of DNA
fragments into a vector can be used to construct expression vectors containing
a
chimeric gene containing appropriate transcriptional/translational control
signals and
protein coding sequences. These methods can include in vitro recombinant DNA
and
synthetic techniques and in vivo recombinants (genetic recombination).
Expression of
nucleic acid sequences encoding protein, or domains, derivatives, fragments or
homologs thereof, can be regulated by a second nucleic acid sequence so that
the
genes or fragments thereof are expressed in a host transformed with the
recombinant
DNA molecule(s). For example, expression of the proteins can be controlled by
any
promoter/enhancer known in the art. In a specific embodiment, the promoter is
not
native to the genes for a desired protein. Promoters which can be used include
but are
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not limited to the SV40 early promoter (Bernoist and Chambon, Nature 290:304-
310
(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma
virus
(Yamamoto et al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter
(Wagner etal., Proc. Natl. Acad. Set USA 78:1441-1445 (1981)), the regulatory
sequences of the metallothionein gene (Brinster et al., Nature 296:39-42
(1982));
prokaryotic expression vectors such as the fl-lactamase promoter (Jay et al.,
(1981)
Proc. Natl. Acad Sci. USA 78:5543) or the tac promoter (DeBoer etal., Proc.
Natl.
Acad. Sci. USA 80:21-25 (1983)); see also "Useful Proteins from Recombinant
Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors
containing the nopaline synthetase promoter (1errar-Estrella et al., Nature
303:209-
213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Garder et al.,
Nucleic Acids Res. 9:2871 (1981)), and the promoter of the photosynthetic
enzyme
ribulose bisphosphate carboxylase (Herrera-Estrella etal., Nature 310:115-120
(1984)); promoter elements from yeast and other fungi such as the Ga14
promoter, the
alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the
alkaline
phosphatase promoter, and the following animal transcriptional control regions
that
exhibit tissue specificity and have been used in transgenic animals: elastase
I gene
control region which is active in pancreatic acinar cells (Swift et al., Cell
38:639-646
(1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);
MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is
active in pancreatic beta cells (Hanahan et al., Nature 315:115-122 (1985)),
immunoglobulin gene control region which is active in lymphoid cells
(Grosschedl et
al., Cell 38:647-658 (1984); Adams etal., Nature 3/8:533-538 (1985); Alexander
et
al., MoL Cell Biol. 7:1436-1444 (1987)), mouse mammary tumor virus control
region
which is active in testicular, breast, lymphoid and mast cells (Leder et al.,
Cell
45:485-495 (1986)), albumin gene control region which is active in liver
(Pinckert et
al., Genes and Devel. /:268-276 (1987)), alpha-fetoprotein gene control region
which
is active in liver (Krumlauf et al., Mol. Cell. BioL 5:1639-1648 (1985);
Hammer et
al., Science 235:53-58 1987)), alpha-1 antitrypsin gene control region which
is active
in liver (Kelsey etal., Genes and Devet 1:161-171 (1987)), beta globin gene
control
region which is active in myeloid cells (Mogram etal., Nature 3/5:338-340
(1985);
Kollias etal., Cell 46:89-94 (1986)), myelin basic protein gene control region
which
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is active in oligodendrocyte cells of the brain (Readhead etal., Cell
48:7037712 _
(1987)), myosin light chain-2 gene control region which is active in skeletal
muscle
(Sani, Nature 3/4:283-286 (1985)), and gonadotrophic releasing hormone gene
control region which is active in gonadotrophs of the hypothalamus (Mason et
al.,
Science 234:1372-1378(1986)).
In a specific embodiment:a vector is used that contains a promoter operably
linked to nucleic acids encoding a desired protein, or a domain, fragment,
derivative
or homolog, thereof, one or more origins of replication, and optionally, one
or more
selectable markers (e.g., an 'antibiotic resistance gene). For example,
vectors and
systems for expression of the protease domains of the protease proteins
include the
well known Pichia vectors (available, for example, from Invitrogen, San Diego,
CA),
particularly those designed for secretion of the encoded proteins. Exemplary
plasmid
vectors for transformation of E.coli cells, include, for example, the pQE
expression
vectors (available from Qiagen, Valencia, CA; see also literature published by
Qiagen
describing the system). pQE vectors have a phage T5 promoter (recognized by E.
colt
RNA polymerase) and a double lac operator repression module to provide tightly
regulated, high-level expression of recombinant proteins in E. coli, a
synthetic
ribosomal binding site (RBS II) for efficient translation, a 6XHis tag coding
sequence,
to and Ti transcriptional terminators, ColE1 origin of replication, and a beta-
lactamase gene for conferring ampicillin resistance. The pQE vectors enable
placement of a 6xHis tag at either the N- or C-terminus of the recombinant
protein.
Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiple
cloning
sites for all three reading frames and provide for the expression of N-
terminally
6xHis-tagged proteins. Other exemplary plasmid vectors for transformation of
E. coli
cells, include, for example, the pET expression vectors (see, U.S patent
4,952,496;
available from NOVAGEN, Madison, WI).
Such plasmids include pET I la, which contains the T7lac
promoter, T7 terminator, the inducible E. coli lac operator, and the lac
repressor gene;
pET 12a-c, which Contains the T7 promoter, T7 terminator, and the E. coli ompT
secretion signal; and pET 15b and pET19b (NOVAGEN, Madison, WI), which
contain a His-Tagml leader sequence for use in purification with a His column
and a
thrombin cleavage site that permits cleavage following purification over the
column,
the T7-lac promoter region and the T7 terminator.
*Trademark
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2. Expression
Proteins, such as any set forth herein including any protease trap
polypeptides
or variants thereof, or selected proteases or catalytically active portions
thereof, can
be produced by any method known to those of skill in the art including in vivo
and in
vitro methods. Desired proteins can be expressed in any organism suitable to
produce
the required amounts and forms of the proteins, such as for example, needed
for
administration and treatment. Expression hosts include prokaryotic and
eukaryotic
organisms such as E.coli, yeast, plants, insect cells, mammalian cells,
including
human cell lines and transgenic animals. Expression hosts can differ in their
protein
production levels as well as the types of post-translational modifications
that are
present on the expressed proteins. The choice of expression host can be made
based
on these and other factors, such as regulatory and safety considerations,
production
costs and the need and methods for purification.
Many expression vectors are available and known to those of skill in the art
and can be used for expression of proteins. The choice of expression vector
will be
influenced by the choice of host expression system. In general, expression
vectors can
include transcriptional promoters and optionally enhancers, translational
signals, and
transcriptional and translational termination signals. Expression vectors that
are used
for stable transformation typically have a selectable marker which allows
selection
and maintenance of the transformed cells. In some cases, an origin of
replication can
be used to amplify the copy number of the vector.
Proteins, such as for example any variant protease provided herein or any
protease trap polypeptide or variant thereof, also can be utilized or
expressed as
protein fusions. For example, a protease fusion can be generated to add
additional
functionality to a protease. Examples of protease fusion proteins include, but
are not
limited to, fusions of a signal sequence, a tag such as for localization, e.g.
a his6 tag or
a myc tag, or a tag for purification, for example, a GST fusion, and a
sequence for
directing protein secretion and/or membrane association.
In one embodiment, a protease can be expressed in an active form. In another
embodiment, a protease is expressed in an inactive, zymogen form. a.
Prokaryotic Cells
Prokaryotes, especially E.coli, provide a system for producing large amounts
of proteins. Transformation of E.coli is simple and rapid technique well known
to
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those of skill in the art. Expression vectors for E.coli can contain inducible
promoters, such promoters are useful for inducing high levels of protein
expression
and for expressing proteins that exhibit some toxicity to the host cells.
Examples of
inducible promoters include the lac promoter, the trp promoter, the hybrid tac
promoter, the T7 and SP6 RNA promoters and the temperature regulated XPL
promoter.
Proteins, such as any provided herein, can be expressed in the cytoplasmic
environment of E.coli. The cytoplasm is a reducing environment and for some
molecules, this can result in the formation of insoluble inclusion bodies.
Reducing
agents such as dithiothreotol and P-mercaptoethanol and denaturants, such as
guanidine-HCl and urea can be used to resolubilize the proteins. An
alternative
approach is the expression of proteins in the periplasmic space of bacteria
which
provides an oxidizing environment and chaperonin-like and disulfide isomerases
and
can lead to the production of soluble protein. Typically, a leader sequence is
fused to
the protein to be expressed which directs the protein to the periplasm. The
leader is
then removed by signal peptidases inside the periplasm. Examples of
periplasmic-
targeting leader sequences include the pelB leader from the pectate lyase gene
and the
leader derived from the alkaline phosphatase gene. In some cases, periplasmic
expression allows leakage of the expressed protein into the culture medium.
The
secretion of proteins allows quick and simple purification from the culture
supernatant. Proteins that are not secreted can be obtained from the periplasm
by
osmotic lysis. Similar to cytoplasmic expression, in some cases proteins
can.become
insoluble and denaturants and reducing agents can be used to facilitate
solubilization
and refolding. Temperature of induction and growth also can influence
expression
levels and solubility, typically temperatures between 25 C and 37 C are used.
Typically, bacteria produce aglycosylated proteins. Thus, if proteins require
glycosylation for function, glycosylation can be added in vitro after
purification from
host cells.
b. Yeast Cells
Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,
Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are well known
yeast
expression hosts that can be used for production of proteins, such as any
described
herein. Yeast can be transformed with episomal replicating vectors or by
stable
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chromosomal integration by homologous recombination. Typically, inducible
promoters are used to regulate gene expression. Examples of such promoters
include
GAL1, GAL7 and GALS and metallothionein promoters, such as CUP], A0X1 or
other Pichia or other yeast promoter, Expression vectors often include a
selectable
marker such as LEU2, TRP I, HIS3 and URA3 for selection and maintenance of the
transformed DNA. Proteins expressed in yeast are often soluble. Co-expression
with
chaperonins such as Bip and protein disulfide isomerase can improve expression
levels and solubility. Additionally, proteins expressed in yeast can be
directed for
secretion using secretion signal peptide fusions such as the yeast mating type
alpha-
factor secretion signal from Saccharomyces cerevisae and fusions with yeast
cell
surface proteins such as the Aga2p mating adhesion receptor or the Arxula
adeninivorans glucoamylase. A protease cleavage site such as for the Kex-2
protease,
can be engineered to remove the fused sequences from the expressed
polypeptides as
they exit the secretion pathway. Yeast also is capable of glycosylation at Asn-
X-
Ser/Thr motifs.
c. Insect Cells
Insect cells, particularly using baculovirus expression, are useful for
expressing polypeptides such as modified proteases or modified protease trap
polypeptides. Insect cells express high levels of protein and are capable of
most of
the post-translational modifications used by higher eukaryotes. Baculovirus
have a
restrictive host range which improves the safety and reduces regulatory
concerns of
eukaryotic expression. Typical expression vectors use a promoter for high
level
expression such as the polyhedrin promoter of baculovirus. Commonly used
baculovirus systems include the baculoviruses such as Auto grapha californica
nuclear
polyhedrosis virus (AcNPV), and the bombyx mori nuclear polyhedrosis virus
(BmNPV) and an insect cell line such as Sf9 derived from Spodoptera
frugiperda,
Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1). For high-level
expression, the nucleotide sequence of the molecule to be expressed is fused
immediately downstream of the polyhedrin initiation codon of the virus.
Mammalian
secretion signals are accurately processed in insect cells and can be used to
secrete the
expressed protein into the culture medium. In addition, the cell lines
Pseudaletia
umpuncta (A7S) and Danaus plexippus (DpN1) produce proteins with glycosylation
patterns similar to mammalian cell systems.
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An alternative expression system in insect cells is the use of stably
transformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells
(Drosophila
melanogaster) and C7 cells (Aedes a/bopictus) can be used for expression. The
Drosophila metallothionein promoter can be used to induce high levels of
expression
in the presence of heavy metal induction with cadmium or copper. Expression
vectors
are typically maintained by the use of selectable markers such as neomycin and
hygromycin.
d. Mammalian Cells
Mammalian expression systems can be used to express proteins including
modified proteases or catalytically active portions thereof, or protease trap
polypeptides or variants thereof. Expression constructs can be transferred to
mammalian cells by viral infection such as adenovirus or by direct DNA
transfer such
as liposomes, calcium phosphate, DEAE-dextran and by physical means such as
electroporation and microinjection. Expression vectors for mammalian cells
typically
include an mRNA cap site, a TATA box, a translational initiation sequence
(Kozak
consensus sequence) and polyadenylation elements. Such vectors often include
transcriptional promoter-enhancers for high-level expression, for example the
SV40
promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long
terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are
active
in many cell types. Tissue and cell-type promoters and enhancer regions also
can be
used for expression. Exemplary promoter/enhancer regions include, but are not
limited to, those from genes such as elastase I, insulin, immunoglobulin,
mouse
mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta
globin,
myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone
gene
control. Selectable markers can be used to select for and maintain cells with
the
expression construct. Examples of selectable marker genes include, but are not
limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-
guanine
phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate
reductase and thymidine kinase. Fusion with cell surface signaling molecules
such as
TCR-c and Fc,RI-y can direct expression of the proteins in an active state on
the cell
surface.
Many cell lines are available for mammalian expression including mouse, rat
human, monkey, chicken and hamster cells. Exemplary cell lines include but are
not
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limited to CHO, Balb/3T3, HeLa, MT2, mouse NSO (nonsecreting) and other
myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes,
fibroblasts, Sp2/0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines
also are available adapted to serum-free media which facilitates purification
of
secreted proteins from the cell culture media. One such example is the serum
free
EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84:332-42.)
e. Plants
Transgenic plant cells and plants can be used to express proteins such as any
described herein. Expression constructs are typically transferred to plants
using direct
DNA transfer such as microprojectile bombardment and PEG-mediated transfer
into
protoplasts, and with agrobacterium-mediated transformation. Expression
vectors can
include promoter and enhancer sequences, transcriptional termination elements
and
translational control elements. Expression vectors and transformation
techniques are
usually divided between dicot hosts, such as Arabidopsis and tobacco, and
monocot
hosts, such as corn and rice. Examples of plant promoters used for expression
include
the cauliflower mosaic virus promoter, the nopaline syntase promoter, the
ribose
bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters.
Selectable markers such as hygromycin, phosphomannose isomerase and neomycin
phosphotransferase are often used to facilitate selection and maintenance of
transformed cells. Transformed plant cells can be maintained in culture as
cells,
aggregates (callus tissue) or regenerated into whole plants. Transgenic plant
cells also
can include algae engineered to produce proteases or modified proteases (see
for
example, Mayfield et al. (2003) PNAS /00:438-442). Because plants have
different
glycosylation patterns than mammalian cells, this can influence the choice of
protein
produced in these hosts.
3. Purification Techniques
Method for purification of polypeptides, including protease polypeptides or
other proteins, from host cells will depend on the chosen host cells and
expression
systems. For secreted molecules, proteins are generally purified from the
culture
media after removing the cells. For intracellular expression, cells can be
lysed and the
proteins purified from the extract. When transgenic organisms such as
transgenic
plants and animals are used for expression, tissues or organs can be used as
starting
material to make a lysed cell extract. Additionally, transgenic animal
production can
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include the production of polypeptides in milk or eggs, which can be
collected, and if
necessary, the proteins can be extracted and further purified using standard
methods
in the art.
In one example, proteases can be expressed and purified to be in an inactive
form (zymogen form) or alternatively the expressed protease can be purified
into an
active form, such as a two-chain form, by autocatalysis to remove the
proregion.
Typically, the autoactivation occurs during the purification process, such as
by
incubating at room temperature for 24-72 hours. The rate and degree of
activation is
dependent on protein concentration and the specific modified protease, such
that for
example, a more dilute sample can need to be incubated at room temperature for
a
longer period of time. Activation can be monitored by SDS-PAGE (e.g., a 3
kilodalton shift) and by enzyme activity (cleavage of a fluorogenic
substrate).
Typically, a protease is allowed to achieve >75% activation before
purification.
Proteins, such as proteases or protease-trap polypeptides, can be purified
using
standard protein purification techniques known in the art including but not
limited to,
SDS-PAGE, size fraction and size exclusion chromatography, ammonium sulfate
precipitation and ionic exchange chromatography, such as anion exchange.
Affinity
purification techniques also can be utilized to improve the efficiency and
purity of the
preparations. For example, antibodies, receptors and other molecules that bind
proteases or protease trap polypeptides can be used in affinity purification.
Expression constructs also can be engineered to add an affinity tag to a
protein such
as a myc epitope, GST fusion or His6 and affinity purified with myc antibody,
glutathione resin and Ni-resin, respectively. Purity can be assessed by any
method
known in the art including gel electrophoresis and staining and
spectrophotometric
techniques.
4. Fusion Proteins
Fusion proteins containing a variant protease provided herein and one or more
other polypeptides also are provided. Pharmaceutical compositions containing
such
fusion proteins formulated for administration by a suitable route are
provided. Fusion
proteins are formed by linking in any order the modified protease and another
polypeptide, such as an antibody or fragment thereof, growth factor, receptor,
ligand
and other such agent for the purposes of facilitating the purification of a
protease,
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altering the pharmacodynamic properties of a protease by directing the
protease to a
targeted cell or tissue, and/or increasing the expression or secretion of a
protease.
Within a protease fusion protein, the protease polypeptide can correspond to
all or a
catalytically active portion thereof of a protease protein. In some
embodiments, the
protease or catalytically active portion thereof is a modified protease.
Fusion proteins
provided herein retain substantially all of their specificity and/or
selectivity for any
one or more of the desired target substrates. Generally, protease fusion
polypeptides
retain at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% substrate
specificity and/or selectivity compared with a non-fusion protease, including
96%,
97%, 98%, 99% or greater substrate specificity compared with a non-fusion
protease.
Linkage of a protease polypeptide and another polypeptide can be effected
directly or indirectly via a linker. In one example, linkage can be by
chemical
linkage, such as via heterobifunctional agents or thiol linkages or other such
linkages.
Fusion of a protease to another polypeptide can be to the N- or C- terminus of
the
protease polypeptide. Non-limiting examples of polypeptides that can be used
in
fusion proteins with a protease provided herein include, for example, a GST
(glutathione S-transferase) polypeptide, Fe domain from an immunoglobulin, or
a
heterologous signal sequence. The fusion proteins can contain additional
components, such as E. colt maltose binding protein (MBP) that aid in uptake
of the
protein by cells (see, International PCT application No. WO 01/32711).
A protease fusion protein can be produced by standard recombinant
techniques. For example, DNA fragments coding for the different polypeptide
sequences can be ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation,
restriction enzyme digestion to provide for appropriate termini, filling-in of
cohesive
ends as appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and
enzymatic ligation. In another embodiment, the fusion gene can be synthesized
by
conventional techniques including automated DNA synthesizers. Alternatively,
PCR
amplification of gene fragments can be carried out using anchor primers that
give rise
to complementary overhangs between two consecutive gene fragments that can
subsequently be annealed and reamplified to generate a chimeric gene sequence
(see,
e.g., Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, 1992). Moreover, many expression vectors are commercially
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available that already encode a fusion moiety (e.g., a GST polypeptide). A
protease-
encoding nucleic acid can be cloned into such an expression vector such that
the
fusion moiety is linked in-frame to the protease protein.
5. Nucleotide Sequences
Nucleic acid molecules encoding modified proteases are provided herein.
Nucleic acid molecules include allelic variants or splice variants of any
encoded
protease, or catalytically active portion thereof. In one embodiment, nucleic
acid
molecules provided herein have at least 50, 60, 65, 70, 75, 80, 85, 90, 91,
92, 93, 94,
95, or 99% sequence identity or hybridize under conditions of medium or high
stringency along at least 70% of a full-length of any nucleic acid encoded
wild-type
protease, or catalytically active portion thereof. In another embodiment, a
nucleic
acid molecule can include those with degenerate codon sequences of any of the
proteases or catalytically active portions thereof such as those provided
herein.
Nucleic acid molecules, or fusion proteins containing a catalytically active
portion of a nucleic acid molecule, operably-linked to a promoter, such as an
inducible promoter for expression in mammalian cells also are provided. Such
promoters include, but are not limited to, CMV and SV40 promoters; adenovirus
promoters, such as the E2 gene promoter, which is responsive to the HPV E7
oncoprotein; a PV promoter, such as the PBV p89 promoter that is responsive to
the
PV E2 protein; and other promoters that are activated by the HIV or PV or
oncogenes.
Modified proteases provided herein, also can be delivered to the cells in gene
transfer vectors. The transfer vectors also can encode additional other
therapeutic
agent(s) for treatment of the disease or disorder, such as coagulation
disorders or
cancer, for which the protease is administered. Transfer vectors encoding a
protease
can be used systemically, by administering the nucleic acid to a subject. For
example,
the transfer vector can be a viral vector, such as an adenovirus vector.
Vectors
encoding a protease also can be incorporated into stem cells and such stem
cells
administered to a subject such as by transplanting or engrafting the stem
cells at sites
for therapy. For example, mesenchymal stem cells (MSCs) can be engineered to
express a protease and such MSCs engrafted at a tumor site for therapy.
I. PREPARATION, FORMULATION AND ADMINISTRATION OF
SELECTED PROTEASE POLYPEPTIDES
1. Compositions and Delivery
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Compositions of selected proteases, such as for example selected mutant u-PA
polypeptides, can be formulated for administration by any route known to those
of
skill in the art including intramuscular, intravenous, intradermal,
intraperitoneal
injection, subcutaneous, epidural, nasal, oral, rectal, topical, inhalational,
buccal (es.,
sublingual), and transdermal administration or any route. Selected proteases
can be
administered by any convenient route, for example by infusion or bolus
injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and
intestinal mucosa, etc.) and can be administered with other biologically
active agents,
either sequentially, intermittently or in the same composition. Administration
can be
local, topical or systemic depending upon the locus of treatment. Local
administration to an area in need of treatment can be achieved by, for
example, but
not limited to, local infusion during surgery, topical application, e.g., in
conjunction
with a wound dressing after surgery, by injection, by means of a catheter, by
means of
a suppository, or by means of an implant. Administration also can include
controlled
release systems including controlled release formulations and device
controlled
release, such as by means of a pump. The most suitable route in any given case
depends on a variety of factors, such as the nature of the disease, the
progress of the
disease, the severity of the disease the particular composition which is used.
Various delivery systems are known and can be used to administer selected
proteases, such as but not limited to, encapsulation in liposomes,
microparticles,
microcapsules, recombinant cells capable of expressing the compound, receptor
mediated endocytosis, and delivery of nucleic acid molecules encoding selected
proteases such as retrovirus delivery systems.
Pharmaceutical compositions containing selected proteases can be prepared.
Generally, pharmaceutically acceptable compositions are prepared in view of
approvals for a regulatory agency or other agency prepared in accordance with
generally recognized pharmacopeia for use in animals and in humans.
Pharmaceutical
compositions can include carriers such as a diluent, adjuvant, excipient, or
vehicle
with which an isoform is administered. Such pharmaceutical carriers can be
sterile
liquids, such as water and oils, including those of petroleum, animal,
vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame
oil. Water is
a typical carrier when the pharmaceutical composition is administered
intravenously.
Saline solutions and aqueous dextrose and glycerol solutions also can be
employed as
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liquid carriers, particularly for injectable solutions. Compositions can
contain along
with an active ingredient: a diluent such as lactose, sucrose, dicalcium
phosphate, or
carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium
stearate and
talc; and a binder such as starch, natural gums, such as gum acaciagelatin,
glucose,
molasses, polvinylpyrrolidine, celluloses and derivatives thereof, povidone,
crospovidones and other such binders known to those of skill in the art.
Suitable
pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monosteamte, talc, sodium
chloride,
dried skim milk, glycerol, propylene, glycol, water, and ethanol. A
composition, if
desired, also can contain minor amounts of wetting or emulsifying agents, or
pH
buffering agents, for example, acetate, sodium citrate, cyclodextrine
derivatives,
sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate,
and
other such agents. These compositions can take the form of solutions,
suspensions,
emulsion, tablets, pills, capsules, powders, and sustained release
formulations. A
composition can be formulated as a suppository, with traditional binders and
carriers
such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and other such agents. Examples of
suitable pharmaceutical carriers are described in "Remington's Pharmaceutical
Sciences" 18th Ed., E. W. Martin (ed.), Mack Publishing Co., Easton, PA. Such
compositions will contain a therapeutically effective amount of the compound,
generally in
purified form, together with a suitable amount of carrier so as to provide the
form for
proper administration to the patient. The formulation should suit the mode of
administration.
Formulations are provided for administration to humans and animals in unit
dosage forms, such as tablets, capsules, pills, powders, granules, sterile
parenteral
solutions or suspensions, and oral solutions or suspensions, and oil water
emulsions
containing suitable quantities of the compounds or pharmaceutically acceptable
derivatives thereof. Pharmaceutically therapeutically active compounds and
derivatives thereof are typically formulated and administered in unit dosage
forms or
multiple dosage forms. Each unit dose contains a predetermined quantity of
therapeutically active compound sufficient to produce the desired therapeutic
effect,
in association with the required pharmaceutical carrier, vehicle or diluent.
Examples
of unit dose forms include ampoules and syringes and individually packaged
tablets or
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capsules. Unit dose forms can be administered in fractions or multiples
thereof. A
multiple dose form is a plurality of identical unit dosage forms packaged in a
single
container to be administered in segregated unit dose form. Examples of
multiple dose
forms include vials, bottles of tablets or capsules or bottles of pints or
gallons. Hence,
.. multiple dose form is a multiple of unit doses that are not segregated in
packaging.
Dosage forms or compositions containing active ingredient in the range of
0.005% to 100% with the balance made up from non-toxic carrier can be
prepared.
For oral administration, pharmaceutical compositions can take the form of, for
example, tablets or capsules prepared by conventional means with
pharmaceutically
acceptable excipients such as binding agents (e.g., pregelatinized maize
starch,
polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,
lactose,
microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g.,
magnesium stearate, talc or silica); disintegrants (e.g., potato starch or
sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can
be coated
.. by methods well-known in the art.
Pharmaceutical preparation also can be in liquid form, for example, solutions,
syrups or suspensions, or can be presented as a drug product for
reconstitution with
water or other suitable vehicle before use. Such liquid preparations can be
prepared
by conventional means with pharmaceutically acceptable additives such as
suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated
edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles
(e.g., almond
oil, oily esters, or fractionated vegetable oils); and preservatives (e.g.,
methyl or
propyl-p-hydroxybenzoates or sorbic acid).
Formulations suitable for rectal administration can be provided as unit dose
suppositories. These can be prepared by admixing the active compound with one
or
more conventional solid carriers, for example, cocoa butter, and then shaping
the
resulting mixture.
Formulations suitable for topical application to the skin or to the eye
include
ointments, creams, lotions, pastes, gels, sprays, aerosols and oils. Exemplary
carriers
include vaseline, lanoline, polyethylene glycols, alcohols, and combinations
of two or
more thereof. The topical formulations also can contain 0.05 to 15, 20, 25
percent by
weight of thickeners selected from among hydroxypropyl methyl cellulose,
methyl
cellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly (alkylene glycols),
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poly/hydroxyalkyl, (meth)acrylates or poly(meth)acrylamides. A topical
formulation
is often applied by instillation or as an ointment into the conjunctival sac.
It also can
be used for irrigation or lubrication of the eye, facial sinuses, and external
auditory
meatus. It also can be injected into the anterior eye chamber and other
places. A
topical formulation in the liquid state also can be present in a hydrophilic
three-
dimensional polymer matrix in the form of a strip or contact lens, from which
the
active components are released.
For administration by inhalation, the compounds for use herein can be
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, dichlorotetrafluoroethane, carbon dioxide or other
suitable
gas. In the case of a pressurized aerosol, the dosage unit can be determined
by
providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g.,
gelatin, for use in an inhaler or insufflator can be formulated containing a
powder mix
of the compound and a suitable powder base such as lactose or starch.
Formulations suitable for buccal (sublingual) administration include, for
example, lozenges containing the active compound in a flavored base, usually
sucrose
and acacia or tragacanth; and pastilles containing the compound in an inert
base such
as gelatin and glycerin or sucrose and acacia.
Pharmaceutical compositions of selected proteases can be formulated for
parenteral administration by injection, e.g., by bolus injection or continuous
infusion.
Formulations for injection can be presented in unit dosage form, e.g., in
ampules or in
multi-dose containers, with an added preservative. The compositions can be
suspensions, solutions or emulsions in oily or aqueous vehicles, and can
contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient can be in powder form for reconstitution
with a
suitable vehicle, e.g., sterile pyrogen-free water or other solvents, before
use.
Formulations suitable for transdermal administration are provided. They can
be provided in any suitable format, such as discrete patches adapted to remain
in
intimate contact with the epidermis of the recipient for a prolonged period of
time.
Such patches contain the active compound in optionally buffered aqueous
solution of,
for example, 0.1 to 0.2M concentration with respect to the active compound.
Formulations suitable for transdermal administration also can be delivered by
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iontophoresis (see, e.g., Pharmaceutical Research 3(6), 318 (1986)) and
typically take
the form of an optionally buffered aqueous solution of the active compound.
Pharmaceutical compositions also can be administered by controlled release
formulations and/or delivery devices (see, e.g., in U.S. Patent Nos.
3,536,809;
.. 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719;
4,687,610;
4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476;
5,674,533 and 5,733,566).
In certain embodiments, liposomes and/or nanoparticles also can be employed
with selected protease administration. Liposomes are formed from phospholipids
that
.. are dispersed in an aqueous medium and spontaneously form multilamellar
concentric
bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally
have
diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation
of
small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 =
angstroms containing an aqueous solution in the core.
Phospholipids can form a variety of structures other than liposomes when
dispersed in water, depending on the molar ratio of lipid to water. At low
ratios, the
liposomes form. Physical characteristics of liposomes depend on pH, ionic
strength
and the presence of divalent cations. Liposomes can show low permeability to
ionic
and polar substances, but at elevated temperatures undergo a phase transition
which
markedly alters their permeability. The phase transition involves a change
from a
closely packed, ordered structure, known as the gel state, to a loosely
packed, less-
ordered structure, known as the fluid state. This occurs at a characteristic
phase-
transition temperature and results in an increase in permeability to ions,
sugars and
drugs.
Liposomes interact with cells via different mechanisms: endocytosis by
phagocytic cells of the reticuloendothelial system such as macrophages and
neutrophils; adsorption to the cell surface, either by nonspecific weak
hydrophobic or
electrostatic forces, or by specific interactions with cell-surface
components; fusion
with the plasma cell membrane by insertion of the lipid bilayer of the
liposome into
the plasma membrane, with simultaneous release of liposomal contents into the
cytoplasm; and by transfer of liposomal lipids to cellular or subcellular
membranes, or
vice versa, without any association of the liposome contents. Varying the
liposome
formulation can alter which mechanism is operative, although more than one can
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operate at the same time. Nanocapsules can generally entrap compounds in a
stable
and reproducible way. To avoid side effects due to intracellular polymeric
overloading, such ultrafine particles (sized around 0.1 1.1m) should be
designed using
polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate
nanoparticles that meet these requirements are contemplated for use herein,
and such
particles can be easily made.
Administration methods can be employed to decrease the exposure of selected
proteases to degradative processes, such as proteolytic degradation and
immunological intervention via antigenic and immunogenic responses. Examples
of
such methods include local administration at the site of treatment. Pegylation
of
therapeutics has been reported to increase resistance to proteolysis, increase
plasma
half-life, and decrease antigenicity and immunogenicity. Examples of
pegylation
methodologies are known in the art (see for example, Lu and Felix, Int. J
Peptide
Protein Res., 43: 127-138, 1994; Lu and Felix, Peptide Res., 6: 142-6, 1993;
Felix et
al., Int. J. Peptide Res., 46 : 253-64, 1995; Benhar etal., J Biol. Chem.,
269: 13398-
404, 1994; Brumeanu etal., J Immunol., 154: 3088-95, 1995; see also, Caliceti
etal.
(2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003)
Pharmacotherapy
23 (8 Pt 2):35-8S). Pegylation also can be used in the delivery of nucleic
acid
molecules in vivo. For example, pegylation of adenovirus can increase
stability and
gene transfer (see, e.g., Cheng et al. (2003) Pharm. Res. 20(9): 1444-51).
Desirable blood levels can be maintained by a continuous infusion of the
active agent as ascertained by plasma levels. It should be noted that the
attending
physician would know how to and when to terminate, interrupt or adjust therapy
to
lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions.
Conversely, the attending physician would also know how to and when to adjust
treatment to higher levels if the clinical response is not adequate
(precluding toxic
side effects).
Pharmaceutical compositions can be administered, for example, by oral,
pulmonary, parental (intramuscular, intraperitoneal, intravenous (IV) or
subcutaneous
injection), inhalation (via a fine powder formulation), transdermal, nasal,
vaginal,
rectal, or sublingual routes of administration and can be formulated in dosage
forms
appropriate for each route of administration (see, e.g., International PCT
application
Nos. WO 93/25221 and WO 94/17784; and European Patent Application 613,683).
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A selected protease is included in the pharmaceutically acceptable carrier in
an
amount sufficient to exert a therapeutically useful effect in the absence of
undesirable
side effects on the patient treated. Therapeutically effective concentration
can be
determined empirically by testing the compounds in known in vitro and in vivo
systems, such as the assays provided herein.
The concentration of a selected protease in the composition depends on
absorption, inactivation and excretion rates of the complex, the
physicochemical
characteristics of the complex, the dosage schedule, and amount administered
as well
as other factors known to those of skill in the art. The amount of a selected
protease
to be administered for the treatment of a disease or condition, for example
cancer or
angiogenesis treatment can be determined by standard clinical techniques. In
addition,
in vitro assays and animal models can be employed to help identify optimal
dosage
ranges. The precise dosage, which can be determined empirically, can depend on
the
route of administration and the seriousness of the disease.
A selected protease can be administered at once, or can be divided into a
number of smaller doses to be administered at intervals of time. Selected
proteases
can be administered in one or more doses over the course of a treatment time
for
example over several hours, days, weeks, or months. In some cases, continuous
administration is useful. It is understood that the precise dosage and
duration of
treatment is a function of the disease being treated and can be determined
empirically
using known testing protocols or by extrapolation from in vivo or in vitro
test data. It
is to be noted that concentrations and dosage values also can vary with the
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 the concentration
ranges
set forth herein are exemplary only and are not intended to limit the scope or
use of
compositions and combinations containing them. The compositions can be
administered hourly, daily, weekly, monthly, yearly or once. The mode of
administration of the composition containing the polypeptides as well as
compositions containing nucleic acids for gene therapy, includes, but is not
limited to
intralesional, intraperitoneal, intramuscular and intravenous administration.
Also
included are infusion, intrathecal, subcutaneous, liposome-mediated, depot-
mediated
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administration. Also included, are nasal, ocular, oral, topical, local and
otic delivery.
Dosages can be empirically determined and depend upon the indication, mode of
administration and the subject. Exemplary dosages include from 0.1, 1, 10,
100, 200
and more mg/day/kg weight of the subject.
2. In vivo Expression of Selected Proteases and Gene Therapy
Selected proteases can be delivered to cells and tissues by expression of
nucleic acid molecules. Selected proteases can be administered as nucleic acid
molecules encoding a selected protease, including ex vivo techniques and
direct in
vivo expression.
a. Delivery of Nucleic Acids
Nucleic acids can be delivered to cells and tissues by any method known to
those of skill in the art.
i. Vectors ¨ Episomal and Integrating
Methods for administering selected proteases by expression of encoding
.. nucleic acid molecules include administration of recombinant vectors. The
vector can
be designed to remain episomal, such as by inclusion of an origin of
replication or can
be designed to integrate into a chromosome in the cell. Recombinant vectors
can
include viral vectors and non-viral vectors. Non-limiting viral vectors
include, for
example, adenoviral vector, herpes virus vectors, retroviral vectors, and any
other
viral vector known to one of skill in the art. Non-limiting non-viral vectors
include
artificial chromosomes or liposomes or other non-viral vector. Selected
proteases also
can be used in ex vivo gene expression therapy using viral and non-viral
vectors. For
example, cells can be engineered to express a selected protease, such as by
integrating
a selected protease encoding-nucleic acid into a genomic location, either
operatively
linked to regulatory sequences or such that it is placed operatively linked to
regulatory
sequences in a genomic location. Such cells then can be administered locally
or
systemically to a subject, such as a patient in need of treatment.
A selected protease can be expressed by a virus, which is administered to a
subject in need of treatment. Virus vectors suitable for gene therapy include
adenovirus, adeno-associated virus, retroviruses, lentiviruses and others
noted above.
For example, adenovirus expression technology is well-known in the art and
adenovirus production and administration methods also are well known.
Adenovirus
serotypes are available, for example, from the American Type Culture
Collection
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(ATCC, Rockville, MD). Adenovirus can be used ex vivo, for example, cells are
isolated from a patient in need of treatment, and transduced with a selected
protease-
expressing adenovirus vector. After a suitable culturing period, the
transduced cells
are administered to a subject, locally and/or systemically. Alternatively,
selected
protease-expressing adenovirus particles are isolated and formulated in a
pharmaceutically-acceptable carrier for delivery of a therapeutically
effective amount
to prevent, treat or ameliorate a disease or condition of a subject.
Typically,
adenovirus particles are delivered at a dose ranging from 1 particle to 1014
particles
per kilogram subject weight, generally between 106 or 108 particles to 1012
particles
per kilogram subject weight. In some situations it is desirable to provide a
nucleic
acid source with an agent that targets cells, such as an antibody specific for
a cell
surface membrane protein or a target cell, or a ligand for a receptor on a
target cell.
ii. Artificial Chromosomes and Other Non-viral Vector
Delivery Methods
The nucleic acid molecules can be introduced into artificial chromosomes and
other non-viral vectors. Artificial chromosomes (see, e.g., U.S. Patent No.
6,077,697 and PCT International PCT application No. WO 02/097059) can be
engineered to encode and express the isoform.
= iii. Liposomes and Other Encapsulated Forms and
Administration of Cells Containing Nucleic Acids
The nucleic acids can be encapsulated in a vehicle, such as a liposome, or
introduced into cells, such as a bacterial cell, particularly an attenuated
bacterium or
introduced into a viral vector. For example, when liposomes are employed,
proteins
that bind to a cell Surface membrane protein associated with endocytosis can
be used
for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments
thereof
tropic for a particular cell type, antibodies for proteins which undergo
internalization
in cycling, and proteins that target intracellular localization and enhance
intracellular
half-life.
b. In vitro and Ex vivo Delivery
For ex vivo and in vivo methods, nucleic acid molecules encoding the selected
protease is introduced into cells that are from a suitable donor or the
subject to be
treated. Cells into which a nucleic acid can be introduced for purposes of
therapy
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include, for example, any desired, available cell type appropriate for the
disease or
condition to be treated, including but not limited to epithelial cells,
endothelial cells,
keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T
lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes, granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., such as stem cells obtained from
bone
marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources
thereof.
For ex vivo treatment, cells from a donor compatible with the subject to be
treated, or cells from the subject to be treated, are removed, the nucleic
acid is
introduced into these isolated cells and the modified cells are administered
to the
subject. Treatment includes direct administration, such as or, for example,
encapsulated within porous membranes, which are implanted into the patient
(see, e.g.
U.S. Pat. Nos. 4,892,538 and 5,283,187). Techniques suitable for the transfer
of
nucleic acid into mammalian cells in vitro include the use of liposomes and
cationic
lipids (e.g., DOTMA, DOPE and DC-Chol) eleetroporation, microinjection, cell
fusion, DEAE-dextran, and calcium phosphate precipitation methods. Methods of
DNA delivery can be used to express selected proteases in vivo. Such methods
include liposome delivery of nucleic acids and naked DNA delivery, including
local
and systemic delivery such as using electroporation, ultrasound and calcium-
phosphate delivery. Other techniques include microinjection, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene transfer and
spheroplast fusion.
In vivo expression of a selected protease can be linked to expression of
additional molecules. For example, expression of a selected protease can be
linked
with expression of a cytotoxic product such as in an engineered virus or
expressed in
a cytotoxic virus. Such viruses can be targeted to a particular cell type that
is a target
for a therapeutic effect. The expressed selected protease can be used to
enhance the
cytotoxicity of the virus.
In vivo expression of a selected protease can include operatively linking a
selected protease encoding nucleic acid molecule to specific regulatory
sequences
such as a cell-specific or tissue-specific promoter. Selected proteases also
can be
expressed from vectors that specifically infect and/or replicate in target
cell types
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and/or tissues. Inducible promoters can be used to selectively regulate
selected
protease expression.
c. Systemic, Local and Topical Delivery
Nucleic acid molecules, as naked nucleic acids or in vectors, artificial
chromosomes, liposomes and other vehicles can be administered to the subject
by
systemic administration, topical, local and other routes of administration.
When
systemic and in vivo, the nucleic acid molecule or vehicle containing the
nucleic acid
molecule can be targeted to a cell.
Administration also can be direct, such as by administration of a vector or
cells that typically targets a cell or tissue. For example, tumor cells and
proliferating
cells can be targeted cells for in vivo expression of selected proteases.
Cells used for
in vivo expression of an isofonn also include cells autologous to the patient.
Such
cells can be removed from a patient, nucleic acids for expression of a
selected
protease introduced, and then administered to a patient such as by injection
or
.. engraftment.
2. Combination Therapies
Any of the selected protease polypeptides, and nucleic acid molecules
encoding selected protease polypeptides described herein can be administered
in
combination with, prior to, intermittently with, or subsequent to, other
therapeutic
.. agents or procedures including, but not limited to, other biologics, small
molecule
compounds and surgery. For any disease or condition, including all those
exemplified
above, for which other agents and treatments are available, selected protease
polypeptides for such diseases and conditions can be used in combination
therewith.
For example, selected protease polypeptides provided herein for the treatment
of a
proliferative disease for example, cancer, can be administered in combination
with,
prior to, intermittently with, or subsequent to, other anti-cancer therapeutic
agents, for
example chemotherapeutic agents, radionuclides, radiation therapy, cytokines,
growth
factors, photosensitizing agents, toxins, anti-metabolites, signaling
modulators, anti-
cancer antibiotics, anti-cancer antibodies, angiogenesis inhibitors, or a
combination
thereof. In a specific example, selected protease polypeptides provided herein
for the
treatment of thrombotic diseases can be administered in combination with,
prior to,
intermittently with, or subsequent to, other anticoagulant agents including,
but not
limited to, platelet inhibitors, vasodilators, fibrolytic activators, or other
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anticoagulants. Exemplary anticoagulants include heparin, coumarin, hinidin,
aspirin,
naproxen, meclofenamic acid, ibuprofen, indomethacin, phenylbutazare,
ticlopidine,
streptokinase, urokinase, and tissue plasminogen activator.
3. Articles of Manufacture and Kits
Pharmaceutical compounds of selected protease polypeptides for nucleic acids
encoding selected protease polypeptides, or a derivative or a biologically
active
portion thereof can be packaged as articles of manufacture containing
packaging
material, a pharmaceutical composition which is effective for treating the
disease or
disorder, and a label that indicates that selected protease polypeptide or
nucleic acid
molecule is to be used for treating the disease or disorder.
The articles of manufacture provided herein contain packaging materials.
Packaging materials for use in packaging pharmaceutical products are well
known to
those of skill in the art. See, for example, U.S. Patent Nos. 5,3,23,907,
5,052,558 and
5,033,252. Examples of
pharmaceutical packaging materials include, but are not limited to, blister
packs,
bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles,
and any
packaging material suitable for a selected formulation and intended mode of
administration and treatment. A wide array of formulations of the compounds
and
compositions provided herein are contemplated as are a variety of treatments
for any
. 20 target-mediated disease or disorder.
Selected protease polypeptides and nucleic acid molecules also can be
= provided as kits. Kits can include a pharmaceutical composition described
herein and
an item for administration. For example a selected protease can be supplied
with a
device for administration, such as a syringe, an inhaler, a dosage cup, a
dropper, or an
applicator. The kit can, optionally, include instructions for application
including
dosages, dosing regimens and instructions for modes of administration. Kits
also can
include a pharmaceutical composition described herein and an item for
diagnosis. For
example, such kits can include an item for measuring the concentration, amount
or
activity of the selected protease in a subject.
J. EXEMPLARY METHODS OF TREATMENT WITH SELECTED
PROTEASE POLYPEPTIDES
The selected protease polypeptides provided herein that cleave particular
targets and nucleic acid molecules that encode the selected proteases provided
herein
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can be used for treatment of any disease or condition associated with a
protein
containing the target sequence or for which a protease that cleaves the target
sequence
is employed. For example, selected uPA polypeptides engineered to cleave tPA
target
substrates, such as plasminogen, can be used for treatment of any disease or
condition
associated with the tPA target substrate or for which tPA polypeptides are
employed.
Exemplary diseases associated with a tPA target substrate include tbrombolytic
diseases where treatment with a selected protease provided herein can promote
cleavage of plasminogen to its active protease form plasmin, and induce
dissolution of
a blot clot.
Selected protease polypeptides have therapeutic activity alone or in
combination with other agents. The selected protease polypeptides provided
herein
are designed to exhibit improved properties over competing binding proteins.
Such
properties, for example, can improve the therapeutic effectiveness of the
polypeptides.
This section provides exemplary uses of and administration methods. These
described therapies are exemplary and do not limit the applications of
selected
protease polypeptides.
The selected protease polypeptides provided herein can be used in various
therapeutic as well as diagnostic methods that are associated with a protein
containing
the target sequence. Such methods include, but are not limited to, methods of
treatment of physiological and medical conditions described and listed below.
Selected protease polypeptides provided herein can exhibit improvement of in
vivo
activities and therapeutic effects compared to competing binding proteins or a
protease that cleaves the particular target, including lower dosage to achieve
the same
effect, a more sustained therapeutic effect and other improvements in
administration
and treatment. Examples of therapeutic improvements using selected protease
polypeptides include, but are not limited to, better target tissue penetration
(e.g. tumor
penetration), higher effectiveness, lower dosages, fewer and/or less frequent
administrations, decreased side effects and increased therapeutic effects.
Notably,
because the selected proteases can cleave and inactivate high numbers of the
target
substrate, the selected proteases offer substantial therapeutic amplification.
In particular, selected protease polypeptides, are intended for use in
therapeutic methods in which a protease that cleaves the particular target has
been
used for treatment. Such methods include, but are not limited to, methods of
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treatment of diseases and disorders, such as, but not limited to, blood
coagulation
disorders, including thrombolytic disorders and disseminated intravascular
coagulation, cardiovascular diseases, neurological disorders, proliferative
diseases,
such as cancer, inflammatory diseases, autoimmune diseases, viral infection,
bacterial
infection, respiratory diseases, gastrointestinal disorders, and metabolic
diseases.
Treatment of diseases and conditions with selected protease polypeptides can
be effected by any suitable route of administration using suitable
formulations as
described herein including, but not limited to, intramuscular, intravenous,
intradermal,
intraperitoneal injection, subcutaneous, epidural, nasal oral, rectal,
topical,
inhalational, buccal (e.g., sublingual), and transdermal administration. If
necessary, a
particular dosage and duration and treatment protocol can be empirically
determined
or extrapolated. For example, exemplary doses of wild-type protease
polypeptides
that cleave similar sequences can be used as a starting point to determine
appropriate
dosages. For example, a dosage of a recombinant tPA polypeptide can be used as
a
guideline for determining dosages of selected uPA polypeptides that cleave tPA
targets.
Dosage levels and regimens can be determined based upon known dosages
and regimens, and, if necessary can be extrapolated based upon the changes in
properties of the selected protease polypeptides and/or can be determined
empirically
based on a variety of factors. Factors such as the level of activity and half-
life of the
selected protease polypeptides in comparison to other similar proteases can be
used in
making such determinations. Particular dosages and regimens can be empirically
determined. Other such factors include body weight of the individual, general
health,
age, the activity of the specific compound employed, sex, diet, time of
administration,
.. rate of excretion, drug combination, the severity and course of the
disease, and the
patient's disposition to the disease and the judgment of the treating
physician. The
active ingredient, the selected protease polypeptide, typically is combined
with a
pharmaceutically effective carrier. The amount of active ingredient that can
be
combined with the carrier materials to produce a single dosage form or multi-
dosage
form can vary depending upon the host treated and the particular mode of
administration.
The effect of the selected protease polypeptides on the treatment of a disease
or amelioration of symptoms of a disease can be monitored using any diagnostic
test
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known in the art for the particular disease to be treated. Upon improvement of
a
patient's condition, a maintenance dose of a compound or compositions can be
administered, if necessary; and the dosage, the dosage form, or frequency of
administration, or a combination thereof can be modified. In some cases, a
subject
can require intermittent treatment on a long-term basis upon any recurrence of
disease
symptoms or based upon scheduled dosages. In other cases, additional
administrations can be required in response to acute events such as
hemorrhage,
trauma, or surgical procedures.
In some examples, variants of the selected protease proteins that function as
either protease agonists (i.e., mimetics) or as protease antagonists are
employed.
Variants of the selected protease polypeptide can be generated by mutagenesis
(e.g.,
discrete point mutation or truncation of the protease protein). An agonist of
the
selected protease polypeptide can retain substantially the same, or a subset
of, the
biological activities of the naturally occurring form of the selected protease
polypeptide. An antagonist of the selected protease polypeptide can inhibit
one or
more of the activities of the naturally occurring form of the selected
protease
polypeptide by, for example, cleaving the same target protein as the selected
protease
polypeptide. Thus, specific biological effects can be elicited by treatment
with a
variant of limited function. In one embodiment, treatment of a subject with a
variant
having a subset of the biological activities of the naturally occurring form
of the
selected protease polypeptide has fewer side effects in a subject relative to
treatment
with the naturally occurring form of the selected protease polypeptide.
The following are some exemplary diseases or conditions for which selected
proteases can be used as a treatment agent alone or in combination with other
agents.
Exemplary targets for selection of proteases are for illustrative purposes and
not
intended to limit the scope of possible targets for use in the methods
provided herein.
1. Exemplary Methods
of Treatment for Selected uPA Polypeptides
That Cleave tPA Targets
Selected uPA polypeptides that cleave tPA target sequences are useful in
therapeutic applications for use in ameliorating thrombotic disorders
including both
acute and chronic conditions. Acute conditions include among others both heart
attack and stroke while chronic situations include those of arterial and deep
vein
thrombosis and restenosis. The selected uPA polypeptides can be used as
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thrombolytic therapeutic agents for ameliorating the symptoms of such
conditions.
Therapeutic compositions include the polypeptides, cDNA molecules alone or
part of
a viral vector delivery system or other vector-based gene expression delivery
system,
presented in a liposome delivery system and the like. A composition for use as
a
thrombolytic therapeutic agent generally is a physiologically effective amount
of the
selected uPA polypeptides in a pharmaceutically suitable excipient. Depending
on the
mode of administration and the condition to be treated, the thrombolytic
therapeutic
agents are administered in single or multiple doses. One skilled in the art
will
appreciate that variations in dosage depend on the condition to be treated.
Selected uPA polypeptides provided herein that inhibit or antagonize blood
coagulation can be used in anticoagulant methods of treatment for ischemic
disorders,
such as a peripheral vascular disorder, a pulmonary embolus, a venous
thrombosis,
deep vein thrombosis (DVT), superficial thrombophlebitis (SVT), arterial
thrombosis,
a myocardial infarction, a transient ischemic attack, unstable angina, a
reversible
ischemic neurological deficit, an adjunct thrombolytic activity, excessive
clotting
conditions, reperfusion injury, sickle cell anemia or stroke disorder. In
patients with
an increased risk of excessive clotting, such as DVT or SVT, during surgery,
protease
inactive selected uPA polypeptides provided herein can be administered to
prevent
excessive clotting in surgeries, such as, but not limited to heart surgery,
angioplasty,
lung surgery, abdominal surgery, spinal surgery, brain surgery, vascular
surgery, or
organ transplant surgery, including transplantation of heart, lung, pancreas,
or liver.
In some cases treatment is performed with selected uPA polypeptides alone. In
some
cases, selected uPA polypeptides are administered in conjunction with
additional
anticoagulation factors as required by the condition or disease to be treated.
tPA is the only therapy for acute thromboembolic stroke, which is approved
by the Food.and Drug Administration (FDA). tPA and variants thereof are
commercially available and have been approved for administration to humans for
a
variety of conditions. For example alteplase (Activase , Genentech, South San
Francisco, Calif.) is recombinant human tPA. Reteplase (Retavase , Rapilysin0;
Boehringer Mannheim, Roche Centoror) is a recombinant non-glycosylated form of
human tPA in which the molecule has been genetically engineered to contain 355
of
the 527 amino acids of the original protein. Tenecteplase (TNKase , Genentech)
is a
527 amino acid glycoprotein derivative of human tPA that differs from
naturally
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occurring human tPA by having three amino acid substitutions. These
substitutions
decrease plasma clearance, increase fibrin binding (and thereby increase
fibrin
specificity), and increase resistance to plasminogen activator inhibitor-1
(PAI-1).
Anistreplase (Eminase , SmithKline Beecham) is yet another commercially
available
human tPA. Selected uPA polypeptides provided herein with specificity toward
tPA
targets can be similarly modified and prescribed for any therapy that is
treatable with
tPA.
a. Thrombotic Diseases and Conditions
Thrombotic diseases are characterized by hypercoagulation, or the
deregulation of hemostasis in favor of development of blot clots. Exemplary
thrombotic diseases and conditions include arterial thrombosis, venous
thrombosis,
venous thromboembolism, pulmonary embolism, deep vein thrombosis, stroke,
ischemic stroke, myocardial infarction, unstable angina, atrial fibrillation,
renal
damage, percutaneous translumenal coronary angioplasty, disseminated
intravascular
coagulation, sepsis, artificial organs, shunts or prostheses, and other
acquired
thrombotic diseases, as discussed below. Typical therapies for thrombotic
diseases
involve anticoagulant therapies, including inhibition of the coagulation
cascade.
The selected uPA polypeptides provided herein and the nucleic acids encoding
the selected uPA polypeptides provided herein can be used in anticoagulant
therapies
for thrombotic diseases and conditions, including treatment of conditions
involving
intravascular coagulation. The selected uPA polypeptides provided herein the
can
inhibit blood coagulation can be used, for example, to control, dissolve, or
prevent
formation of thromboses. In a particular embodiment, the selected uPA
polypeptides
herein, and nucleic acids encoding selected uPA polypeptides can be used for
treatment of an arterial thrombotic disorder. In another embodiment, the
selected uPA
polypeptides herein, and nucleic acids encoding modified selected uPA
polypeptides
can be used for treatment of a venous thrombotic disorder, such as deep vein
thrombosis. In a particular embodiment, the selected uPA polypeptides herein,
and
nucleic acids encoding selected uPA polypeptides can be used for treatment of
an
ischemic disorder, such as stroke. Examples of therapeutic improvements using
selected uPA polypeptides include for example, but are not limited to, lower
dosages,
fewer and/or less frequent administrations, decreased side effects, and
increased
therapeutic effects. Selected uPA polypeptides can be tested for therapeutic
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effectiveness, for example, by using animal models. For example mouse models
of
ischemic stroke, or any other known disease model for a thrombotic disease or
condition, can be treated with selected uPA polypeptides (Dodds, Ann NY Acad
Sci
516: 631-635(1987)). Progression of disease symptoms and phenotypes is
monitored
to assess the effects of the selected uPA polypeptides. Selected uPA
polypeptides
also can be administered to animal models as well as subjects such as in
clinical trials
to assess in vivo effectiveness in comparison to placebo controls.
=i. Arterial Thrombosis
Arterial thrombi form as a result of a rupture in the arterial vessel wall.
Most
often the rupture occurs in patients with vascular disease, such as
atherosclerosis. The
arterial thrombi usually form in regions of disturbed blood flow and at sites
of rupture
due to an atherosclerotic plaque, which exposes the thrombogenic
subendothelium to
platelets and coagulation proteins, which in turn activate the coagulation
cascade.
Plaque rupture also can produce further narrowing of the blood vessel due to
hemorrhage into the plaque. Nonocclusive thrombi can become incorporated into
the
vessel wall and can accelerate the growth of atherosclerotic plaques.
Formation
arterial thrombi can result in ischemia either by obstructing flow or by
embolism into
the distal microcirculation. Anticoagulants and drugs that suppress platelet
function
and the coagulation cascade can be effective in the prevention and treatment
of
arterial thrombosis. Such classes of drugs are effective in the treatment of
arterial
thrombosis. Arterial thrombosis can lead to conditions of unstable angina and
acute
myocardial infarction. Selected uPA polypeptides provided herein that inhibit
coagulation can be used in the treatment and/or prevention of arterial
thrombosis and
conditions, such as unstable angina and acute myocardial infarction.
ii. Venous Thrombosis and Thromboembolism
Venous thrombosis is a condition in which a blood clot forms in a vein due to
imbalances in the signals for clot formation versus clot dissolution,
especially in
instances of low blood flow through the venous system. Results of thrombus
formation can include damage to the vein and valves of the vein, though the
vessel
wall typically remains intact. The clots can often embolize, or break off, and
travel
through the blood stream where they can lodge into organ areas such as the
lungs
(pulmonary embolism), brain (ischemic stroke, transient ischemic attack),
heart (heart
attack/myocardial infarction, unstable angina), skin (purpura fiilminans), and
adrenal
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gland. In some instances the blockage of blood flow can lead to death.
Patients with
a tendency to have recurrent venous thromboembolism are characterized as
having
thrombophilia. Risk factors for developing thromboembolic disease include
trauma,
immobilization, malignant disease, heart failure, obesity, high levels of
estrogens, leg
paralysis, myocardial infarction, varicose veins, cancers, dehydration,
smoking, oral
contraceptives, and pregnancy. Genetic studies of families with thrombophilia
have
shown inheritable high levels coagulation factors, including FVIII, FIX, and
FXI
(Lavigne et al. J. Thromb. Haemost. 1:2134-2130 (2003)).
Deep vein thrombosis (DVT) refers the formation of venous blot clot in the
deep leg veins. The three main factors that contribute to DVT are injury to
the vein
lining, increased tendency for the blood to clot and slowing of blood flow.
Collectively, these factor are called Virchow's triad. Veins can become
injured during
trauma or surgery, or as a result of disease condition, such as Buerger's
disease or
DIC, or another clot. Other contributing factors to development of DVT are
similar to
that of more general thromboembolic diseases as discussed above. The clot that
forms in DVT causes only minor inflammation, thus, allowing it to break loose
into
the blood stream more easily. Often the thrombus can break off as a result of
minor
contraction of the leg muscles. Once the thrombus becomes an embolus it can
become lodged into vessels of the lungs where is can cause a pulmonary
infarction.
Patients with high levels of active FIX in their bloodstream are at an
increased risk of
developing deep vein thrombosis (Weltermann et al. J. Thromb. Haemost. 1(1):
16-18
(2003)).
Thromboembolic disease can be hereditary, wherein the disease is caused by
hereditary abnormalities in clotting factors, thus leading to the imbalance in
hemostasis. Several congenital deficiencies include antithrombin III, protein
C,
protein S, or plasminogen. Other factors include resistance to activated
protein C
(also termed APC resistance or Factor V leiden effect, in which a mutation in
factor V
makes it resistant to degradation by protein C), mutation in prothrombin,
dysfibrinogemia (mutations confer resistance to fibrinolysis), and
hyperhomocysteinemia. Development of thromboembolic disease in younger
patients
is most often due to the congenital defects described above and is called
Juvenile
Thrombophilia.
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Treatments for venous thromboembolic disease and DVT typically involve
anticoagulant therapy, in which oral doses of heparin and warfarin are
administered.
Heparin is usually infused into patients to control acute events, followed by
longer
term oral anticoagulant therapy with warfarin to control future episodes.
Other
therapies include direct thrombin inhibitors, inhibitors of platelet function,
such as
aspirin and dextran, and therapies to counteract venous stasis, including
compression
stockings and pneumatic compression devices. Selected uPA polypeptides
provided
herein that inhibit blood coagulation can be used in anticoagulant therapies
for
thromboembolic disease and/or DVT. In some embodiments, selected uPA
polypeptides provided herein that inhibit blood coagulation can be used in
prevention
therapies thromboembolic disease and/or DVT in patients exhibiting risk
factors for
thromboembolic disease and/or DVT.
(a) Ischemic stroke
Ischemic stroke occurs when the blood flow to the brain is interrupted,
wherein the sudden loss of circulation to an area of the brain results in a
corresponding loss of neurologic function. In contrast to a hemorrhagic
stroke, which
is characterized by intracerebral bleeding, an ischemic stroke is usually
caused by
thrombosis or embolism. Ischemic strokes account for approximately 80% of all
strokes. In addition to the causes and risk factors for development a
thromboembo-
lism as discussed above, processes that cause dissection of the cerebral
arteries (e.g.,
trauma, thoracic aortic dissection, arteritis) can cause thrombotic stroke.
Other causes
include hypoperfusion distal to a stenotic or occluded artery or hypoperfusion
of a
vulnerable watershed region between 2 cerebral arterial territories.
Treatments for
ischemic stroke involve anticoagulant therapy for the prevention and treatment
of the
condition. Selected uPA polypeptides provided herein that inhibit coagulation
can be
used in the treatment and/or prevention or reduction of risk of ischemic
stroke.
iii. Acquired Coagulation Disorders
Acquired coagulation disorders are the result of conditions or diseases, such
as
vitamin K deficiency, liver disease, disseminated intravascular coagulation
(DIC), or
development of circulation anticoagulants. The defects in blood coagulation
are the
result of secondary deficiencies in clotting factors caused by the condition
or disease.
For example, production of coagulation factors from the liver is often
impaired when
the liver is in a diseased state. Along with decreased synthesis of
coagulation factors,
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fibrinolysis becomes increased and thrombocytopenia (deficiency in platelets)
is
increased. Decreased production of coagulation factors by the liver also can
result
from fulminant hepatitis or acute fatty liver of pregnancy. Such conditions
promote
intravascular clotting which consumes available coagulation factors. Selected
uPA
polypeptides provided herein can be used in the treatment of acquired
coagulation
disorders in order to alleviate deficiencies in blood clotting factors.
(a) Disseminated Intravascular Coagulation
(DIC)
Disseminated intravascular coagulation (DIC) is a disorder characterized by a
widespread and ongoing activation of coagulation. In DIC, there is a loss of
balance
between thrombin activation of coagulation and plasmin degradation of blot
clots.
Vascular or microvascular fibrin deposition as a result can compromise the
blood
supply to various organs, which can contribute to organfailure. In sub-acute
or
chronic DIC, patients present with a hypercoagulatory phenotype, with
thromboses
from excess thrombin formation, and the symptoms and signs of venous
thrombosis
can be present. In contrast to acute DIC, sub-acute or chronic DIC is treated
by
methods of alleviating the hyperthrombosis, including heparin, anti-thrombin
III and
activated protein C treatment. The selected uPA polypeptides provided herein
and the
nucleic acids encoding the selected uPA polypeptides provided herein can be
used in
therapies for sub-acute or chronic DIC. In one embodiment, the sub-acute or
chronic
DIC polypeptides herein, and nucleic acids encoding the selected uPA
polypeptides
can be used in combination with other anticoagulation therapies. Selected uPA
polypeptides can be tested for therapeutic effectiveness, for example, by
using animal
models. Progression of disease symptoms and phenotypes is monitored to assess
the
effects of the selected uPA polypeptides. Selected uPA polypeptides also can
be
administered to animal models as well as subjects such as in clinical trials
to assess in
vivo effectiveness in comparison to placebo controls.
(b) Bacterial Infection and Periodontitis
Systemic infection with microorganisms, such as bacteria, is commonly
associated with DIC. The upregulation of coagulation pathways can be mediated
in
part by cell membrane components of the microorganism (lipopolysaccharide or
endotoxin) or bacterial exotoxins (e.g. staphylococcal alpha toxin) that cause
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inflammatory responses leading to elevated levels of cytokines. The cytokines,
in
turn, can influence induction of coagulation.
Bacterial pathogens, such as Porphyrus gingivalis, are well-known as
causative agents for adult periodontitis. The Porphyrus gingivalis bacterium
produces
arginine-specific cysteine proteinases that function as virulence factors
(Grenier et al.
J. Clin. Microbiol. 25:738-740 (1987), Smalley et al. Oral Microbiol. Immunol.
4:178-181 (1989), Marsh, et al. FEMS Microbiol. 59:181-185 (1989), and Potempa
et
al. J Biol. Chem. 273:21648-21657 (1998). Porphyrus gingivalis generated two
proteinases that are referred to as 50 kDa and 95 kDa gingipains R (RgpB and
HRgpA, respectively). The protease can proteolytically cleave and hence
activate
coagulation factors. During bacterial infection release of the gingipains R
into the
blood stream can thus lead to uncontrolled activation of the coagulation
cascade
leading to overproduction of thrombin and increase the possibility of inducing
disseminated intravascular coagulation (DIC). The large increases in thrombin
concentrations can furthermore contribute alveolar bone resorption by
osteoclasts at
sites of periodontitis.
The selected uPA polypeptides provided herein that inhibit blood coagulation,
and nucleic acids encoding selected uPA polypeptides can be used in treatment
of
periodontitis. Selected uPA polypeptides can be tested for therapeutic
effectiveness
for airway responsiveness in periodontitis models. Such models are available
in
animals, such as nonhuman primates, dogs, mice, rats, hamsters, and guinea
pigs
(Weinberg and Bral, I of Periodontology 26(6), 335-340). Selected uPA
polypeptides also can be administered to animal models as well as subjects
such as in
clinical trials to assess in vivo effectiveness in comparison to placebo
controls.
b. Other tPA Target-associated Conditions
The selected uPA polypeptides provided herein also can be used in treatment
of neurological conditions for which tPA had been implicated. tPA is thought
to
regulate physiological processes that include tissue remodeling and plasticity
due to
the ability of tPA to hydrolyze extracellular matrix proteins and other
substrates
(Gravanis and Tsirska (2004) Glia 49:177-183). Patients who have experienced
events such as stroke or injury (e.g., due to accident or surgery) often
suffer from
neurological damage that can be treatable with selected uPA polypeptides
provided
herein, The selected uPA polypeptides provided herein can be useful for
treating'
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subjects suffering from a variety of neurological diseases and conditions
including,
but not limited to, neurodegenerative diseases such as multiple sclerosis,
amyotrophic
lateral sclerosis, subacute sclerosing panencephalitis, Parkinson's disease,
Huntington's disease, muscular dystrophy, and conditions caused by nutrient
deprivation or toxins (e.g., neurotoxins, drugs of abuse). Additionally,
selected uPA
polypeptides can be useful for providing cognitive enhancement and/or for
treating
cognitive decline, e.g., "benign senescent forgetfulness", "age-associated
memory
impairment", "age-associated cognitive decline", etc. (Petersen et al., J
Immunological
Meth. 257:107-116 (2001)), and Alzheimer's disease.
Selected uPA polypeptides can be tested using any of a variety of animal
models for injury to the nervous system. Models that can be used include, but
are not
limited to, rodent, rabbit, cat, dog, or primate models for thromboembolic
stroke
(Krueger and Busch, Invest. Radiol. 37:600-8 (2002); Gupta and Briyal, Indian
J
Physiol. Pharmacol. 48:379-94 (2004)), models for spinal cord injury (Webb et
al.,
Vet. Rec. 155:225-30 (2004)), etc. The methods and compositions also can be
tested
in humans. A variety of different methods, including standardized tests and
scoring
systems, are available for assessing recovery of motor, sensory, behavioral,
and/or
cognitive function in animals and humans. Any suitable method can be used. In
one
example, the American Spinal Injury Association score, which has become the
principal instrument for measuring the recovery of sensory function in humans,
could
be used. See, e.g., Martinez-Arizala, J Rehabil. Res. Dev. 40:35-9 (2003),
Thomas and
Noga, J Rehabil. Res. Dev. 40:25-33 (2003), Kesslak and Keirstead, J Spinal
Cord
Med 26:323-8 (2003) for examples of various scoring systems and methods.
Preferred dose ranges for use in humans can be established by testing the
agent(s) in
tissue culture systems and in animal models taking into account the efficacy
of the
agent(s) and also any observed toxicity.
c. Diagnostic Methods
Selected uPA polypeptides provided herein can be used in diagnostic methods
including, but not limited to, diagnostic assays to detect fibrin and fibrin
degradation
products that have altered activities. The assays are thus indicated in
thrombotic
conditions. Other diagnostic applications, include kits containing antibodies
against
the selected uPA polypeptides and are familiar to one of ordinary skill in the
art.
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2. Exemplary Methods of Treatment for Selected Protease
Polypeptides That Cleave VEGF or VEGFR Targets
Vascular endothelial growth factor (VEGF) is a cytokine that binds and
signals through a specific cell surface receptor (VEGFR) to regulate
angiogenesis, the
process in which new blood vessels are generated from existing vasculature.
Pathological angiogenesis describes the increased vascularization associated
with
disease and includes events such as the growth of solid tumors (McMahon,
(2000)
Oncologist. 5 Suppl 1:3-10), macular degeneration and diabetes. In cancer,
solid
tumors require an ever-increasing blood supply for growth and metastasis.
Hypoxia
or oncogenic mutation increases the levels of VEGF and VEGF-R mRNA in the
tumor and surrounding stromal cells leading to the extension of existing
vessels and
formation of a new vascular network. In wet macular degeneration, abnormal
blood
vessel growth forms beneath the macula. These vessels leak blood and fluid
into the
macula damaging photoreceptor cells. In diabetes, a lack of blood to the eyes
also can
lead to blindness. VEGF stimulation of capillary growth around the eye leads
to
disordered vessels which do not function properly.
Three tyrosine kinase family receptors of VEGF have been identified (VEGF-
R-1/Flt-1, VEGF-R-2/F1k-1/KDR, VEGF-R-3/Flt-4). KDR (the mouse homolog is
Flk-1) is a high affinity receptor of VEGF with a Kd of 400-800 pM
(Waltenberger,
(1994)J Biol Chem. 269(43):26988-95) expressed exclusively on endothelial
cells.
VEGF and KDR association has been identified as a key endothelial cell-
specific
signaling pathway required for pathological angiogenesis (Kim, (1993) Nature.
362
(6423):841-4; Millauer, (1994) Nature. 367 (6463):576-9; Yoshiji, (1999)
Hepatology. 30(5): 1179-86). Dimerization of the receptor upon ligand binding
causes
autophosphorylation of the cytoplasmic domains, and recruitment of binding
partners
that propagate signaling throughout the cytoplasm and into the nucleus to
change the
cell growth programs. Treatment of tumors with a soluble VEGF-R2 inhibits
tumor
growth (Lin, (1998) Cell Growth Difftr. 9(1):49-58), and chemical inhibition
of
phosphorylation causes tumor cells to become apoptotic (Shaheen, (1999) Cancer
Res. 59(21):5412-6).
Signaling by vascular endothelial growth factor (VEGF) and its receptors is
implicated in pathological angiogenesis and the rapid development of tumor
vasculature in cancer. Drugs that block this signaling pathway prevent the
growth and
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maintenance of tumor blood supply, and lead to the systematic death of the
tumor.
The recent success of the anti-VEGF antibody AVASTINTm in patients with
metastatic colon cancer has validated VEGF as a target for anti-angiogenic
therapy of
cancer. Despite these encouraging results, tumor progression has still
occurred =
despite anti-VEGF treatment. The mechanisms of antibody affecting VEGF
function
and how the antibody impedes tumor growth are unknown. Knock down experiments
show that blocking VEGF function blocks angiogenesis. Thus the inhibition of
angiogenic signaling through VEGFR-2 represents an underdeveloped therapeutic
area ideal for the development of engineered proteases with novel targeting.
Therapies targeting the VEGF receptors and Flk-1/KDR specifically have
inhibited pathological angiogenesis and shown reduction of tumor size in
multiple
mouse models of human and mouse solid tumors (Prewett, (1999) Cancer Res.
59(20):5209-18; Fong, (1999) Neoplasia 1(1):31-41. Erratum in: (1999)
Neoplasia
1(2):183) alone and in combination with cytotoxic therapies (Klement, (2000) J
Clin
.. Invest. 105(8):R15-24). Studies with small molecule inhibitors and
antibodies validate
the VEGF receptor family as a potent anti-angiogenesis target but more
effective
therapeutics are still needed.
VEGFR is composed of an extracellular region of seven immunoglobin (Ig)-
like domains, a transmembrane region, and two cytoplasmic tyrosine kinase
domains.
The first three Ig-like domains have been shown to regulate ligand binding,
while
domains 4 through 7 have a role in inhibiting correct dimerization and
signaling in the
absence of ligand. As a target for selective proteolysis by engineered
proteases, it has
the following promising target characteristics: a labile region of amino acids
accessible to proteolysis; high sequence identity between the human, rat and
mouse
species; down regulation of signaling upon cleavage; and proteolytic
generation of
soluble receptors able to non-productively bind ligand. Several regions of
VEGF-R2
are available for specific proteolysis including the stalk region before the
transmembrane region and unstructured loop between Ig-like domains. In one
example, serine-like proteases provided herein can be engineered to cleave
specific
target receptors between their transmembrane and cytokine or growth factor
binding
domains (e.g. VEGFR). The stalk regions that function to tether protein
receptors to
the surface of a cell or loop regions are thereby disconnected from the
globular
domains in a polypeptide chain.
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a. Angiogenesis, Cancer, and Other Cell Cycle Dependent
Diseases or Conditions
Exemplary selected proteases provided herein cleave a VEGF or VEGFR
which are responsible for modulation of angiogenesis. Where the cell surface
.. molecule is a VEGFR signaling in tumor angiogenesis, cleavage prevents the
spread
of cancer. For example, cleavage of a cell surface domain from a VEGFR
molecule
can inactivate its ability to transmit extracellular signals, especially cell
proliferation
signals. Without angiogenesis to feed the tumor, cancer cells often cannot
proliferate.
In one embodiment, a selected protease provided herein is therefore used to
treat
cancer. Also, cleavage of VEGFR can be used to modulate angiogenesis in other
pathologies, such as macular degeneration, inflammation and diabetes. In one
embodiment, cleaving a target VEGF or VEGFR protein involved in cell cycle
progression inactivates the ability of the protein to allow the cell cycle to
go forward.
Without the progression of the cell cycle, cancer cells can not proliferate.
Therefore,
the selected proteases provided herein which cleave VEGF or VEGFR are used to
treat cancer and other cell cycle dependent pathologies.
Selected proteases provided herein also can cleave soluble proteins that are
responsible for tumorigenicity. Cleaving VEGF polypeptide prevents signaling
through the VEGF receptor and decreases angiogenesis, thus decreasing disease
in
which angiogenesis plays a role, such as cancer, macular degeneration,
inflammation
and diabetes. Further, VEGF signaling is responsible for the modulation of the
cell
cycle in certain cell types. Therefore, the selected proteases provided herein
which
cleave VEGF are useful in the treatment of cancer and other cell cycle
dependent
pathologies.
b. Combination Therapies with Selected Proteases That
Cleave VEGF or VEGFR
In one embodiment, treatment of a pathology, such as a cancer, involves
administration to a subject in need thereof therapeutically effective amounts
of a
protease that specifically cleaves and inactivates the signaling of the
VEGFNEGFR-2
complex, such as in combination with at least one anti-cancer agent.
Antiangiogenic
therapy has proven successful against both solid cancers and hematological
malignancies. (See, e.g., Ribatti et al. (2003) J Hematother Stem Cell Res.
12(1), 11-
22). Therefore, compositions provided herein as antiangiogenic therapy can
facilitate
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- the treatment-of both hematological and sold tissue malignancies.
Compositions and
methods of treatment provided herein can be administered alone or in
combination
with any other appropriate anti-cancer treatment known to one skilled in the
art. For
example, the selected proteases provided herein can be administered in
combination
with or in place of AVASTINTm in any therapy where AVASTINTm administration
provides therapeutic benefit.
In one embodiment, the anti-cancer agent is at least one chemotherapeutic
agent. In a related embodiment, the administering of the protease is in
combination
with at least one radiotherapy. Administration of the combination therapy will
attenuate the angiogenic signal and create a pool of soluble receptor that
lowers free
VEGF levels. In a specific embodiment, a selected protease provided herein has
an in
vitro specificity that matches a critical region of the receptor, the Flk-UKDR
stalk,
over a six amino acid region.
The selected protease polypeptides provided herein can be administered in a
composition containing more than one therapeutic agent. The therapeutic agents
can
be the same or different, and can be, for example, therapeutic radionuclides,
drugs,
hormones, hormone antagonists, receptor antagonists, enzymes or proenzymes
activated by another agent, autocrines, cytokines or any suitable anti-cancer
agent
known to those skilled in the art. In one embodiment, the anti-cancer agent co-
administered with the selected protease polypeptide is AVASTINTm. Other
therapeutic agents useful in the methods provided herein include toxins, anti-
DNA,
anti-RNA, radiolabeled oligonucleotides, such as antisense oligonucleotides,
anti-
protein and anti-chromatin cytotoxic or antimicrobial agents. Other
therapeutic
agents are known to those skilled in the art, and the use of such other
therapeutic
agents in accordance with the method provided herein is specifically
contemplated.
The antitumor agent can be one of numerous chemotherapy agents such as an
alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, an
antibody, an
anti-cancer biological, GleeveCc, colchicine, a vinca alkaloid, L-
asparaginase,
procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide.
Suitable agents are those agents that promote depolarization of tubulin or
prohibit
tumor cell proliferation. Chemotherapeutic agents contemplated include, but
are not
limited to, anti-cancer agents listed in the Orange Book of Approved Drug
Products
With Therapeutic Equivalence Evaluations, as compiled by the Food and Drug
*Trademark
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Administration and the U.S. Department of Health and Human Services (see e.g.
Nightingale S.L. (1999), "Electronic 'Orange Book'," JAMA, 281:1580). In
addition
to the above chemotherapy agents, the serine protease-like proteases provided
herein
also can be administered together with radiation therapy treatment. Additional
treatments known in the art are contemplated.
The therapeutic agent can be a chemotherapeutic agent. Chemotherapeutic
agents are known in the art and include at least the taxanes, nitrogen
mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic
acid analogs,
pyrimidine analogs, purine analogs, vinca alkaloids, antibiotics, enzymes,
platinum
coordination complexes, substituted urea, methyl hydrazine derivatives,
adrenocortical suppressants, or antagonists. More specifically, the
chemotherapeutic
agents can be one or more agents chosen from the non-limiting group of
steroids,
progestins, estrogens, antiestrogens, or androgens. Even more specifically,
the
chemotherapy agents can be azaribine, bleomycin, bryostatin-1, busulfan,
cannustine,
chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine, dacarbazine,
dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin,
ethinyl
estradiol, etoposide, fluorouracil, fluoxymesterone, gemcitabine,
hydroxyprogesterone
caproate, hydroxyurea, L-asparaginase, leucovorin, lomustine, mechlorethaminei
'medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,
methotrexate, methotrexate, mithramycin, mitomycin, mitotane, phenyl butyrate,
prednisone, procarhazine, sernustine streptozocin, tamoxifen, taxanes, taxol,
testosterone propionate, thalidomide, thioguanine, thiotepa, uracil mustard,
vinblastine, or vincristine. The use of any combinations of chemotherapy
agents also
= is contemplated. The administration of the chemotherapeutic agent can be
before,
= during or after the administration of the serine protease-like mutein
polypeptide.
= 25 Other suitable therapeutic agents for use in
combination or for co-
administration with the selected protease polypeptides provided herein are
selected
from the group consisting of radioisotope, boron addend, immunomodulator,
toxin,
photoactive agent or dye, cancer chemotherapeutic drug, antiviral drug,
antifungal
drug, antibacterial drug, antiprotozoal drug and chemosensitizing agent (See,
U.S. Pat.
Nos. 4,925,648 and 4,932,412). Suitable chemotherapeutic agents are described,
for
example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack
Publishing Co. 1995), and in Goodman and Gilman's THE PHARMACOLOGICAL
BASIS OF THERAPEUTICS (Goodman et al., Eds. Macmillan Publishing Co., New
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York, 190 and 2001 editions). Other suitable chemotherapeutic agents, such as
experimental drugs., are known to those of skill in the art. Moreover a
suitable
therapeutic radioisotope is selected from the group consisting of ax-
emitters,13-
emitters, 7-emitters, Auger electron emitters, neutron capturing agents that
emit a-
particles and radioisotopes that decay by electron capture. Preferably, the
radioisotope
of223 A I"Au, "P, "11,1311,9 Y, "6Re, I"Re,
is selected from the group consisting
"Cu, I"Lu, 213131, 1013, and 2"At.
Where more than one therapeutic agent is used in combination with the
selected proteases provided herein, they can be of the same class or type or
can be
from different classes or types. For example, the therapeutic agents can
comprise
different radionuclides, or a drug and a radionuclide.
In another embodiment, different isotopes that are effective over different
distances as a result of their individual energy emissions are used as first
and second
therapeutic agents in combination with the proteases provided herein. Such
agents
can be used to achieve more effective treatment of tumors, and are useful in
patients
presenting with multiple tumors of differing sizes, as in normal clinical
circumstances.
Few of the available isotopes are useful for treating the very smallest tumor
deposits and single cells. In these situations, a drug or toxin can be a more
useful
therapeutic agent for co-administration with a protease provided herein.
Accordingly,
in some embodiments, isotopes are used in combination with non-isotopic
species
such as drugs, toxins, and neutron capture agents and co-administered with a
protease
provided herein. Many drugs and toxins are known which have cytotoxic effects
on
cells, and can be used in combination with the proteases provided herein. They
are to
be found in compendia of drugs and toxins, such as The Merck Index: An
Encyclopedia of
Chemicals, Drugs and Biologicals, 12th Ed., Budavari S. et al., Eds.; Merck &
Co.:
Whitehouse Station, NJ, 1996; Bruntion, L.B., Lazo, J.S., & Parker, K.L.
(Eds.), 2005,
Goodman and Gilman 'S the pharmacological basis of therapeutics, 11th Ed., New
York:
McGraw-Hill, and in the references cited above.
'30 Drugs that interfere with intracellular protein synthesis also
can be used in
combination with a protease in the therapeutic the methods herein; such drugs
are
known to those skilled in the art and include puromycin, cycloheximide, and
ribonuclease.
The therapeutic methods provided herein can be used for cancer therapy. It is
well known that radioisotopes, drugs, and toxins can be conjugated to
antibodies or
antibody fragments which specifically bind to markers which are produced by or
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associated with cancer cells, and that such antibody conjugates can be used to
target
the radioisotopes, drugs or toxins to tumor sites to enhance their therapeutic
efficacy
and minimize side effects. Examples of these agents and methods are reviewed
in
Wawrzynczak and Thorpe (in Introduction to the Cellular and Molecular Biology
of
Cancer, L. M. Franks and N. M. Teich, eds, Chapter 18, pp. 378-410, Oxford
University Press. Oxford, 1986), in Immunoconjugates: Antibody Conjugates in
Radioimaging and Therapy of Cancer (C. W. Vogel, ed., 3-300, Oxford University
Press, N.Y., 1987), in Dillman, R. 0. (CRC Critical Reviews in
Oncology/Hematology 1:357, CRC Press, Inc., 1984), in Pastan et al. (Cell
47:641,
1986) in Vitetta et al. (Science 238:1098-1104, 1987) and in Brady et al.
(Int. J. Rad.
Oncol. Biol. Phys. 13:1535-1544, 1987). Other examples of the use of
immunoconjugates for cancer and other forms of therapy have been disclosed,
inter
alia, in U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744,
4,460,459, 4,460,561 4,624,846, 4,818,709, 4,046,722, 4,671,958, 4,046,784,
.. 5,332,567, 5,443,953, 5,541,297, 5,601,825, 5,635,603, 5,637,288,
5,677,427,
5,686,578, 5,698,178, 5,789,554, 5,922,302, 6,187,287, and 6,319,500.
Additionally, the treatment methods provided herein include those in which a
selected protease herein is used in combination with other compounds or
techniques
for preventing, mitigating or reversing the side effects of certain cytotoxic
agents.
Examples of such combinations include, e.g., administration of IL-1 together
with an
antibody for rapid clearance, as described in e.g., U.S. Pat. No. 4,624,846.
Such
administration can be performed from 3 to 72 hours after administration of a
primary
therapeutic treatment with a granzyme B mutein or MT-SP1 mutein in combination
with an anti-cancer agent (e.g., with a radioisotope, drug or toxin as the
cytotoxic
component). This can be used to enhance clearance of the conjugate, drug or
toxin
from the circulation and to mitigate or reverse myeloid and other
hematopoietic
toxicity caused by the therapeutic agent.
In another example, and as noted above, cancer therapy can involve a
combination of more than one tumoricidal agent, e.g., a drug and a
radioisotope, or a
radioisotope and a Boron-10 agent for neutron-activated therapy, or a drug and
a
biological response modifier, or a fusion molecule conjugate and a biological
response modifier. The cytokine can be integrated into such a therapeutic
regimen to
maximize the efficacy of each component thereof.
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Similarly, certain antileukemic and antilymphoma antibodies conjugated with
radioisotopes that are 13 or a-emitters can induce myeloid and other
hematopoietic side
effects when these agents are not solely directed to the tumor cells. This is
observed
particularly when the tumor cells are in the circulation and in the blood-
forming
organs. Concomitant and/or subsequent administration of at least one
hematopoietic
cytokine (e.g., growth factors, such as colony stimulating factors, such as G-
CSF and
GM-CSF) is preferred to reduce or ameliorate the hematopoietic side effects,
while
augmenting the anticancer effects.
It is well known in the art that various methods of radionuclide therapy can
be
used for the treatment of cancer and other pathological conditions, as
described, e.g.,
in Harbert, "Nuclear Medicine Therapy", New York, Thieme Medical Publishers,
1087, pp. 1-340. A clinician experienced in these procedures will readily be
able to
adapt the cytokine adjuvant therapy described herein to such procedures to
mitigate
any hematopoietic side effects thereof. Similarly, therapy with cytotoxic
drugs, co-
administered with a protease mutein, can be used, e.g., for treatment of
cancer,
infectious or autoimmune diseases, and for organ rejection therapy. Such
treatment is
governed by analogous principles to radioisotope therapy with isotopes or
radiolabeled antibodies. Thus, the ordinary skilled clinician will be able to
adapt the
description of cytokine use to mitigate marrow suppression and other such
hematopoietic side effects by administration of the cytokine before, during
and/or
after the primary anti-cancer therapy.
3. Exemplary Methods of Treatment for Selected MT-SP1
Polypeptides That Cleave Complement Protein Targets
The protease polypeptides and nucleic acid molecules provided herein can be
used for treatment of any condition for which activation of the complement
pathway
is implicated, particularly inflammatory conditions including acute
inflammatory
conditions, such as septic shock, and chronic inflammatory conditions, such as
Rheumatoid Arthritis (RA). Acute and inflammatory conditions can be manifested
as
an immune-mediated disease such as for example autoimmune disease or tissue
injury
caused by immune-complex-mediated inflammation. A complement-mediated
inflammatory condition also can be manifested as a neurodegenerative or
cardiovascular disease that have inflammatory components. This section
provides
exemplary uses of, and administration methods for, proteases. These described
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therapies are exemplary and do not limit the applications of proteases. Such
methods
include, but are not limited to, methods of treatment of physiological and
medical
conditions described and listed below. Such methods include, but are not
limited to,
methods of treatment of sepsis, Rheumatoid arthritis (RA),
membranoproliferative
glomerulonephritis (MPGN), lupus erythematosus, Multiple Sclerosis (MS),
Myasthenia gravis (MG), asthma, inflammatory bowel disease, respiratory
distress
syndrome, immune complex (IC)-mediated acute inflammatory tissue injury, multi-
organ failure, Alzheimer's Disease (AD), Ischemia-reperfusion injuries caused
by
events or treatments such as myocardial infarct (MI), stroke, cardiopulmonary
bypass
(CPB) or coronary artery bypass graft, angioplasty, or hemodialysis, or
Guillan Barre
syndrome.
Treatment of diseases and conditions with proteases can be effected by any
suitable route of administration using suitable formulations as described
herein
including, but not limited to, subcutaneous injection, oral and transdermal
administration. If necessary, a particular dosage and duration and treatment
protocol
can be empirically determined or extrapolated. For example, exemplary doses of
recombinant and native protease polypeptides can be used as a starting point
to
determine appropriate dosages. Modified proteases that have more specificity
and/or
selectivity compared to a wildtype or scaffold protease can be effective at
reduced
dosage amounts and or frequencies. Dosage levels can be determined based on a
variety of factors, such as body weight of the individual, general health,
age, the
activity of the specific compound employed, sex, diet, time of administration,
rate of
excretion, drug combination, the severity and course of the disease, and the
patient's
disposition to the disease and the judgment of the treating physician. The
amount of
active ingredient that can be combined with the carrier materials to produce a
single
dosage form with vary depending upon the host treated and the particular mode
of
administration.
Upon improvement of a patient's condition, a maintenance dose of a
compound or compositions can be administered, if necessary; and the dosage,
the
dosage form, or frequency of administration, or a combination thereof can be
modified. In some cases, a subject can require intermittent treatment on a
long-term
basis upon any recurrence of disease symptoms.
a. Immune-mediated Inflammatory Diseases
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Proteases and modified proteases selected in the method described herein,
including but not limited to variant MT-SP1 proteases provided herein, can be
used to
treat inflammatory diseases. Inflammatory diseases that can be treated with
proteases
include acute and chronic inflammatory diseases. Exemplary inflammatory
diseases
include central nervous system diseases (CNS), autoimmune diseases, airway
hyper-
responsiveness conditions such as in asthma, rheumatoid arthritis,
inflammatory
bowel disease, and immune complex (IC)-mediated acute inflammatory tissue
injury.
Experimental autoimmune encephalomyelitis (EAE) can serve as a model for
multiple sclerosis (MS) (Piddlesden et al., (1994)J Immunol 152:5477). EAE can
be
induced in a number of genetically susceptible species by immunization with
myelin
and myelin components such as myelin basic protein, proteolipid protein and
myelin
oligodendrocyte glycoprotein (MOG). For example, MUG-induced EAE
recapitulates essential features of human MS including the chronic, relapsing
clinical
disease course the pathohistological triad of inflammation, reactive gliosis,
and the
formation of large confluent demyelinated plaques. Proteases and modified
proteases
can be assessed in EAE animal models. Proteases are administered, such as by
daily
intraperitoneal injection, and the course and progression of symptoms is
monitored
compared to control animals. The levels of inflammatory complement components
that can exacerbate the disease also can be measured by assaying serum
complement
activity in a hemolytic assay and by assaying for the deposition of complement
components, such as for example Cl, C3 and C9.
Complement activation modulates inflammation in diseases such as
rheumatoid arthritis (RA) (Wang et at, (1995) PNAS 92:8955). Proteases and
modified proteases, including variant MT-SP1 polypeptides provided herein, can
be
used to treat RA. For example, proteases can be injected locally or
systemically.
Proteases can be dosed daily or weekly. PEGylated proteases can be used to
reduce
immunogenicity. In one example, type II collagen-induced arthritis (CIA) can
be
induced in mice as a model of autoimmune inflammatory joint disease that is
histologically similar to RA characterized by inflammatory synovitis, pannus
formation, and erosion of cartilage and bone. To induce CIA, bovine type II
collagen
(B-Cu) in the presence of complete Freund's adjuvant can be injected
intradermally at
the base of the tail. After 21 days, mice can be reimmunized using the
identical
protocol. To examine the effects of a protease or modified protease, including
MT-
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SP1 polypeptides, 3 weeks following the initial challenge with B-CII, a
protease or
control can be administered intraperitoneally twice weekly for 3 weeks. Mice
can be
sacrificed 7 weeks following the initial immunization for histologic analysis.
To
assess the therapeutic affect of a protease on established disease, a protease
can be
administered daily for a total of 10 days following the onset of clinical
arthritis in one
or more limbs. The degree of swelling in the initially affected joints can be
monitored
by measuring paw thickness using calipers. In both models, serum can be drawn
from
mice for hemolytic assays and measurement of complement markers of activation
such as for example C5a and C5b-9. In another example, primate models are
available for RA treatments. Response of tender and swollen joints can be
monitored
in subjects treated with protease polypeptides and controls to assess protease
treatment.
Proteases or modified proteases, including but not limited to variant MT-SP1
polypeptides provided herein, can be used to treat immune complex (IC)-
mediated
acute inflammatory tissue injury. IC-mediated injury is caused by a local
inflammatory response against IC deposition in a tissue. The ensuing
inflammatory
response is characterized by edema, neutrophila, hemorrhage, and finally
tissue
necrosis. IC-mediated tissue injury can be studied in an in vivo Arthus (RPA)
reaction. Briefly, in the RPA reaction, an excess of antibody (such as for
example
rabbit IgG anti-chicken egg albumin) is injected into the skin of animals,
such as for
example rats or guinea pigs, that have previously been infused intravenously
with the
corresponding antigen (i.e. chicken egg albumin) (Szalai etal., (2000)J
Immunol
164:463). Immediately before the initiation on an RPA reaction, a protease, or
a
bolus control, can be administered at the same time as the corresponding
antigen by
an intravenous injection via the right femoral vein. Alternatively, a protease
can be
administered during the initial hour of the RPA reaction, beginning
immediately after
injection of the antigen and just before dermal injection of the antibody. The
effects
of a protease on the generation of complement-dependent IC-mediated tissue
injury
can be assessed at various times after initiation of RPA by collecting blood
to
determine the serum hemolytic activity, and by harvesting the infected area of
the skin
for quantitation of lesion size.
Therapeutic proteases, such as those described herein including variant MT-
SP1 polypeptides provided herein, can be used to treat sepsis and severe
sepsis that
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can result in lethal shock. A model of complement-mediated lethal shock can be
used
to test the effects of a protease as a therapeutic agent. In one such example,
rats can
be primed with a trace amount of lipopolysaccharide (LPS), followed by the
administration of a monoclonal antibody against a membrane inhibitor of
complement
(anti-Crry) (Mizuno M et al., (2002) Int Arch Allergy Immunol 127:55). A
protease
or control can be administered at any time during the course of initiation of
lethal
shock such as before LPS priming, after LPS priming, or after anti-Crry
administration and the rescue of rats from lethal shock can be assessed.
b. Neurodegenerative disease
Complement activation exacerbates the progression of Alzheimer's disease
(AD) and contributes to neurite loss in AD brains. Proteases and modified
proteases
described herein, including but not limited to variant MT-SP1 polypeptides
provided
herein, can be used to treat AD. Mouse models that mimic some of the
neuropathological and behavioral features of AD can be used to assess the
therapeutic
effects of proteases. Examples of transgenic mouse models include introducing
the
human amyloid precursor protein (APP) or the presenilin 1 (PS1) protein with
disease-producing mutations into mice under the control of an aggressive
promoter.
These mice develop characteristics of AD including increases in beta-amyloid
plaques
and dystrophic neurites. Double transgenic mice for APP and PSI mutant
proteins
develop larger numbers of fibrillar beta-amyloid plaques and show activated
glia and
complement factors associated with the plaque. Proteases can be administered,
such
as by daily intraperitoneal or intravenous injections, and the course and
progression of
symptoms is monitored compared to control animals.
c. Cardiovascular disease
Proteases and modified proteases described herein, including but not limited
to
variant MT-SP1 proteases provided herein, can be used to treat cardiovascular
disease. Proteases can be used in the treatment of cardiovascular diseases
including
ischemia reperfusion injury resulting from stroke, myocardial infarction,
cardiopulmonary bypass, coronary artery bypass graft, angioplasty, or
hemodialysis.
.. Proteases also can be used in the treatment of the inflammatory response
associated
with cardiopulmonary bypass that can contribute to tissue injury. Generally, a
protease can be administered prior to, concomitantly with, or subsequent to a
treatment or event that induces a complement-mediated ischemia reperfusion
injury.
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In one example, a protease can be administered to a subject prior to the
treatment of a
subject by a complement-mediated, ischemic-injury inducing event, such as for
example coronary artery bypass graft of angioplasty.
Effects of a protease on treatment of ischemia reperfusion injury can be
assessed in animal models of the injury. In one such model, myocardial
ischemia is
induced in rabbits that have had an incision made in their anterior
pericardium by
placing a 3-0 silk suture around the left anterior descending (LAD) coronary
artery 5-
8 min from its origin and tightening the ligature so that the vessel becomes
completely
occluded (Buerke et al, (2001)J Immunol 167:5375). A protease, such as for
example a variant MT-SP I polypeptide provided herein, or a control vehicle
such as
saline, can be given intravenously in increasing doses as a bolus 55
minutes.after the
coronary occlusion (i.e. 5 minutes before reperfusion). Five minutes later
(i.e. after a
total of 60 minutes of ischemia) the LAD ligature can be untied and the
ischemic
myocardium can be reperfused for 3 hours. At the end of the reperfusion
period, the
ligature around the LAD is tightened. Effects of a protease on ischemia injury
can be
analyzed by assessing effects on myocardial necrosis, plasma creatine kinase
levels,
and markers of neutrophil activation such as for example myeloperoxidase
activity
and superoxide radical release.
In another model of complement-mediated myocardial injury sustained upon
perfusion of isolated mouse hearts with Krebs-Henseleit buffer containing 6%
human
plasma, treatment with proteases or modified proteases can be used to limit
tissue
damage to the heart. In such an example, the buffer used to perfuse the hearts
can be
supplemented with varying doses of proteases, such as but not limited to
variant
proteases including MT-SP I polypeptides polypeptides provided herein. The
perfused hearts can be assayed for deposition of human C3 and C5b-9, coronary
artery perfusion pressure, end-diastolic pressure, and heart rate.
Proteases and modified proteases, such as for example variant MT-SF1
polypeptides provided herein, can be used as therapeutics prior to or
following
Cardiopulmonary Bypass (CPB) or coronary artery bypass graft to inhibit the
.. inflammatory immune response that often follows bypass and that can
contribute to
tissue injury. An in vitro recirculation of whole blood in an extracorporeal
bypass
circuit can be used to stimulate platelet and leukocyte changes and complement
activation induced by CPB (Rinder etal. (1995)J. Clin. Invest. 96:1564). In
such a
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model, addition of a protease or modified protease or control buffer, in
varying doses,
can be added to a transfer pack already containing blood from a healthy donor
and
porcine heparin, just prior to addition of the blood to the extracorporeal
circuit. Blood
samples can be drawn at 5, 15, 30, 45, 60, 75, and 90 minutes after
recirculation and
assayed for complement studies such as for example hemolytic assays and/or
complement activation assays to measure for C5a, C3a, and/or sC5b-9. A
pretreatment sample of blood drawn before its addition to the extracorporeal
circuit
can be used as a control. Flow cytometry of blood samples can be performed to
determine levels of adhesion molecules on populations of circulating
leukocytes (i.e.
neutrophils) in the blood such as for example CD1lb and P-selectin levels.
K. EXAMPLES
The following examples are included for illustrative purposes only and are not
intended to limit the scope of the invention.
EXAMPLE 1
Mutant PA!-! Inhibitors
A. Expression and purification of mutant PAI-1 inhibitors
The pPAIST7HS, recombinant plasmid carrying the cDNA of human PAI-1
(encoding mature PAI-1 containing an N-terminal Met as set forth in SEQ ID
NO:396), was used as template to introduce modifications into the amino acid
sequence of the PAI-1 reactive center loop. Plasmid pPAISTHS is a derivative
of
plasmid pPAIST7 lacking the Hindlll site at nucleotide pair 1 and the Sall
site at
nucleotide pair 2106. Plasmid pPAIST7 was generated as described (Franke et
al.
(1990) Biochimic et Biophysica Acta 1037: 16-23). Briefly, the PAI-1 cDNA
clone
pPAI-11RB was cleaved with restriction endonucleases ApaLl and PflMI, and the
1127 bp fragment of PAI-1 containing 2 bp of the codon for residue 1 of PAI-1
and
the full coding sequence for residues 2-376 of the 379-residue protein was
purified by
gel electrophoresis. Synthetic linkers were constructed to reconstruct both
ends of the
PAI-1 cDNA coding sequence and to introduce an ATG protein synthesis
initiation
codon immediately before the triplet encoding the first residue of mature PAI-
1
generating a mature PAI-1 having a sequence of amino acids set forth in SEQ ID
NO:396. In addition, to facilitate insertion of the cDNA coding region into
plasmid
pBR322, the linkers were designed to generate EcoRI and Hindlll restriction
endonuclease sites at the 5' and 3' termini, respectively, of the PAI-1 cDNA
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fragment. The synthetic linkers are as follows: N-terminus, 5'AATTCTATGG-3'
(SEQ ID NO:392) and 5'- TGCACCATAG-3' (SEQ ID NO:393); C-terminus, 5'-
ATGGAACCCTGAA- 3' (SEQ ID NO:394) and 5'- AGCTTCAGGGTTCCATCAC-
3' (SEQ ID NO:395). The linkers were treated with polynucleotide kinase before
use.
The synthetic linkers (10 bp at the 5' end and 13 bp at the 3' end) were then
ligated
with the 1127 bp ApaLl-Pf1MIDNA fragment, digested with EcoRI and HindHI and
the 1146 bp EcoRI-HindlII fragment was isolated by gel electrophoresis and
cloned
into EcoRI and HindHI cleaved pBR322 (SEQ ID NO:377).
To initiate construction of the pPAIST-7 expression plasmid, the subclone in
the pBR322 vector was cleaved with EcoRI and the linear plasmid was
dephosphorylated using bacterial alkaline phosphatase. Using a 360 bp EcoRI
DNA
fragment from pC5A-48 containing the trp promoter and ribosome binding site
the
pPAIST-7 was generated following standard ligation.
To generate the pPAIST7HS prokaryotic expression vector (Shubeita et al.
(1990)J Biol. Chem., 265: 18379-18385), the pPAIST7 was partially digested
with
HindlII to linearize the plasmid, blunt-ended with the Klenow fragment of
E.coli
DNA polymerase land ligated to eliminate the upstream HindlII site. Deletion
of
sequences in pPAIST7 downstream of the PAI-1 coding sequences between the
Hinall and Sall sites and elimination of the Sall site was accomplished by
sequential
partial Sall digestion, complete HindIll digestion, blunt-ending with the
Klenow
fragment of E.coli DNA polymerase I, and ligation.
Mutagenesis reaction was carried out using the Multi site mutagenesis kit
(Stratagene) following conditions specified by the supplier. Mutagenesis of
amino
acids in wild-type PAI-1 at positions P4-P1' in the reactive center loop
corresponding
to amino acids VSARM (SEQ ID NO:378) were made. A mutant PAI-1 was made
(PAI-1/RRAR) containing replacement of the wild-type amino acid sequence
VSARM with RRARM (SEQ ID NO:379) sequences in the PAI-1 reactive center
loop from the P4 to P1' positions. The sequence of the RRARM mutagenic primer
was: 5'-CCACAGCTGTCATAAGGAGGGCCAGAATGGCCCCCGAGGAGATC-
3' (SEQ ID NO:380). A second mutant PAI-1 was made (PAI-1/69) containing
replacement of the wild-type amino acid sequence VSARM with PFGRS (SEQ ID
NO:389) sequences in the PA!-1 reactive center loop from the P4 to P I '
positions.
The sequence for the PFGRS mutagenic primer was:
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5' CCACAGCTGTCATACCCTTCGGCAGAAGCGCCCCCGAGGAGATC-3'
(SEQ ID NO:390). Following mutagenesis, the DNA isolated from the
transformants
was fully sequenced to confirm the presence of desired mutations and the
absence of
any additional mutations.
The mutants PAI-1/RRAR and PAL-1/69 were expressed as fusion proteins
utilizing N-terminal poly histidine residues present in the pPAIST7HS vector.
The
expression and purification of mutant PAI-Is (i.e. PAI-1/RRAR and PAI-1/69)
were
based on methods as described in Ke etal. (J Biol. Chem., 272: 16603-16609
(1997)).
Expression of wild type and mutated variants of PAI-1 was accomplished by
transforming 0.114 DNA of pPAIST7HS vector encoding mutant PAI-1 into the
E. colt strain BL21[DE3]pLyss (Novagen), which synthesizes Ti RNA polymerase
in
the presence of isopropyl-1-thio-13-D-galactopyranoside. Bacterial cultures
were
grown at 37 C with vigorous shaking to an absorbance A595 of 1.1-1.3, and
isopropyl-
1-thio-13-D-galactopyranoside was added to a final concentration of 1 mM to
induce
the synthesis of T7 RNA polymerase and the production of PAT-1 proteins.
Cultures
were grown for an additional 1-2 h at 37 C and then shifted to 30 C for 2-6 h.
Cells
were pelleted by centrifugation at 8000 x g for 20 min at 4 C and resuspended
in
40 ml of cold start buffer (20 mM sodium acetate, 200 mM NaC1, and 0.01% Tween
20, pH.5.6). The cell suspension was disrupted in a French pressure cell
(Aminco),
and cellular debris was removed by ultracentrifugation for 25 min at 32,000 x
g.
Purification of soluble, active mutant PAI-1 was performed by injecting the
lysate of E. coli containing soluble form of PAI-URRAR or PA1=-1 /69 onto an
XK-26
column (Pharmacia Biotech Inc) packed with CM-50 Sephadex (Pharmacia, see
e.g.,
Sancho et al. (1994) Eur. J. Biochem. 224, 125-134). The column was washed
with
5 colummvoiumes of start buffer (20 mM sodium acetate, 200 mM NaCI, and 0.01%
Tween 20, pH 5.6), and PAT-1 proteins were eluted using a 0.2-1.8 M linear
gradient =
of NaC1 in the same buffer. Peak fractions were collected, pooled, and
concentrated
using a centriplus 30 concentrator (Amicon). The concentrated fractions were
used
for activity measurement.
B. PAI-1 Activity Measurements
1. Active Site Titration Against Standard Trypsin
Active concentration of PAT-URRAR and PAI-1/69 was determined by active
site titration against standard trypsin as described by Olson et at. (J. Biol.
Chem., 270:
*Trademark
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30007 (1995)). Briefly, sequential additions of concentrated inhibitor (0.5 ¨
6.0 uM)
were made to solutions of 1 MM 13-trypsin (Sigma) and 10 M p-aminobenzamidine
probe (Sigma). Binding was monitored from the decrease in fluorescence
accompanying the displacement of the bound probe from the enzyme active site
as the
inhibitor bound. After addition of each concentrated inhibitor a 1-2 minute
equilibration time was allowed before assessment of fluorescence changes at
excitation and emission wavelengths of 325 run and 345 nm, respectively, to
maximize the difference between bound and free probe fluorescence. Control
titrations of just the probe with the inhibitor in the absence of the trypsin
enzyme were
performed to correct for background fluorescence. Inhibitor-enzyme titrations
were
fit by linear regression analysis.
2. Titration of standardized t-PA preparations
Mutant PAI-ls were also titrated against standardized t-PA preparations to
assess activity. The inhibitory activity of wild-type PAL-1 or mutant PAI-1
was
measured by a direct chromogenic assay using tPA (American Diagnostics, Inc,
100
U/ug) and the chromogenic tPA substrate H-D-Ile-Pro-Arg-para-nitroaniline
substrate
(S-2288, Chromogenix). Serially diluted PAI-1 (0.1 ¨4.0 Mg) were incubated
with t-
PA in a microtiter plate for a fixed time (typically, 20-60 minutes) at room
temperature and the residual activity of tPA was measured by the addition of
the
chromogenic substrate S-2288 to a final concentration of 0.5 mM. The residual
activity of t-PA following incubation with increasing concentrations of
inhibitor was
assessed by measuring the absorbance at 405 nm.
EXAMPLE 2
Construction of phage display libraries of u-PA variants
A. Cloning of wild-type u-PA into phagemid
To demonstrate functional display of the protease domain of u-PA on phage,
high fidelity PCR was performed using primers 496 and 497, Pfu DNA polymerase
and pCMV4 plasmid (SEQ ID NO:373) containing c-DNA of full length u-PA gene
(SEQ ID NO:474) as a template. The primers used in the PCR amplification were
as
_______________________________________________________ follows: 496, 5'-
ACGTGOCCCAGGCGOCC CAGTGTGGCCAAAAG-3' (SEQ
ID NO:374); 497, 5'-TCCTGGCCGOCCTGOCCGAGCAGGCCATTCTC-3' (SEQ
ID NO:375). Both the primers carry restriction sites (underlined) for SfiI
enzyme at
the 5'terminus. After purification and SfiI digestion, the PCR product was
ligated to
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Sfi/ digested phagemid vector, pComb3H (SEQ ID NO:376) (Andris-Widhopf et al.
(2000)J Immunol Methods, 28: 159-81). The phagemid was used for monovalent
display of wild type or mutant u-PA (see below). This construct contains
sequences
encoding the C-terminal region of the glIIp gene of fd phage. The presence of
two Sfil
restriction sites in the vector with two different recognition sequences was
exploited
for designing the above-mentioned primers. The PCR products amplified using
primers 496 and 497 enabled directional cloning of the u-PA protease domain
cDNA
(SEQ ID NO:475) into the phagemid (see below for ligation conditions). In the
final
construct, the PCR products containing sequences of the u-PA protease domain
were
cloned in the middle of OmpA and gIIIp sequences to display wild-type or
mutant u-
PA as an N-terminal gllIp fusion.
B. Construction of mutant u-PA and phage display libraries of
mutant u-PA
To construct u-PA phage display libraries, error-prone PCR amplification was
carried out using the pCMV4/u-PA plasmid containing cDNA of the uPA gene as
template, as set forth above, for 25 cycles in 100 111 reaction mixtures using
reagents
supplied with the PCR Diversify mutagenesis Kit (Clontech). Appropriate PCR
conditions were followed to set up three different PCR reactions to amplify
only the
protease domain of the uPA gene (SEQ ID NO:475) using primers 496 and 497 from
above by varying the amounts of manganese (MN2+) or dGTP as described by the
manufacturer, to achieve 0.2, 0.5, or 0.9% of mutation rate incorporation into
the
cDNA. The amplified PCR products (805 bp, SEQ ID NO:475) were purified using a
PCR purification kit (Qiagen) followed by Sfil enzyme digestion. The Sfil
digested
PCR products from each mutatgenesis reaction were used to generate three
different
libraries.
For library construction, the pComb3H vector (SEQ ID NO:376) was digested
with S'fif enzyme and the larger DNA fragment from this reaction was gel
purified
using a "Gel slice kit" (Qiagen). Three separate ligation reactions were
carried out to
construct three libraries with PCR products containing 0.2, 0.5 and 0.9%
mutation
rates. At least 1 u.g of the gel purified vector was mixed with SfiI digested
PCR
products (1:2 ratio) and the ligation mixtures were incubated overnight at 18
C. Next,
the ligation mixtures were purified using Qiagen mini elute kit (Qiagen) and
the DNA
was finally eluted into 60 n1 of.Milli Q purified water. The purified DNA was
electroporated into 400 p1 of E. coli XL Bluel electroporation competent cells
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(Stratagene) using gene pulser (Bio Rad). The cells were then transferred to
10 ml of
SOC medium (Invitrogen) and incubated at 37 C in a shaker for 1 hr followed by
plating on large LB agar plates ( 245 mm X 245 mm) supplemented with
carbenicillin
(75 ug/mL). After overnight incubation of plates at 30 C the transfonnants
were
scraped using a cell scraper and the resulting cultures were grown in 2X YT
medium
supplemented with carbenicillin (75 g/ml) at 37 C shaking for 2 hours. The
cultures
were infected with helper phage (VCS M13) at an MOI (multiplicity of
infection) of 5
for amplification of the libraries. After 1 hour of growth at 37 C shaking,
the cultures
were supplemented with kanamycin to final concentrations of 3 tig/mL and grown
overnight at 30 C with shaking. The cells were harvested and the phage
particles
present in the supernatant were precipitated using a PEG-NaCI solution.
Simultaneously, to calculate the diversity of each library, an aliquot of the
electroporated cells were plated on LB agar plates supplemented with
carbenicillin
(100 g/m1) and after overnight incubation at 37 C the colonies were counted.
The
same methods were used to generate successive generations of u-PA phage
display
libraries for further improvement of identified u-PA variants against PAI-
1/RRAR
inhibitor. The DNA of u-PA variants identified from the previous libraries was
used
as template for construction of next generation libraries.
The catalytic efficiencies of wild type or u-PA phage libraries generated
using
random mutagenesis were analyzed using the indirect plasminogen activation
assay.
Briefly, 5 I of u-PA phage (typically ¨5x1012 cfu), 0.2 M Lys-plasminogen
(American Diagnostic) and 0.62 mM Spectrozyme PL (American Diagnostica) were
present in a total volume of 100 .1. Assays were performed in microtiter
plates, and
the optical density at 405 nm was read every 30 seconds for 1 h in a Molecular
Devices Thermomax. Reactions were performed at 37QC. Inhibition of u-PA or u-
PA
variant phage also was assessed. Briefly, 5 I of u-PA phages (typically
¨5x1012cfu)
were mixed with wt-PAI-1 (0.1 M) or mutant PAI-1/RRAR inhibitor (1.0 M) and
incubated for 30 min at room temperature followed by addition of 0.62 mM
Spectrozyme PL, 0.2 M Lys-plasminogen (American Diagnostics, Inc.). The
assays
were read as mentioned above.
Example 3
Selection of variant u-PA from u-PA phage libraries against mutant
inhibitor
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A. Selection of u-PA phage/ mutant PAI-1 complexes
Mutant PAI-1/RRAR inhibitor or PAI-1/69 were used as the "bait substrate"
in the panning experiment to isolate altered u-PA phage/s with improved
reactivity
towards the mutant substrate sequence from large, combinatorial u-PA phage
display =
libraries described in Example 2 above. In brief, 5 I 2 X 1012 to ¨ 1 X1013)
u-PA
phage, containing an equal representation of u-PA phage from all three u-PA
phage
libraries (0.2, 0.5 and 0.9% mutagenesis frequency), was mixed with 5 pi bait
substrate (from 0.1 to 1.0 M PAI-1(RRAR or PAI-1/69) and 10 I 10X indirect
buffer pH 7.4 (0.5 M Tris, 1.0 M NaC1, 10 mM EDTA, 0.1% Tween 80) in a total
volume of 100 1 (i.e. reaction contained 80 I H20). The reaction was
incubated for
varying times at room temperature (typically 1 hour, however, incubation time
was
adjusted to control the stringency of the solution).
The u-PA phage-PAI-1 inhibitor complexes were captured using CuSO4
activated sepharose. Pre-activation of the chelating sepharose (200 .1;
Pharmacia)
was aceomplished by treatment with CuSO4 (100 mM). The CuSO4 activated
sepharose was blocked with 0.5% BSA in PBS buffer for 1 hour at room
temperature.
100 gl of BSA blocked sepharose beads was added to 100 I of the above panning
mixture reaction in the presence of 800 I of binding buffer (0.5 M NaCI, 20
mM
Tris, 20 mM immidozole, pH7.4) to capture the His-tagged PAT-1 inhibitor-u-PA
phage complexes. The incubation was continued for another 1 hour at room
temperature. Next, the panning mixture was centrifuged for 1 min at 2000 rpm
and
the sepharose beads containing bound u-PA phage-PAI-I inhibitor complexes were
washed with 1 ml of binding buffer to remove unbound phages. The washing step
was
repeated 5-10 times and the beads containing bound complexes were transferred
to
new tubes after each wash to avoid any potential "carry over" of non-specific
phages.
The bound u-PA phage-PAI-1/RRAR complexes were eluted in 100 ttl of
elution buffer (0.5M EDTA). A 95' I aliquot of the eluted phages was used to
infect 1
ml of XL-1 Blue E. coli cells (0.6 OD) to calculate the output of the
libraries
(indicating the number of phage obtained after selection). The infected
bacteria were
plated on large plates (245 mm X 245 mm) containing carbenicillin (75 g/m1)
to
generate a library for the next round of selection.. Finally, the remaining
output
library (i.e. selected phage) was used to prepare individual phage clones for
screening
and/or generating a new library for the next round of selection. The
concentrations of
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bait substrate used in the selection and times of incubation of the library
with the bait
substrate were adjusted according to the desired stringency level. For
example,
conditions could be chosen such that <1%, 2%, 5%, 10%, or greater than 10% of
the
u-PA activity of the library was inhibited by the "bait PAI-1" containing the
bait
substrate sequence. Typically, the first round of selection was carried out
using
higher (e.g., 0.5 M) concentrations of bait PAI-1 and for successive rounds
of
selection the bait serpin concentration and incubation time with libraries
were
reduced. In addition, multiple stringencies can be used in parallel at each
round of
selection. Based on the quality of the output (e.g., signal to noise ratio of
the phage
output in paired +/- bait serpin selections (see description below)), and the
quality of
the resulting "hits" based on functional analysis, the phage output from one
or more
of these selections can be carried forward into the next round(s) of
selection.
In parallel, control experiments were performed using the above-mentioned
conditions for selection of phage from the u-PA library without bait and the
phage
from this control experiment was compared with the output of the library
selections in
the presence of bait serpin substrate. The cfu from the output of library
selected in the
presence of bait was normally in the range of 104 to 105 higher than the
output
obtained from the control selection. If higher background was observed with
the
control selection, the panning was repeated using more stringent conditions
such as
reducing incubation times, increasing concentrations of reactants (library,
bait or
beads) and increasing number and time of washing the selected phage bound to
chelating sepharose (up to a factor of 10 or more).
B. Screening of u-PA phages with increased reactivity and catalytic
efficiency towards new substrate sequences
An aliquot of eluted u-PA phages (5 I) was mixed with XL-1Blue E. coil
cells (100 I) for infection and incubated at 37 C for 1 hr. The infected E.
coil cells
were then plated on LB agar plates supplemented with carbenicillin (100
g/m1).
After overnight incubation at 37 C, individual colonies were picked for phage
preparations. The cells were grown in 2 ml of 2x YT supplemented with
carbenicillin
(100 g/m1) and tetracycline (10 g/ml) and phage preparation was performed as
described (Sambrook, Jet al (1989) Molecular Cloning, A laboratory manual,
Cold
spring Harbor laboratory). The identified phage were tested in the following
assays
to assess activity. The individual phage preparations were used in the
indirect
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plasminogen activation assay to identify active phages, and the active phage
preparations were used for inhibition assays.
1. Indirect Plasminogen Activation Assay
The individual phage preparations were used in the indirect plasminogen
activation assay to identify active u-PA phages. Briefly, 5 ill of u-PA phage
(typically ¨5x1012 cfu), 0.2 itM Lys-plasminogen (American Diagnostic) and
0.62
mM Spectrozyme PL (American Diagnostica) were present in a total volume of 100
1. Assays were performed in microtiter plates, and the optical density at 405
ttm was
read every 30 seconds for 1 h in a Molecular Devices Thermomax. Reactions were
performed at 37 C.
2. Inhibition of u-PA phage by mutant PAI-1/RRAR
The phage that were identified as active in the indirect plasminogen activity
assay were further tested for inhibition by mutant PAI-1/RRAR. Briefly, for
the
inhibition assays, 5 p.1 of active u-PA phages (typically-5x1012 cfu) were
added in
duplicate wells of a microtiter plate followed by addition of a fixed
concentration of
mutant PAT-1 (e.g., 1.01.1M ) to one well and phosphate buffered saline (PBS)
to the
duplicate well. After mixing, the reaction was allowed to continue for a fixed
time
(e.g., 30 min) at room temperature followed by addition of 0.62 mM Spectrozyme
PL,
0.2 pM Lys-plasminogen (American Diagnostics, Inc.). For control experiments,
) 20 wild-type u-PA phage was assessed under the same conditions. The
plates were read
at 405 nm in a spectrophotometer for 2 hrs. The selected u-PA phages that
exhibited
improved sensitivity for PAI-1/RRAR or PAI-1/69 as compared with wild-type u-
PA
= phage were selected for further analysis and subjected to DNA sequencing.
3. Peptide Substrate Screening
In addition, to identify variants of u-PA phage with improved catalytic
efficiency the individual phage clones were screened against an Ac-RRAR-AMC
substrate. For the assay, a fixed volume of phage supernatants (e.g., 35 p.1)
was mixed
with 75 p.M Ac-RRAR-AMC substrate in IX indirect assay buffer (50 mM Tris, 100
mM NaCl, 1 mM EDTA, 0.01% Tween 80) in a total volume of 100 pl. The assay
was carried out in 96-well or 384-well black assay plates (Corning) and read
at 380-
450 nm for 2 hrs in spectrophotometer (Molecular Devices) reader.
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To confirm their improvement, the positive u-PA phages identified after
inhibitor and peptide substrate screenings were re-screened using the assays
set forth
above.
C. Identification of
Selected u-PA Mutants and Optimization of Identified
Mutants
Positive phage clones were mixed with XL-1Blue E. coil cells for infection as
mentioned above and the cultures were grown overnight with shaking at 37 C.
Plasmid DNA was purified from the overnight culture using a plasmid
preparation kit
(Qiagen). The DNA was sent for custom sequencing using the following primers:
535- 5'-CAGCTATCGCGATTGCAG-3' (SEQ ID NO:381) ; 5542-
5'GTGCGCAGCCATCCCGG-3' (SEQ ID NO:382). Amino acid residues altered in
the mutant u-PA genes were identified after analyzing the sequencing data.
Table 10 below sets forth variants of u-PA identified from selection of
variant
u-PA from u-PA phage libraries against a PAI-1/RRAR mutant inhibitor. The
mutations set forth in Table 10 below are with chymotrypsin numbering. The
numbers in parentheses indicate the number of times the mutants were
identified in
the phage selection method. Based on results from activity assays, the best
variants of
u-PA phages are highlighted by underline. Amino acid sequences of a mature u-
PA
preproprotein (SEQ ID NO:433) containing the designated mutations are set
forth in
any of SEQ ID NOS: 434-445.
Table 10
u-PA Pbage Mutant Mutation site/s Amino acid/s modified
SEQ
libraries Vs name (Chymotrypsin ID
PAI-1/RRAR Number) NO:
AR73 (2) 30 Phe ¨ Ileu 434
Selection I
AR81 73, 89 Leu ¨ Ala, lieu - Val 435
AR1 73 Leu-Pro 436
AR3 217 Arg-Cys 437
AR4 (3) 155 Leu-Pro 438
AR7 75, 89, 138 Ser-Pro, Ileu-
Val, Ileu-Thr 439
Selection AR32 137 Glu-Gly 440
11 & III AR36 72, 155 Arg-Gly, Leu-Pro 441
AR37 133 Gly-Asp 442
AR66 160 Val-Ala 443
AR24 38 Val-Asp 444
AR85 132, 160 Phe-Leu, Val-Ala 445
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Amino acid residues altered in the mutant u-PA gene exhibiting increased
sensitivity against PAI-1/69 inhibitor following selection against the PAI-
1/69
inhibitor were identified after analyzing the sequencing data as set forth
above. The
mutants identified from the first generation protease phage display library
(I) are set
forth below in Table 11. Subsequent generations of protease phage display
libraries
were created using the method as set forth above in Example 2B using the PCR
Diversify mutagenesis kit and primers 496 and 497. For the generation II phage
display library, the u-PA mutant u-PA/Ic containing a mutation corresponding
to
F3OV based on chymotrypsin numbering was used as a template for the
mutagenesis
reaction. The mutants identified from the second generation protease phage
display
library (II) are set forth in the Table 11 below. For the generation III phage
display
library, the u-PA mutant u-PA-Ifb or u-PA-Jib mutant containing mutations
corresponding to F30V/ Y61(A)H or F30V/ K82E, respectively, based on
chymotrypsin numbering were used as templates for the mutagenesis reaction.
The
mutants identified from the third generation protease phage display library
(III) are set
forth in Table 11 below. For the generation IV phage display library, the u-PA
mutant u-PA/IIIa containing mutations corresponding to F30V/K82EN159A based
on chymotrypsin numbering was used as a template for the mutagenesis reaction.
The
mutants identified from the fourth generation protease phage display library
(IV) are
set forth in Table 11 below. The numbers in parentheses indicate the number of
times
u-PA phage were selected for that had the same mutation. The underline
indicates the
new mutations acquired by the mutant. Amino acid sequences of a mature u-PA
preproprotein (SEQ ID NO:433) containing the designated mutations are set
forth in
any of SEQ ID NOS: 460-472,
Table 11
SEQ
Phage Mutant
Mutation sites Amino acids modified
ID
libraries name
NO:
u-PA/Ia 21 Phe-Val
460
u-PA/Ib 24 Ile-Leu 461
u-PATIc (2) 30 Phe-Val
462
u-PA/Id 30 Phe-Leu
463
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II u-PA/1Ia 30, 61(A) Tyr-His
464
u-PA/IIb 30, 82 Lys-Glu
465
u-PA/1Ic 30, 156 Lys-Thr
466
jj u-PA/IIIa (8) 30, 82, 159
Val-Ala .. 467
u-PA/IIIb 30, 82, 39, 159 Thr-Ala, Val-Ala
468
u-PA/Illc 30, 82, 158 159 Thr-Ala, Val-Ala
469
u-PA/Illd (2) 30, 6IA, 92 Lys-Glu
470
pi u-PA/IVa 30, 82, 159, 80, 89, 187
Glu-Gly, Ile-Val, Lys-Glu 471
u-PA/IVb 30, 82, 159, 80, 84, 89, 187
Glu-Gly, Glu-Lys, Ile-Val, Lys-Glu 472
1. Optimization and recombination of amino acid
resides 30 and 155
in Focused Phage Display Libraries against PAI-1/RRAR inhibitor
To enrich the sensitivity of u-PA variants against the PAI-1/RRAR inhibitor,
amino acid 30 and amino acid 155 based on chymotrypsin numbering (identified
as
hot spots in the first selections as set forth in Table 10 above) were
targeted for
randomization and recombination using the following primers, respectively:
TC30- 5'GCCCTGGNNSGCGGCCATC- 3' (SEQ ID NO:383)
TC155- 5'GGAGCAGNNSAAAATGACTG- 3' (SEQ ID NO:384)
Mutagenesis was performed using the Quick Change multi site-directed
mutagenesis kit (Stratagene) following conditions described by the
manufacturer. In
brief, after phosphorylation of the primers using T4 polynucelotide kinase
(New
England Biolabs) following conditions described by the manufacturer, three
different
reactions were performed for randomization of residues 30 and 155: 1) 30
individually; 2) 155 individually; and 30 plus 155 together. The DNA
construct,
pComb3H/u-PA variant (pARF 81), carrying mutations at residues L73A and I89V
as
compared to the corresponding wild type u-PA protease domain sequences, was
used
as a template in the mutagenesis reaction using primers TC30 (SEQ ID NO:383)
and
TC155 (SEQ ID NO: 384) individually for randomization of positions 30 and 155
respectively. In another reaction, these two primers were used to randomize
positions
and 155 together in the pARF 81 variant DNA. Mutagenesis reaction was carried
out using the Multi site mutagenesis kit (Stratagene) following conditions
specified by
the supplier. After mutagenesis, the reaction products were transformed into
XL-1
Blue E. coli cells for library construction (See Example 2 above). The
amplified u-PA
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phage libraries were used for selection of improved variants of u-PA as set
forth in
Example 3A and 3B above.
Table 12 below sets forth variants of u-PA identified from selection against a
PAI-1/RRAR mutant inhibitor Of variant u-PA from focused u-PA phage libraries
where all variants had background mutations at amino acid residues L73A and
I89V
based on chymotrypsin numbering. The mutations set forth in Table 12 below are
with chymotrypsin numbering. The numbers in parentheses indicate the number of
times the mutants were identified in the phage selection method. Based on
results
from activity assays, the best variants of u-PA phages are highlighted by
underline.
Amino acid sequences of a mature u-PA preproprotein (SEQ ID NO:433) containing
the designated mutations are set forth in any of SEQ ID NOS: 446-459.
Table 12
Focused
SEQ
u-PA libraries Mutant
30(Phe) 155 (Leu) ID
Vs Name
NO
PAI-1/RRAR
ARF2 Thr 446
ARF6 Leu 447
ARF11 Val 448
ARF17 Gly 449
ARF16 Leu 450
ARF33 Val 451
Selection I ARF35 Met 452
& II ARF36 Met 453
ARF37 Met 454
ARF43 Leu 455
ARF47 Val 456
ARF48 Leu Met 457
ARF103 Leu 458
ARF115 Gly Met 459
EXAMPLE 4
Expression of modified u-PA enzymes by transient transfection of COS
cells.
For the expression of variant u-PA enzymes in a mammalian expression
system, the positive clones identified from phage display results were used as
a
template (pComb3H carrying mutant u-PA protease domain sequences) for PCR
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amplification of cDNA encoding the selected u-PA variant gene. Overlap
extension
PCR (Ho, S et al (1989) Gene 77, 51-59) was carried out using the following
primers.
717-5' -TTTCAGTGTGGCCAAAAG -3' (SEQ ID NO:385);
718, 5'-CAGAGTCT1TTGGCCACA ¨3' (SEQ ID NO:386);
850,5' ¨GGGGTACCGCCACCATGAGAGCCCTGCTGGCGCGC-3' (SEQ ID
NO:387);
851,5' ¨GCTCTAGATCATCAGAGGGCCAGGCCATTCTCT-3' (SEQ ID
NO:388). The primers 850 and 851 carry sequences for Kpnl and Xbal restriction
enzymes (underlined) respectively.
PCR was performed in two steps to accomplish full-length amplification of
mutated cDNA u-PA gene as described below. In the first step, PCR was carried
out
in a 100 pi reaction to amplify a 500 bp product (corresponding to the EGF and
Kringle domains of u-PA) using Pfu DNA polymerase, primers 850, 718 and pCMV4
containing the full-length uPA gene (SEQ ID NO:474) as template. Similarly,
another
PCR was carried out to amplify the mutant u-PA protease domain (800 bp, i.e.
corresponding to mutant sequences as compared to the wild-type sequence set
forth in
SEQ ID NO:475) using primers 851 and 717 with the appropriate mutant u-PA-
pComb3H as template. These two PCR products were gel purified and used in the
next round of PCR amplification. In the second step, the gel purified PCR
products
(5 1 each) were used as templates in 100111 reaction mixture with primers 850
and
851. The primers 717 and 718 have overlapping complementary sequences that
allowed the amplification of the full-length u-PA cDNA (1.3 kb) in the second
step
PCR. The PCR product was purified using a PCR purification kit (Qiagen) and
then
digested with Kpnl and Xbal restriction enzymes. After purification using
QIAquick*
columns (Qiagen), the full-length u-PA gene was ligated with the pCMV4
mammalian expression vector (SEQ ID NO:373) that had been previously digested
with Kpnl and Xbal. The ligation mixture was electroporated into E. coli XL-I
Blue
cells and plated on LB plates supplemented with carbenicillin (100 pg/m1).
After
overnight incubation of plates' at 37 C, individual colonies were picked up
and grown
in 2 ml LB medium for plasmid purification. The plasmids were used for
sequencing
the entire u-PA gene using the following sequencing primers. UPAF1-
5'ATGAGAGCCCTGCTGGCGCGCC-3' (SEQ ID NO:476) and
UPAF2- 5'GGAAAAGAGAATTCTACCG-3' (SEQ ID NO:477).
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Mutant u-PA clones with the correct mutations, without any additional
mutation(s), were prepared in large quantities using Midi Plasmid preparation
kit
(Qiagen) and used for electroporation into COS-1 cells using a Bio-Rad Gene
Pulser.
2014 of cDNA, 100 pig of carrier DNA, and approximately 107 COS-1 cells were
placed into a 0.4-cm cuvette, and electroporation was performed at 320 V, 960
microfarads, and Si = oo (Tachias et al. (1995)J Biol. Chem., 270: 18319-
18322).
Following electroporation, the transfected cells were incubated overnight at
37 C in
DMEM medium (Irvine Scientific) containing 10% fetal calf serum and 5 mM
sodium
butyrate. Cells were then washed with serum free medium and incubated in DMEM
for 48 hat 37 C. After incubation with serum-free media, conditioned media was
collected and used for further characterization.
EXAMPLE 5
Characterization of Purified Mutant u-PAs
A. Measurement of enzyme concentration
The single-chain form of the mutant u-PA enzymes in conditioned media was
converted into the corresponding two-chain enzyme by treatment with plasmin-
sepharose (Calbiochem). The concentration of active u-PA in these media was
measured by active site titration with a standard PAI-1 inhibitor preparation
that had
been previously titrated against a trypsin primary standard as described in
Example 1
above. Total enzyme concentrations were measured by enzyme-linked
immunosorbent assay following the protocols of laboratory manual, Harlow et al
(1998) Using Antibodies, Cold Spring Harbor Laboratory. The ratio of these
concentrations yields the fraction of u-PA variant that is active in each
media.
B. Direct Chromogenic -Assay of u-PA
Direct assays of u-PA activity utilized the substrate carbobenzoxy-L-7-
glutamyl (a-t-butoxy)-glycyl-arginine-p-nitroanilide monoacetate salt (Cbo-L-
(y)-
Glu(a-t-f3u0)-Gly-Arg-pNA AcOH; Spectrozyme uPA, American diagnostica)
(Madison et al. (1995)J Biol. Chem., 270:7558-7562). Enzyme activity was
determined by measuring the increase in absorbance of the free (pNA) generated
per
unit time at an absorbance of 405 nm. Kinetic assays were performed over time
using
enzyme concentrations between 6 and 8 n.M. The concentration of Spectrazyme
uPA was varied from 25 to 150 M in assays of two-chain u-pA, and from 25 to
200
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_ p.M in assays of the protease domain of u-PA. Reactions were performed in
96¨well
microtiter plates and reaction rates were assessed by measurement of
absorbance at
405 nm every 30 seconds for up to 2 hours using a Spectromax M2 or M5 plate
reader
(Molecular Devices). The kinetic constants kcal, Km, and kcm/Km (specificity
constant)
were calculated by graphing the inverse of the substrate concentration versus
the
inverse of the velocity of absorbance at 0D405, and fitting to the Lineweaver-
Burk
equation (1/velocity=(K.N.)(1/[S]) + 1N,,õõ; where Vmax=[E]*kcm).
Table 13 below set forth the results of kinetic analysis of mutants of u-PA,
identified as exhibiting increased sensitivity against PAI-1/69 inhibitor, in
a direct
assay of u-PA enzyme activity. The results show that each of the mutant u-PAs
identified have a decreased enzyme activity as compared to wild-type u-PA as
determined from the measurement of the specificity constant for cleavage
(kcat/Km)
of the Spectrozyme uPA substrate. In the Table, the variants tested are those
identified in Table 11 above following selection from successive generations
(Ito IV)
of u-PA phage display libraries.
TABLE 13:
u-PA mutants Km kcal kcatfic Mutant/Wt u-PA
=
(mm) 01/44-I
u-PA/Ic (30) 0.745 22.3 2.9 x 105 0.30
u-PA/Ilb (30, 82) 0.299 11 3.6 x 105 0.38
u-PA/IIIa (30, 82, 159) 0.239 11.6 4.8 x 105 0.50
u-PA/IIIb (30, 39, 82, 159) 0.212 9.6 4.5 x 105 0.47
u-PA/IVa (30, 80, 82, 89, 159, 187) 0.177 10 5.6 x 105 0.58
u-PA/IVb (30, 80, 82, 84, 89, 159, 187) 0.321 11 3.4 x 105 0.35
Wild type u-PA 0.174 16.6 9.5 x 105 1.0 =
C. Kinetic analysis of u-PA variants using flourogenic substrate
Direct assays for measuring activity of the u-PA variants against the RRAR
target substrate sequence were performed utilizing an Ac-RRAR-AMC substrate.
The
use of 7-amino-4-methylcoumarin (AMC) fluorogenic peptide substrate is a
routine
method for the determination of protease specificity (Zimmerman etal. (1977)
Anal
Biochem, 78:47-51; Harris etal. (2000) PNAS, 97:7754-7759). Specific cleavage
of
the anilide bond frees the fluorogenic AMC leaving group, providing an
efficient
means to determine the cleavage rates for individual substrates. The
substrates were
*Trademark
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serially diluted from 0.05 to 12.0 mM and incubated Mille presence of protease
(9- 25
nM) in a Costar 96-well black half-area assay plate. The fluorescence from the
free
AMC leaving group was measured in a fluorescence spectrophotometer (Molecular
Devices Gemini XPS) at an excitation wavelength (380 am) and emission
wavelength
(450 am) with reference to an AMC standard. The rate of increase in
fluorescence
was measured over 30 minutes with readings taken at 30 second intervals. The
kinetic constants k, Km, and km,t/Km (specificity constant) were calculated by
graphing the inverse of the substrate concentration versus the inverse of the
velocity
of substrate cleavage, and fitting to the Lineweaver-Burk equation
(1/velocity=(Km/Võ,m,)(1/[S]) + 1/Vm,,,,; where Vmax=[E]*kcat).
Table 14 below sets forth the results of the kinetic analysis of the u-PA
variants ARF2 and ARF36 againsi the Ac-RRAR-AMC substrate. The results show
that the specificity constant for the RRAR substrate for the selected u-PA
protease
variants are increased about or more than 10-fold as compared to wild-type u-
PA.
TABLE 14:
Mutants Km ( M) Icat/Kni s-1) Improvement of K,/Km
mutant/Wt u-PA
u-PA/ARF2 546 434 11.7
u-PA/ARF36 614 357 9.6
Wt u-PA 381 37 1.0
D. Kinetic analysis of plasminogen activation using an indirect
chromogenie
assay
An indirect chromogenic assay was performed to determine the activities of
the wild-type and mutant u-PA produced as purified protein preparations
(Madison et
al. (1989)Nature, 339: 721-724; Madison etal. (1990), Bio/. Chem., 265: 21423--
21426). In this assay, free p-nitroaniline is released from the chromogenie
substrate
Spectrozyme PL (H-D-norleucylheXahydrotyrosyl-lysine-p-nitroanilide diacetate
salt,
American Diagnostics, Inc.) by the action of plasmin generated by the action
of u-PA
on plasminogen. The release of free p-nitroaniline. was measured
spectrophotometrically at 0D405 rim.
For the assay, 100 jil reaction mixtures containing 0.25-1 ng of the uPA
enzymes to be tested, 0.62 mM Spectrozyme PL, 0.2 M Lys-plasminogen (American
*Trademark
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Diagnostics, Inc.), were combined in a buffer containing 50 mM Tris-HCL (pH
7.5),
0.1 M NaC1, 1.0 mM EDTA and 0.01% (v/v) Tween 80. The reaction was incubated
at 37 C in 96-well, flat-bottomed microtiter plates (Costar, Inc.) and the
optical
density at 405 nm (0D405) was read every 30 s for 1 h in a Molecular Devices
.. Thermomax. The kinetic constants kcat, Km, and kcat/Km (specificity
constant) were
calculated as described earlier (Madison, E. L (1989)Nature 339, 721-724).
Table 15 below sets forth the results of kinetic analysis of mutants of u-PA,
identified as exhibiting increased sensitivity against PAI-1/69 inhibitor, in
an indirect
assay of u-PA enzyme activity. The results show that each of the mutant u-PAs
identified have a decreased enzyme activity as compared to wild-type u-PA as
determined from the indirect measurement of the specificity constant for
cleavage
(kcat/Km) of cleavage of the Spectrozyme PL substrate. In the Table, the
variants
tested are those identified in Table 11 above following selection from
successive
generations (Ito IV) of u-PA phage display libraries.
TABLE 15:
u-PA mutants Km ( M) k55 (s-I)
k.catIK.("1-1'1) Mutant/Wt u-PA
u-PA/Ic 9.01 24.7 2.7x 106 0.24
u-PA/Hb 8.6 22.6 2.6 x 106 0.23
u-PA/IIIa 6.31 37.6 5,9x 106 0.53
u-PA/IIIb 9.3 29.8 3.2 x 106 0.29
u-PA/IVa 7.03 38.7 5.5 x 106 0.50
u-PA/IVb 7.5 45.3 6.0 x 106 0.54
Wild type u-PA 6.03 70.1 1.1 x 107 1.0
E. Kinetic analysis of inhibition of mutant u-PA enzymes by wild-type
PAI-1
and mutant PAI-1
The second order rate constants (ki) for inhibition of mutant and wild type u-
PA (positive control) were determined using pseudo-first order (ki <2 x 106)
or
second order (ki >2 x 106) conditions. For each enzyme, the concentrations of
enzyme and inhibitor (mutant PAI-1) were chosen to yield several data points
for
which the residual enzymatic activity varied between 20 and 80% of initial
activity.
Kinetic measurements on the rate of interaction of wild-type and mutant u-PA
with
wild-type and mutant PAL-1 was performed at 24 C in 0.1 M Tris-HCI buffer (pH
7.4) containing 0.1 mM EDTA and 0.1% (v/v) Tween 20. The indirect chromogenic
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assay as described in Part D above was used to determine the residual enzyme
activity
remaining as a function of time.
The rate constants for inhibition of wild-type or mutant u-PA by PAI-1 were
under pseudo-first order conditions for an excess of PAI-1 over u-PA as
described
.. previously (see e.g., Holmes etal. (1987) Biochemistry, 26: 5133-5140;
Beatty et al.
(1980)]. Biol. Chem., 255:3931-3934; Madison et al. (1990) PNAS, 87: 3530-
3533;
Madison etal. (1993) Methods Enzymol., 223:249-271). Briefly, purified wild-
type
or mutant u-PA (3-50 fmol) were incubated at room temperature for 0 to 120
minutes
with wild-type or mutant PAI-1 (35-1330 fmol). Following incubation, the
mixtures
.. were diluted and the residual enzymatic activity was determined in a
standard
chromogenic assay as set forth in D above. Data were analyzed by plotting In
(residual activity/initial activity) versus time and determining the slope of
the
resulting straight line. Pseudo-first order rate constants were then derived
by dividing
the slope by the concentration of the inhibitor in the reaction.
For second order reactions, equimolar concentrations of wild-type or mutant
u-PA and wild-type or mutant PAI-1 were mixed directly in microtiter plate
wells and
preincubated at room temperature for periods of time varying from 0 to 30 min.
Following preincubation, the mixtures were quenched with an excess of
neutralizing
anti-PAI antibodies and residual enzymatic activity was measured in the
indirect
.. chromogenic assay. The indirect, chromogenic assays were compared with
control
reactions containing no PAI-1 or to which PAT-1 was added after preincubation
and
addition of anti-PAT-1 antibody, plasminogen and Spectrozyme PL to the
reaction
=
mixture. Data were analyzed by plotting In (residual activity/initial
activity) versus
time and determining the slope of the resulting straight line. Second order
rate
.. constants were then derived by dividing the slope by the concentration of
the inhibitor
in the reaction.
Table 16 below sets forth second order rate constants for inhibition of u-PA
variants by PAI-1/ RRAR inhibitor. The results show that the variants ARF2 and
ARF36 have about a 20-fold improvement in specificity for the PAI-1/RRAR
inhibitor substrate as assessed by the increased ki rate constant for
inhibition as
compared with wild-type u-pA.
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=
TABLE 16:
u-PA variants 2nd order rate .. Improvement
constant (M's') Mutant/Wt-uPA
u-PA/ARF2 1.6 x 105 18.3
u-PA/ARF36 1.7x 105 20.2
Wt u-PA 8.7x 103 1.0
Tables 17 and 18 below are the results of second order rate constants of
inhibition for wild-type (Table 17) or mutant PAI-1/69 inhibitor (Table 18) by
wild-
type u-PA or u-PA variants selected against the PAI-1/69 inhibitor. The
variant u-
PAs set forth in each of Tables 17 and 18 are those identified from one (I) to
four (IV)
successive rounds of a u-PA phage library selection as depicted in Table 11
above.
The results in Table 17 show that some of the mutant u-PAs (i.e. u-PA/IIIa, u-
PA/IIIb,
and u-PA/IVa) have a slightly increased second order rate constant for
inhibition as
compared to wild-type u-PA and the mutants u-PA/Ic, u-PA/IIb, and u-PA/IVb
have a
decreased second order rate constant for inhibition as compared to wild-type u-
PA.
The results in Table 18 show that the second order rate constant for
inhibition is
dramatically increased for each of the selected u-PA variants for inhibition
by the
mutant PAI-1/69 inhibitor. The results show that that each of the selected
variants
have a greater than 13-fold improvement in specificity for the PAI-1/69
inhibitor
substrate, with variants u-PA/IIIb, u-PA/IVa,.and u-PA/IVb each exhibiting
close to
or more than a 40-fold improvement in specificity.
TABLE 17:
u-PA mutants Sensitivity
2nd order rate factor
constant (M' s') (Mutant/Wt u-
PA)
u-PA/Ic 2.4x106 0.3
u-PA/IIb 2.9x106 0.3
u-PA/IIIa 9.7x106 1.2
u-PA/I1lb 1.3x107 1.6
u-PA/IVa 2.7x107 1.6
u-PA/IVb 6.8x106 0.8
Wild type u-PA 7.5x106 1.0
TABLE 18:
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u-PA mutants 2" order Increased sensitivity factor
rate (Mutant/Wt u-PA)
constant
(Iv-1 -1
s )
u-PA/Ic 1.0x105 13.7
u-PA/IIb 1.1x105 15.3
u-PA/IIIa 1.8x105 24.5
u-PA/IIIb 2.8x105 37.5
u-PA/IVa 2.7x105 35.9
u-PA/IVb = 3.2x105 42.6
Wild type u-PA 7.5x103 1.0
EXAMPLE 6
Selection of variant MT-SP1 from MT-SP1 phage libraries against
mutant AT3 inhibitor
A mutant antithrombin III (AT3) inhibitor (SEQ ID NO:5) containing a hexa-
peptide sequence in the reactive site loop (RSL) residues P4-P3-P2-PI-P1'-P2'
of a
wildtype AT3 corresponding to amino acid residues IAGRSL (SEQ ID NO: 478) was
mutated to contain a substitution in these residues to SLGRKI (SEQ ID NO:479),
corresponding to the amino acid residues of a complement C2 cleavage sequence.
The mutant AT3SLGR-KI
was used as the "bait substrate" in the protease selection
experiment to isolate phage with improved reactivity towards the mutant
substrate
sequence from a large, combinatorial MT-SP1 phage display library. In brief,
for
analysis of the first generation selection, 5 I of a 1:100 SM1 3 X 1013)
MT-SP I
phage library that is a low mutagenic frequency library (i.e., 0.2-0.5%
mutagenesis
frequency) that has enzymatic activity was combined in equal representation
with 5 I
of a 1:100 SM2 (-- 3 X 1012) MT-SP1 phage library that contains a higher
mutagenesis frequency (i.e. 0.9%). The phage libraries were mixed with 5 id
heparin
(5 ng/ 1; from stock of porcine intestinal mucosa), 5 I bait AT3sLGR-1(1
substrate
(ranging in concentrations from 0 (i.e. 5 pl H20), 0.018 M, 0.18 p.M, 1.8 M,
or 18
M) in the presence of 5 I (18 M) wildtype, plasma purified AT3 and 5 I 10X
MTSP activity buffer (0.5 M Tris HCI, pH 8, 0.3 M NaCl, 0.1% Tween 30) in a
total
volume of 50 1 (i.e. reaction contained 20 1 H20). The reaction was
incubated for
4.5 hours at 37 C.
The MTSP-1 phage-AT3 inhibitor complexes were captured using CuSO4
activated sepharose. 200 1 of chelating sepharose (Pharmacia) was pre-
activated
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with CuSO4 (100 mM). The CuSO4 activated sepharose (100 I) was blocked with 2
ml 0.5% BSA in PBS buffer for 1 hour at room temperature. The beads were
harvested from the blocking solution by pelleting at 6500 rpm for 60 sec.
followed by
resuspension in 450 I of binding buffer (0.5 M NaC1, 100 M Tris pH 8, 10 mM
Imidazole, 0.1% Tween 20). 50 1 of the above panning mixture reaction was
added
to the CuSO4 activated sepharose beads to capture the His-tagged AT3 inhibitor-
MTSP-1 phage complexes. The incubation was continued for another 1 hour at
room
temperature. Next, the panning mixture was centrifuged for 1 min at 2000 rpm
and
the sepharose beads containing bound AT3 phage-MTSP-1 inhibitor complexes were
washed with 500 I of binding buffer to remove unbound phages. The washing
step
was repeated 5 times and the beads containing bound complexes were transferred
to
= new tubes after each wash to avoid any potential "carry over" of non-
specific phages.
-
The bound MT-SP1 phage- AT3SLGRK1 complexes were eluted in 100 I of
elution buffer (0.5 M EDTA, pH 8.0). A 50 I aliquot of the eluted phages was
used
to infect 3 ml of TG I E. coli actively growing cells (A600= 0.5; 0.5-0D= ¨1.5
X 108
colonies/ml) for 20 minutes at 37 C. The infected bacteria were plated on
large plates
(245 mm X 245 mm) containing carbenicillin (75 ug/m1) and incubated at 30 C
overnight. The next morning the plates were harvested. The colonies on each
plate
were counted and compared to the background plate that contained no AT3SLGR-KI
inhibitor. The results of the colony counts are set forth in Table 19.
TABLE 19: Results of First Generation Selection
Concentration of AT3SLGR-KI Colonies from first round
in reaction of selection
18 M AT3sL1R-m Lawn
1.8 M AT3s""I 6088
0.18 M AT3sL"-Ki 1712
0.018 M AT3s1A1R-KI 3700
0 M AT38u1K-KI 840
The colonies from the AT3SLGR-10_ containing plates were scraped into 25 ml
2YT supplemented with carbenicillin (¨ 100 g/ml) after the plates had been
placed
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in the cold room for 2 hours to firm up the agar. 20 ml of the 2YT bacteria
containing
medium was added to 500 ml 2YT containing carbenicllin and the A600 was
determined to be 0.13. The bacteria were grown to an OD= ¨0.5 and then ¨ 1
X101
to 2.6 X 1013 cfu/ml of helper phage (VS MI3) were added (in ¨150 111-200 I)
for
amplification of the libraries. After 1 hour of growth at 37 C shaking, the
cultures
were supplemented with kanamycin to final concentrations of 3 p.g/mL and grown
overnight at 30 C with shaking. The cells were harvested and the phage
particles
present in the supernatant were precipitated using a PEG-NaC1 solution. For
analysis
of the second generation of MT-SP1 phage selection, the conditions were
similar to
the first generation, except that reaction against the AT381-GR-1(1 bait
substrate was for
only 27 minutes instead of 4.5 hours to enhance the stringency of the
selection.
Following elution of the bound MT-SP1 phage- AT3stoR-ki complexes as
described above, 50 I aliquot of the eluted phages were used to infect 3 ml
of TG1 E.
coli actively growing cells (A600= 0.5; 0.5 OD= ¨1.5 X 108 colonies/ml) for 20
minutes at 37 C. The infected bacteria were plated on large plates (245 mm X
245
mm) containing carbenicillin (75 Orli) and incubated at 30 C overnight. The
next
morning the plates were harvested. The colonies on each plate were counted and
compared to the background plate that contained no AT381-GR-K1 inhibitor. The
results
of the colony counts are set forth in Table 20.
TABLE 20: Results of Second Generation Selection
Concentration of Colonies from Enrichment
AT3stzn-Ki in reaction
second round of Ratio Compared
selection; to Background
(background)
18 MM AT3stuit-ki 2476 (165) 15:1
1.8 M AT3sL3K-KI 1750 (90) 19:1
0.18 M AT3sLuR-KI 2012(110) 18:1
0.018 M AT3sLuR-KI 1824 (89) 21:1
The colonies were picked for further characterization. The cells were grown
in 2 ml of 2x YT supplemented with carbenicillin (100 gimp and tetracycline
(10
g/m1) and phage preparation were performed as described (Sambrook, J eta!
(1989)
Molecular Cloning, A laboratory manual, Cold spring Harbor laboratory). The
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selected phage were tested for enzymatic activity against an Ac-SLGR-ACC
substrate. Further, the selected phage were selected for resistance to
inactivation by
wildtype AT3 or Plasma.
EXAMPLE 7
Expression and purification of Mutant AT3 Inhibitors
A. Generation of variant AT3
Mutant AT3 proteins for use as protease trap "bait substrates" were created by
introducing modifications into the amino acid sequence of the AT3 reactive
center
loop (RCL) by using the coding region of human antithrombin III (AT3) gene
(SEQ
'10 ID NO.: 612, purchased from Origene Technologies, Catalog I# TC110831;
) as a
template. The AT3 cDNA was amplified by PCR using the forward primer having
the sequence of nucleic acids set forth in SEQ ID NO.: 626:
GTCACTGACTGACGTGGATCCCACGGGAGCCCTGTGGACATC (which
contains a stuffer sequence (shown in bold above), a BamH1 site (shown in
italics),
and a portion that hybridizes to the AT3 gene (shown in plain text)), and a
reverse
primer having the sequence of nucleic acids set forth in SEQ ID NO.: 628:
GTAGCCAACCCTTGTGTTAAGGGAGGCGGAAGCCATCACCACCATCACCA
CTAAGAATTC. Following amplification, the cDNA was subcloned into the
pAcGP67b baculovirus transfer vector (BD Biosciences SEQ ID NO.: 494) using
restriction sites Barn and EcoRl. Either a C-terminal 6xHis-tag (SEQ ID NO.:
496) or
a C-terminal FLAG-tag (DYKDDDDK; SEQ ID NO.: 495) was added during this
subcloning step so that AT3 mutants later could be isolated by affinity
purification.
The nucleotide and amino acid sequence of the cloned AT3 fusion protein,
containing
AT3 fused to the 6xHis tag using a four amino acid GGGS linker (SEQ ID
NO.:620)
are set forth in SEQ ID NOs.:613 and 614, respectively.
To make mutant AT3 bait substrates for isolating target proteases with various
specificities, mutagenesis reactions were carried out using the Quikchangee
site-
directed mutagenesis kit (Stratagene) following the conditions specified by
the
supplier to introduce amino acid residues of target cleavage sequences in
place of the
wild-type AT3 reactive center loop (RCL) sequence, IAGRSL (SEQ ID NO.: 478)
(amino acid residues 422-427 of the precursor AT3 polypeptide sequence set
forth in
RECTIFIED SHEET (RULE 91) ISA/EP
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SEQ ID NO.: 5; and amino acid residues 390-395 of the mature AT3 polypeptide
sequence set forth in SEQ ID NO.: 493).
One such mutant, AT3 (AT3/ RRVR-KE) (SEQ ID NO.: 497), was made by
replacing amino acid residues of the wild-type IAGRSL amino acid sequence with
amino acid residues RRVRKE (SEQ ID NO. :498) from a targeted VEGFR2 cleavage
sequence. Another mutant, AT3 (AT3/ SLGR-KI) (SEQ ID NO.: 499), was made by
replacing the wild-type IAGRSL amino acid sequence with amino acid residues
SLGRKI (SEQ ID NO. :479) from a targeted complement C2 protein cleavage
sequence.
For the Quikchange PCR, the Wild-type AT3 RCL primer had the following
sequence of nucleic acids, which is set forth in SEQ ID NO: 630:
GCTGCAAGTACCGCTGTTGTGATTGCTGGCCGTTCGCTAAACCCCAACAG
GGTGACTTTC. The Complement C2 target sequence primer had the following
sequence of nucleic acids, which is set forth in SEQ ID NO.: 632:
GCTGCAAGTACCGCTGTTGTGTCGTTAGGCCGTAAAA TTAACCCCAACAGGGTGA
CTTTC. The VEGFR2 Target sequence primer had the following sequence of nucleic
acids, which is set forth in SEQ ID NO.: 634:
GCTGCAAGTACCGCTGTTGTGCGCCGTGTGCGCAAAGAAAACCCCAACAG
GGTGACTTTC.
Vectors containing the wild-type AT3 cDNA and vectors containing the
mutant AT3 cDNA were each transformed and amplified in XL-I -Blue
supercompetent cells (Stratagene). Plasmid DNA was purified from the cells
using
the Qiagen Plasmid Maxi Kit (Qiagen) following the conditions specified by the
supplier.
B. Expression of AT3 mutants
Sf9 insect cells were used to express and purify both His-tagged and FLAG-
tagged wild-type and mutant AT3 proteins using the AT3-containing pAcGP67b
transfer vectors described above. Sf9 cells were adapted for growth in SF900
II
serum-free medium (Invitrogen) and grown to 85-90% confluence in 35 mm dishes.
Cells were transfected using the FlashBac baculovirus expression system
(Oxford
Expression Technologies) following the conditions and protocol specified by
the
supplier. 500 ng of the AT3 transfer vector and 500 ng of the FlashBac
recombination vector were pre-incubated for 20 min with 5 I of Cellfectin
RECTIFIED SHEET (RULE 91) ISNEP
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transfection reagent (Invitrogen) in 1 ml SF900 II serum-free media without
antibiotics, then applied drop-wise to cells. Five (5) hours after
transfection, cells
were centrifuged and resuspended in 2 mL SF900 II serum-free medium with
antibiotics (antibiotic/antimycotie solution; Cellgro) and were incubated at
28 C for 4
days. Virus was expanded in Sf9 cells to a maximum titer of 1 x 106 pfu/mL, as
determined by plaque assay. Recombinant AT3 then was expressed using the High
Five (BTI-1N5B1-4) insect cell line (Invitrogen) and Excell'm 405 serum-free
media (JRH Biosciences). Cells were infected at a multiplicity of infection
(MOI)
between 0.1 and 1 and grown in 300 mL culture volumes in 1 L Erlenmeyer flasks
for
4-5 days, shaking at 125 RPM on an orbital shaking platform.
C. Affinity-based purification of wild-type and mutant AT3 proteins
For affinity-based purification of His-tagged AT3 proteins, supernatants from
the cultures from Example 78 were cleared by centrifugation and filtration
using a
0.45 1.1M filter and dialyzed into a buffer containing 50 mM Sodium Phosphate
pH
7.5, 300 mM NaCl. Protein was purified by column chromatography using the
BioLogic DuoflowTm chromatography apparatus (Bio-Rad) and 10 mL of TALON
cobalt metal affinity resin (Clontech). The resin-bound His-tagged protein was
eluted
with a linear gradient of 50 mM Sodium Phosphate pH 7.5, 300 mM NaC1 and 50 mM
Sodium Phosphate pH 6.5, 300 mM NaC1, 150 mM Imidazole. Fractions containing
protein were combined and dialyzed into AT3 storage buffer (50 mM Sodium
Phosphate pH 6.5, 300 mM NaC1, 5% glycerol). To demonstrate that the purified
AT3 preparation contained active protein, an MT-SP1 inhibition (active site
titration)
assay, as described herein, in Example 14 below, was performed in order to
measure
the ability of the dialyzed AT3 to inhibit the ability of MT-SP1 to cleave a
substrate.
The reaction mixture from this MT-SP I inhibition assay was assessed
kinetically for
cleavage of 0.4 mM Ac-RQAR-ACC (Acetyl-Arg-Gln-Ala-Arg-ACC) substrate
(custom synthesis) on a SpectraMax MS (SpectraMax M.5 Microplate Reader,
Molecular Devices) (Molecular Devices, Inc). The "ACC" in the name of this
substrate refers to the 7-amino-4-carbameiylmethylcoumarin leaving group. The
ACC
leaving group was detected at wavelengths of Excitation (Ex) = 380, Emission
(Em) --
450 and cutoff (c/o) = 435. Total yield of purified His-tagged AT3 protein was
approximately 1-3 mg/L.
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For affinity-based purification of FLAG-tagged AT3 proteins, supernatants from
the cultures were cleared by centrifugation and filtration as described above.
Cleared
supernatant was dialyzed into Tris-buffered Saline (TBS) pH 7.4 and added to a
fresh
1 L Erlenmeyer flask in a total volume of 300 mL. 2 mL of pre-equilibrated
anti-
FLAG M2 affinity gel (Sigma) was added, and the total volume was incubated on
an
orbital shaking platform at 125 RPM for 3 hours at 4 C. Resin-bound FLAG-
tagged
AT3 protein was collected by gravity using a fitted 20 mL chromatography
column
(Bio-Rad). The resin was washed a first time with 5 mL of TBS, once with 5 mL
of
TBST (TBS with 0.1% Tween-20), and a second time with 5 mL of TBS. The AT3
protein then was eluted by adding 10 mL TBS containing 0.2 mg/mL FLAG peptide
(Sigma). Eluate was concentrated and dialyzed into AT3 storage buffer (50 mM
Sodium Phosphate pH 6.5, 300 mM NaC1, 5% glycerol) and activity was assayed as
above, using a matriptase (MT-SP1) inhibition (active site titration) assay as
described
above and in Example 14A below. Total yield of purified FLAG-tagged AT3
protein
was approximately 0.5-1 mg/L.
These purified FLAG- and His-tagged AT3 proteins were used as protease
traps for identification of proteases recognizing particular target site
sequences, for
example, in the methods described in the following Examples.
EXAMPLE 8
Construction of phage-display libraries of protease variants
A. Cloning of wild-type and C122S MT-SP1 protease domain (B-chain) into
the pMal-C2 phagemid display vector
cDNA (SEQ ID NO.: 504) encoding a mature MT-SP I protease domain
(MT-SP I B-chain) (SEQ ID NO.: 505), which contains amino acids 615-854 of the
full-length MT-SP1 protein set forth in SEQ ID NO.: 253, was cloned, using the
restrictions sites Ndel and HindIII, into a pMal-C2 vector (SEQ ID NO.: 615)
(New
England Biolabs), which contains an STII leader sequence
(TGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTTTTCTATTG
CTACAAACGCGTATGCA (SEQ ID NO.: 636) to facilitate secretion, and nucleic
acids encoding a C-terminal domain of filamentous phage M13 GeneIII (SEQ ID
NO.:616). The MT-SP1 protease domain cDNA was inserted between the leader
sequence and the GeneIII domain so that the final construct contained the
sequence of
nucleic acids set forth in SEQ ID NO.: 510, which encodes an MT-SP1 N-terminal
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GeneIII fusion protein. The encoded amino acid sequence (SEQ ID NO.: 506) of
this
fusion protein is set forth below, with the STII leader sequence (SEQ ID NO.:
511) in
plain text, the mature MT-SP1 domain in bold and the C-terminal Genelli (SEQ
ID
NO.: 512) domain in italics. The * indicates the presence of a stop codon in
the
nucleic acid sequence encoding the protein.
SEQ ID NO.: 506:
mkkniafllasmfvfsiatnayavvggtdadegewpwqvslhalgqghiegaslispnwlvsaahcyi
ddrgfrysdptqwtaflglhdqsqrsapgvqerrlkriishpffndftfdydiallelekpaeyssinvr
piclpdashvfpagkaiwvtgwghtqyggtgalilqkgeirvinqttcenllpqqitprmmcvgfisg
gvdscqgdsggpIssyeadgrifqagyvswgdgcaqrnkpgyytrIplfrdwikentgvsgssgggs
egggsegggsegggsgggsgsgdfdyelcmanankgamtenadenalqsdakgkIdsvaidygaaide
igdvsglangngatgdfagsnsgmaqvgdgdnsplmnnfrqylpslpqsvecrpfvfsagkpyefsidcdk
inlfrgyfaftlyvatfmyvfstfanilrnkes*
In addition to the wild-type MT-SP1 B-chain fusion protein, an MT-SP1 B-
chain variant (CB469) - GeneIII fusion protein was generated using the method
described above. The CB469 variant amino acid sequence, set forth in SEQ ID
NO.:
507, was generated by substituting a serine for the cysteine at position 122
(based on
chymotrypsin numbering) of the wild-type MT-SP1 protease domain sequence,
which
is shown in the sequence above in italics and set forth in SEQ ID NO.: 505.
This
CB469 sequence was cloned into the pMal-C2 vector as described above. In order
to
achieve improved display, the phagemid vector containing nucleic acids
encoding this
variant MT-SP I fusion protein was used to generate MT-SP I mutant phage
display
libraries as described in Example 8B below.
B. Mutagenesis of protease domains for the generation of mutant phage
display libraries
Generation of phage display libraries containing mutated protease domains
was done using standard error-prone PCR mutagenesis protocols that are known
in the
art (Matsumura etal., Methods Mol Biol. 2002;182:259-67; Cirino etal., Methods
Mol Biol. 2003;231:3-9) as exemplified below.
1. Mutagenesis of B-chain MT-SP1 fusion proteins
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For the construction of mutant MT-SP1-containing fusion proteins, the MT-
SP1 CB469 cDNA was amplified from the pMal-C2 vector by error-prone PCR using
the Diversify PCR Random Mutagenesis Kit (BD Biosciences, Clonetech) and
following conditions suggested by the supplier to obtain five (5) mutations
per
kilobase. The MT-SP I forward primer used in this PCR, having the sequence of
nucleic acids set forth in SEQIDNO.:508):
GCGCAGATATCGTACCGCATATGAAAAAGAil TA TCGCA 17 ________ I C77', was
designed to hybridize within the STII leader sequence (with the residues shown
in
italics) and contained a Nde restriction site sequence (shown in bold). The MT-
SP1
reverse Primer, having the sequence of nucleic acids set forth in
SEQIDNO.:509:
GTGCATGCTGACTGACTGAGCTCCCGCT7'ACCCCAGTGT7'CTC, was designed
to hybridize within the 3' portion of the sequence encoding the MT-SP I
protease
domain (with the residues shown in italics) and contained a Sad l restriction
site
sequence (shown in bold).
2. Purification of mutagenesis products
Taq polymerase binds tightly to DNA and thus is not completely removed by
the Qiagen PCR purification kit; and its presence may interfere with
downstream
restriction digests of PCR products (Crowe et al., Nucleic Acids Res. 1991
January
11; 19(1): 184; Wybranietz et al., Biotechniques (1998) 24, 578-580). Thus, to
remove Taq polymerase from the amplified wild-type and mutant PCR products
from
Example 8B(1), prior to their purification, the following were added to the
reaction: 5
mM EDTA, 0.5% SDS, 50 ng/id proteinase K. To eliminate Taq polymerase, the
mixture was incubated at 65 C for 15 minutes.
To ensure that there was no wild-type template remaining (which could
potentially interfere with the selection methods described below), the PCR
product =
was purified to remove template DNA. To separate the.vector from the PCR
product,
samples were loaded onto a 1% agarose gel in the presence of 10X Orange G Gel
Loading Buffer (New England Biolabs) which does not co-migrate with the PCR
product or ladder. The PCR product was excised from the gel using a scalpel.
The
excised product was purified using either the QIAquick Gel Extraction Kit
protocols
(Qiagen) or the ZymocleanT" Gel Extraction Kit (Zymoclean CA, Cat # D400I)
following the conditions specified by the supplier.
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For the Qiagen Gel Extraction kit, the excised gel fragment was solublized in
QG Buffer and slowly passed through a Qiagen QG column, each of which has a
binding capacity of about 5 ¨ 10 pig, and does not hold more than 2 mL. A
sufficient
number of columns was used to accommodate the full volume of starting
material.
Columns were centrifuged in collector tubes at 14,000 rpm to remove any
residual
Buffer QG. 0.7 mL Buffer PE was added to each column and samples were
incubated
for 2-5 minutes. Columns then were centrifuged twice, for an additional 1
minute
each, at 13,000 rpm to remove all residual PE buffer. Samples were transferred
into a
new 1.5mL microcentrifuge tube, followed by addition of 504 H20 and incubation
.. for two minutes. Bound DNA was eluted by centrifugation at 7000 rpm.
Typical
yield was between 30% and 60% of the starting amount of sample.
For the Zymoclean TM Gel Extraction Kit, either one or more Zymoclean TM Gel
Extraction Kit columns (each of which has a 5 jig maximum binding capacity),
or one
or more columns from the Zymoclean TM DNA Clean & Concentrator' m Kit (each of
which has a 25 jig capacity) were used, depending on the amount of starting
material.
Using this kit, the excised DNA fragment was transferred to a 1.5 ml
microcentrifuge
tube, followed by addition of three (3) volumes of ADB BufferTM to each volume
of
agarose excised from the gel. Samples were incubated at 37-55 C for 5-10
minutes
until the gel slice had completely dissolved. The dissolved agarose solution
was
transferred to a Zymo-Spin 1TM Column in a Collection Tube and centrifuged at
at
least 10,000 rpm for 30-60 seconds. Flow through from the column was
discarded.
200 piL Wash Buffer was added to the column and centrifuged at at least 10,000
rpm
for 30 seconds. Flow-through was discarded and the wash step repeated. 50 1.PL
of
water was added directly to the column matrix. The minimum elution volume was
10
piL for the ZymocleanTM Gel Extraction column and 35 mL for the DNA Clean &
ConcentratorTM column. The column was placed into a 1.5 ml tube and
centrifuged
at at least 10,000 rpm for 30-60 seconds to elute DNA.
After elution using one of these two methods, samples were pooled and DNA
concentration was assessed by measuring absorbance of the sample at 260 rim in
a 70
piL UV cuvette, using a spectrofluorometer. DNA concentration was calculated
according to the following equation: 1A260 = 50 ng/pilds DNA.
C. Construction of protease mutant phage libraries using the pMal-C2
phagemid vector
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For construction of phage display libraries expressing mutant protease _.
domains, digested PCR products, such as those obtained from the protease PCR
mutagenesis and purification described in Example 8A and B above, were ligated
into
the pMal-C2 phagemid vector described above. For this process, the vector was
digested using Ndel and Sac 1, and the product gel purified and combined in a
ligation
reaction (described below) with the purified, restriction-digested PCR
products. The
molecular weight (MW) of the Ndel/Sacl digested, gel purified pSTII-g3 pMal-C2
phagemid is 5835 base pairs (bp); the MW of the Ndel/Sacl digested MT-SPI PCR
product is 806 bp. Typically, for a 2 mL ligation reaction, 7.58 ug cut vector
was
about 1 nM of vector and 3.14 g cut MT-SP I product was about 3 nM insert
product.
For ligation of the MT-SP I products, 40 I (3 nM, 3.14 jig) of the digested,
purified product was mixed with: 40 I (1 rilvf, 7.58 g) of digested,
purified vector;
1510 I H20; 4000 5X T4 DNA Ligase Buffer (Gibco); and 10 I (10 units) T4 DNA
Ligase (New England Biolabs). The ligationreaction was carried out overnight
at
16 C or at room temperature for 4 hours in a 2 mL volume. After ligation, the
DNA
Ligase was heat-inactivated by incubating the ligation-reaction mixture at 65
C for 15
minutes followed by addition of 4 mL ZymoResearch DNA Binding Buffer. This
sample then was added, 800 plat a time, to a 25 g ZymoResearch Column. The
column was washed twice with 600 I ZymoResearch Wash Buffer and eluted with
50 I, water, which had been pre-warmed to 42 C. Percent DNA recovery was =
assessed by measuring absorbance of diluted elution sample (3 I elution in 70
1
H20) at 260 nm using a spectrofluorometer. For example, for the 3 mL ligation
above, if the A260 corresponded to 0.12 -= 140 ng/ 1, then the total yield was
determined to be about 7 g (which is about 70% recovery of DNA from the
ligation).
The ligation product was electroporated into XL-1 blue cells, which can
accommodate approximately 500 ng,/ I by electroporation. 7.5 I of a 140 ng/ I
ligation was added per 200 I XL-1 Blue Cells (Stratagene). The cells were
added to
a 0.2 cm gap cuvette and electroporated in a Gene Pulsar (Bio-Rad, CA) using
the
following conditions: Voltage (V) = 2500, Capacitance = 25 (uF), Resistance =
200
ohms. Immediately following electroporation, 1 mL SOC medium (Invitrogen) was
added to the cuvette. The cells then were transferred to 25 mL SOC medium and
incubated at 37 C for 20 minutes. Following this incubation five (5) small
aliquots
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(100 microliter) of serial 100-fold dilutions of the cells were made and
plated on small
2YT Carbenicillin agar plates and incubated overnight at 37 C for counting the
number of colonies (representative of the number of clones from the library
generated
by electroporation). In one method, the remaining culture volume was
centrifuged to
pellet the cells followed by resuspension in 12 mL SOC buffer and plating on
large
agar plates (245 mm X 245 mm) supplemented with Carbenicillin (75 ug/ml) and
grown overnight at 30 C. Alternatively, to prepare phage stock from the
library, the
cells were added directly to 500 mL 2YT medium supplemented with Carbenicillin
(75 ug/ml) with M13K07 helper phage at lx 1010 CFU/mL and grown overnight at
37 C.
EXAMPLE 9
Selection of variant protease domains from protease domain phage libraries
using mutant AT3 protease traps
A. Selection of MT-SP1 phage/mutant AT3 complexes and ELISA-based readout
assay
In this example, a mutant AT3 inhibitor containing the complement C2 target
cleavage sequence SLGRKI, as described in Example 7 above, was used as the
"bait
substrate" in a panning experiment designed both to isolate and provide a
readout for
the presence of mutant MT-SP1-bearing phages with improved reactivity toward
the
target cleavage sequence. Phages were isolated from large, combinatorial MT-
SP1
phage display libraries produced as described in Examples 8A, 8B and 8C above
using the following procedure.
1. Interaction with and cleavage of AT3 variants by mutant MT-SP1-
bearing phages
Phage-bound MT-SP1 mutants from phage libraries were first selected using
mutant AT3 having the target cleavage site as follows. Each cleavage reaction
was
carried out in duplicate wells of a 96-well polystyrene plate (Nunc Maxysorp)
for 60
minutes at 37 C, by incubating the following reaction components in a 70 I,
volume:
I MT-SP I library phage at 3.14E12 CFU/ml; 7 L 10X MT-SP1 Activity Buffer.
30 (0.5 M Tris-HC1, pH 7.4, 1M, NaCI, 1% Tween20); 7 1AL 10 M Low
Molecular
Weight Heparin (BD Biosciences); 14 L H20; and 7 L of His-tagged mutant AT3-
SLGRKI. Individual cleavage reactions were carried out with the following
different
AT3-SLGRKI concentrations: 100 nM, 33 nM, 11 nM, 3.3 nM, 1.1 nM and 0.33 M.
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=
Each reaction was terminated with the addition of 2 iL100 mg/ml of the
protease
inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride (Pefabloc, Roche
Diagnostics).
40 L of a 0.55% BSA, 0.275% Tween 20 solution then was added and mixed
thoroughly by pipetting up and down.
2. Capture of MT-SP1 phage-AT3 complexes using anti-His antibodies
Meanwhile, wells in another 96-well polystyrene plate (Nunc Maxysorp) were
coated for 1 hour with shaking at room temperature with 100 .1, of 5 ng/11
Streptavidin (Pierce) in 0.2 M Carbonate Buffer (pH 9, Pierce). The wells then
were
washed three times with 250 ILL PBST, blocked with 200 I, of 0.2% BSA in PBS
for
2 hours at room temperature, incubated for 1 hour at room temperature with 100
I of
5ng/ I biotinylated Anti-6HIS antibody to capture His-tagged AT3 mutants, and
washed thoroughly with PBST. Each mutant MT-SP1 phage sample from the AT3
cleavage reaction in Example 9A(1) then was added in duplicate to the coated
96 well
plate and incubated for 1 hour at room temperature. Plates then were washed 14
times
with 250 L PBST.
3. ELISA-based readout for selection of MT-SP1 phage-AT3 complexes
After washing, the first of the two rows in the rnicrotiter plate was used to
carry out an ELISA (Enzyme-Linked Immunoassay)-based assay to obtain a readout
for phage capture. To each well in this row, 100 L of a 1:5000 dilution of an
HRP
conjugate anti M13 phage antibody (GE Healthcare) was added and allowed to
bind
for one hour. The wells in this row then were washed 8 times using a
Skanwasher*
plate washer (Molecular Devices), followed by addition of 100 L TMB/Peroxide
substrate solution (Pierce) and incubation at room temperature for 5 minutes.
This
reaction was quenched with 100 L 2M H2SO4 and assayed on a SpectraMax plate
reader (Molecular Devices) for absorbance at 450 nM. This readout was used as
a
surrogate for the presence of phage-AT3 complexes. In this assay, a
concentration-
dependent increase in absorbance Was observed (based on increasing
concentrations
of AT3-SLGRKI bait used in the cleavage reaction in Example 9A(1)). Further,
when
the process was performed using successive rounds of panning as described
herein,
increased absorbance was observed after each round, indicating that this
panning
method could successively enrich the phage pool for target cleavage site
affinity.
These data suggest that this method can be used to select, from a phage
library,
mutant protease domain fusion proteins having affinity for a particular target
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sequence. The ELISA assay provided a method for obtaining a readout for this
selection and enrichment, using the same 96-well plate that was used for
affinity-
based capture and subsequent elution described in Example 9A(4) below.
4. Elution of selected MT-SP1 phage
The second of the two duplicate rows of the microtiter plate from Example
9A(2) then was used to elute specifically bound phages for use in subsequent
purification and screening. To each well in this row, 100 L 100 mM HCL was
added and incubated for 5 minutes to elute the specifically bound phage. The
resulting phage eluate was added to a separate well containing 33 1..tL of 1M
Tris pH 8
for acid neutralization prior to infection. The 133 L of neutralized phage
mix then
was added to 1 ml of XL-1 Blue cells growing at an 0D600 of 0.5, which then
were
incubated for 20 minutes at 37 C and plated out onto 2xYT agar plates (245 mm
x
245 mm) supplemented with Carbenicillin. These cells were used in subsequent
screening, sequencing and purification methods as described in the Examples
below.
This selection and ELISA-based assay method was also used to select and
assess uPA mutants from uPA libraries, such as those described in Examples 2
and 3
above.
B. Purification of selected protease domain-bearing phages
This Example describes a method for isolation of phage supernatants that had
been selected using bait inhibitors. Titers (cfu/mL) of selected phages, such
as those
recovered in Example 9A above, were determined. Phage stocks were diluted with
PBS and used to infect E. coli XL-1 Blue cells, growing at an 0D600 of 0.5 or
approximately 2.1 x 108 cells/mL. The desired infectivity range was 1000 ¨
2000
colonies per plate. Infected cells were plated onto 2YT agar supplemented with
100i.tg/mL Carbenicillin in square 245mm x 245 mm polystyrene BioAssay
(Corning)
dishes and allowed to grow for 16-20 hours or until colony size were roughly
2mm in
diameter. Using an automated Colony Picker, individual colonies were picked
and
dispersed into wells in a 96 well polypropylene plate, each well containing
150-170
uL of 2YT medium supplemented with 10014/mL Carbenicillin and 12 g/mL
Tetracycline. Control wells were inoculated with cells infected with either
template
phage (phage having a protease domain that had been used as the template for
mutagenesis) or with phage containing fusion proteins containing inactive
protease
variants. The inoculated plates were sealed with an air-permeable membrane,
placed
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into a HiGro (GeneMachines) incubator and shaken at 400 rpm at 37 C for 14-20
hours.
After the incubation, to obtain log-phase cells, 100 pl from each well was
used to inoculate a well in a deep well 96 well plate containing 1 mL 2YT
medium
supplemented with 100 ug/mL Carbenicillin and 12 pg/mL Tetracycline. The deep
well plate then was sealed with an air-permeable membrane and placed in the
HiGro
incubator with shaking at 400 rpm at 37 C with oxygen aeration until the cell
density
reached an 0D600 of between 0.4 and 0.6. A typical incubation period was
between
4 and 5 hours. After incubation, 100 L of a helper phage stock was added to
each
well and the plate sealed and shaken again at 400 rpm for 5-10 minutes at 37
C. After
the 5-10 minutes of shaking, the plate was incubated at 37 C in a static state
without
shaking for 30-45 minutes. Shaking then was resumed at 400 rpm for 15-30
minutes '
at 37 C. Following shaking, 100 uL kanamycin solution (4001g/mL) was added to
each well to yield a final concentration of 33.3 pg/mL in each well. The plate
was
resealed, and shaken at 400 rpm at 37 C for 12 ¨ 16 hours. To pellet the
cells, the
plate then was centrifuged at 3500 ¨ 4500 rpm for 20 minutes at 4 C. After
centrifugation, supernatants, which contained isolated phage, were either used
immediately for screening as described in the Examples below, or first were
stored at
4 C.
C. Polyethylene Glycol (PEG) Precipitation of protease domain phage
supernatants
This Example describes a method for removing potentially contaminating .
background protease activity (to which some characterization assays described
herein
below are sensitive) in purified selected phage supernatants using
Polyethylene
Glycol precipitation. In this method, after rescuing the MT-SP1-bearing phage
supernatants (such as those selected and eluted in Example 9A) overnight (12-
16
hours) with helper phage samples Were centrifuged for 20 minutes at 4 C at
3500-
4500 rpm and 1000 iL of supernatant was removed from each well and transferred
to
a well in another 96 well deep well plate.
For precipitation, 250 [IL of a solution containing 20% PEG (by volume) in
2.5M NaCI was added to each well. The plate was sealed and mixed by vigorous
inversion, and then placed in an ice-water bath and left static for 1-2 hours.
The plate
then was centrifuged for 60 minutes at 4500 rpm or for 90 minutes at 3500 rpm.
The
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supernatant solution from each well was decanted out and the plate was patted
dry and
allowed to drain for 20-30 minutes. The resultant precipitate was resuspended
using
PBS at a final volume equivalent to 20% of the original phage supernatant
volume
(200uL) to yield a 5-fold concentrate. This material either was used
immediately in
assays described below, or stored at 4 C until ready for testing.
EXAMPLE 10
Screening of protease domain-bearing phages having increased reactivity and
catalytic efficiency towards target substrate sequences
Individual phage preparations, such as those described in Example 9B and 9C,
were used in various assays to determine their specificity and/or activity.
A. Analysis of phages expressing protease domain variants by monitoring
inhibition of fluorogenic peptide hydrolysis by bait proteins
As one approach for assessing mutant protease-bearing phage clones, a
biochemical inhibition assay can be performed comparing the ability of an
inhibitor
(bait serpin) to inhibit the activity of the selected variant protease domain
with its
ability to inhibit the activity of the template protease domain (i.e., the
"parental"
protease) that was originally used in phagemid library construction. With this
approach, the ability of mutant protease-bearing phages, such as those
recovered in
Example 9 above, to cleave a fluorogenic substrate containing a target
substrate
sequence is assessed in the presence and in the absence of a given
concentration of
inhibitor bait, and compared to the ability of the template protease domain to
cleave
the same sequence in the presence of the same bait. The use of fluorogenic
peptide
substrates is a routine method for the determination of protease specificity
(Zimmerman et al. (1977) Anal Biochem, 78:47-51; Harris et al. (2000) PNAS,
97:7754-7759).
For analysis of inhibition of MT-SP I B-chain mutants compared to inhibition
of MT-SP1 B-chain template (used for mutation in Example 8B(1)), the variant
AT3
(with a desired target sequence) can be used as the inhibitor and Ac-RQAR-ACC
can
be used as the substrate. In this substrate, specific cleavage of the anilide
bond frees
the fluorescent ACC leaving group, providing an efficient means to determine
the
cleavage rates for individual substrates.
In one example of such an assay, the ability of uPA protease-bearing phages,
such
as those recovered in Example 3 above, to cleave the fluorogenic substrate Ac-
AGR-
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AMC (SEQ ID NO.: 617) was assessed in the presence and in the absence of a
given
concentration of PAT bait. As described above, the use of such a 7-amino-4-
methylcoumarin (AMC) fluorogenic peptide substrate is a routine method for the
determination of protease specificity. In this example, 354 of phage
supernatant (such
as that obtained as described in Example 3(A)) was transferred to both a
designated assay
well and a designated control well in a 384-well Polypropylene plate (CoStar,
#3658). 35
1_, of 2x Indirect Assay Buffer containing the same PAI bait used in the
selection was
added to the assay wells. The concentration of bait was the same as used in
the selection.
354 of 2x Indirect Assay Buffer (without inhibitor) was added to the
corresponding
control wells. The plates were incubated at 37 C for 60 minutes. Following
the mixing
of the phage with inhibitor or control buffer, 101AL of an AGR-AMC fluorogenic
peptide
substrate, diluted to a final assay concentration of 60 p.M in Indirect Assay
Buffer, was
added the wells. Fluorescence was measured using a Molecular Devices
SpectraMax
Plate Reader with Excitation at 380 nm, Emission set at 460 nm, using the
kinetic read
mode for one hour. Clones showing enhanced inhibition of the target substrate
with
respect to the template protease were further analyzed.
B. Analysis of variant protease activity using fluorogenic peptide substrates
To directly assess the activity and specificity of MT-SP1 mutants, an assay
was performed using the fluorogenic peptide substrates Ac-RQAR-ACC (having the
native autocatalytic cleavage sequence recognized by wild-type MT-SP1) and Ac-
SLGR-ACC (Acetyl-Ser-Leu-Gly-Arg-ACC) having the C2 target site cleavage
sequence). As noted above, the ACC in the names of these substrates represents
7-
amino-4-carbamoylmethylcoumarin, which is the fluorescent leaving group. Also
as
noted above, the use of fluorogenic peptide substrates is a routine method for
the
determination of protease specificity (Zimmerman et al. (1977) Anal Biochem,
78:47-
51; Harris et al. (2000) PNAS, 97:7754-7759). In this example, specific
cleavage of
the anilide bond frees the fluorescent ACC leaving group, providing an
efficient
means to determine the cleavage rates for individual substrates. In this
method, 35
of 2x Indirect Assay Buffer was added to all test wells. 35 p.L of phage
supernatant
(isolated as described in Example 9B) or re-suspended PEG precipitated phage
(isolated as described in Example 9C) was added to each of the designated
wells.
After addition of the phage, the plate was centrifuged at 2000 rpm for 1
minute to
remove air bubbles. 104 of each of the Peptide Substrates ( Ac-SLGR-ACC (final
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assay concentration = 125 uM)) and Ac-RQAR-ACC (final assay concentration = 60
uM)) was diluted with 1X Indirect Assay Buffer and then added individually to
appropriate wells. The rate of hydrolysis (ROH), measured as Relative
Fluorescence
Units/second (RFU/s)); indicative of substrate cleavage, was monitored over
time
using a SpectraMax M5 Microplate Reader (Molecular Devices), using the
kinetic
read mode.
EXAMPLE 11
Production, selection, assessment and identification of MT-SP1 mutants
A. Fluorogenic assay of B-chain MT-SP1 mutants from phagemid library
This example describes a fluorogenic assay that was carried out to analyze the
activity and specificity of MT-SP1 protease domain-bearing phages produced
using
library produced as described in Example 8, The MT-SP1 library was prepared as
described in Example 8 above, using the native B-chain of MT-SP1 that has a
serine
in place of the cysteine at position 122 based on chymotrypsin numbering (SEQ
ID
NO.: 507) as a template. The library was prepared as described above, using
the
pMal-C2 vector and error-prone PCR conditions recommended by the supplier to
achieve an approximate mutagenesis rate of 0.5%. The yield from this
mutagenesis
reaction was 4 x 109 recombinants.
Selection of phages based on the rate of interaction with and cleavage of the
variant AT3 containing the target substrate sequence (in place of the native
RCL; as
described in Example 7) was carried out as described in Example 9A using a
polypropylene 96 well format. For this selection, lx 1012 recombinant phages
were
mixed with 3.3 nM variant AT3 carrying an SLGRICI RCL sequence for 30 minutes.
After washing, phage were eluted as described in Example 9A above, and used to
infect XL-1 blue cells as described in example 9B.
Successive rounds of selection were performed to enrich for rapid interaction
with and cleavage of the target substrate sequence using methods provided and
described herein. For example, clones selected in the first round were
subjected to a
second round of selections as described herein, using 3.3, 1.1, and 0.33 nM
AT3 for
one hour as described in Example 9A(3).
Following the first round of selection, phage supernatant was prepared as in
Example 9C using PEG precipitation from selected clones. Phage clones were
screened using the method of Example 10(B) above, using, as fluorogenic
substrates,
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both Ac-SLGR-ACC (containing C2 target cleavage site sequence) and Ac-RQAR-
ACC (containing the native cleavage sequence for the native MT-SP1). For each
clone, the rate of fluorescence (ROF) determined from the Ac-SLGR-ACC assay
was
compared to the ROF determined from the Ac-RQAR-ACC assay as a means to
compare the activity of each mutated MT-SP1 protease domain on the native
substrate
sequence to its activity on the target substrate sequences. The ROFs in the
mutant
MT-SP1 assays also were compared to the ROFs in the template (C122S) MT-SP I
assay. The results obtained with individual clones are shown in Table 21
below,
which lists clone numbers, and lists the rate of fluorescence as RFU/s
(relative
fluorescence units per second).
Table 21: Screening of mutant MT-SP1 protease domain-bearing phage selected
for cleavage rate of AT3-SLGRKI
Mutant MT- Ac-SLGR-
Ac-RQAR-
ACC
SP1 Clone ACC Rate
Rate
Number (RFU/sec.)
(RFU/sec.)
Template 1.85 15.6
CPC-0019595 7.1 33
CPC-0023085 0.8 2
CPC-0023230 1.3 4
CPC-0023401 3.9 12
CPC-0023949 0.7 2
CPC-0024129 3.8 15
CPC-0024153 2.5 6
CPC-0024527 4.3 12
CPC-0024715 3.2 12
CPC-0025366 1.3 1
CPC-0025387 6.8 14
CPC-0025533 6.9 23
CPC-0025582 1.7 3
CPC-0025720 2.5 5
CPC-0025866 1,2 4
CPC-0025876 4.0 8
CPC-0025890 10.6 33
CPC-0025941 1.0 4
CPC-0025974 9.3 41
CPC-0026100 14.8 25
CPC-0026122 6.5 31
CPC-0026125 17.4 84
CPC-0026200 7.0 21
CPC-0026219 i 8.0 23
CPC-0026232 7.1 15
CPC-0026597 11.0 34
CPC-0026727 0.8 2
CPC-0026761 7.8 25
CPC-0027290 3.9 12
CPC-0027306 11.0 50
CPC-0027309 8.3 50
CPC-0027326 9.1 54
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Ac-SLGR-
Mutant MT- ACC Ac-RQAR-
SPI Clone ACC Rate
Rate
Number (RFU/sec.)
(RFU/sec.)
CPC-0027335 12.3 57
CPC-0027369 2.3 11
CPC-0027399 13.4 99
CPC-0027484 2.5 12
CPC-0027516 3.4 17 ,
CPC-0027617 1.4 7
CPC-0027706 0.4 1
CPC-0027718 2.1 7
CPC-0027797 5.5 15
CPC-0027841 2.7 9
CPC-0028017 5.1 16
CPC-0028333 5.6 17
CPC-0028341 5.5 26
B. Identification of selected MT-SP1 mutant phages by DNA sequencing
This Example describes a method used for identification of positive phage
clones that were prepared as described in the previous Examples and selected
based
on results from a fluorogenic assay, such as the one described in Example 108
above.
For this method, individual clones were mixed with XL-1Blue E. coli cells for
infection and the cultures grown overnight shaking at 37*C. Plasmid DNA was
purified from the overnight culture using a plasmid preparation kit (Qiagen),
and the
DNA sent out for sequencing for identification of the mutants.
In one example of this method, the amino acid sequences of selected B-chain
MT-SP1 mutants from Example 11A above were identified using the steps outlined
above. The sequencing primer that was used for identification of these clones
is set
forth in SEQ ID NO.: 618: 5'GGTGTTTTCACGAGCACTTC3'. The results
obtained by analyzing the sequencing data are set forth in Table 22 below.
This table
lists only mutants with residues found to be mutated in more than one isolate.
Table
22 lists the amino acid mutations/positions for each clone compared with the
wild-
type MT-SP1 B-chain sequence (SEQ ID NO.: 505), which were determined by
analysis of sequencing data. Amino acid numbering is according to chymotrypsin
numbering. SEQ ID NOs. also are listed for both the sequence of amino acid
residues
that encodes the MT-SP1 protease domains (B-chains) containing the indicated
amino
acid mutations and also for the sequence of amino acid residues that encodes
the full-
length MT-SP1 protein having the same mutations.
Table 22: Selected MT-SP1-mutants
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Mutant MT- SEQ ID NO SEQ ID NO
Amino Acid Mutation (Chymotrypsin
SP1 Clone (protease (full-length).:
Numbering)
Number domain).:
CPC-0019595 C122S/1136T/N164D/T166A/F184(A)L/D217V 516 537
CPC-0023085 141F/C122S 517 538
CPC-0024153 141F/C122S/A126TN244G 518 539
CPC-0025366 D23E/141Ffr98P/C122S/T1441 519 540
CPC-0025387 141F/C122S 520 541
CPC-0025582 141F/C122S/L171FN244G 512 542
CPC-0025720 C122S/1-1143R/Q175R 522 543
CPC-0025876 141F/C122S/L171F 523 544
CPC-0025974 C122S/R230W 524 545
CPC-0026100 141F/C122S/1154VN244G 525 546
CPC-0026232 141F/L52M/C122S/V129D/Q221(A)L 526 547
CPC-0027399 F99L/C122S 527 548
CPC-0027706 F97Y/C122S/1136V/Q192H/S2011 528 549
CPC-0027797 H71R/C122S/P131S/D217V 529 550
CPC-0028017 C122S/D217V 530 551
CPC-0028333 T651UF93L/F97Y/C122S/D217V 531 552
EXAMPLE 12
Preparation and characterization of large quantities of selected phage-bound
MT-SP1 protease
A. Large-scale preparation of MT-SP1 phage
This Example describes preparation of larger quantities of selected MT-SP I
protease domain-bearing phages for analysis and subsequent use of selected
protease
domains in downstream methods, such as in vitro translation in whole MT-SP1
proteases. For this Example, single phage-bearing colonies, selected as in
Example 11
above, were grown overnight in 2YT medium supplemented with Carbenicillin, at
a
final concentration of 50 tig/mL, and tetracycline, at a final concentration
of
121.1g/mL, in small sterile Corning Orange Capped Erlenmeyer Flasks overnight.
To
make glycerol stocks, 85 60% glycerol was added to 500 1 of each culture
followed by storage at -80 C. The remaining volume of each culture was added
to a
2L widemouth baffled flask containing 500 ml 2YT medium supplemented with
Carbenicillin and Tetracycline. Alternatively, this step was performed in a
500 mL
flask in a 50 mL volume. The culture was grown until an 0D600 of 0.5 was
reached.
M13K07 Helper Phage was added to the culture to yield lEl CFU/ml and the
culture incubated for 1 hour at 37 C. Kanamycin was added to a final
concentration
of 30 g/mL. The cultures with helper phage were rescued overnight by
incubation at
37 C. Following overnight culture, samples were centrifuged at 6000 rpm for 15
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minutes. One volume PEG/NaCl (20% PEG 8K/1.5 M NaCI) solution was added per
volumes of culture. The sample then was stirred at 4 C for 20 minutes. Samples
were centrifuged at 10,000 rpm for 20 minutes and supernatants removed. After
a
second centrifugation step at 10,000 rpm, the pellet was resuspended in 5 mL
(for the
5 initial 500 mL volume) or 1 mL (for the initial 50 mL volume) PBS.
Precipitated
cells that were not resuspended were removed by brief centrifugation at 14,000
rpm
for 2 minutes. Glycerol was added, at 10% by volume, to the supernatant
containing
the resuspended cells. The cells were frozen at -80 C.
B. Assay of prepared phage using ACC and QF substrates
This Example describes a fluorogenic assay used to assess activity and
specificity of the phages prepared in Example 12A. PEG-precipitated mutant MT-
SPI -bearing phage clones, prepared using the 50 mL volume culture as
described in
Example 12A, were normalized to 1E13 particles/ml. The phage were then assayed
enzymatically using the ACC fluorogenic and QF (Quenched Fluorescence)
substrates
as follows. 54 phage (at 1E13 particles/m1) was added to each well in a black
Costar
polypropylene half-well microtiter plate (Corning) along with 5 AL 10X MT-SP1
assay buffer, 35 AL H20 and 5 L substrate in a total volume of 50 L. The
substrates used in individual wells were: Ac-SLGR-ACC (120 uM final
concentration), Ac-RQAR-ACC (60 uM final concentration), or the following
quenched-fluorescence substrates: SLGR-KT, and RQAR-SA (both used at 0.625 uM
=
final concentration). The Ac-SLGR-ACC substrate was used to assess cleavage,
by
the mutant MT-SP1 clones, of the target (complement C2) cleavage sequence,
while
the Ac-RQAR-ACC substrate was used to assess cleavage of the native target
cleavage sequence for MT-SP1. Likewise, the SLGR-KI substrate was used to
assess
cleavage of the target (complement C2) cleavage sequence, while the RQAR-SA
substrate was used to assess cleavage of the native target cleavage sequence
for MT-
SP 1. The ratio of these two cleavage rates was one quantitative measure of
the
specificity of the selected proteases for the targeted, new=cleavage sequence.
Comparison of these ratios for a selected variant and the corresponding
original
= scaffold (i.e., parental) protease indicated whether the selected protease
exhibited
enhanced selectivity towards the targeted, new cleavage sequence. For the ACC
Readout, the SpectraMax plate reader was set for excitation at 380 nM and to
detect
emission at 460 nM, with a 435 nIVI cutoff. For the QF readout, the SpectraMax
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. plate reader was set for-excitation at 490 nM, to detect emission at
520nM, with a
cutoff of 515 nM. The results of this assay are set forth in Table 23 below.
As
above, SEQ ID NOs. are listed for both the sequence of amino acid residues
that
encodes the MT-SF1 protease domains (B-chains) containing the indicated amino
acid
mutations and also for the sequence of amino acid residues that encodes the
full-
length MT-SP1 protein having the same mutations, as determined by sequencing,
as
described in Example 1113 above. RFUr(relative fluorescence units) numbers=
correspond to the rate of hydrolysis observed in a 60 minute reaction at 37 C
for each
substrate.
=
Table 23: Kinetic assay of selected WIT-SPi protease domain-bearing phage
clones =
Mutant Amino Acid SEQ ID SEQ ID Ac- Ac-
SLGR- RQAR-
MT-SPI Mutation NO. NO. SLGR- RQAR-
KI SA
Clone (Chymotrypsin (protease (lull- ACC = ACC
(RFU/s) (RFU/s) .
Number Numbering) domain): letigitb): (RFU/s) (RFU/s) _
Template C122S 507 515 2.4 23.5 0.12 = 0.11
CPC- -141T/C122S/P 532 553 . 10.9 = .56.8
0.36 0.48
0028341 73S/Q209L
CPC- F97L/C122S/F2 6.2 37.2 0.21 0.19
0033634 34L 533 554
CPC- . .3.4 11.0 0.20 0.18
534 555 =
0028971 C122S/Q175R
CPC- 2.0 9.5 0.07 0.05
535 556
0027484 N95K/C122S
CPC- 36 557 0.5 1.0 0.02 0.01
5
0028993 Y60(G)S/C122S
* Relative fluorescence units/second (Rate of hydrolysis) =
EXAMPLE 13
Expression of Selected MT-SP1 Mutant proteins using in vitro translation
This example describes the expression of MT-SP1 protease domains selected
and screened as in the Examples described above, that are not part of a gene
III fusion
protein.
A. Subeloning of MT-SP1 sequence into a modified IVEX vector
In order to express MT-SP I protease domains selected on phage, as described
= in the Examples above, that are not synthesized as gene III fusion
proteins, the coding
= region for the MT-SP1 protease domain containing the N-terminal
activation
sequence and a C-terminal 6xHis tag was cloned into the pIVEX.2.3d RTS in
vitro
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=
- translation vector (Roche; SEQ ID NO.: 559)) using the Ndel and
Xhol restriction
sites. The full N-terminal amino acid sequence of the pIVEX.2.3d.MT-SP1
preceding
the RQAR cleavage site is set forth in SEQ ID NO.: 558:
MEKTRHHHHHHSGSDCGLRSFTRQAR. Residues encoding MT-SPI B-chain =
proteins, which had been selected using phagemid libraries as described above,
,assayed using fiuorogenic screening methods as described in Example 10B and
11 A,
and sequenced as described in Example 11B, were subcloned into the.
pIVEX.2.3dIVIT-SP1 vector using the internal SphI and BsrGI restriction sites.
Phagemid selectads having mutations in the MT-SP1 sequence that were outside
these internal sites were created for use in this method by PCR mutagenesis.
B. Expression of MT-SP1 by in vitro translation
= Expression of MT-SP1 protease domains using min vitro translation kit,
the
RTS 100 E. coil Disulfide kit (Roche Applied Science), was performed using
conditions specified by the supplier with the following optimizations: The
components of the 50 I reaction solution were modified to include 12 1 of
amino
acid mix, 10 I of reaction mix, 12 1 of lysate, along with the addition of 5
1 of 1 M
Hepes pH 8 buffer, 2.5 p112 nM Tween-20, 2.5 Al of Protein Disulfide Isomerase
(PD!), and 6 gl of the chaperone RTS GroE Supplement (Roche Applied Science).
The 1 mL Feeding mix was also modified to include 168 1 of amino. acid mix,
24 I
of methionine, 608 p.1 of feeding mix, 100 Al of 1 M Hepes pH 8,50 Al of 12 nM
Tween-20, and 50 I of water. The in vitro translation.(IVT) reaction was
incubated
on a plate shaker at 30 C for 18 hours.
C. Purification of His-tagged MT-SP1 =
. Following the in vitro translation ([Vi) reaction, insoluble
protein was cleared
by centrifugation and transferred to a fresh tube. The cleared supernatant
(with a
volume approximately 45 pl) was brought up to a final volume of 1 mL in 50 mM
= Sodium Phosphate pH 7.5, 300 mM NaCI. The solution-was applied to 300 Al
of pre-
equilibrated TALON resin.in a 2 mL flitted chromatography column (Clontech)
and
allowed to flow through by gravity. The column was washed with 3 mL of a
solution
containing 50 mM Sodium Phosphate pH 7, 300 mM NaCI and 7.5 mM Imidazole;
and eluted with 600 pl'of a solution containing 50 mM Sodium Phosphate pH 6.5,
300
mM NaCI and 75 mM Imidazole. Eluate was dialyzed into phosphate-buffered
saline
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with 0.1% Tween-20 (PBST) and concentrated to 20 /LL. Typically, the yield of
purified protease was approximately 70%.
EXAMPLE 14
Characterization of mutated MT-SP1 protease domains
This Example describes the characterization of the mutated mutant MT-SP1
protease domains that were produced as in Example 13 above.
A. Active site titration of IVT reactions
To assess protease activity, active site titration of in vitro-translated
mutant
MT-SP I protease domains was performed on cleared supernatant with the MT-SP I
inhibitor M84R Ecotin, as described (Takeuchi et al, (1999) PNAS 96,11054-
11061).
For this assay, IVT protein was diluted to a final concentration of 1:10,000
in IX MT-
SPI activity buffer and incubated with 15 nM Ecotin in 1:2.5 serial dilutions
for 1
hour at 30 C. The reaction was assessed kinetically for cleavage of 0.4 niM Ac-
RQAR-ACC substrate on a SpectraMax M5 Microplate Reader (Molecular Devices,
Inc). The ACC leaving group was detected at wavelengths of Excitation (Ex) =
380,
Emission (Em) = 450 and cutoff (c/o) = 435. The assay points showing
fractional
activity between 20% and 80% uninhibited activity was plotted on a graph of
activity
vs. Ecotin concentration, and a line plotted though the points. The x-
intercept of the
line was used to establish the active concentration of the IVT protease. The
reaction
was graphed, with the linear part of the curve representing the active
concentration of
the IVT protease. Thus, the active site concentration (set forth for several
mutants in
Table 24 below; Active Site Conc.) was determined using Active Site Titration.
B. Assay of IVT MT-SP1 protease domain mutants with ACC and QF substrates
Several IVT-produced MT-SP1 phage selectants were assessed for increased
specificity for the mutant RCL cleavage site over the native RQAR MT-SP1
cleavage
site by quenched fluorescence (QF) kinetic enzyme assays. IVT supernatants,
cleared
as described in. Example 13C above, were diluted 1:10,000 in 1X MT-SP1
activity
bufferand incubated with 6.25 AM of either the native RQAR-SL QF substrate or
the
mutant RCL C2 cleavage substrate; SLGR-KI. Cleavage was assessed using a
SpectraMax MS Microplate Reader (Molecular Devices) with wavelengths of Ex =
490, Em = 520 and c/o = 515. The relative specificity of the IVT-produced
protease
for the RCL target sequence over the native sequence was calculated using the
ratio of
the RFU/s (Relative fluorescent units per second) for SLGR-K1 and RQAR-SL. The
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results are set forth in Table 24 below. In the column labeled_SEQ ID NO.:,
the SEQ
ID NOs. setting forth the amino acid sequence of the protease domains
containing the
indicated amino acid mutations are listed first; and the SEQ ID NOs. setting
forth the
sequence of amino acid residues that encodes the full-length MT-SP I
containing the
5 indicated amino acid mutations is shown in parentheses. RFU numbers
indicate the
measured relative fluorescence units (rate of hydrolysis) for each substrate.
Table 24: Kinetic assay of mutant MT-SP1 protease domains
Mutant Amino Acid Ac- Ac- Ac-
SEQ Active Ac-
RQAR-
MT-SPI Mutation SLGR RQAR
SLGR-
ID Site SA QF
Clone (Chymotrypsin -ACC -ACC KI QF
NO.: Conc. RFU/s
Number Numbering) =RFU/s RFU/s
RFU/s
CPC- CI22S/H143R/ 522
3.9 0.10 0.46 0.05
0.04
0025720 Q175R (543)
CPC- 141F/C122S/L1 523
2.5 0.10 0.39 0.00
0.01
0025876 7IF (544)
CPC- 527
4.2 0.55 9.84 0.04
009
0027399 F99UC122S (548)
CPC- H71R/C122S/P 529
3.7 0.31 1.62 0.14
0.19
0027797 131S/D217V (550)
CPC- 530
4.7 1.42 7.29 0.38
0.50
0028017 C122S/D217V (551)
T651C/F9311F97 531
CPC- Y/C122S/D217 3.6 1.05 = 5.97
0.37 0.51
0028333 V (552)
507
3.5 0.22 3.58 0.05
0.06
Template C122S (515)
EXAMPLE 15
10 Expression of
Selected MT-SP1 Mutant proteins as purified protein
A. Transfer of MT-SP1protease domain into pQE vector
A subset of MT-SP1 protease domain-bearing phage clones assayed in the
previous Examples was selected for transfer of the MT-SP1 protease domain
sequence into a pQE30expression vector that was previously modified for
expression
15 of wild-type MT-SP I protease domain. The InFusion DryDown PCR Cloning
Kit
= (Clonetech) was used to transfer selected clones into pQE30-MT-SP1 (SEQ
ID NO.:
624) using conditions specified by the supplier and as described by Benoit et
al.
(2006), Protein Expression & Purification 45:66-71. For this process, a
portion of the
phage clone DNA encoding the MT-SP1 protease.domain was amplified by
20 polymerase chain reaction (PCR) with the pQE-Insert-F2 forward primer:
TTCACGAGACAGGCTCGTOTTGTTGGGGGCACGGAT (SEQ ID NO.: 560)
and pQE-Insert-R3 reverse primer:
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. .
CAGCTAATTAAGCTTATTATACCCCAGTGTTCTC ____________ Fri (SEQ ID NO.: 561),
each cairying non-annealing 5' tails. Plasmid pQE30-MT-SP1 without the
protease
domain of MT-SP I was linearized using PCR with the forward primer: pQE-Linear-
F2: ACGAGCCTGTCTCGTGAATGACCGCAGCCC (SEQ ID NO.: 562) and
=
reverse primer: pQE-Linear-R1:
TAATAAGCTTAATTAGCTGAGCTTGGACTCC (SEQ ID NO.: 563) followed
by treatment of both the donor and acceptor PCR products with DpnI enzyme. For
each linearizing primer sequence set forth above, the 18-nt long homology
region, a
non-annealing 5' primer tail, is shown in bold. Both acceptor and donor DNA
were
then mixed together, and the InFusion reaction was nimbi a 10 AL volume using
"conditions specified by the supplier. 2 AL of the reaction mix was
transformed into
50 AL of E. coli TOPIOF' competent cells (Invitrogen, Carlsbad, CA). Colonies
were
selected on LB agar plates supplemented with 100 ppm Carbenicillin. Plasmid
DNA.
was isolated from selected clones, and sequenced using the forward primer: MT-
SP1-
5F: GGAGAAACCGGCAGAGTAC (SEQ ID NO.: 564) and reverse primer MT-
SPI-5R: GGTTCTCGCAGGTGGTCTG (SEQ ID NO.: 565) to verify correct
transfer. These primers are fully annealing.
B. Expression, refolding and purification of mutated MT-SPI protease domains
Plasmids encoding the protease domain of MT-SPI variants in the pQE30
. 20 vector (Qiagen) described in Example 15B above were transformed into BL21-
Gold(DE3) E. coli cells (Stratagene). Small starter cultures containing I mL
LB
supplemented with 100 AgImL Carbenicillin were inoculated frem colonies and
incubated for between 8 and 10 hours at 37 C. 100 AL of this culture was used
to
inoculate 50 mL of 2xYT medium supplemented with 100 Ag/mL Carbenicillin and
grown overnight. In 50 mL conical tubes (Coming), the cells were harvested by
centrifugation then lysed. Inclusion bodies (TB) were isolated with BugBuster
Reagent (Novagen) using the conditions specified by the supplier. The IB
pellets were
solubilized with 1 mL of a denaturing solution containing 100 mM Tris pH 8, 6
M
GdmHCL and 20 mM DTT. After removal of any insoluble material by
centrifugation
in microcentrifuge tubes (20,000 x g for 10 min), the sUpematant was diluted
into 40
mL refolding solution, containing 1.5 M arginine, 100 mM Tris pH 8, 150 mM
NaCI,
5 mM reduced glutathione and 50 AM oxidized glutathione, in 50 mL conical
tubes
(Corning). The tubes were placed horizontally on a Nutator platform (Fisher
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SP1 variants then
were extensively dialyzed at room temperature against 25 mM Tris pH 8, 25 mM
NaC1 for 3-4 days Following the removal of arginine during dialysis, MT-SP1
protease domain variants were able to activate.
Crude preparations of activated MT-SF1 protease domain variants were then
chromatographed on a 5 mL HiTrap"' Q HP column (GE Healthcare) attached to an
AKTA system operating in an automated mode enabling the processing of up to
seven
variants per round. The running buffer was 25 mM Bis-Tris pH 6.5 and purified
MT-
SP I was eluted within a 50 mL gradient to 350 mM NaCI. Active fractions were
pooled, then buffer exchanged into PBS +20 mM benzamidine and concentrated to
0.5-10 mg/mL using Amicon-Ultra 15 devices (Millipore) with a MWCO of 10 kDa.
Finally, aliquots were flash-frozen in liquid nitrogen and stored at 40 C.
EXAMPLE 16
Preparation of biotinylated mutant PAL inhibitor baits
This Example describes methods that were used to express and purify mutant
PAI inhibitor proteins, tagged with biotin for capture on streptavidin coated
surfaces,
for use in selecting variant OA proteases from uPA libraries. These mutant PAI
inhibitors are also useful for selection of some variant MT-SP I proteases
from MT-
SP1 libraries, depending on the MT-SP I variant and the RCL sequence used in
the
PAI.
A. N-terminal Biotinylation of 6xHis-PAI-1
For biotinylation of 6xHis-PAI-1 or reactive center loop variants thereof,
wild-
type His-tagged PAT-1 (SEQ ID NO.: 625) and His-tagged PAI-1 variants, as
described herein in Example 1, were transformed into the Rosetta-2 (DE3)pLysS
host
strain (Novagen). Expression was carried out essentially as described (Blouse,
G. E.,
Perron, M. J., Thompson, J. H., Day, D. E., Link, C. A., and Shore, J. D.
(2002)
Biochemistry 41(40), 11997-12009), with the following modifications. Induction
was
carried out for three hours at 30 C in 2XYT medium supplemented with 0.2%
glucose, 100 ug/mL Carbenicillin and 10 ug/mL chloramphenicol (Cm). The active
fraction of 6xHis-PAI-1 then was purified from the cell lysates as described
(Blouse,
G. E., Perron, M. J., Kvassman, J. 0., Yunus, S., Thompson, J. H., Betts, R.
L.,
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Lutter, L. C., and Shore, J. D. (2003) Biochemistry 42(42), 12260-12272;
Kvassman,
J.-0., and Shore, J. D. (1995) Fibrinolysis 9, 215-221).
6xHis-PAI-1 variants were preferentially biotinylated at the N-terminus using
the disulfide cleavable reagent EZ-Link NHS-SS-PE04-Biotin (PIERCE, Rockford,
IL #21442). Reactions were carried out at p1-16.2, for 4 hours, at 4 C on ice
in a
buffer containing 50 mM NaPi/300 mM NaCl/1 mM EDTA. The reaction was
initiated by the addition of a 5-fold molar excess of biotinylation reagent
dissolved in
DMSO. The final concentration of DMSO in the reaction was maintained at below
1%. The Biotinylation reaction was quenched by the addition of 0.5 M Iris/1.0
NaC1/10 mM EDTA, pH 7.4 to a final Tris concentration of 20 mM. Excess
biotinylation reagent was removed by extensive dialysis against a storage
buffer
containing 50 mM NaPi/300 mM NaC1/1 mM EDTA, pH 6.2. The concentration of
PAI-1 in the resulting solution was confirmed using an extinction coefficient
of 0.93
mL mg"' cm.' (see: Kvassman, J.-0., and Shore, J. D. (1995) Fibrinolysis
9, 215-
.
221). The extent of biotinylation was determined using the EZ-Quant HABA/Avidn
kit (PIERCE, Rockford, IL #28005), following the supplier-specified
conditions, and
was typically between 1.0 and 1.2 moles biotin per 1 mole PAI-1 variant.
B. in vivo biotinylation of PAIs: Biotinylation of BRS-TEV-OptiPAI-l"' in
vivo:
This Example describes a method that was used to in vivo biotinylate PAL. In
this Example, an appropriate recognition sequence was incorporated into the
gene
encoding the bait molecule so that the biotin-tagging of the bait could be
accomplished in growing cells, instead of being carried out with purified bait
in vitro.
A gene encoding stable PAT-1 protein (PAI-Istab, having the sequence of amino
acid
residues set forth in SEQ ID NO.: 567), which has N1501-1, K154T, Q319L and
M354I mutations (Berkenpas, M. B., Lawrence, D. A., and Ginsburg, D. (1995)
EMBO J. 14(13), 2969-2977), was designed to contain the following regions in
the
following order: 1) Start codon, 2) Biotin Recognition Sequence (BRS), 3)
Tobacco
Etch Virus Protease Sequence (TEV) 4) PAI coding sequence 5) stop codon; with
Escherichia coil codon optimization. This synthetic PAT-Istab gene was cloned
into
the commercial expression vector pET21Ta (Novagen, Madison, WI) (SEQ ID NO.:
566) using XbaI and HindIII restriction enzymes resulting in plasmid pCAT0002
(SEQ ID NO: 619), which expressed Optimized PAI-1 (OptiPAI- I ; encoded by the
amino acid sequence set forth in SEQ ID NO.: 621, in which amino acid residue
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positions 3-17 and 20-26 correspond to the BRS and TEV sites, respectively)
using
the T7 expression system. Plasmid pCAT0002 was then co-transformed into E.
coli
BL21-Gold (DE3) competent cells (Stratagene, San Diego, CA) carrying the
plasmid
pBirA (described in Asai et al., (1999) J. Biol. Chem. 274:20079-20078), which
overexpresses the E coil biotin ligase, BirA. Transformants were selected on
Luria-
Bertani (LB) agar plates supplemented with 100 ug/mL Carbenicillin and 10
gg/mL
chloramphenicol (Cm).
Expression of BRS-TEV-OptiPAI-1 stab (SEQ ID NO.: 621) and reactive center
loop variant's thereof was carried out essentially as described (Blouse, G.
E., Perron,
M. J., Thompson, J. H., Day, D. E., Link, C. A., and Shore, J. D. (2002)
Biochemistry
41(40), 11997-12009), with the following modifications. Induction was
initiated by
the addition of 0.1 mM IPTG and 0.1 mM D-biotin for three hours at 30 C in
2XYT
medium supplemented with 0.2% glucose, 100 i.tg/mL Carbenicillin and 10 [tg/mL
chloramphenicol (Cm). The active fraction of BRS-TEV-OptiPAI-1gab was
.. subsequently purified from the cell lysates as described (see: Blouse, G.
E., Perron,
M. J., Kvassman, J. 0., Yunus, S., Thompson, J. H., Betts, R. L., Lutter, L.
C., and
Shore, J. D. (2003) Biochemistry 42(42), 12260-12272; and Kvassman, J.-0., and
Shore, J. D. (1995) Fibrinolysis 9, 215-221) or by selection by chromatography
on
monomeric avidin (PIERCE, Rockford, IL #20227), following the conditions
.. specified by the supplier, with the following modifications. The binding
buffer
contained 50 mM Tris/100 mM NaCl/1 mM EDTA/0.01% tween-80 and had a pH of
7.4; and a competitive elution buffer was used that contained this binding
buffer plus
2 mM D-biotin. Biotin from the competitive elution step was removed by
extensive
dialysis against a storage buffer containing 50 mM NaPi/300 mM NaCl/1 mM EDTA,
pH 6.2. The PAI-1 concentration was confirmed using an extinction coefficient
of
0.93 mL mg-1 em-1 as described (Kvassman, J.-0., and Shore, J. D. (1995) =
Fibrinolysis 9,215-221).
C. in vitro Biotinylation of NU OptiPAI-e"
This Example sets forth methods for N-terminal Biotinylation of a PA!
variant. The methods described in this Example were carried out to incorporate
an
appropriate reactive group into the gene encoding the PAI bait molecule, such
that the
tagging of the bait with biotin could be accomplished after the protein had
been
purified, allowing position-specific labeling of the bait. In this Example,
since native
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PAI does not contain any cysteine residues, a cysteine codon was added to the
DNA
encoding the PAI gene to create a Cys-containing PAI that could then be
reacted with
Cys-reactive biotinylation reagents.
The N-terminal BRS-TEV sequence of OptiPAI-1, in plasmid pCAT0002
= 5 described above, was deleted with simultaneous introduction of
VIC mutation using
-ie
the QuikChange-XL mutagenesis Kit (Stratagene, San Diego, CA), according to
supplier specifications, resulting in plasmid pCAT0051 (SEQ ID NO.: 623)
expressing VIC OptiPAI-1'1' protein (SEQ ID NO.: 622). Plasmid pCAT0051 was
transformed into E. coil BL21(DE3) pLysS competent cells (Stratagene, San
Diego,
CA). Transformants were selected on Luria-Bertani (LB) agar plates
supplemented
with 100 ug/mL Carbenicillin and 10 ug/mL chloramphenicol (Cm).
Expression of the VIC OptiPAI-l' protein and reactive center loop variants
thereof Was carried out essentially as described (Blouse, G. E., Perron, M.
J.,
Thompson, J. H., Day, D. E., Link, C. A., and Shore, J. D. (2002) Biochemistry
41(40), 11997-12009), with the following modifications. Induction was
initiated by
the addition of 0.1 mM IPTG for three hours at 30 C in 2XYT medium that was
supplemented with 0.2% glucose, 100 ug/mL Carbenicillin and 10 ug/mL
chloramphenicol. The active fraction of the VIC OptiPAI-rab or variant thereof
was
purified from the cell lysates as described (Blouse, G. E., Perron, M. J.,
Kvassman, J.
0., Yunus, S., Thompson, J. H., Betts, R. L., Lutter, L. C., and Shore, J. D.
(2003)
Biochemistry 42(42), 12260-12272; and Kvassman, J.-0., and Shore, J. D. (1995)
Fibrinolysis 9, 215-221).
The VIC OptiPAI-1 proteins and variants were biotinylated at the engineered
N-terminal cysteine residue using the thiol-reactive and reversible
biotinylation
reagent, EZ-Link Biotin-HPDP (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-
propionamide) (PIERCE, Rockford, IL #21341). Biotin conjugation was
accomplished according to the supplier specifications, with some
modifications, as
follows. Stock solutions of biotin-HPDP were prepared at 5 mg/mL in anhydrous
DMF (9.3 mM). VIC OptiPAI-rab was rapidly desalted on G-25 gel filtration
column, from which it was eluted into a conjugation buffer containing 50 mM
NaPi/150 mM NaCU1 mM EDTA/0.01% Tween-80, pH 7.4. Biotinylation reactions
were initiated by the addition of a 10-fold molar excess of stock Biotin-HPDP.
The
final concentration of dimethylformamide (DMF) was maintained below 2-3%.
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Reactions were carried out for 4 hours at 25 C and the reaction progress
followed using release of the pyridine-2-thione leaving group at 343 rim.
Excess
biotinylation reagent was removed by extensive dialysis against a storage
buffer
containing 50 mM NaPi/300 mM NaCl/1 mM EDTA, pH 6.2. The PAI-1
concentration was confirmed using an extinction coefficient of 0.93 rnL mg cm-
I as
described (Kvassman, 3.-0., and Shore, .1. D. (1995) Fibrinolysis 9, 215-221).
The
= extent of biotinylation was typically 1:0-1.2 moles of biotin per mole of
PM-1
variant using the EZ-Quant HABA/Avidin kit and the release of pyridin-2-thione
(PIERCE, Rockford, IL #28005).
EXAMPLE 17
Screening of MT-SP1 variants from E. coil culture supernatants and periplasmic
extracts
= This Example describes two methods, each used as an alternative to
screening
the activity of MT-SP1 variants on phage by assaying either the protein from
the E.
coil periplasmic space or the protein from E. coil cell culture medium.
For both methods, 1 mL cultures were prepared as follows. ImL of 2YT
medium supplemented with 10Oug/mL Carbenicillin and 12ug/mL Tetracycline were
dispensed into each well of a 96 well deep well plate, and inoculated with
10p.L'of
XL-1 Blue cells that had been infected with MT-SIT protease domain-bearing
phage
' overnight as described in Example 14 above, from a 96 well master plate. The
deep
well plate was sealed with an air-permeable membrane and placed in a HiGro
shaker
incubator with shaking at 400 rpm at 37 C with oxygen aeration until the cell
density
reached 0.4 ¨0.6 0D600 (usually 4-5 hours of shaking). At that point IPTG was
added to a final concentration of 0.5 mM, and growth with shaking was
continued
overnight. The following day, the plate was centrifuged at 3600 rpm for 20min
to
pellet the cells.
A. Screening of MT-SP1 variants from Preiplasmic preps
The methods in this example were used to assay the full length MT-SP1-gene
III fusion proteins, and enzymatically active cleavage products of the fusion
proteins,
.30 that had been transported into the E. coil periplasmic space. After the
centrifugation at
3600 rpm, the culture supernatant was discarded and the cell pellet was used
to
release periplasmic proteins using either of the following conditions:
Condition 1:
The cell pellets were resuspended in 150 AL cold phosphate buffered saline
(PBS);
RECTIFIED SHEET (RULE 91) ISA/EP
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_
the suspension was transferred a 96 well PCR plate; followed by one step
freeze
thawing (20 min at -80 C / 10 min in a room temperature water bath); or
Condition 2:
The cell pellets were resuspended in 150 uL of 3% BugBuster Protein Extraction
Reagent (Novagen); the suspension was transferred to a 96 well PCR plate; and
the
suspension was incubated at room temperature for 30 min. Next, the cell
suspensions
were centrifuged for 20min at 3600 rpm at 4 C and the supernatants containing
periplasmic proteins were carefully removed without disturbing the pellet.
Further, the
periplasmic extracts were used to determine enzyme activity of the MT-SP I
variants
using appropriate substrates as described in Example 10, Section B.
B. Screening of MT-SP1 variants from supernatant preps
The methods in this example were used to assay the full length MT-SP1-gene
III fusion proteins, and catalytically active fragments of the fusion protein,
that had
diffused from the periplasm and into the bacterial cell culture media. In this
example,
after centrifugation in the 1 mL culture, 10 iL of the culture supernatant
were
removed and assayed using the protease assay described in Example 10, Section
B,
except an additional 25 1., of assay buffer was added to the reaction.
Since modifications will be apparent to those of skill in this art, it is
intended
that this invention be limited only by the scope of the appended claims.
CA 02791144 2013-02-27
236a
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description
contains a
sequence listing in electronic form in ASCII text format (file: 51205-113D Seq
2012-09-27 vl.txt).
A copy of the sequence listing in electronic form is available from the
Canadian
Intellectual Property Office.
