U.S. patent application number 13/506845 was filed with the patent office on 2013-06-27 for protease screening methods and proteases identified thereby.
This patent application is currently assigned to Torrey Pines Institute for Molecular Studies and Catalyst Biosciences, Inc.. The applicant listed for this patent is Edwin L. Madison. Invention is credited to Edwin L. Madison.
Application Number | 20130164820 13/506845 |
Document ID | / |
Family ID | 39217879 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130164820 |
Kind Code |
A9 |
Madison; Edwin L. |
June 27, 2013 |
PROTEASE SCREENING METHODS AND PROTEASES IDENTIFIED THEREBY
Abstract
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, such as a serpin, an alpha macroglobulins or a p35
family protein or modified serpins and modified p35 family members
or modified alpha macroglobulins, that, upon cleavage of the
substrate, traps the protease by forming a stable complex. Also
provided are modified proteases.
Inventors: |
Madison; Edwin L.; (San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Madison; Edwin L. |
San Francisco |
CA |
US |
|
|
Assignee: |
Torrey Pines Institute for
Molecular Studies and Catalyst Biosciences, Inc.
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120301945 A1 |
November 29, 2012 |
|
|
Family ID: |
39217879 |
Appl. No.: |
13/506845 |
Filed: |
May 18, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11825627 |
Jul 5, 2007 |
8211428 |
|
|
13506845 |
|
|
|
|
60818804 |
Jul 5, 2006 |
|
|
|
60818910 |
Jul 5, 2006 |
|
|
|
Current U.S.
Class: |
435/219 ;
435/252.3; 435/254.11; 435/254.2; 435/320.1; 435/325; 435/348;
435/419; 506/11; 536/23.2 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 11/06 20180101; A61P 31/04 20180101; A61P 43/00 20180101; A61P
19/02 20180101; C12N 9/64 20130101; A61P 21/02 20180101; A61P 37/02
20180101; A61P 7/02 20180101; A61P 25/00 20180101; A61P 37/06
20180101; C07K 2319/23 20130101; C12N 15/1037 20130101; A61P 9/08
20180101; C07K 2319/02 20130101; A61P 35/00 20180101; C07K 2319/21
20130101; C07K 2319/42 20130101; C12N 9/6462 20130101; A61P 3/10
20180101; A61P 9/14 20180101; A61P 21/04 20180101; A61K 38/00
20130101; C07K 2319/00 20130101; A61P 7/08 20180101; C12Y 304/21109
20130101; G01N 33/6842 20130101; A61P 25/28 20180101; C12N 9/6408
20130101; C12Y 304/21073 20130101; A61P 17/06 20180101; A61P 1/02
20180101; A61P 13/12 20180101; A61P 17/02 20180101; A61P 27/02
20180101; A61P 7/00 20180101; A61P 9/10 20180101; A61P 1/04
20180101; A61P 9/00 20180101; C07K 2319/30 20130101; C07K 2319/41
20130101; C12Q 1/37 20130101; A61P 1/00 20180101; C12N 9/50
20130101 |
Class at
Publication: |
435/219 ; 506/11;
536/23.2; 435/320.1; 435/252.3; 435/254.2; 435/254.11; 435/419;
435/348; 435/325 |
International
Class: |
C12N 9/50 20060101
C12N009/50; C12N 15/57 20060101 C12N015/57; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 1/15 20060101
C12N001/15; C40B 30/08 20060101 C40B030/08; C12N 15/58 20060101
C12N015/58 |
Claims
1. A method for identifying or selecting a protease or
catalytically active portion thereof that cleaves a cleavage
sequence in a protein target substrate, comprising: a) contacting a
collection comprising a plurality of different mutant proteases
and/or catalytically active portions thereof that comprise the
mutations(s) with a protease trap polypeptide, wherein: the
protease trap polypeptide comprises a reactive site containing a
cleavage sequence for the protein target substrate, or is modified
in its reactive site by amino acid replacement(s), deletion(s), or
substitution(s) to include the cleavage sequence of the protein
target substrate; contacting is effected under conditions for
cleavage of amino acids in the cleavage sequence in the protease
trap polypeptide by a protease or a catalytically active portion
thereof in the collection to form stable complexes containing the
protease trap polypeptide covalently linked to a protease or
catalytically active portion thereof; upon cleavage of amino acids
in the cleavage sequence in the protease trap polypeptide by a
protease or catalytically active portion thereof in the collection,
the protease trap polypeptide forms a stable complex with a
protease or catalytically active portion thereof in the collection;
b) separating the complexed proteases from uncomplexed members of
the collection; and c) identifying or selecting the protease or
catalytically active portion thereof in the complex, thereby
identifying or selecting a protease or catalytically active portion
thereof that cleaves the protein target substrate.
2. The method of claim 1, wherein the protease trap polypeptide is
a serpin or modified serpin, an alpha macroglobulin or a modified
member of the alpha macroglobulin family, or, a p35 protein or a
modified member of the p35 family.
3. The method of claim 1, wherein the mutant proteases that are
members of the collection comprise one or more of amino acid
replacements, deletions or insertions in the primary sequence of an
unmodified protease.
4. The method of claim 3, wherein the unmodified proteases or
catalytically active portions thereof are serine and/or cysteine
proteases, or catalytically active portions thereof.
5. The method of claim 4, wherein a protease or catalytically
active portion thereof is selected from granzyme B, testisin,
trypstase beta 1, kallikrein hk5, corin, kallikrein 12, DESC1
oritesase, trypstase gamma 1, kallikrein hK14, hyaluronan-binding
serine protease, tryptase, kallikrein hK15, trypsin, neutrophil
elastase, mannan-binding lectin-associated serine protease-3,
cathepsin G, myeloblastin, granzyme A, granzyme M, chymase,
granzyme K, granzyme H, chymotrypsin B, pancreatic elastase,
pancreatic endopeptidase E, pancreatic elastaste II,
enteropeptidase, chymotrypsin C, prostasin, kallikrein 1,
kallikrein hK2, kallikrein 3, mesotrypsin, Factor XII, plasma
kallikrein KLK3, factor XI, factor IX, factor VII, factor Xa,
thrombin, protein C, acrosin, hepsin, hepatocyte growth factor
activator, urinary plasminogen activator (uPA), tissue plasminogen
activator (tPA), plasmin, neurosin, neurotrypsin, neuropsin,
kallikrein hK10, epitheliasin, prostase, chymopasin, kallikrein 11,
membrane type serine protease 1 (MT-SP1), spinesin, cathepsin L,
cathepsin V, cathepsin K, cathepsin S, cathepsin F, cathepsin B,
papain, cruzain, subtilisin, thermitase, C5a peptidase,
fervidolysin, lactocepin, furin, kexin, caspase-1, caspase-3,
caspase-7, caspase-6, caspase-2, caspase-4, caspase-5, caspase-8,
caspase-9, caspase-10, caspase-11, caspase-12, caspase-13 or
caspase-14.
6. The method of claim 1, wherein the collection contains at least
5, 10, 50, 100, 500, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 or more
different members.
7. The method of claim 1, wherein the proteases are provided as
catalytically active portions of a protease.
8. The method of claim 1, wherein: the protease trap polypeptide is
labeled for detection or separation; and separation is effected by
capture of complexes containing the detectable protease trap
polypeptide with a protease or catalytically active portion
thereof.
9. The method of claim 8, wherein the protease trap polypeptide is
labeled with biotin.
10. The method of claim 1, wherein the protease or catalytically
active portions thereof are displayed on a solid support, on a cell
surface, or on the surface of a microorganism.
11. The method of claim 1, wherein the proteases or catalytically
active portions thereof in the complexes are displayed on a
microorganism, and wherein following identification or selection of
a protease or catalytically active portion thereof in the
collection, the microorganism displaying the protease or
catalytically active portion is grown to thereby amplify the
nucleic acid molecules that encode the identified or selected
protease or catalytically active portion thereof to produce a
second collection of proteases and/or catalytically active portions
thereof.
12. The method of claim 1, wherein: at least two different protease
trap polypeptides are contacted with the collection; and at least
one of the protease trap polypeptides is detectably labeled to
effect capture of stable complexes containing the detectable
protease trap polypeptide and a protease or catalytically active
portion thereof.
13. The method of claim 12, wherein: only one protease trap
polypeptide is detectably labeled; and the other or others are each
present in excesses compared to the detectably labeled protease
trap polypeptide.
14. The method of claim 1, wherein the protease trap polypeptide is
a serpin selected from plasminogen activator inhibitor-1 (PAI-1),
antithrombin III (AT3), and modified forms thereof, wherein the
modified form contains one or more amino acid replacements,
deletions, or substitutions in the reactive site in the protease
trap polypeptide to include a cleavage sequence of the protein
target substrate.
15. The method of claim 1, wherein the target substrate is a
protein involved in the etiology of a disease or disorder.
16. The method of claim 1, wherein the target substrate is selected
from Interleukin-5 (IL-5), IL-5 receptor, IL-1, IL-1 receptor,
IL-13, IL-13 receptor, IL-12, IL-12 receptor, IL-4, IL-4 receptor,
tumor necrosis factor (TNF), TNF receptor, C--C chemokine receptor
type 5 (CCR5), CXC chemokine receptor 4 (CXCR4), gp120, gp141, CD4,
RSV fusion protein, hemagglutinin, B7, CD28, IgE, IgE receptor,
CD2, CD3, CD40, IL-2, IL-2 receptor, vascular endothelial growth
factor (VEGF), fibroblast growth factor (FGF), epidermal growth
factor (EGF), transforming growth factor (TGF), HER2, C--C
chemokine receptor type 1 (CCR1), CXC chemokine receptor 3 (CXCR3),
C--C chemokine receptor type 3 (CCR3), Src, Akt, Bcl-2, BCR-Abl,
GSK-3, Cdk-2, Cdk-4, EGFR, VEGFR-1, VEGFR-2 or a complement
protein.
17. The method of claim 1, further comprising: after identifying a
first protease or proteases, preparing a second collection of
proteases, wherein an identified first protease is used as a
template to make further mutations in the protease sequence or
catalytically active portion thereof such that members of the
second collection contain polypeptides having mutations of the
identified first proteases and additional mutations; then
contacting the second collection with a second protease trap
polypeptide that is either identical or different from the first
protease trap polypeptide used to isolate the first protease or
proteases, wherein: the second protease trap polypeptides is a
serpin, alpha macroglobulin, p35 family protein or a modified form
thereof that comprises in the reactive site amino acid residues
containing a cleavage sequence of the protein target substrate; and
upon cleavage of the amino acid residues in the second protease
trap polypeptide by a protease or catalytically active portion
thereof in the collection, covalent complexes of the protease trap
polypeptide with the protease or catalytically active portion
thereof in the collection are produced; and identifying a second
protease(s) or catalytically active portion(s) thereof from the
collection in the complex, whereby the second identified
protease(s) has greater activity or specificity towards the target
substrate than the first identified protease.
18. The method of claim 1, wherein in step a) the method further
comprises adding a competitor to the reaction between a protease
trap polypeptide and a protease or catalytically active portion
thereof to thereby enhance selectivity of identified protease(s) or
catalytically active portion(s).
19. The method of claim 18, wherein the competitor is a cell or
tissue extract, a biological fluid, a wild-type form of a protease
trap polypeptide or a variant of a protease trap polypeptide.
20. The method of claim 18, wherein the competitor is human serum
or plasma.
21. The method of claim 1, further comprising screening proteases
or catalytically active portions thereof that formed complexes to
assess the substrate specificity of these proteases or
catalytically active portions thereof for the cleavage sequence of
the target substrate compared to a non-target substrate.
22. A modified urinary plasminogen activator (u-PA) polypeptide or
catalytically active fragment thereof, comprising an amino acid
replacement in an unmodified u-PA polypeptide at a position
corresponding to a position selected from among positions 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, whereby substrate specificity or activity is altered
compared to the u-PA polypeptide not containing the amino acid
replacement(s).
23. The modified u-PA polypeptide or catalytically active fragment
thereof of claim 22, wherein the unmodified u-PA polypeptide
comprises the sequence of amino acids set forth in SEQ ID NO:433,
or a sequence of amino acids that exhibits at least 85% sequence
identity to the u-PA polypeptide set forth in SEQ ID NO:433.
24. The modified u-PA polypeptide or catalytically active fragment
thereof of claim 22, comprising an amino acid replacement selected
from among F21V, I24L, F30I, F30V, F30L, F30T, F30G, F30M, V38D,
T39A, Y61(A)H, R72G, L73A, L73P, S75P, E80G, K82E, E84K, I89V,
K92E, F132L, G133D, E137G, I138T, L155P, L155V, L155M, K156Y,
T158A, V159A, V160A, K187E, and R217c, based on chymotrypsin
numbering.
25. The modified u-PA polypeptide or catalytically active fragment
thereof of claim 22, wherein the u-PA polypeptide or catalytically
active portion thereof contains amino acid replacements selected
from among L73A/I89V; S75P/I89V/I138T; R72G/L155P; F132L/V160A;
L73A/I89V/F30T; L73A/I89V/F30L; L73A/I89V/F30V; L73A/I89V/F30G;
L73A/I89V/L155V; L73A/I89V/F30M; L73A/I89V/L155M;
L73A/I89V/F30L/L155M; L73A/I89V/F30G/L155M; F30V/Y61(A)H;
F30V/K82E; F30V/K156T; F30V/K82E/V159A; F30V/K82E/T39A/V159A;
F30V/K82E/T158A/V159A; F30V/Y61(A)H/K92E;
F30V/K82E/V159A/E80G/I89V/K187E; and
F30V/K82E/V159A/E80G/E84K/I89V/K187E.
26. The modified u-PA polypeptide or catalytically active fragment
thereof of claim 22, whereby substrate specificity to VEFGR-2, or a
sequence thereof, is altered.
27. The modified u-PA polypeptide or catalytically active fragment
thereof of claim 22, whereby substrate specificity to a t-PA
substrate, or sequence thereof, is altered.
28. A pharmaceutical composition, comprising a modified u-PA
polypeptide or catalytically active fragment thereof of claim
22.
29. A nucleic acid molecule, comprising a sequence of nucleotides
that encodes a modified u-PA polypeptide or catalytically active
fragment thereof of claim 22.
30. A vector, comprising the nucleic acid molecule of claim 29.
31. An isolated cell, comprising the vector of claim 30.
32. A modified membrane type serine protease 1 (MT-SP I)
polypeptide or a catalytically active portion thereof, comprising:
an amino acid replacement in an unmodified MT-SP 1 at a position
corresponding to a position selected from among positions 23, 52,
60(g), 65, 71, 93, 95, 126, 129, 131, 136, 144, 154, 164, 166,
184(a), 201, 209, 230, 234, and 244, based on chymotrypsin
numbering, whereby substrate specificity or activity is altered
compared to the MT-SP1 polypeptide not containing the amino acid
replacement(s); or an amino acid replacement selected from among
T65K, F97Y, F97L, T98P, F99L, H143R, L171F, P173S and Q192H, based
on chymotrypsin numbering, whereby substrate specificity or
activity is altered compared to the MT-SP1 polypeptide not
containing the mutation(s).
33. The modified MT-SP1 polypeptide or catalytically active
fragment thereof of claim 32, wherein the unmodified MT-SP1
polypeptide is selected from among: an MT-SP1 polypeptide having
the sequence of amino acids set forth in SEQ ID NO:253 or 507, or a
sequence of amino acids that exhibits at least 85% sequence
identity to SEQ ID NO:253 or 507; and a catalytically active
portion of an MT-SP1 having the sequence of amino acids set forth
in SEQ ID NO: 505 or 517 or a sequence of amino acids that exhibits
at least 85% sequence identity to SEQ ID NO:505 or 517.
34. The modified MT-SP1 polypeptide or catalytically active
fragment thereof of claim 32, comprising an amino acid replacement
at a position set forth in a), wherein the amino acid replacement
is selected from among D23E, L52M, Y60(g)s, H71R, F93L, N95K,
A126T, V129D, P131S, I136T, I136V, T144I, I154V, N164D, T166A,
F184(a)L, S201I, Q209L, R230W, F234L, and V244G.
35. The modified MT-SP1 polypeptide or catalytically active
fragment thereof of claim 32, comprising a further amino acid
replacement selected from among D23E, I41F, I41T, L52M, Y60(g)s,
T65K, H71R, F93L, N95K, F97Y, F97L, T98P, F99L, A126T, V129D,
P131S, I136T, I136V, H143R, T144I, I154V, N164D, T166A, L171F,
P173S, Q175R, F184(a)L, Q192H, S201I, Q209L, D217V, Q221(a)L,
R230W, F234L, and V244G.
36. The modified MT-SP1 polypeptide or catalytically active
fragment thereof of claim 32, wherein the MT-SP1 polypeptide or
catalytically active portion thereof contains amino acid
replacements selected from among I136T/N164D/T166A/F184(A)L/D217V;
I41F/A126T/V244G; D23E/I41F/T98P/T144I; I41F/L171F/V244G;
H143R/Q175R; I41F/I154V/V244G; I141F/L52M/V129D/Q221(A)L;
F97Y/I136V/Q192H/S201I; H71R/P131S/D217V; T65K/F93L/F97Y/D217V;
I41T/P173S/Q209L; F97L/F234L and I41F/L171F.
37. A pharmaceutical composition, comprising a modified MT-SP1
polypeptide of claim 32.
38. A nucleic acid molecule, comprising a sequence of nucleotides
that encodes a modified u-PA polypeptide or catalytically active
fragment thereof of claim 32.
39. A vector, comprising the nucleic acid molecule of claim 38.
40. An isolated cell, comprising the vector of claim 39.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of allowed U.S. patent
application Ser. No. 11/825,627, to Edwin Madison, entitled
"Protease Screening Methods and Proteases Identified Thereby,"
filed Jul. 5, 2007, which claims priority to U.S. Provisional
Application Ser. No. 60/818,804, to Edwin Madison, entitled
"Protease Screening Methods and Proteases Identified Thereby,"
filed Jul. 5, 2006, and to U.S. Provisional Application Ser. No.
60/818,910, to Edwin Madison, entitled "Modified
Urinary-Plasminogen Activator (u-PA) Proteases," filed Jul. 5,
2006. The subject matter of the above-noted applications are
incorporated by reference in their entirety.
[0002] This application is related to International Application No.
PCT/US2007/015571 to Edwin Madison, entitled "Protease Screening
Methods and Proteases Identified Thereby," filed Jul. 5, 2007,
which also claims priority to U.S. Provisional Application Ser. No.
60/818,804 and to U.S. Provisional Application Ser. No.
60/818,910.
[0003] This application also is related to U.S. application Ser.
No. 10/677,977, filed Oct. 2, 2003 and published as U.S.
Application No. US-2004-0146938 on Jul. 29, 2004, entitled "Methods
of Generating and Screening for Proteases with Altered Specificity"
to J Nguyen, C Thanos, S Waugh-Ruggles, and C Craik, and to
corresponding published International PCT Application No.
WO2004/031733, published Apr. 15, 2004, which claim benefit to U.S.
Provisional Application Ser. No. 60/415,388 filed Oct. 2, 2002.
[0004] This application also is related to U.S. application Ser.
No. 11/104,110, filed Apr. 12, 2005, and issued as U.S. Pat. No.
7,939,304 on May 10, 2011, entitled "Cleavage of VEGF and VEGF
Receptor by Wild-Type and Mutant MTSP-1" to J Nguyen and S
Waugh-Ruggles, and to corresponding published International PCT
Application No. WO2005/110453, published Nov. 24, 2005, which claim
benefit to U.S. Provisional Application Ser. No. 60/561,720 filed
Apr. 12, 2004.
[0005] This application also is related to U.S. application Ser.
No. 11/104,111, now abandoned, filed Apr. 12, 2005, and published
as U.S. Application No. US-2006-0024289 on Feb. 2, 2006, entitled
"Cleavage of VEGF and VEGF Receptor by Wild-Type and Mutant
Proteases" to J Nguyen and S Waugh-Ruggles, and to corresponding
published International PCT Application No. WO2005/100556,
published Oct. 27, 2005, which claim benefit to U.S. Provisional
Application Ser. No. 60/561,671 filed Apr. 12, 2004.
[0006] This application also is related to U.S. Provisional
Application Ser. No. 60/729,817 filed Oct. 21, 2005, entitled
"Modified Proteases that Inhibit Complement Activation" to Edwin L.
Madison. This application also is related to U.S. application Ser.
No. 11/584,776, now abandoned, filed Oct. 20, 2006, entitled
"Modified Proteases that Inhibit Complement Activation" to Edwin L.
Madison, Jack Nguyen, Sandra Waugh Ruggles and Christopher Thanos,
and to corresponding published International PCT Application No.
WO2007/047995, published Apr. 26, 2007, which each claim benefit to
U.S. Provisional Application No. 60/729,817.
[0007] The subject matter of each of the above-noted related
applications is incorporated by reference in its entirety.
Incorporation by Reference of Sequence Listing Provided on Compact
Discs
[0008] An electronic version on compact disc (CD-R) of the Sequence
Listing is filed herewith in duplicate (labeled Copy #1 and Copy
#2), the contents of which are incorporated by reference in their
entirety. The computer-readable file on each of the aforementioned
compact discs, created on May 18, 2012, is identical, 1,810
kilobytes in size, and titled 4902BSEQ.001.txt.
FIELD OF THE INVENTION
[0009] 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
[0010] 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.
[0011] 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.
[0012] 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
[0013] 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 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 and/or 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
(NiSO.sub.4), cobalt chloride (CoCl.sub.2), copper sulphate
(CuSO.sub.4) and zinc chloride (ZnCl.sub.2). The metal chelating
agent can be conjugated to a solid support, such as for example, on
beads such as sepharose beads or magnetic beads.
[0014] 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 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] The collection of proteases used in the methods provided
herein are any collection of proteases or catalytically active
portions thereof and include members with at least, about, or equal
to 5, 10, 50, 100, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6 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.
[0020] 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.
[0021] 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 urokinase plasminogen
activator (u-PA) proteases, tissue plasminogen activator (t-PA)
proteases, or MT-SP1 proteases.
[0022] 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 (PAI-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, a t-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.
[0023] 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
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.
[0024] 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.
[0025] 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.
[0026] 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
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.
[0027] 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)
contacting 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.
[0028] 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.
[0029] 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
proteolytically 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).
[0030] 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.
[0031] 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.
[0032] 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.
[0033] Kits containing the combinations are provided. Typically the
kits contain the packaged components and, optionally, additional
reagents and instructions for performing the methods.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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 contains one or more modifications in positions
selected from among 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, F30L, T39A, Y61(A)H, E80G, K82E, E84K,
I89V, K92E, K156T, T158A, 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/K82E/V159A; F30V/K82E/T39A/V159A; F30V/K82E/T158A/V159A;
F30V/Y61(a)H/K92E; F30V/K82E/V159A/E80G/I89V/K187E; and
F30V/K82E/V159A/E80G/E84K/189V\K187E.
[0038] 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, S75P, F132L, G133D,
E137G, I138T, L155P, 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 F30I, F30T, F30L, F30V, F30G,
F30M, and I89V. 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; S75P/I89V/1138T; E137G;
R72G/L155P; G133D; V160A; V38D; F132L/V160A; L73A/I89V/F30T;
L73A/I89V/F30L; L73A/I89V/F30V; L73A/I89V/F30G; L73A/I89V/L155V;
L73A/I89V/F30M; L73A/I89V/L155M; L73A/I89V/F30L/L155M; and
L73A/I89V/F30G/L155M.
[0039] 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 F30I, F30T, F30G, and
F30M.
[0040] 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, I41F, I41T, L52M, Y60(g)s, T65K, H71R, F93L, N95K,
F97Y, F97L, T98P, F99L, A126T, V129D, P131S, I136T, I136V, H143R,
T1441, I154V, N164D, T166A, L171F, P173S, F184(a)L, Q192H, S201I,
Q209L, Q221(a)L, R230W, F234L, and V244G, 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-SP1 having a sequence of amino acids set forth in
SEQ ID NO:505. In other examples, the modifications are in an
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 I41F, 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
I136T/N164D/T166A/F184(A)L/D217V; 141F; 141F/A126T/V244G;
D23E/I41F/T98P/T144I; I41F/L171F/V244G; H143R/Q175R; I41F/L171F;
R230W; I41F/I154V/V244G; I41F/L52M/V129D/Q221(A)L; F99L;
F97Y/I136V/Q192H/S201I; H71R/P131S/D217V; D217V;
T65K/F93L/F97Y/D217V; I41T/P173S/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.
[0041] Also provided are pharmaceutical compositions containing the
modified proteases, including modified serine proteases, such as
the modified u-PA polypeptides or modified MT-SP1 polypeptides. The
pharmaceutical compositions contain pharmaceutically acceptable
excipients, and can be formulated for any suitable route of
administration, including, but not limited to, systemic, oral,
nasal, pulmonary, local and topical administration. Also provided
are kits containing any of the pharmaceutical compositions, a
device for administration of the composition and, optionally,
instructions for administration.
[0042] 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.
[0043] 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 of t-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.
[0044] 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.
[0045] 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 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.
[0046] 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
[0047] FIG. 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 (E1). This is followed by
cleavage of the P1-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.sup.#). 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
[0048] A. Definitions
[0049] B. Method for Screening Proteases
[0050] C. Protease Trap Polypeptides [0051] 1. Serpins: Structure,
Function, and Expression [0052] 2. Protease Catalysis, Inhibitory
Mechanism of Serpins, and Formation of Acyl Enzyme Intermediate
[0053] a. Exemplary Serpins [0054] i. PAI-1 [0055] ii. Antithrombin
(AT3) [0056] 3. Other Protease Trap Polypeptides [0057] a. p35
[0058] b. Alpha Macroglobulins (aM) [0059] 4. Protease Trap
Polypeptide Competitors [0060] 5. Variant Protease Trap
Polypeptides
[0061] D. Proteases [0062] 1. Candidate Proteases [0063] a. Classes
of Proteases [0064] i. Serine Proteases [0065] (a) Urokinase-type
Plasminogen Activator (u-PA) [0066] (b) Tissue Plasminogen
Activator (t-PA) [0067] (c) MT-SP1 [0068] ii. Cysteine
Proteases
[0069] E. Modified Proteases and Collections for Screening [0070]
1. Generation of Variant Proteases [0071] a. Random Mutagenesis
[0072] b. Focused Mutagenesis [0073] 2. Chimeric Forms of Variant
Proteases [0074] 3. Combinatorial Libraries and Other Libraries
[0075] a. Phage Display Libraries [0076] b. Cell Surface Display
Libraries [0077] c. Other Display Libraries
[0078] F. Methods of Contacting, Isolating, and Identifying
Selected Proteases [0079] 1. Iterative Screening [0080] 2.
Exemplary Selected Proteases
[0081] G. Methods of Assessing Protease Activity and
Specificity
[0082] H. Methods of Producing Nucleic Acids Encoding Protease Trap
Polypeptides (i.e. Serpins) or Variants Thereof or
Proteases/Modified Proteases [0083] 1. Vectors and Cells [0084] 2.
Expression [0085] a. Prokaryotic Cells [0086] b. Yeast Cells [0087]
c. Insect Cells [0088] d. Mammalian Cells [0089] e. Plants [0090]
3. Purification Techniques [0091] 4. Fusion Proteins [0092] 5.
Nucleotide Sequences
[0093] I. Preparation, Formulation and Administration of Selected
Protease Polypeptides [0094] 1. Compositions and Delivery [0095] 2.
In vivo Expression of Selected Proteases and Gene Therapy [0096] a.
Delivery of Nucleic Acids [0097] i. Vectors--Episomal and
Integrating [0098] ii. Artificial Chromosomes and Other Non-viral
Vector Delivery Methods [0099] iii. Liposomes and Other
Encapsulated Forms and Administration of Cells Containing Nucleic
Acids [0100] b. In vitro and Ex vivo Delivery [0101] c. Systemic,
Local and Topical Delivery [0102] 2. Combination Therapies [0103]
3. Articles of Manufacture and Kits
[0104] J. Exemplary Methods of Treatment with Selected Protease
Polypeptides [0105] 1. Exemplary Methods of Treatment for Selected
uPA Polypeptides That Cleave tPA Targets [0106] a. Thrombotic
Diseases and Conditions [0107] i. Arterial Thrombosis [0108] ii.
Venous Thrombosis and Thromboembolism [0109] (a) Ischemic stroke
[0110] iii. Acquired Coagulation Disorders [0111] (a) Disseminated
Intravascular Coagulation (DIC) [0112] (b) Bacterial Infection and
Periodontitis [0113] b. Other tPA Target-associated Conditions
[0114] c. Diagnostic Methods [0115] 2. Exemplary Methods of
Treatment for Selected Protease Polypeptides That Cleave VEGF or
VEGFR Targets [0116] a. Angiogenesis, Cancer, and Other Cell Cycle
Dependent Diseases or Conditions [0117] b. Combination Therapies
with Selected Proteases That Cleave VEGF or VEGFR [0118] 3.
Exemplary Methods of Treatment for Selected MT-SP1 Polypeptides
that cleave complement protein targets [0119] a. Immune-mediated
Inflammatory Diseases [0120] b. Neurodegenerative Disease [0121] c.
Cardiovascular Disease
[0122] K. Examples
A. DEFINITIONS
[0123] 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. All patents,
patent applications, published applications and publications,
Genbank sequences, databases, websites and other published
materials referred to throughout the entire disclosure herein,
unless noted otherwise, are incorporated by reference in their
entirety. 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
internet can come and go, but equivalent information can be found
by searching the internet. Reference thereto evidences the
availability and public dissemination of such information.
[0124] As used herein, a "protease trap" or "protease trap
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.
[0125] 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 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.
[0126] 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
P.sub.n-P.sub.15-P.sub.14-P.sub.13 . . .
P.sub.4-P.sub.3-P.sub.2-P.sub.1-P.sub.1'-P.sub.2'-P.sub.3' . . .
Pn'-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 PAI-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 (SEQ ID NO: 378) amino acid residues to RRARM (SEQ ID NO:
379) or PFGRS (SEQ ID NO: 389).
[0127] 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 P1-P1' position in the cleavage sequence of a
substrate.
[0128] 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 P1-P1' scissile bond
sequence.
[0129] 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, or portions thereof containing the variant RSL sequence,
can be used to select for proteases with altered substrate
specificity.
[0130] 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 (k.sub.4) and substrate (k.sub.5) pathways (such as
is depicted in FIG. 1), which is represented by the stoichiometry
of inhibition (SI=1+k.sub.5/k.sub.4); good inhibitors have the
value of 1 because most of the serpin molecules partition into
complex formation and k.sub.5/k.sub.4 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.
[0131] As used herein, catalytic efficiency or kcat/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.
[0132] 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.
[0133] 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.
[0134] 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, 10.sup.4, 10.sup.5 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.
[0135] 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 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.
[0136] As used herein, a "template 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.
[0137] 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).
[0138] 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.
[0139] 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.
[0140] 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 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.
[0141] As used herein, desired specificity with reference to
substrate specificity refers to cleavage specificity for a
predetermined or preselected or otherwise targeted substrate.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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, the microorganism is introduced into an appropriate
host and grown or cultured so that more displayed protease or
proteolytically active portion is produced.
[0146] 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.
[0147] 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.
[0148] As used herein, a disease or condition that is treated by
administration of t-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.
[0149] 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.
[0150] 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.
[0151] 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 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] As used herein, "S1-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 S1-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.
[0161] As used herein, the terms "P1-P4" and "P1'-P4'" refer to the
residues in a substrate peptide that specifically interact with the
S1-S4 residues and S1'-S4' residues, respectively, and are cleaved
by the protease. P1-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, S1, S1', S2',
S3', . . . , Sj). The cleavage is catalyzed between P1 and P1.'
[0162] 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 S1'-S4' amino acids make up
the substrate sequence binding pocket and facilitate substrate
recognition by interaction with P1-P4 and P1'-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 S1-S4 and S1'-S4' positions. If the amino acids in any
one or more of the S1, S2, S3, S4, S1', S2', S3' and S4' sub-sites
interact with or recognize any one or more of the amino acids in
the P1, P2, P3, P4, P1', 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'.
[0163] 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 S1-S4, S1'-S4' residues in a protease.
[0164] 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 S1-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.
[0165] 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.
[0166] 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
Asp.sup.102, His.sup.57, and Ser.sup.195. These residues are
critical for the catalytic efficiency of a serine protease.
[0167] 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 P1-P4 and P1'-P4' amino acids in a substrate, where cleavage
occurs after the P1 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 (SEQ ID NO: 637), 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 (SEQ ID NO: 638).
[0168] 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.
[0169] 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-P1-P1'-P2'-P3' . . . , and
cleavage occurs between the P1 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 P1-P4 sequence of RQAR (SEQ ID NO: 513). An activating cleavage
also is cleavage whereby a 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.
[0170] 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 or
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.
[0171] 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.
[0172] 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 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.
[0173] 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-SP1, 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 conserved amino 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 C1 of the mature
chymotrypsin polypeptide. Thus, amino acid C168 in u-PA also is C1
based on chymotrypsin numbering. Using such a chymotrypsin
numbering standard, amino acid L244 in the precursor u-PA 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-SP1 (SEQ ID NO:253),
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 Asp60b or
Arg60c.
[0174] 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 (k.sub.cat/K.sub.m), which is a
measure of the affinity of a protease for its substrate and the
efficiency of the enzyme.
[0175] As used herein, a specificity constant for cleavage is
(k.sub.cat/K.sub.m), wherein K.sub.m is the Michaelis-Menton
constant ([S] at one half V.sub.max) and K.sub.cat is the
V.sub.max/[E.sub.T], where E.sub.T is the final enzyme
concentration. The parameters k.sub.cat, K.sub.m and
k.sub.cat/K.sub.m 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=(K.sub.m/V.sub.max)(1/[S])+1/V.sub.max; where
V.sub.max[E.sub.T]k.sub.cat). 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, k.sub.cat 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 S1-S4 pockets of a protease for a concomitant P1-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.
[0176] 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 k.sub.cat/K.sub.m. For example, a protease
that has a specificity constant of 2.times.10.sup.6 M.sup.-1
sec.sup.-1 for a target substrate and 2.times.10.sup.4 M.sup.-1
sec.sup.-1 for a non-target substrate is more specific for the
target substrate. Using the specificity constants from above, the
protease has a substrate specificity ratio of 100 for the target
protease.
[0177] 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.
[0178] 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.
[0179] 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
cleavage of 2.times.10.sup.6 M.sup.-1 sec.sup.-1 for a target
substrate and 2.times.10.sup.4 M.sup.-1 sec.sup.-1 for any other
one of more substrates, the protease is more selective for the
former target substrate.
[0180] 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.
[0181] 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.
[0182] 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 proteolysis 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.
[0183] 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-SP1
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.
[0184] 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.
[0185] As used herein, the residues of naturally occurring
.alpha.-amino acids are the residues of those 20 .alpha.-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.
[0186] As used herein, non-naturally occurring amino acids refer to
amino acids that are not genetically encoded.
[0187] 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.
[0188] As used herein, a peptide refers to a polypeptide that is
from 2 to 40 amino acids in length.
[0189] 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.
[0190] 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 .alpha.-carbon has a side chain).
[0191] 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. NH.sub.2 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: 3557-3559 (1968), and adopted 37
C.F.R..sctn..sctn.1.821-1.822, abbreviations for amino acid
residues are shown in Table 1:
TABLE-US-00001 TABLE 1 Table of Correspondence SYMBOL 1-Letter
3-Letter AMINO ACID Y Tyr Tyrosine G Gly Glycine F Phe
Phenylalanine M Met Methionine A Ala Alanine S Ser Serine I Ile
Isoleucine L Leu Leucine T Thr Threonine V Val Valine P Pro proline
K Lys Lysine H His Histidine Q Gln Glutamine E Glu glutamic acid Z
Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine D Asp aspartic
acid N Asn asparagines B Asx Asn and/or Asp C Cys Cysteine X Xaa
Unknown or other
[0192] 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
(Table 1) and modified and unusual amino acids, such as those
referred to in 37 C.F.R. .sctn..sctn.1.821-1.822, and incorporated
herein by reference. 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 NH.sub.2 or to a
carboxyl-terminal group such as COOH.
[0193] As used herein, "naturally occurring amino acids" refer to
the 20 L-amino acids that occur in polypeptides.
[0194] 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.
[0195] 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).
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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
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.
[0200] 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).
[0201] "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
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)).
[0202] 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 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;
Carillo et al. (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 be 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.
[0203] 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. Sci. USA 85:2444 (other programs include the GCG
program package (Devereux, J., et al., Nucleic Acids Research
12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J
Molec Biol 215:403 (1990)); Guide to Huge Computers, Martin J.
Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al.
(1988) SIAM J Applied Math 48:1073). For example, the BLAST
function of the National Center for Biotechnology Information
database can be used to determine identity. Other commercially or
publicly available programs include, DNAStar "MegAlign" program
(Madison, Wis.) and the University of Wisconsin Genetics Computer
Group (UWG) "Gap" program (Madison Wis.). 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 similarity 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 comparison
matrix of Gribskov et al. (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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.times.SSPE, 0.1% SDS, 65.degree. C., and at medium
stringency are 0.2.times.SSPE, 0.1% SDS, 50.degree. 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] As used herein, species variants refer to variants in
polypeptides among different species, including different mammalian
species, such as mouse and human.
[0214] 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.
[0215] 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.
[0216] 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 enkephalin 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).
Morphine, 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.
[0217] 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.
[0218] 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 transcription. Promoter sequences are
commonly, but not always, found in the 5' non-coding region of
genes.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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
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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] As used herein, inhibit and inhibition refer to a reduction
in an activity relative to the uninhibited activity.
[0239] 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.
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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.
[0246] As used herein, the term "subject" refers to an animal,
including a mammal, such as a human being.
[0247] As used herein, a patient refers to a human subject.
[0248] 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.
[0249] As used herein, prevention or prophylaxis refers to methods
in which the risk of developing disease or condition is
reduced.
[0250] 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.
[0251] 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.
[0252] Administration 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.
[0253] 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.
[0254] As used herein, a single dosage formulation refers to a
formulation for direct administration.
[0255] 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.
[0256] 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.
[0257] 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.
[0258] As used herein, a cellular extract or lysate refers to a
preparation or fraction which is made from a lysed or disrupted
cell.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 bases" means "about 5 bases" and also "5
bases."
[0263] 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.
[0264] As used herein, the abbreviations for any protective groups,
amino acids and other compounds, are, unless indicated otherwise,
in accord with their common usage, recognized abbreviations, or the
IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972)
Biochem. 11:1726).
B. METHOD FOR SCREENING PROTEASES
[0265] 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
[0266] 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.
[0267] 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 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.
[0268] 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.
[0269] 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 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.
[0270] 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.
[0271] 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 unreacted protease
trap polypeptides.
[0272] 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.
[0273] 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.
[0274] The methods provided herein can be multiplexed, such as by
including two or more differentially labeled or differentially
identifiable protease trap polypeptides.
[0275] 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.
[0276] 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.
[0277] 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 and/or to optimize the
specificity or activity of an existing protease lead.
C. PROTEASE TRAP POLYPEPTIDES
[0278] 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
(P1-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 acylation of the enzyme) while at
the same time forming very tight (i.e. covalent) complexes that are
easily isolated from collection mixtures.
[0279] 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.
[0280] 1. SERPINS: Structure, Function, and Expression
[0281] 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 et al. (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. 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 et al.
(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).
[0282] 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 alpha1-antitrypsin and have a conserved
tertiary structure. Structurally, serpins are made up of three 13
sheets (A, B, and C) and 8-9 .alpha.-helices (A-I), which are
organized into an upper 13-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 et al.
(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 .beta.-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 .beta.-sheet A to
become strand four of the six-stranded .beta.-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 .beta.-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 only
converted to a more stable structure following protease cleavage
(Law et al. (2006) Genome Biology, 7:216).
[0283] 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 . . .
P.sub.n-P.sub.3-P.sub.2-P.sub.1-P.sub.1'-P.sub.2'-P.sub.3'-P'.sub.n
. . . , where the reactive site is the scissile bond between
P.sub.1 and P.sub.1'. For mature .alpha.1-antitrypsin, cleavage at
the P1-P1' bond occurs at the Met.sub.358-Ser.sub.359 bond
(corresponding to amino acids Met.sub.382 and Ser.sub.389 of the
sequence of amino acids set forth in SEQ ID NO:1). The
corresponding binding site for the residues on the protease are . .
. S.sub.n-S.sub.3-S.sub.2-S.sub.1-S.sub.1', S.sub.2', S.sub.3',
S.sub.n'- . . . 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.
[0284] 2. Protease Catalysis, Inhibitory Mechanism of Serpins, and
Formation of Acyl Enzyme Intermediate
[0285] 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 P1-P1' peptide bond of
the substrate is cleaved. 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 H is
residue in the catalytic center of a serine protease provides a
general base and accepts the OH group of the reactive Ser.
[0286] 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
et al. (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., FIG. 1; Zhou et al. (2001) J.
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 et al. (2001), J. Biol. Chem., 276:
33293-33296). The serine residue (for serine proteases), in the
active site of the protease attacks the P1-P1' bond, facilitating
cleavage of the peptide bond and formation of a covalent ester
linkage between the serine residue and the backbone carbonyl of the
P1 residue. After the RSL is cleaved, the RSL inserts into n-sheet
A of the serpin molecule. The first residue to insert is P14 (i.e.
amino acid 345 in mature .alpha.1-antitrypsin, which corresponds to
amino acid position T369 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 et al. (2005) Mol. 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 FIG. 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 P1 side chain is removed from the S1
pocket. The net result of the conformational rearrangements is
trapping of the acyl enzyme intermediate (Silverman et al. (2001),
J. Biol. Chem., 276: 33293-33296).
[0287] 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).
[0288] 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
SCCA1 (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 et al. (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 et al. (2001) J.
Biol. Chem., 276:33293-33296; Schick et al. (1998) Biochemistry,
37:5258-5266).
[0289] 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 FIG. 1 as the
formation of an inhibited complex versus the non-inhibitory pathway
(Lawrence et al. (2000), J. 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 inhibitor-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
PAI-1 to each of uPA, tPA, and thrombin; mutation to neutral amino
acids (i.e. His, Tyr, Gln, 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 et al. (2000), J. Biol. Chem.,
275: 5839-5844).
[0290] 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 et al. (2001) J. 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.
.alpha.2-antiplasmin, C1-inhibitor, and CrmA). An
.alpha.2-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 et al. (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.
[0291] a. Exemplary Serpins
[0292] 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.
TABLE-US-00002 TABLE 2 Exemplary Serpins Mature Nick- Poly- SEQ
Serpin Protein name name Function Acc. # peptide (aa) ID NO Extra-
SERPINA1 Alpha-1- AIAT Inhibits elastase P01009 25-418 1 cellular
antitrypsin inhibitory SERPINA2 Alpha-1- A1AU May be a pseudogene
P20848 22-420 2 Serpins antitrypsin- related protein SERPINF2
Alpha-2- A2AP Inhibits plasmin and P08697 40-491 3 antiplasmin
trypsin SERPINA3 Alpha-1- AACT Inhibits neutrophil P01011 24-423 4
antichymotrypsin cathepsin G and mast cell cymase SERPINC1
Antithrombin- ANT3 Regulates blood P01008 33-464 5 III coagulation
cascade; thrombin and factor Xa inhibitor SERPIND1 Heparin cofactor
HEP2 Regulates blood P05546 20-499 6 II coagulation cascade;
thrombin inhibitor SERPING1 Plasma protease IC1 Regulation of
complement P05155 23-500 7 C1 inhibitor activation, blood
coagulation, fibrinolysis and the generation of kinins; C1 esterase
inhibitor SERPINA5 Plasma serine protease IPSP, Inhibits activated
protein P05154 20-406 8 inhibitor, Protein PAI-3 C and plasminogen
C inhibitor activators SERPINA4 Kallistatin KAIN, Inhibits
amidolytic and P29622 21-427 9 PI4 kininogenase activities of human
tissue kallikrein SERPINII Neuroserpin NEUS, Formation or
reorganization Q99574 17-410 10 PII2 of synaptic connections and
synaptic plasticity; inhibitor of tPA, uPA, and plasmin SERPINE1
Plasminogen activator PAI1 Regulation of fibrinolysis; P05121
24-402 11 inhibitor-1 inhibitor of thrombin, uPA, tPA, and plasmin
SERPIN12 Myoepithelium- PII4 Inhibition of cancer O75830 19-405 12
derived serine metastasis proteinase inhibitor SERPINA10 Protein
Z-dependent ZP1 Inhibits factor Z Q9UK55 22-444 13 protease
inhibitor and XI SERPINE2 Protease nexin I, PI7, Inhibition of uPA
and P07093 20-398 14 glia-derived GDN, tPA nexin precursor PN-1
Intra- SERPINB1 Leukocyte elastase ILEU Inhibition of neutrophil
P30740 1-379 15 cellular inhibitor, monocytes protease inhibitory
neutrophil elastase Serpins inhibitor SERPINB2 Plasminogen
activator PAI2 Tissue-type plasminogen P05120 1-415 16 inhibitor-2
activator, intracellular signaling; inhibition of uPA SERPINB6
Placental thrombin PTI6 Inhibits thrombin P35237 1-376 17 inhibitor
SERPINB10 Bomapin SB10, Haematopoiesis, inhibition P48595 1-397 18
P110 of thrombin and trypsin SERPINB11 epipin SB11 Q96P15 1-392 19
SERPINB12 Yukopin SB12 Inhibits trypsin Q96P63 1-405 20 and plasmin
SERPINB13 Headpin SB13, Proliferation or Q9UIV8 1-391 21 P113
differentiation of keratinocytes, inhibition of cathepsins L and K
SERPINB3 Squamous cell SCC1 Modulates immune P29508 1-390 22
carcinoma antigen 1 response towards tumors, inhibition of
cathepsins L, K, S and V, and papain SERPINB4 Squamous cell SCC2
Modulates immune P48594 1-390 23 carcinoma antigen 2 response
towards tumors, inhibition of Cathepsin G and chymase SERPINB7
Megsin SPB7 Maturation of O75635 1-390 24 megakaryocytes SERPINB8
Cytoplasmic SPB8, Inhibition of Furin P50452 1-374 25
antiproteinase 8 PI8 SERPINB9 Cytoplasmic SPB9, Granzyme B
inhibitor P50453 1-376 26 antiproteinase 9 PI9 SERPINB6 Proteinase
inhibitor-6, PI6, Inhibition of cathepsin P35237 1-376 27 placental
thrombin PTI G, inhibits thrombin inhibitor Non- SERPINA8
Angiotensinogen ANGT Blood pressure regulation, P01019 34-485 28
inhibitory hormone precursor serpins SERPINA6 Corticosteroid- CBG
Hormone carrier P08185 23-405 29 binding globulin (glucocorticoids
and progestins), cortisol binding SERPINH1 47 kDa heat HS47
Molecular chaperone P29043 18-417 30 shock protein for collagen
SERPINF1 Pigment PEDF Induces neuronal P36955 20-418 31 epithelium-
differentiation derived factor in retinoblastoma cells; inhibitor
of angiogenesis SERPINB5 Maspin MASP Tumor suppressor, P36952 1-375
32 prevents metastasis SERPINH2 Collagen-binding SIH2 Molecular
chaperone P50454 19-418 33 protein 2 for collagen SERPINA7
Thyroxine-binding THBG Thyroid hormone transport, P05543 21-415 34
protein thyroxine binding SERPINA9 Germinal center GCET1
Maintenance of Q86WD7 24-417 35 B-cell expressed naive B cells
transcript 1 protein SERPINA12 Vaspin Insulin-sensitizing Q8IW75
21-414 36 adipocytokine SERPINA11 Q86U17 20-422 37 SERPINA13 Q6UXR4
22-307 38
[0293] 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 of t-PA is the serpin PAI-1, a
glycoprotein of approximately 50 kD (Pannekoek et al. (1986) EMBO
J., 5:2539-2544; Ginsberg et al., (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 C1 esterase
inhibitor and alpha-2-antiplasmin with tPA are orders of magnitude
lower than that of PAI-1 (Ranby et al. (1982) Throm. Res.,
27:175-183; Hekman et al. (1988) Arch. Biochem. Biophys.,
262:199-210), these serpins nevertheless inhibit tPA (see e.g.,
Lucore et al. (1988) Circ. 77:660-669).
TABLE-US-00003 TABLE 3 Serine Protease Cognate Serpin Inhibitor
Activated protein C Protein C inhibitor PAI-1 C1 esterase C1
esterase inhibitor Cathepsin G Alpha-1-antitrypsin
Alpa-1-antichymotrypsin Chymase Alpha-1-antichymotrypsin
Chymotrypsin Alpha-1-antichymotrypsin Alpha-2-antiplasmin
Contrapsin Coagulation Factors (VIIa, Xa, Antithrombin III XIa,
XIIa) C1 esterase inhibitor Elastase Alpha-1-antitrypsin Kallikrein
C1 esterase inhibitor Alpha-1-antitrypsin Plasmin
Alpha-2-antiplasmin Thrombin Antithrombin III Heparin cofactor II
tPA PAI-1, PAI-2, PAI-3 Trypsin Alpha-1-antitrypsin Growth hormone
regulated protein Trypsin-like protease Protease nexin I u-PA
PAI-1, PAI-2, PAI-3
[0294] 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 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.
[0295] i. PAI-1
[0296] Exemplary of serpins used in the protease selection methods
is plasminogen activator inhibitor-1 (PAI-1), 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 fibrinolysis due to the activation of
plasminogen. PAI-1 has a second order rate constant for t-PA and
u-PA of about 2.times.10.sup.7 M.sup.-1s.sup.-1. 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.
[0297] 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 P1-P1' reactive site located at Arg.sup.346-Met.sup.347 (i.e.
corresponding to amino acids Arg.sup.369 and Met.sup.370 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 .beta.-sheet A to form strand four of the
.beta.-sheet (i.e. s4A), and residues P3 to P10' form an extended
loop at the surface of the molecule (De Taeye et al. (2003) J Biol.
Chem., 278: 23899-23905). Thus, active PAI-1 is relatively unstable
at 37.degree. 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, guanidinium chloride, and
urea (Declerek et al. (1992) J. Biol. Chem., 267: 11693-11696) and
heat (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).
[0298] 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. Pat. Nos. 5,866,413; 5,728,564; 5,550,042;
5,486,602; 5,304,482).
[0299] ii. Antithrombin (AT3)
[0300] 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 et al. (2006) Genome
Biology, 7(216): 1-11).
[0301] 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 the reactive site P1-P1' occurs between amino acids
Arg.sup.425-Ser.sup.426.
[0302] 3. Other Protease Trap Polypeptides
[0303] 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.
[0304] a. p35
[0305] 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 et al.
(2003) J. Biol. Chem. 278(7):5455-5461). Cleavage of the
P.sub.1-P.sub.1' bond of p35 (at the caspase cleavage site
DQMD.sup.87; SEQ ID NO: 639) 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.
[0306] 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 TVTD.sup.94
(SEQ ID NO: 640).
[0307] 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, caspase
recognition is found at amino acid positions 84-87 (DQMD.sup.87;
SEQ ID NO: 639). 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.
[0308] 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.
[0309] b. Alpha Macroglobulins (aM)
[0310] 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 bait 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 the RBD sequence
facilitates the removal of the aM-protease complex from
circulation.
[0311] 4. Protease Trap Competitors
[0312] 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.
[0313] 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-P1' 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 provided
herein. Example 6 exemplifies such a strategy where a plasma
purified AT3 serpin is used as a competitor against the modified
serpin AT3.sup.SLGR-KI.
[0314] 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.
[0315] 5. Variant Protease Trap Polypeptides
[0316] 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.
[0317] 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 .alpha.1-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-US-00004 TABLE 4 RSL LOOP SEQUENCE ALIGNMENT* SEQ ID SERPIN
RSL loop sequence NO .sub.343 P.sub.15 P.sub.10 P.sub.4
P.sub.1P.sub.1' P.sub.5'.sub.363 Manduca sexta serpin 1B
EGAEAAAANAFGIVPKSLILY 397 Manduca sexta serpin 1K
EGAEAAAANAFKITTYSFHFV 398 .alpha.1-antichymotrypsin
EGTEASAATAVKITLLSALVE 399 Antithrombin-III EGSEAAASTAVVIAGRSLNPN
400 PAI-II EGTEAAAGTGGVMTGRTGHGG 401 .alpha.1-antitrypsin
KGTEAAGAMFLEAIPMSIPPE 402 PAI-I SGTVASSSTAVIVSARMAPEE 403 PAI-III
SGTRAAAATGTIFTFRSARLN 404 Ovalbumin AGREVVGSAEAGVDAASVSEE 405
*adapted from Ye et al. (2001) Nature Structural Biology 8: 979
[0318] 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.
[0319] 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 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.
[0320] 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.
[0321] 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 Ser. No. 60/729,817). For example, the
target protein can be one involved in rheumatoid arthritis (i.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 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-US-00005 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/IL-12R Immunological disorders Cytokine
IL-4/IL-4R Asthma Cytokine TNF/TNFR Asthma, Crohn's 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
Hemaglutinin Influenza infection Fusion Protein RSV fusion RSV
infection Fusion Protein protein B7/CD28 Graft-v-host disorder,
Immune Receptor rheumatoid arthritis, transplant rejection,
diabetes mellitus IgE/IgER Graft-v-host disorder, Antibody receptor
transplant rejection CD2, CD3, CD4, Graft-v-host disorder, Immune
Receptor CD40 transplant rejection, psoriasis IL-2/IL-2R Autoimmune
disorders, Cytokine graft-v-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 Bcl-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 VEGFR-2 Receptor
Complement Inflammatory diseases Immune molecules
[0322] 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. P1-P4 positions) to match the extended
substrate specificity of proteases, but can be longer or shorter.
For example, the P4-P1 amino acid residues for a cleavage sequence
in complement factor C2 is 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 P1' 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.
[0323] 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-US-00006 TABLE 6 Cleavage Sequence for Exemplary Target
Substrates (P4-P1 residues) Target Cleavage sequence TNF-.alpha.
AEAK (406) TNF-R1 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)
[0324] 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 of t-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).
[0325] 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., provisional application Ser. No. 60/729,817).
Example 6 to Examples 15 exemplify selection of an MT-SP 1 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.
[0326] 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 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.
[0327] 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.
[0328] Thus, in selection, for example, of a u-PA protease, or
variant thereof, that exhibits increased substrate specificity for
the VEGFR cleavage sequence RRVR (SEQ ID NO: 489), 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 (SEQ ID NO: 378), 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.
[0329] 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, 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.
[0330] 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
[0331] 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 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.
[0332] 1. Candidate Proteases
[0333] 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 et al. (1996) J. 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 to 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.
[0334] Exemplary proteases that can be used, and/or modified to be
used, in the selection method provided herein are described, and
include truncated polypeptides thereof that include a catalytically
active portion. Exemplary candidate proteases are listed in Table 7
and described herein (see e.g., www.merops.sanger.ac.uk). 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 the Table. 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.
[0335] 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 any one 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 silico 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-US-00007 TABLE 7 Exemplary Candidate Proteases Nt. A.A. A.A.
Signal/ Peptidase unit Protease Merops Nt. ACC. SEQ ACC. SEQ
propeptide (active site Type Code Name NO: ID NO: NO: ID NO:
sequence residues) Serine S01.010 granzyme B, M17016 39 P10144 40
1-18/19-20 21-247 Protease: human-type (64, 108, Chymo- 203)
trypsin S01.011 Testisin NM_006799 41 NP_006790 42 1-19/20-41
42-288 family (v1) (82, 137, NM_144956 43 NP_659205 44 238) (v2)
NM_144957 45 NP_659206 46 (v3) S01.015 trypstase beta 1 NM_003294
47 NP_003285 48 1-18/19-30 31-274 (Homo sapiens) (74, 121, (III)
224) S01.017 kallikrein hk5 NM_012427 49 NP_036559 50 1-22/ 67-292
(108, 153, 245) S01.019 Corin NM_006587 51 NP_006578 52 802-1037
(843, 892, 985) S01.020 kallikrein 12 NM_019598 53 NP_062544 54
1-17/ 22-248 (v1) (843, 892, NM_145894 55 NP_665901 56 985) (v2)
NM_145895 57 NP_665902 58 (v3) S01.021 DESC1 oritease AF064819 59
AAF04328 60 191-422 (231, 276, 372) S01.028 tryptase gamma 1
NM_012467 61 NP_036599 62 1-19/ 38-272 (78, 125, 222) S01.029
kallikrein hK14 NM_022046 63 Q9P0G3 64 1-18/19-24 25-249 (67, 111,
204) S01.033 hyaluronan-binding NM_004132 65 NP_004123 66 1-23/
314-557 serine protease (362, 411, (HGF activator- 509) like
protein) S01.034 transmembreane NM_019894 67 NP_063947 68 205-436
protease, NM_183247 69 NP_899070 70 (245, 290, serine 4 387)
S01.054 tryptase delta 1 NM_012217 71 Q9BZJ3 72 1-18/19-30 31-235
(Homo sapiens) (74, 121, 224) S01.074 Marapsin NM_031948 73
NP_114154 74 1-22/23-34 35-279 (75, 124, 229) S01.075 Tryptase
homologue 2 BC036846 75 AAN04055 76 37-281 (Homo sapiens) (77, 126,
231) S01.076 Tryptase homologue 3 Putative 77 78 67-304 (Homo
sapiens) Only (107, 213, AC005570 259) (Cosmid 407D8) S01.079
transmembrane NM_024022 79 NP_076927 80 217-451 protease, serine 3
(vA) (257, 304, NM_032401 81 NP_115777 82 401) (vB) NM_032404 83
NP_115780 84 (vC) NM_032405 85 NP_115781 86 (vD) S01.081 kallikrein
hK15 NM_023006 87 NP_075382 88 1-16/17-21 22-256 (Homo sapiens)
(v1) (62, 106, NM_138563 89 NP_612630 90 209) (v2) NM_138564 91
NP_612631 92 (v3) NM_017509 93 NP_059979 94 (v4) S01.085
Mername-AA031 BC035384 95 AAH35384 96 1-241 peptidase (deduced (56,
101, from ESTs by MEROPS) 195) S01.087 membrane-type AB048796 97
BAB39741 98 321-556 mosaic serine (361, 409, protease 506) S01.088
mername-AA038 Putative CAC12709 99 10-142 peptidase Only (50, 101)
AL136097 (RP11-62C3 clone) S01.098 mername-AA128 Putative 100
AAH41609 101 33-202 peptidase (deduced Only (50, 152) from ESTs by
MEROPS) BC041609 S01.127 cationic trypsin NM_002769 102 NP_002760
103 1-15/16-23 24-246 (Homo sapiens-type 1) (63, 107, (cationic)
200) S01.131 Neutrophils NM_001972 104 NP_001963 105 1-27/28-29
30-249 elastase (70, 117, 202) S01.132 mannan-binding AF284421 106
AAK84071 107 1-19/ 449-710 lectin-associated (497, 553, serine
protease-3 664) S01.133 cathepsin G NM_001911 108 NP_001902 109
1-18/19-20 21-245 (64, 108, 201) S01.134 myeloblastin NM_002777 110
NP_002768 111 1-25/26-27 28-250 (proteinase 3) (71, 118, 203)
S01.135 granzyme A NM_006144 112 NP_006135 113 1-26/27-28 29-261
(CTLA3) (69, 114, 212) S01.139 granzyme M NM_005317 114 NP_005308
115 1-23/24-25 26-256 (66, 111, 207) S01.140 chymase NM_001836 116
NP_001827 117 1-19/21-21 22-247 (human-type) (66, 110, 203) S01.143
tryptase alpha NM_003294 118 NP_003285 119 1-18/19-30 31-274 (1)
(74, 121, 224) S01.146 granzyme K NM_002104 120 NP_002095 121
1-24/26-26 27-261 (67, 116, 214) S01.147 granzyme H NM_033423 122
NP_219491 123 1-18/19-20 21-246 (CTLA1) (64, 108, 202) S01.152
chymotrypsin B M24400 124 P17538 125 1-18 34-263 (75, 120, 213)
S01.153 pancreatic NM_001971 126 NP_001962 127 1-8/9-18 19-256
elastase (63, 111, 206) S01.154 pancreatic NM_005747 128 NP_005738
129 1-15/16-28 29-270 endopeptidase (73, 123, E (A) 217) S01.155
pancreatic M16652 130 AAA52380 131 1-16/7-28 29-269 elastase II
(73, 121, (IIA) 216) S01.156 Enteropeptidase NM_002772 132
NP_002763 133 785-1019 (825, 876, 971) S01.157 chymotrypsin C
NM_007272 134 NP_009203 135 1-16/17-29 30-268 (74, 121, 2161
S01.159 Prostasin NM_002773 136 NP_002764 137 1-29/30-32 45-288
(85, 134, 238) S01.160 kallikrein 1 NM_002257 138 NP_002248 139
1-18/19-24 25-261 (65, 120, 214) SO1.161 kallikrein hK2 NM_005551
140 NP_005542 141 1-18/19-24 25-260 (Homo sapiens) (v1) (65, 120,
NM_001002231 142 NP_001002231 143 213) (v2) NM_001002232 144
NP_001002232 145 (v3) S01.162 kallikrein 3 NM_001648 146 NP_001639
147 1-17/18-24 25-260 (v1) (v1) (65, 120, NM_001030047 148
NP_001025218 149 213) (v3) (v3) NM_001030048 150 NP_001025219 151
(v4) (v4) NM_001030049 152 NP_001025220 153 (v5) (v5) NM_001030050
154 NP_001025221 155 (v6) (v6) S01.174 Mesotrypsin NM_002771 156
NP_002762 157 1-24/ 24-246 (63, 107, 200) S01.205 pancreatic
NM_007352 158 NP_031378 159 1-15/16-28 29-270 endopeptidase E (73,
123, form B (B) 217) S01.206 pancreatic elastase NM_015849 160
NP_056933 161 1-16/17-28 29-269 II form B (Homos (73, 121, sapiens)
(IIB) 216) S01.211 coagulation NM_000505 162 NP_000496 163 1-19/
373-615 factor XIIa (412, 461, 563) S01.212 plasma NM_000892 164
NP_000883 165 1-19/ 391-628 kallikrein (434, 483, (KLK3) 578)
S01.213 coagulation NM_000128 166 NP_000119 167 1-18/ 388-625
factor XIa (v1) (v1) (431, 480, (HAF) NM_019559 168 NP_062505 169
575) (v2) (v2) S01.214 coagulation NM_000133 170 NP_000124 171
1-28/29-46 227-461 factor IXa (267, 315, 411) S01.215 coagulation
NM_000131 172 NP_000122 173 1-20/21-60 213-454 factor VIIa (v1)
(v1) (253, 302, NM_019616 174 NP_062562 175 404) (v2) (v2) S01.216
coagulation NM_000504 176 NP_000495 177 1-31/32-40 235-469 factor
Xa (276, 322, 419) S01.217 Thrombin NM_000506 178 NP_000497 179
1-24/25-43 364-620 (406, 462, 568) S01.218 protein C NM_000312 180
NP_000303 181 1-32/33-42 212-452 (activated) (253, 299, 402)
S01.223 Acrosin NM_001097 182 NP_001088 183 1-19 43-292 (88, 142,
240) S01.224 Hepsin NM_182983 184 NP_892028 185 163-407 (v1) (203,
257, NM_002151 186 NP_002142 187 353) (v2) S01.228 hepatocyte
growth NM_001528 188 NP_001519 189 1-35/36-372 408-648 factor
activator (447, 497, (HGFA) 598) S01.231 u-plasminogen NM_002658
190 NP_002649 191 1-20/ 179-426 activator (uPA) (224, 275, 376)
S01.232 t-plasminogen NM_000930 192 NP_000921 193 1-23/24-32
311-562 activator (tPA) (v1) (v1) and 33-35 (357, 406, NM_000931
194 NP_000922 195 513) (v2) (v2) NM_033011 196 NP_127509 197 (v3)
(v3) S01.233 Plasmin NM_000301 198 NP_000292 199 1-19/20-97 581-810
(622, 665, 760) S01.236 Neurosin NM_002774 200 NP_002765 201
1-16/17-21 22-244 (vA) (62, 106, NM_001012964 202 NP_001012982 203
197) (vB) NM_001012965 204 NP_001012983 205 (vC) NM_001012966 206
NP_001012984 207 (vD) S01.237 Neurotrypsin NM_003619 208 NP_003610
209 1-20/ 631-875 (676, 726, 825) S01.242 tryptase beta 2 NM_024164
210 NP_077078 211 1-30/ 31-268 (Homo sapiens) (I) S01.244 Neuropsin
NM_007196 212 NP_009127 213 1-28/29-32 33-258 (v1) (v1) (73, 120,
NM_144505 214 NP_653088 215 212) (v2) (v2) NM_144506 216 NP_653089
217 (v3) (v3) NM_144507 218 NP_653090 219 (v4) (v4) S01.246
kallikrein hK10 NM_002776 220 NP_002767 221 1-30/ 35-276 (Homo
sapiens) (v1) (86, 137, NM_145888 222 NP_665895 223 229) (v2)
S01.247 Epitheliasin NM_005656 224 NP_005647 225 256-491
(296, 345, 441) S01.251 Prostase NM_004917 226 NP_004908 227
1-26/27-30 31-254 (71, 116, 207) S01.252 Brain serine NM_022119 228
NP_071402 229 1-32 50-292 proteinase 2 (90, 141, 242) S01.256
Chymopasin NM_001907 230 NP_001898 231 1-18/19-33 34-264 (75, 121,
214) S01.257 kallikrein 11 NM_006853 232 NP_006844 233 1-50/51-53
22-250 (v1) (v1) (62, 110, NM_144947 234 NP_659196 235 203) (v2)
(v2) S01.258 anionic trypsin NM_002770 236 NP_002761 237 1-15/16-23
24-246 (Homo sapiens) (II) (63, 107, (TRY2, TRY8, 200) TRYP2)
S01.291 LOC144757 Putative 238 AAH48112 239 78-319 peptidase
BC048112 (122, 171, (Homo sapiens) 268) S01.292 Mername-AA169
BN000133 240 CAD67985 241 1-19 175-406 peptidase (215, 260, 356)
S01.294 Mername-AA171 Putative 242 peptidase No DNA S01.298
Mername-AA174 Putative AAC80208 243 24-246 peptidase (TRY6) no DNA
seq (63, 107, 200) S01.299 Mername-AA175 NM_198464 244 NP_940866
245 68-302 peptidase (108, 156, 250) S01.300 stratum corneum
NM_005046 246 NP_005037 247 1-22/23-29 30-250 chymotryptic (v1)
(70, 112, enzyme (SCCE) NM_139277 248 NP_644806 249 205) (v2)
S01.301 trypsin-like enzyme, NM_004262 250 NP_004253 251 187-471
respiratory (227, 272, (Transmembrane 368) protease, serine 11D)
S01.302 Matripase AF118224 252 AAD42765 253 615-855 (MTSP1) (656,
711, 805) S01.306 kallikrein hK13 NM_015596 254 NP_056411 255 1-16/
36-263 (76, 124, 218) S01.307 kallikrein hK9 NM_012315 256
NP_036447 257 1-15/ 23-250 (human (63, 111, numbering) 204) S01.308
Mername-AA035 NM_153609 258 NP_705837 259 49-283 peptidase (89,
140, 234) S01.309 umbilical vein NM_007173 260 NP_009104 261 1-23/
95-383 proteinase (175, 246, 316) S01.311 LCLP proteinase Peptide
P34168 262 1-26 (LCLP (N- fragment (0) terminus)) No DNA S01.313
Spinesin NM_030770 263 NP_110397 264 218-455 (258, 308, 405)
S01.318 Mername-AA178 NM_183062 265 NP_898885 266 1-33/ 53-288
peptidase (93, 143, 238) S01.320 Mername-AA180 BN000120 267
CAD66452 268 1-23/ 52-301 peptidase (92, 142, 240) S01.322
Mername-AA182 BN000128 269 CAD67579 270 1-17/ 8-298 peptidase (87,
139, 237) S01.414 Mername-AA122 Putative 271 BAC11431 272 1-177
peptidase AK075142 (12, 64, (deduced from 168) ESTs by MEROPS)
Cysteine C01.032 Cathepsin L X12451 273 P07711 274 1-17/18-113
113-333 protease: (132, 138, Papain 276, 300) family C01.009
Cathepsin V U13665 275 O60911 276 1-17/18-113 114-334 (132, 138,
277, 301) C01.036 Cathepsin K S93414 277 P43235 278 1-15/16-114
115-329 (133, 139, 276, 296) C01.034 Cathepsin S AJ007331 279
P25774 280 1-16/17-114 115-331 (133, 139, 278, 298) C01.018
Cathepsin F M14221 281 Q9UBX1 282 1-19/20-270 271-484 (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
1-18/19-133 135-342 (158, 292, 308) C01.075 Cruzain Y14734 287
P25779 288 123-467/ 124-334 (Cruzapain) (147, 284, 304, Serine
S08.001 Subtilisin Carlsberg X03341 290 P00780 289 1-29/30-105
111-370 Protease: precursor (137, 168, subtilisin 325) family
S08.002 Subtilisin (Alkaline P07518 291 6-266 mesentericopeptidase)
(32, 64, 221) S08.003 Subtilisin Savinase P29600 292 6-260
(Alkaline protease) (32, 62, 215) S08.007 Thermitase P04072 293
13-264 (38, 71, 225) S08.009 Thermophilic serine L29506 295 Q45670
294 1-24/25-121 134-391 proteinase precursor (160, 193, (Ak.1
protease) 347) S08.020 C5a peptidase J05229 297 P15926 296 1-31/
120-339, precursor 458-560 (130, 193, 512) S08.021 fervidolysin
AY035311 299 AAK61552 298 164-457 S08.035 Subtilisin J M64743 301
P29142 300 1-29/30-106 112-372 precursor (138, 170, 327) S08.036
Subtilisin E K01988 303 P04189 302 1-23/24-106 112-372 precursor
(138, 170, 327) S08.037 Subtilisin DY P00781 304 6-257 (32, 63,
220) S08.054 Proteinase K X14689 306 P06873 305 1-15/16-105 134-373
precursor (144, 174, (Endopeptidase K) 329) S08.050 Alkaline serine
M25499 308 P16588 307 1-21/22-141 155-412 exoprotease A (180, 213,
precursor 363) S08.060 Epidermin leader X62386 310 P30199 309
1-23/24-? 123-451 peptide-processing (149, 194 serine protease 402)
epiP precursor S08.063 Membrane-bound AF078105 312 Q9Z2A8 311
1-14/18-186 179-473 transcription (218, 249, factor site 1 338,
414) protease precursor (Site-1 protease, Subtilisin/kexin- isozyme
1, SKI-1) S08.066 Alkaline proteinase M87516 314 Q03420 313
1-20/21-120 121-409 precursor (ALP) (161, 192, 353) S08.094
Extracellular M13469 316 P09489 315 1-27/ 50-389 serine protease
(76, 112, precursor 341) S08.090 Tripeptidyl- AF035251 318 Q9V6K1
317 5-509 peptidase II (131, 359, (TPP-II) 549) S08.114 Minor
extracellular M76590 320 P29141 319 1-28/29-160 163-365, protease
vpr 487-586 precursor (189, 233, 534) S08.116 PIII-type proteinase
J04962 322 P15292 321 1-33/34-187 190-379, precursor (Lactocepin)
584-628 (217, 281, 620) S08.048 Furin-like protease 1, M81431 324
P30430 323 1-?/?-309 376-646 isoform 1-CRR precursor (372, 413,
587) S08.070 Kexin precursor M22870 326 P13134 325 1-19/20-109
149-445 (KEX2 protease) and 110-113 (175, 213, (Kex2-like 385)
endoprotease 1, dKLIP-1) S08.071 Furin precursor X17094 328 P09958
327 1-24/25-107 131-421 (PACE) (153, 194, 295, 368) S08.075
Subtilisin/kexin- M80482 330 P29122 329 1-63/64-149 182-473 like
protease PACE4 (205, 246, (Proprotein convertase 347, 420)
subtilisin/kexin type 6 precursor) S08.079 Calcium-dependent X56955
332 P23916 331 254-521 protease precursor (233, 270, (Trypsin) 466)
Cysteine C14.001 Caspase-1 precursor U14647 334 P43527 333 /1-118
120-404 protease: (Interleukin-1 (236, 284) caspase beta
convertase, family IL-1BC) C14.002 Cell death protein 3 L29052 336
P42573 335 235-495 precursor (315, 358) C14.003 Caspase-3 precursor
U13737 338 P42574 337 /1-9 10-277 (Apopain, Cysteine (121, 163)
protease) C14.004 Caspase-7 precursor U39613 340 P55210 339 /1-23
29-303 (ICE-like apoptotic (144, 186) protease 3, Apoptotic
protease Mch-3) C14.005 Caspase-6 precursor U20536 342 P55212 341
/1-23 24-292 (Apoptotic protease (121, 163) Mch-2) C14.006
Caspase-2 precursor D28492 344 P29594 343 /1-169 170-432 (ICH-1
protease, (277, 320) NEDD2 protein) C14.007 Caspase-4 precursor
Z48810 346 P49662 345 /1-80 93-377 (ICH-2 protease, TX (210, 258)
protease) C14.008 Caspase-5 precursor U28015 348 P51878 347 /1-120
134-418 (ICH-3 protease, TY (251, 299) protease, ICE(rel)-III)
C14.009 Caspase-8 precursor X98172 350 Q14790 349 /1-216 193-479
(FADD-like ICE, (317, 360) ICE-like apoptotic protease 5) C14.010
Caspase-9 precursor U56390 352 P55211 351 117-416 (ICE-LAP6,
Apoptotic (237, 287) protease Mch-6 C14.011 Caspase-10 precursor
U60519 354 Q92851 353 /1-219 243-514 (358, 401) C14.012 Caspase-11
(Caspase-4 U59463 356 P70343 355 /1-80 89-373 precursor) (206, 254)
C14.013 Caspase-12 precursor Y13090 358 O08736 357 133-419 (250,
298) C14.015 Caspase precursor Y12261 360 O01382 359 /1-28 (169,
211) (insect) (drICE) C14.016 Caspase-1 precursor AF001464 362
O02002 361 /1-33 (154, 196) (insect) C14.017 Caspase-13 precursor
AF078533 364 O75601 363 (210, 258) C14.018 Caspase-14 precursor
AF097874 366 P31944 365 1-242 (89, 132) C14.019 Caspase Nc
precursor AF104357 368 Q9XYF4 367 /1-134 (271, 318) (NEDD2-like
caspase DRONC) C14.026 MALT lymphoma AF130356 370 Q9UDY8 369
337-523 translocation (415, 464) protein 1 paracaspase
(paracaspase) C14.971 CASP8 and FADD-like U85059 372 O15519 371
260-433 apoptosis regulator (315, 363) precursor (c-FLIP)
[0336] a. Classes of Proteases
[0337] 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.
[0338] 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.
[0339] 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 et al. (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
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.
[0340] 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, S1, S2 . . . Sn
towards the N-terminus of the substrate, and S1', S2' . . . Sn'
towards the C-terminus. The residues they accommodate are numbered
P1, P2 . . . Pn, and P1', P2' . . . Pn', respectively. The cleavage
of a target protein is catalyzed between P1 and P1' where the amino
acid residues from the N to C terminus of the polypeptide substrate
are labeled (Pi, . . . , P3, P2, P1, P P2', P3', . . . Pj) and
their corresponding binding recognition pockets on the protease are
labeled (Si, . . . , S3, S2, S1, S1', S2', S3', . . . , Sj)
(Schecter and Berger (1967) Biochem Biophys Res Commun 27:157-162).
Thus, P2 interacts with S2, P1 with S1, P1' with S1', etc.
Consequently, the substrate specificity of a protease comes from
the S1-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 between 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 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 et al., (2000) Nat Struct Biol. 7(9):762-5; Cameron et al.,
(1993) J Biol. Chem. 268:11711; Cameron et al., (1994) J Biol.
Chem. 269: 11170).
[0341] i. Serine Proteases
[0342] 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 C1r,
C1s, and the C3 convertases play a role in complement
activation.
[0343] 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.
[0344] 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 cancer invasion and metastasis.
The activity and/or expression of some proteases have been shown to
correlate with tumor progression and development.
[0345] Over 20 families (denoted S1-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 N D et al. (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).
[0346] 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, C1 esterase, cathepsin G, chymase, and proteases of the
blood coagulation cascade including kallikrein, thrombin, and
Factors VIIa, 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. et al., (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 al. (1986) Hosp. Prac.,
21: 131-137).
[0347] 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 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.
[0348] 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.
[0349] 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).
[0350] 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 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 S1 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 S1 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 S1-S4
pocket affect the specificity or selectivity of a protease for a
target substrate.
TABLE-US-00008 TABLE 8 The structural determinants for various
serine proteases Residues that Determine Specificity S4 S2 S1
Cys168 S3 60's Cys191 171 174 180 215 Cys182 192 218 99 57 loop 189
190 226 Cys220 Granzyme B Leu Tyr Glu Tyr 14 Arg Asn Ile His 6 Gly
Ser Arg No Granzyme A Asn Val Met Phe 17 Asn Leu Arg His 7 Asp Ser
Gly Yes Granzyme M Arg Ser Met Phe 15 Lys Arg Leu His 8 Ala Pro Pro
Yes Cathepsin G Phe Ser Gln Tyr 13 Lys Ser Ile His 6 Ala Ala Glu No
MT-SP1 Leu Gln Met Trp 13 Gln Asp Phe His 16 Asp Ser Gly Yes
Neutrophil -- -- -- Tyr 5 Phe Gly Leu His 10 Gly Val Asp Yes
elastase Chymase Phe Arg Gln Tyr 12 Lys Ser Phe His 6 Ser Ala Ala
No Alpha-tryptase Tyr Ile Met Trp 22 Lys Glu Ile His 9 Asp Ser Gly
Yes Beta-tryptase(I) Tyr Ile Met Trp 22 Gln Glu Val His 9 Asp Ser
Gly Yes Beta-tryptase (II) Tyr Ile Met Trp 22 Lys Glu Thr His 9 Asp
Ser Gly Yes Chymotrypsin Trp Arg Met Trp 13 Met Ser Val His 7 Ser
Ser Gly Yes Easter Tyr Ser Gln Phe 16 Arg Thr Gln His 14 Asp Ser
Gly Yes Collagenase Tyr Ile -- Phe 12 Asn Ala Ile His 8 Gly Thr Asp
Yes 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 Gln Met Tyr 13 Arg Pro Phe His 11 Asp Ala Ala Yes
kallikrein Plasmin Glu Arg Glu Trp 15 Gln Leu Thr His 11 Asp Ser
Gly Yes Trypsin Tyr Lys Met Trp 13 Gln Tyr Leu His 6 Asp Ser Gly
Yes Thrombin Thr Ile Met Trp 13 Glu Glu Leu His 16 Asp Ala Gly Yes
tPA Leu Thr Met Trp 15 Gln Leu Tyr His 11 Asp Ala Gly Yes uPA His
Ser Met Trp 15 Gln Arg His His 11 Asp Ser Gly yes
[0351] (a) Urokinase-type Plasminogen Activator (u-PA)
[0352] 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.
[0353] 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 1-49), 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.
[0354] 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 PAI-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.
[0355] 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 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).
[0356] (b) Tissue Plasminogen Activator (t-PA)
[0357] 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.
[0358] 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 of t-PA display
similar activity. (Strandberg et al. (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
Arg.sup.275-Ile.sup.276 (corresponding to Arg.sup.310-Ile.sup.311
in the sequence of amino acids set forth in SEQ ID NO:193)
resulting in the generation of the active two-chain form of t-PA.
The two-chain polypeptide contains an A and a B chain that are
connected by an interchain disulfide bond.
[0359] The mature t-PA contains 16 disulfide bridges and is
organized into five distinct domains (Gething et al. (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.
[0360] 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 of
t-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 et al. (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 of t-PA in
the blood supports t-PA as the primary fibrinolytic activator of
plasminogen, while u-PA is the primary cellular activator of
plasminogen.
[0361] (c) MT-SP1
[0362] 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.
[0363] 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-SP1 are known. For example, a mouse homolog of
MT-SP1 has been identified and is called epithin.
[0364] MT-SP1 contains a transmembrane domain, two CUB domains,
four LDLR repeats, and a serine protease domain (or peptidase S1
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.
[0365] An MT-SP 1 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.
[0366] MT-SP1 belongs to the peptidase S1 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
.beta. 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 (SEQ ID NO: 641), VVGG
(SEQ ID NO: 642), or IVGG (SEQ ID NO: 643), where the first amino
acid in this quartet is numbered according to the chymotrypsin
numbering and given the designation Ile16. This numbering does not
reflect the length of the precursor sequence).
[0367] 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 Gln at P3, small
residues at P2, Arg or Lys at P1, and Ala at P1'. Effective
substrates contain Lys-Arg-Ser-Arg (SEQ ID NO: 644) in the P4 to P1
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 (sc-uPA), the proform of
MT-SP1, and hepatocyte growth factor (HGF), conform to the cleavage
sequence for MT-SP1 specific substrates.
[0368] 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; SEQ ID NO: 645), 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).
[0369] 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 51 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 P1-Lys
residues is mediated by Ser.sup.190 whose side chain provides an
additional hydrogen bond acceptor to stabilize the buried
.alpha.-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 Phe.sup.99 benzyl group. The substrate amino
acids at positions P3 (for either Gln 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 Gln.sup.192 or alternatively, the P3 side
chain can extend into the S4 sub-site to form a hydrogen bond with
Phe.sup.97 thereby weakening the inter-main chain hydrogen bonds
with Gly.sup.216. 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-SP1 (based on
chymotrypsin numbering) that contribute to extended specificity for
substrate binding include: 146 and 151 (S1'); 189, 190, 191, 192,
216, 226 (S1); 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).
[0370] ii. Cysteine Proteases
[0371] 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.
[0372] 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 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 P1 residue in serine proteases).
[0373] 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 .about.160,000 tetrapeptide sequences.
[0374] Overall, P1 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.
[0375] 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
selectivity of a protease for a target substrate including a
complement protein target substrate.
TABLE-US-00009 TABLE 9 The structural determinants for various
cysteine proteases Residues that Determine Specificity Active Site
Residues S3 S2 S1 25 159 175 61 66 66 133 157 160 205 19 20 158
Cathepsin L Cys His Asn Glu Gly Gly Ala Met Gly Ala Gln Gly Asp
Cathepsin V Cys His Asn Gln Gly Gly Ala Leu Gly Ala Gln Lys Asp
Cathepsin K Cys His Asn Asp Gly Gly Ala Leu Ala Leu Gln Gly Asn
Cathepsin S Cys His Asn Lys Gly Gly Gly Val Gly Phe Gln Gly Asn
Cathepsin F Cys His Asn Lys Gly Gly Ala Ile Ala Met Gln Gly Asp
Cathepsin B Cys His Asn Asp Gly Gly Ala Gly Ala Glu Gln Gly Gly
Papain Cys His Asn Tyr Gly Gly Val Val Ala Ser Gln Gly Asp Cruzain
Cys His Asn Ser Gly Gly Ala Leu Gly Glu Gln Gly Asp
E. MODIFIED PROTEASES AND COLLECTIONS FOR SCREENING
[0376] 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. Appln. 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.
[0377] 1. Generation of Variant Proteases
[0378] 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, 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.
[0379] a. Random Mutagenesis
[0380] Random mutagenesis methods include, for example, use of E.
coli XL1red, 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., et al. (1997)
Gene 188:35-39), the use of dNTP analogs (Zaccolo, M., et al.
(1996) J. 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.
[0381] b. Focused Mutagenesis
[0382] 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
residues. In this example, residues that form the S1-S4 pocket of a
protease (where the protease is in contact with the P1-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 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 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 the Kunkle method, Current Protocols in Molecular Biology,
John Wiley and Sons, Inc., Media Pa.). 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.
[0383] 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 et al.,
(2001) Biochemistry 40(34):10038; Hopfner et al., Structure Fold
Des. 1999 7(8):989; Friedrich et al., (2002) J Biol Chem
277(3):2160; Waugh et al., (2000) Nat Struct Biol. 7(9):762). For
example, focused mutations of MT-SP1 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 (51); 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 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.
[0384] 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.
[0385] 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.
[0386] 2. Chimeric Forms of Variant Proteases
[0387] 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.
[0388] 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,
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.
[0389] 3. Combinatorial Libraries and Other Libraries
[0390] 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, Curr. Opin. Chem. Biol.,
2(3):363-71 (1998); Lam, Anticancer Drug Des., 12(3):145-67 (1997);
Blaney and Martin, Curr. Opin. Chem. Biol., 1(1):54-9 (1997); and
Schultz and Schultz, Biotechnol. Prog., 12(6):729-43 (1996)).
[0391] 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 al. (2000) Appl Environ
Microbiol. 66: 788 793; MacBeath, G. P. et al. (1998) Science
279:1958-1961; Soumillion, P. L. et al. (1994) Appl. Biochem.
Biotechnol. 47:175-189, Wang, C. I. et al. (1996). Methods Enzymol.
267:52-68, U.S. Pat. Nos. 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.
[0392] 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 al.
(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 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 al.,
(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.
[0393] a. Phage Display Libraries
[0394] 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, f1, T7, and
.lamda. 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 (gIIIp; 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.
[0395] 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 et al., 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 et al. (1998) Immunotechnology
4:1-20; Hoogenboom et al. (2000) Immunol Today 2:371-8; Fuchs et
al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum
Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins
et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al.
(1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods
Enzymol. 267:129-49; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
[0396] 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; Armstrong et al. (1996)
Academic Press, Kay et al., Ed. pp. 35-53; Corey et al. (1993) Gene
128(1):129-34; Cwirla et al. (1990) Proc Natl Acad Sci USA
87(16):6378-82; Fowlkes et al. (1992) Biotechniques 13(3):422-8;
Hoogenboom et al. (1991) Nuc Acid Res 19(15):4133-7; McCafferty et
al. (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).
[0397] 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 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 phage 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. Amp.sup.R or Kan.sup.R (for ampicillin or kanamycin
resistance, respectively) for the selection of cells that are
infected by a member of the library.
[0398] 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.
[0399] 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 methods are particularly
useful in situations where the fusion protein is less tolerated by
the phage.
[0400] Phage display systems typically utilize filamentous phage,
such as M13, fd, and f1. 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 (gIIIp), 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).
[0401] 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.
[0402] 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 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).
[0403] 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
tet 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 et al. (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 et al. (1979)
Proc. Nat. Acad. Sci. U.S.A., 76:106-110; J. D. Windass et al.
(1982) Nucl. Acids. Res., 10:6639-57; R. Crowl et al. (1985) Gene,
38:31-38; Brosius (1984) Gene 27: 161-172; Amanna and Brosius,
(1985) Gene 40: 183-190; Guzman et al. (1992) J. Bacteriol., 174:
7716-7728; Haldimann et al. (1998) J. Bacteriol., 180:
1277-1286).
[0404] 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
depression, e.g., inactivation of a repressor molecule.
[0405] A regulatable promoter sequence also can be indirectly
regulated. Examples of promoters that can be engineered for
indirect regulation include: the phage lambda P.sub.R, P.sub.L,
phage T7, SP6, and T5 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 T7 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 T7 RNA polymerase and a regulatory sequence (e.g., the
lac promoter) that is regulated by an environmental parameter. The
activity of the T7 RNA polymerase also can be regulated by the
presence of a natural inhibitor of RNA polymerase, such as T7
lysozyme.
[0406] In another configuration, the lambda P.sub.L 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 P.sub.L
promoter from repression.
[0407] 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.
[0408] 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.
[0409] b. Cell Surface Display Libraries
[0410] 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 coli.
[0411] 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-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 .beta.-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. Sci. 745:372-382 (1994) and
Georgiou G. et al. 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.
[0412] In another example, variant proteases can be fused to
autotransporter domains of proteins such as the N gonorrhoeae IgA 1
protease, Serratia marcescens serine protease, the Shigella
flexneri VirG protein, and the E. coli adhesin AIDA-I (Klauser et
al. EMBO J. 1991-1999 (1990); Shikata S, et al. J Biochem.
114:723-731 (1993); Suzuki T et al. J Biol. Chem. 270:30874-30880
(1995); and Maurer J et al. J Bacteria 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 .beta.-lactamase, have been successful
expressed on the surface of E. coli using this system (Lattemann C
T et al. J. Bacteriol. 182(13): 3726-3733 (2000)).
[0413] Bacteria can be recombinantly engineered to express a fusion
protein, such a membrane fusion protein. Nucleic acids encoding the
variant proteases can be fused 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.
[0414] 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. Appl.
Microbiol. Biotechnol. 64: 625-635 (2004)).
[0415] 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. U.S. Pat. No.
5,789,208 and WO 03/029456).
[0416] c. Other Display Libraries
[0417] 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.
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. et al. (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)
[0418] 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
[0419] 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.
[0420] 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.
[0421] 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 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.
[0422] 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) J 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 M13 phage
(e.g., New England Biolabs, MA) and a standard ELISA (see e.g.,
Ausubel et al. (1987) Current Protocols in Molecular Biology, John
Wiley & Sons, New York).
[0423] 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 polyhistidine (e.g., His 6-tag) and capture
using a metal chelating agent such as, but not limited to, nickel
sulphate (NiSO.sub.4), cobalt chloride (CoCl.sub.2), copper
sulphate (CuSO.sub.4), or zinc chloride (ZnCl.sub.2). 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), 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).
[0424] 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.
[0425] 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 protease, for example,
to measure protease activity. Such a procedure is described in
Example 17.
[0426] 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.
[0427] 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.
[0428] 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 precipitated 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.
[0429] 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 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.
[0430] 1. Iterative Screening
[0431] 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 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.
[0432] 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.
[0433] 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 (SEQ ID NO: 379), and subsequent rounds of panning are
performed against a protease trap polypeptide containing the VEGFR2
cleavage sequence RRVR (SEQ ID NO: 489).
[0434] 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 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.
[0435] 2. Exemplary Selected Proteases
[0436] Provided herein are variant u-PA and MT-SP 1 polypeptides
identified in the methods provided herein as having an altered
and/or improved substrate specificity. Such variant u-PA and MT-SP
1 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.
[0437] a. Variant u-PA Polypeptides
[0438] 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 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).
[0439] 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, I24, F30, V38, T39, Y61(A),
R72, L73, S75, E80, K82, E84, I89, K92, F132, G133, E137, I138,
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, F30L, F30T, F30G, F30M,
V38D, T39A, Y61(A)H, R72G, L73A, L73P, S75P, E80G, K82E, E84K,
I89V, K92E, F132L, G133D, E137G, I138T, L155P, L155V, L155M, K156Y,
T158A, 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.
[0440] 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,
F30L, F30V, F300, F30M, R72G, L73A, L73P, S75P, I89V, 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/189V; L73P; R217c; L155P;
S75P/I89V/1138T; E137G; R72G/L155P; G133D; V160A; V38D;
F132L/V160A; L73A/I89V/F30T; L73A/I89V/F30L; L73A/I89V/F30V;
L73A/I89V/F30G; L73A/I89V/L155V; L73A/I89V/F30M; L73A/I89V/L155M;
L73A/I89V/F30L/L155M; and L73A/I89V/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/189V; L155P;
R72G/L155P; F132L/V160A; L73A/I89V/F30T; L73A/I89V/L155V;
L73A/I89V/L155M; and L73A/I89V/F30L/L155M, based on chymotrypsin
numbering.
[0441] 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, 124L, F30V,
F30L, T39A, Y61(A)H, E80G, K82E, E84K, I89V, K92E, K156T, 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; 124L;
F30V; F30L; F30V/Y61(A)H; F30V/K82E; F30V/K156T; F30V/K82E/V159A;
F30V/K82E/T39A/V159A; F30V/K82E/T158A/V159A; F30V/Y61(A)H/K92E;
F30V/K82E/V159A/E80G/I89V/K187E; and
F30V/K82E/V159A/E80G/E84K/I89V/K187E, 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:460-472,
or fragments thereof of contiguous amino acids containing the
mutation and having catalytic activity.
[0442] 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 et al. (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 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.
[0443] b. Variant MT-SP1 Polypeptides
[0444] In 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-SP 1 selection methods provided herein
include, for example, AT3 modified in its native P4-P2' residues
IAGRSL (SEQ ID NO:478) with amino acid residues for a complement
protein C2 cleavage sequence SLGRKI (SEQ ID NO:479).
[0445] 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, I41, L52, Y60(g), T65, H71, F93, N95, F97,
I98, F99, A126, V129, P131, I136, H143, T144I, I154, N164, T166,
L171, P173, Q175, F184(a), Q192, 5201, Q209, 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, 141F, 141T, L52M, Y60(g)s, T65K, H71R,
F93L, N95K, F97Y, F97L, T98P, F99L, A126T, V129D, P131S, I136T,
I136V, H143R, T144I, I154V, N164D, T166A, L171F, P173S, Q175R,
F184(a)L, Q192H, S201I, 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 of 141F, F97Y, L171F,
Q175R, D217V and V244G, for example, any one or more of 141F, F97Y,
L171F and V244G.
[0446] 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 I136T/N164D/T166A/F184(A)L/D217V; I41F; I41F/A126T/V244G;
D23E/I41F/T98P/T144I; 141F/L171F/V244G; H143R/Q175R; I41F/L171F;
R230W; I41F/I154V/V244G; I41F/L52M/V129D/Q221(A)L; F99L;
F97Y/I136V/Q192H/S201I; 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-SP1 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-SP1 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.
[0447] In particular, modified u-PA polypeptides having the
following amino acid modifications are provided: L73A/I89V; L155P;
R72G/L155P; F132L/V160A; L73A/I89V/F30T; L73A/I89V/L155V;
L73A/I89V/L155M; and L73A/I89V/F30L/L155M, based on chymotrypsin
numbering.
G. METHODS OF ASSESSING PROTEASE ACTIVITY AND SPECIFICITY
[0448] 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.
[0449] 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
assessed 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.
[0450] 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.
[0451] 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 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.
[0452] 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
polypeptide, 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 k.sub.cat, K.sub.m
and k.sub.cat/K.sub.m 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=(K.sub.mN.sub.max)(1/[S])+1/V.sub.max; where
V.sub.max=[ET]k.sub.cat). The second order rate constant or
specificity constant (k.sub.cat/K.sub.m) is a measure of how well a
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.
[0453] 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 50 mM 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 quantified
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.
[0454] 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.
[0455] 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.
[0456] 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.
[0457] In specific embodiments, comparison 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 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
[0458] Polypeptides set forth herein, including protease trap
polypeptides or protease polypeptides or catalytically active
portions thereof, including modified u-PA polypeptides or modified
MT-SP I 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.
[0459] 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.
[0460] 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
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.
[0461] 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 pharmacokinetics of
a product of a synthetic gene.
[0462] 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., 6.times.His, HHHHH; SEQ ID
NO:496) or Flag Tag (DYKDDDDK; SEQ ID NO:495).
[0463] 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 pCMV4, pBR322 or pUC
plasmid derivatives or the Bluescript vector (Stratagene, La Jolla,
Calif.). The insertion into a cloning vector can, for 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, Calif.). 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.
[0464] 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.
[0465] 1. Vectors and Cells
[0466] 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.
[0467] 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.
[0468] 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 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.
[0469] 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.
[0470] 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.
[0471] 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 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 et al., Proc. Natl. Acad. Sci. 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 .beta.-lactamase promoter (Jay et al., (1981) Proc. Natl.
Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., 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 (Herrar-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 et al., 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 et al., 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 et al.,
Nature 318:533-538 (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. 1:268-276
(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 et al., Genes and Devel.
1:161-171 (1987)), beta globin gene control region which is active
in myeloid cells (Mogram et al., Nature 315:338-340 (1985); Kollias
et al., Cell 46:89-94 (1986)), myelin basic protein gene control
region which is active in oligodendrocyte cells of the brain
(Readhead et al., Cell 48:703-712 (1987)), myosin light chain-2
gene control region which is active in skeletal muscle (Sani,
Nature 314:283-286 (1985)), and gonadotrophic releasing hormone
gene control region which is active in gonadotrophs of the
hypothalamus (Mason et al., Science 234:1372-1378 (1986)).
[0472] 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, Calif.),
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, Calif.; see also literature published by Qiagen
describing the system). pQE vectors have a phage T5 promoter
(recognized by E. coli 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
6.times.His tag coding sequence, t.sub.0 and T1 transcriptional
terminators, ColE1 origin of replication, and a beta-lactamase gene
for conferring ampicillin resistance. The pQE vectors enable
placement of a 6.times.His 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
6.times.His-tagged proteins. Other exemplary plasmid vectors for
transformation of E. coli cells, include, for example, the pET
expression vectors (see, U.S. Pat. No. 4,952,496; available from
NOVAGEN, Madison, Wis.; see, also literature published by Novagen
describing the system). Such plasmids include pET 11a, 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, Wis.), which
contain a His-Tag.TM. 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.
[0473] 2. Expression
[0474] 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.
[0475] 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.
[0476] 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 his.sub.6 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.
[0477] In one embodiment, a protease can be expressed in an active
form. In another embodiment, a protease is expressed in an
inactive, zymogen form.
[0478] a. Prokaryotic Cells
[0479] 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 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 .lamda.PL promoter.
[0480] 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 .beta.-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.degree. C. and
37.degree. 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.
[0481] b. Yeast Cells
[0482] 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
chromosomal integration by homologous recombination. Typically,
inducible promoters are used to regulate gene expression. Examples
of such promoters include GAL1, GAL7 and GAL5 and metallothionein
promoters, such as CUP1, AOX1 or other Pichia or other yeast
promoter. Expression vectors often include a selectable marker such
as LEU2, TRP1, 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.
[0483] c. Insect Cells
[0484] 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 Autographa califormica
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 unipuncta
(A7S) and Danaus plexippus (DpN1) produce proteins with
glycosylation patterns similar to mammalian cell systems.
[0485] 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
albopictus) 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.
[0486] d. Mammalian Cells
[0487] 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-.zeta. and
Fc.sub..epsilon.RI-.gamma. can direct expression of the proteins in
an active state on the cell surface.
[0488] Many cell lines are available for mammalian expression
including mouse, rat human, monkey, chicken and hamster cells.
Exemplary cell lines include but are not limited to CHO, Balb/3T3,
HeLa, MT2, mouse NS0 (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.)
[0489] e. Plants
[0490] 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 100:438-442). Because plants have different
glycosylation patterns than mammalian cells, this can influence the
choice of protein produced in these hosts.
[0491] 3. Purification Techniques
[0492] 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 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.
[0493] 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.
[0494] 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 His.sub.6 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.
[0495] 4. Fusion Proteins
[0496] 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,
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.
[0497] 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, Fc domain from an immunoglobulin, or a
heterologous signal sequence. The fusion proteins can contain
additional components, such as E. coli maltose binding protein
(MBP) that aid in uptake of the protein by cells (see,
International PCT application No. WO 01/32711).
[0498] 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 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.
[0499] 5. Nucleotide Sequences
[0500] 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.
[0501] 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.
[0502] 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
[0503] 1. Compositions and Delivery
[0504] 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 (e.g., 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.
[0505] 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.
[0506] 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 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,
polyinylpyrrolidine, 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 monostearate, 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" by E. W. Martin. 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.
[0507] 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 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.
[0508] 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.
[0509] 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).
[0510] 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.
[0511] 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),
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.
[0512] 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.
[0513] 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.
[0514] 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.
[0515] 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.
[0516] 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
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.
[0517] Pharmaceutical compositions also can be administered by
controlled release formulations and/or delivery devices (see, e.g.,
in U.S. Pat. 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).
[0518] 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 .mu.m. 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.
[0519] 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.
[0520] 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 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 .mu.m) 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.
[0521] 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
et al., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., J
Immunol., 154: 3088-95, 1995; see also, Caliceti et al. (2003) Adv.
Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003) Pharmacotherapy
23 (8 Pt 2):3S-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).
[0522] 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).
[0523] 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).
[0524] 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.
[0525] 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.
[0526] 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 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.
[0527] 2. In Vivo Expression of Selected Proteases and Gene
Therapy
[0528] 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.
[0529] a. Delivery of Nucleic Acids
[0530] Nucleic acids can be delivered to cells and tissues by any
method known to those of skill in the art.
[0531] i. Vectors--Episomal and Integrating
[0532] 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.
[0533] 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 (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.
[0534] ii. Artificial Chromosomes and Other Non-viral Vector
Delivery Methods
[0535] The nucleic acid molecules can be introduced into artificial
chromosomes and other non-viral vectors. Artificial chromosomes
(see, e.g., U.S. Pat. No. 6,077,697 and PCT International PCT
application No. WO 02/097059) can be engineered to encode and
express the isoform.
[0536] iii. Liposomes and Other Encapsulated Forms and
Administration of Cells Containing Nucleic Acids
[0537] 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.
[0538] b. In Vitro and Ex Vivo Delivery
[0539] 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
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.
[0540] 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) electroporation, 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.
[0541] 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.
[0542] 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 and/or tissues. Inducible promoters can be used
to selectively regulate selected protease expression.
[0543] c. Systemic, Local and Topical Delivery
[0544] 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.
[0545] 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 isoform 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.
[0546] 2. Combination Therapies
[0547] 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
anticoagulants. Exemplary anticoagulants include heparin, coumarin,
hirudin, aspirin, naproxen, meclofenamic acid, ibuprofen,
indomethacin, phenylbutazare, ticlopidine, streptokinase,
urokinase, and tissue plasminogen activator.
[0548] 3. Articles of Manufacture and Kits
[0549] 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.
[0550] 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. Pat. Nos. 5,323,907, 5,052,558 and
5,033,252, each of which is incorporated herein in its entirety.
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 target-mediated disease or
disorder.
[0551] 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
[0552] The selected protease polypeptides provided herein that
cleave particular targets and nucleic acid molecules that encode
the selected proteases provided herein 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 thrombolytic 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.
[0553] 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.
[0554] 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.
[0555] 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 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.
[0556] 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.
[0557] 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.
[0558] 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 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.
[0559] 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.
[0560] 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.
[0561] 1. Exemplary Methods of Treatment for Selected uPA
Polypeptides that Cleave tPA Targets
[0562] 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 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.
[0563] 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.
[0564] 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.RTM., Genentech, South San
Francisco, Calif.) is recombinant human tPA. Reteplase
(Retavase.RTM., Rapilysin.RTM.; 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.RTM., Genentech) is a 527 amino acid glycoprotein
derivative of human tPA that differs from naturally 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.RTM., 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.
[0565] a. Thrombotic Diseases and Conditions
[0566] 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.
[0567] 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 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.
[0568] i., Arterial Thrombosis
[0569] 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.
[0570] ii. Venous Thrombosis and Thromboembolism
[0571] 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
fulminans), and adrenal 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)).
[0572] 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)).
[0573] 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.
[0574] 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.
[0575] (a) Ischemic Stroke
[0576] 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 thromboembolism 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.
[0577] iii. Acquired Coagulation Disorders
[0578] 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, 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.
[0579] (a) Disseminated Intravascular Coagulation (DIC)
[0580] 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 organ failure. 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.
[0581] (b) Bacterial Infection and Periodontitis
[0582] 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 inflammatory
responses leading to elevated levels of cytokines. The cytokines,
in turn, can influence induction of coagulation.
[0583] 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.
[0584] 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, J. 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.
[0585] b. Other tPA Target-Associated Conditions
[0586] 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 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.
[0587] 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.
[0588] c. Diagnostic Methods
[0589] 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.
[0590] 2. Exemplary Methods of Treatment for Selected Protease
Polypeptides That Cleave VEGF or VEGFR Targets
[0591] 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.
[0592] Three tyrosine kinase family receptors of VEGF have been
identified (VEGF-R-1/Flt-1, VEGF-R-2/Flk-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 .mu.M (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 Differ. 9(1):49-58), and chemical inhibition of
phosphorylation causes tumor cells to become apoptotic (Shaheen,
(1999) Cancer Res. 59(21):5412-6).
[0593] 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 maintenance of tumor
blood supply, and lead to the systematic death of the tumor. The
recent success of the anti-VEGF antibody AVASTIN.TM. 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.
[0594] 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.
[0595] 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.
[0596] a. Angiogenesis, Cancer, and Other Cell Cycle Dependent
Diseases or Conditions
[0597] 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.
[0598] 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.
[0599] b. Combination Therapies with Selected Proteases That Cleave
VEGF or VEGFR
[0600] 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 VEGF/VEGFR-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 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 AVASTINT.TM. in any
therapy where AVASTINT.TM. administration provides therapeutic
benefit.
[0601] 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-1/KDR stalk, over a six amino acid
region.
[0602] 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 AVASTIN.TM.. 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.
[0603] 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,
Gleevec, 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 Administration and the U.S. Department of Health
and Human Services. 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.
[0604] 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, carmustine, 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, mechlorethamine,
medroprogesterone acetate, megestrol acetate, melphalan,
mercaptopurine, methotrexate, methotrexate, mithramycin, mitomycin,
mitotane, phenyl butyrate, prednisone, procarbazine, semustine
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.
[0605] 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 York, 1980 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,
.beta.-emitters, .gamma.-emitters, Auger electron emitters, neutron
capturing agents that emit .alpha.-particles and radioisotopes that
decay by electron capture. Preferably, the radioisotope is selected
from the group consisting of .sup.225Ac, .sup.198Au, .sup.32P,
.sup.131I, .sup.131I, .sup.90Y, .sup.186Re, .sup.188Re, .sup.67Cu,
.sup.177Lu, .sup.213Bi, .sup.10B, and .sup.211At.
[0606] 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.
[0607] 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.
[0608] 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, Goodman and Gilman, and the like, and in the
references cited above.
[0609] 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.
[0610] 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 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. O. (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.
[0611] 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.
[0612] 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.
[0613] Similarly, certain antileukemic and antilymphoma antibodies
conjugated with radioisotopes that are .beta. of .alpha.-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.
[0614] 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.
[0615] 3. Exemplary Methods of Treatment for Selected MT-SP1
Polypeptides That Cleave Complement Protein Targets
[0616] 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
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.
[0617] 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.
[0618] 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.
[0619] a. Immune-mediated Inflammatory Diseases
[0620] 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.
[0621] 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, MOG-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 C1, C3 and
C9.
[0622] Complement activation modulates inflammation in diseases
such as rheumatoid arthritis (RA) (Wang et al., (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-CII) 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-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.
[0623] 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 et al., (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.
[0624] Therapeutic proteases, such as those described herein
including variant MT-SP1 polypeptides provided herein, can be used
to treat sepsis and severe sepsis that 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.
[0625] b. Neurodegenerative Disease
[0626] 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 PS1 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.
[0627] c. Cardiovascular Disease
[0628] 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. 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.
[0629] 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 mm 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-SP1
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.
[0630] 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-SP1 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.
[0631] Proteases and modified proteases, such as for example
variant MT-SP1 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
et al. (1995) J. Clin. Invest. 96:1564). In such a 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 CD11b and P-selectin
levels.
K. EXAMPLES
[0632] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
Mutant PAI-1 Inhibitors
A. Expression and Purification of Mutant PAI-1 Inhibitors
[0633] 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 HindIII site at nucleotide pair 1 and the SalI 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 ApaLI 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 HindIII restriction endonuclease
sites at the 5' and 3' termini, respectively, of the PAI-1 cDNA
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 ApaLI-PflMI DNA fragment, digested with EcoRI and HindIII
and the 1146 bp EcoRI-HindIII fragment was isolated by gel
electrophoresis and cloned into EcoRI and HindIII cleaved pBR322
(SEQ ID NO:377).
[0634] 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.
[0635] To generate the pPAIST7HS prokaryotic expression vector
(Shubeita et al. (1990) J Biol. Chem., 265: 18379-18385), the
pPAIST7 was partially digested with HindIII to linearize the
plasmid, blunt-ended with the Klenow fragment of E. coli DNA
polymerase I and ligated to eliminate the upstream HindIII site.
Deletion of sequences in pPAIST7 downstream of the PAI-1 coding
sequences between the HindIII and SalI sites and elimination of the
SalI site was accomplished by sequential partial SalI digestion,
complete HindIII digestion, blunt-ending with the Klenow fragment
of E. coli DNA polymerase I, and ligation.
[0636] 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 PAI-1 reactive center loop from the P4 to P1'
positions. The sequence for the PFGRS mutagenic primer was:
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.
[0637] The mutants PAI-1/RRAR and PAI-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-1s (i.e. PAI-1/RRAR and PAI-1/69) were based on methods as
described in Ke et al. (J. Biol. Chem., 272: 16603-16609 (1997)).
Expression of wild type and mutated variants of PAI-1 was
accomplished by transforming 0.1 .mu.g DNA of pPAIST7HS vector
encoding mutant PAI-1 into the E. coli strain BL21[DE3]pLys.sup.s
(Novagen), which synthesizes T7 RNA polymerase in the presence of
isopropyl-1-thio-.beta.-D-galactopyranoside. Bacterial cultures
were grown at 37.degree. C. with vigorous shaking to an absorbance
A.sub.595 of 1.1-1.3, and
isopropyl-1-thio-.beta.-D-galactopyranoside was added to a final
concentration of 1 mM to induce the synthesis of T7 RNA polymerase
and the production of PAI-1 proteins. Cultures were grown for an
additional 1-2 h at 37.degree. C. and then shifted to 30.degree. C.
for 2-6 h. Cells were pelleted by centrifugation at 8000.times.g
for 20 min at 4.degree. C. and resuspended in 40 ml of cold start
buffer (20 mM sodium acetate, 200 mM NaCl, 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.times.g.
[0638] Purification of soluble, active mutant PAI-1 was performed
by injecting the lysate of E. coli containing soluble form of
PAI-1/RRAR or PAI-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
column volumes of start buffer (20 mM sodium acetate, 200 mM NaCl,
and 0.01% Tween 20, pH 5.6), and PAI-1 proteins were eluted using a
0.2-1.8 M linear gradient of NaCl 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
[0639] 1. Active Site Titration Against Standard Trypsin
[0640] Active concentration of PAI-1/RRAR and PAI-1/69 was
determined by active site titration against standard trypsin as
described by Olson et al. (J. Biol. Chem., 270: 30007 (1995)).
Briefly, sequential additions of concentrated inhibitor (0.5-6.0
.mu.M) were made to solutions of 1 .mu.M .beta.-trypsin (Sigma) and
10 .mu.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 nm 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.
[0641] 2. Titration of Standardized t-PA Preparations
[0642] Mutant PAI-1s were also titrated against standardized t-PA
preparations to assess activity. The inhibitory activity of
wild-type PAI-1 or mutant PAI-1 was measured by a direct
chromogenic assay using tPA (American Diagnostics, Inc, 100
U/.mu.g) and the chromogenic tPA substrate
H-D-Ile-Pro-Arg-para-nitroaniline substrate (S-2288, Chromogenix).
Serially diluted PAI-1 (0.1-4.0 .mu.g) 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
[0643] A. Cloning of Wild-Type u-PA into Phagemid
[0644] 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'-ACGTGGCCCAGGCGGCCTTTCAGTGTGGCCAAAAG-3' (SEQ ID
NO:374); 497, 5'-TCCTGGCCGGCCTGGCCGAGCAGGCCATTCTC-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 SfiI 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 gIIIp gene
of fd phage. The presence of two SfiI 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 gIIIp fusion.
B. Construction of Mutant u-PA and Phage Display Libraries of
Mutant u-PA
[0645] 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 .mu.l 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 SfiI enzyme digestion. The SfiI digested PCR products
from each mutatgenesis reaction were used to generate three
different libraries.
[0646] For library construction, the pComb3H vector (SEQ ID NO:376)
was digested with SfiI 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 mg of the gel purified vector was mixed with SfiI
digested PCR products (1:2 ratio) and the ligation mixtures were
incubated overnight at 18.degree. C. Next, the ligation mixtures
were purified using Qiagen mini elute kit (Qiagen) and the DNA was
finally eluted into 60 .mu.l of Milli Q purified water. The
purified DNA was electroporated into 400 .mu.l of E. coli XL Blue1
electroporation competent cells (Stratagene) using gene pulser (Bio
Rad). The cells were then transferred to 10 ml of SOC medium
(Invitrogen) and incubated at 37.degree. C. in a shaker for 1 hr
followed by plating on large LB agar plates (245 mm.times.245 mm)
supplemented with carbenicillin (75 .mu.g/mL). After overnight
incubation of plates at 30.degree. C. the transformants were
scraped using a cell scraper and the resulting cultures were grown
in 2.times.YT medium supplemented with carbenicillin (75 .mu.g/ml)
at 37.degree. 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.degree. C. shaking, the cultures were supplemented with
kanamycin to final concentrations of 3 .mu.g/mL and grown overnight
at 30.degree. C. with shaking. The cells were harvested and the
phage particles present in the supernatant were precipitated using
a PEG-NaCl 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 .mu.g/ml) and
after overnight incubation at 37.degree. 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.
[0647] 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 .mu.l of u-PA
phage (typically .about.5.times.10.sup.12 cfu), 0.2 .mu.M
Lys-plasminogen (American Diagnostic) and 0.62 mM Spectrozyme PL
(American Diagnostica) were present in a total volume of 100 .mu.l.
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 37.degree. C. Inhibition of
u-PA or u-PA variant phage also was assessed. Briefly, 5 .mu.l of
u-PA phages (typically .about.5.times.10.sup.12 cfu) were mixed
with wt-PAI-1 (0.1 .mu.M) or mutant PAI-1/RRAR inhibitor (1.0
.mu.M) and incubated for 30 min at room temperature followed by
addition of 0.62 mM Spectrozyme PL, 0.2 .mu.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
[0648] A. Selection of u-PA Phage/Mutant PAI-1 Complexes
[0649] 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 .mu.l
(.about.2.times.10.sup.12 to .about.1.times.10.sup.13) 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 .mu.l bait substrate (from 0.1 to 1.0 .mu.M PAI-1/RRAR
or PAI-1/69) and 10 .mu.l 10.times. indirect buffer pH 7.4 (0.5 M
Tris, 1.0 M NaCl, 10 mM EDTA, 0.1% Tween 80) in a total volume of
100 .mu.l (i.e. reaction contained 80 .mu.l H.sub.2O). 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).
[0650] The u-PA phage-PAI-1 inhibitor complexes were captured using
CuSO.sub.4 activated sepharose. Pre-activation of the chelating
sepharose (200 .mu.l; Pharmacia) was accomplished by treatment with
CuSO.sub.4 (100 mM). The CuSO.sub.4 activated sepharose was blocked
with 0.5% BSA in PBS buffer for 1 hour at room temperature. 100
.mu.l of BSA blocked sepharose beads was added to 100 .mu.l of the
above panning mixture reaction in the presence of 800 .mu.l of
binding buffer (0.5 M NaCl, 20 mM Tris, 20 mM immidozole, pH7.4) to
capture the His-tagged PAI-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-1 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.
[0651] The bound u-PA phage-PAI-1/RRAR complexes were eluted in 100
.mu.l of elution buffer (0.5M EDTA). A 95 .mu.A aliquot of the
eluted phages was used to infect 1 ml of XL-1 Blue E. coli cells
(0.60D) 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.times.245 mm) containing
carbenicillin (75 .mu.g/ml) 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 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 .mu.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.
[0652] 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
10.sup.4 to 10.sup.5 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
[0653] An aliquot of eluted u-PA phages (5 .mu.l) was mixed with
XL-1Blue E. coli cells (100 .mu.l) for infection and incubated at
37.degree. C. for 1 hr. The infected E. coli cells were then plated
on LB agar plates supplemented with carbenicillin (100 .mu.g/ml).
After overnight incubation at 37.degree. C., individual colonies
were picked for phage preparations. The cells were grown in 2 ml of
2.times.YT supplemented with carbenicillin (100 .mu.g/ml) and
tetracycline (10 .mu.g/ml) and phage preparation was performed as
described (Sambrook, J et 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 plasminogen activation
assay to identify active phages, and the active phage preparations
were used for inhibition assays.
[0654] 1. Indirect Plasminogen Activation Assay
[0655] The individual phage preparations were used in the indirect
plasminogen activation assay to identify active u-PA phages.
Briefly, 5 .mu.l of u-PA phage (typically .about.5.times.10.sup.12
cfu), 0.2 .mu.M Lys-plasminogen (American Diagnostic) and 0.62 mM
Spectrozyme PL (American Diagnostica) were present in a total
volume of 100 .mu.l. 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
37.degree. C.
[0656] 2. Inhibition of u-PA Phage by Mutant PAI-1/RRAR
[0657] 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 .mu.l of
active u-PA phages (typically.about.5.times.10.sup.12 cfu) were
added in duplicate wells of a microtiter plate followed by addition
of a fixed concentration of mutant PAI-1 (e.g., 1.0 .mu.M) 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 .mu.M Lys-plasminogen (American Diagnostics,
Inc.). For control experiments, 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.
[0658] 3. Peptide Substrate Screening
[0659] 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 .mu.l) was mixed with 75
.mu.M Ac-RRAR-AMC substrate in 1.times. indirect assay buffer (50
mM Tris, 100 mM NaCl, 1 mM EDTA, 0.01% Tween 80) in a total volume
of 100 .mu.l. 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.
[0660] 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
[0661] Positive phage clones were mixed with XL-1Blue E. coli cells
for infection as mentioned above and the cultures were grown
overnight with shaking at 37.degree. 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.
[0662] 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-US-00010 TABLE 10 Mutation u-PA Phage site/s libraries Vs
Mutant (Chymotrypsin Amino acid/s SEQ PAI-1/RRAR name Number)
modified ID NO: Selection I AR73 (2) 30 Phe - Ileu 434 AR81 73, 89
Leu - Ala, 435 Ileu - Val Selection AR1 73 Leu-Pro 436 II & III
AR3 217 Arg-Cys 437 AR4 (3) 155 Leu-Pro 438 AR7 75, 89, 138
Ser-Pro, Ileu-Val, 439 Ileu-Thr AR32 137 Glu-Gly 440 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
[0663] 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 F30V 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-IIb or u-PA-IIb 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/K82E/V159A 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-US-00011 TABLE 11 Phage Mutant Amino acids SEQ libraries name
Mutation sites modified ID NO: I u-PA/Ia 21 Phe-Val 460 u-PA/Ib 24
Ile-Leu 461 u-PA/Ic (2) 30 Phe-Val 462 u-PA/Id 30 Phe-Leu 463 II
u-PA/IIa 30, 61(A) Tyr-His 464 u-PA/IIb 30, 82 Lys-Glu 465 u-PA/IIc
30, 156 Lys-Thr 466 III u-PA/IIIa (8) 30, 82, 159 Val-Ala 467
u-PA/IIIb 30, 82, 39, 159 Thr-Ala, Val-Ala 468 u-PA/IIIc 30, 82,
158, Thr-Ala, Val-Ala 469 159 u-PA/IIId (2) 30, 61A, 92 Lys-Glu 470
IV u-PA/IVa 30, 82, 159, 80, Glu-Gly, Ile-Val, 471 89, 187 Lys-Glu
u-PA/IVb 30, 82, 159, 80, Glu-Gly, Glu-Lys, 472 84, 89, 187
Ile-Val, Lys-Glu
[0664] 1. Optimization and Recombination of Amino Acid Resides 30
and 155 in Focused Phage Display Libraries against PAI-1/RRAR
Inhibitor
[0665] 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:
TABLE-US-00012 (SEQ ID NO: 383) TC30- 5'GCCCTGGNNSGCGGCCATC- 3'
(SEQ ID NO: 384) TC155- 5'GGAGCAGNNSAAAATGACTG- 3'
[0666] 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 polynucleotide 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 189V 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 30 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 phage
libraries were used for selection of improved variants of u-PA as
set forth in Example 3A and 3B above.
[0667] 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 189V 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-US-00013 TABLE 12 Focused u-PA libraries SEQ Vs Mutant ID
PAI-1/RRAR Name 30(Phe) 155 (Leu) NO Selection ARF2 Thr -- 446 I
& II ARF6 Leu -- 447 ARF11 Val -- 448 ARF17 Gly -- 449 ARF16
Leu -- 450 ARF33 -- Val 451 ARF35 Met -- 452 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
[0668] 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 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'-CAGAGTCTTTTGGCCACA-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 KpnI and XbaI restriction
enzymes (underlined) respectively.
[0669] 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 .mu.l 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 .mu.l each) were used as templates in 100
.mu.l 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 KpnI and XbaI 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 KpnI and
XbaI. The ligation mixture was electroporated into E. coli XL-1
Blue cells and plated on LB plates supplemented with carbenicillin
(100 .mu.g/ml). After overnight incubation of plates at 37.degree.
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).
[0670] 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. 20 .mu.g of cDNA, 100
.mu.g of carrier DNA, and approximately 10.sup.7 COS-1 cells were
placed into a 0.4-cm cuvette, and electroporation was performed at
320 V, 960 microfarads, and .OMEGA.=.infin. (Tachias et al. (1995)
J. Biol. Chem., 270: 18319-18322). Following electroporation, the
transfected cells were incubated overnight at 37.degree. 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 h at 37.degree. 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
[0671] 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
[0672] Direct assays of u-PA activity utilized the substrate
carbobenzoxy-L-.gamma.-glutamyl
(.alpha.-t-butoxy)-glycyl-arginine-p-nitroanilide monoacetate salt
(Cbo-L-(.gamma.)-Glu(.alpha.-t-.beta.uO)-Gly-Arg-pNA AcOH;
Spectrozyme.RTM. 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 nM.
The concentration of Spectrazyme.RTM. uPA was varied from 25 to 150
.mu.M in assays of two-chain u-pA, and from 25 to 200 .mu.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 k.sub.cat, K.sub.m, and k.sub.cat/K.sub.m
(specificity constant) were calculated by graphing the inverse of
the substrate concentration versus the inverse of the velocity of
absorbance at OD.sub.405, and fitting to the Lineweaver-Burk
equation (1/velocity=(K.sub.m/V.sub.max)(1/[S])+1/V.sub.max; where
V.sub.max=[E]*k.sub.cat).
[0673] 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.RTM. uPA substrate. In the
Table, the variants tested are those identified in Table 11 above
following selection from successive generations (I to IV) of u-PA
phage display libraries.
TABLE-US-00014 TABLE 13 K.sub.m k.sub.cat k.sub.cat/K.sub.m Mutant/
u-PA mutants (mM) (s-1) (M-1 s-1) Wt u-PA u-PA/Ic (30) 0.745 22.3
2.9 .times. 10.sup.5 0.30 u-PA/IIb (30, 82) 0.299 11 3.6 .times.
10.sup.5 0.38 u-PA/IIIa (30, 82, 159) 0.239 11.6 4.8 .times.
10.sup.5 0.50 u-PA/IIIb (30, 39, 82, 159) 0.212 9.6 4.5 .times.
10.sup.5 0.47 u-PA/IVa (30, 80, 82, 89, 0.177 10 5.6 .times.
10.sup.5 0.58 159, 187) u-PA/IVb (30, 80, 82, 84, 89, 0.321 11 3.4
.times. 10.sup.5 0.35 159, 187) Wild type u-PA 0.174 16.6 9.5
.times. 10.sup.5 1.0
C. Kinetic Analysis of u-PA Variants Using Fluorogenic
Substrate
[0674] 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 et al. (1977) Anal
Biochem, 78:47-51; Harris et al. (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
serially diluted from 0.05 to 12.0 mM and incubated in the 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 nm) and emission
wavelength (450 nm) 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.sub.cat,
K.sub.m, and k.sub.cat/K.sub.m (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=(K.sub.m/V.sub.max)(1/[S])+1/V.sub.max; where
V.sub.max=[E]*k.sub.cat).
[0675] Table 14 below sets forth the results of the kinetic
analysis of the u-PA variants ARF2 and ARF36 against 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-US-00015 TABLE 14 Improvement of K.sub.cat/K.sub.m Mutants
K.sub.m (.mu.M) K.sub.cat/K.sub.m (M.sup.-1 s.sup.-1) 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
Chromogenic Assay
[0676] 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 et al. (1990) J Biol. Chem., 265: 21423-21426). In this
assay, free p-nitroaniline is released from the chromogenic
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 OD.sub.405
nm.
[0677] For the assay, 100 .mu.l reaction mixtures containing 0.25-1
ng of the uPA enzymes to be tested, 0.62 mM Spectrozyme PL, 0.2
.mu.M Lys-plasminogen (American Diagnostics, Inc.), were combined
in a buffer containing 50 mM Tris-HCL (pH 7.5), 0.1 M NaCl, 1.0 mM
EDTA and 0.01% (v/v) Tween 80. The reaction was incubated at
37.degree. C. in 96-well, flat-bottomed microtiter plates (Costar,
Inc.) and the optical density at 405 nm (OD405) was read every 30 s
for 1 h in a Molecular Devices Thermomax. The kinetic constants
k.sub.cat, K.sub.m, and k.sub.cat/K.sub.m (specificity constant)
were calculated as described earlier (Madison, E. L (1989) Nature
339, 721-724).
[0678] 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.RTM.
PL substrate. In the Table, the variants tested are those
identified in Table 11 above following selection from successive
generations (I to IV) of u-PA phage display libraries.
TABLE-US-00016 TABLE 15 u-PA mutants K.sub.m (.mu.M)
k.sub.cat.sup.(s-1) k.sub.cat/K.sub.m.sup.(M-1 s-1) Mutant/Wt u-PA
u-PA/Ic 9.01 24.7 2.7 .times. 10.sup.6 0.24 u-PA/IIb 8.6 22.6 2.6
.times. 10.sup.6 0.23 u-PA/IIIa 6.31 37.6 5.9 .times. 10.sup.6 0.53
u-PA/IIIb 9.3 29.8 3.2 .times. 10.sup.6 0.29 u-PA/IVa 7.03 38.7 5.5
.times. 10.sup.6 0.50 u-PA/IVb 7.5 45.3 6.0 .times. 10.sup.6 0.54
Wild type u-PA 6.03 70.1 1.1 .times. 10.sup.7 1.0
E. Kinetic Analysis of Inhibition of Mutant u-PA Enzymes by
Wild-Type PAI-1 and Mutant PAI-1
[0679] 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.times.10.sup.6) or second order
(ki>2.times.10.sup.6) 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 PAI-1 was performed at 24.degree. C.
in 0.1 M Tris-HCl buffer (pH 7.4) containing 0.1 mM EDTA and 0.1%
(v/v) Tween 20. The indirect chromogenic assay as described in Part
D above was used to determine the residual enzyme activity
remaining as a function of time.
[0680] 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
et al. (1987) Biochemistry, 26: 5133-5140; Beatty et al. (1980) J.
Biol. Chem., 255:3931-3934; Madison et al. (1990) PNAS, 87:
3530-3533; Madison et al. (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.
[0681] 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 PAI-1 was added after preincubation and
addition of anti-PAI-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.
[0682] 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.
TABLE-US-00017 TABLE 16 2.sup.nd order rate Improvement u-PA
variants constant (M.sup.-1 s.sup.-1) Mutant/Wt-uPA U-PA/ARF2 1.6
.times. 10.sup.5 18.3 U-PA/ARF36 1.7 .times. 10.sup.5 20.2 Wt u-PA
8.7 .times. 10.sup.3 1.0
[0683] 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-US-00018 TABLE 17 2.sup.nd order rate constant Sensitivity
factor u-PA mutants (M.sup.-1 s.sup.-1) (Mutant/Wt u-PA) u-PA/Ic
2.4 .times. 10.sup.6 0.3 u-PA/IIb 2.9 .times. 10.sup.6 0.3
u-PA/IIIa 9.7 .times. 10.sup.6 1.2 u-PA/IIIb 1.3 .times. 10.sup.7
1.6 u-PA/IVa 2.7 .times. 10.sup.7 1.6 u-PA/IVb 6.8 .times. 10.sup.6
0.8 Wild type u-PA 7.5 .times. 10.sup.6 1.0
TABLE-US-00019 TABLE 18 2.sup.nd order rate constant Increased
sensitivity factor u-PA mutants (M.sup.-1 s.sup.-1) (Mutant/Wt
u-PA) u-PA/Ic 1.0 .times. 10.sup.5 13.7 u-PA/IIb 1.1 .times.
10.sup.5 15.3 u-PA/IIIa 1.8 .times. 10.sup.5 24.5 u-PA/IIIb 2.8
.times. 10.sup.5 37.5 u-PA/IVa 2.7 .times. 10.sup.5 35.9 u-PA/IVb
3.2 .times. 10.sup.5 42.6 Wild type u-PA 7.5 .times. 10.sup.3
1.0
Example 6
Selection of variant MT-SP1 from MT-SP1 Phage Libraries Against
Mutant AT3 Inhibitor
[0684] 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-P1-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 AT3.sup.SLGR-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 .mu.l of a 1:100 SM1
(.about.3.times.10.sup.13) MT-SP1 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 .mu.l of a 1:100 SM2 (.about.3.times.10.sup.12) MT-SP1 phage
library that contains a higher mutagenesis frequency (i.e. 0.9%).
The phage libraries were mixed with 5 .mu.l heparin (5 ng/.mu.l;
from stock of porcine intestinal mucosa), 5 .mu.l bait
AT3.sup.SLGR-KI substrate (ranging in concentrations from 0 (i.e. 5
.mu.l H.sub.2O), 0.018 .mu.M, 0.18 .mu.M, 1.8 .mu.M, or 18 .mu.M)
in the presence of 5 .mu.l (18 .mu.M) wildtype, plasma purified AT3
and 5 .mu.l 10.times.MTSP activity buffer (0.5 M Tris HCl, pH 8,
0.3 M NaCl, 0.1% Tween 30) in a total volume of 50 .mu.l (i.e.
reaction contained 20 .mu.l H.sub.2O). The reaction was incubated
for 4.5 hours at 37.degree. C.
[0685] The MTSP-1 phage-AT3 inhibitor complexes were captured using
CuSO.sub.4 activated sepharose. 200 .mu.l of chelating sepharose
(Pharmacia) was pre-activated with CuSO.sub.4 (100 mM). The
CuSO.sub.4 activated sepharose (100 .mu.l) 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 .mu.l of binding buffer
(0.5 M NaCl, 100 M Tris pH 8, 10 mM Imidazole, 0.1% Tween 20). 50
.mu.l of the above panning mixture reaction was added to the
CuSO.sub.4 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 .mu.l 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.
[0686] The bound MT-SP1 phage-AT3.sup.SLGR-KI complexes were eluted
in 100 .mu.l of elution buffer (0.5 M EDTA, pH 8.0). A 50 .mu.l
aliquot of the eluted phages was used to infect 3 ml of TG1 E. coli
actively growing cells (A600=0.5; 0.5 OD=.about.1.5.times.10.sup.8
colonies/ml) for 20 minutes at 37.degree. C. The infected bacteria
were plated on large plates (245 mm.times.245 mm) containing
carbenicillin (75 .mu.g/ml) and incubated at 30.degree. 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 AT3.sup.SLGR-KI inhibitor. The results of the
colony counts are set forth in Table 19.
TABLE-US-00020 TABLE 19 Results of First Generation Selection
Concentration of AT3.sup.SLGR-KI Colonies from first round in
reaction of selection 18 .mu.M AT3.sup.SLGR-KI Lawn 1.8 .mu.M
AT3.sup.SLGR-KI 6088 0.18 .mu.M AT3.sup.SLGR-KI 1712 0.018 .mu.M
AT3.sup.SLGR-KI 3700 0 .mu.M AT3.sup.SLGR-KI 840
[0687] The colonies from the AT3.sup.SLGR-KI containing plates were
scraped into 25 ml 2YT supplemented with carbenicillin (.about.100
.mu.g/ml) after the plates had been placed 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
A.sub.600 was determined to be 0.13. The bacteria were grown to an
OD=.about.0.5 and then .about.1.times.10.sup.10 to
2.6.times.10.sup.13 cfu/ml of helper phage (VS M13) were added (in
.about.150 .mu.l-200 .mu.l) for amplification of the libraries.
After 1 hour of growth at 37.degree. C. shaking, the cultures were
supplemented with kanamycin to final concentrations of 3 .mu.g/mL
and grown overnight at 30.degree. C. with shaking. The cells were
harvested and the phage particles present in the supernatant were
precipitated using a PEG-NaCl 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
AT3.sup.SLGR-KI bait substrate was for only 27 minutes instead of
4.5 hours to enhance the stringency of the selection.
[0688] Following elution of the bound MT-SP1 phage-AT3.sup.SLGR-KI
complexes as described above, 50 .mu.l aliquot of the eluted phages
were used to infect 3 ml of TG1 E. coli actively growing cells
(A600=0.5; 0.5 OD=.about.1.5.times.10.sup.8 colonies/ml) for 20
minutes at 37.degree. C. The infected bacteria were plated on large
plates (245 mm.times.245 mm) containing carbenicillin (75 .mu.g/ml)
and incubated at 30.degree. 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 AT3.sup.SLGR-KI
inhibitor. The results of the colony counts are set forth in Table
20.
TABLE-US-00021 TABLE 20 Results of Second Generation Selection
Colonies from second round of Enrichment Concentration of
selection; Ratio Compared AT3.sup.SLGR-KI in reaction (background)
to Background 18 .mu.M AT3.sup.SLGR-KI 2476 (165) 15:1 1.8 .mu.M
AT3.sup.SLGR-KI 1750 (90) 19:1 0.18 .mu.M AT3.sup.SLGR-KI 2012
(110) 18:1 0.018 .mu.M AT3.sup.SLGR-KI 1824 (89) 21:1
[0689] The colonies were picked for further characterization. The
cells were grown in 2 ml of 2.times.YT supplemented with
carbenicillin (100 .mu.g/ml) and tetracycline (10 .mu.g/ml) and
phage preparation were performed as described (Sambrook, J et al
(1989) Molecular Cloning, A laboratory manual, Cold spring Harbor
laboratory). The 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
[0690] 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 ID NO.:
612, purchased from Origene Technologies, Catalog # 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: GTCACTGACTGACGTGGA TCCCACGGGAGCCCTGTGGACATC (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 EcoRI. Either a C-terminal
6.times.His-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 6.times.His tag
using a four amino acid GGGS linker (SEQ ID NO.:620) are set forth
in SEQ ID NOs.:613 and 614, respectively.
[0691] To make mutant AT3 bait substrates for isolating target
proteases with various specificities, mutagenesis reactions were
carried out using the Quikchange.RTM. 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 SEQ ID NO.: 5; and amino acid
residues 390-395 of the mature AT3 polypeptide sequence set forth
in SEQ ID NO.: 493).
[0692] 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.
[0693] For the Quikchange.RTM. 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: GCTGCAAGTACCGCTGTTGTGTCGTTAGGCCGTAAAATTAACCCCAACAGGGTGA
CTTTC. The VEGFR2 Target sequence primer had the following sequence
of nucleic acids, which is set forth in SEQ ID NO.: 634:
GCTGCAAGTACCGCTGTTGTGCGCCGTGTGCGCAAAGAAAACCCCAACAG GGTGACTTTC.
[0694] Vectors containing the wild-type AT3 cDNA and vectors
containing the mutant AT3 cDNA were each transformed and amplified
in XL-1-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
[0695] 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.RTM. 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.RTM. recombination vector were
pre-incubated for 20 min with 5 .mu.l of Cellfectin.RTM.
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/antimycotic solution; Cellgro) and were incubated at
28.degree. C. for 4 days. Virus was expanded in Sf9 cells to a
maximum titer of 1.times.10.sup.6 pfu/mL, as determined by plaque
assay. Recombinant AT3 then was expressed using the High Five.RTM.
(BTI-TN5B1-4) insect cell line (Invitrogen) and Excell3 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
[0696] For affinity-based purification of His-tagged AT3 proteins,
supernatants from the cultures from Example 7B were cleared by
centrifugation and filtration using a 0.45 .mu.M 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 Duoflow3 chromatography apparatus (Bio-Rad) and 10 mL
of TALON.RTM. 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 NaCl and 50 mM Sodium
Phosphate pH 6.5, 300 mM NaCl, 150 mM Imidazole. Fractions
containing protein were combined and dialyzed into AT3 storage
buffer (50 mM Sodium Phosphate pH 6.5, 300 mM NaCl, 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-SP 1 to cleave a substrate. The reaction mixture from this
MT-SP1 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.RTM. M5 (SpectraMax.RTM. M5 Microplate
Reader, Molecular Devices) (Molecular Devices, Inc). The "ACC" in
the name of this substrate refers to the
7-amino-4-carbamoylmethylcoumarin 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.
[0697] 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.degree. 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 NaCl, 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.
[0698] 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
[0699] A. Cloning of Wild-Type and C122S MT-SP1 Protease Domain
(B-Chain) into the pMal-C2 Phagemid Display Vector
[0700] cDNA (SEQ ID NO.: 504) encoding a mature MT-SP1 protease
domain (MT-SP1 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 Nde1 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
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 GeneIII (SEQ ID NO.: 512) domain
in italics. The * indicates the presence of a stop codon in the
nucleic acid sequence encoding the protein.
TABLE-US-00022 SEQ ID NO.: 506:
mkkniafllasmfvfsiatnayavvggtdadegewpwqvslhalgqghicgaslispnwlvsaahcyi
ddrgfrysdptqwtaflglhdqsqrsapgvqerrlkriishpffndftfdydiallelekpaeyssmvr
piclpdashvfpagkaiwvtgwghtqyggtgalilqkgeirvinqttcenllpqqitprmmcvgflsg
gvdscqgdsggplssveadgrifqagvvswgdgcaqrnkpgvytrlplfrdwikentgvsgssgggs
egggsegggsegggsgggsgsgdfdyekmanankgamtenadenalqsdakgkldsvatdygaaidgf
igdvsglangngatgdfagsnsqmaqvgdgdnsplmnnfrqylpslpqsvecrpfvfsagkpyefsidcdk
inlfrgvfafllyvatfmyvfstfanilrnkes*
[0701] 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-SP1 fusion protein was used to generate MT-SP1 mutant
phage display libraries as described in Example 8B below.
B. Mutagenesis of Protease Domains for the Generation of Mutant
Phage Display Libraries
[0702] 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 et al.,
Methods Mol. Biol. 2002; 182:259-67; Cirino et al., Methods Mol.
Biol. 2003; 231:3-9) as exemplified below.
[0703] 1. Mutagenesis of B-Chain MT-SP1 Fusion Proteins
[0704] 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.RTM. PCR Random
Mutagenesis Kit (BD Biosciences, Clonetech) and following
conditions suggested by the supplier to obtain five (5) mutations
per kilobase. The MT-SP1 forward primer used in this PCR, having
the sequence of nucleic acids set forth in SEQ ID NO.:508):
GCGCAGATATCGTACCGCATATGAAAAA GAATATCGCATTTCTT, 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 SEQ ID NO.:509:
GTGCATGCTGACTGACTGAGCTCCCGCTTACCCCAGTGTTCTC, was designed to
hybridize within the 3' portion of the sequence encoding the MT-SP1
protease domain (with the residues shown in italics) and contained
a Sac1 restriction site sequence (shown in bold).
[0705] 2. Purification of Mutagenesis Products
[0706] 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 Jan. 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/.mu.l proteinase K. To eliminate Taq polymerase, the mixture was
incubated at 65.degree. C. for 15 minutes.
[0707] 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 10.times. 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.RTM. Gel Extraction Kit protocols (Qiagen) or
the Zymoclean3 Gel Extraction Kit (Zymoclean CA, Cat # D4001)
following the conditions specified by the supplier.
[0708] For the Qiagen Gel Extraction kit, the excised gel fragment
was solubilzed in QG Buffer and slowly passed through a Qiagen QG
column, each of which has a binding capacity of about 5-10 .mu.g,
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 1.5 mL microcentrifuge tube, followed by addition of 50 .mu.L
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.
[0709] For the Zymoclean3 Gel Extraction Kit, either one or more
Zymoclean3 Gel Extraction Kit columns (each of which has a 5 .mu.g
maximum binding capacity), or one or more columns from the
Zymoclean3 DNA Clean & Concentrator3 Kit (each of which has a
25 .mu.g 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 Buffer3 to each volume of agarose excised from the
gel. Samples were incubated at 37-55.degree. C. for 5-10 minutes
until the gel slice had completely dissolved. The dissolved agarose
solution was transferred to a Zymo-Spin I3 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 .mu.L 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
.mu.L of water was added directly to the column matrix. The minimum
elution volume was 10 .mu.L for the Zymoclean3 Gel Extraction
column and 35 mL for the DNA Clean & Concentrator3 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.
[0710] After elution using one of these two methods, samples were
pooled and DNA concentration was assessed by measuring absorbance
of the sample at 260 nm in a 70 .mu.L UV cuvette, using a
spectrofluorometer. DNA concentration was calculated according to
the following equation: 1A260=50 ng/.mu.l ds DNA.
C. Construction of Protease Mutant Phage Libraries Using the
pMal-C2 Phagemid Vector
[0711] 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 NdeI and SacI, 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 NdeI/SacI digested, gel purified pSTII-g3 pMal-C2
phagemid is 5835 base pairs (bp); the MW of the NdeI/SacI digested
MT-SP1 PCR product is 806 bp. Typically, for a 2 mL ligation
reaction, 7.58 .mu.g cut vector was about 1 nM of vector and 3.14
.mu.g cut MT-SP1 product was about 3 nM insert product.
[0712] For ligation of the MT-SP1 products, 40 .mu.l (3 nM, 3.14
.mu.g) of the digested, purified product was mixed with: 40 .mu.l
(1 nM, 7.58 .mu.g) of digested, purified vector; 1510 .mu.l
H.sub.2O; 400 .mu.l 5X T4 DNA Ligase Buffer (Gibco); and 10 .mu.l
(10 units) T4 DNA Ligase (New England Biolabs). The ligation
reaction was carried out overnight at 16.degree. 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.degree. C. for 15 minutes followed by addition of 4
mL ZymoResearch DNA Binding Buffer. This sample then was added, 800
.mu.l at a time, to a 25 .mu.g ZymoResearch Column. The column was
washed twice with 600 .mu.l ZymoResearch Wash Buffer and eluted
with 50 .mu.L water, which had been pre-warmed to 42.degree. C.
Percent DNA recovery was assessed by measuring absorbance of
diluted elution sample (3 .mu.l elution in 70 .mu.l H2O) at 260 nm
using a spectrofluorometer. For example, for the 3 mL ligation
above, if the A260 corresponded to 0.12=140 ng/.mu.l, then the
total yield was determined to be about 7 .mu.g (which is about 70%
recovery of DNA from the ligation).
[0713] The ligation product was electroporated into XL-1 blue
cells, which can accommodate approximately 500 ng/.mu.l by
electroporation. 7.5 .mu.l of a 140 ng/.mu.l ligation was added per
200 .mu.l 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.degree. C. for 20 minutes. Following this
incubation five (5) small aliquots (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.degree. 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.times.245 mm) supplemented with
Carbenicillin (75 ug/ml) and grown overnight at 30.degree. 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 M13KO7 helper phage at
1.times.10.sup.10 CFU/mL and grown overnight at, 37.degree. 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
[0714] 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.
[0715] 1. Interaction with and Cleavage of AT3 Variants by Mutant
MT-SP1-Bearing Phages
[0716] 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.degree. C., by incubating the following reaction components in a
70 .mu.L volume: 35 .mu.l MT-SP1 library phage at 3.14E.sup.12
CFU/ml; 7 .mu.L 10.times. MT-SP1 Activity Buffer (0.5 M Tris-HCl,
pH 7.4, 1M, NaCl, 1% Tween20); 7 .mu.L 10 .mu.M Low Molecular
Weight Heparin (BD Biosciences); 14 .mu.L H.sub.2O; and 7 .mu.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. Each reaction was
terminated with the addition of 2 .mu.L 100 mg/ml of the protease
inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride (Pefabloc,
Roche Diagnostics). 40 .mu.L of a 0.55% BSA, 0.275% Tween 20
solution then was added and mixed thoroughly by pipetting up and
down.
[0717] 2. Capture of MT-SP1 Phage-AT3 Complexes Using Anti-His
Antibodies
[0718] Meanwhile, wells in another 96-well polystyrene plate (Nunc
Maxysorp) were coated for 1 hour with shaking at room temperature
with 100 .mu.L of 5 ng/.mu.l Streptavidin (Pierce) in 0.2 M
Carbonate Buffer (pH 9, Pierce). The wells then were washed three
times with 250 .mu.L PBST, blocked with 200 .mu.L of 0.2% BSA in
PBS for 2 hours at room temperature, incubated for 1 hour at room
temperature with 100 .mu.l of 5 ng/.mu.l 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 .mu.L PBST.
[0719] 3. ELISA-Based Readout for Selection of MT-SP1 Phage-AT3
Complexes
[0720] After washing, the first of the two rows in the microtiter
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 .mu.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 .mu.L TMB/Peroxide substrate solution
(Pierce) and incubation at room temperature for 5 minutes. This
reaction was quenched with 100 .mu.L 2M H2SO4 and assayed on a
SpectraMax.RTM. 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 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.
[0721] 4. Elution of Selected MT-SP1 Phage
[0722] 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 .mu.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 .mu.L of 1M Tris
pH 8 for acid neutralization prior to infection. The 133 .mu.L of
neutralized phage mix then was added to 1 ml of XL-1 Blue cells
growing at an OD600 of 0.5, which then were incubated for 20
minutes at 37.degree. C. and plated out onto 2.times.YT agar plates
(245 mm.times.245 mm) supplemented with Carbenicillin. These cells
were used in subsequent screening, sequencing and purification
methods as described in the Examples below.
[0723] 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
[0724] 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 OD600 of 0.5 or
approximately 2.1.times.10.sup.8 cells/mL. The desired infectivity
range was 1000-2000 colonies per plate. Infected cells were plated
onto 2YT agar supplemented with 100 .mu.g/mL Carbenicillin in
square 245 mm.times.245 mm polystyrene BioAssay (Corning) dishes
and allowed to grow for 16-20 hours or until colony size were
roughly 2 mm 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 100 .mu.g/mL Carbenicillin and 12 .mu.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 into a HiGro (GeneMachines) incubator and shaken at 400 rpm
at 37.degree. C. for 14-20 hours.
[0725] After the incubation, to obtain log-phase cells, 100 .mu.L
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 .mu.g/mL Tetracycline. The deep well plate
then was sealed with an air-permeable membrane and placed in the
HiGro.RTM. incubator with shaking at 400 rpm at 37.degree. C. with
oxygen aeration until the cell density reached an OD600 of between
0.4 and 0.6. A typical incubation period was between 4 and 5 hours.
After incubation, 100 .mu.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.degree. C. After the 5-10 minutes of shaking, the
plate was incubated at 37.degree. C. in a static state without
shaking for 30-45 minutes. Shaking then was resumed at 400 rpm for
15-30 minutes at 37.degree. C. Following shaking, 100 .mu.L
kanamycin solution (400 .mu.g/mL) was added to each well to yield a
final concentration of 33.3 .mu.g/mL in each well. The plate was
resealed, and shaken at 400 rpm at 37.degree. C. for 12-16 hours.
To pellet the cells, the plate then was centrifuged at 3500-4500
rpm for 20 minutes at 4.degree. 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.degree. C.
C. Polyethylene Glycol (PEG) Precipitation of Protease Domain Phage
Supernatants
[0726] 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.degree. C. at 3500-4500 rpm and
1000 .mu.L of supernatant was removed from each well and
transferred to a well in another 96 well deep well plate.
[0727] For precipitation, 250 .mu.L of a solution containing 20%
PEG (by volume) in 2.5M NaCl 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 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
(200 uL) to yield a 5-fold concentrate. This material either was
used immediately in assays described below, or stored at 4.degree.
C. until ready for testing.
Example 10
Screening of Protease Domain-Bearing Phages Having Increased
Reactivity and Catalytic Efficiency Towards Target Substrate
Sequences
[0728] 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
[0729] 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).
[0730] For analysis of inhibition of MT-SP1 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.
[0731] 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-AMC (SEQ ID NO.:
617) was assessed in the presence and in the absence of a given
concentration of PAI 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, 35 .mu.L 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 .mu.l of 2.times. 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. 35 .mu.L of 2.times. Indirect Assay Buffer
(without inhibitor) was added to the corresponding control wells.
The plates were incubated at 37.degree. C. for 60 minutes.
Following the mixing of the phage with inhibitor or control buffer,
10 .mu.L of an AGR-AMC fluorogenic peptide substrate, diluted to a
final assay concentration of 60 .mu.M in Indirect Assay Buffer, was
added the wells. Fluorescence was measured using a Molecular
Devices SpectraMax.RTM.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
[0732] 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 .mu.L of 2.times. Indirect Assay Buffer was added to all
test wells. 35 .mu.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. 10 .mu.L of each of the Peptide
Substrates (Ac-SLGR-ACC (final assay concentration=125 .mu.M)) and
Ac-RQAR-ACC (final assay concentration=60 uM)) was diluted with
1.times. 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.RTM.
M5 Microplate Reader (Molecular Devices), using the kinetic read
mode.
Example 11
Production, Selection, Assessment and Identification of MT-SP1
Mutants
[0733] A. Fluorogenic Assay of B-Chain MT-SP1 Mutants from Phagemid
Library
[0734] 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.times.10.sup.9 recombinants.
[0735] 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, 1.times.10.sup.12 recombinant
phages were mixed with 3.3 nM variant AT3 carrying an SLGRKI 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.
[0736] 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).
[0737] 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, 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-SP1 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-US-00023 TABLE 21 Screening of mutant MT-SP1 protease
domain-bearing phage selected for cleavage rate of AT3-SLGRKI
Mutant MT- Ac-SLGR- Ac-RQAR- SP1 Clone ACC Rate ACC 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 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 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
[0738] 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 10B above. For this
method, individual clones were mixed with XL-1Blue E. coli cells
for infection and the cultures grown overnight shaking at
37.degree. 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.
[0739] 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-US-00024 TABLE 22 Selected MT-SP1-mutants SEQ SEQ Mutant MT-
ID NO ID NO SP1 Clone Amino Acid Mutation (protease (full- Number
(Chymotrypsin Numbering) domain).: length).: CPC-0019595
C122S/I136T/N164D/T166A/ 516 537 F184(A)L/D217V CPC-0023085
I41F/C122S 517 538 CPC-0024153 I41F/C122S/A126T/V244G 518 539
CPC-0025366 D23E/I41F/T98P/C122S/ 519 540 T144I CPC-0025387
I41F/C122S 520 541 CPC-0025582 I41F/C122S/L171F/V244G 512 542
CPC-0025720 C122S/H143R/Q175R 522 543 CPC-0025876 I41F/C122S/L171F
523 544 CPC-0025974 C122S/R230W 524 545 CPC-0026100
I41F/C122S/I154V/V244G 525 546 CPC-0026232 I41F/L52M/C122S/V129D/
526 547 Q221(A)L CPC-0027399 F99L/C122S 527 548 CPC-0027706
F97Y/C122S/I136V/Q192H/ 528 549 S201I CPC-0027797
H71R/C122S/P131S/D217V 529 550 CPC-0028017 C122S/D217V 530 551
CPC-0028333 T65K/F93L/F97Y/C122S/ 531 552 D217V
Example 12
Preparation and Characterization of Large Quantities of Selected
Phage-Bound MT-SP1 Protease
A. Large-Scale Preparation of MT-SP1 Phage
[0740] This Example describes preparation of larger quantities of
selected MT-SP 1 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 .mu.g/mL, and
tetracycline, at a final concentration of 12 .mu.g/mL, in small
sterile Corning Orange Capped Erlenmeyer Flasks overnight. To make
glycerol stocks, 85 .mu.L 60% glycerol was added to 500 .mu.l of
each culture followed by storage at -80.degree. 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 OD600 of
0.5 was reached.
[0741] M13KO7 Helper Phage was added to the culture to yield
1E.sup.10 CFU/ml and the culture incubated for 1 hour at 37.degree.
C. Kanamycin was added to a final concentration of 30 .mu.g/mL. The
cultures with helper phage were rescued overnight by incubation at
37.degree. C. Following overnight culture, samples were centrifuged
at 6000 rpm for 15 minutes. One volume PEG/NaCl (20% PEG 8K/1.5 M
NaCl) solution was added per 5 volumes of culture. The sample then
was stirred at 4.degree. 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 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.degree. C.
B. Assay of Prepared Phage Using ACC and QF Substrates
[0742] This Example describes a fluorogenic assay used to assess
activity and specificity of the phages prepared in Example 12A.
PEG-precipitated mutant MT-SP1-bearing phage clones, prepared using
the 50 mL volume culture as described in Example 12A, were
normalized to 1E.sup.13 particles/ml. The phage were then assayed
enzymatically using the ACC fluorogenic and QF (Quenched
Fluorescence) substrates as follows. 5 .mu.L phage (at 1E.sup.13
particles/ml) was added to each well in a black Costar
polypropylene half-well microtiter plate (Corning) along with 5
.mu.l 10.times.MT-SP1 assay buffer, 35 .mu.L H.sub.2O and 5 .mu.L
substrate in a total volume of 50 .mu.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-KI, 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-SP1. 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.RTM. plate
reader was set for excitation at 380 nM and to detect emission at
460 nM, with a 435 nM cutoff. For the QF readout, the
SpectraMax.RTM. plate reader was set for excitation at 490 nM, to
detect emission at 520 nM, 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-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, as determined by sequencing, as described in
Example 11B above. RFU (relative fluorescence units) numbers
correspond to the rate of hydrolysis observed in a 60 minute
reaction at 37.degree. C. for each substrate.
TABLE-US-00025 TABLE 23 Kinetic assay of selected MT-SP1 protease
domain-bearing phage clones SEQ ID NO. SEQ ID NO. Ac-SLGR- Ac-RQAR-
Mutant MT-SP1 Amino Acid Mutation (protease (full- ACC ACC SLGR-KI
RQAR-SA Clone Number (Chymotrypsin Numbering) domain): length):
(RFU/s) (RFU/s) (RFU/s) (RFU/s) Template C122S 507 515 2.4 23.5
0.12 0.11 CPC-0028341 I41T/C122S/P173S/Q209L 531 553 10.9 56.8 0.36
0.48 CPC-0033634 F97L/C122S/F234L 533 554 6.2 37.2 0.21 0.19
CPC-0028971 C122S/Q175R 534 555 3.4 11.0 0.20 0.18 CPC-0027484
N95K/C122S 535 556 2.0 9.5 0.07 0.05 CPC-0028993 Y60(G)S/C122S 536
557 0.5 1.0 0.02 0.01 * RFU/s = Relative fluorescence units/second
(Rate of hydrolysis)
[0743] Expression of Selected MT-SP1 Mutant Proteins Using In Vitro
Translation
[0744] 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. Subcloning of MT-SP1 Sequence into a Modified IVEX Vector
[0745] In order to express MT-SP1 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 6.times.His tag was cloned into the pIVEX.2.3d RTS in
vitro translation vector (Roche; SEQ ID NO.: 559)) using the NdeI
and XhoI 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-SP1 B-chain proteins, which had been selected using
phagemid libraries as described above, assayed using fluorogenic
screening methods as described in Example 10B and 11A, and
sequenced as described in Example 11B, were subcloned into the
pIVEX.2.3d.MT-SP1 vector using the internal SphI and BsrGI
restriction sites. Phagemid selectants 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
[0746] Expression of MT-SP1 protease domains using an in vitro
translation kit, the RTS 100 E. coli Disulfide kit (Roche Applied
Science), was performed using conditions specified by the supplier
with the following optimizations: The components of the 50 .mu.l
reaction solution were modified to include 12 .mu.l of amino acid
mix, 10 .mu.l of reaction mix, 12 .mu.l of lysate, along with the
addition of 5 p. 1 of 1 M Hepes pH 8 buffer, 2.5 .mu.l 12 nM
Tween-20, 2.5 .mu.l of Protein Disulfide Isomerase (PDI), and 6
.mu.l of the chaperone RTS GroE Supplement (Roche Applied Science).
The 1 mL Feeding mix was also modified to include 168 .mu.l of
amino acid mix, 24 .mu.l of methionine, 608 .mu.l of feeding mix,
100 .mu.l of 1 M Hepes pH 8, 50 .mu.l of 12 nM Tween-20, and 50
.mu.l of water. The in vitro translation (IVT) reaction was
incubated on a plate shaker at 30.degree. C. for 18 hours.
C. Purification of His-Tagged MT-SP1
[0747] Following the in vitro translation (IVT) reaction, insoluble
protein was cleared by centrifugation and transferred to a fresh
tube. The cleared supernatant (with a volume approximately 45
.mu.l) was brought up to a final volume of 1 mL in 50 mM Sodium
Phosphate pH 7.5, 300 mM NaCl. The solution was applied to 300
.mu.l of pre-equilibrated TALON.RTM. 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 NaCl and 7.5 mM Imidazole; and
eluted with 600 .mu.l of a solution containing 50 mM Sodium
Phosphate pH 6.5, 300 mM NaCl and 75 mM Imidazole. Eluate was
dialyzed into phosphate-buffered saline with 0.1% Tween-20 (PBST)
and concentrated to 20 .mu.L. Typically, the yield of purified
protease was approximately 70%.
Example 14
Characterization of Mutated MT-SP1 Protease Domains
[0748] 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
[0749] To assess protease activity, active site titration of in
vitro-translated mutant MT-SP1 protease domains was performed on
cleared supernatant with the MT-SP1 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 1.times.MT-SP1 activity buffer and incubated with 15 nM Ecotin
in 1:2.5 serial dilutions for 1 hour at 30.degree. C. The reaction
was assessed kinetically for cleavage of 0.4 mM Ac-RQAR-ACC
substrate on a SpectraMax.RTM. 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
[0750] 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 1.times.MT-SP1 activity
buffer and incubated with 6.25 .mu.M of either the native RQAR-SL
QF substrate or the mutant RCL C2 cleavage substrate, SLGR-KI.
Cleavage was assessed using a SpectraMax.RTM. M5 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-KI and RQAR-SL. The 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 1 containing the indicated amino acid
mutations is shown in parentheses. RFU numbers indicate the
measured relative fluorescence units (rate of hydrolysis) for each
substrate.
TABLE-US-00026 TABLE 24 Kinetic assay of mutant MT-SP1 protease
domains Active Ac-SLGR- Ac-RQAR- Ac-SLGR- Ac-RQAR- Mutant MT-SP1
Amino Acid Mutation SEQ ID Site ACC ACC KI QF SA QF Clone Number
(Chymotrypsin Numbering) NO.: Conc. RFU/s RFU/s RFU/s RFU/s
CPC-0025720 C122S/H143R/Q175R 522 (543) 3.9 0.10 0.46 0.05 0.04
CPC-0025876 I41F/C122S/L171F 523 (544) 2.5 0.10 0.39 0.00 0.01
CPC-0027399 F99L/C122S 527 (548) 4.2 0.55 9.84 0.04 0.09
CPC-0027797 H71R/C122S/P131S/D217V 529 (550) 3.7 0.31 1.62 0.14
0.19 CPC-0028017 C122S/D217V 530 (551) 4.7 1.42 7.29 0.38 0.50
CPC-0028333 T65K/F93L/F97Y/C122S/D217V 531 (552) 3.6 1.05 5.97 0.37
0.51 Template C122S 507 (515) 3.5 0.22 3.58 0.05 0.06
Example 15
Expression of Selected MT-SP1 Mutant Proteins as Purified
Protein
[0751] A. Transfer of MT-SP1 Protease Domain into pQE Vector
[0752] 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 of wild-type MT-SP1 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 polymerase
chain reaction (PCR) with the pQE-Insert-F2 forward primer:
TTCACGAGACAGGCTCGTGTTGTTGGGGGCACGGAT (SEQ ID NO.: 560) and
pQE-Insert-R3 reverse primer: CAGCTAATTAAGCTTATTATACCCCAGTGTTCTCTTT
(SEQ ID NO.: 561), each carrying non-annealing 5' tails. Plasmid
pQE30-MT-SP1 without the protease domain of MT-SP1 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 run in a 10
.mu.L volume using conditions specified by the supplier. 2 .mu.l,
of the reaction mix was transformed into 50 .mu.L of E. coli
TOP10F' competent cells (Invitrogen, Carlsbad, Calif.). 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-SP1-5R: GGTTCTCGCAGGTGGTCTG
(SEQ ID NO.: 565) to verify correct transfer. These primers are
fully annealing.
B. Expression, Refolding and Purification of Mutated MT-SP1
Protease Domains
[0753] Plasmids encoding the protease domain of MT-SP1 variants in
the pQE30 vector (Qiagen) described in Example 15B above were
transformed into BL21-Gold(DE3) E. coli cells (Stratagene). Small
starter cultures containing 1 mL LB supplemented with 100 .mu.g/mL
Carbenicillin were inoculated from colonies and incubated for
between 8 and 10 hours at 37.degree. C. 100 .mu.L of this culture
was used to inoculate 50 mL of 2.times.YT medium supplemented with
100 .mu.g/mL Carbenicillin and grown overnight. In 50 mL conical
tubes (Corning), the cells were harvested by centrifugation then
lysed. Inclusion bodies (IB) were isolated with BugBuster.RTM.
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.times.g for 10 min), the supernatant
was diluted into 40 mL refolding solution, containing 1.5 M
arginine, 100 mM Tris pH 8, 150 mM NaCl, 5 mM reduced glutathione
and 50 .mu.M oxidized glutathione, in 50 mL conical tubes
(Corning). The tubes were placed horizontally on a Nutator platform
(Fisher Scientific) at 4.degree. C. for 3-4 days. The refolded, not
yet activated, MT-SP1 variants then were extensively dialyzed at
room temperature against 25 mM Tris pH 8, 25 mM NaCl for 3-4 days
Following the removal of arginine during dialysis, MT-SP1 protease
domain variants were able to activate.
[0754] Crude preparations of activated MT-SP1 protease domain
variants were then chromatographed on a 5 mL HiTrap3 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-SP1 was eluted within a 50 mL gradient to 350 mM NaCl. 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 -80.degree. C.
Example 16
Preparation of Biotinylated Mutant PAI Inhibitor Baits
[0755] 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 uPA proteases from uPA libraries. These mutant PAI
inhibitors are also useful for selection of some variant MT-SP1
proteases from MT-SP1 libraries, depending on the MT-SP1 variant
and the RCL sequence used in the PAI.
A. N-Terminal Biotinylation of 6.times.His-PAI-1
[0756] For biotinylation of 6.times.His-PAI-1 or reactive center
loop variants thereof, wild-type His-tagged PAI-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.degree. C. in 2XYT medium supplemented with 0.2%
glucose, 100 ug/mL Carbenicillin and 10 ug/mL chloramphenicol (Cm).
The active fraction of 6.times.His-PAI-1 then was purified from the
cell lysates as described (Blouse, G. E., Perron, M. J., Kvassman,
J. O., Yunus, S., Thompson, J. H., Betts, R. L., Lutter, L. C., and
Shore, J. D. (2003) Biochemistry 42(42), 12260-12272; Kvassman,
J.-O., and Shore, J. D. (1995) Fibrinolysis 9, 215-221).
[0757] 6.times.His-PAI-1 variants were preferentially biotinylated
at the N-terminus using the disulfide cleavable reagent EZ-Link
NHS-SS-PEO.sub.4-Biotin (PIERCE, Rockford, Ill. #21442). Reactions
were carried out at pH 6.2, for 4 hours, at 4.degree. 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 Tris/1.0 NaCl/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 NaCl/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.sup.-1 cm.sup.-1
(see: Kvassman, J.-O., and Shore, J. D. (1995) Fibrinolysis 9,
215-221). The extent of biotinylation was determined using the
EZ-Quant HABA/Avidn kit (PIERCE, Rockford, Ill. #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-1.sup.stab In Vivo:
[0758] This Example describes a method that was used to in vivo
biotinylate PAI. 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 PAI-1 protein (PAI-1.sup.stab, having
the sequence of amino acid residues set forth in SEQ ID NO.: 567),
which has N150H, 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 coli codon optimization.
This synthetic PAI-1.sup.stab gene was cloned into the commercial
expression vector pET21-a (Novagen, Madison, Wis.) (SEQ ID NO.:
566) using XbaI and HindIII restriction enzymes resulting in
plasmid pCAT0002 (SEQ ID NO: 619), which expressed Optimized PAI-1
(OptiPAI-1; encoded by the amino acid sequence set forth in SEQ ID
NO.: 621, in which amino acid residue 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,
Calif.) carrying the plasmid pBirA (described in Asai et al.,
(1999) J. Biol. Chem. 274:20079-20078), which overexpresses the E.
coli biotin ligase, BirA. Transformants were selected on
Luria-Bertani (LB) agar plates supplemented with 100 ug/mL
Carbenicillin and 10 .mu.g/mL chloramphenicol (Cm).
[0759] Expression of BRS-TEV-OptiPAI-1.sup.stab (SEQ ID NO.: 621)
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 and 0.1 mM D-biotin for three hours at 30.degree. C. in 2XYT
medium supplemented with 0.2% glucose, 100 .mu.g/mL Carbenicillin
and 10 .mu.g/mL chloramphenicol (Cm). The active fraction of
BRS-TEV-OptiPAI-1.sup.stab was subsequently purified from the cell
lysates as described (see: Blouse, G. E., Perron, M. J., Kvassman,
J. O., Yunus, S., Thompson, J. H., Betts, R. L., Lutter, L. C., and
Shore, J. D. (2003) Biochemistry 42(42), 12260-12272; and Kvassman,
J.-O., and Shore, J. D. (1995) Fibrinolysis 9, 215-221) or by
selection by chromatography on monomeric avidin (PIERCE, Rockford,
Ill. #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.sup.-1 cm.sup.-1 as described (Kvassman, J.-O., and
Shore, J. D. (1995) Fibrinolysis 9, 215-221).
C. In Vitro Biotinylation of V1C OptiPAI-1.sup.stab
[0760] This Example sets forth methods for N-terminal Biotinylation
of a PAI 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 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.
[0761] The N-terminal BRS-TEV sequence of OptiPAI-1, in plasmid
pCAT0002 described above, was deleted with simultaneous
introduction of V1C mutation using the QuikChange-XL mutagenesis
Kit (Stratagene, San Diego, Calif.), according to supplier
specifications, resulting in plasmid pCAT0051 (SEQ ID NO.: 623)
expressing V1C OptiPAI-1.sup.stab protein (SEQ ID NO.: 622).
Plasmid pCAT0051 was transformed into E. coli BL21(DE3) pLysS
competent cells (Stratagene, San Diego, Calif.). Transformants were
selected on Luria-Bertani (LB) agar plates supplemented with 100
ug/mL Carbenicillin and 10 ug/mL chloramphenicol (Cm).
[0762] Expression of the V1C OptiPAI-1.sup.stab 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.degree. 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 V1C OptiPAI-1.sup.stab or variant thereof
was purified from the cell lysates as described (Blouse, G. E.,
Perron, M. J., Kvassman, J. O., Yunus, S., Thompson, J. H., Betts,
R. L., Lutter, L. C., and Shore, J. D. (2003) Biochemistry 42(42),
12260-12272; and Kvassman, J.-O., and Shore, J. D. (1995)
Fibrinolysis 9, 215-221).
[0763] The V1C 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, Ill. #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-1.sup.stab was rapidly desalted on G-25 gel filtration
column, from which it was eluted into a conjugation buffer
containing 50 mM NaPi/150 mM NaCl/1 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%.
[0764] Reactions were carried out for 4 hours at 25.degree. C. and
the reaction progress followed using release of the
pyridine-2-thione leaving group at 343 nm. 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
mL cm.sup.-1 as described (Kvassman, J.-O., and Shore, J. D. (1995)
Fibrinolysis 9, 215-221). The extent of biotinylation was typically
1.0-1.2 moles of biotin per mole of PAI-1 variant using the
EZ-Quant HABA/Avidin kit and the release of pyridin-2-thione
(PIERCE, Rockford, Ill. #28005).
Example 17
Screening of MT-SP1 Variants from E. coli Culture Supernatants and
Periplasmic Extracts
[0765] 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. coli periplasmic space
or the protein from E. coli cell culture medium.
[0766] For both methods, 1 mL cultures were prepared as follows. 1
mL of 2YT medium supplemented with 100 ug/mL Carbenicillin and 12
ug/mL Tetracycline were dispensed into each well of a 96 well deep
well plate, and inoculated with 10 .mu.L of XL-1 Blue cells that
had been infected with MT-SP1 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.degree. C. with oxygen aeration until the cell density
reached 0.4-0.6 OD600 (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 20 min to pellet the cells.
A. Screening of MT-SP1 Variants from Periplasmic Preps
[0767] 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, that had been transported
into the E. coli 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 .mu.L cold phosphate buffered saline (PBS); the
suspension was transferred a 96 well PCR plate; followed by one
step freeze thawing (20 min at -80.degree. 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 20 min at 3600 rpm
at 4.degree. 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-SP1 variants using appropriate substrates as
described in Example 10, Section B.
B. Screening of MT-SP1 Variants from Supernatant Preps
[0768] 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 .mu.L of the
culture supernatant were removed and assayed using the protease
assay described in Example 10, Section B, except an additional 25
.mu.L of assay buffer was added to the reaction.
[0769] 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.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130164820A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20130164820A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References