U.S. patent application number 11/584776 was filed with the patent office on 2007-04-26 for modified proteases that inhibit complement activation.
Invention is credited to Edwin L. Madison, Jack Nguyen, Sandra Waugh Ruggles, Christopher Thanos.
Application Number | 20070093443 11/584776 |
Document ID | / |
Family ID | 37686157 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070093443 |
Kind Code |
A1 |
Madison; Edwin L. ; et
al. |
April 26, 2007 |
Modified proteases that inhibit complement activation
Abstract
Provided are methods for and compounds for modulating the
complement system. In particular, compounds are provided that
inhibit complement activation and compounds are provided that
promote complement activation. The compounds are therapeutics by
virtue of their effects on the complement system. Hence, the
compounds that inhibit complement activation can be used for
treatment of ischemic and reperfusion disorders, including
myocardial infarction and stroke, sepsis, autoimmune diseases,
inflammatory diseases and diseases with an inflammatory component,
including Alzheimer's Disease and other neurodegenerative
disorder.
Inventors: |
Madison; Edwin L.; (San
Francisco, CA) ; Nguyen; Jack; (San Francisco,
CA) ; Ruggles; Sandra Waugh; (Sunnyvale, CA) ;
Thanos; Christopher; (San Francisco, CA) |
Correspondence
Address: |
Stephanie Seidman;Fish & Richardson P.C.
12390 El Camino Real
San Diego
CA
92130-2081
US
|
Family ID: |
37686157 |
Appl. No.: |
11/584776 |
Filed: |
October 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60729817 |
Oct 21, 2005 |
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|
Current U.S.
Class: |
514/44R ;
435/455 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
9/10 20180101; A61P 11/06 20180101; A61P 31/04 20180101; C12Y
304/21109 20130101; A61P 37/02 20180101; A61P 37/00 20180101; A61P
13/12 20180101; A61P 29/00 20180101; C12N 9/6421 20130101; C07K
2319/00 20130101; A61P 19/02 20180101; A61K 38/49 20130101; C12N
9/6424 20130101; A61K 38/482 20130101; A61P 9/00 20180101; A61P
31/00 20180101; A61P 25/28 20180101; A61P 1/00 20180101; A61P 21/04
20180101; C12N 9/6467 20130101; A61P 25/00 20180101 |
Class at
Publication: |
514/044 ;
435/455 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/09 20060101 C12N015/09 |
Claims
1. A method of modulating complement activation, comprising
contacting a non-complement protease with any one or more target
substrates of a complement pathway, whereby a target substrate
protein is cleaved such that complement activation in a pathway
comprising the target substrate is altered.
2. The method of claim 1, wherein contacting the non-complement
protease with any one or more target substrates of a complement
pathway occurs in vitro.
3. The method of claim 1, wherein contacting the non-complement
protease with any one or more target substrates of a complement
pathway occurs in vivo.
4. The method of claim 1, wherein contacting the non-complement
protease with any one or more target substrates of a complement
pathway occurs ex vivo.
5. The method of claim 1, wherein complement activation is
inhibited.
6. The method of claim 1, wherein complement activation is
increased.
7. The method of claim 1, wherein the complement pathway is
selected from among one or more of the classical, alternative and
lectin pathways of complement.
8. The method of claim 1, wherein the target substrate is any one
or more of C1q, C2, C3, iC3, C4, iC4, C5, C6, C7, C8, C9, MBL,
Factor B, Factor D, Factor P, MASP-1, MASP-2, C1r, C1s, C4b, C4a,
C2b, C2a, C3b, C3a, Ba and Bb.
9. The method of claim 1, wherein the target substrate comprises a
sequence of amino acids set forth in any one of SEQ ID NOS: 298,
299, 300, 302, 304, 305, 306, 311, 312, 313, 314, 315, 316, 317,
318, 319, 320, 321, 322, 326, 328, 330, 332, 334, 335, 338, 340,
and 344, or comprises a fragment thereof that exhibits a complement
activity.
10. The method of claim 1 wherein the target substrate is a
ficolin.
11. The method of claim 10, wherein the target substrate comprises
a sequence of amino acids set forth in any of SEQ ID NOS:
660-662.
12. The method of claim 1, wherein the non-complement protease
comprises modifications at any one or more amino acid residues
compared to an unmodified or scaffold protease, wherein the
modified amino acid residue(s) increases one or both of specificity
for a target substrate or activity towards a target substrate.
13. The method of claim 12, wherein the unmodified or scaffold
protease is any one of a serine protease, a cysteine protease, an
aspartic protease, a threonine protease, or a metallo-protease.
14. The method of claim 13, wherein the scaffold protease is
selected from among proteases selected from among granzyme B,
granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil elastase,
chymase, alpha-tryptase, beta-trypsase I or II, chymotrypsin,
collagenase, factor XII, factor XI, factor CII, factor X, thrombin,
protein C, u-plasminogen activator (u-PA), t-plasminogen activator
(t-PA), plasmin, plasma kallikrein, chymotrypsin, trypsin, a
cathepsin, papain, cruzain, a metalloprotease and allelic
variations, isoforms and catalytically active portions thereof.
15. The method of claim 14, wherein the scaffold protease comprises
a sequence of amino acids set forth in any one of SEQ ID NOS: 2, 4,
8, 77, 79, 83, 85, 87, 89, 93, 99, 117, 119, 121, 123, 132, 134,
138, 142, 144, 146, 148, 162, 166, 168, 170, 172, 174, 176, 178,
180, 182, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 218,
220, 222, 224, 226, 238, 248, 250, 260, 262, 280, 282, 373, 375,
377, 379, 381, 383, 385, 387, 547, 549, and 551 and catalytically
active portions thereof.
16. The method of claim 14, wherein the scaffold protease is an
MT-SP1 protease.
17. The method of claim 16, wherein an MT-SP1 protease or a
catalytically active portion thereof comprises a sequence of amino
acids set forth in SEQ ID NO: 2 or 10, or is an allelic or species
variant thereof.
18. The method of claim 16, wherein the target substrate is C2 or
C3.
19. The method of claim 16, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from a protease having a modification in position 224
and/or position 146, based on chymotrypsin numbering.
20. The method of claim 18, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from a protease having a modification in position 224
and/or position 146, based on chymotrypsin numbering.
21. The method of claim 16, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from a protease having a modification in position 151,
based on chymotrypsin numbering.
22. The method of claim 18, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from a protease having a modification in position 151,
based on chymotrypsin numbering.
23. The method of claim 21, wherein the modification in an MT-SP1
protease or catalytically active portion thereof correspond to a
protease having a modification selected from among
I41T/Y146D/G151L/K224F, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R, AND
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146D/G151L/K224N,
Y146D/G151L/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N based on chymotrypsin numbering.
24. The method of claim 16, wherein the modifications in the MT-SP1
protease or a catalytically active portion thereof are in any one
or more amino acids that contribute to extended substrate
specificity or secondary sites of interaction.
25. The method of claim 24, wherein the modifications in a MT-SP1
protease or a catalytically active portion thereof correspond to
any one or more of amino acid positions 97, 146, 192, and 224 of an
MT-SP1 protease, based on chymotrypsin numbering.
26. The method of claim 25, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
correspond to any one or more of amino acids F97, Y146, Q192, and
K224 of the MT-SP1 protease, based on chymotrypsin numbering.
27. The method of claim 26, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from any one or more of F97D, F97E, F97A, F97W, Y146N,
Y146D, Y146E, Y146A, Y146W, Y146R, Q192R, Q192V, K224A, and
K224F.
28. The method of claim 16, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
corresponds to a modified MT-SP1 polypeptide having a sequence of
amino acids as set forth in any of SEQ ID NOS: 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69, 404-418, 419-447,
524-533, 552-659, or 663-710.
29. The method of claim 27, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
Y146D/K224F or Y146E.
30. The method of claim 28, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
correspond to a modified MT-SP1 polypeptide having a sequence of
amino acids as set forth in SEQ ID NOS: 596 or 650.
31. The method of claim 16, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from among F97N, F97D, F97E, F99Y, F99V, F99W, D217A,
D217V, F97A, F97W, F99A, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R,
W215F, W215Y, Q192V, Q192R, Q192F, K224A, K224F, M180E,
Y146D/K224F, D96A, Y146E/K224N, I41T/Y146E/Q175D/K224R,
I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N,
I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F,
I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Q221aD,
Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R,
I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/L224L,
G147E, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175L/K224L,
Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A,
Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y,
Y146D/Q175F/K224L, Y146D/Q175F/K225L, Y146D/Q221aL/K224S,
I41E/Y146D/K224L, Y146D/D217F/K224L, Y146D/D217F/K224L,
H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T,
I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F,
I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F,
I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F;
I41T/Y146D/Q175D/K224F, Q192F/K224F, Y146D/Q192A/K224F,
Q192V/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R,
I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R,
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F,
I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N,
Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N,
Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering.
32. The method of claim 31, wherein the MT-SP1 protease cleaves a
target substrate at a substrate recognition site.
33. The method of claim 32, wherein the target substrate is C2 or
C3.
34. The method of claim 33, wherein the target substrate is C2 and
the substrate recognition site includes a sequence of amino acids
of SLGR (SEQ ID NO:392).
35. The method of claim 1, wherein the non-complement protease
cleaves a substrate recognition site of a target substrate.
36. The method of claim 35, wherein the non-complement protease
cleaves a Factor I substrate recognition site.
37. A method for treating a subject with a complement-mediated
disorder, comprising administering a non-complement protease,
whereby the non-complement protease cleaves any one or more target
substrates of a complement pathway such that complement activation
in a pathway comprising the target substrate is altered.
38. The method of claim 37, wherein complement activation is
inhibited.
39. The method of claim 38, wherein the inhibition of complement
activation leads to a reduction of inflammatory symptoms associated
with a complement-mediated disorder selected from among an
inflammatory disorder, a neurodegenerative disorder and a
cardiovascular disorder.
40. The method of claim 39, wherein the complement-mediated
disorder is selected from among 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), and Ischemia-reperfusion injury.
41. The method of claim 39, wherein the complement-mediated
disorder is Guillan-Barre syndrome.
42. The method claim 40, wherein the ischemia-reperfusion injury is
caused by an event or treatment selected from among myocardial
infarct (MI), stroke, angioplasty, coronary artery bypass graft,
cardiopulmonary bypass (CPB), and hemodialysis.
43. The method of claims 37, wherein the complement-mediated
disorder results from a treatment of a subject.
44. The method of claim 43, wherein administering a non-complement
protease is effected prior to treatment of a subject.
45. The method of claim 37, wherein administering a non-complement
protease is effected by contacting a body fluid or tissue sample in
vitro, ex vivo, or in vivo with a non-complement protease.
46. The method of claim 43, wherein the treatment results in
complement-mediated ischemia-reperfusion injury.
47. The method of claim 46, wherein the treatment is angioplasty or
coronary artery bypass graft.
48. The method of claim 37, wherein the complement pathway is one
or more of the classical, alternative and lectin pathways.
49. The method of claim 37, wherein the target substrate is any one
or more of C1q, C2, C3, iC3, C4, iC4, C5, C6, C7, C8, C9, MBL,
Factor B, Factor D, Factor P, MASP-1, MASP-2, C1r, C1s, C4b, C4a,
C2b, C2a, C3b, C3a, Ba and Bb.
50. The method of any of claim 37, wherein the target substrate
comprises a sequence of amino acids set forth in any one of SEQ ID
NOS: 298, 299, 300, 302, 304, 305, 306, 311, 312, 313, 314, 315,
316, 317, 318, 319, 320, 321, 322, 326, 328, 330, 332, 334, 335,
338, 340, or 344, or comprises a fragment thereof that exhibits a
complement activity.
51. The method of claims 37, wherein the target substrate is
ficolin.
52. The method of claim 49, wherein the non-complement protease
comprises modifications at any one or more amino acid residues
compared to an unmodified or scaffold protease, wherein the
modified amino acid residue(s) increases one or both of specificity
for a target substrate or activity towards a target substrate.
53. The method of claim 52, wherein the unmodified or scaffold
protease is any one of a serine protease, cysteine protease,
aspartic protease, threonine protease, or metallo-protease.
54. The method of claim 53, wherein the scaffold protease is is
selected from among proteases selected from among granzyme B,
granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil elastase,
chymase, alpha-tryptase, beta-trypsase I or II, chymotrypsin,
collagenase, factor XII, factor XI, factor CII, factor X, thrombin,
protein C, u-plasminogen activator (u-PA), t-plasminogen activator
(t-PA), plasmin, plasma kallikrein, chymotrypsin, trypsin, a
cathepsin, papain, cruzain, a metalloprotease and allelic
variations, isoforms and catalytically active portions thereof.
55. The method of claim 54, wherein the scaffold protease comprises
a sequence of amino acids as set forth in any one of SEQ ID NOS: 2,
4, 8, 77, 79, 83, 85, 87, 89, 93, 99, 117, 119, 121, 123, 132, 134,
138, 142, 144, 146, 148, 162, 166, 168, 170, 172, 174, 176, 178,
180, 182, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 218,
220, 222, 224, 226, 238, 248, 250, 260, 262, 280, 282, 373, 375,
377, 379, 381, 383, 385, 387, 547, 549, and 551 and catalytically
active portions thereof.
56. The method of claim 54, wherein the scaffold protease is an
MT-SP1 protease.
57. The method of claim 56, wherein an MT-SP1 protease or a
catalytically active portion thereof comprises a sequence of amino
acids set forth in SEQ ID NO: 2 or 10, or is an allelic or species
variant thereof.
58. The method of claim 56, wherein the target substrate is C2 or
C3.
59. The method of claim 56, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof comprises
a modification in position 224, based on chymotrypsin
numbering.
60. The method of claim 58, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof comprises
a modification in position 224, based on chymotrypsin
numbering.
61. The method of claim 56, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof comprises
a modification in position 151, based on chymotrypsin
numbering.
62. The method of claim 58, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof comprises
a modification in position 151, based on chymotrypsin
numbering.
63. The method of claim 51, wherein the modification in an MT-SP1
protease or catalytically active portion thereof is selected from
among I41T/Y146D/G151L/K224F, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R, AND
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146D/G151L/K224N,
Y146D/G151L/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N based on chymotrypsin numbering.
64. The method of claim 56, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are in
any one or more amino acids that contribute to extended substrate
specificity or secondary sites of interaction.
65. The method of claim 64, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
correspond to any one or more of amino acid positions 97, 146, 192,
and 224 of an MT-SP1 protease, based on chymotrypsin numbering.
66. The method of claim 65, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
correspond to any one or more of amino acids F97, Y146, Q192, and
K224 of the MT-SP1 protease, based on chymotrypsin numbering.
67. The method of claim 66, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from any one or more of F97D, F97E, F97A, F97W, Y146N,
Y146D, Y146E, Y146A, Y146W, Y146R, Q192R, Q192V, K224A, and
K224F.
68. The method of claim 56, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
corresponds to a modified MT-SP1 polypeptide having a sequence of
amino acids as set forth in any of SEQ ID NOS: 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69, 404-418, 419-447,
524-533, 552-659, or 663-710.
69. The method of claim 67, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
Y146D/K224F or Y146E.
70. The method of claim 68, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof
correspond to a modified MT-SP1 polypeptide having a sequence of
amino acids as set forth in SEQ ID NO:596 or 650.
71. The method of claim 56, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from among F97N, F97D, F97E, F99Y, F99V, F99W, D217A,
D217V, F97A, F97W, F99A, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R,
W215F, W215Y, Q192V, Q192R, Q192F, K224A, K224F, M180E,
Y146D/K224F, D96A, Y146E/K224N, I41T/Y146E/Q175D/K224R,
I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N,
I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F,
I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Q221aD,
Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R,
I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/L224L,
G147E, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175L/K224L,
Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A,
Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y,
Y146D/Q175F/K224L, Y146D/Q175F/K225L, Y146D/Q221aL/K224S,
I41E/Y146D/K224L, Y146D/D217F/K224L, Y146D/D217F/K224L,
H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T,
I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F,
I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F,
I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F,
I41T/Y146D/Q175D/K224F, Q192F/K224F, Y146D/Q192A/K224F,
Q192V/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R,
I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R,
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F,
I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N,
Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N,
Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering.
72. The method of claim 71, wherein the MT-SP1 protease cleaves a
substrate recognition site of the target substrate.
73. The method of claim 72, wherein the target substrate is C2 or
C3.
74. The method of claim 73, wherein the target substrate is C2 and
the substrate recognition site includes a sequence of amino acids
of SLGR (SEQ ID NO:392).
75. The method of claim 37, wherein the non-complement protease
cleaves a substrate recognition site of the target substrate.
76. The method of claim 75, wherein the non-complement protease
cleaves a Factor I substrate recognition site.
77. The method of claim 37, wherein the non-complement protease or
a catalytically active portion thereof is administered in
combination with a second agent for treating a complement-mediated
disorder.
78. The method of claim 77, wherein the second agent is an
anti-inflammatory agent or an anticoagulant.
79. The method of claim 78, wherein the second agent is selected
from among any one or more of a NSAID, antimetabolite,
corticosteroid, analgesic, cytotoxic agent, pro-inflammatory
cytokine inhibitor, anti-inflammatory cytokines, B cell targeting
agents, compounds targeting T antigens, adhesion molecule blockers,
chemokines receptor antagonists, kinase inhibitors, PPAR-.gamma.
ligands, complement inhibitors, heparin, warfarin, acenocoumarol,
phenindione, EDTA, citrate, oxalate, argatroban, lepirudin,
bivalirudin, and ximelagatran.
80. A combination, comprising: (a) a non-complement protease that
cleaves any one or more complement target substrates of a
complement pathway such that complement activation in a pathway
comprising the target substrate is altered; and (b) a second agent
or agents for treating a complement-mediated disorder.
81. The combination of claim 80, wherein the second agent or agents
is an anti-inflammatory agent(s) or anticoagulant(s).
82. The combination of claim 81 wherein, the anti-inflammatory
agent(s) is selected from among any one or more of a NSAID,
antimetabolite, corticosteroid, analgesic, cytotoxic agent,
pro-inflammatory cytokine inhibitor, anti-inflammatory cytokines, B
cell targeting agents, compounds targeting T antigens, adhesion
molecule blockers, chemokines receptor antagonists, kinase
inhibitors, PPAR-.gamma. ligands, complement inhibitors, heparin,
warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate,
argatroban, lepirudin, bivalirudin, and ximelagatran.
83. The combination of claim 80, wherein complement activation is
inhibited.
84. A modified non-complement protease, comprising modifications in
any one or more amino acids of a scaffold protease, wherein: the
modified amino acid residue(s) increases one or both of specificity
for a target substrate or activity towards a target substrate,
wherein the target substrate is a complement protein.
85. The modified non-complement protease of claim 84, wherein the
target substrate is any one or more of C1q, C2, C3, iC3, C4, iC4,
C5, C6, C7, C8, C9, MBL, Factor B, Factor D, Factor P, MASP-1,
MASP-2, C1r, C1s, C4b, C4a, C2b, C2a, C3b, C3a, Ba, or Bb.
86. The modified non-complement protease of claim 84, wherein the
target substrate comprises a sequence of amino acids set forth in
any one SEQ ID NOS: 298, 299, 300, 302, 304, 305, 306, 311, 312,
313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 326, 328, 330,
332, 334, 335, 338, 340, and 344, or comprises a fragment thereof
that exhibits a complement activity.
87. The modified non-complement protease of claim 84, wherein the
target substrate is a ficolin.
88. The modified non-complement protease of claim 87, wherein the
target substrate comprises a sequence of amino acids set forth in
any of SEQ ID NOS:660-662.
89. The modified non-complement protease of claim 84, wherein the
scaffold protease is selected from among a serine protease,
cysteine protease, aspartic protease, threonine protease, and
metallo-protease.
90. The modified non-complement protease of claim 89, wherein the
scaffold protease is selected from among granzyme B, granzyme A,
granzyme M, cathepsin G, MT-SP1, neutrophil elastase, chymase,
alpha-tryptase, beta-trypsase I or II, chymotrypsin, collagenase,
factor XII, factor XI, factor CII, factor X, thrombin, protein C,
u-plasminogen activator (u-PA), t-plasminogen activator (t-PA),
plasmin, plasma kallikrein, chymotrypsin, trypsin, a cathepsin,
papain, cruzain, a metalloprotease and allelic variations, isoforms
and catalytically active portions thereof.
91. The modified non-complement protease of claim 90, wherein the
scaffold protease comprises a sequence of amino acids as set forth
in any one of SEQ ID NOS: 2, 4, 8, 77, 79, 83, 85, 87, 89, 93, 99,
117, 119, 121, 123, 132, 134, 138, 142, 144, 146, 148, 162, 166,
168, 170, 172, 174, 176, 178, 180, 182, 190, 192, 194, 196, 198,
200, 202, 204, 206, 208, 218, 220, 222, 224, 226, 238, 248, 250,
260, 262, 280, 282, 373, 375, 377, 379, 381, 383, 385, 387, 547,
549, and 551 and catalytically active portions thereof.
92. The modified non-complement protease of claim 90, wherein the
scaffold protease is an MT-SP1 protease.
93. The modified non-complement protease of claim 92, wherein the
MT-SP1 protease or a catalytically active portion thereof comprises
a sequence of amino acids set forth in SEQ ID NO: 2 or 10.
94. The modified non-complement protease of claims 92, wherein the
target substrate is C2 or C3.
95. The modified non-complement protease of claim 92, comprising at
least two or more modifications, wherein one modification is at
position 146 and the second modification is at position 224, based
on chymotrypsin numbering, provided that: (i) where the protease
includes only two modifications, the protease does not include
Y146D and K224F as the two modification; and (ii) where the
protease contains three modifications, the protease does not
include F99V or I or L or T with Y146D and K224F.
96. The modified non-complement protease of claim 94, comprising at
least two or more modifications, wherein one modification is a
position 146 and the second is at position 224, based on
chymotrypsin numbering, provided that: (i) where the protease
includes only two modifications, the protease does not include
Y146D and K224F as the two modification; and (ii) where the
protease contains three modifications, the protease does not
include F99V or I or L or T with Y146D and K224F.
97. The modified non-complement protease of claim 92, comprising at
least one modification at position 151, based on chymotrypsin
numbering.
98. The modified non-complement protease of claim 94, comprising at
least one modification at position 151, based on chymotrypsin
numbering.
99. The modified non-complement protease of claim 97, wherein
modification(s) in an MT-SP1 or catalytically active portion are
selected from among I41T/Y146D/G151L/K224F,
I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L,
I41T/Y146D/G151L/Q175D/K224R, AND I41T/Y146D/G151L/Q175D/K224N,
I41T/Y146D/G151L/K224N, Y146D/G151L/K224N,
I41T/Y146D/G151L/Q175K/K224F, I41T/Y146D/G151L/Q175R/K224F,
I41T/Y146D/G151L/Q175H/K224F, I41T/Y146D/G151L/Q175Y/K224F,
I41T/Y146D/G151L/Q175K/K224N, I41T/Y146D/G151L/Q175R/K224N,
I41T/Y146D/G151L/Q175H/K224N, and I41T/Y146D/G151L/Q175Y/K224N
based on chymotrypsin numbering.
100. The modified non-complement protease of claim 99, wherein
modification(s) in an MT-SP1 or catalytically active portion
correspond to modification of I41T/I46D/G151L/K224F, based on
chymotrypsin numbering.
101. The modified non-complement protease of claim 92, wherein
modification(s) in an MT-SP1 protease or a catalytically active
portion thereof are selected from any one or more of D96A, D96V,
D96F, D96F, D96S, D96T, F99S, F99G, Q174H, Q174A, Q174V, Q174F,
Q174R, Q174K, Q174L, Q174Y, Q192L, Q192I, Q192E, Q192K, Q192Y,
D217Q, D217N, D217H, K224A, based on chymotrypsin numbering.
102. The modified non-complement protease of claim 92, wherein
modification(s) in an MT-SP1 protease or a catalytically active
portion thereof are selected from among Y146E/K224N,
I41T/Y146E/Q175D/K224R, I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N,
I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F,
I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Q221aD,
Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R,
I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/L224L,
G147E, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175L/K224L,
Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A,
Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y,
Y146D/Q175F/K224L, Y146D/Q175F/K225L, Y146D/Q221aL/K224S,
I41E/Y146D/K224L, Y146D/D217F/K224L, Y146D/D217F/K224L,
H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T,
I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F,
I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F,
I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F,
I41T/Y146D/Q175D/K224F, Q192F/K224F, Y146D/Q192A/K224F,
Q192V/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R,
I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R,
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F, and
I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N,
Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N,
Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering.
103. The modified non-complement protease of claim 92, wherein the
modification(s) in an MT-SP1 protease or a catalytically active
portion thereof correspond to a modified MT-SP1 polypeptide having
a sequence of amino acids as set forth in any one of SEQ ID NOS:
41-51, 56, 57, 60-64, 67, 69, 419-429, 431, 434, 435, 438-442, 445,
524, 525, 527-530, 532, 533, 552-659, or 663-710.
104. The modified non-complement protease of claim 92, wherein the
modification(s) in an MT-SP1 protease or a catalytically active
portion thereof correspond to a modified MT-SP1 polypeptide having
a sequence of amino acids as set forth in any one of SEQ ID NOS:
596 or 650.
105. The modified non-complement protease of claim 103, wherein the
MT-SP1 protease cleaves a substrate recognition site of the target
substrate.
106. The modified non-complement protease of claim 105, wherein the
target substrate is C2 or C3.
107. The modified non-complement protease of claim 106, wherein the
target substrate is C2 and the substrate recognition site includes
a sequence of amino acids of SLGR (SEQ ID NO:392).
108. A pharmaceutical composition, comprising a modified
non-complement protease of claim 84.
109. A combination, comprising: (a) a pharmaceutical composition of
claim 108; and (b) a second agent or agents for treating a
complement-mediated disorder.
110. The pharmaceutical composition of claim 108, further
comprising a pharmaceutically acceptable excipient.
111. The pharmaceutical composition of 110, wherein the
pharmaceutical composition is formulated for systemic, oral, nasal,
pulmonary, local, or topical administration.
112. A kit, comprising the pharmaceutical composition of claim 108,
a device for administration of the composition and, optionally,
instructions for administration.
113. A nucleic acid molecule, comprising a sequence of nucleotides
that encodes any one of the modified non-complement proteases of
claim 84.
114. A vector, comprising the nucleic acid molecule of claim
113.
115. A cell, comprising the vector of claim 114.
116. A method of treatment, comprising administering to a subject a
nucleic acid molecule of claim 113.
117. The method of treatment of claim 116, wherein the nucleic acid
molecule is introduced into a vector for administration.
118. The method of treatment of claim 117, wherein the vector is an
expression vector.
119. The method of treatment of claim 118, wherein the vector is
episomal.
120. The method of treatment of claim 117, wherein the expression
vector is selected from among an adenovirus vector, an
adeno-associated virus vector, EBV, SV40, cytomegalovirus vector,
vaccinia virus vector, herpesvirus vector, a retrovirus vector, a
lentivirus vector, or an artificial chromosome.
121. The method of treatment of claims 117, wherein the nucleic
acid is administered in vivo or ex vivo.
122. The method of treatment of claim 121, wherein ex vivo
treatment comprises administering the nucleic acid into a cell in
vitro, followed by administration of the cell into the subject.
123. The method of treatment of claim 122, wherein the cell is from
a suitable donor or from the subject to be treated.
124. The method of treatment of claim 116, wherein the subject is a
human.
125. A fusion protein, comprising a catalytically active portion of
a protease of claim 84 that is fused to a non-protease
polypeptide.
126. The method of claim 16, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from a protease having a modification in position 41,
based on chymotrypsin numbering.
127. The method of claim 18, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof are
selected from a protease having a modification in position 41,
based on chymotrypsin numbering.
128. The method of claim 56, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof comprises
a modification in position 41, based on chymotrypsin numbering.
129. The method of claim 58, wherein the modification(s) in an
MT-SP1 protease or a catalytically active portion thereof comprises
a modification in position 41, based on chymotrypsin numbering.
130. The modified non-complement protease of claim 92, comprising
at least one modification at position 41, based on chymotrypsin
numbering.
131. The modified non-complement protease of claim 94, comprising
at least one modification at position 41, based on chymotrypsin
numbering.
Description
RELATED APPLICATIONS
[0001] Benefit of priority is claimed to U.S. provisional
application Ser. No. 60/729,817, filed Oct. 21, 2005, entitled
"MODIFIED PROTEASES THAT INHIBIT COMPLEMENT ACTIVATION," to Edwin
Madison. The subject matter of this application is incorporated by
reference in it entirety.
[0002] This application is related to International PCT application
No. (Attorney Docket No. 19049-003WO1/4903PC), filed Oct. 20, 2006,
entitled "MODIFIED PROTEASES THAT INHIBIT COMPLEMENT ACTIVATION,"
to Edwin Madison, Jack Nguyen, Sandra Waugh Ruggles and Christopher
Thanos, which also claims priority to U.S. Provisional Application
Ser. No. 60/729,817. This application also is related to U.S.
application Ser. No. 10/677,977, filed Oct. 02, 2003, entitled
Methods of Generating and Screening for Proteases with Altered
Specificity; to U.S. application Ser. No. 11/104,110, filed Apr.
12, 2005, entitled Cleavage of VEGF and VEGF Receptor by Wild-Type
and Mutant MTSP-1; and to U.S. application Ser. No. 11/104,111,
filed Apr. 12, 2005, entitled Cleavage of VEGF and VEGF Receptor by
Wild-Type and Mutant Protease.
[0003] The subject matter of each of the above-noted applications
is incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT
DISCS
[0004] 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 Oct. 20, 2006, is identical, 2,075
kilobytes in size, and titled 4903SEQ.001.txt.
FIELD OF INVENTION
[0005] Provided are methods for and compounds for modulating the
complement system. In particular, compounds are provided that
inhibit complement activation and compounds are provided that
promote complement activation. The compounds are therapeutics by
virtue of their effects on the complement system. Hence, the
compounds that inhibit complement activation can be used for
treatment of ischemic and reperfusion disorders, including
myocardial infarction and stroke, sepsis, autoimmune diseases,
inflammatory diseases and diseases with an inflammatory component,
including Alzheimer's Disease and other neurodegenerative
disorders.
BACKGROUND
[0006] The complement (C) system is part of the immune system and
plays a role in eliminating invading pathogens and in initiating
the inflammatory response. The complement system of humans and
other mammals involves more than 30 soluble and membrane-bound
proteins that participate in an orderly sequence of reactions
resulting in complement activation. The blood complement system has
a wide array of functions associated with a broad spectrum of host
defense mechanisms including anti-microbial and anti-viral actions.
Products derived from the activation of C components include the
non-self recognition molecules C3b, C4b and C5b, as well as the
anaphylatoxins C3a, C4a and C5a that influence a variety of
cellular immune responses. These anaphylatoxins also act as
pro-inflammatory agents.
[0007] The complement system is composed of an array of enzymes and
non-enzymatic proteins and receptors. Complement activation occurs
by one of three primary modes known as the "classical" pathway, the
"alternative" pathway and the "lectin" pathway (see FIG. 1). These
pathways can be distinguished by the process that initiates
complement activation. The classical pathway is initiated by
antibody-antigen complexes or aggregated forms of immunoglobulins;
the alternative pathway is initiated by the recognition of
structures on microbial and cell surfaces; and the lectin pathway,
which is an antibody-independent pathway, is initiated by the
binding of mannan binding lectin (MBL, also designated mannose
binding protein) to carbohydrates such as those that are displayed
on the surface of bacteria or viruses. Activation of the cascades
results in production of complexes involved in proteolysis or cell
lysis and peptides involved in opsonization, anaphylaxis and
chemotaxis.
[0008] The complement cascade, which is a central component of an
animal's immune response, is an irreversible cascade. Numerous
protein cofactors regulate the process. Inappropriate regulation,
typically inappropriate activation, of the process is a facet of or
can occur in a variety of disorders that involve inappropriate
inflammatory responses, such as those observed in acute and chronic
inflammatory diseases. These diseases and disorders include
autoimmune diseases, such as rheumatoid arthritis and lupus,
cardiac disorders and other inflammatory diseases, such as sepsis
and ischemia-reperfusion injury.
[0009] Because of the involvement of the complement pathways in a
variety of diseases and conditions, components of the complement
pathways are targets for therapeutic intervention, particularly for
inhibition of the pathway. Examples of such therapeutics include
synthetic and natural small molecule therapeutics, antibody
inhibitors, and recombinant soluble forms of membrane complement
regulators. There are limitations to strategies for preparing such
therapeutics. Small molecules have short half-lives in vivo and
need to be continually infused to maintain complement inhibition
thereby limiting their role, especially in chronic diseases.
Therapeutic antibodies result in an immune response in a subject,
and thus can lead to complications in treatment, particularly
treatments designed to modulate immune responses. Thus, there
exists an unmet need for therapeutics for treatment of
complement-mediated diseases and diseases in which complement
activation plays a role. These include acute and chronic
inflammatory diseases. Accordingly, among the objects herein, it is
an object to provide such therapeutics to target the activation of
the complement cascade and to provide therapeutics and methods of
treatment of diseases.
SUMMARY
[0010] Provided herein are therapeutics and methods that target the
activation of the complement cascade and methods of treatment of
diseases, including acute and chronic inflammatory diseases. The
therapeutics are non-complement proteases that target complement
pathway substrates. Included among the non-complement proteases are
unmodified proteases that cleave their native substrate as well as
a complement substrate and also proteases modified to have
increased selectivity or substrate specificity for a target
substrate. The modified proteases can exhibit reduced or altered
activity with respect to their native substrates.
[0011] Among the methods provided herein are methods of modulating
complement activation by contacting a non-complement protease with
any one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 20, 25, 30 or more target substrates of a complement
pathway, whereby a target substrate protein is cleaved such that
complement activation in a pathway comprising the target substrate
is altered. Uses of proteases for treatment and/or for forumation
of medicaments also are provided. Target substrates for these
methods and for any of the methods and uses provide herein are
complement proteins, including: C1q, C2, C3, iC3, C4, iC4, C5, C6,
C7, C8, C9, MBL, Factor B, Factor D, Factor P, MASP-1, MASP-2, C1r,
C1s, C4b, C4a, C2b, C2a, C3b, C3a, Ba, Bb and ficolin. Contacting
can be effected ex vivo, in vitro and/or in vivo. Exemplary targets
include any of those having a sequence of amino acids set forth in
any of SEQ ID NOS: 298, 299, 300, 302, 304, 305, 306, 311, 312,
313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 326, 328, 330,
332, 334, 335, 338, 340, 344, 660-662 and a fragment of any of the
targets that exhibits a complement pathway activity, or allelic or
species variants thereof or polypeptides having 60, 70, 80, 85, 90,
95, 96, 97, 98, 99% or more sequence identity.
[0012] For all and for any methods and uses provided herein, the
target substrates can be present in a body fluid or tissue sample,
or can be a collection of target substrates or any other
composition containing such substrates. Depending upon the target
substrate(s), complement activation can be inhibited or activated.
The methods target one or more any complement pathway. Thus, the
complement pathway modulated can be selected from among one or more
of the classical, alternative and lectin pathways of complement.
The non-complement proteases contain modifications at any one or
more amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 15, 20, 25, 30, 35 or more residues, compared to an unmodified
or scaffold protease. The modified amino acid residue(s) increases
one or both of specificity for a target substrate or activity
towards a target substrate. Exemplary unmodified or scaffold
proteases include any one of a serine protease, a cysteine
protease, an aspartic protease, a threonine protease and a
metallo-protease, such as, for example, granzyme B, granzyme A,
granzyme M, cathepsin G, MT-SP1, neutrophil elastase, chymase,
alpha-tryptase, beta-trypsase I or II, chymotrypsin, collagenase,
factor XII, factor XI, factor CII, factor X, thrombin, protein C,
u-plasminogen activator (u-PA), t-plasminogen activator (t-PA),
plasmin, plasma kallikrein, chymotrypsin, trypsin, a cathepsin,
papain, cruzain, a metalloprotease and allelic variations, isoforms
and catalytically active portions thereof. For example, the
scaffold protease comprises a sequence of amino acids set forth in
any one of SEQ ID NOS: 2, 4, 8, 77, 79, 83, 85, 87, 89, 93, 99,
117, 119, 121, 123, 132, 134, 138, 142, 144, 146, 148, 162, 166,
168, 170, 172, 174, 176, 178, 180, 182, 190, 192, 194, 196, 198,
200, 202, 204, 206, 208, 218, 220, 222, 224, 226, 238, 248, 250,
260, 262, 280, 282, 373, 375, 377, 379, 381, 383, 385, 387, 547,
549, and 551 and catalytically active portions thereof, allelic and
species variants thereof and polypeptides having 60, 70, 80, 85,
90, 95, 96, 97, 98, 99% or more sequence identity. MT-SP1 or a
fragment thereof, such as the polypeptide sequence set forth in SEQ
ID NO: 2 and 10, respectively, is exemplary of a scaffold protease.
C2 or C3 proteins of a complement pathway(s) are exemplary target
substrates of an MT-SP1 protease, modified MT-SP1 protease, or
catalytically active portions thereof.
[0013] Modification of an MT-SP1 protease or a catalytically active
portion thereof include modification(s) at positions 146, 224, 41,
and/or 151, based on chymotrypsin numbering. Such modified MT-SP1
proteases include those with any of the following modifications:
I41T/Y146D/G151L/K224F, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R, AND
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146D/G151L/K224N,
Y146D/G151L/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering. In
particular, a modified MT-SP1 contains amino acid modifications
I41T/Y146D/G151L/K224F.
[0014] The modifications can be in any one or more amino acids that
contribute to extended substrate specificity or secondary sites of
interaction, such as, for example, modifications in an MT-SP1
protease or a catalytically active portion thereof that correspond
to any one or more of amino acid positions 97, 146, 192, and 224 of
an MT-SP1 protease, based on chymotrypsin numbering. Exemplary of
such modifications are one or more of F97, Y146, Q192, and K224 of
the MT-SP1 protease, based on chymotrypsin numbering, such as F97D,
F97E, F97A, F97W, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, Q192R,
Q192V, K224A, and K224F. Exemplary of such modified MT-SP1
proteases including those polypeptides having a sequence of amino
acids set forth in any of SEQ ID NOS: 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 14, 38, and 40 and 405-418. Other examples of a
modified MT-SP1 protease or a catalytically active portion thereof
include amino acid modifications Y146D/K224F or Y146E, such as
those corresponding to a modified MT-SP1 polypeptide having a
sequence of amino acids as set forth in SEQ ID NOS:12, 404, 28 or
412.
[0015] MT-SP1 protease and catalytically active portion thereof
include polypeptides that contain one or more of the following
modifications: F97N, F97D, F97E, F99Y, F99V, F99W, D217A, D217V,
F97A, F97W, F99A, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, W215F,
W215Y, Q192V, Q192R, Q192F, K224A, K224F, M180E, Y146D/K224F, D96A,
Y146E/K224N, I41T/Y146E/Q175D/K224R, I41T/Y146D/K224F,
I41T/Y146E/Q175D/K224N, I41T/Y146E/G151L/Q175D/K224L,
Y146E/Q221aE/K224F, I41T/Y146E/G151L/Q175D/K224R,
I41T/Y146E/G151L/Q175D/K224N, Q221aD, Y146E/K224R,
Y146E/Q175D/K224N, Y146D/K224R, I41T/Y146E/G151L/Q175D/K224F,
Y146E/Q175D/K224R, Y146E/L224L, G147E, Y146D/Q175D/K224R,
Y146D/Q175L/K224L, Y146D/Q175L/K224L, Y146D/Q175W/K224L,
Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A, Y146D/Q175H/K224L,
Y146D/Q175Y/K224L, Y146E/K224Y, Y146D/Q175F/K224L,
Y146D/Q175F/K225L, Y146D/Q221aL/K224S, I41E/Y146D/K224L,
Y146D/D217F/K224L, Y146D/D217F/K224L, H143V/Y146D/K224F,
Y146E/K224F, Y146A/K224F, Y146E/K224T, I41T/Y146E/K224L,
I41F/Y146D/K224F, I41L/Y146D/K224F, I41T/Y146D/G151L/K224F,
I41A/Y146D/K224F, I41E/Y146D/K224F, I41D/Y146D/K224L,
I41D/Y146D/K224F, Y146N/K224F, I41T/Y146D/Q175D/K224F, Q192F/K224F,
Y146D/Q192A/K224F, Q192V/K224F, I41T/Y146D/Q175D/K224L,
I41T/Y146D/Q175D/K224R, I41T/Y146D/Q175D/K224N,
I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L,
I41T/Y146D/G151L/Q175D/K224R, I41T/Y146D/G151L/Q175D/K224N,
I41T/Y146E/Q175D/K224F, I41T/Y146E/Q175D/K224L,
I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N, Y146D/Q175D/K224N,
Y146D/G151L/K224N, Y146D/Q175R/K224N, Y146D/Q175K/K224N,
Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering.
Exemplary of such modified MT-SP1 proteases, or catalytically
active portions thereof, include those polypeptides having a
sequence of amino acids set forth in any of SEQ ID NOS: 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69, 404-418,
419-447, 524-533, 552-659, or 663-710. In particular, a modified
MT-SP1 protease, or a catalytically active portion thereof, is one
having a sequence of amino acids set forth in SEQ ID NOS: 596 or
650.
[0016] For all methods and uses provided herein, the modification
can be selected such that the modified protease, such as MT-SP1,
cleaves a substrate recognition site of the target substrate.
Target substrate are any of the complement pathway polypeptides
noted above and known to those of skill in the art, including for
example, C2 and/or C3. For example, where the target substrate is
C2 the substrate recognition site includes a sequence of amino
acids of SLGR (SEQ ID NO:392).
[0017] Other recognition sites targeted in the methods and uses
provided herein include a Factor I substrate recognition site, such
as LPSR (SEQ ID NO:388), SLLR (SEQ ID NO:389), or HRGR (SEQ ID NO:
390). Modifications in the MT-SP1 protease or a catalytically
active portion can correspond to any one or more of amino acid
positions 174, 217, 96, 192, 146, or 99 of an MT-SP1 protease,
based on chymotrypsin numbering, such as any one or more of amino
acids Q174, D217, D96, Q192, Y146, and F99 of a MT-SP1 protease,
based on chymotrypsin numbering. Exemplary of such modifications
are modifications selected from among one ore more of: Q174H,
D217Q, D217N, D217H, D96A, D96V, D96F, D96S, D96T, Q192L, Q192I,
Q192F, Y146F, F99A, F99V, F99S, and F99G (see, e.g., the
polypeptides having a sequence of amino acids set forth in any of
SEQ ID NOS: 41-57 or 419-435.
[0018] Another recognition site includes the sequence of amino
acids LPSR. Exemplary of a modified protease modified for
recogniztion thereof are: an MT-SP1 protease or a catalytically
active portion thereof having modifications at sites corresponding
to any one or more of amino acid positions 174, 180, 215, 192, or
99 of an MT-SP1 protease, based on chymotrypsin numbering, such as,
for example, any one or more of amino acids Q174, M180, W215, Q192,
or F99 of a MT-SP1 protease, based on chymotrypsin numbering.
Exemplary of such modifications is any one or more of Q174F, Q174V,
Q174L, Q174Y, M180E, W215F, W215Y, Q192K, Q192R, Q192Y, and F99Y
(see, e.g., modified MT-SP1 polypeptides having a sequence of amino
acids as set forth in any of SEQ ID NOS: 36, 58, 59, 61, 62, 69,
416, 436, 437, 439, 440, 447, or 524-533).
[0019] Another substrate recognition site for use in the methods
herein includes the sequence of amino acids HRGR. Exemplary of a
protease with modifications to recognize such sites are an MT-SP1
protease or a catalytically active portion thereof that has
modifications that correspond(s) to modifications at any one or
more of amino acid positions 215, 174, 217, 192 and 99 of an MT-SP1
protease, based on chymotrypsin numbering, such as, for example,
W215, Q174, D217, Q192 and F99 of an MT-SP1 protease, based on
chymotrypsin numbering. Exemplary thereof ar modification selected
from among: any one or more of W215F, W215Y, Q174A, Q174V, Q174F,
Q174R, Q174K, D217A, D217V, Q192E, F99W and F99Y (e.g., an MT-SP1
protease or a catalytically active portion thereof corresponding to
a modified MT-SP1 polypeptide containing a sequence of amino acids
as set forth in any of SEQ ID NOS: 58-69 and 436-447.
[0020] Methods for treatment of complement-mediated disorders and
disorders whose symptoms are ameliorated by modulating a complement
pathway, including one or more of the classical, alternative and
lectin pathways, are provided. In practicing the methods, one ore
more non-complement proteases is/are contacted with one ore more
target substrates, such as by administration in vitro, in vivo or
ex vivo, whereby the non-complement protease cleaves any one or
more target substrates of a complement pathway such that complement
activation in a pathway comprising the target substrate is altered.
Uses of the non-complement proteases for treatment of such diseases
and disorders and/or for formulation of medicaments for such
treatment also are provided. Modulation includes inhibition or
enhancement (increasing) complement activation. Inhibition of of
complement activation can lead to a reduction in inflammatory
symptoms associated with a complement-mediated disorder. Exemplary
of inflammatory disorders are neurodegenerative disorders and
cardiovascular disorders, such as, 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.
Complement-mediated disorders can result from a treatment of a
subject. Ischemia-reperfusion injury can be caused by an event or
treatment selected from among myocardial infarct (MI), stroke,
angioplasty, coronary artery bypass graft, cardiopulmonary bypass
(CPB), and hemodialysis.
[0021] The methods of treatment provided herein can be effected by
administering to a subject a non-complement protease effected prior
to treatment of the subject for the manifested disorder. As noted
administering can be effected by contacting a body fluid or tissue
sample in vitro, ex vivo, or in vivo with a non-complement
protease. Complement-mediated ischemia-reperfusion injury is
exemplary of such disorders. The treatment causing such disorder is
angioplasty or coronary artery bypass graft.
[0022] As noted above, in any of the methods and uses provide
herein, target substrates include one or more of C1q, C2, C3, iC3,
C4, iC4, C5, C6, C7, C8, C9, MBL, Factor B, Factor D, Factor P,
MASP-1, MASP-2, C1r, C1s, C4b, C4a, C2b, C2a, C3b, C3a, Ba, Bb and
ficolin, such as a substrate that contains a sequence of amino
acids set forth in any one of SEQ ID NOS: 298, 299, 300, 302, 304,
305, 306, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,
322, 326, 328, 330, 332, 334, 335, 338, 340, or 344, or is fragment
thereof that exhibits a complement activity.
[0023] In these methods and uses and all methods and uses provided
herein, the non-complement protease can include modifications at
any one or more amino acid residues compared to an unmodified or
scaffold protease, wherein the modified amino acid residue(s)
increases one or both of specificity for a target substrate or
activity towards a target substrate. Unmodified or scaffold
protease include any one of a serine protease, cysteine protease,
aspartic protease, threonine protease, or metallo-protease, such as
granzyme B, granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil
elastase, chymase, alpha-tryptase, beta-trypsase I or II,
chymotrypsin, collagenase, factor XII, factor XI, factor CII,
factor X, thrombin, protein C, u-plasminogen activator (u-PA),
t-plasminogen activator (t-PA), plasmin, plasma kallikrein,
chymotrypsin, trypsin, a cathepsin, papain, cruzain, a
metalloprotease and allelic variations, isoforms and catalytically
active portions thereof. Exemplary are those that contain or have a
sequence of amino acids as set forth in any one of SEQ ID NOS: 2,
4, 8, 77, 79, 83, 85, 87, 89, 93, 99, 117, 119, 121, 123, 132, 134,
138, 142, 144, 146, 148, 162, 166, 168, 170, 172, 174, 176, 178,
180, 182, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 218,
220, 222, 224, 226, 238, 248, 250, 260, 262, 280, 282, 373, 375,
377, 379, 381, 383, 385, 387, 547, 549, and 551 and catalytically
active portions thereof. MT-SP1 is an exemplary scaffold protease
provided herein and is as described above. Cleavage can be targeted
to the recognition sequences as described above. Any modified
MT-SP1 described herein can be used in the methods of treatment
including such as any MT-SP1 described above. Exemplary of modified
MT-SP1 polypeptides or catalytically active portions thereof for
use in the treatment provided herein include any having a sequence
of amino acids set forth in any of 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, 34, 36, 38, 40-69, 404-418, 419-447, 524-533, 552-659,
or 663-710. In particular, an MT-SP1 polypeptide or catalytically
active portion thereof for use in the treatments provided herein
has a sequence of amino acids set forth in SEQ ID NOS:596 or
650.
[0024] In any or all of the methods and uses provided herein, a
non-complement protease or a catalytically active portion thereof
can be administered in combination with a second agent or treatment
for treating a complement-mediated disorder or any other disorder.
The second agent or treatment can be administered simultaneously,
sequentially or intermittently with the non-complement protease.
The second agent can be administered as a separate composition or
in the same composition as the non-complement protease. Exemplary
of second agents are anti-inflammatory agent and anticoagulants,
such as, but not limited to, any one or more of an NSAID,
antimetabolite, corticosteroid, analgesic, cytotoxic agent,
pro-inflammatory cytokine inhibitor, anti-inflammatory cytokines, B
cell targeting agents, compounds targeting T antigens, adhesion
molecule blockers, chemokines receptor antagonists, kinase
inhibitors, PPAR-.gamma. ligands, complement inhibitors, heparin,
warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate,
argatroban, lepirudin, bivalirudin, and ximelagatran.
[0025] Also provided are combinations of the non-complement
proteases and other elements, such as reagents, second agents, and
devices and containers for administering the proteases and/o agents
and any other elements. The combinations can be for practicing or
effecting the methods and uses provided herein. Hence provided, for
example, are combinations of elements that include: (a) a
non-complement protease that cleaves any one or more complement
target substrates of a complement pathway such that complement
activation in a pathway comprising the target substrate is altered;
and (b) a second agent or agents for treating a complement-mediated
disorder, such as, but not limited to anti-inflammatory agent(s) or
anticoagulant(s), such as, for example, any one or more of a NSAID,
antimetabolite, corticosteroid, analgesic, cytotoxic agent,
pro-inflammatory cytokine inhibitor, anti-inflammatory cytokines, B
cell targeting agents, compounds targeting T antigens, adhesion
molecule blockers, chemokines receptor antagonists, kinase
inhibitors, PPAR-.gamma. ligands, complement inhibitors, heparin,
warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate,
argatroban, lepirudin, bivalirudin, and ximelagatran.
[0026] As noted, the combinations are for practicing or effecting
any of the methods herein for modulating a complement pathway, such
as one or more of the classical, alternative, or lectin pathways of
complement. Target substrates and scaffold proteases include any of
those set forth above. For example, scaffold proteases include any
set forth Table 14, and allelic variations, isoforms and
catalytically active portions of the proteases set forth in Table
14. Exemplary of such proteases, include any that has or contains a
sequence of amino acids set forth in any one of SEQ ID NOS: 2, 4,
8, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101,
103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,
128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,
154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,
180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,
206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,
232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,
258, 260, 262, 264, 266, 268, 269, 270, 272, 274, 276, 278, 280,
282, 284, 286, 287, 289, 291, 293, 295, 297, 373, 375, 377, 379,
381, 383, 385, 387, 544, 545, 547, 549, and 551, or a catalytically
active portion thereof or an allelic or species variant thereof. In
some examples, the protease used in the combinations is an MT-SP1
or catalytically active portion thereof, such as an MT-SP1 set
forth in SEQ ID NO: 2 or 10, and the variants noted above. As noted
above, MT-SP1 protease and catalytically active portion thereof
include polypeptides that contain one or more of the following
modifications: F97N, F97D, F97E, F99Y, F99V, F99W, D217A, D217V,
F97A, F97W, F99A, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, W215F,
W215Y, Q192V, Q192R, Q192F, K224A, K224F, M180E, Y146D/K224F, D96A,
Y146E/K224N, I41T/Y146E/Q175D/K224R, I41T/Y146D/K224F,
I41T/Y146E/Q175D/K224N, I41T/Y146E/G151L/Q175D/K224L,
Y146E/Q221aE/K224F, I41T/Y146E/G151L/Q175D/K224R,
I41T/Y146E/G151L/Q175D/K224N, Q221aD, Y146E/K224R,
Y146E/Q175D/K224N, Y146D/K224R, I41T/Y146E/G151L/Q175D/K224F,
Y146E/Q175D/K224R, Y146E/L224L, G147E, Y146D/Q175D/K224R,
Y146D/Q175L/K224L, Y146D/Q175L/K224L, Y146D/Q175W/K224L,
Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A, Y146D/Q175H/K224L,
Y146D/Q175Y/K224L, Y146E/K224Y, Y146D/Q175F/K224L,
Y146D/Q175F/K225L, Y146D/Q221aL/K224S, I41E/Y146D/K224L,
Y146D/D217F/K224L, Y146D/D217F/K224L, H143V/Y146D/K224F,
Y146E/K224F, Y146A/K224F, Y146E/K224T, I41T/Y146E/K224L,
I41F/Y146D/K224F, I41L/Y146D/K224F, I41T/Y146D/G151L/K224F,
I41A/Y146D/K224F, I41E/Y146D/K224F, I41D/Y146D/K224L,
I41D/Y146D/K224F, Y146N/K224F, I41T/Y146D/Q175D/K224F, Q192F/K224F,
Y146D/Q192A/K224F, Q192V/K224F, I41T/Y146D/Q175D/K224L,
I41T/Y146D/Q175D/K224R, I41T/Y146D/Q175D/K224N,
I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L,
I41T/Y146D/G151L/Q175D/K224R, I41T/Y146D/G151L/Q175D/K224N,
I41T/Y146E/Q175D/K224F, I41T/Y146E/Q175D/K224L,
I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N, Y146D/Q175D/K224N,
Y146D/G151L/K224N, Y146D/Q175R/K224N, Y146D/Q175K/K224N,
Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering.
Exemplary of such modified MT-SP1 proteases, or catalytically
active portions thereof, include those polypeptides having a
sequence of amino acids set forth in any of SEQ ID NOS: 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69, 404-418,
419-447, 524-533, 552-659, or 663-710. In particular, a modified
MT-SP1 protease, or a catalytically active portion thereof, is one
having a sequence of amino acids set forth in SEQ ID NOS: 596 or
650. In some cases, the modifications in an MT-SP1 protease or a
catalytically active portion thereof include modifications of any
one or more amino acids that contribute to extended substrate
specificity or secondary sites of interaction. Exemplary of this
include, but are not limited to, any that correspond to any one or
more of amino acid positions 97, 146, 192, and 224 of an MT-SP1
protease, based on chymotrypsin numbering. Exemplary of such
modifications are modifications in any one or more of amino acids
F97, Y146, Q192, and K224 of the MT-SP1 protease, based on
chymotrypsin numbering, such as modification(s) in an MT-SP1
protease or a catalytically active portion thereof are selected
from any one or more of F97D, F97E, F97A, F97W, Y146N, Y146D,
Y146E, Y146A, Y146W, Y146R, Q192R, Q192V, K224A, and K224F (e.g.,
an MT-SP1 protease or a catalytically active portion thereof
corresponds to a modified MT-SP1 polypeptide having a sequence of
amino acids as set forth in any of SEQ ID NOS: 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 14, 38, and 40 and 405-418 and/or in an
MT-SP1 protease or a catalytically active portion thereof with
modifications at positions Y146D and K224F or Y146E, such as those
modified MT-SP1 polypeptides having a sequence of amino acids as
set forth in SEQ ID NO: 12, 404, 28 or 412).
[0027] In the combinations and methods and uses provided herein,
the non-complement protease can cleave a substrate recognition site
of the target substrate. Exemplary recognition sites are set forth
above.
[0028] Also provided are particular modified non-complement
proteases. For example provided are non-complement proteases
containing modifications in any one or more amino acids of a
scaffold protease, where the modified amino acid residue(s)
increases one or both of specificity for a target substrate or
activity towards a target substrate, where the target substrate is
a complement protein, such as any target substrate and mixture
thereof and source thereof set forth above. The modified
non-complement protease includes any suitable scaffold including
any noted above, such as a scaffold protease selected from among
granzyme B, granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil
elastase, chymase, alpha-tryptase, beta-trypsase I or II,
chymotrypsin, collagenase, factor XII, factor XI, factor CII,
factor X, thrombin, protein C, u-plasminogen activator (u-PA),
t-plasminogen activator (t-PA), plasmin, plasma kallikrein,
chymotrypsin, trypsin, a cathepsin, papain, cruzain, a
metalloprotease and allelic variations, isoforms and catalytically
active portions thereof. Target substrates include any set forth
above, such as, but not limited to C2 and/or C3.
[0029] Exemplary modified non-complement proteases include those
based on an MT-SP1 scaffold (full-length or a catalytically active
portion thereof). The MT-SP1 scaffold can include one, or at least
two or more modifications, where one modification is at position
146 and the second modification is at position 224, based on
chymotrypsin numbering, provided that: [0030] (i) where the
protease includes only two modifications, the protease does not
include Y146D and K224F as the two modification; and (ii) where the
protease contains three modifications, the protease does not
include F99V or I or L or T with Y146D and K224F. For example, such
modified non-complement protease can one containing at least two or
more modifications, where one modification is a position 146 and
the second is at position 224, based on chymotrypsin numbering,
provided that: (i) where the protease includes only two
modifications, the protease does not include Y146D and K224F as the
two modification; and [0031] (ii) where the protease contains three
modifications, the protease does not include F99V or I or L or T
with Y146D and K224F. An MT-SP1 protease, or catalytically portion
thereof, also includes a modified protein containing one, or at
least two or more modifications at position 141 and/or position 41.
Exemplary of any of the above modified MT-S P1 proteases or
catalytically active portions thereof include a modified MT-SP1
containing modifications of any of I41T/Y146D/G151L/K224F,
I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L,
I41T/Y146D/G151L/Q175D/K224R, AND I41T/Y146D/G151L/Q175D/K224N,
I41T/Y146D/G151L/K224N, Y146D/G151L/K224N,
I41T/Y146D/G151L/Q175K/K224F, I41T/Y146D/G151L/Q175R/K224F,
I41T/Y146D/G151L/Q175H/K224F, I41T/Y146D/G151L/Q175Y/K224F,
I41T/Y146D/G151L/Q175K/K224N, I41T/Y146D/G151L/Q175R/K224N,
I41T/Y146D/G151L/Q175H/K224N, and I41T/Y146D/G151L/Q175Y/K224N
based on chymotrypsin numbering.
[0032] Exemplary of such modified protease include an MT-SP1
protease or a catalytically active portion thereof selected from
any one or more of D96A, D96V, D96F, D96F, D96S, D96T, F99S, F99G,
Q174H, Q174A, Q174V, Q174F, Q174R, Q174K, Q174L, Q174Y, Q192L,
Q192I, Q192E, Q192K, Q192Y, D217Q, D217N, D217H, K224A, based on
chymotrypsin numbering. Exemplary are MT-SP1 proteases that contain
or have a sequence of amino acids as set forth in any one of SEQ ID
NOS: 41-51, 56, 57, 60-64, 67, 419-429, 431, 434, 435, 438-442, or
445.
[0033] Exemplary modified MT-SP1 proteases, or catalytically active
portion thereof, also include any having any of the following
modifications: Y146E/K224N, I41T/Y146E/Q175D/K224R,
I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N,
I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F,
I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Q221aD,
Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R,
I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/L224L,
G147E, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175L/K224L,
Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A,
Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y,
Y146D/Q175F/K224L, Y146D/Q175F/K225L, Y146D/Q221aL/K224S,
I41E/Y146D/K224L, Y146D/D217F/K224L, Y146D/D217F/K224L,
H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T,
I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F,
I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F,
I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F,
I41T/Y146D/Q175D/K224F, Q192F/K224F, Y146D/Q192A/K224F,
Q192V/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R,
I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R,
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F, and
I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N,
Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N,
Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering.
Exemplary of such proteases are any having a sequence of amino
acids set forth in any of SEQ ID NOS: 41-51, 56, 57, 60-64, 67, 69,
419-429, 431, 434, 435, 438-442, 445, 524, 525, 527-530, 532, 533,
552-659, or 663-710. In particular, an modified MT-SP1 protease or
catalytically active portion thereof has a sequence of amino acids
set forth in SEQ ID NOS: 596 or 650. Included among the modified
MT-SP1 proteases, or catalytically active protions thereof,
provided herein are those that cleave a target substrate,
typically, at a substrate recognition site in the target substrate.
Exemplary of target substrates include C2 or C3. Cleavage of C2 can
be at a substrate recognition site SLGR (SEQ ID NO:392) in C2.
[0034] Among the modified non-complement protease that contain
modifications in any one or more amino acids of a scaffold
protease, where the modified amino acid residue(s) increases one or
both of specificity for a target substrate or activity towards a
target substrate, wherein the target substrate is a complement
protein that are provided are such modified non-complement protease
that not cleave a VEGF or VEGFR or that exhibit a reduction in any
cleavage activity of a VEGF or VEGFR or that exhibit greater
substrate specificity or activity for a target substrate such as a
complement protein, than for VEGF or VEGFR with the modification
compared to without the modification. For example, the
non-complement protease cleaves a complement protein with at least
or about 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold,
50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, or more greater
specificity or activity than it cleaves VEGF or VEGFR. The target
substrates include complement proteins, such as, but not limited to
any one or more of C1q, C2, C3, iC3, C4, iC4, C5, C6, C7, C8, C9,
MBL, Factor B, Factor D, Factor P, MASP-1, MASP-2, C1r, C1s, C4b,
C4a, C2b, C2a, C3b, C3a, Ba, Bb and ficolin. Exemplary target
substrates include, but are not limited to target substrates
containing a sequence of amino acids set forth in any one of SEQ ID
NOS: 298, 299, 300, 302, 304, 305, 306, 311, 312, 313, 314, 315,
316, 317, 318, 319, 320, 321, 322, 326, 328, 330, 332, 334, 335,
338, 340 and 344 or a fragment thereof that exhibits a complement
activity.
[0035] Scaffold proteases include any protease, including, a serine
protease, cysteine protease, aspartic protease, threonine protease,
or metallo-protease, such as any set forth in Table 14, and allelic
and species variants thereof, isoforms and catalytically active
portions, and modified forms thereof that have 60, 65, 70, 75, 80,
85, 90, 95, 96, 97, 98, 99% or more sequence identity to any
provided in the Table or elsewhere herein, such as a scaffold
protease that contains a sequence of amino acids as set forth in
any one of SEQ ID NOS: 2, 4, 8, 71, 73, 75, 77, 79, 81, 83, 85, 87,
89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140,
142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,
220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,
246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 269,
270, 272, 274, 276, 278, 280, 282, 284, 286, 287, 289, 291, 293,
295, 297, 373, 375, 377, 379, 381, 383, 385, 387, 544, 545, 547,
549, and 551, and catalytically active portions thereof. The
scaffolds can be used for preparing modified proteases and in any
method or use herein. As discussed above, MT-SP1 or a catalytically
active fragment thereof is exemplary of such proteases. An
exemplary MT-SP1 protease or portion thereof has the sequence of
amino acids set forth in SEQ ID NO: 2 or 10. Modification include
any that alter substrate specificity or selectivity, particularly
those that increase substrate specificity or selectivity for a
complement protein. Exemplary of such modifications are MT-SP1
proteases or a catalytically active portions thereof with any of
the following modifications D96A, D96V, D96F, D96F, D96S, D96T,
F97D, F97E, F97A, F97W, F99A, F99S, F99G, F99W, F99Y, Y146N, Y146D,
Y146E, Y146A, Y146W, Y146R, Q174H, Q174A, Q174V, Q174F, Q174R,
Q174K, Q174L, Q174Y, M180E, Q192R, Q192V, Q192L, Q192I, Q192F,
Q192E, Q192K, Q192Y, W215F, W215Y, D217Q, D217N, D217H, D217A,
D217V, K224A, Y146E/K224N, I41T/Y146E/Q175D/K224R,
I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N,
I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F,
I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Q221aD,
Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R,
I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/L224L,
G147E, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175L/K224L,
Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A,
Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y,
Y146D/Q175F/K224L, Y146D/Q175F/K225L, Y146D/Q221aL/K224S,
I41E/Y146D/K224L, Y146D/D217F/K224L, Y146D/D217F/K224L,
H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T,
I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F,
I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F,
I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F,
I41T/Y146D/Q175D/K224F, Q192F/K224F, Y146D/Q192A/K224F,
Q192V/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R,
I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F,
I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R,
I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F, and
I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N,
Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N,
Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F,
I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F,
I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N,
I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and
I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering. and
allelic and species variants and isoforms thereof and variants with
60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence
identity therewith and including the corresponding modification.
For example, modified MT-SP1 polypeptide containing a sequence of
amino acids as set forth in any of SEQ ID NOS: 41-51, 56, 57,
60-64, 67, 69, 419-429, 431, 434, 435, 438-442, 445, 524, 525,
527-530, 532, 533, 552-659, or 663-710.
[0036] Also provided are pharmaceutical compositions containing any
of the modified non-complement proteases or elements of the
combinations provided herein. The pharmaceutical compositions
include, as needed, pharmaceutically acceptable excipients and
other components. The compositions are formulated for any desired
or suitable route of administration, including, but not limited to
systemic, oral, nasal, pulmonary, local, or topical
administration.
[0037] Kits are provided. The kits can be used in practicing the
methods. Kits containing the combinations are provided. Kits
containing the pharmaceutical compositions also are provided. The
kits also can contain devices for administration of the composition
and/or proteases and, optionally, instructions for administration
and other reagents and products employed in the methods.
[0038] Also provided are nucleic acid molecules that encode any of
the the modified non-complement proteases. Included among these are
nucleic acid molecules that encode or that hybridize under medium
or high stringency to any nucleic acid that encodes any of the
polypeptides set forth in any of SEQ ID NOS: 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69, 404-418, 419-447,
524-533, 552-659, or 663-710. Also included among the nucleic acid
molecules are those selected from among:
[0039] a) a nucleic acid molecule comprising a sequence of
nucleotides set forth in any of SEQ ID NOS: 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 451-455, 457-462, 464-479, and
534-538;
[0040] b) a nucleic acid molecule comprising at least 60, 65, 70,
75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence
identity to any of SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27,
29, 31, 33, 35, 37, 451-455, 457-462, 464-479, 534-538;
[0041] c) a nucleic acid that hybridizes under conditions of medium
or high stringency along at least 70% of its full length to a
nucleic acid molecule comprising a sequence of nucleotides set
forth in any of SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 451-455, 457-462, 464-479, 534-538;
[0042] d) a nucleic acid molecule that comprises degenerate codons
of a), b), or c); or
[0043] e) a nucleic acid molecule comprising splice variants or
allelic variants of any of SEQ ID NOS: 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 451-455, 457-462, 464-479, 534-538 or
any of a)-d), or. nucleic acid molecule is selected from among:
[0044] a) a nucleic acid molecule comprising a sequence of
nucleotides set forth in any of SEQ ID NOS: 480-493, 495-499,
501-506, 508-523, 539-543;
[0045] b) a nucleic acid molecule comprising at least 60, 65, 70,
75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence
identity to any of SEQ ID NOS: 480-493, 495-499, 501-506, 508-523,
539-543;
[0046] c) a nucleic acid that hybridizes under conditions of medium
or high stringency along at least 70% of its full length to a
nucleic acid molecule comprising a sequence of nucleotides set
forth in any of SEQ ID NOS: 480-493, 495-499, 501-506, 508-523,
539-543;
[0047] d) a nucleic acid molecule that comprises degenerate codons
of a), b), or c); or
[0048] e) a nucleic acid molecule comprising splice variants or
allelic variants of any of SEQ ID NOS: 480-493, 495-499, 501-506,
508-523, 539-543 or any of a)-d).
[0049] Vectors containing the nucleic acid molecules. Vectors
include eukaryotic and prokaryotic expression vectors, including
mammalian and yeast vectors. Cells containing the nucleic acid
molecules and/or vectors also are provided. Exemplary expression
vector include but are not limited to: an adenovirus vector, an
adeno-associated virus vector, EBV, SV40, cytomegalovirus vector,
vaccinia virus vector, herpesvirus vector, a retrovirus vector, a
lentivirus vector and an artificial chromosome. Methods for
production or preparation of the encoded non-complement proteases
are provided. The vectors or nucleic acid molecules are introduced
into cells and cultured under conditions, whereby the protease is
expressed. The nucleic acid molecule can include sequence encoded a
signal sequence to direct trafficking of the expressed protease,
such as a signal sequence for secretion. The expressed proteases
can be purified by routine methods known to those of skill in the
art.
[0050] Methods of treatment by administering to a subject a nucleic
acid molecule, vector or cell are provided. The diseases treated
include any mediated by or involving a complement protein or the
complement pathway, such as diseases with an underlying
inflammatory component or pathology. Vectors include an expression
vector that integrated into a host cell's chromosome or a vector
that remains episomal. Administration can be in vivo or ex vivo. Ex
vivo treatment includes administering the nucleic acid into a cell
in vitro, followed by administration of the cell into the subject.
The cell can be from a suitable (compatible) donor or from the
subject, such as a human, to be treated.
[0051] Also provided are fusion proteins containing a catalytically
active portion of any of the non-complement proteases hat is fused
to a non-protease polypeptide. Fusion can be by insertion into the
non-protease polypeptide or linkage at either end.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 depicts an overview of the classical, lectin, and
alternative complement pathways and the activation of the terminal
complement complex, the membrane attack complex (MAC). In
particular, the figure depicts many of the more than 30 proteins
that participate in the complement cascade, their action within the
cascade, and where applicable, their points of convergence among
the complement pathways. For example, the three pathways converge
upon the generation of a C3 convertase, which cleaves C3 to form a
C5 convertase yielding the formation of the MAC complex. The figure
also depicts the generation of many of the complement cleavage
products. All proteins depicted in the pathways can serve as
substrate targets.
DETAILED DESCRIPTION
Outline
A. Definitions
B. Target: Complement
[0053] 1. Nomenclature
[0054] 2. Pathways of Complement Initiation [0055] a. Classical
[0056] b. Alternative [0057] c. Lectin
[0058] 3. Complement-mediated effector functions [0059] a.
Complement-mediated lysis: Membrane Attack Complex [0060] b.
Inflammation [0061] c. Chemotaxis [0062] d. Opsonization [0063] e.
Activation of the Humoral Immune Response
[0064] 4. Complement Receptors
[0065] 5. Complement Regulation [0066] a. Factor I
[0067] 6. Complement-Mediated Disease [0068] a. Disease mediated by
complement activation [0069] i. Rheumatoid Arthritis [0070] ii.
Sepsis [0071] iii. Multiple Sclerosis [0072] iv. Alzheimer's
Disease [0073] v. Ischemia-Reperfusion Injury [0074] b. Disease
mediated by complement deficiency C. Proteases
[0075] 1. Classes of proteases [0076] a. Serine Proteases [0077] i.
MT-SP1 [0078] ii. Granzyme B [0079] b. Cysteine Proteases [0080] c.
Aspartic Proteases [0081] d. Metalloproteases [0082] e. Threonine
Proteases D. Scaffold Proteases
[0083] 1. Modified Scaffold Proteases [0084] a. Rational
Modification [0085] i. Synthesis of Positional Scanning Libraries
and Screening using Fluorescence [0086] b. Empirical
Modification
[0087] 2. Methods of assessing specificity
[0088] 3. Protease polypeptides [0089] a. MT-SP1 polypeptides E.
Assays to Assess or Monitor Modified Protease Activity on
Complement-Mediated Functions
[0090] a. Protein Detection [0091] i. SDS-PAGE [0092] ii. Enzyme
Immunoassay [0093] iii. Radial Immunodiffusion (RID)
[0094] b. Hemolytic assays
F. Methods of Producing Nucleic Acids Encoding Modified Proteases
and Methods of Producing Modified Protease Polypeptides
[0095] 1. Vectors and Cells
[0096] 2. Expression [0097] a. Prokaryotes [0098] b. Yeast [0099]
c. Insect cells [0100] d. Mammalian cells [0101] e. Plants
[0102] 3. Purification Techniques
[0103] 4. Fusion Proteins
[0104] 5. Nucleotide sequences
G. Methods of Using: Formulations/Packaging/Administration
[0105] 1. Administration of modified protease polypeptides
[0106] 2. Administration of nucleic acids encoding modified
protease polypeptide (gene therapy)
H. Therapeutic Uses
[0107] 1. Immune-mediated Inflammatory Disease
[0108] 2. Neurodegenerative Disease
[0109] 3. Cardiovascular Disease
I. Combination Therapies
J. Examples
A. DEFINITIONS
[0110] 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.
[0111] As used herein, MBL (mannose binding lectin) also is
designated mannose-binding protein (MBP).
[0112] As used herein, complement activation refers to the
sequential activation of serum components C1 through C9, initiated
by a variety of activators including, for example, antigen-antibody
complex, lipopolysaccharide, or microbial polysaccharides, and
producing an inflammatory response via any pathway.
[0113] As used herein, a "complement protein" or a "complement
component" is a protein of the complement system that functions in
the host defense against infections and in the inflammatory
process. Complement proteins constitute target substrates for the
proteases and modified proteases provided herein.
[0114] Complement proteins are a group of interacting blood
proteins and glycoproteins found in all vertebrates. There are at
least 30 soluble plasma proteins in addition to cell surface
receptors that bind complement reaction products and that occur on
inflammatory cells and cells of the immune system. In addition,
there are regulatory membrane proteins that protect host cells from
accidental complement attack. Complement proteins include those
that function in the classical pathway, for example, C2, those that
function in the alternative pathway, for example, Factor B, and
those that function in the lectin pathway, for example MASP-1.
Among the complement proteins are proteases that participate in the
complement pathways. In addition, as used herein, complement
proteins include any of the "cleavage products" (also referred to
as "fragments") that are formed upon activation of the complement
cascade. Also included among complement proteins are inactive or
altered forms of complement proteins, such as iC3 and
C3a-desArg.
[0115] Thus, complement proteins include, but are not limited to:
C1q, C1r, C1s, C2, C3, C3a, C3b, C3c, C3dg, C3g, C3d, C3f, iC3,
C3a-desArg, C4, C4a, C4b, iC4, C4a-desArg, C5, C5a, C5a-des-Arg,
C6, C7, C8, C9, MASP-1, MASP-2, MBL, Factor B, Factor D, Factor H,
Factor I, CR1, CR2, CR3, CR4, properdin, C1Inh, C4bp, MCP, DAF,
CD59 (MIRL), clusterin and HRF and allelic and species variants of
any complement protein.
[0116] As used herein, a "native" form of a complement protein is
one which can be isolated from an organism such as a vertebrate in
the absence of complement activation, and which has not been
intentionally modified by man in the laboratory. Examples of native
complement proteins include C1q, C1r, C1s, C2, C3, C4, Factor B,
Factor D, properdin, C5, C6, C7, C6, and C9.
[0117] Generally, native complement proteins are inactive and
acquire activity upon activation. Activation can require activation
cleavage, maturation cleavage and/or complex formation with other
proteins. An exception to this is Factor I and Factor D which have
enzymatic activity in their native form. In some examples,
activation of a native complement protein occurs following cleavage
of the protein. For example, complement zymogens such as C2 and
Factor B are proteases which are themselves activated by protease
cleavage such that cleavage of C2 by the protease C1s generates C2b
which associates with C4b to form the proteolytically active C4b2b
(C3 convertase) and cleavage of Factor B by the protease Factor D
generates Bb which associates with C3b to form the proteolytically
active alternative C3 convertase, C3bBb. In another example,
cleavage of an inactive native complement protein results in
changes in the structural stability of a protein resulting in
activation of the protein. For example, C3 and C4 contain an
internal thioester bond which in the native protein is stable, but
can become highly reactive and activated following conformational
changes that result from cleavage of the protein. Thus, the
cleavage products of C3 and C4 are biologically active. Activation
of C3 and C4 also can occur spontaneously in the absence of
cleavage. It is the spontaneous conversion of the thioester bond in
native C3 that is an initiating event of the alternative pathway of
complement. In other example, activation of a native complement
protein occurs following the release of a complexed regulatory
molecule that inhibits the activity of an otherwise active native
complement protein. For example, C1inh binds to and inactivates C1s
and C1r, unless they are in complex with C1q.
[0118] As used herein, maturation cleavage is a general term that
refers to any cleavage required for activation of a zymogen. This
includes cleavage that leads to a conformational change resulting
in activity (i.e. activation cleavage). It also includes cleavage
in which a critical binding site is exposed or a steric hindrance
is exposed or an inhibitory segment is removed or moved.
[0119] As used herein, altered form of a complement protein refers
to a complement protein that is present in a non-native form
resulting from modifications in its molecular structure. For
example, C3 reaction of the thioester with water can occur in the
absence of convertase cleavage, giving a hydrolyzed inactive form
of C3 and C4 termed iC3 and iC4. In another example, anaphylatoxins
including C3a, C5a, and C4a can be desarginated by carboxypeptidase
N into more stable, less active forms.
[0120] As used herein, a "fragment" or "cleavage product" of a
complement protein is a subset of a complement protein that
contains a portion of the polypeptide sequence of a native
complement protein. A fragment of a complement protein usually
results following the activation of any one or more, such 1, 2 or
3, of the complement cascades. Generally, a fragment results from
the proteolytic cleavage of a native complement protein. For
example, Factor B is enzymatically cleaved by Factor D, resulting
in two fragments: Ba which constitutes the N-terminal portion of B;
and Bb which constitutes the C-terminal portion and contains the
serine protease site. A fragment of a complement protein also
results from the proteolytic cleavage of another fragment of a
complement protein. For example, C3b, a fragment generated from the
cleavage of C3, is cleaved by Factor I to generate the fragments
iC3b and C3f. Generally cleavage products of complement proteins
are biologically active products and function as cleavage effector
molecules of the complement system. Hence a fragment or portion of
complement protein includes cleavage products of complement
proteins and also portions of the proteins that retain or exhibit
at least one activity.
[0121] As used herein, "cleavage effector molecules" or "cleavage
effector proteins" refers to the active cleavage products generated
as a result of the triggered-enzyme cascade of the complement
system. A cleavage effector molecule, a fragment or cleavage
product resulting from complement activation can contribute to any
of one or more of the complement-mediated functions or activities,
which include opsonization, anaphylaxis, cell lysis and
inflammation. Examples of cleavage or effector molecules include,
but are not limited to, C3a, C3b, C4a, C4b, C5a, C5b-9, and Bb.
Cleavage effector molecules of the complement system, by virtue of
participation in the cascade, exhibit activities that include
stimulating inflammation, facilitating antigen phagocytosis, and
lysing some cells directly. Complement cleavage products promote or
participate in the activation of the complement pathways.
[0122] As used herein, anaphylatoxins (such as, for example, C3a,
C4a or C5a) are cleavage effector proteins that trigger
degranulation of (release of substances from) mast cells or
basophils, which participate in the inflammatory response,
particularly as part of defense against parasites. If the
degranulation is too strong, it can cause allergic reactions.
Anaphylatoxins also indirectly mediate spasms of smooth muscle
cells (such as bronchospasms), an increase in permeability of blood
capillaries, and chemotaxis.
[0123] As used herein, chemotaxis refers to receptor-mediated
movement of leukocytes towards a chemoattractant typically in the
direction of the increasing concentration thereof, such as in the
direction of increasing concentration of an anaphylatoxin.
[0124] As used herein, opsonization refers to the alteration of the
surface of a pathogen or other particle so that it can be ingested
by phagocytes. A protein that binds or alters the surface of a
pathogen is termed an opsonin. Antibody and complement proteins
opsonize extracellular bacteria for uptake and destruction by
phagocytes such as neutrophils and macrophages.
[0125] As used herein, cell lysis refers to the breaking open of a
cell by the destruction of its wall or membrane. Hemolysis of red
blood cells is a measure of cell lysis.
[0126] 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 proteases refer 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.
[0127] 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. A number
of complement proteins are zymogens; they are inactive, but become
cleaved and activated upon the initiation of the complement system
following infection. Zymogens, generally, are inactive and can be
converted to mature active polypeptides by catalytic or
autocatalytic cleavage of the proregion from the zymogen.
[0128] 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.
[0129] As used herein, an activation sequence refers to a sequence
of amino acids in a zymogen that is 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] As used herein, a catalytically 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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 subsites are labeled (Si, . . . , S3, S2, S1, S1', S2',
S3', . . . , Sj). The cleavage is catalyzed between P1 and P1'.
[0138] 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' subsites
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; others proteases can have extended recognition beyond
P4-P2'.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] As used herein, a catalytic triad of a serine or cysteine
protease refers to a combination of 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
nucleophile 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.
[0143] 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, 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.
[0144] As used herein, target substrate refers to a substrate that
is cleaved by a protease. Typically, the target substrate 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. For example, for
purposes herein in which complement inactivation is intended, a
target substrate is any one or more, such as for example, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or more, complement proteins, or
any portion or fragment thereof of a complement protein containing
a cleavage sequence recognized by a protease. Such target
substrates can be purified proteins, or can be present in a
mixture, such as a mixture in vitro or a mixture in vivo. Mixtures
can include, for example, blood or serum, or other tissue fluids.
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. The proteases can be
modified to exhibit greater substrate specificity for a target
substrate.
[0145] 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. An activating cleavage also is cleavage
whereby a protein is cleaved into one or more proteins that
themselves have activity. For example, the complement system 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. Thus, cleavage of C3 by convertase into
C3a and C3b is an activation cleavage.
[0146] 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 complement proteins that is an inhibitory cleavage results in
the concomitant reduction or inhibition of any one or more of the
classical, lectin, or alternative functional pathways of
complement. 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.
[0147] As used herein, reference to a protease that cleaves VEGF or
a VEGFR refers to a protease that is modified to cleave VEGF or a
VEGFR or that in its native form cleaves VEGF or a VEGFR to thereby
reduce or inactivate signaling of the VEGF or VEGFR complex,
particularly cell proliferation signaling that can be manifested as
a biological effect such as angiogenesis, particular undesired
angiogenesis. Cleavage of VEGF or VEGFR by a protease can be
determined by assaying for the activity of a VEGF or VEGFR using
any method or assay known to one of skill in the art to assess VEGF
or VEGFR function.
[0148] As used herein, reference to a protease (modified or
unmodified) that does not cleave VEGF or a VEGFR refers to a
protease that does not reduce or inactivate signaling of the VEGF
or a VEGFR complex. In particular, for purposes herein, the
protease has greater substrate specificity or activity to a target
substrate (i.e. a complement protein), such as or about 1-fold,
1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold,
200-fold, 300-fold, 400-fold or more, than for a VEGF or a VEGFR
protein or a peptide substrate that contains the corresponding
cleavage sequence (i.e. RRVR). For purposes herein, comparison of
cleavage of a complement protein with a VEGF or VEGFR protein or
peptide substrate is under the same reaction conditions as a
protease cleaves a complement protein.
[0149] As used herein, the "scaffold" or "protease scaffold" refers
to a prototype protease that can be modified to alter its target
specificity. Scaffolds include wildtype proteases, allelic variants
and isoforms. They can serve as the starting material for
modification to produce a protease that has a targeted
specificity.
[0150] 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 scaffold 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 scaffold protease, or can be a catalytically active
portion thereof of a modified full length scaffold protease as long
as the modified protease contains modifications in regions that
alter the activity or substrate specificity of the protease and the
protease is proteolytically active. Generally, these mutations
change the specificity and activity of the scaffold proteases for
cleavage of any one or more of the complement proteins. 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.
[0151] As used herein, chymotrypsin numbering refers to the amino
acid numbering of a mature chymotrypsin polypeptide of SEQ ID NO:8.
Alignment of a protease domain of another protease, such as for
example the protease domain of MT-SP1, can be made with
chymotrypsin. In such an instance, the amino acids of MT-SP1 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 the serine protease domain of MT-SP1 (SEQ ID NO:10)
with mature chymotrypsin, V at position 1 in MT-SP1 is given the
chymotrypsin numbering of V16. Subsequent amino acids are numbered
accordingly. In one example, an F at amino acid position 708 of
full-length MT-SP1 (SEQ ID NO:2) or at position 94 of the protease
domain of MT-SP1 (SEQ ID NO:10), 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.
[0152] As used herein, "inhibiting complement activation" or
"complement inactivation" refers to the reduction or decrease of a
complement-mediated function or activity of any one or more of the
complement pathways by a protease or in the activity of any of the
proteins in a pathway. A function or activity of complement can
occur in vitro or in vivo. Exemplary functions of complement that
can be assayed and that are described herein include hemolytic
assays, and assays to measure any one or more of the complement
effector molecules such as by SDS PAGE followed by Western Blot or
Coomassie Brilliant Blue staining or by ELISA. A protease can
inhibit complement activation by 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or more. In other embodiments, complement activation
is inhibited by a protease by 40%, 50%, 60%, 70%, 80%, 85%, 90%,
95% or 99.9% compared to the activity of complement in the absence
of a protease.
[0153] As used herein, specificity for a target substrate refers to
a preference for cleavage of a target substrate by a protease
compared to a another substrate, referred to as a non-target
substrate. Specificity is reflected in the 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.
[0154] 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.
[0155] As used herein, substrate specificity refers to the
preference of a protease for one target substrate over another.
Substrate specificity can be measured as a ration of specificity
constants.
[0156] 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.-1sec.sup.-1 for a target substrate and 2.times.10.sup.4
M.sup.-1sec.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.
[0157] As used herein, preference or substrate specificity 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.
[0158] As used herein, altered specificity refers to a change in
substrate specificity of a modified protease compared to a starting
scaffold 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 scaffold
protease (herein referred to as a non-target substrate). Typically,
modified proteases provided herein exhibit increased substrate
specificity for any one or more of the complement proteins compared
to the substrate specificity of a scaffold protease. For example, a
modified 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 scaffold 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 complement
proteins.
[0159] 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.-1sec.sup.-1 for a target
substrate and 2.times.10.sup.4 M.sup.-1sec.sup.-1 for any other one
of more substrates, the protease is more selective for the former
target substrate.
[0160] 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.
[0161] As used herein, a functional activity with reference to a
complement protein refers to a complement-mediated function
including, but not limited to, anaphylaxis, opsonization,
chemotaxis, or cell lysis. Nonlimiting assays for testing
activities of complement include hemolysis of red blood cells, and
detection of complement effector molecules such as by ELISA or
SDS-PAGE.
[0162] 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.
[0163] 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. A functional activity of a complement protein
target substrate by a protease can be measured by assessing an IC50
in a complement assay such as red blood cell lysis, or other such
assays known by one of skill in the art or provided herein to
assess complement activity. 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 complement protein as
compared to in the absence of the protease.
[0164] As used herein, serine proteases 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 and inflammation, as well as
functioning 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.
[0165] As used herein, a complement protease refers to a protease
that is involved in the generation and amplification of complement
cascade reactions in any of the complement pathways. These
proteases include the serine protease factor I, factor D,
MBL-associated serine protease (MASP)-2, MASP-1, C1s, C1r, factor
B, C2, and the convertases and any other protease that occurs in a
complement pathway whereby complement activation is effected. In
particular, complement proteases are any unmodified complement
proteases, including factor I, factor D, MASP-2, MASP-1, C1s, C1r,
factor B and C2.
[0166] As used herein, a non-complement protease is any protease
that is not normally part of any one or more of the complement
pathways.
[0167] As used herein, MT-SP1 refers to a serine protease that is
part of the S1 peptidase family of serine proteases (also
containing trypsin and chymotrypsin) based on the location of the
Ser, His, and Lys active site residues. MT-SP1 is characterized by
a transmembrane domain, two CUB domains, four LDLR repeats, and a
serine protease domain (or peptidase S1 domain) that is highly
conserved among all members of the peptidase S1 family of serine
proteases, such as for example with chymotrypsin. The sequence of
an exemplary MT-SP1 is set forth in SEQ ID NO: 2. The protease
domain occurs between and includes amino acids 615-854.
[0168] Reference to an MT-SP1 protease includes a full-length
MT-SP1 or any catalytically active portion thereof and includes
allelic variants and species variants and variants encoded by
splice variants. An MT-SP1 protease occurs as a single chain
zymogen, and as an activated two-chain polypeptide. Reference to
MT-SP1 includes active single-chain and two-chain forms thereof. Of
particular interest are MT-SP1 proteases of mammalian, including
human, origin. An MT-SP1 protease also can include those of rat or
mouse origin. Those of skill in this art recognize that, in
general, single amino acid substitutions in non-essential regions
of a polypeptide do not substantially alter biological activity
(see, e.g., Watson et al. Molecular Biology of the Gene, 4th
Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Sequences
of encoding nucleic molecules and the encoded amino acid sequences
of exemplary MT-SP1 proteases of human origin and/or catalytically
active domains thereof are set forth in SEQ ID NOS: 1, 2, 9 and 10.
Exemplary MT-SP1 polypeptides of non-human origin are those having
amino acid sequences such as in mice (Mus musculus, SEQ ID NO: 449)
and rats (Rattus norvegicus, SEQ ID NO: 450). Herein, an MT-SP1
protease can be a scaffold MT-SP1.
[0169] As used herein, reference to a "catalytically active portion
thereof" of an MT-SP1 protease refers to the protease domain, or
any fragment or portion thereof that retains protease activity. For
example, a catalytically active portion of an MT-SP1 can be an
MT-SP1 protease domain including an isolated single chain form of
the protease domain or an activated two-chain form.
[0170] As used herein, a modified MT-SP1 protease refers to a
protease that exhibits altered activity, such as altered substrate
specificity, compared to the scaffold or unmodified form. Such
proteases include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, or more modifications (i.e. changes in
amino acids) compared to a scaffold MT-SP1 such that an activity,
such as substrate specificity or selectivity, of the MT-SP1
protease for cleaving a complement protein is altered. A modified
MT-SP1 can be a full-length scaffold MT-SP1, or can be a portion
thereof of a full length scaffold protease, as long as the modified
protease contains modifications in regions that alter the activity
or substrate specificity of the protease and the protease is
proteolytically active. A modified MT-SP1 protease also can include
other modifications in regions that do not impact on substrate
specificity of the protease. Hence, a modified MT-SP1 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 MT-SP1. A
modified full-length MT-SP1 protease or a catalytically active
portion thereof of a modified MT-SP1 can include proteases that are
fusion proteins as long as the fusion protein possesses the target
specificity.
[0171] 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.
[0172] 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.
[0173] As used herein, non-naturally occurring amino acids refer to
amino acids that are not genetically encoded.
[0174] 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.
[0175] As used herein, a peptide refers to a polypeptide that is
from 2 to 40 amino acids in length.
[0176] 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.
[0177] 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).
[0178] As used herein, the "amino acids," which occur in the
various amino acid sequences appearing herein, are identified
according to their well-known, three-letter or one-letter
abbreviations (see Table 1). The nucleotides, which occur in the
various DNA fragments, are designated with the standard
single-letter designations used routinely in the art.
[0179] 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: 3552-3559 (1969), 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
[0180] 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.
[0181] As used herein, "naturally occurring amino acids" refer to
the 20 L-amino acids that occur in polypeptides.
[0182] 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.
[0183] 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).
[0184] 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.
[0185] 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.
[0186] As used herein, the term ortholog means a polypeptide or
protein obtained from one species that is the functional
counterpart of a polypeptide or protein from a different species.
Sequence differences among orthologs are the result of
speciation.
[0187] 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.
[0188] As used herein, a protease polypeptide is a polypeptide
having an amino acid sequence corresponding to any one of the
scaffold or modified proteases described herein.
[0189] 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).
[0190] "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 exist a number of methods to
measure identity between two polynucleotide or polypeptides, the
term "identity" is well known to skilled artisans (Carrillo, H.
& Lipman, D., SIAM J Applied Math 48:1073 (1988)).
[0191] As used herein, by 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%,
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;
Carrillo et al. (1988) SIAM J Applied Math 48:1073). By sequence
homology, the number of conserved amino acids is determined by
standard alignment algorithm 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.
[0192] 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 (Altschul, S. F., et al.,
J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
Bishop, ed., Academic Press, San Diego, 1994, and Carrillo 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 the 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.
[0193] 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.
[0194] 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.
[0195] 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 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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, 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] For purposes herein, amino acid substitutions, deletions
and/or insertions, can be made in any of the proteases and protease
domains thereof provided that the resulting protein exhibits
protease activity or other activity (or, if desired, such changes
can be made to eliminate activity). Modifications can be made by
making conservative amino acid substitutions and also
non-conservative amino acid substitutions. For example, amino acid
substitutions that desirably or advantageously alter properties of
the proteins can be made. In one embodiment, mutations that prevent
degradation of the polypeptide can be made. Many proteases cleave
after basic residues, such as R and K; to eliminate such cleavage,
the basic residue is replaced with a non-basic residue. Interaction
of the protease with an inhibitor can be blocked while retaining
catalytic activity by effecting a non-conservative change at the
site of interaction of the inhibitor with the protease. Other
activities also can be altered. For example, receptor binding can
be altered without altering catalytic activity.
[0205] Amino acid substitutions contemplated include conservative
substitutions, such as those set forth in Table 2, which do not
eliminate proteolytic activity. As described herein, substitutions
that alter properties of the proteins, such as removal of cleavage
sites and other such sites also are contemplated; such
substitutions are generally non-conservative, but can be readily
effected by those of skill in the art.
[0206] Suitable conservative substitutions of amino acids are known
to those of skill in this art and can be made generally without
altering the biological activity, for example enzymatic activity,
of the resulting molecule. Those of skill in this art recognize
that, in general, single amino acid substitutions in non-essential
regions of a polypeptide do not substantially alter biological
activity (see, e.g., Watson et al. Molecular Biology of the Gene,
4th Edition, 1987, The Benjamin/Cummings Pub. co., p. 224). Also
included within the definition, is the catalytically active
fragment of a serine protease, particularly a single chain protease
portion. Conservative amino acid substitutions are made, for
example, in accordance with those set forth in TABLE 2 as follows:
TABLE-US-00002 TABLE 2 Original residue Conservative substitution
Ala (A) Gly; Ser; Abu Arg (R) Lys; orn Asn (N) Gln; His Cys (C) Ser
Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I)
Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr;
Ile Ornithine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T)
Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu; Met
[0207] Other substitutions also are permissible and can be
determined empirically or in accord with known conservative
substitutions.
[0208] 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.
[0209] 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 of
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.
[0210] 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 equal
to 20%, 10% or 5% of the volume of the protease protein
preparation.
[0211] 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.
[0212] As used herein, production by recombinant means by using
recombinant DNA methods refers to the use of the well known methods
of molecular biology for expressing proteins encoded by cloned
DNA.
[0213] As used herein, vector (or plasmid) refers to discrete
elements that are used to introduce 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.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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 Coomassie blue.
[0222] 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.
[0223] 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 (such as,
but not limited to, conservative changes such as those set forth in
Table 2, above) 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.
[0224] 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.
[0225] As used herein, a "chimeric protein" or "fusion protein"
protease refers to a polypeptide operatively-linked to a different
polypeptide. A chimeric or fusion protein provided herein can
include one or more proteases or a portion thereof, such as single
chain protease domains thereof, and one or more other polypeptides
for any one or more of a transcriptional/translational control
signals, signal sequences, a tag for localization, a tag for
purification, part of a domain of an immunoglobulin G, and/or a
targeting agent. These chimeric or fusion proteins include those
produced by recombinant means as fusion proteins, those produced by
chemical means, such as by chemical coupling, through, for example,
coupling to sulfhydryl groups, and those produced by any other
method whereby at least one protease, or a portion thereof, is
linked, directly or indirectly via linker(s) to another
polypeptide.
[0226] As used herein, operatively-linked when referring to a
fusion protein refers to a protease polypeptide and a non-protease
polypeptide that are fused in-frame to one another. The
non-protease polypeptide can be fused to the N-terminus or
C-terminus of the protease polypeptide.
[0227] As used herein, a targeting agent, is any moiety, such as a
protein or effective portion thereof, that provides specific
binding of the conjugate to a cell surface receptor, which in some
instances can internalize bound conjugates or portions thereof. A
targeting agent also can be one that promotes or facilitates, for
example, affinity isolation or purification of the conjugate;
attachment of the conjugate to a surface; or detection of the
conjugate or complexes containing the conjugate.
[0228] As used herein, an antibody conjugate refers to a conjugate
in which the targeting agent is an antibody.
[0229] As used herein, derivative or analog of a molecule refers to
a portion derived from or a modified version of the molecule.
[0230] 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.
[0231] As used herein, a complement-mediated disorder is any
disorder in which any one or more of the complement proteins plays
a role in the disease, either due to an absence or presence of the
protein. A complement-mediated disorder is one that is due to a
deficiency in a complement protein. A complement-mediated disorder
also is one that is due to the presence of any one or more of the
complement proteins and the continued activation of the complement
pathway.
[0232] 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.
[0233] 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.
[0234] As used herein, treatment means any manner in which the
symptoms of a condition, disorder or disease are ameliorated or
otherwise beneficially altered. Treatment also encompasses any
pharmaceutical use of the compositions herein.
[0235] 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.
[0236] As used herein, prevention or prophylaxis refers to methods
in which the risk of developing disease or condition is
reduced.
[0237] As used herein an effective amount of a compound or
composition for treating a particular disease is an amount that is
sufficient to ameliorate, or in some manner reduce the symptoms
associated with the disease. Such amount can be administered as a
single dosage or can be administered according to a regimen,
whereby it is effective. The amount can cure the disease but,
typically, is administered in order to ameliorate the symptoms of
the disease. Typically, repeated administration is required to
achieve a desired amelioration of symptoms.
[0238] As used herein, "therapeutically effective amount" or
"therapeutically effective dose" refers to an agent, compound,
material, or composition containing a compound that is at least
sufficient to produce a therapeutic effect. 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.
[0239] As used herein, administration of a non-complement protease,
such as a modified non-complement protease, refers to any method in
which the non-complement protease is contacted with its substrate.
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 non-complement 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.
[0240] As used herein, an anticoagulant is a drug that helps
prevent the clotting (coagulation) of blood. These drugs tend to
prevent new clots from forming or an existing clot from
enlarging.
[0241] 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.
[0242] As used herein, "patient" or "subject" to be treated
includes humans and human or non-human animals. Mammals include;
primates, such as humans, chimpanzees, gorillas and monkeys;
domesticated animals, such as dogs, horses, cats, pigs, goats,
cows; and rodents such as mice, rats, hamsters and gerbils.
[0243] As used herein, a combination refers to any association
between two or among more items. The association can be spacial or
refer to the use of the two or more items for a common purpose.
[0244] As used herein, a composition refers to any mixture of two
or more products or compounds (e.g., agents, modulators,
regulators, etc.). It can be a solution, a suspension, liquid,
powder, a paste, aqueous or non-aqueous formulations or any
combination thereof.
[0245] 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.
[0246] 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.
[0247] As used herein, a "kit" refers to a packaged combination,
optionally including reagents and other products and/or components
for practicing methods using the elements of the combination. For
example, kits containing 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 are provided. Kits
optionally include instructions for use.
[0248] As used herein, a cellular extract refers to a preparation
or fraction which is made from a lysed or disrupted cell.
[0249] As used herein, an agent is said to be randomly selected
when the agent is chosen randomly without considering the specific
sequences involved in the association of a protein alone or with
its associated substrates, binding partners and/or other
components. An example of randomly selected agents is the use of a
chemical library or a peptide combinatorial library, or a growth
broth of an organism or conditioned medium.
[0250] As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutically active
compound is modified such that the active compound is regenerated
by metabolic processes. The prodrug can be designed to alter the
metabolic stability or the transport characteristics of a drug, to
mask side effects or toxicity, to improve the flavor of a drug or
to alter other characteristics or properties of a drug. By virtue
of knowledge of pharmacodynamic processes and drug metabolism in
vivo, those of skill in this art, once a pharmaceutically active
compound is known, can design prodrugs of the compound (see, e.g.,
Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford
University Press, New York, pages 388-392).
[0251] As used herein, a peptidomimetic is a compound that mimics
the conformation and certain stereochemical features of a
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,
polypeptides in which one or more peptidic bonds that form the
backbone of a polypeptide are replaced with bioisosteres are
peptidomimetics.
[0252] As used herein, antibody includes antibody fragments, such
as Fab fragments, which are composed of a light chain and the
variable region of a heavy chain.
[0253] As used herein, a receptor refers to a molecule that has an
affinity for a particular ligand. Receptors can be
naturally-occurring or synthetic molecules. Receptors also can be
referred to in the art as anti-ligands.
[0254] As used herein, primer refers to an oligonucleotide
containing two or more deoxyribonucleotides or ribonucleotides,
typically more than three, from which synthesis of a primer
extension product can be initiated. Experimental conditions
conducive to synthesis include the presence of nucleoside
triphosphates and an agent for polymerization and extension, such
as DNA polymerase, and a suitable buffer, temperature and pH.
[0255] As used herein, animal includes any animal, such as, but 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.
[0256] As used herein, genetic therapy or gene therapy involves the
transfer of heterologous nucleic acid, such as DNA, into certain
cells, target cells, of a mammal, particularly a human, with a
disorder or condition for which such therapy is sought. The nucleic
acid, such as DNA, is introduced into the selected target cells,
such as directly or in a vector or other delivery vehicle, in a
manner such that the heterologous nucleic acid, such as DNA, is
expressed and a therapeutic product encoded thereby is produced.
Alternatively, the heterologous nucleic acid, such as DNA, can in
some manner mediate expression of DNA that encodes the therapeutic
product, or it can encode a product, such as a peptide or RNA that
in some manner mediates, directly or indirectly, expression of a
therapeutic product. Genetic therapy also can be used to deliver
nucleic acid encoding a gene product that replaces a defective gene
or supplements a gene product produced by the mammal or the cell in
which it is introduced. The introduced nucleic acid can encode a
therapeutic compound, such as a protease or modified protease, that
is not normally produced in the mammalian host or that is not
produced in therapeutically effective amounts or at a
therapeutically useful time. The heterologous nucleic acid, such as
DNA, encoding the therapeutic product can be modified prior to
introduction into the cells of the afflicted host in order to
enhance or otherwise alter the product or expression thereof.
Genetic therapy also can involve delivery of an inhibitor or
repressor or other modulator of gene expression.
[0257] As used herein, heterologous nucleic acid is nucleic acid
that is not normally produced in vivo by the cell in which it is
expressed or that is produced by the cell but is at a different
locus or expressed differently or that mediates or encodes
mediators that alter expression of endogenous nucleic acid, such as
DNA, by affecting transcription, translation, or other regulatable
biochemical processes. Heterologous nucleic acid is generally not
endogenous to the cell into which it is introduced, but has been
obtained from another cell or prepared synthetically. Heterologous
nucleic acid can be endogenous, but is nucleic acid that is
expressed from a different locus or altered in its expression.
Generally, although not necessarily, such nucleic acid encodes RNA
and proteins that are not normally produced by the cell or in the
same way in the cell in which it is expressed. Heterologous nucleic
acid, such as DNA, also can be referred to as foreign nucleic acid,
such as DNA. Thus, heterologous nucleic acid or foreign nucleic
acid includes a nucleic acid molecule not present in the exact
orientation or position as the counterpart nucleic acid molecule,
such as DNA, is found in a genome. It also can refer to a nucleic
acid molecule from another organism or species (i.e.,
exogenous).
[0258] Any nucleic acid, such as DNA, that one of skill in the art
would recognize or consider as heterologous or foreign to the cell
in which the nucleic acid is expressed is herein encompassed by
heterologous nucleic acid; heterologous nucleic acid includes
exogenously added nucleic acid that also is expressed endogenously.
Examples of heterologous nucleic acid include, but are not limited
to, nucleic acid that encodes traceable marker proteins, such as a
protein that confers drug resistance, nucleic acid that encodes
therapeutically effective substances, such as anti-cancer agents,
enzymes and hormones, and nucleic acid, such as DNA, that encodes
other types of proteins, such as antibodies. Antibodies that are
encoded by heterologous nucleic acid can be secreted or expressed
on the surface of the cell in which the heterologous nucleic acid
has been introduced.
[0259] As used herein, a therapeutically effective product for gene
therapy is a product that is encoded by heterologous nucleic acid,
typically DNA, that, upon introduction of the nucleic acid into a
host, a product is expressed that ameliorates or eliminates the
symptoms, manifestations of an inherited or acquired disease or
that cures the disease. Also included are biologically active
nucleic acid molecules, such as RNAi and antisense.
[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 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, recitation that a polypeptide consists
essentially of a recited sequence of amino acids means that only
the recited portion, or a fragment thereof, of the full-length
polypeptide is present. The polypeptide can optionally, and
generally will, include additional amino acids from another source
or can be inserted into another polypeptide. For example, for
purposes 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.
[0262] 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. Target: Complement
[0263] The complement system and its components are the target
substrates for modified proteases as provided herein. The proteases
are modified or selected or identified to cleave one or more
components of the system and to thereby provide a way to modulate
the activity of the system. Such proteases can serve as
therapeutics or as candidate therapeutics to modulate the activity
of the complement system.
[0264] The complement system is part of the immune system and plays
a role in the elimination of invading foreign organisms and
initiates inflammatory responses. There are over 30 soluble and
cell-membrane proteins that are part of the complement system.
These proteins function not only in the antibody-mediated immune
response, but also in the innate immune response to recognize and
kill pathogens such as bacteria, virus-infected cells, and
parasites. Complement proteins are produced constitutively by
macrophages and hepatocytes, and are present in the circulation as
inactive molecules. Several complement proteins are pro-enzyme
proteases (termed zymogens) that are themselves activated by
proteolytic cleavage to become effector proteases that cut peptide
bonds in other complement proteins to activate them in turn. Since
each activated protease can activate many substrate molecules, the
initial activation is rapidly amplified to produce millions of
effector molecules (a cascade). The complement system constitutes
an irreversible cascade of proteolytic events whose termination
results in the formation of multiple effector molecules that
stimulate inflammation, facilitate antigen phagocytosis, and lyse
some cells directly, and, thus, can serve as a therapeutic point of
intervention for treatment of a variety of disorders that share a
common pathology or include this system in the etiology or
pathology.
[0265] There are three distinct pathways through which complement
can be activated on the pathogen surface: the classical pathway,
the alternative pathway, and the lectin pathway. These pathways are
distinct in that the components required for their initiation are
different, but the pathways ultimately generate the same set of
effector molecules (see, e.g., FIG. 1). Likewise, the early events
of each pathway are governed by a similar mechanism of
triggered-enzyme cascades in which inactive complement zymogens are
cleaved to yield two fragments, the larger of which is an active
serine protease. The active protease is retained at the pathogen
surface so that the subsequent complement zymogen is cleaved and
activated to continue the proteolytic cascade of complement
activation. The second fragment generated upon zymogen cleavage is
a smaller peptide fragment which can act as a soluble mediator of
complement functioning as an opsonin or proinflammatory
mediator.
[0266] 1. Nomenclature
[0267] The complement pathway contains over 30 soluble mediators
(see Table 3), some of which are generated from the cleavage of
inactive protein zymogens to yield two fragments. Table 3 depicts
exemplary native complement proteins and provides a description of
their polypeptide sequence, including the location in the
polypeptide of encoded complement fragments thereof. For example,
SEQ ID NO: 315 encodes a C5 complement protein and also encodes a
C5a fragment of a complement protein, encoded by residues 678-751
of C5, that exhibits complement activity upon its generation
following cleavage of C5 by a C5 convertase. The native components
of complement are designated by a C followed by a number such as
C1, C2, etc. . . . . The numbering of the complement components is
based on the order of their discovery rather than the order of the
sequence of reactions within the complement cascade. As a result,
the sequence of reactions of the complement cascades is C1, C4, C2,
C3, C5, C6, C7, C8, and C9. Following activation, the products of
the cleavage reactions are designated by adding lower case letters,
the larger fragment generally being designated "b" and the smaller
fragment as "a" (i.e. C4 is cleaved to generate C4b and C4a). In
some instances C2a is designated as the larger cleavage product,
although more generally C2b is considered the larger cleavage
product. Consequently, the C3 convertase C3b2b is sometimes
referred to as C3b2a. Inactive complement cleavage products are
designated with an "i" (i.e. iC3b). The protein zymogen components
specific to the alternative pathway of complement are not
designated by a C, but are rather designated by different capital
letters such as Factor B, which upon cleavage becomes Bb or Ba. The
two initiating protease zymogens of the lectin pathway are
designated as MASP-1 and MASP-2. TABLE-US-00003 TABLE 3 Complement
Proteins Amino Gene acid SEQ ID Entry Name AC # Name length
Description NO C1QA_HUMAN P02745 C1QA 245 Complement C1q
subcomponent, A chain precursor 298 C1QB_HUMAN P02746 C1QB 251
Complement C1q subcomponent, B chain precursor 299 C1QC_HUMAN
P02747 C1QG, C1QC 245 Complement C1q subcomponent, C chain
precursor 300 C1R_HUMAN P00736 C1R 705 Complement C1r subcomponent
precursor (Complement 301; 302 component 1, r subcomponent)
[Contains: Complement C1r subcomponent heavy chain (aa: 18-463);
Complement C1r subcomponent light chain (aa: 464-705)] C1S_HUMAN
P09871 C1S 688 Complement C1s subcomponent precursor (C1 esterase)
303, 304 [Contains: Complement C1s subcomponent heavy chain (aa:
16-437); Complement C1s subcomponent light chain (aa: 438-688)]
C4BB_HUMAN P20851 C4BPB 252 C4b-binding protein beta chain
precursor 305 C4BP_HUMAN P04003 C4BPA 597 C4b-binding protein alpha
chain precursor (C4bp) (Proline- 306 C4BP rich protein) (PRP)
CFAI_HUMAN P05156 IF 583 Complement factor I precursor (EC
3.4.21.45) (C3B/C4B 307, 308 inactivator) [Contains: Complement
factor I heavy chain (aa: 19-335); Complement factor I light chain
(340-583)] CLUS_HUMAN P10909 CLU 449 Clusterin precursor
(Complement-associated protein SP- 309 40, 40) (Complement
cytolysis inhibitor) (CLI) (NA1/NA2) (Apolipoprotein J) (Apo-J)
(Testosterone-repressed prostate message 2) (TRPM-2) [Contains:
Clusterin beta chain (ApoJalpha) (Complement cytolysis inhibitor a
chain) (aa: 23-227); Clusterin alpha chain (ApoJbeta) (Complement
cytolysis inhibitor b chain) (aa: 228-449)] CO2_HUMAN P06681 C2 752
Complement C2 precursor (EC 3.4.21.43) (C3/C5 310, 311 convertase)
CO3_HUMAN P01024 C3 1663 Complement C3 precursor [Contains:
Complement C3 beta 312 chain (aa: 23-667); Complement C3 alpha
chain (aa: 672-1663); C3a anaphylatoxin (aa: 672-748); Complement
C3b alpha' chain (aa: 749-1663); Complement C3c fragment (aa:
749-954); Complement C3dg fragment (aa: 955-1303); Complement C3g
fragment (aa: 955-1001); Complement C3d fragment (aa: 1002-1303);
C3f fragment (aa: 1304-1320)] CO4_HUMAN P01028 C4A and 1744
Complement C4 precursor [Contains: Complement C4 beta 313 C4B chain
(aa: 20-675); Complement C4 alpha chain (aa: 680-1446); C4a
anaphylatoxin (680-756); C4b (aa: 757-1446); Complement C4 gamma
chain (aa: 1454-1744)] CO5_HUMAN P01031 C5 1676 Complement C5
precursor [Contains: Complement C5 beta 314 chain (aa: 19-673);
Complement C5 alpha chain (aa: 678-1676); C5a anaphylatoxin (aa:
678-751); Complement C5 alpha' chain (aa: 752-1676)] CO6_HUMAN
P13671 C6 934 Complement component C6 precursor 315 CO7_HUMAN
P10643 C7 843 Complement component C7 precursor 316 CO8A_HUMAN
P07357 C8A 584 Complement component C8 alpha chain precursor 317
(Complement component 8 alpha subunit) CO8B_HUMAN P07358 C8B 591
Complement component C8 beta chain precursor 318 (Complement
component 8 beta subunit) CO8G_HUMAN P07360 C8G 202 Complement
component C8 gamma chain precursor 319 CO9_HUMAN P02748 C9 559
Complement component C9 precursor [Contains: 320 Complement
component C9a (aa: 22-265); Complement component C9b (aa: 266-559)]
CR1_HUMAN P17927 CR1, 2039 Complement receptor type 1 precursor
(C3b/C4b receptor) 321 C3BR (CD35 antigen) CR2_HUMAN P20023 CR2,
1033 Complement receptor type 2 precursor (Cr2) (Complement 322
C3DR C3d receptor) (Epstein-Barr virus receptor) (EBV receptor)
(CD21 antigen) DAF_HUMAN P08174 DAF, 381 Complement
decay-accelerating factor precursor (CD55 323 CD55, CR antigen)
IC1_HUMAN P05155 SERPING 500 Plasma protease C1 inhibitor precursor
(C1 Inh) (C1Inh) 324 1, C1IN, C1NH MASP1_HUMAN P48740 MASP1, 699
Complement-activating component of Ra-reactive factor 325, 326
CRARF, precursor (EC 3.4.21.--) (Ra-reactive factor serine protease
(V1); CRARF1, p100) (RaRF) (Mannan-binding lectin serine protease
1) 327, 328 PRSS5 (Mannose-binding protein associated serine
protease) (V2); (MASP-1) [Contains: Complement-activating component
329, 330 of Ra-reactive factor heavy chain (aa: 20-448); (V3)
Complement-activating component of Ra-reactive factor light chain
(aa: 449-699)] MASP2_HUMAN O00187 MASP2 686 Mannan-binding lectin
serine protease 2 precursor (EC 331, 332 3.4.21.--)
(Mannose-binding protein associated serine (V1); protease 2)
(MASP-2) (MBL-associated serine protease 2) 333, 334 [Contains:
Mannan-binding lectin serine protease 2 A chain (V2) (aa: 16-444);
Mannan-binding lectin serine protease 2 B chain (aa: 445-686)]
MBL2_HUMAN P11226 MBL2, 248 Mannose-binding protein C precursor
(MBP-C) (MBP1) 335 MBL (Mannan-binding protein) (Mannose-binding
lectin) MCP_HUMAN P15529 MCP 392 Membrane cofactor protein
precursor (CD46 antigen) 336 (Trophoblast leukocyte common antigen)
(TLX) CFAB_HUMAN P00751 BF 764 Complement factor B precursor (C3/C5
convertase) 337, 338 (Properdin factor B) (Glycine-rich beta
glycoprotein) (GBG) (PBF2) [Contains: Complement factor B Ba
fragment (aa: 26-259); Complement factor B Bb fragment (aa:
260-764)] CFAD_HUMAN P00746 DF 253 Complement factor D precursor
(C3 convertase activator) 339, 340 (Properdin factor D) (Adipsin)
CFAH_HUMAN P08603 CFH, HF, 1231 Complement factor H precursor (H
factor 1) 341, 342 HF1 PROP_HUMAN P27918 PFC 469 Properdin
precursor (Factor P) 343, 344 FCN2_HUMAN Q15485 FCN2 313 Ficolin-2
(collagen/fibrinogen domain-containing protein 2; 660 ficolin B;
serum lectin p35; L-Ficolin FCN1_HUMAN O00602 FCN1 326 Ficolin-1
(collagen/fibrinogen domain-containing protein 1; 661 Ficolin A;
M-Ficolin) FCN3_HUMAN O75636 FCN3 299 Ficolin-3
(collagen/fibrinogen domain-contain 3; 662 collagen/fibrinogen
domain-containing lectin 3 p35; Hakata antigen; Factor-H)
[0268] 2. Pathways of Complement Initiation
[0269] The pathways of complement are distinct in that they rely on
different molecules and mechanisms for their initiation, but the
pathways are similar in that they converge to generate the same set
of effector molecules. The convergence point of the C pathways is
the cleavage of C3 by C3 convertase (a C3 activating enzyme).
Convertase is a general name used for a complement enzyme that
converts an inactive complement protein into an active one. For
example, C3 convertase converts inactive C3 to active C3a and C3b.
Different enzyme complexes have C3 convertase activity. For
example, in the classical pathway C4b2b acts as a C3 convertase,
whereas in the alternative pathway, C3bBb is a C3 convertase (see
Table 4). Cleavage of C3 generates C3b, which acts as an opsonin
and as the main effector molecule of the complement system for
subsequent complement reactions, and C3a, which is a peptide
mediator of inflammation. The addition of C3b to each C3 convertase
forms a C5 convertase to generate C5a and C5b. C5a, like C3a, is a
peptide mediator of inflammation. C5b mediates the "late" events of
complement activation initiating the sequence of reactions
culminating in the generation of the membrane attack complex (MAC).
Although the three pathways produce different C3 and C5
convertases, all of the pathways produce the split products of C3
and C5 and form MAC. TABLE-US-00004 TABLE 4 Complement Cascades
Alternative Pathway Classical Pathway Lectin Pathway Activators
Pathogen surface antigen-bound IgM and Pathogens via recognition
molecules IgG; non-immune of carbohydrates on LPS, teichoic acid,
molecules surface zymosan C3 convertase C3bBb C4b2b C4b2b C5
convertase C3bBb3b C4b2b3b C4b2b3b MAC C5678poly9 C5678poly9
C5678poly9 anaphylatoxins C3a, C5a C3a, C4a, C5a C3a, C4a, C5a
[0270] a. Classical Pathway
[0271] C1q is the first component of the classical pathway of
complement. C1q is a calcium-dependent binding protein associated
with the collectin family of proteins due to an overall shared
structural homology (Malhotra R et al., Clin Exp Immunol. 1994,
97(2):4-9; Holmskov et al. Immunol Today. 1994, 15(2):67-74).
Mannose binding lectin (MBL), the first component of the lectin
pathway, also is a member of the collectin family. Collectins are
named because they contain a collagen-like and lectin domain. The
amino-terminal collagen-like region of the collectin structure
interacts with cell surface receptors and confers structural
stability to the protein. The carboxy-terminal regions of the
collectin structure have a calcium-dependent lectin activity. The
lectin domain mediates the interaction of the collectins with a
wide variety of pathogens due to the recognition of carbohydrate
moieties on the surface of pathogens. Collectins, often called
pattern recognition molecules, generally function as opsonins to
target pathogens for phagocytosis by immune cells. In contrast to
conventional collectins, such as MBL, the carboxy-terminal globular
recognition domain of C1q does not have lectin activity but can
serve as a "charged" pattern recognition molecule due to marked
differences in the electrostatic surface potential of its globular
domains (Gaboriaud et al. J. Biol. Chem., 2003, 278(47):
46974-46982).
[0272] C1q initiates the classical pathway of complement in two
different ways. First, the classical pathway is activated by the
interaction of C1q with immune complexes (i.e. antigen-antibody
complexes or aggregated IgG or IgM antibody) thus linking the
antibody-mediated humoral immune response with complement
activation. When the Fab portion (the variable region) of IgM or
IgG binds antigen, the conformation of the Fc (constant) region is
altered, allowing C1q to bind. C1q must bind at least 2 Fc regions
to be activated, so it takes two IgG molecules to activate C1q.
Serum IgM is a pentamer of five IgM molecules with five Fc regions,
so IgM activates complement most efficiently. IgA, IgE and IgD do
not bind C1q and cannot activate complement. C1q, however, also is
able to activate complement in the absence of antibody thereby
functioning in the innate or immediate immune response to
infection. Besides antibody, complement activation also is achieved
by the interaction of C1q with non-immune molecules such as
polyanions (bacterial lipopolysaccharides, DNA, and RNA), certain
small polysaccharides, viral membranes, C reactive protein (CRP),
serum amyloid P component (SAP), and bacterial, fungal and virus
membrane components.
[0273] C1q is part of the C1 complex which contains a single C1q
molecule bound to two molecules each of the zymogens C1r and C1s.
Binding of more than one of the C1q globular domains to a target
surface (such as aggregated antibody or a pathogen), causes a
conformational change in the (C1r:C1s).sub.2 complex which results
in the activation of the C1r protease to cleave C1s to generate an
active serine protease. Active C1s cleaves subsequent complement
components C4 and C2 to generate C4b and C2b, which together form
the C3 convertase of the classical pathway. The C3 convertase
cleaves C3 into C3b, which covalently attaches to the pathogen
surface and acts as an opsonin, and C3a, which stimulates
inflammation. Some C3b molecules associate with C4b2b complexes
yielding C4b2b3b which is the classical cascade C5 convertase.
Table 5 summarizes the proteins involved in the classical pathway
of complement. TABLE-US-00005 TABLE 5 Proteins of the Classical
Pathway Native Component Active Form Function of the Active Form C1
C1q Binds directly to pathogen surfaces (C1q:(C1r:C1s).sub.2) or
indirectly to antibody bound to pathogens C1r Cleaves C1s to an
active protease C1s Cleaves C4 and C2 C4 C4b Binds to pathogen and
acts as an opsonin; binds C2 for cleavage by C1s C4a Peptide
mediator of inflammation C2 C2b Active enzyme of classical pathway
C3/C5 convertase; cleaves C3 and C5 C2a Precursor of vasoactive C2
kinin C3 C3b Binds to pathogen surfaces and acts as an opsonin;
initiates amplification via the alternative pathway; binds C5 for
cleavage by C2b C3a Peptide mediator of inflammation
[0274] b. Alternative Pathway
[0275] The alternative pathway is initiated by foreign pathogens in
the absence of antibody. Instead, the initiation of complement by
the alternative pathway occurs through the spontaneous hydrolysis
of C3 into C3b. A small amount of C3b is always present in body
fluids, due to serum and tissue protease activity. Host self-cells
normally contain high levels of membrane silica acid which
inactivate C3b if it binds, but bacteria contain low external
sialic acid and thereby bind C3b without inactivating it. C3b on
pathogen surfaces is recognized by the protease zymogen Factor B.
Factor B is cleaved by Factor D. Factor D is the only activating
protease of the C system that circulates as an active enzyme rather
than as a zymogen, but since Factor B is the only substrate for
Factor D the presence of low levels of an active protease in normal
serum is generally safe for the host. Cleavage of Factor B by
Factor D yields the active product Bb which can associate with C3b
to form C3bBb, the C3 convertase of the alternative pathway.
Similar to the classical pathway, the C3 convertase produces more
C3b and C3a from C3. C3b covalently attaches to the pathogen
surface and acts as an opsonin, while C3a stimulates inflammation.
Some C3b joins the complex to form C3bBb3b, the alternative pathway
C5 convertase. C3bBb3b is stabilized by the plasma protein
properdin or Factor P which binds to microbial surfaces and
stabilizes the convertase. Table 6 summarizes the proteins involved
in the alternative pathway of complement. TABLE-US-00006 TABLE 6
Proteins of the Alternative Pathway Native Component Active Form
Function of the Active Form C3 C3b Binds to pathogen surface, binds
Factor B for cleavage by Factor D Factor B Ba Small fragment of
Factor B, unknown function Bb Active enzyme of the C3 convertase
and C5 convertase Factor D D Plasma serine protease, cleaves Factor
B when it is bound to C3b to Ba and Bb Factor P P Plasma proteins
with affinity for C3bBb (properdin) convertase on bacterial cells;
stabilizes convertase
[0276] c. Lectin
[0277] The lectin pathway (also referred to as the MBL pathway) is
initiated following recognition and binding of pathogen-associated
molecular patterns (PAMPs; i.e. carbohydrates moieties) by lectin
proteins. Examples of lectin proteins that activate the lectin
pathway of complement include mannose binding lectin (MBL) and
ficolins (i.e. L-ficolin, M-ficolin, and H-ficolin). As mentioned
above, MBL is a member of the collectin family of proteins and
thereby exists as an oligomer of subunits composed of identical
polypeptide chains each of which contains a cysteine-rich, a
collagen-like, a neck, and a carbohydrate-recognition or lectin
domain. MBL acts as a pattern recognition molecule to recognize
carbohydrate moieties, particularly neutral sugars such as mannose
or N-acetylglucosamine (GlcNAc) on the surface of pathogens via its
globular lectin domain in a calcium-dependent manner. Besides a
role in the complement system, MBL also acts as an opsonin to
facilitate the phagocytosis of bacterial, viral, and fungal
pathogens by phagocytic cells. In addition, other initiators of the
lectin pathway include the ficolins including L-ficolin, M-ficolin,
and H-ficolin (see e.g., Liu et al. (2005) J Immunol., 175:3150-6).
Similar to MBL, ficolins recognize carbohydrate moieties such as,
for example, N-acetyl glucosamine and mannose structures.
[0278] The activation of the alternative pathway by MBL or ficolins
is analogous to activation of the classical pathway by C1q whereby
a single lectin molecule interacts with two protease zymogens. In
the case of the lectin proteins, the zymogens are MBL-associated
serine proteases, MASP-1 and MASP-2, which are closely homologous
to the C1r and C1s zymogens of the classical pathway. Upon
recognition of a PAMP by a lectin protein, such as for example by
binding to a pathogen surface, MASP-1 and MASP-2 are activated to
cleave C4 and C2 to form the MBL cascade C3 convertase. C3b then
joins the complex to form the MBL cascade C5 convertase. MASP
activation is implicated not only in responses to microorganisms,
but in any response that involves exposing neutral sugars,
including but not limited to tissue injury, such as that observed
in organ transplants. Like the alternative cascade, the MBL cascade
is activated independent of antibody; like the classical cascade,
the MBL cascade utilizes C4 and C2 to form C3 convertase. Table 7
summarizes the proteins involved in the lectin pathway of
complement. TABLE-US-00007 TABLE 7 Proteins of the Lectin Pathway
Native Component Active Form Function of the Active Form MBL MBL
Recognizes PAMPs, such as on pathogen surfaces (e.g., via
recognition of carbohydrates) Ficolins L-Ficolin; M- Recognizes
PAMPs, such as on pathogen Ficolin, or H- surfaces (e.g., via
recognition of Ficolin carbohydrates) MASP-1 MASP-1 Cleaves C4 and
C2 MASP-2 MASP-2 Cleaves C4 and C2
[0279] 3. Complement-Mediated Effector Functions
[0280] Regardless of which initiation pathway is used, the end
result is the formation of activated fragments of complement
proteins (e.g. C3a, C4a, and C5a anaphylatoxins and C5b-9 membrane
attack complexes). These fragments mediate several functions
including leukocyte chemotaxis, activation of macrophages, vascular
permeability and cellular lysis (Frank, M. and Fries, L.
Complement. In Paul, W. (ed.) Fundamental Immunology, Raven Press,
1989). A summary of some effector functions of complement products
are listed in Table 8. TABLE-US-00008 TABLE 8 Complement Effector
Molecules and Functions Product Activity C2b (prokinin)
accumulation of body fluid C3a (anaphylatoxin) basophil and mast
cell degranulation; enhanced vascular permeability; smooth muscle
contraction; Induction of suppressor T cells C3b and its products
opsonization; Phagocyte activation C4a (anaphylatoxin) basophil
& mast cell activation; smooth muscle contraction; enhanced
vascular permeability C4b opsonization C5a (anaphylatoxin; basophil
& mast cell activation; enhanced vascular permeability; smooth
chemotactic factor) muscle contraction; chemotaxis; neutrophil
aggregation; oxidative metabolism stimulation; stimulation of
leukotriene release; induction of helper T-cells C5b67 chemotaxis;
attachment to other cell membranes and lysis of bystander cells
C5b6789 (C5b-9) lysis of target cells
[0281] a. Complement-Mediated Lysis: Membrane Attack Complex
[0282] The final step of the complement cascade by all three
pathways is the formation of the membrane attack complex (MAC)
(FIG. 1). C5 can be cleaved by any C5 convertase into C5a and C5b.
C5b combines with C6 and C7 in solution, and the C5b67 complex
associates with the pathogen lipid membrane via hydrophobic sites
on C7. C8 and several molecules of C9, which also have hydrophobic
sites, join to form the membrane attack complex, also called
C5b6789 or C5b-9. C5b-9 forms a pore in the membrane through which
water and solutes can pass, resulting in osmotic lysis and cell
death. If complement is activated on an antigen without a lipid
membrane to which the C5b67 can attach, the C5b67 complex can bind
to nearby cells and initiate bystander lysis. A single MAC can lyse
an erythrocyte, but nucleated cells can endocytose MAC and repair
the damage unless multiple MACs are present. Gram negative
bacteria, with their exposed outer membrane and enveloped viruses,
are generally susceptible to complement-mediated lysis. Less
susceptible are Gram positive bacteria, whose plasma membrane is
protected by their thick peptidoglycan layer, bacteria with a
capsule or slime layer around their cell wall, or viruses which
have no lipid envelope. Likewise, the MAC can be disrupted by
proteins that bind to the complex before membrane insertion such as
Streptococcal inhibitor of complement (SIC) and clusterin.
Typically, the MAC helps to destroy gram-negative bacteria as well
as human cells displaying foreign antigens (virus-infected cells,
tumor cells, etc.) by causing their lysis and also can damage the
envelope of enveloped viruses.
[0283] b. Inflammation
[0284] Inflammation is a process in which blood vessels dilate and
become more permeable, thus enabling body defense cells and defense
chemicals to leave the blood and enter the tissues. Complement
activation results in the formation of several proinflammatory
mediators such as C3a, C4a, and C5a. The intact anaphylatoxins in
serum or plasma are quickly converted into the more stable, less
active C3a-desArg, C4a-desArg, or C5a-desArg forms, by
carboxypeptidase N. C3a, C4a and C5a, and to a lesser extent their
desArg derivatives, are potent bioactive polypeptides, termed
anaphylatoxins because of their inflammatory activity.
Anaphylatoxins bind to receptors on various cell types to stimulate
smooth muscle contraction, increase vascular permeability, and
activate mast cells to release inflammatory mediators. Among the
three anaphylatoxins, C5a is the most potent. C5a primarily acts on
white blood cells, and in particular neutrophils. C5a stimulates
leukocyte adherence to blood vessel walls at the site of infection
by stimulating the increased expression of adhesion molecules so
that leukocytes can squeeze out of the blood vessels and into the
tissues, a process termed diapedesis. C5a also stimulates
neutrophils to produce reactive oxygen species for extracellular
killing, proteolytic enzymes, and leukotrienes. C5a also can
further amplify the inflammatory process indirectly by inducing the
production of chemokines, cytokines, and other proinflammatory
mediators. C5a also interacts with mast cells to release
vasodilators such as histamine so that blood vessels become more
permeable. C3a also interacts with white blood cells, with major
effects on eosinophils suggesting a role for C3a in allergic
inflammation. C3a induces smooth muscle contraction, enhances
vascular permeability, and causes degranulation of basophils and
release of histamine and other vasoactive substances. C2a can be
converted to C2 kinin, which regulates blood pressure by causing
blood vessels to dilate.
[0285] Although technically not considered an anaphylatoxin, iC3b,
an inactive derivative of C3b, functions to induce leukocyte
adhesion to the vascular endothelium and induce the production of
the pro-inflammatory cytokine IL-1 via binding to its cell surface
integrin receptors. C5b-9 also indirectly stimulates leukocyte
adhesion, activation, and chemotaxis by inducing the expression of
cell adhesion molecules such as E-selectin, and inducing
interleukin-8 secretion (Bhole et al. (2003) Crit Care Med 31(1):
97-104). C5b-9 also stimulates the release of secondary mediators
that contribute to inflammation, such as for example prostaglandin
E.sub.2, leukotriene B.sub.4, and thromboxane.
[0286] Conversion of the human complement components C3 and C5 to
yield their respective anaphylatoxin products has been implicated
in certain naturally occurring pathologic states including:
autoimmune disorders such as systemic lupus erythematosus,
rheumatoid arthritis, malignancy, myocardial infarction,
Purtscher's retinopathy, sepsis and adult respiratory distress
syndrome. In addition, increased circulating levels of C3a and C5a
have been detected in certain conditions associated with iatrogenic
complement activation such as: cardiopulmonary bypass surgery,
renal dialysis, and nylon fiber leukaphoresis. Elevated levels of
C4a anaphylatoxin is associated with the autoimmune disorders
mentioned above.
[0287] c. Chemotaxis
[0288] Chemotaxis is a process by which cells are directed to
migrate in response to chemicals in their environment. In the
immune response, a variety of chemokines direct the movement of
cells, such as phagocytic cells, to sites of infection. For
example, C5a is the main chemotactic factor for circulating
neutrophils, but also can induce chemotaxis of monocytes.
Phagocytes will move towards increasing concentrations of C5a and
subsequently attach, via their CR1 receptors, to the C3b molecules
attached to the antigen. The chemotactic effect of C5a, observed
with basophils, eosinophils, neutrophils, and mononuclear
phagocytes, is active at concentrations as low as 10.sup.-10M.
[0289] d. Opsonization
[0290] An important action of complement is to facilitate the
uptake and destruction of pathogens by phagocytic cells. This
occurs by a process termed opsonization whereby complement
components bound to target bacteria interact with complement
receptors on the surface of phagocytic cells such as neutrophils or
macrophages. In this instance, the complement effector molecules
are termed opsonins. Opsonization of pathogens is a major function
of C3b and C4b. iC3b also functions as an opsonin. C3a and C5a
increase the expression of C3b receptors on phagocytes and increase
their metabolic activity.
[0291] C3b and, to a lesser extent, C4b help to remove harmful
immune complexes from the body. The C3b and C4b attach the immune
complexes to CR1 receptors on erythrocytes. The erythrocytes then
deliver the complexes to fixed macrophages within the spleen and
liver for destruction. Immune complexes can lead to a harmful Type
III hypersensitivity
[0292] e. Activation of the Humoral Immune Response
[0293] Activation of B cells requires ligation of the B cell
receptor (BCR) by antigen. It has been shown, however, that
complement plays a role in lowering the threshold for B cell
responses to antigen by up to 1000-fold. This occurs by the binding
of C3d or C3dg, complement products generated from the breakdown
fragments of C3, to CR2 receptors on B-lymphocytes which can
co-ligate with the BCR. Co-ligation occurs when antigenic
particles, such as for example immune complexes, opsonized with C3d
binds the CR2 receptor via C3d as well as the BCR through antigen.
Co-ligation of antigen complexes also can occur when C3d binds to
antigens enhancing their uptake by antigen presenting cells, such
as dendritic cells, which can then present the antigen to B cells
to enhance the antibody response. Mice deficient in CR2 display
defects in B cell function that result in reduced levels of natural
antibody and impaired humoral immune responses.
[0294] 4. Complement Receptors
[0295] The recognition of complement effector molecules by cells
for the initiation of effector functions such as chemotaxis and
opsonization is mediated by a diverse group of complement
receptors. The complement receptors are distributed on a wide range
of cell types including erythrocytes, macrophages, B cells,
neutrophils, and mast cells. Upon binding of a complement component
to the receptor, the receptors initiate an intracellular signaling
cascade resulting in cell responses such as stimulating
phagocytosis of bacteria and secreting inflammatory molecules from
the cell. For example, the complement receptors CR1 and CR2 which
recognize C3b, C4b, and their products are important for
stimulating chemotaxis. CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are
integrins that are similarly important in phagocytic responses but
also play a role in leukocyte adhesion and migration in response to
iC3b. The C5a and C3a receptors are G protein-coupled receptors
that play a role in many of the pro-inflammatory-mediated functions
of the C5a and C3a anaphylatoxins. For example, receptors for C3a,
C3aR, exist on mast cells, eosinophils, neutrophils, basophils and
monocytes and are directly involved in the pro-inflammatory effects
of C3a.
[0296] 5. Complement Regulation
[0297] Although the complement system is beneficial to the host by
protecting against foreign pathogens, the production of
inflammatory mediators can be toxic and damaging leading to a wide
variety of inflammatory disease conditions as discussed below.
Likewise, although most of the active proteases of the complement
system are zymogens that only become activated locally upon
cleavage, nearly all components of complement are spontaneously
activated at low rates in serum and thus their activity needs to be
minimized. Consequently, regulatory proteins of the complement
system have been identified. Their primary functions are to
regulate the activity of complement activating molecules for
prevention of excessive complement activation and autolytic
destruction of host tissues. These complement regulators are either
soluble plasma proteins or integral membrane proteins expressed on
a variety of cell types. The former include C4b binding protein
(C4bp) and Factor H. The latter include the C3b/C4b receptor
(Complement receptor 1, CR1, CD35), membrane cofactor protein (MCP,
CD46), and decay accelerating factor (DAF, CD55). These proteins
possess many structural similarities. Each is composed of multiple
short consensus repeats (SCRs) of approximately 60 amino acids in
length having conserved cysteine, glycine and proline residues. The
genes encoding these proteins have been localized to chromosome 1
and are collectively known as the regulators of complement
activation (RCA) gene cluster (Hourcade et al. (1989) Adv. Immunol.
45:381).
[0298] C1 inhibitor (C1INH) is a serine proteinase inhibitor or
serpin which dissociates activated C1r and C1s from C1q, limiting
the time the complex is active. C1INH also blocks spontaneous
activation of C1 by plasma proteases. Deficiency in C1INH is
associated with serious sudden edema (swelling) called
Angioneurotic Edema. Several inhibitory proteins dissociate the C3
and C5 convertases and promote degradation of C4b and C3b by Factor
I, a plasma protease. Factor I circulates in an active form but it
is only able to cleave C3b and C4b when they are bound to a
cofactor protein. Factor I cleaves C3b leading to the production of
iC3b, C3c, C3d, C3f, and C3dg thereby permanently inactivating C3b,
although the degradation products can act as effector molecules,
since, for example, iC3b acts as an opsonin. C4b is inactivated
upon cleavage into C4c and C4d. The inhibitory proteins that serve
as cofactors for Factor I include plasma proteins C4 binding
protein that dissociates classical C3 convertase, and Factor H that
dissociates alternative C3 convertase, and membrane proteins
Complement Receptor 1 (CR1), Decay Accelerating Factor (DAF), and
Membrane Cofactor Protein (MCP) that inhibit the activity of both
pathways. Cofactors for Factor I regulate its activity. For
example, human cells produce Factor H that binds to C3b and allows
Factor I to inactivate C3b. On the other hand, substances such as
LPS on bacterial cells, which otherwise do not express Factor I
cofactors, facilitate the binding of Factor B to C3b and this
protects the C3b from inactivation by Factor I.
[0299] Other membrane and plasma proteins block the formation of
MAC on host cells to prevent the inappropriate insertion of MAC
into membranes. Several plasma proteins, such as the soluble
protein C8.beta., bind to the C5b67 complex and inhibit its
insertion into the cell membrane. Host cell membranes also contain
a membrane-bound protein called HRF (CD59, protectin) which
inhibits the binding of C9 to C5b678 to prevent formation of the
membrane attack complex on autologous or allogenic cells.
[0300] Factor I
[0301] Factor I (fI) is one of several serine proteases (also
including factor D, MBL-associated serine protease (MASP)-2, C1s,
C1r, factor B, and C2) of the complement system that play a role in
the generation and amplification of the complement cascade
reactions. All of the complement serine proteases share domain
homology with the trypsin family and share some of the structural
attributes that determine substrate specificity. The C-terminus of
Factor I is made up of a trypsin-like serine protease light chain
that, based on homology to other serine proteases, contains the
residues that form the His-Asp-Ser catalytic triad. Additionally,
residues are present that define the specificity pocket (D.sup.501)
and the extended substrate binding site S.sup.527, W.sup.528, and
G.sup.529 (based on numbering of the mature protein in the absence
of the signal peptide, see for example, Tsiftsoglou et al., (2005)
Biochemistry 44:6239).
[0302] Factor I plays a role in modulating complement activation by
cleaving C3b and C4b, components of the C3 convertase in the
classical, alternative, and lectin pathways thereby inactivating
the pathways. Cleavage of fI substrates, C3b and C4b, requires a
conformational change in the substrates caused by the formation of
a thioester bond. For example, proteolytic activation of C3 to C3b
by convertase results in a conformational change from the latent
form to the C3b form which leads to reaction of an intramolecular
thiolester with nucleophiles, such as water, thereby rendering C3b
susceptible to fl cleavage (Ogata et al., (1998) J Immunol
161:4785). Reaction of the thioester with water can occur in the
absence of convertase cleavage, giving a hydrolyzed inactive form
of C3 and C4 termed iC3 and iC4. For example, the iC3 species is a
mimic of C3b; iC3 is sensitive to fI cleavage and can substitute
for C3b in the C3 and C5 convertases. Generally, cleavage of the
C3b and C4b substrates by Factor I requires the formation of a
ternary complex with a cofactor protein, such as factor H or
C4-binding protein, and MCP. Cleavage of synthetic substrates by
Factor I, however, does not require the presence of cofactors
(Tsiftsoglou et al., (2004) J Immunol 173:367-375). Cleavage by fI
is restricted to cleavage of arginyl bonds in the substrate. fI
cleavage sites in C3 are LPSR (SEQ ID NO: 388) and SLLR (SEQ ID NO:
389) and a cleavage site in C4 is HRGR (SEQ ID NO:390).
[0303] 6. Complement-Mediated Diseases and Disorder
[0304] By virtue of the pivotal role of the complement system in
the etiology of diseases and disorders, the system can serve as a
point of therapeutic intervention in such diseases and disorders.
The proteases provided herein target this system and permit
modulation thereof.
[0305] The skilled artisan understands the role of the complement
system in disease processes and is aware of a variety of such
diseases. The following is a discussion of exemplary diseases and
the role of the complement system in their etiology and pathology.
Modulation of the complement system by the proteases provided
herein can serve to treat such diseases. Diseases can involve
complement activation or inhibition.
[0306] a. Disease Mediated by Complement Activation
[0307] The complement cascade is a dual-edged sword, causing
protection against bacterial and viral invasion by promoting
phagocytosis and inflammation. Conversely, even when complement is
functioning normally, it can contribute to the development of
disease by promoting local inflammation and damage to tissues.
Thus, pathological effects are mediated by the same mediators that
are responsible for the protective roles of complement. For
example, the anaphylactic and chemotactic peptide C5a drives
inflammation by recruiting and activating neutrophils, C3a can
cause pathological activation of other phagocytes, and the membrane
attack complex can kill or injure cells. In one example, such as in
many autoimmune diseases, complement produces tissue damage because
it is activated under inappropriate circumstances such as by
antibody to host tissues. In other situations, complement can be
activated normally, such as by septicemia, but still contributes to
disease progression, such as in respiratory distress syndrome.
Pathologically, complement can cause substantial damage to blood
vessels (vasculitis), kidney basement membrane and attached
endothelial and epithelial cells (nephritis), joint synovium
(arthritis), and erythrocytes (hemolysis) if it is not adequately
controlled.
[0308] Complement has a role in immuno-pathogenesis of a number of
disorders, including autoimmune diseases such as rheumatoid
arthritis (see, e.g., Wang et al. (1995) Proc. Natl. Acad. Sci.
U.S.A. 92:8955-8959; Moxley et al. (1987) Arthritis &
Rheumatism 30:1097-1104), lupus erythematosus (Wang et al. (1996)
Proc. Natl. Acad. Sci. U.S.A. 90:8563-8568; and Buyon et al. (1992)
Arthritis Rheum. 35:1028-1037) and acute glomerulonephritis (Couser
et al. (1995) J Am Soc Nephrol. 5:1888-1894). Other pathologies
that involve activation of the complement system include sepsis
(see, e.g., Stove et al. (1996) Clin Diag Lab Immunol 3:175-183;
Hack et al. (1989) Am. J. Med. 86:20-26), respiratory distress
syndrome (see, e.g., Zilow et al. (1990) Clin. Exp. Immunol.
79:151-157; and Stevens et al. (1986) J. Clin. Invest.
77:1812-1816), multiorgan failure (see, e.g., Hecke et al. (1997)
Shock 7:74; and Heideman et al. (1984) J. Trauma 24:1038-1043) and
ischemia-reperfusion injury such as occurs in cardiovascular
disease such as stroke or myocardial infarct (Austen W G et al.
(2003) Int J Immunopathol Pharm 16(1):1-8). Some exemplary examples
of complement-mediated disease are described below.
[0309] i. Rheumatoid Arthritis
[0310] Rheumatoid arthritis (RA) is a chronic inflammatory illness.
It is an autoimmune disease in which the immune system attacks
normal tissue components as if they were invading pathogens. The
inflammation associated with rheumatoid arthritis primarily attacks
the linings of the joints. The membranes lining the blood vessels,
heart, and lungs also can become inflamed. RA is characterized by
activated B cells and plasma cells that are present in inflamed
synovium, and in established disease lymphoid follicles and
germinal centers. This results in high levels of local
immunoglobulin production and the deposition of immune complexes,
which can include IgG and IgM rheumatoid factors, in the synovium
and in association with articular cartilage which can serve as
initiators of the complement cascade. Elevated levels of complement
components, such as C3a, C5a, and C5b-9 have been found within the
inflamed rheumatoid joints. These complement components can
exacerbate the inflammation associated with RA by inducing a
variety of proinflammatory activities such as for example,
alterations in vascular permeability, leukocyte chemotaxis, and the
activation and lysis of multiple cell types.
[0311] ii. Sepsis
[0312] Sepsis is a disease caused by a serious infection, such as a
bacterial infection, leading to a systemic inflammatory response.
The bacterial cell wall component, lipopolysaccharide, is often
associated with sepsis, although other bacterial, viral, and fungal
infections can stimulate septic symptoms. Septic shock often
results if the natural immune system of the body is unable to
defend against an invading microorganism such that, for example,
the pro-inflammatory consequences of the immune response is
damaging to host tissues. The early stages of sepsis are
characterized by excessive complement activation resulting in
increased production of complement anaphylatoxins, such as C3a,
C4a, and C5a which act to increase vascular permeability, stimulate
superoxide production from neutrophils and stimulate histamine
release. The actions of C5a can contribute to a productive immune
response to a bacterial infection, but if left unregulated, C5a
also can be severely damaging. In an E. coli-induced model of
inflammation, blockade of C5a improved the outcome of septic
animals by limiting C5a-mediated neutrophil activation that can
lead to neutrophil-mediated tissue injury.
[0313] The continued impairment of the innate immune response to a
bacterial infection often leads to chronic sepsis or septic shock,
which can be life-threatening. In the late stage of sepsis, it is
the "dormant" activity of neutrophils, as opposed to the
hyperactivity that occurs in the early phases, that contributes to
continued disease. In the late stage, the major functions of
neutrophils including chemotaxis, respiratory burst activity, and
ability for bacterial killing are reduced. Complement, and in
particular C5a, also play a role in the later stages of sepsis.
Excessive production of C5a during sepsis is associated with the
"deactivation" of blood neutrophils, a process that has been linked
to C5a-induced downregulation of its own receptor, C5aR, on
neutrophils (Guo et al. (2003) FASEB J 13:1889). The reduced levels
of C5aR on neutrophils correlates with a diminished ability of
blood neutrophils to bind C5a, impaired chemotactic responses, a
loss of superoxide productions, and impaired bactericidal activity.
C5aR levels, however, can begin to "recover" at later stages of
sepsis and correlate with instances of beneficial disease
outcome.
[0314] iii. Multiple Sclerosis
[0315] Multiple sclerosis (MS) and its animal model experimental
allergic encephalomyelitis (EAE) are inflammatory demyelinating
diseases of the central nervous system (CNS). In MS, inflammation
of nervous tissue causes the loss of myelin, a fatty material which
acts as a sort of protective insulation for the nerve fibers in the
brain and spinal cord. This demyelination leaves multiple areas of
scar tissue (sclerosis) along the covering of the nerve cells,
which disrupts the ability of the nerves to conduct electrical
impulses to and from the brain, producing the various symptoms of
MS. MS is mediated by activated lymphocytes, macrophages/microglia
and the complement system. Complement activation can contribute to
the pathogenesis of these diseases through its dual role: the
ability of activated terminal complex C5b-9 to promote
demyelination and the capacity of sublytic C5b-9 to protect
oligodendrocytes (OLG) from apoptosis.
[0316] iv. Alzheimer's Disease
[0317] Alzheimer's disease (AD) is characterized by tangles
(abnormal paired helical filaments of the protein tau, which
normally binds to microtubules) and plaques (extracellular deposits
composed primarily of beta-amyloid protein) within the brain.
Although, it is not entirely clear the what the precise cause of AD
is, chronic neuroinflammation in affected regions of AD brains
suggest that proinflammatory mediators can play a role. The tangles
and plaques within an AD brain are deposited with activated
complement fragments, such as for example, C4d and C3d. Likewise,
dystrophic neurites in AD brain can be immunostained for MAC,
indicating autocatalytic attack of these neurites and concomitant
neurite loss in AD. Activation of complement in AD occurs by an
antibody-independent mechanism induced by aggregated beta-amyloid
protein. Further, the complement cascade can be activated by the
pentraxins, C-reactive protein (CRP), and amyloid P (AP) which are
all upregulated in AD (McGeer et al., (2002) Trends Mol Med 8:519).
The activation of complement in AD, marked by increases in
complement mediators, is not adequately controlled by a
compensatory upregulation of complement regulatory proteins such
as, for example, CD59. Thus, the proinflammatory consequences of
complement activation exacerbates AD disease progression and likely
contributes to neurite destruction.
[0318] v. Ischemia-Reperfusion Injury
[0319] Ischemia-reperfusion injury is the injury sustained after an
ischemic event and subsequent restoration of blood flow and results
from the inflammatory response to a hypoxic insult.
Ischemia-reperfusion damage can be acute as during cardiac surgery
procedures, such as for example following open heart surgery or
angioplasty, or chronic as with congestive heart failure or
occlusive cardiovascular disease. Examples of injuries that can
cause ischemia-reperfusion injury include myocardial infarct (MI)
and stroke. The initiation of an inflammatory response is likely
caused by the increase in tissue oxygen levels that occur with
reperfusion and the concomitant accumulation of metabolites that
can generate oxygen free radicals which are immunostimulatory. It
is associated with a variety of events including severity of
myocardial infarction, cerebral ischemic events, intestinal
ischemia, and many aspects of vascular surgery, cardiac surgery,
trauma, and transplantation. The injury is manifested by
inflammatory events of the innate immune system, particularly
activation of the complement system, in response to newly altered
tissue as non-self. As such ischemia-reperfusion injury is
characterized by tissue edema caused by increased vascular
permeability, and an acute inflammatory cell infiltrate caused by
influx of polymorphonuclear leukocytes.
[0320] Activation of the complement system plays a role in the
inflammatory events of ischemia-reperfusion injury. The ischemia
injury results in alterations of the cell membrane, affecting
lipids, carbohydrates, or proteins of the external surface such
that these exposed epitopes are altered and can act as neo-antigens
(modified self antigens). Circulating IgM recognize and bind the
neo-antigens to form immune complexes on the injured cell surface.
The antigen-antibody complexes formed are classic activators of the
classical pathway of complement, although all pathways are likely
involved in some way to the exacerbating effects of the injury. The
involvement of the classical pathway of complement to
ischemia-reperfusion injury is evidenced by mice genetically
deficient in either C3 or C4 that display equal protection from
local injury in a hindlimb and animal model of injury (Austen et
al. (2003) Int J Immunopath Pharm 16:1). Conversely, in a kidney
model of ischemia injury, C3-, C5-, and C6-deficient mice were
protected whereas C4-deficient mice were not, suggesting the
importance of the alternative complement pathway (Guo et al. (2005)
Ann Rev Immunol 23:821). Mediators induced upon complement
activation initiate an inflammatory response directed at the cell
membrane at the site of local injury.
[0321] A major effector mechanism of complement in
ischemia-reperfusion injury is the influx and activation of
neutrophils to the inflamed tissue by complement components, such
as for example C5a. Activation of neutrophils results in increased
production of reactive oxygen species and the release of lysosomal
enzymes in local injured organs which ultimately results in
apoptosis, necrosis, and a loss or organ function. The generation
of the terminal MAC, C5b-9, also contributes to local tissue injury
in ischemia-reperfusion injury.
[0322] b. Disease Mediated by Complement Deficiencies
[0323] The development of disease also can occur due to the absence
of complement components that are important for controlling
infection. Complement deficiencies are linked with frequent
infections and immune complex diseases. Deficiencies have been
identified in all of the complement factors except C9, including
Factor D and properdin. Deficiencies also have been identified in
the complement regulatory proteins C1INH, Factor I, Factor H, DAF,
and HRF.
[0324] In general, deficiencies in complement components result in
increased bacterial infections due to reduced opsonization and
phagocytosis. Typically, deficiencies in complement components that
function as opsonins, such as for example C3b, result in increased
susceptibility to infection. For example, whereas individuals
deficient in any of the late components of complement are
relatively unaffected, individuals lacking C3 or any of the
molecules that catalyze C3b deposition show increased
susceptibility to infection by a wide range of extracellular
bacteria. Likewise, people deficient in MBL, which normally
functions as a traditional opsonin and as the initiator of the
lectin pathway of complement following recognition of foreign
pathogens, have increased susceptibility to infection, particularly
during early childhood. The role of deficiencies in the late
components of complement, including C5-C9 that are involved in the
formation of the membrane attack complex, to bacterial infection is
more limited. Deficiencies in C5-C9 have only been shown to be
associated with susceptibility to infection by Neisseria species,
the bacteria that causes gonorrhea and bacterial meningitis.
[0325] Another consequence of complement deficiency is immune
complex disease. Immune complex disease is caused by
complement-mediated inflammation in response to persisting
antigen-antibody complexes in the circulation and the tissues.
Since the early components of the classical complement pathway
initiate complement in response to the recognition of
antigen-antibody complexes, deficiencies of these early components,
such as for example C1q, can cause significant pathology in
autoimmune disease such as systemic lupus erythematosus.
[0326] Deficiencies in complement regulatory proteins such as
Factor H, DAF, and HRF also can result in complement-mediated
disease. For example, uncontrolled complement activation can result
in depletion of complement proteins resulting in an increased
infection by bacteria, particularly ubiquitous pyogenic bacteria.
This is the case in genetic factor I deficiency where factor I is
not present and unable to inhibit the activation of the C3
convertase. Other examples include the complement regulatory
proteins DAF or HRF, which normally function to protect a person's
own cell surfaces from complement activation, but when deficient
result in the destruction of host red blood cells resulting in the
disease paroxysmal nocturnal hemoglobinuria. Deficiencies in
C1-inhibitor causes the disease hereditary angioneurotic edema
which is a result of the unregulated activity of serine proteinase
enzymes including the complement components C1r and C1s, as well as
other serine proteinases such as factor XIIa and kallikrein. The
result of the unregulated activity of these serine proteinases is
the production of a variety of vasoactive mediators, such as C2
kinin that is produced by the activity of C1s and C2a, resulting in
fluid accumulation in the tissues and epiglottal swelling that can
lead to suffocation.
C. Proteases
[0327] Provided herein are proteases and methods of using the
proteases to cleave (thereby inactivating) proteins involved in
disease processes. Typically, a protease provided herein is a
non-complement protease that does not normally participate in the
complement pathways. Exemplary proteases provided herein cleave any
one or more proteins or components of the complement pathway and
allelic variants thereof. Cleavage of a complement protein can be
an activating cleavage whereby the activity of the complement
pathway is enhanced, such as by cleavage of a zymogen to an
activated form of a protease or cleavage of a complement protein
into its cleavage effector molecules. Cleavage of a complement
protein also can be an inhibitory cleavage whereby the activity of
the complement protein is diminished. Provided herein are proteases
that cleave a complement protein in an inhibitory manner, thereby
inhibiting complement activation of any one or more of the
complement pathways. The proteases provided herein can be used for
modulating complement activation. A protease provided herein can
cleave any one or more complement proteins in vitro or in vivo
thereby affecting complement activation in vitro or in vivo.
[0328] A protease can be any portion of a full-length protease as
long as the portion of the protease retains proteolytic activity.
For example, a protease can include only the protease domain of a
polypeptide or any catalytically active portion thereof. The
protease domain can include a single chain protease domain thereof
and can be a fusion protein or a conjugate as long as the resulting
fusion protein or conjugate retains proteolytic activity.
[0329] If a protease, or portion thereof, recognizes a substrate
sequence within a target protein or proteins, such as for example a
complement protein, (i) that would alter the function i.e. by
inactivation of the target protein(s) upon catalysis of peptide
bond hydrolysis, and (ii) the target proteins(s) is a point of
molecular intervention for a particular disease or diseases, than
the engineered protease has a therapeutic effect via a
proteolysis-mediated inactivation event. Complement activities that
can be altered include, but are not limited to, hemolysis of red
blood cells and/or the generation of effector complement cleavage
products such as but not limited to C3a, C3b, C4a, C5a, C5b-9, and
Bb. Biological activities of complement can be altered in vitro or
in vivo. Generally, a complement activity is altered by a protease
at least 0.1, 0.5, 1, 2, 3, 4, 5, or 10 fold compared to the
absence of a protease. Typically, a biological activity is altered
10, 20, 50, 100 or 1000 fold or more compared to the activity in
the absence of the protease. For purposes herein with reference to
complement activity, a protease modulates complement activation or
a complement-mediated activity.
[0330] A protease provided herein can be from any one or more of
the serine, cysteine, aspartic, metallo-, or threonine classes or
proteases. A protease can be tested to determine if it cleaves any
one or more of the complement proteins and/or it can be used as a
scaffold to make modifications in any one or more of the amino acid
residues that modulates specificity towards a target substrate
and/or modulates an activity of a target substrate. Exemplary
classes of proteases and amino acid determinants that contribute to
substrate specificity are described below.
[0331] 1. Classes of Proteases
[0332] 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 a
target. Proteases are classified based on the way they attack the
protein, either exo- or endo-proteases. Proteinases or
endopeptidases attack inside the protein to produce large peptides.
Peptidases or exopeptidases attack ends or fragments of protein to
produce small peptides and amino acids. The peptidases are
classified on their action pattern: aminopeptidase cleaves amino
acids from the amino end: carboxypeptidase cleaves amino acids from
the carboxyl end, dipeptidyl peptidase cleaves two amino acids;
dipeptidase splits a dipeptide, and tripeptidase cleaves an amino
acid from a tripeptide. Most proteases are small from 21,000 to
45,000 Daltons. Many proteases are synthesized and secreted as
inactive forms called zymogens and subsequently activated by
proteolysis. This changes the architecture of the active site of
the enzyme.
[0333] 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 could affect (i.e. enhance
specificity/selectivity) the ability of a protease to cleave a
substrate.
[0334] 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, P1', 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) 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 can be changed in one pocket without
affecting the specificity of the other pocket.
[0335] 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). A protease can be tested
to determine if it cleaves any one or more of the complement
proteins and/or it can be used as a scaffold to make modifications
in any one or more of the amino acid residues that modulates
specificity towards a complement protein target substrate and/or
modulates an activity of a complement protein target substrate. To
make a modified protease with an altered substrate recognition
profile, the amino acids in the three-dimensional structure that
contribute to the substrate selectivity (specificity determinants)
can be targeted for mutagenesis. Exemplary proteases include, but
are not limited to, any protease such as a serine, cysteine,
aspartic, metallo-, or threonine protease as described below and
provided herein.
[0336] a. Serine Proteases
[0337] Serine proteases (SPs), which include secreted enzymes and
enzymes sequestered in cytoplasmic storage organelles, have a
variety of physiological roles, including roles 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 as discussed above.
[0338] 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.
[0339] 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.
[0340] 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 (R-group colored blue below). 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.
[0341] 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
MT-SP1, are Asp102, His57, and Ser195. 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).
[0342] 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 9 with numbering based on the to 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-00009 TABLE 9 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- Tyr Ile Met Trp 22 Lys Glu Ile His 9 Asp Ser
Gly Yes tryptase Beta- Tyr Ile Met Trp 22 Gln Glu Val His 9 Asp Ser
Gly Yes tryptase(I) Beta- Tyr Ile Met Trp 22 Lys Glu Thr His 9 Asp
Ser Gly Yes tryptase (II) Chymo- Trp Arg Met Trp 13 Met Ser Val His
7 Ser Ser Gly Yes trypsin 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
[0343] i. MT-SP1
[0344] Exemplary of the scaffold protease contemplated for use in
modulating complement activation or as a scaffold for further
modification to increase its activity in modulating the complement
pathway is membrane-type serine protease MT-SP1 (also called
matriptase, TADG-15, suppressor of tumorigenicity 14, ST14); see
SEQ ID NOS: 1, 2 and GenBank Accession Nos: AF118224 and AAD42765;
(1999) J. Biol. Chem. 274:18231-18236; U.S. Pat. No. 5,792,616;
see, also Takeuchi (1999) Proc. Natl. Acad. Sci. U.S.A.
96:11054-1161. The protein designated herein as an exemplary
scaffold is a 855 amino acid MT-SP1 protease (see SEQ ID NOS: 1 and
2). The nucleic acid molecule whose sequence is set forth in SEQ ID
NO: 1 (see, also Genbank AF118224) encodes the 855 amino acid
MT-SP1 (SEQ ID NO: 2, GenBank AAD42765).
[0345] It is 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.
[0346] MT-SP1 is highly expressed or active in prostate, breast,
and colorectal cancers and it may 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.
[0347] MT-SP1 contains a transmembrane domain, two CUB domains,
four LDLR repeats, and a serine protease domain (or peptidase S1
domain) between amino acids 615-854 (set forth as SEQ ID NOS:9 and
10), which is highly conserved among all members of the peptidase
S1 family of serine proteases, such as for example with
chymotrypsin (SEQ ID NOS:7 and 8). 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.
[0348] 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. 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 proteases
interact with a substrate in a conserved beta sheet manner. Up to 6
conserved hydrogen bonds can occur between the substrate and
enzyme. All serine proteases of the chymotrypsin family have a
conserved region at their N-terminus of the protease domain that is
necessary for catalytic activity (i.e. IIGG, VVGG, or IVGG, where
the first amino acid in this quartet is numbered according to the
chymotrypsin numbering and given the designation Ile16. This
numbering does not reflect the length of the precursor
sequence).
[0349] 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 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.
[0350] 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
subsites and an exposed 60 loop. Similarly, MT-SP1 does not
indiscriminately cleave peptide substrates at accessible Lys or Arg
residues, but requires recognition of additional residues
surrounding the scissile peptide bond. This requirement for an
extended primary sequence highlights the specificity of MT-SP1 for
its substrates. For example, although MT-SP1 cleaves proteinase
activated receptor-2 (PAR-2) (displaying a P4 to P1 target sequence
of Ser-Lys-Gly-Arg), the enzyme does not activate proteins closely
related to this substrate such as PAR-1, PAR-3, and PAR-4 that do
not display target sequences matching the extended MT-SP1
specificity near the scissile bond (see Friedrich et al. (2002) J
Biol Chem 277: 2160).
[0351] The protease domain of MT-SP1 (see, e.g, SEQ ID NOS: 9 and
10) 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 NOS: 1 and 2 at RQAR followed by the residues
VVGG). The S1 pocket of MT-SP1 and trypsin are similar with good
complementarity for Lys as well as Arg P1 residues, thereby
accounting for some similarities in substrate cleavage with
trypsin. The accommodation of the 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. Association of 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). Table 10 summarizes
the residues in MT-SP1 for some of the amino acid positions
important for specificity interactions with a targeted substrate.
Typically, modification of an MT-SP1 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. TABLE-US-00010
TABLE 10 Structural determinants for MT-SP1 substrate cleavage
Residues that Determine Specificity S4 S2 S1 Cys168 S3 60's loop
Cys191 171 174 180 215 Cys182 192 218 99 57 (58-64) 189 190 226
Cys220 Leu Gln Met Trp 13 Gln Asp Phe His 16 Asp Ser Gly yes
[0352] ii. Granzyme B
[0353] Granzyme B also is exemplary of the scaffold proteases
contemplated for use in modulating complement activation or for
further modification to increase its activity in modulating a
complement pathway. Granzyme B is a serine protease (S1-type)
necessary for target cell lysis in cell-mediated immune responses.
Granzyme B is linked to an activation cascade of caspases
(aspartate-specific cysteine proteases) responsible for apoptosis
execution and cleaves caspase-3, caspase-7, caspase-9 and
caspase-10 to give rise to active enzymes mediating apoptosis.
Granzyme B (SEQ ID NO:3, GenBank #:M17016) encodes a 247 amino acid
polypeptide (SEQ ID NO: 4, GenBank #:P10144). The precursor
granzyme B polypeptide has a signal sequence and propeptide
activation peptide at amino acids 1 to 20. The mature granzyme B
protein is characterized by a peptidase S1 or protease domain at
amino acids 21-245.
[0354] Granzyme B is a member of the family of chymotrypsin fold
serine proteases, and has greater than 50% identity to other
members of the granzyme family including granzymes C-G, cathepsin
G, and rat mast cell protease II. The protein is a sandwich of two
six stranded, anti-parallel beta-barrel domains connected by a
short alpha-helix.
[0355] A substrate cleavage site of wildtype granzyme B has a
consensus recognition site of I/V (P4)-E/Q/M (P3)-P/T (P2)-D (P1).
These amino acids line the P1-P4 pocket of the substrate for
recognition and cleavage by granzyme B. Generally granzyme B has a
preference for cleaving after Asp in its consensus recognition.
[0356] The structural determinants for granzyme B substrate
cleavage have been identified by the three-dimensional structure of
rat granzyme B (SEQ ID NOS: 5 and 6) in complex with ecotin (IEPD),
a macromolecular inhibitor with a substrate-like binding loop
(Waugh et al., (2000) Nature Struct. Biol 7:762). The catalytic
triad is composed of Asp102, His57 and Ser195. The surface loops
are numbered according to the additions and deletions compared to
alpha-chymotrypsin and represent the most variable regions of this
structural family. Other structural determinants of specificity
include Lys 41, Ile99, Arg192, Asn218, Tyr215, Tyr174, Leu172,
Arg226, and Tyr151, by chymotrypsin numbering. The other members of
the granzyme family of serine proteases share only two of these
amino acids with granzyme B. They are Tyr215 and Leu172, two
residues that vary very little across the entire structural family.
This indicates that while the sequence identity of the granzymes is
high, their substrate specificities are very different. The
structural determinants for granzyme B substrate specificity are
listed in Table 11 with chymotrypsin numbering. Typically,
modification of a granzyme B protease to alter any one or more of
the amino acids in the extended specificity binding pocket, or
other secondary sites of interactions, affects the specificity or
selectivity of a protease for a target substrate including a
complement protein target substrate. TABLE-US-00011 TABLE 11
Structural determinants for Granzyme B substrate cleavage Residues
that Determine Specificity S4 S2 S1 Cys.sup.168 S3 60's Cys.sup.191
171 174 180 215 Cys.sup.182 192 218 99 57 loop 189 190 226
Cys.sup.220 Leu Tyr Glu Tyr 14 Arg Asn Ile His 6 Gly Ser Arg no
[0357] The importance of granzyme B structural determinants to
specificity has been profiled using a combinatorial substrate
library to determine the effect of a mutation on extended
specificity. Mutation of Ile99, Arg192, Asn218 and Tyr174 to the
amino acid alanine have shown that Ile99 contributes to P2
specificity, Asn218 and Arg192 to P3 specificity, and Tyr174 to P4
specificity. Since the P1 specificity of a protease represents the
majority of its specificity, the modifications do not destroy
unique specificity of granzyme B towards P1 aspartic acid amino
acids but modulate specificity in the extended P2 to P4 sites. For
the P3 and P4 subsites, mutations at Tyr174, Arg192 and Asn218 did
not significantly affect the specificity. Y174A increases the
activity towards Leu at P4, but the rest of the amino acids
continue to be poorly selected. R192A and N218A both broaden the
specificity at P3. Instead of a strong preference for glutamic
acid, Ala, Ser, Glu and Gin are introduced into a modified
protease. The overall activity (k.sub.cat/K.sub.m) of the mutant is
less than 10% below the wild-type activity toward an ideal
wild-type substrate, N-acetyl-Ile-Glu-Pro-Asp-AMC
(7-amino-4-methylcoumarin) (Ac-IEPD-AMC). A greater effect is
observed at the P2 subsite. In wildtype granzyme B, the preference
is broad with a slight preference for Pro residues. I99A narrows
the P2 specificity to Phe and Tyr residues. Phe narrows specificity
by nearly 5 times over the average activity of other amino acids at
this position. Within the chymotrypsin family of serine proteases,
more than a dozen proteases have a small residue at this structural
site, either an asparagine, serine, threonine, alanine or glycine.
From this group, two proteases have been profiled using
combinatorial substrate libraries, (plasma kallikrein and plasmin),
and both show strong preferences towards Phe and Tyr. These two
results suggest that any serine protease that is mutated to an Asn,
Ser, Thr, Gly or Ala at position 99 will show the same hydrophobic
specificity found in plasma kallikrein, plasmin and the I99A
granzyme B mutant.
[0358] P2 specificity determinants can be expanded to the
contrasting mutation and substrate preference. For example, nearly
two dozen chymotrypsin-fold serine proteases have an aromatic amino
acid at position 99. Four of these proteases have been profiled
using combinatorial substrate libraries: human granzyme B, tissue
type plasminogen activator, urokinase type plasminogen activator,
and membrane type serine protease 1. All but granzyme B have a
preference for serine, glycine and alanine amino acids at the
substrate P2 position.
[0359] b. Cysteine Proteases
[0360] 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.
[0361] 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).
[0362] 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.
[0363] 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 bulkly
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.
[0364] 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 12. 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-00012 TABLE 12 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
[0365] c. Aspartic Proteases
[0366] Aspartate proteases include the digestive enzyme pepsin,
some proteases found in lysosomes, the kidney enzyme renin, and the
HIV-protease. Two aspartate residues participate in acid/base
catalysis at the active site. In the initial reaction, one
aspartate accepts a proton from an active site H.sub.2O, which
attacks the carbonyl carbon of the peptide linkage. Simultaneously,
the other aspartate donates a proton to the oxygen of the peptide
carbonyl group. They can exhibit a variety of specificities, but
typically cleave between two hydrophobic amino acids. Well defined
S4, S3, S2, S1, S1', S2', S3', and S4' subsite pockets for the
amino acid side chains of the substrate are hallmarks for these
enzymes (see e.g. Brinkworth et al., (2001) J Biol Chem
276:38844).
[0367] Exemplary aspartic proteases include retroviral proteases,
such as the human immunodeficiency virus, type 1 (HIV-1) PR, or the
avian myeloblastosis/Rous sarcoma virus (AMV/RSV) PR (Cameron et
al., (1993) J Biol Chem. 268:11711). The PRs possess substrate
binding pockets that contain at least seven subsites (S4-S3') which
interact with 7 amino acids of the substrate (P4-P3') (Cameron et
al., (1993) J Biol Chem. 268:11711; Cameron et al., (1994) J Biol
Chem. 269: 11170). Residues that contribute to substrate
specificity of the AMV/RSV PR include P62, I42, M73, R105', H7',
Q63, R10', D41, I64 (S4); H65, V104', R105', G106', Q63, R10',
L35', D37', G39, D41, G66, I67, I108', R111 (S3); I42, I44, H65,
M73, A100, A40, D41, I64, G66, I67', I108 (S2); H65, V104', R105',
G106', S107', R10', L35', D37', D37, G39, G66, I67, I108' (S1);
H65', V104, R105, G106, S107, R10, L35, D37, D37', G39', G66',
I67', I108 (S1'), I42', I44', H65', M73', A100', V104', A40', D41',
I64', G66', I67, I108' (S2'); and S38', H65', V104, R105, G106,
Q63', R10, L35, G39', D41', I64', G66', I67', I108, R111' (S3'),
where the amino acid residues in the second subunit of the dimer
are indicated by a prime. Residues that contribute to substrate
specificity of the HIV-1 PR include D30, V56, P81', R8', D29, I47
(S4); G48, T80', P81' V82', R8', L23', D25', G27, D29, G49, I50,
I84', R87 (S3); D30, V32, G48, V56, L76, A28, D29, I47, G49, I50',
I84 (S2); G48, T80', P81', V82', N83', R8', L23', D25', D25, G27,
G49, I50, I84' (S1); G48', T80, P81, V82, N83, R8, L23, D25, D25',
G27', G49', I50', I84 (S1'); D30', V32', G48', V56', L76', T80'
(S2'); and R8, L23, G27', D29', I47', G49', I50', I84, R87' (S3'),
where the amino acid residues in the second subunit of the dimer
are indicated by a prime. Typically, modification of an aspartic
protease, such as for example a retroviral protease, to alter any
one or more of the amino acids in the extended specificity binding
pocket, or other secondary sites of interaction, affects the
specificity or selectivity of a protease for a target substrate
including a complement protein target substrate.
[0368] d. Metalloproteases
[0369] Metalloproteases (also called Zinc proteases) include the
digestive enzymes carboxypeptidases, various matrix
metalloproteases (MMPs) that are secreted by cells, ADAMs (a
disintegrin and metalloprotease domain), and lysosomal proteases.
These enzymes, including ADAMs and MMPs have roles in embryonic
development, cell growth and proliferation, inflammatory responses,
wound repair, multiple sclerosis, arthritis, and cancer progression
and metastasis (Manzetti et al., (2003) J of Computer-Aided Mol.
Design, 17: 551). Some MMPs (e.g., collagenase) are involved in
degradation of the extracellular matrix during tissue remodeling.
For example, many of these enzymes can cleave components of the
basement membrane and extracellular matrix. Some MMPs have roles in
cell signaling relating to their ability to release cytokines or
growth factors, such as TNF.alpha., TGF.beta., and interleukins,
from the cell surface by cleavage of membrane-bound
pre-proteins.
[0370] A zinc binding motif at the active site of a metalloprotease
includes two histidine residues whose imidazole side-chains are
ligands to the Zn.sup.++. During catalysis, the Zn.sup.++ promotes
nucleophilic attack on the carbonyl carbon by the oxygen atom of a
water molecule at the active site. An active site base (a glutamate
residue in carboxypeptidase) facilitates this reaction by
extracting a proton from the attacking water molecule. Generally,
these enzymes have a common zinc binding motif (HExxHxxGxxH) in
their active site, and a conserved methionine turn following the
active site. Mutation of any one of the histidines ablates
catalytic activity. The active site specificity differs between
metalloproteases to accommodate different peptide backbones of
substrates around the scissile bond. A crucial molecular
determinant of MMP substrate specificity is the side chain of the
amino acid at the P1' position. Thus, the S1' subsite is important
in determining the peptide bond preference for cleavage. For
example, the small S1' pocket of MMP-1 and MMP-7 promotes a
preference for small hydrophobic residues while other MMPs have
large S1' pockets (Overall et al., (2002) Mol Biotech 22:51). The
S2 position also is a molecular determinant of specificity. For
example, between MMP-2 and MMP-9, the S2 sub-site is one of the few
differences between the catalytic clefts of the MMPs where the
presence of Glu.sup.412 in MMP-2 versus Asp.sup.410 in MMP-9 play
important roles in altering substrate specificity. In fact, among
the larger MMP family the Glu.sup.412 position is highly variable
where it is occupied by acidic residues, large hydrophobic
residues, and even glycine. In contrast, most of the residues
surrounding the S2 subsite are strictly conserved among all MMPs
(Chen et al., (2003) J Biol Chem 278:17158). Other molecular
determinants of specificity are described in Table 13 below (see
e.g., Manzetti et al., (2003) J Computer-Aided Mol Design 17:551).
Typically, modification of a metalloprotease, such as for example a
MMP or ADAM 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-00013 TABLE 13 The structural
determinants for various metalloproteases S4 S3 S2 S1 S1' S2' S3'
S4' MMP-3 F210 F210 H166 L164 V163 L164 L164 L222 F83 A169 H211
V198 P221 ADAM9 F317 V318 V318 M315 I344 N373 S374 G310 V318 M315
H351 H357 A313 T312 F333 H357 N356 N373 ADAM10 P391 V332 V332 L329
L327 V326 N366 T421 V332 P391 H392 H392 T379 W331 N387 A418
[0371] e. Threonine
[0372] Threonine proteases include the proteasome hydrolase. The
proteasome is a large barrel-shaped protein complex made up of
alpha and beta subunits. The beta subunits supply the catalytic
machinery found within the two central rings of the complex.
Typically, the mechanism of catalysis of the catalytically active
beta subunit involves a conserved N-terminal threonine at each
active site. The beta subunits become activated when the N-terminus
is cleaved off, making threonine the N-terminal residue such that
catalytic threonines are exposed at the lumenal surface. Hydrolysis
is initiated by attack of an amide bond by the hydroxyl nucleophile
on the catalytic machinery. The structural determinants of
specificity of the beta subunits of the proteasome have been
determined, such as, for example by using libraries of
peptide-based covalent inhibitors of the proteasome (see e.g.,
Groll et al., (2002) Chem Biol 9:655; Zhang et al., (2003) EMBO J,
22:1488).
D. Scaffold Proteins
[0373] Scaffold proteins are provided. Scaffold proteins include
any wild-type protease so so long as they are non-complement
proteases, and also include allelic or species variants, or
catalytically active protions thereof. The scaffold proteases can
be used to target (i.e. cleave) any one or more complement pathway
substrates. Typically, such cleavage results in inactivation of a
complement pathway. In some instances, such cleavage can result in
activation of complement. Hence, such scaffold proteases can be
used as therapeutics by targeting complement pathway substrates to,
for example, inhibit complement activation which is associated with
the etiology of various diseases or disorders. Scaffold proteins
also are any proteins that can be modified to cleave a target
substrate. Among them are scaffold proteases, whose target
substrate specificity can be modified. Scaffold proteins, including
proteases, can be modified in any one or more amino acids such that
the resulting protease exhibits altered specificity or selectivity
for any one or more protein components of the complement pathway
and/or modulates an activity of a complement protein or pathway.
For example, a modified protease can have an altered substrate
specificity such that the modified protease preferentially cleaves
a targeted substrate component of the complement pathway compared
to a non-targeted substrate, such as for example a native substrate
of a wildtype scaffold protease. In one embodiment, the specificity
can be increased compared to the specificity of a wildtype or
scaffold protease for a targeted substrate. In another example, a
modified protease can exhibit a selectivity for a complement
component such that the ability of a modified protease to cleave a
particular substrate is greater than any other target substrate for
which the modified protease also can exhibit specificity.
Additionally, a modified protease can cleave a target substrate,
such as for example any one or more proteins of a complement
pathway, and modulate an activity of a complement pathway.
[0374] Exemplary scaffold proteases that can be used to cleave any
one or more complement protein or can be used as a template to make
modifications in the protease to increase substrate specificity or
activity towards any one or more of the complement proteins are
described. Protease scaffolds include any non-complement protease
that is any one of the serine, cysteine, aspartic, metallo-, or
threonine classes of proteases. Exemplary scaffold proteases are
listed in Table 14 and described herein. Protease scaffolds include
allelic variant and isoform of any one protein, including the
scaffold protease polypeptides exemplified in any of SEQ ID NOS: 2,
4, 8, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,
152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,
204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,
230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254,
256, 258, 260, 262, 264, 266, 268, 269, 270, 272, 274, 276, 278,
280, 282, 284, 286, 287, 289, 291, 293, 295, 297, 373, 375, 377,
379, 381, 383, 385, 387, 544,545, 547, 549, and 551.
[0375] A scaffold protein or scaffold 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. Table 14 sets forth
exemplary scaffold proteases (see also 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. TABLE-US-00014
TABLE 14 Exemplary Scaffold Proteases Peptidase unit Nucl. AC
Protein SEQ ID Signal/Propep. (active site Merops Code Name Gene
NO: Synonym AC NO: NO: sequence residues) S01.010 granzyme GZMB
M17016 HLP, P10144 3, 4 1-18/19-20 21-247 B, human- CCPI, (64, 108,
203) type CGL1, CSPB, SECT, CGL-1, CSP-B, CTLA1, CTSGL1 S01.011
Testisin PRSS21 NM_006799 ESP-1, NP_006790 70, 71 1-19/20-41 42-288
(v1) TEST1 NP_659205 (V1); 72, (82, 137, 238) NM_144956 NP_659206
73 (V2); (v2) 74, 75 NM_144957 (V2) (v3) S01.015 trypstase TPSB1
NM_003294 TPS1, NP_003285 76, 77 1-18/19-30 31-274 beta 1 TPS2,
(74, 121, 224) (Homo TPSAB1, sapiens) alpha II (III) S01.017
kallikrein KLK5 NM_012427 SCTE, NP_036559 78, 79 1-22/ 67-292 hk5
KLKL2, (108, 153, 245) KLK-L2 S01.019 Corin NM_006587 CRN,
NP_006578 80, 81 802-1037 ATC2, (843, 892, 985) Lrp4, TMPRSS10
S01.020 kallikrein KLK12 NM_019598 KLK-L5 NP_062544 82, 83 1-17/
22-248 12 (v1) NP_665901 (V1); 84, (843, 892, 985) NM_145894
NP_665902 85 (V2); (v2) 86, 87 NM_145895 (V3) (v3) S01.021 DESC1
AF064819 AAF04328 88, 89 191-422 oritease (231, 276, 372) S01.028
tryptase TPSG1 NM_012467 TMT, NP_036599 90, 91 1-19/ 38-272 gamma 1
trpA, (78, 125, 222) PRSS31 S01.029 kallikrein KLK14 NM_022046
KLK-L6 NP_071329 92, 93 1-18/19-24 25-249 hK14 (67, 111, 204)
S01.033 hyaluronan- HABP2 NM_004132 FSAP, NP_004123 94, 95 1-23/
314-557 binding HABP, (362, 411, 509) serine PHBP, protease HGFAL
(HGF activator- like protein) S01.034 transmembrane TMPRSS4
NM_019894 MT-SP2, NP_063947 96, 97 205-436 protease, (v1) TMPRSS3
NP_899070 (V1); 98, (245, 290, 387) serine 4 NM_183247 99 (V2) (v2)
S01.054 tryptase TPSD1 NM_012217 MCP7L1, NP_036349 100, 101
1-18/19-30 31-235 delta 1 MMCP- (74, 121, 224) (Homo 7L, sapiens)
MGC95428 S01.074 Marapsin NM_031948 PRSS27, NP_114154 102, 103
1-22/23-34 35-279 CAPH2 (75, 124, 229) S01.075 Tryptase BC036846
PRSS33, AAN04055 104, 105 37-281 homologue EOS (77, 126, 231) 2
(Homo sapiens) S01.076 Tryptase Putative 106, 107 67-304 homologue
Only (107, 213, 259) 3 (Homo AC005570 sapiens) (Cosmid 407D8)
S01.077 tryptase chromosome 21 (Homo sapiens) S01.079 transmembrane
TMPRSS3 NM_024022 DFNB8, NP_076927 108, 109 217-451 protease, (vA)
DFNB10, NP_115777 (VA); 110, (257, 304, 401) serine 3 NM_032401
ECHOS1, NP_115780 111 (vB); (vB) TADG12 NP_115781 112, 113
NM_032404 (vC); 114, (vC) 115 (vD) NM_032405 (vD) S01.081
kallikrein NM_023006 ACO, NP_075382 116, 117 1-16/17-21 22-256 hK15
(v1) HSRNASPH NP_612630 (v1); 118, (62, 106, 209) (Homo NM_138563
NP_612631 119 (v2); sapiens) (v2) NP_059979 120, 121 NM_138564
(v3); 122, (v3) 123 (v4) NM_017509 (v4) S01.085 Mername- BC035384
FLJ16649, AAH35384 124, 125 1-241 AA031 MGC35022, (56, 101, 195)
peptidase TRYX3, (deduced UNQ2540 from ESTs by MEROPS) S01.087
membrane- AB048796 BAB39741 126, 127 321-556 type mosaic (361, 409,
506) serine protease S01.088 mername- Putative CAC12709 128 10-142
AA038 Only (50, 101) peptidase AL136097 (RP11- 62C3 clone) S01.098
mername- Putative AAH41609 129, 130 33-202 AA128 Only (50, 152)
peptidase BC041609 (deduced from ESTs by MEROPS) S01.127 cationic
PRSS1 NM_002769 TRP1, NP_002760 131, 132 1-15/16-23 24-246 trypsin
TRY1, (63, 107, 200) (Homo TRY4, sapiens- TRYP1 type 1) (cationic)
S01.131 Neutrophils ELA2 NM_001972 NE, HLE, NP_001963 133, 134
1-27/28-29 30-249 elastase HNE, (70, 117, 202) PMN-E S01.132
mannan- AF284421 AAK84071 135, 136 1-19/ 449-710 (497, binding 553,
664) lectin- associated serine protease-3 S01.133 cathepsin G CTSG
NM_001911 CG, NP_001902 137, 138 1-18/19-20 21-245 MGC23078 (64,
108, 201) S01.134 myeloblastin PRTN3 NM_002777 MBT, P29, NP_002768
139, 140 1-25/26-27 28-250 (proteinase ACPA, (71, 118, 203) 3)
AGP7, PR- 3, C- ANCA S01.135 granzyme A GZMA NM_006144 HFSP,
NP_006135 141, 142 1-26/27-28 29-261 CTLA3 (69, 114, 212) S01.139
granzyme M GZMM NM_005317 MET1, NP_005308 143, 144 1-23/24-25
26-256 LMET1 (66, 111, 207) S01.140 chymase CMA1 NM_001836 CYH,
NP_001827 145, 146 1-19/21-21 22-247 (human- MCT1 (66, 110, 203)
type) S01.143 tryptase TPS1 NM_003294 TPS1, NP_003285 147, 148
1-18/19-30 31-274 alpha (1) TPS2, (74, 121, 224) TPSB1, alpha II
S01.146 granzyme K GZMK NM_002104 TRYP2 NP_002095 149, 150
1-24/26-26 27-261 (67, 116, 214) S01.147 granzyme H GZMH NM_033423
CCP-X, NP_219491 151, 152 1-18/19-20 21-246 CGL-2, (64, 108, 202)
CSP-C, CTLA1, CTSGL2 S01.152 chymotrypsin B CTRB1 M24400 CTRB,
P17538 7, 8 1-18 34-263 MGC88037 (75, 120, 213) S01.153 pancreatic
ELA1 NM_001971 NP_001962 153, 154 1-8/9-18 19-256 elastase (63,
111, 206) S01.154 pancreatic NM_005747 ELA3 NP_005738 155, 156
1-15/16-28 29-270 endopeptidase (73, 123, 217) E (A) S01.155
pancreatic M16652 AAA52380 157, 158 1-16/7-28 29-269 elastase II
(73, 121, 216) (IIA) S01.156 Enteropeptidase PRSS7 NM_002772 ENTK
NP_002763 159, 160 785-1019 (825, 876, 971) S01.157 chymotrypsin C
NM_007272 CLCR NP_009203 161, 162 1-16/17-29 30-268 (74, 121, 216)
S01.159 Prostasin PRSS8 NM_002773 NP_002764 163, 164 1-29/30-32
45-288 (85, 134, 238) S01.160 kallikrein 1 KLK1 NM_002257 hK1,
NP_002248 165, 166 1-18/19-24 25-261 KLKR, (65, 120, 214) Klk6
SO1.161 kallikrein KLK2 NM_005551 hK2, NP_005542 167, 168
1-18/19-24 25-260 hK2 (Homo (v1) KLK2A2, NP_001002231 (v1); 169,
(65, 120, 213) sapiens) NM_001002231 MGC12201 NP_001002232 170
(v2); (v2) 171, 172 NM_001002232 (v3) (v3) S01.162 kallikrein 3
KLK3 NM_001648 APS, PSA, NP_001639 173, 174 1-17/18-24 25-260 (v1)
hK3, NP_001025218 (v1); 175, (65, 120, 213) NM_001030047 KLK2A1
NP_001025219 176 (v3); (v3) NP_001025220 177, 178 NM_001030048
NP_001025221 (v4); 179, (v4) 180 (v5); NM_001030049 181, 182 (v5)
(v6) NM_001030050 (v6) S01.174 Mesotrypsin PRSS3 NM_002771 MTG,
NP_002762 183, 184 1-24/ 24-246 TRY3, (63, 107, 200) TRY4, PRSS4
S01.205 pancreatic ELA3B NM_007352 NP_031378 185, 186 1-15/16-28
29-270 endopeptidase (73, 123, 217) E form B (B) S01.206 pancreatic
NM_015849 MGC97052 NP_056933 187, 188 1-16/17-28 29-269 elastase II
(73, 121, 216) form B (Homos sapiens) (IIB) S01.211 coagulation F12
NM_000505 HAF NP_000496 189, 190 1-19/ 373-615 factor XIIa (412,
461, 563) S01.212 plasma KLKB1 NM_000892 KLK3 NP_000883 191, 192
1-19/ 391-628
kallikrein (434, 483, 578) S01.213 coagulation F11 NM_000128 FXI
NP_000119 193, 194 1-18/ 388-625 factor XIa (v1) NP_062505 (v1);
195, (431, 480, 575) NM_019559 196 (v2) (v2) S01.214 coagulation F9
NM_000133 FIX, PTC NP_000124 197, 198 1-28/29-46 227-461 factor IXa
HEMB, (267, 315, 411) GLA domain S01.215 coagulation F7 NM_000131
NP_000122 199, 200 1-20/21-60 213-454 factor VIIa (v1) NP_062562
(v1); 201, (253, 302, 404) NM_019616 202 (v2) (v2) S01.216
coagulation F10 NM_000504 FX, FXA NP_000495 203, 204 1-31/32-40
235-469 factor Xa (276, 322, 419) S01.217 Thrombin F2 NM_000506 PT
NP_000497 205, 206 1-24/25-43 364-620 (406, 462, 568) S01.218
protein C PROC NM_000312 PROC1, NP_000303 207, 208 1-32/33-42
212-452 (activated) protein C (253, 299, 402) S01.223 Acrosin ACR
NM_001097 NP_001088 209, 210 1-19 43-292 (88, 142, 240) S01.224
Hepsin HPN NM_182983 TMPRSS1 NP_892028 211, 212 163-407 (v1)
NP_002142 (v1); 213, (203, 257, 353) NM_002151 214 (v2) (v2)
S01.228 hepatocyte HGFAC NM_001528 HGFA NP_001519 215, 216
1-35/36-372 408-648 growth (447, 497, 598) factor activator S01.231
u- PLAU NM_002658 ATF, NP_002649 217, 218 1-20/ 179-426 plasminogen
UPA, (224, 275, 376) activator URK, uPA (uPA) S01.232 t- PLAT
NM_000930 TPA, TPA, NP_000921 219, 220 1-23/24-32 311-562
plasminogen (v1) NP_000922 (v1); 221, and 33-35 (357, 406, 513)
activator NM_000931 DKFZp686 NP_127509 222 (V2), (tPA) (v2) I03148
223, 224 NM_033011 (V3) (v3) S01.233 Plasmin PLG NM_00301 DKFZp779
NP_000292 225, 226 1-19/20-97 581-810 M0222 (622, 665, 760) S01.236
Neurosin KLK6 NM_002774 hK6, Bssp, NP_002765 227, 228 1-16/17-21
22-244 (vA) Klk7, NP_001012982 (vA); 229, (62, 106, 197)
NM_001012964 SP59, NP_001012983 230 (vB); (vB) ZYME, NP_001012984
231, 232 NM_001012965 PRSS9, (vC); 233, (vC) PRSS18, 234 (vD)
NM_001012966 MGC9355, (vD) NEUROSIN S01.237 Neurotrypsin PRSS12
NM_003619 BSSP3, NP_003610 235, 236 1-20/ 631-875 BSSP-3, (676,
726, 825) MGC12722, MOTOPSIN S01.242 tryptase TPSB1 NM_024164 TPS2,
NP_077078 237, 238 1-30/ 31-268 beta 2 TPSB1, (Homo tryptaseC
sapiens) (I) S01.244 Neuropsin KLK8 NM_007196 NP, HNP, NP_009127
239, 240 1-28/29-32 33-258 (v1) NRPN, NP_653088 (v1); 241, (73,
120, 212) NM_144505 PRSS19, NP_653089 242 (v2), (v2) TADG14
NP_653090 243, 244 NM_144506 (v3); 245, (v3) 246 (v4) NM_144507
(v4) S01.246 kallikrein KLK10 NM_002776 NES1, NP_002767 247, 248
1-30/ 35-276 hK10 (v1) PRSSL1 NP_665895 (v1); 249, (86, 137, 229)
(Homo NM_145888 250 (v2) sapiens) (v2) S01.247 Epitheliasin TMPRSS2
NM_005656 PRSS10 NP_005647 251, 252 256-491 (296, 345, 441) S01.251
Prostase KLK4 NM_004917 ARM1, NP_004908 253, 254 1-26/27-30 31-254
EMSP, (71, 116, 207) PSTS, EMSP1, KLK-L1, PRSS17 S01.252 Brain
serine NM_022119 BSSP-4 NP_071402 255, 256 1-32 50-292 proteinase 2
MGC9599, (90, 141, 242) SP001LA, hBSSP-4 S01.256 Chymopasin CTRL
NM_001907 CTRL1, NP_001898 257, 258 1-18/19-33 34-264 MGC70821 (75,
121, 214) S01.257 kallikrein KLK11 NM_006853 TLSP, NP_006844 259,
260 22-250 1I 3 (v1) PRSS20, 4 (v1); 261, 1-50/51-53 (62, 110, 203)
NM_144947 MGC33060 NP_659196 262 (v2) (v2) S01.258 anionic PRSS2
NM_002770 TRY2, NP_002761 263, 264 1-15/16-23 24-246 trypsin TRY8,
(63, 107, 200) (Homo TRYP2 sapiens) (II) S01.291 LOC144757 Putative
MGC57341 AAH48112 265, 266 78-319 peptidase BC048112 (122, 171,
268) (Homo sapiens) S01.292 Mername- BN000133 CAD67985 267, 268
1-19 175-406 AA169 (215, 260, 356) peptidase S01.294 Mername-
Putative 269 AA171 No DNA peptidase S01.298 Mername- Putative TRY6
AAC80208 270 24-246 AA174 no DNA (63, 107, 200) peptidase seq
S01.299 Mername- NM_198464 NP_940866 271, 272 68-302 AA175 (108,
156, 250) peptidase S01.300 stratum KLK7 NM_005046 SCCE, NP_005037
273, 274 1-22/23-29 30-250 corneum (v1) PRSS6 NP_644806 (v1); 275,
(70, 112, 205) chymotryptic NM_139277 276 (v2) enzyme (v2) S01.301
trypsin-like NM_004262 HAT NP_004253 277, 278 187-471 enzyme, (227,
272, 368) respiratory (Homo sapiens) S01.302 Matripase ST14
AF118224 HAI, AAD42765 1, 2 615-855 MTSP1, (656, 711, 805) SNC19,
MT-SP1, MTSP-1, PRSS14, TADG-15 S01.306 kallikrein KLK13 NM_015596
KLKL4, NP_056411 279, 280 1-16/ 36-263 hK13 KLK-L4, (76, 124, 218)
DKFZP58 6J1923 S01.307 kallikrein KLK9 NM_012315 KLKL3, NP_036447
281, 282 1-15/ 23-250 hK9 KLK-L3 (63, 111, 204) (human numbering)
S01.308 Mername- NM_153609 NP_705837 283, 284 49-283 AA035 (89,
140, 234) peptidase S01.309 umbilical NM_007173 SIG13, NP_009104
285, 286 1-23/ 95-383 vein SPUVE, (175, 246, 316) proteinase
ZSIG13, MGC5107 S01.311 LCLP Peptide P34168 287 1-26 proteinase
fragment (0) (LCLP (N- No DNA terminus)) S01.313 Spinesin TMPRSS5
NM_030770 NP_110397 288, 289 218-455 (258, 308, 405) S01.318
Mername- MPN2 NM_183062 NP_898885 290, 291 1-33/ 53-288 AA178 (93,
143, 238) peptidase S01.320 Mername- OVTN BN000120 CAD66452 292,
293 1-23/ 52-301 AA180 (92, 142, 240) peptidase S01.322 Mername-
OVCH1 BN000128 CAD67579 294, 295 1-17/ 8-298 AA182 (87, 139, 237)
peptidase S01.414 Mername- Putative BAC11431 296, 297 1-177 AA122
AK075142 (12, 64, 168) peptidase (deduced from ESTs by MEROPS)
C01.032 Cathepsin L CTSL Y14734 P07711 372, 373 1-17/18-113 113-333
(132, 138, 276, 300) C01.009 Cathepsin V CTSL2 U13665 O60911 374,
375 1-17/18-113 114-334 (132, 138, 277, 301) C01.036 Cathepsin K
CTSK S93414 P43235 376, 377 1-15/16-114 115-329 (133, 139, 276,
296) C01.034 Cathepsin S CTSS AJ007331 P25774 378, 379 1-16/17-114
115-331 (133, 139, 278, 298) C01.018 Cathepsin F CTSF M14221 Q9UBX1
380, 381 1-19/20-270 271-484 (289, 295, 431, 451) C01.060 Cathepsin
B CTSB M15203 P07858 382, 383 1-17/18-79 80-331 (102, 108, 278,
298) C01.001 Papain M84342 P00784 384, 385 1-18/19-133 135-342
(158, 292, 308) C01.075 Cruzain Y14734 P25779 386, 387 123-467/
124-334 (Cruzapain) (147, 284, 304, A02.001 HIV-1 HIV-1 P03366 544
protease retropepsin; (aa 500-598) HIV-1 PR A02.015 RSV avian
P03322 545 protease myeloblastosis (aa 578-701) virus retropepsin;
avian sarcoma virus endopeptidase; retropepsin M10.005 Matrix MMP3
X05232 collagenase CAA28859.1 546, 547 metalloprotease-3 activating
protein; MMP-3; stromelysin 1; transin M12.209 ADAM9 ADAM9
NM_003816 MDC9 NP_003807 548, 549 endopeptidase M12.210 ADAM10
ADAM10 NM_003816 MADM NP_001101 550, 551 endopeptidase
[0376] In some embodiments, the protease scaffold is a granzyme B,
granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil elastase,
chymase, alpha-tryptase, beta-trypsase I or II, chymotrypsin,
collagenase, factor XII, factor XI, factor CII, factor X, thrombin,
protein C, u-plasminogen activator (u-PA), t-plasminogen activator
(t-PA), plasmin, plasma kallikrein, chymotrypsin, trypsin, a
cathepsin, papain, cruzain, a metalloprotease and allelic
variations, isoforms and catalytically active portions thereof.
Such proteases can be used in the methods provided herein to target
one or more target substrates of a complement pathway. Such
proteases also can be modified to have altered specificity or
selectivity for any one or more protein components of the
complement pathway and/or to modulate an activity of a complement
protein or pathway. The proteases or modified proteases can be used
in the methods provided herein to modulate complement activation,
and hence can be used as therapeutic to treat any
complement-mediated disease or disorder. In some embodiments, the
protease scaffold is MT-SP1. Modifications of amino acids in MT-SP1
can be made to alter the specificity and/or selectivity for a
complement protein target substrate.
[0377] 1. Modified Scaffold Proteases
[0378] Virtually every aspect of a protease can be re-engineered,
including the enzyme substrate sequence specificity,
thermostability, pH profile, catalytic efficiency, oxidative
stability, and catalytic function. Provided herein are modified
proteases that exhibit increased specificity and/or selectivity to
any one or more complement proteins compared to a scaffold
protease. Proteases can be modified using any method known in the
art for modification of proteins. Such methods include
site-directed and random mutagenesis. Assays such as the assays for
biological function of complement activation provided herein and
known in the art can be used to assess the biological function of a
modified protease to determine if the modified protease targets a
substrate for cleavage and inactivation. Exemplary methods to
identify a protease and the modified proteases are provided
herein.
[0379] For example, any of a variety of general approaches for
protein-directed evolution based on mutagenesis can be employed.
Any of these, alone or in combination can be used to modify a
polypeptide such as a protease to achieve altered specificity
and/or selectivity to a target substrate. Such methods include
random mutagenesis, where the amino acids in the starting protein
sequence are replaced by all (or a group) of the 20 amino acids
either in single or multiple replacements at different amino acid
positions on the same molecule, at the same time. Another method,
restricted random mutagenesis, introduces either all or some of the
20 amino acids or DNA-biased residues. The bias is based on the
sequence of the DNA and not on that of the protein in a stochastic
or semi-stochastic manner, respectively, within restricted or
predefined regions of the protein known in advance to be involved
in the biological activity being "evolved." Exemplary methods for
generating modified proteases are described in related U.S.
application Ser. No. 10/677,977, herein incorporated by reference
in its entirety. Additionally, any method known in the art can be
used to modify or alter a protease polypeptide sequence.
[0380] Among the modified polypeptides provided herein are
proteases with one or more modifications compared to a scaffold
protease. Modified protease polypeptides include 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. Modified proteases provided herein retain
their protease ability but display altered (i.e. enhanced)
specificity towards any one or more complement protein target
substrates compared to a natural substrate of the protease and/or
display an altered selectivity for any one or more proteins of the
complement system. A modified protease specific for any one or more
of the complement proteins can be generated rationally or
empirically by: (a) rationally targeting sites that complement a
cleavage sequence of a target complement substrate recognized by a
known protease, such as for example, complement Factor I or, (b)
empirically testing a library of modified proteases in functional
assays for inactivation of the complement cascade.
[0381] a. Rational Modification
[0382] Methods are provided to rationally modify a protease to
increase the specificity and/or selectivity to a target substrate,
such as to any one or more complement proteins. In such a method, a
cleavage sequence of the target substrate is known. 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 or helices), as determined by atomic structure or
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 inactivate the
protein, based on its known function. Cleavage sequences are e.g.,
four residues in length to match the extended substrate specificity
of many proteases, but can be longer or shorter.
[0383] Factor I is a serine protease that functions as a natural
regulator of complement activation by cleaving C3b and C4b. Factor
I cleaves and inactivates C3b and C4b after activation by the
convertase on C3 and C4, respectively, and release of C3a and C4a.
The peptide cleavage sequences recognized by Factor I include LPSR
(SEQ ID NO: 388) and SLLR (SEQ ID NO: 389) in C3 and HRGR (SEQ ID
NO: 390) in C4 (see e.g., Davis et al., (1982) Biochemistry
21:5745); Harrison et al., (1980) Mol. Immunology 17:9; Kai et al.,
(1980) J Immunol. 125:2409). Provided herein are methods to
rationally design the specificity binding pocket of a protease to
recognize and specifically cleave Factor I substrates, including C3
and C4 as well as iC3, C3b, iC4, and C4b, thereby inhibiting
complement activation. Products that cleave the Factor I substrates
are provided.
[0384] As an example, modified proteases with altered specificity
for a Factor I target sequence are generated by a structure-based
design approach. Each protease has a series of amino acids that
line the active site pocket and make direct contact with a
substrate. The amino acids that line the active site pocket of a
protease are designated S1-S4 and the respective substrate contact
sites are designated P1-P4. The identity of the amino acids that
contain the S1-S4 pocket of the active site determines the
substrate specificity of that particular pocket. Amino acids that
form the substrate binding pocket of exemplary proteases are
described herein. Generally, the substrate specificity of a
protease is known or can be determined, 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). In
one example, the amino acids that participate in the S1-S4
substrate binding pocket of MT-SP1 are as follows (based on
chymotrypsin numbering): 189, 190, 191, 192, 216 and 226 (S1); 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, the amino
acids in a papain family protease that contribute to P2 specificity
(standard papain numbering) include amino acids 66-68, 133, 157,
160, and/or 215. Modifying any one or more of the amino acids that
make up the S1-S4 active site of a protease would alter the
substrate specificity of that protease. For example, a mutation at
position 99 in the S2 pocket of a serine protease, such as for
example an MT-SP1 protease, to a smaller amino acid confers a
preference for larger hydrophobic residues in the P2 substrate
position. Using this process of selective mutagenesis, proteases
with substrate specificities to a Factor I cleavage sequence can be
generated.
[0385] In one embodiment, point mutations can be made in the amino
acids that contribute to the specificity binding pocket of a
protease, particularly in any one or more S1-S4 amino acid residues
that contribute to P1-P4 substrate specificity. Generally, the
amino acid residues that contribute to the P1-P4 specificity of a
protease can be rationally replaced so that the target substrate
cleavage site recognized by Factor I is produced. In one example, a
protease can be modified to recognize the target cleavage sequence
LPSR/KI where any one or more amino acids in the S4 position can be
modified to recognize a leucine at the P4 position, any one or more
amino acids in the S3 position can be modified to recognize a
proline at the P3 position, any one or more amino acids in the S2
position can be modified to recognize a serine at the P2 position,
and any one or more amino acids in the S1 position can be modified
to recognize an arginine at the P1 position. In another example, a
protease can be modified to recognize the target cleavage sequence
SLLR/SE where any one or more amino acids in the S4 position can be
modified to recognize a serine at the P4 position, any one or more
amino acids in the S3 position can be modified to recognize a
leucine at the P3 position, any one or more amino acids in the S2
position can be modified to recognize a leucine at the P2 position,
and any one or more amino acids in the S1 position can be modified
to recognize an arginine at the P1 position. In an additional
example, a protease can be modified to recognize the target
cleavage sequence HRGR/TL where any one or more amino acids in the
S4 position can be modified to recognize a histidine at the P4
position, any one or more amino acids in the S3 position can be
modified to recognize an arginine at the P3 position, any one or
more amino acids in the S2 position can be modified to recognize a
glycine at the P2 position, and any one or more amino acids in the
S1 position can be modified to recognize an arginine at the P1
position. In some cases, mutations in serine proteases have shown
that each of the sub-sites that form the active site (S1-S4)
function independently of one another, such that modification of
specificity at one sub-site has little influence on specificity at
adjacent sub-sites. Thus, engineering substrate specificity and/or
selectivity throughout the extended binding site can be
accomplished in a step-wise manner.
[0386] For example, a protease with low specificity for a residue
at a particular binding site or for a particular sequence is
altered in its specificity by making point mutations in the
substrate sequence binding pocket. In some cases, the resulting
mutant has a greater than 1.5, 2, 5, 8, 10-fold or greater increase
in specificity at a site for a particular sequence than does
wildtype. In another embodiment, the resulting mutant has a greater
than 100-fold increase in specificity at a site for a particular
sequence than does wildtype. In another embodiment, the resulting
mutant has an over 1000-fold increase in specificity at a site or
for a particular sequence than does a wildtype.
[0387] In one exemplary embodiment, wildtype MT-SP1 protease having
a P1-P4 preference for a target cleavage sequence of Arg/Lys at P4,
basic residues or Gin at P3, small residues at P2, and Arg or Lys
at P1 can be modified so that the Factor I cleavage sequence of
LPSR, SLLR, or HRGR is recognized by an MT-SP1 protease (see Table
15). In such an example, the S1 position of the modified MT-SP1 is
unchanged since the arginine residue at the P1 site is conserved
between the target substrate cleavage site of MT-SP1 and the Factor
I cleavage sites. Amino acid residues in any one of more of the
S2-S4 sub-sites of MT-SP1 can be modified alone or in combination
to increase the specificity and/or selectivity for a Factor I
cleavage sequence. For example, to modify an MT-SP1 set forth in
SEQ ID NO:2 or 10 to have increased specificity and/or selectivity
for a SLLR Factor I cleavage sequence in C3b, modifications in the
S4 position of MT-SP1 to recognize a serine in the P4 position of
the substrate can include amino acid modifications Q174H, D217Q,
D217N, D217H, D96A, D96V, D96F, D96S, and/or D96T, based on
chymotrypsin numbering; modification in the S3 position of MT-SP1
to recognize a leucine in the P3 position of the substrate can
include amino acid modifications Q192L, Q192I, Q192F, and/or Y146F,
based on chymotrypsin numbering; and/or modifications in the S2
position of MT-SP1 to recognize a leucine in the P2 position of the
substrate can include amino acid modifications F99A, F99V, F99S,
and/or F99G, based on chymotrypsin numbering. In another example,
to modify an MT-SP1 set forth in SEQ ID NO: 2 or 10 to have
increased specificity and/or selectivity for a LPSR Factor I
cleavage sequence in C3b, modifications in the S4 position of
MT-SP1 to recognize a leucine in the P4 position of the substrate
can include amino acid modifications W215F, W215Y, Q174F, Q174V,
Q174L, Q174Y, and/or M180E, based on chymotrypsin numbering;
modifications in the S3 position of MT-SP1 to recognize a proline
in the P3 position of the substrate can include amino acid
modifications Q192K, Q192R, Q192R, based on chymotrypsin numbering;
and/or modifications in the S2 position of MT-SP1 to recognize a
serine in the P2 position of the substrate can include amino acid
modifications F99Y, based on chymotrypsin numbering. In an
additional example, to modify an MT-SP1 set forth in SEQ ID NO: 2
or 10 to have increased specificity and/or selectivity for a HRGR
Factor I cleavage sequence in C4b, modifications in the S4 position
of MT-SP1 to recognize a histidine in the P4 position of the
substrate can include amino acid modifications W215F, W215Y, Q174A,
Q174V, Q174F, Q174R, and/or Q174K, based on chymotrypsin numbering;
modifications in the S3 position of MT-SP1 to recognize an arginine
in the P3 position of the substrate can include amino acid
modifications D217A, D217V, and/or Q192E, based on chymotrypsin
numbering; and/or modification is the S2 position of MT-SP1 to
recognize a glycine in the P2 position of the substrate can include
amino acid modifications F99W, F99Y, and/or F99D, based on
chymotrypsin numbering. Exemplary modifications of an MT-SP1
protease scaffold are summarized in Table 15. Combinations of
modifications of the noted positions also are contemplated. Any
method known in the art to effect mutation of any one or more amino
acids in a target protein can be employed. Methods include standard
site-directed mutagenesis (using e.g., a kit, such as kit such as
QuikChange available from Stratagene) of encoding nucleic acid
molecules, or by solid phase polypeptide synthesis methods.
TABLE-US-00015 TABLE 15 Exemplary modifications in MT-SP1 to alter
target specificity to Factor I cleavage sequence S4 S3 S2 D96 Q174
M180 W215 D217 Y146 Q192 F99 SLLR A, V, F, S, T H Q, N, H F L, I, F
A, V, S, G LPSR F, V, L, Y E F, Y K, R, Y Y HRGR A, V, F, R, K F, Y
A, V E W, Y
[0388] i. Synthesis of Positional Scanning Libraries and Screening
using Fluorescence
[0389] A protease, modified at any one or more of the S1-S4
subsites can be verified for P1-P4 substrate specificity at any
given sub-site using a positional scanning synthetic combinatorial
library (PS-SCL) containing a combinatorial fluorogenic substrate
library (Harris et al., (2000) PNAS 97:7754; US 2004/0175777; US
2004/0146938). A PS-SCL strategy allows for the rapid and facile
determination of proteolytic substrate specificity at any one or
more S1-S4 active site sub-sites. A PS-SCL strategy involves the
use of libraries of peptides whereby one position in the library is
held constant, while the remaining positions are composed of all
combinations of amino acids used to prepare the library. The use of
a combinatorial fluorogenic peptide substrate library, such as for
example a 7-amino-4-methylcoumarin (AMC) fluorogenic peptide
substrate or a 7-amino-4-carbamoylmethylcoumarin (ACC) fluorogenic
peptide substrate, can be used to assay for the activity of a
modified protease whereby a fluorogenic moiety is released from a
peptide substrate upon action of the protease. Those of skill in
the art will appreciate that these methods provide a wide variety
of alternative library formats. In one example, a protease can be
profiled with a P1-diverse library. A P1-diverse tetrapeptide
library contains ACC- or AMC-fluorogenic tetrapeptides whereby the
P1 position is systematically held constant while the P2, P3, and
P4 positions contain an equimolar mixture of any one or more of the
20 amino acids. An ACC P1-fixed library allows for the verification
of the P4, P3, and P2 specificities of any one of the modified
proteases. In another example, fixing the P2-position as a large
hydrophobic amino acid can circumvent preferential internal
cleavage by papain-fold proteases and lead to proper register of
the substrate sequence. Determination and consideration of
particular limitations relevant to any particular enzyme or method
of substrate sequence specificity determination are within the
ability of those of skill in the art.
[0390] Those of skill in the art will recognize that many methods
exist to prepare the peptides. In an exemplary embodiment, the
substrate library is screened by attaching a fluorogenically tagged
substrate to a solid support. In one example, the fluorogenic
leaving group from the substrate peptide is synthesized by
condensing an N-Fmoc coumarin derivative, to acid-labile Rink
linker to provide ACC resin (Backes et al., (2000) Nat Biotechnol.
18:187). Fmoc-removal produces a free amine. Natural, unnatural and
modified amino acids can be coupled to the amine, which can be
elaborated by the coupling of additional amino acids. In an
alternative embodiment, the fluorogenic leaving group can be
7-amino-4-methylcoumarin (AMC) (Harris et al., (2000) PNAS
97:7754). After the synthesis of the peptide is complete, the
peptide-fluorogenic moiety conjugate can be cleaved from the solid
support, or alternatively, the conjugate can remain tethered to the
solid support.
[0391] Typically, a method of preparing a fluorogenic peptide or a
material including a fluorogenic peptide includes: (a) providing a
first conjugate containing a fluorogenic moiety covalently bonded
to a solid support; (b) contacting the first conjugate with a first
protected amino acid moiety and an activating agent, thereby
forming a peptide bond between a carboxyl group and the amine
nitrogen of the first conjugate; (c) deprotecting, thereby forming
a second conjugate having a reactive amine moiety; (d) contacting
the second conjugate with a second protected amino acid and an
activating agent, thereby forming a peptide bond between a carboxyl
group and the reactive amine moiety; and (e) deprotecting, thereby
forming a third conjugate having a reactive amine moiety. In an
exemplary embodiment, the method further includes: (f) contacting
the third conjugate with a third protected amino acid and an
activating agent, thereby forming a peptide bond between a carboxyl
group and the reactive amine moiety; and (e) deprotecting, thereby
forming a fourth conjugate having a reactive amine moiety.
[0392] For amino acids that are difficult to couple (e.g., Ile,
Val, etc.), free, unreacted amine can remain on the support and
complicate subsequent synthesis and assay operations. A specialized
capping step employing the 3-nitrotriazole active ester of acetic
acid in DMF efficiently acylates the remaining aniline. The
resulting acetic-acid capped coumarin that can be present in
unpurified substrate sequence solution is generally not a protease
substrate sequence.
[0393] Solid phase peptide synthesis in which the C-terminal amino
acid of the sequence is attached to an insoluble support followed
by sequential addition of the remaining amino acids in the sequence
is an exemplary method for preparing the peptide backbone of the
polypeptides provided herein. Techniques for solid phase synthesis
are described by Narany and Merrifield, Solid-Phase Peptide
Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology.
Vol. 2; Special Methods in Peptide Synthesis, Part A., Gross and
Meienhofer, eds. Academic press, N.Y., (1980); and Stewart et al.,
(1984) Solid Phase Peptide Synthesis, 2.sup.nd etd. Pierce Chem.
Co., Rockford, Ill. Solid phase synthesis is most easily
accomplished with commercially available peptide synthesizers
utilizing Fmoc or t-BOC chemistry.
[0394] For example, peptide synthesis can be performed using well
known Fmoc synthesis chemistry. For example, the side chains of
Asp, Ser, Thr, and Tyr are protected using t-butyl and the side
chain of Cys residue using S-trityl and S-t-butylthio, and Lys
residues are protected using t-Boc, Fmoc and 4-methyltrityl.
Appropriately protected amino acid reagents are commercially
available or can be prepared using art-recognized methods. The use
of multiple protecting groups allows selective deblocking and
coupling of a fluorophore to any particular desired side chain.
Thus, for example, t-Boc deprotection is accomplished using TFA in
dichloromethane. Fmoc deprotection is accomplished using, for
example, 20% (v/v) piperidine in DMF or N-methylpyrolidone, and
4-methyltrityl deprotection is accomplished using, for example, 1
to 5% (v/v) TFA in water or 1% TFA and 5% triisopropylsilane in
DCM. A-t-butylthio deprotection is accomplished using, for example,
aqueous mercaptoethanol (10%). Removal of t-buyl, t-boc, and
S-trityl groups is accomplished using, for example
TFA:phenol:water:thioaniso:ethanedithio (85:5:5:2.5:2.5), or
TFA:phenol:water (95:5:5).
[0395] Diversity at any particular position or combination of
positions can be introduced using a mixture of at least two, six,
12, 20 or more amino acids to grow the peptide chain. The mixtures
of amino acids can include any useful amount of a particular amino
acid in combination with any useful amount of one or more different
amino acids. In one embodiment, the mixture is an isokinetic
mixture of amino acids (a mixture in appropriate ratios to allow
for equal molar reactivity of all components).
[0396] Modified proteases can be combined to acquire the
specificity of multiple modified proteases. A mutation at one
residue of a scaffold, which produces specificity at one site, is
combined in the same protease with another mutation at another site
on the scaffold to make a combined specificity protease. Any number
of mutations at discrete sites on the same scaffold can be used to
create a combined specificity protease.
[0397] Modified proteases, such as for example a modified MT-SP1
protease, that match the desired specificity profile, can then be
assayed using individual fluorogenic peptide substrates
corresponding to the desired cleavage sequence. A method of
assaying for a modified protease that can cleave any one or more of
the Factor I cleavage sequences includes: (a) contacting a peptide
fluorogenic sample (containing a Factor I 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 an
ACC- or AMC-tetrapeptide such as Ac-SLLR-AMC or Ac-HRGR-AMC can be
made and incubated with a modified protease and activity of the
protease can be assessed by assaying for release of the fluorogenic
moiety.
[0398] Assaying for a protease in a solution simply requires adding
a quantity of the stock solution of 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.
[0399] While detection of fluorogenic compounds can be accomplished
using a fluorometer, detection also 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.
[0400] 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.
[0401] Modified proteases also are assayed to ascertain that they
will cleave the desired sequence when presented in the context of
the full-length protein. The target substrate proteins containing
Factor I cleavage sites are in the C3 and C4 sequences,
specifically in C3b and C4b which are generated from C3 and C4,
respectively, following convertase activation. Factor I also
cleaves iC3 and iC4 which are altered species forms of C3 and C4.
Methods to assess cleavage of a target protein are described herein
and/or are well known in the art. In one example, a purified
complement protein, C3b, C4b, iC3, or iC4, can be incubated in the
presence or absence of a modified 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 activity of the target protein also is assayed,
such as, for example in a hemolysis assay, using methods described
herein or that are well known in the art, to verify that its
function has been destroyed by the cleavage event.
[0402] b. Empirical Modification
[0403] A library of modified proteases 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). The library of modified proteases can be tested in
functional assays of complement activation to determine if they are
"Hits" for inhibiting complement activation. The target complement
substrate of the modified protease can be identified, and the
peptide cleavage sequence can be determined.
[0404] In one example, any one or more amino acids of a protease
are mutated using any standard 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 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 mutagenic oligonucleotide primer is
synthesized which contains either NNS or NNK-randomization at the
desired codon. The 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.
[0405] 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). In
one example, mutations of MT-SP1 can be in any one or more residues
(based on chymotrypsin numbering) that contribute to substrate
specificity including 195, 102, 57 (the catalytic triad); 189, 190,
191, 192, 216 and 226 (S1); 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.
[0406] To identify those modified proteases that target any one or
more of the complement proteins, a library of modified proteases
generated from a protease scaffold, such as for example an MT-SP1
scaffold, are tested in functional assays of complement activation.
Assays for complement activation are described herein and can
include any one or more of hemolytic assays and/or assays to detect
activation products of one or more of the complement cascades. For
example, enzyme immunoassays or ELISAs can be used to detect the
presence of cleavage products of complement activation such as for
example C4a, C5a, C3b, C3d, and C5-b9. Modified proteases that
inhibit the activation of complement (such as by increasing CH50
levels as determined by a hemolytic assay or decreasing the
detection of a complement cleavage product) can be identified as a
"Hit". In one embodiment, combinations of "Hits" can be made to
further increase the specificity and/or selectivity of a protease
for inhibiting complement activation.
[0407] Modified proteases, such as for example a modified MT-SP1,
that are identified as "Hits" for inhibiting complement activation
in functional assays can be screened to determine the complement
protein target substrate. Assays to detect for cleavage of a
complement protein are described herein. In one example, a purified
complement protein can be incubated in the presence or absence of a
modified protease and analyzed and resolved on an SDS-PAGE gel and
the protein cleavage products can be detected following staining
with a protein stain such as Coomassie Brilliant Blue. Cleavage
products can be excised and the peptide cleavage sequence can be
determined by N-terminal sequencing. Using the identified peptide
cleavage sequence as determined by empirically testing a library of
modified proteases, further modified proteases can be identified
and generated using the rational approach described above for
Factor I cleavage sequences.
[0408] 2. Methods of Assessing Specificity
[0409] Provided herein are methods of assessing substrate
specificity of the resulting scaffold or modified proteases. In one
embodiment, the specificity of any one or more of the S1-S4
sub-sites can be determined using ACC or AMC positional scanning
libraries as discussed above. In another embodiment the specificity
of a scaffold or modified protease for a target substrate compared
to a non-target substrate can be determined using single substrate
kinetic assays, see e.g., Harris, et al. (2000) PNAS, 97:7754. In
specific embodiments, comparison of the specificities of a target
protease and a scaffold protease can be used to determine if the
modified protease exhibits altered, for example, increased,
specificity compared to a scaffold protease.
[0410] 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 scaffold
protease. If the modified protease cleaves fewer target substrates
than the wildtype protease, the modified protease has greater
specificity than the scaffold 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
scaffold protease can be used to assess the fold change in
specificity for a target substrate. The fold change is an increase
in specificity of a modified protease for a target substrate
compared to a scaffold protease that is sufficient to achieve a
predetermined alteration in complement activation or in a
complement-mediated activity. 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-fold or more when compared to the
specificity of a scaffold protein for a target substrate versus a
non-target substrate.
[0411] In one example, a modified protease that matches the desired
specificity profiles, as determined by using positional scanning
substrate libraries, can be assayed using individual peptide
substrates corresponding to the desired target cleavage sequence
compared to a non-target substrate cleavage sequence to determine
the magnitude and change in specificity. 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 such as is described herein, 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 a non-target substrate cleavage
sequence or 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.m/V.sub.max)(1/[S])+1/V.sub.max; where
V.sub.max=[ET]k.sub.cat). The specificity constant
(k.sub.cat/K.sub.m) is a measure of how well a substrate is cut by
a particular protease.
[0412] In one embodiment, a non-target substrate cleavage sequence
can be a native substrate cleavage sequence, such as a cleavage
sequence recognized by wildtype MT-SP1. For example, efficient
auto-activation of MT-SP1 entails recognition and cleavage of an
Arg-Gln-Ala-Arg P4-P1 target sequence. MT-SP1 also can efficiently
activate the proteinase-activated receptor-2 (PAR2), single chain
uPA, and the hepatocyte growth factor/scatter factor. These
extracellular surface-localized proteins display the P4 to P1
target sequences Ser-Lys-Gly-Arg, Pro-Arg-Phe-Lys, and
Lys-Gln-Gly-Arg, respectively, which match closely the MT-SP1
cleavage specificity requirements observed for small peptidic
substrates. In one embodiment, a fluorogenically tagged
tetrapeptide of RQAR (SEQ ID NO: 401), or SLGR (SEQ ID NO: 392), or
PRFK (SEQ ID NO: 402), or KQGR (SEQ ID NO: 403) can be used as a
non-target substrate cleavage sequence.
[0413] In another embodiment, any one or more of the cleavage
sequences of a complement protein can be determined and used as a
desired target cleavage sequence. For example, any one or more of
the Factor I cleavage sequences, such as for example SLLR (SEQ ID
NO: 389), LPSR (SEQ ID NO: 388), and HRGR (SEQ ID NO: 390) can be
used as a fluorogenically tagged tetrapeptide target cleavage
sequence. In another example, the desired cleavage sequence in a
complement protein targeted by any one or more wildtype or modified
protease can be empirically determined by N-terminally sequencing
cleavage products upon cleavage of any one or more of the
complement proteins by a protease. In such an example, any one or
more of the cleavage sequences identified as target cleavage
sequences of a protease provided herein can be used, including
those described in Example 3. Therefore, for example, a
fluorogenically tagged tetrapeptide of GATR (SEQ ID NO: 391), SLGR
(SEQ ID NO: 392), VFAK (SEQ ID NO: 393), REFK (SEQ ID NO: 394),
QHAR (SEQ ID NO: 398), GLAR (SEQ ID NO: 395), RLGR (SEQ ID NO:
396), AEGK (SEQ ID NO: 397), or HRGR (SEQ ID NO: 390) can be used
as a target substrate cleavage sequence.
[0414] In an additional embodiment, a full length complement
protein can be used as a target substrate to assay for protease
specificity compared to a full length native target substrate of a
protease. Further, a full length complement protein can be used to
assess the correlation between substrate specificity and cleavage
by a protease of a full length target substrate versus a four amino
acid P1-P4 substrate cleavage sequence contained within the target
substrate. In one example, a full length C2 protein can be used as
a desired cleavage target of any one or more or the proteases to
assess specificity. In this example, cleavage of C2 by a modified
or scaffold protease can be compared to cleavage of another
full-length substrate, or the cleavage can be compared to a
fluorogenic tetrapeptide cleavage sequence of C2, such as those
described in Example 11 (i.e., GATR, SLGR, or VFAK). 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.
[0415] 3. Protease Polypeptides
[0416] Using the methods described herein, proteases are provided
that cleave any one or more of the complement proteins, whereby
cleavage of the complement protein inhibits complement activation.
As provided herein, a protease polypeptide that cleaves any one or
more of the complement proteins is a non-complement protease. A
protease polypeptide can include the amino acid sequence of a
scaffold protease whose sequence is provided herein, such as in any
one of SEQ ID NOS: 2, 4, 8, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,
91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,
119, 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142,
144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,
170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,
196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,
222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,
248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 269, 270,
272, 274, 276, 278, 280, 282, 284, 286, 287, 289, 291, 293, 295,
297, 373, 375, 377, 379, 381, 383, 385, 387, 544, 545, 547, 549,
and 551, or a catalytically active portion thereof. For example,
the scaffold protease can be a wildtype or prominent form of the
protease. In another embodiment, the scaffold protease can be an
allelic variant of a protease. The scaffold protease is of
mammalian origin, particularly human origin, although the scaffold
protease polypeptide sequence also can be from any one or more of
hamster, mouse, rat, cow, monkey, orangutan, baboon, chimpanzee,
macaque, gibbon or gorilla origin. In other embodiments, the
scaffold protease can be from non-mammalian origin such as from a
plant or parasite.
[0417] In one embodiment, a protease scaffold is modified to have
increased specificity and/or selectivity to any one or more
complement proteins compared to the scaffold protease, while still
encoding a protein that maintains its protease activity. Modified
protease polypeptides include 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. Generally, a modified protease includes any variant in
which residues at a particular position in the sequence have been
substituted by other amino acids, and further include the
possibility of inserting an additional residue or residues between
two residues of the wildtype protease protein as well as the
possibility of deleting one or more residues form the wildtype
protease sequence. Any amino acid substitution, insertion, or
deletion of a wildtype protease sequence is provided herein.
[0418] Provided herein are modified polypeptides that contain a
full length sequence of a scaffold protease, but that contain
modifications in any one or more amino acids that contribute to
substrate specificity and/or selectivity. In one embodiment, the
modified protease polypeptides provided herein have increased
substrate specificity and/or selectivity for any one or more
complement protein compared to a scaffold protease, whereby
cleavage of a complement protein inhibits complement activation. In
another embodiment, a modified protease polypeptide has a greater
specificity for cleavage of a complement protein compared to a VEGF
or VEGFR. In an additional embodiment, the modified proteases
provided herein do not cleave a VEGF or VEGFR. Further, a modified
protease polypeptide provided herein containing modifications in
any one or more amino acids that contribute to substrate
specificity and/or selectivity, also can contain other
modifications in regions that are non-essential to the substrate
specificity of a protease.
[0419] Modified protease polypeptides provided herein also can
contain a catalytically active portion of a full-length scaffold or
unmodified protease. When the polypeptide includes a catalytically
active portion it can include other non-protease portions in
addition thereto as long as the resulting polypeptide exhibits
protease activity at least 1%, 2%, 5%, 10%, 20%, 50%, 100% or more
of the full-length polypeptide. In addition the catalytically
active portion is less than the full-length by at least one amino
acid, and can be less than the full-length protease domain as long
as protease activity is retained. A catalytically active portion of
a protease containing modifications in any one or more amino acids
that contribute to substrate specificity can be an active
single-chain or double-chain form of a scaffold protease. In some
embodiments, a modified protease can be substituted into another
polypeptide, either at the N- or C-terminus, such as in a fusion
protein. In additional embodiments, a modified polypeptide
protease, such as for example a catalytically active portion
thereof of a modified protease, can be inserted to replace the
protease domain from another protease.
[0420] Provided herein are proteases exhibiting increased
specificity and/or selectivity to any one or more complement
proteins having a sequence of amino acids encompassed in any one of
SEQ ID NOS: 298, 299, 300, 302, 304, 305, 306, 311, 312, 313, 314,
315, 316, 317, 318, 319, 320, 321, 322, 326, 328, 330, 332, 334,
335, 338, 340, 344, 660-662 or a fragment thereof that exhibits
complement activity.
[0421] a. MT-SP1 Polypeptides
[0422] Provided herein are MT-SP1 polypeptides that cleave any one
or more of the complement proteins, whereby cleavage of the
complement protein inhibits complement activation. An MT-SP1
polypeptide provided herein can be a full-length MT-SP1 polypeptide
(SEQ ID NO:2) or can be a fragment or partial sequence of
full-length MT-SP1 that exhibits catalytic activity. In one
example, an MT-SP1 polypeptide can be a single-chain protease
domain of MT-SP1 (SEQ ID NO: 10). In another embodiment, an MT-SP1
polypeptide can be any one or more of the allelic variants of
MT-SP1 as set forth in SEQ ID NO:448.
[0423] Also provided herein are modified MT-SP1 polypeptides
containing modifications in any one or more amino acids of a
scaffold MT-SP1 polypeptide using any one of the methods described
herein. In one embodiment, the modifications can be made in a
scaffold MT-SP1 set forth in SEQ ID NO:2, or can be made in any
allelic variant of a wildtype MT-SP1 such as for example any one of
the allelic variants set forth in SEQ ID NO:448. A modified MT-SP1
polypeptide provided herein can constitute a full-length sequence
of an MT-SP1 scaffold, or can constitute a catalytically active
portion thereof of a full-length MT-SP1 scaffold protease. The
modified MT-SP1 exhibits an increase in the specificity and/or
selectivity to any one or more of the complement proteins compared
to a MT-SP1 scaffold, whereby cleavage of the protein inhibits
complement activation.
[0424] Provided herein are modified MT-SP1 polypeptides 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. In one embodiment, a modified MT-SP1
polypeptide includes mutation of any one or more amino acids in the
extended substrate binding pocket of MT-SP1 including, for example,
modification of any one or more amino acid residues of 195, 102, 57
(the catalytic triad); 189, 190, 191, 192, 216 and 226 (S1); 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), based on chymotrypsin numbering.
Modifications in the protease domain of MT-SP1 to alter substrate
specificity and/or selectivity also include modification of any one
or more amino acid residues of 41, 60c, 143, 147, 151 or 221a,
based on chymotrypsin numbering.
[0425] Provided herein are modified MT-SP1 polypeptides where the
following amino acid residues were identified in the protease
domain of MT-SP1 as increasing the specificity and/or selectivity
of cleavage of any one or more complement protein thereby
inhibiting complement activation: 41, 60c, 97, 143, 146, 147, 151,
172, 175, 192, 217, 221a, and 224 based on chymotrypsin numbering.
The modified MT-SP1 polypeptides exhibit increased specificity
and/or selectivity towards any one or more of the complement
components compared to a wildtype MT-SP1 of SEQ ID NO: 2 or a
catalytically active portion thereof set forth in SEQ ID NO:10.
Thus, provided herein are modified MT-SP1 polypeptides exhibiting
increased specificity and/or selectivity towards any one or more
complement components containing a modification at an amino acid
position corresponding to any one or more amino acid residues
selected from among I41, R60c, F97, H143, Y146, G147, G151, L172,
Q175, Q192, D217, Q221a or K224 in an MT-SP1 set forth in SEQ ID
NO: 2 or SEQ ID NO:10, based on chymotrypsin numbering. In one
embodiment, amino acid replacement or replacements correspond to
any of the following positions: I41T, I41A, I41L, I41F, I41D, I41E,
R60.sub.CD, R60.sub.CW, F97D, F97E, F97A, F97W, H143V, Y146N,
Y146D, Y146E, Y146A, Y146W, Y146R, Y146F, G147E, G151L, L172N,
Q175D, Q175E, Q175H, Q175L, Q175F, Q175W, Q175Y, Q175R, Q175K,
Q192A, Q192R, Q192V, Q192F, D217F, Q221.sub.aD, Q221.sub.aL,
Q221.sub.aE, K224A, K224L, K224R, K224N, K224T, K224Y, K224S and
K224F of the protease domain of MT-SP1 based on chymotrypsin
numbering. Table 16 provides non-limiting examples of amino acid
replacements that increase specificity and/or selectivity to any
one or more complement protein, including SEQ ID NOS for exemplary
polypeptide sequences and the encoding nucleic acid sequences.
TABLE-US-00016 TABLE 16 Catalytically Full-length active portion
SEQ ID NOS: SEQ ID NOS: ID # Modification (aa, nt) (aa, nt) CB200
wildtype 1, 2 10, 9 CB12 F97D 406, 482 16, 15 CB13 F97E 407, 483
18, 17 CB16 Y146F 646 592 CB17 L172N 647 593 CB20 Q175D 636 582
CB21 Q175E 635 581 CB31 F97A 408, 484 20, 19 CB32 F97W 409, 485 22,
21 CB40 Y146N 410, 486 24, 23 CB41 Y146D 411, 487 26, 25 CB42 Y146E
412, 488 28, 27 CB43 Y146A 413, 489 30, 29 CB44 Y146W 414, 490 32,
31 CB45 Y146R 415, 491 34, 33 CB62 Q192V 405, 481 14, 13 CB64 Q192R
416, 492 36, 35 CB66 K224A 417, 493 38, 37 CB67 K224F 418, 494 40,
39 CB80 R60.sub.cD 623 569 CB82 R60.sub.cW 621 567 CB268 Q221aD 614
560 CB274 G147E 622 568
[0426] Also provided herein are modified MT-SP1 polypeptides where
the following amino acid residues were identified in the substrate
binding site S1-S4 of MT-SP1 as increasing the specificity and/or
selectivity of cleavage of any one or more complement protein
containing a SLLR/SE Factor I cleavage sequence, thereby inhibiting
complement activation: 96, 174, 217, 146, 192, and 99, based on
chymotrypsin numbering. The modified MT-SP1 polypeptides exhibit
increased specificity and/or selectivity towards a C3b or iC3
complement protein substrate compared to a native target substrate
of wildtype MT-SP1 of SEQ ID NO: 2 or a catalytically active
portion thereof set forth as SEQ ID NO:10. In one embodiment, amino
acid replacement or replacements correspond to any of the following
positions: D96A, D96V, D96F, D96S, D96T, Q174H, D217Q, D217N,
D217H, Q192L, Q192I, Q192F, F99A, F99V, F99S, or F99G of the
protease domain of MT-SP1 based on chymotrypsin numbering. Table 17
provides non-limiting examples of amino acid replacements that
increase specificity and/or selectivity to any one or more
complement protein, including SEQ ID NOS for exemplary polypeptide
sequences and the encoding nucleic acid sequences. TABLE-US-00017
TABLE 17 Full-length Catalytically SEQ ID active portion
Modification NOS: SEQ ID NOS: D96A 423, 499 45, 455 D96V 424, 500
46, 456 D96F 425, 501 47, 457 D96S 426, 502 48, 458 D96T 427, 503
49, 459 Q174H 419, 495 41, 451 D217Q 420, 496 42, 452 D217N 421,
497 43, 453 D217H 422, 498 44, 454 Q192L 428, 504 50, 460 Q192I
429, 505 51, 461 Q192F 430, 506 52, 462 Y146F 431, 507 53, 463 F99A
432, 508 54, 464 F99V 433, 509 55, 465 F99S 434, 510 56, 466 F99G
435, 511 57, 467
[0427] Also provided herein are modified MT-SP1 polypeptides where
the following amino acid residues were identified in the substrate
binding site S1-S4 of MT-SP1 as increasing the specificity and/or
selectivity of cleavage of any one or more complement protein
containing a LPSR/KI Factor I cleavage sequence, thereby inhibiting
complement activation: 174, 180, 215, 192, and 99, based on
chymotrypsin numbering. The modified MT-SP1 polypeptides exhibit
increased specificity and/or selectivity towards a C3b or iC3
complement protein substrate compared to a native target substrate
of wildtype MT-SP1 of SEQ ID NO: 2 or a catalytically active
portion thereof set forth as SEQ ID NO:10. In one embodiment, amino
acid replacement or replacements correspond to any of the following
positions: Q174F, Q174V, Q174L, Q174Y, M180E, W215F, W215Y, Q192K,
Q192R, Q192Y, or F99Y of the protease domain of MT-SP1 based on
chymotrypsin numbering. Table 18 provides non-limiting examples of
amino acid replacements that increase specificity and/or
selectivity to any one or more complement protein, including SEQ ID
NOS for exemplary polypeptide sequences and the encoding nucleic
acid sequences. TABLE-US-00018 TABLE 18 Full-length Catalytically
SEQ ID active portion Modification NOS: SEQ ID NOS: Q174F 440, 516
62, 472 Q174V 439, 515 61, 471 Q174L 529, 539 524, 534 Q174Y 530,
540 525, 535 M180E 531, 541 526, 536 W215F 436, 512 58, 468 W215Y
437, 513 59, 469 Q192K 532, 542 527, 537 Q192R 416, 492 35, 36
Q192Y 533, 543 528, 538 F99Y 447, 523 69, 479
[0428] Also provided herein are modified MT-SP1 polypeptides where
the following amino acid residues were identified in the substrate
binding site S1-S4 of MT-SP1 as increasing the specificity and/or
selectivity of cleavage of any one or more complement protein
containing a HRGR/TL Factor I cleavage sequence, thereby inhibiting
complement activation: 174, 215, 192, 217, and 99 based on
chymotrypsin numbering. The modified MT-SP1 polypeptides exhibit
increased specificity and/or selectivity towards a C4b or iC4
complement protein substrate compared to a native target substrate
of wildtype MT-SP1 of SEQ ID NO: 2 or a catalytically active
portion thereof set forth as SEQ ID NO:10. In one embodiment, amino
acid replacement or replacements correspond to any of the following
positions: W215F, W215Y, Q174A, Q174V, Q174F, Q174R, Q174K, D217A,
D217V, Q192E, F99W, and F99Y of the protease domain of MT-SP1 based
on chymotrypsin numbering. Table 19 provides non-limiting examples
of amino acid replacements that increase specificity and/or
selectivity to any one or more complement protein, including SEQ ID
NOS for exemplary polypeptide sequences and the encoding nucleic
acid sequences. TABLE-US-00019 TABLE 19 Full-length Catalytically
SEQ ID active portion Modification NOS: SEQ ID NOS: W215F 436, 512
58, 468 W215Y 437, 513 59, 469 Q174A 438, 514 60, 470 Q174V 439,
515 61, 471 Q174F 440, 516 62, 472 Q174R 441, 517 63, 473 Q174K
442, 518 64, 474 D217A 443, 519 65, 475 D217V 444, 520 66, 476
Q192E 445, 521 67, 477 F99W 446, 522 68, 478 F99Y 447, 523 69,
479
[0429] In one embodiment, modified proteases can be combined such
as, for example, to acquire the specificity of multiple proteases.
A mutation at one residue of a protease scaffold, which produces
specificity at one site of a substrate sequence, can be combined in
the same protease with another mutation at another site of the
protease scaffold sequence to make a combined specificity protease.
Any number of mutations at discrete sites on the same protease
scaffold an be used to create a combined specificity protease. A
modified protease can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, or more mutations by combining any
two or more mutations identified as contributing to substrate
specificity and/or selectivity of the protease.
[0430] For example, a modified MT-SP1 protease can contain any two
or more mutations, such as any two or more mutations set forth
above, to generate a combined protease. Provided herein are
modified MT-SP1 polypeptides having two or more modifications
corresponding to any of positions 41, 60c, 96, 97, 99, 143, 146,
147, 151, 172, 174, 175, 192, 215, 217, 221a, and 224, based on
chymotrypsin numbering. The modified MT-SP1 polypeptide exhibits
increased specificity and/or selectivity to any one or more
complement components compared to a scaffold or wildtype MT-SP1 of
SEQ ID NO: 2 or a catalytically active portion thereof set forth in
SEQ ID NO: 10. Thus, provided herein are modified MT-SP1
polypeptides exhibiting increased specificity and/or selectivity
towards any one or more complement components containing two or
more modifications at an amino acid position corresponding to any
two or more amino acid residues selected from among I41, R60.sub.C,
D96, F97, F99, H143, Y146, G147, G151, L172, Q174, Q175, Q192,
W215, D217, Q221.sub.a or K224 in an MT-SP1 set forth in SEQ ID NO:
2 or SEQ ID NO:10, based on chymotrypsin numbering. In some
examples, a modified MT-SP1 containing two or more modifications
contains a modification at or or both of position Y146 and K224. In
another example, a modified MT-SP1 containing two or more
modifications contains a modification at position G151. Modified
MT-SP1 polypeptides can be generated using any of the methods
disclosed herein. Table 20 provides non-limiting examples of amino
acid replacements that increase specificity and/or selectivity to
any one or more complement protein, including SEQ ID NOS for
exemplary polypeptide sequences and the encoding nucleic acid
sequences. TABLE-US-00020 TABLE 20 Catalytically Full- active
length portion SEQ ID SEQ ID # Modification NOS: ID NOS: CB155
Y146D/K224F 404, 480 11, 12 CB212 Y146N/K224F 655 601 CB213
Y146E/K224F 642 588 CB214 Y146A/K224F 643 589 CB216 Q192V/K224F 659
605 CB218 Q192F/K224F 657 603 CB219 Y146D/Q192A/K224F 658 604 CB232
Y146E/K224L 620 566 CB235 Y146E/K224A 630 576 CB238 Y146D/K224L 628
574 CB244 Y146D/K224R 617 563 CB245 Y146D/K224N 637 583 CB251
Y146E/K224R 615 561 CB252 Y146E/K224N 606 552 CB255 Y146E/K224T 644
590 CB257 Y146E/K224Y 633 579 CB331 I41D/Y146D/K224L 653 599 CB332
I41E/Y146D/K224L 639 585 CB349 I41D/Y146D/K224F 654 600 CB350
I41E/Y146D/K224F 652 598 CB351 I41T/Y146D/K224F 608 554 CB353
H143V/Y146D/K224F 641 587 CB357 I41T/Y146D/K224L 626 572 CB367
Y146D/Q175D/K224R 624 570 CB373 Y146E/Q175D/K224R 619 565 CB377
Y146E/Q175D/K224N 616 562 CB381 Y146D/Q175H/K224L 631 577 CB383
Y146D/Q175L/K224L 625 571 CB385 Y146D/Q175F/K224L 634 580 CB387
Y146D/Q175W/K224L 627 573 CB388 Y146D/Q175Y/K224L 632 578 CB403
Y146D/D217F/K224L 640 586 CB409 I41A/Y146D/K224F 651 597 CB412
I41L/Y146D/K224F 649 595 CB413 I41F/Y146D/K224F 648 594 CB421
I41T/Y146D/Q175D/K224F 656 602 CB422 I41T/Y146E/Q175D/K224N 609 555
CB423 I41T/Y146E/K224L 645 591 CB450 I41T/I46D/G151L/K224F 650 596
CB451 Y146D/Q221aL/K224S 638 584 CB458 Y146E/Q221aE/K224R 629 575
CB464 Y146E/Q221aE/k224F 611 557 CB476 I41T/Y146D/Q175D/K224L 663
672 CB477 I41T/Y146D/Q175D/K224R 664 673 CB478
I41T/Y146D/Q175D/K224N 665 674 CB480 I41T/Y146D/G151L/Q175D/K224F
666 675 CB481 I41T/Y146D/G151L/Q175D/K224L 667 676 CB482
I41T/Y146D/G151L/Q175D/K224R 668 677 CB483
I41T/Y146D/G151L/Q175D/K224N 669 678 CB484 I41T/Y146E/Q175D/K224F
670 679 CB485 I41T/Y146E/Q175D/K224L 671 680 CB486
I41T/Y146E/Q175D/K224R 607 553 CB487 I41T/Y146E/G151L/Q175D/K224N
613 559 CB488 I41T/Y146E/G151L/Q175D/K224F 618 564 CB489
I41T/Y146E/G151L/Q175D/K224L 610 556 CB490
I41T/Y146E/G151L/Q175D/K224R 612 558 I41T/Y146D/G151L/K224N 681 696
Y146D/Q175D/K224N 682 697 I41T/Y146D/K224N 683 698
Y146D/G151L/K224N 684 699 Y146D/Q175R/K224N 685 700
Y146D/Q175K/K224N 686 701 Y146D/Q175H/K224N 687 702
I41T/Y146D/G151L/Q175K/K224F 688 703 I41T/Y146D/G151L/Q175R/K224F
689 704 I41T/Y146D/G151L/Q175H/K224F 690 705
I41T/Y146D/G151L/Q175Y/K224F 691 706 I41T/Y146D/G151L/Q175K/K224N
692 707 I41T/Y146D/G151L/Q175R/K224N 693 708
I41T/Y146D/G151L/Q175H/K224N 694 709 I41T/Y146D/G151L/Q175Y/K224N
695 710
[0431] Provided herein are modified MT-SP1 polypeptides where the
replacement(s) are made in an MT-SP1 polypeptide scaffold having a
sequence of amino acids set forth in SEQ ID NO:2, where the
modified MT-SP1 polypeptide exhibits increased specificity and/or
selectivity to any one or more complement components compared to
the unmodified protein. Also provided herein are modified MT-SP1
polypeptides containing replacement(s) in an MT-SP1 scaffold having
a sequence of amino acids set forth in SEQ ID NO:10, where the
modified MT-SP1 polypeptide exhibits increased specificity and/or
selectivity to any one or more complement components compared to
the unmodified protein. Such an MT-SP1 scaffold polypeptide is a
catalytically active portions thereof of an MT-SP1 polypeptide.
Exemplary modified MT-SP1 polypeptides provided herein containing
modification(s) of a a catalytically active portion thereof of a
full-length MT-SP scaffold have a sequence of amino acids as set
forth in any one of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36,38, 40-69, 524-528, 552-605, 672-680, or 696-710.
Exemplary modified MT-SP1 polypeptides provided herein containing
modification(s) of a full-length MT-SP1 scaffold have a sequence of
amino acids as set forth in any one of SEQ ID NOS: 404-418-447,
529-533, 606-659, 663-671, or 681-695.
E. Assays to Assess or Monitor Modified Protease Activities on
Complement-Mediated Functions
[0432] A modified protease can exhibit alterations in specificity
and/or selectivity to any one or more complement proteins and
thereby inactivate any one or more complement proteins compared to
the corresponding full-length, scaffold or wildtype form of the
complement protein. Modified proteases retain their protease
activity, but can exhibit an increased specificity and/or
selectivity to any one or more complement proteins. Exemplary
proteases specifically cleave any one or more complement protein
and thereby alter the activity of a complement protein. All such
scaffold or modified proteases with increased specificity and/or
selectivity to any one or more complement protein are candidate
therapeutics.
[0433] Where the protease exhibits an increased specificity and/or
selectivity to any one or more complement protein, in vitro and in
vivo assays can be used to monitor or screen proteases for effects
on complement-mediated functions. Such assays are well known to
those of skill in the art. One of skill in the art can test a
particular scaffold or modified protease for cleavage of any one or
more complement protein and/or test to assess any change in the
effects of a protease on a complement-mediated activity compared to
the absence of a protease. Some such assays are exemplified
herein.
[0434] Exemplary in vitro and in vivo assays are provided herein
for comparison of an activity of a scaffold or modified protease on
the function of any one or more targeted complement proteins. Many
of the assays are applicable to other proteases and modified
proteases. In addition, numerous assays, such as assays for
measuring complement activation, are known to one of skill in the
art. Assays for activities of complement include, but are not
limited to, assays that measure activation products of complement
activation, such as for example the C5b-9 MAC complex, and
generation of any one or more of the complement cleavage products
such as C4a, C5a, C3b, and C3d. Assays to measure complement
activation also include functional assays that measure the
functional activity of specific components of the complement
pathways, such as for example hemolytic assays used to measure
activation of any one of the classical, lectin or alternative
pathways. Assays to assess effects of proteases and modified
proteases on complement proteins and/or complement-mediated
functions include, but are not limited to, SDS-analysis followed by
Western Blot or Coomassie Brilliant Blue staining, enzyme
immunoassays, and hemolytic assays. In one example, in vitro assays
can be performed using purified complement proteins. In another
example, in vivo assays can be performed by testing the serum of a
species, including mammalian or human species, for functional
activation of complement. Exemplary assays are described below.
[0435] a. Protein Detection
[0436] Protein detection is a means to measure individual
complement components in a sample. Complement proteins can be
detected to assess directly the effects of a scaffold or modified
protease on cleavage of the proteins, or alternatively, complement
proteins can be measured as a means to assess for complement
activation. Complement proteins, treated in the presence or absence
of a scaffold or modified protease, can be analyzed by any one or
more assays including SDS-PAGE followed by Coomassie staining or
Western Blot, enzyme immunoassay, immunohistochemistry, flow
cytometry, nephelometry, agar gel diffusion, or radial
immunodiffusion. Exemplary assays for protein detection are
described below.
[0437] i. SDS-PAGE Analysis
[0438] Analysis of complement proteins in the presence or absence
of increasing concentrations of a scaffold or modified protease can
be performed by analysis of proteins on SDS-PAGE followed by
detection of those proteins. In such examples, complement proteins
can be detected by staining for total protein, such as by Coomasie
Brilliant Blue stain, Silver stain, or by any other method known to
one of skill in the art, or by Western Blot using polyclonal or
monoclonal antibodies specific for a specified protein. Typically,
a purified complement protein, such as for example any one or more
of the proteins involved in the complement pathways, can be
incubated in the presence or absence of a scaffold or modified
protease. The treated complement protein can be resolved on an
SDS-PAGE gel followed by a method to detect protein in the gel, for
example, by staining with Coomasie Brilliant blue. The treated
protein can be compared to its cognate full length protein and the
degradation products formed by protease cleavage of the protein can
be determined.
[0439] In another embodiment, a sample, such as for example human
serum or plasma, can be treated in the presence or absence of a
scaffold or modified protease or can be collected after treatment
of an animal or a human with or without a protease. The
protease-treated sample can be analyzed on SDS-PAGE and a specific
complement protein can be detected, such as for example C1q, MBL,
C2, C3, C4, C5, or Factor B, by Western Blot using monoclonal or
polyclonal antibodies against the protein. The cleavage of the
complement protein can be compared to a sample that was not treated
with a protease. Additionally, the sample can be stimulated to
initiate complement activation such as by incubation with IgG which
stimulates activation of the classical pathway or by LPS which
stimulates activation of the alternative pathway. The sample can be
resolved by SDS-PAGE for detection of any one or more of the native
complement proteins to determine the presence or absence of
cleavage products of a specified protein compared to a sample of
the protein not treated with a protease. In such examples, cleavage
effector molecules of native complement proteins also can be
analyzed by Western Blot using monoclonal and polyclonal antibodies
to assess the activation of one or more of the complement pathways.
Examples of complement effector molecules can include, but are not
limited to, C3a, C3d, iC3b, C4d, Bb, and C5-b9. For example, a
decreased expression in a sample of C4d can indicate that a
scaffold or modified protease inhibited the activation of one or
more of the classical or lectin pathway of complement. In another
example, a decreased expression in a sample of Bb can indicate that
a scaffold or modified protease inhibited the activation of the
alternative pathway of complement. The cleavage products of the
effector molecules also can be determined to assess the effects of
increasing concentrations of a scaffold or modified protease on the
cleavage of complement effector molecule themselves.
[0440] ii. Enzyme Immunoassay
[0441] Enzyme immunoassay (EIA; also called enzyme-linked
immunosorbent assay; ELISA) is an assay used to measure the
presence of a protein in a sample. Typically, measurement of the
protein is an indirect measurement of the binding of the protein to
an antibody, which itself is chemically labeled with a detectable
substrate such as an enzyme or fluorescent compound. EIA assays can
be used to measure the effects of scaffold or modified proteases on
complement activation by measuring for the presence of a complement
effector molecule generated following complement activation. In
such examples, a sample, such as for example human serum or plasma,
can be pretreated in the presence or absence of increasing
concentrations of a scaffold or modified protease and subsequently
activated to induce complement activation by incubation with
initiating molecules, or can be collected following treatment of an
animal or a human with a protease. For example, the classical
pathway can be activated by incubation with IgG and the alternative
pathway can be activated by incubation of the sample with LPS. A
complement activation assay specific for the lectin pathway
requires that the classical pathway of complement is inhibited
since the C4/C2 cleaving activity of the lectin pathway is shared
with the classical pathway of complement. Inhibition of the
classical pathway can be achieved using a high ionic strength
buffer which inhibits the binding of C1q to immune complexes and
disrupts the C1 complex, whereas a high ionic strength buffer does
not affect the carbohydrate binding activity of MBL. Consequently,
activation of the lectin pathway can be induced by incubation of a
sample, such as human serum or plasma, with a mannan-coated surface
in the presence of 1 M NaCl.
[0442] Following activation, the sample can be quenched with the
addition of Pefabloc (Roche) and EDTA to minimize continued
activation of the pathways. Samples can be analyzed for the
presence of complement effector molecules by an EIA or ELISA assay.
EIA and ELISA assays for measuring complement proteins are well
known to one skilled in the art. Any complement activation product
can be assessed. Exemplary complement activation products for
measurement of complement activation include iC3b, Bb, C4d, C5b-9,
C3a, C3a-desArg, C4a-desArg, and C5a-desArg. The complement pathway
activated can be determined depending on the complement activation
product measured. For example, measurement of Bb cleavage product
is a unique marker of the alternative pathway.
[0443] In some examples, the EIA can be paired with detection of
the cleaved complement proteins by analysis of the
protease-treated, complement-stimulated sample by SDS-PAGE followed
by Western blot analysis for identification of specific complement
components. Using densitometry software, the cleavage of the
complement product can be compared to the full length complement
component cleaved throughout the assay and the appearance of all
major degradation products and the percentage cleavage can be
determined.
[0444] iii. Radial Immunodiffusion (RID)
[0445] Radial immunodiffusion (RID) is a technique that relies on
the precipitation of immune complexes formed between antibodies
incorporated into agarose gels when it is poured, and antigen
present in a test sample resulting in a circular precipitin line
around the sample well. The diameter of the precipitin ring is
proportional to the concentration of the antibody (or antigen)
present in the test sample. By comparing the diameter of the test
specimen precipitin ring to known standards, a relatively
insensitive estimation of the concentration of specific antibody or
antigen can be achieved. RID can be used to measure the amount of a
complement protein in a sample. For example, a sample such as for
example human serum or plasma, can be treated in the presence or
absence of increasing concentrations of a scaffold or modified
protease. The protease-treated sample can be added to a well of an
agarose gel that has been made to incorporate a polyclonal or
monoclonal antibody against any one of the complement proteins such
as including, but not limited to, C1q, C1r, C1s, C2, C3, C4, C5,
C6, C7, C9, or Factor B. After removal of unprecipitated proteins
by exposure to 0.15 M NaCl, the precipitated protein rings can be
assessed by staining with a protein dye, such as for example
Coomassie Brilliant blue or Crowles double stain.
[0446] b. Hemolytic Assays
[0447] Functional hemolytic assays provide information on
complement function as a whole. This type of assay uses
antibody-sensitized or unsensitized sheep erythrocytes. Hemolytic
assays include the total hemolytic complement assay (CH50), which
measures the ability of the classical pathway and the MAC to lyse a
sheep RBC. It depends on the sequential activation of the classical
pathway components (C1 through C9) to lyse sheep erythrocytes that
have been sensitized with optimal amounts of rabbit anti-sheep
erythrocyte antibodies to make cellular antigen-antibody complexes.
Hemolytic assays also can include an alternative pathway CH50 assay
(rabbit CH50 or APCH50), which measures the ability of the
alternative pathway and the MAC to lyse a rabbit RBC. One CH50
and/or APCH50 unit is defined as the quantity or dilution of serum
required to lyse 50% of the red cells in the test. Typically, to
assess complement activation, a sample, such as for example human
serum or human plasma, can be treated in the presence or absence of
increasing concentrations of a scaffold or modified protease, or
can be collected following treatment of an animal or human in the
presence or absence of a protease. The protease-treated sample can
be subsequently mixed with sheep's red blood cells that have been
activated or sensitized with IgG. A water only sample mixed with
sheep red blood cells can act as a total lysis control in order to
accurately assess percent lysis of the samples analyzed. The
addition of 0.15M NaCl to the sample can be added to stop the
lysing reaction. Lysis of the red blood cells, induced by the
activation of the terminal components of the complement pathway,
can be assessed by measuring the release of hemoglobin. Measurement
can be by optical density (OD) readings of the samples using a
spectrophotometer at an OD of 415 nm.
[0448] In one embodiment, limiting dilution hemolytic assays can be
used to measure functional activity of specific components of
either pathway. In such an assay, a serum source is used that has
an excess of all complement components, but is deficient for the
one being measured in the sample, i.e. a media or serum source is
complement-depleted for a specific protein. The extent of hemolysis
is therefore dependent on the presence of the measured component in
the test sample. In such an assay, a purified complement protein,
such as for example any one of the native complement proteins
including, but not limited to C1q, MBL, C2, C3, C4, or C5 can be
incubated in the presence or absence of increasing concentrations
of a scaffold or modified protease. The protease-treated purified
complement protein can then be mixed with complement-depleted media
or plasma and IgG-activated sheep red blood cells and hemolysis of
the sample can be assessed as described above. In another
embodiment, protease cleavage can be correlated with complement
activation by assaying for hemolytic activity of a protease-treated
sample, and subsequently analyzing the sample on SDS-PAGE gel
followed by staining with a protein stain, such as for example
Coomassie Blue. The purified complement protein treated with the
proteases can be assessed for cleavage and the percentage of the
full length complement component cleaved throughout the assay and
the appearance of all major degradation products can be calculated.
Alternatively, analysis of the protease-treated complement protein
can be by Western blot.
[0449] An alternative to the hemolytic assay, called the liposome
immunoassay (LIA), can be used to assess activation of the
classical pathway. The LIA (Waco Chemicals USA, Richmond, Va.)
utilizes dinitrophenyl (DNP)-coated liposomes that contain the
enzyme glucose-6-phosphate dehydrogenase. When serum is mixed with
the liposomes and a substrate containing anti-DNP antibody,
glucose-6-phosphate, and nicotinamide adenine dinucleotide,
activated liposomes lyse, and an enzymatic colorimetric reaction
occurs which is proportional to total classical complement
activity.
F. Methods of Producing Nucleic Acid Encoding Proteases and Methods
of Producing Protease Polypeptides
[0450] Protease polypeptides, including modified MT-SP1
polypeptides, or domains thereof, 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 protease protein, such as from a cell
or tissue source. Modified proteases can be engineered as described
herein from a scaffold or wildtype protease, such as by
site-directed mutagenesis.
[0451] Proteases 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.
[0452] Methods for amplification of nucleic acids can be used to
isolate nucleic acid molecules encoding a protease, including for
example, polymerase chain reaction (PCR) methods. A nucleic acid
containing material can be used as a starting material from which a
protease-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 protease. For
example, primers can be designed based on expressed sequences from
which a protease is generated. Primers can be designed based on
back-translation of a protease amino acid sequence. Nucleic acid
molecules generated by amplification can be sequenced and confirmed
to encode a protease.
[0453] Additional nucleotide sequences can be joined to a
protease-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 protease-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 sequences such as sequences
specifying protein binding regions also can be linked to
protease-encoding nucleic acid molecules. Such regions include, but
are not limited to, sequences to facilitate uptake of a protease
into specific target cells, or otherwise enhance the
pharmacokinetics of the synthetic gene.
[0454] 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 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 protease 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.
[0455] In specific embodiments, transformation of host cells with
recombinant DNA molecules that incorporate the isolated protease
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.
[0456] 1. Vectors and Cells
[0457] For recombinant expression of one or more of the protease
proteins, the nucleic acid containing all or a portion of the
nucleotide sequence encoding the protease 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.
[0458] Also provided are vectors that contain 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.
[0459] 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
protease or modified protease protein thereof by growing the
above-described cells under conditions whereby the encoded protease
protein is expressed by the cell, and recovering the expressed
protease protein. For purposes herein, the protease can be secreted
into the medium.
[0460] In one embodiment, vectors containing a sequence of
nucleotides that encodes a polypeptide that has protease activity
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 scaffold or
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
nucleic acid encoding a secretion signal, such as the Saccharomyces
cerevisiae .alpha.-mating factor signal sequence or a portion
thereof, or the native signal sequence.
[0461] 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.
[0462] 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 a scaffold or modified protease 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 protease
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 Gal4 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)).
[0463] In a specific embodiment, a vector is used that contains a
promoter operably linked to nucleic acids encoding a scaffold or
modified protease 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). 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 (SEQ ID NO:
345), 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.
[0464] 2. Expression
[0465] Modified proteases can be produced by any method known to
those of skill in the art including in vivo and in vitro methods.
Modified proteases can be expressed in any organism suitable to
produce the required amounts and forms of a modified protease
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.
[0466] Many expression vectors are available and known to those of
skill in the art and can be used for expression of modified
proteases. 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.
[0467] Modified proteases 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.
[0468] In one embodiment, the protease can be expressed in an
active form. In another embodiment, the protease is expressed in an
inactive, zymogen form.
[0469] a. Prokaryotes
[0470] Prokaryotes, especially E. coli, provide a system for
producing large amounts of proteins such as proteases or modified
proteases. 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.
[0471] Modified proteases 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 a modified protease 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.
[0472] b. Yeast
[0473] 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 modified proteases. 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.
[0474] c. Insect Cells
[0475] Insect cells, particularly using baculovirus expression, are
useful for expressing polypeptides such as modified proteases.
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 californica
nuclear polyhedrosis virus (AcNPV), and the Bombyx mori nuclear
polyhedrosis virus (BmNPV) and an insect cell line such as Sf9
derived from Spodoptera frugiperda, Pseudaletia unipuncta (A7S) and
Danaus plexippus (DpN1). For high-level expression, the nucleotide
sequence of the molecule to be expressed is fused immediately
downstream of the polyhedrin initiation codon of the virus.
Mammalian secretion signals are accurately processed in insect
cells and can be used to secrete the expressed protein into the
culture medium. In addition, the cell lines Pseudaletia unipuncta
(A7S) and Danaus plexippus (DpN1) produce proteins with
glycosylation patterns similar to mammalian cell systems.
[0476] 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.
[0477] d. Mammalian Cells
[0478] Mammalian expression systems can be used to express modified
proteases. 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.
[0479] 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.)
[0480] e. Plants
[0481] Transgenic plant cells and plants can be to express modified
proteases. 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 synthase 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 proteases or modified proteases
produced in these hosts.
[0482] 3. Purification Techniques
[0483] Methods for purification of protease polypeptides 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.
[0484] The protease 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 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 (a 3 kilodalton shift) and by enzyme activity
(cleavage of a fluorogenic substrate). Typically, a protease is
allowed to achieve >75% activation before purification.
[0485] Proteases 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 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.
[0486] 4. Fusion Proteases
[0487] Fusion proteins containing a protease 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 scaffold or 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 wildtype or scaffold 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 complement proteins. 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.
[0488] 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 immunoglobulin G, 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).
[0489] 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.
[0490] 5. Nucleotide Sequences
[0491] Nucleic acid molecules encoding scaffold or modified
proteases are provided herein. Nucleic acid molecules include
allelic variants or splice variants of any encoded scaffold
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 the full-length of any nucleic acid encoded
scaffold protease, or catalytically active portion thereof. In
another embodiment, a nucleic acid molecule can include those with
degenerate codon sequences of any of the scaffold proteases or
catalytically active portions thereof such as those provided
herein. Exemplary nucleic acid molecules, encoding scaffold or
modified proteases, or catalytically active portions thereof, have
a sequence of nucleotides as set forth in any of SEQ ID NOS: 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 451-523,
and 534-543.
[0492] 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.
[0493] Scaffold or 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 Rheumatoid Arthritis
or cardiovascular disease, 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.
G. Methods of Using: Formulation/Packaging/Administration
[0494] Pharmaceutical compositions containing a protease or
modified protease produced herein, including MT-SP1 (modified)
polypeptides, modified protease fusion proteins or encoding nucleic
acid molecules, can be formulated in any conventional manner by
mixing a selected amount of the polypeptide with one or more
physiologically acceptable carriers or excipients. Selection of the
carrier or excipient is within the skill of the administering
profession and can depend upon a number of parameters. These
include, for example, the mode of administration (i.e., systemic,
oral, nasal, pulmonary, local, topical or any other mode) and
disorder treated. The pharmaceutical compositions provided herein
can be formulated for single dosage (direct) administration or for
dilution or other modification. The concentrations of the compounds
in the formulations are effective for delivery of an amount, upon
administration, that is effective for the intended treatment.
Typically, the compositions are formulated for single dosage
administration. To formulate a composition, the weight fraction of
a compound or mixture thereof is dissolved, suspended, dispersed or
otherwise mixed in a selected vehicle at an effective concentration
such that the treated condition is relieved or ameliorated.
Pharmaceutical carriers or vehicles suitable for administration of
the compounds provided herein include any such carriers known to
those skilled in the art to be suitable for the particular mode of
administration.
[0495] 1. Administration of Modified Protease Polypeptides
[0496] The polypeptides can be formulated as the sole
pharmaceutically active ingredient in the composition or can be
combined with other active ingredients. The polypeptides can be
targeted for delivery, such as by conjugation to a targeting agent,
such as an antibody. Liposomal suspensions, including
tissue-targeted liposomes, also can be suitable as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art. For example, liposome
formulations can be prepared as described in U.S. Pat. No.
4,522,811. Liposomal delivery also can include slow release
formulations, including pharmaceutical matrices such as collagen
gels and liposomes modified with fibronectin (see, for example,
Weiner et al. (1985) J Pharm Sci. 74(9): 922-5).
[0497] The active compound 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 subject treated. The 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.
[0498] The polypeptides provided herein (i.e. active compounds) can
be administered in vitro, ex vivo, or in vivo by contacting a
mixture, such as a body fluid or other tissue sample, with a
protease polypeptide provided herein, including any of the modified
MT-SP1 proteases provided herein. For example, when administering a
compound ex vivo, a body fluid or tissue sample from a subject can
be contacted with the protease polypeptides that are coated on a
tube or filter, such as for example, a ture or filter in a bypass
machine. When administering in vivo, the active compounds can be
administered by any appropriate route, for example, orally,
nasally, pulmonary, parenterally, intravenously, intradermally,
subcutaneously, or topically, in liquid, semi-liquid or solid form
and are formulated in a manner suitable for each route of
administration.
[0499] The modified protease and physiologically acceptable salts
and solvates can be formulated for administration by inhalation
(either through the mouth or the nose), oral, transdermal,
pulmonary, parenteral or rectal administration. For administration
by inhalation, the modified protease 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 a
therapeutic compound and a suitable powder base such as lactose or
starch.
[0500] For pulmonary administration to the lungs, the modified
protease can be delivered in the form of an aerosol spray
presentation from a nebulizer, turbonebulizer, or
microprocessor-controlled metered dose oral inhaler with the use of
a suitable propellant. Generally, particle size of the aerosol is
small, such as in the range of 0.5 to 5 microns. In the case of a
pharmaceutical composition formulated for pulmonary administration,
detergent surfactants are not typically used. Pulmonary drug
delivery is a promising non-invasive method of systemic
administration. The lungs represent an attractive route for drug
delivery, mainly due to the high surface area for absorption, thin
alveolar epithelium, extensive vascularization, lack of hepatic
first-pass metabolism, and relatively low metabolic activity.
[0501] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets, pills, liquid suspensions,
or capsules prepared by conventional means with pharmaceutically
acceptable excipients such as binding agents (e.g., pregelatinized
maize starch, polyvinylpyrrolidone 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. Liquid preparations for oral administration can take
the form of, for example, solutions, syrups or suspensions, or they
can be presented as a dry product for constitution 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, ethyl alcohol or fractionated vegetable
oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates
or sorbic acid). The preparations also can contain buffer salts,
flavoring, coloring and sweetening agents as appropriate.
[0502] Preparations for oral administration can be formulated for
controlled release of the active compound. For buccal
administration the compositions can take the form of tablets or
lozenges formulated in conventional manner.
[0503] The modified protease polypeptides can be formulated as a
depot preparation. Such long-acting formulations can be
administered by implantation (for example, subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the therapeutic compounds can be formulated with suitable polymeric
or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
[0504] The modified protease 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 ampoules or in multi-dose containers) with an
added preservative. The compositions can take such forms as
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-lyophilized form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.
[0505] The pharmaceutical compositions can be formulated for local
or topical application, such as for topical application to the skin
(transdermal) and mucous membranes, such as in the eye, in the form
of gels, creams, and lotions and for application to the eye or for
intracisternal or intraspinal application. Such solutions,
particularly those intended for ophthalmic use, can be formulated
as 0.01%-10% isotonic solutions and pH about 5-7 with appropriate
salts. The compounds can be formulated as aerosols for topical
application, such as by inhalation (see, for example, U.S. Pat.
Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols
for delivery of a steroid useful for treatment inflammatory
diseases, particularly asthma).
[0506] The concentration of active compound in the drug composition
depends on absorption, inactivation and excretion rates of the
active compound, the dosage schedule, and amount administered as
well as other factors known to those of skill in the art. As
described further herein, dosages can be determined empirically
using comparisons of properties and activities (e.g., cleavage of
one or more complement proteins) of the modified protease compared
to the unmodified and/or native protease.
[0507] The compositions, if desired, can be presented in a package,
in a kit or dispenser device, that can contain one or more unit
dosage forms containing the active ingredient. In some examples,
the composition can be coated on a device, such as for example on a
tube or filter in, for example, a bypass machine. The package, for
example, contains metal or plastic foil, such as a blister pack.
The pack or dispenser device can be accompanied by instructions for
administration. The compositions containing the active agents can
be packaged as articles of manufacture containing packaging
material, an agent provided herein, and a label that indicates the
disorder for which the agent is provided.
[0508] Also provided are compositions of nucleic acid molecules
encoding the protease polypeptides and expression vectors encoding
them that are suitable for gene therapy. Rather than deliver the
protein, nucleic acid can be administered in vivo, such as
systemically or by other route, or ex vivo, such as by removal of
cells, including lymphocytes, introduction of the nucleic therein,
and reintroduction into the host or a compatible recipient.
[0509] 2. Administration of Nucleic Acids Encoding Modified
Protease Polypeptides (Gene Therapy)
[0510] Protease polypeptides can be delivered to cells and tissues
by expression of nucleic acid molecules. Protease polypeptides can
be administered as nucleic acid molecules encoding protease
polypeptides, including ex vivo techniques and direct in vivo
expression. Nucleic acids can be delivered to cells and tissues by
any method known to those of skill in the art. The isolated nucleic
acid can be incorporated into vectors for further manipulation.
Exemplary nucleic acids are any that encode or that hybridize under
medium to high stringency to a nucleic acid that encodes a scaffold
or modified protease, or catalytically active portion thereof
having a sequence of amino acids set forth in any of SEQ ID NOS:
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69,
404-418, 419-447, 524-533, 552-659, or 663-710. Exemplary nucleic
acid molecules, encoding scaffold or modified proteases, or
catalytically active portions thereof, have a sequence of
nucleotides as set forth in any of SEQ ID NOS: 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 451-523, and 534-543.
[0511] Methods for administering protease polypeptides 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. Protease polypeptides also can be used in ex vivo gene
expression therapy using vectors. For example, cells can be
engineered to express a protease polypeptide, such as by
integrating a protease polypeptide 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. Exemplary vectors for in vivo and ex
vivo gene therapy include viral vectors, and non-viral vectors such
as for example, liposomes or artificial chromosomes.
[0512] Viral vectors, including, for example adenoviruses, herpes
viruses, retroviruses EBV, SV40, cytomegalovirus vector, vaccinia
virus vector, and others designed for gene therapy can be employed.
The vectors can remain episomal or can integrate into chromosomes
of the treated subject. A protease polypeptide 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 protease polypeptide-expressing adenovirus vector. After a
suitable culturing period, the transduced cells are administered to
a subject, locally and/or systemically. Alternatively, protease
polypeptide-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. In one embodiment, the disease
to be treated is caused by complement activation. 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.
[0513] The nucleic acid molecules can be introduced into artificial
chromosomes and other non-viral vectors. Artificial chromosomes,
such as ACES (see, Lindenbaum et al. Nucleic Acids Res. 2004 Dec.
7;32(21):e172) can be engineered to encode and express the protease
or modified protease. Briefly, mammalian artificial chromosomes
(MACs) provide a means to introduce large payloads of genetic
information into the cell in an autonomously replicating,
non-integrating format. Unique among MACs, the mammalian satellite
DNA-based Artificial Chromosome Expression System (ACES) can be
reproducibly generated de novo in cell lines of different species
and readily purified from the host cells' chromosomes. Purified
mammalian ACEs can then be re-introduced into a variety of
recipient cell lines where they have been stably maintained for
extended periods in the absence of selective pressure using an ACE
System. Using this approach, specific loading of one or two gene
targets has been achieved in LMTK(-) and CHO cells.
[0514] Another method for introducing nucleic acids encoding the
modified protease polypeptides is a two-step gene replacement
technique in yeast, starting with a complete adenovirus genome
(Ad2; Ketner et al. (1994) Proc. Natl. Acad. Sci. USA 91:
6186-6190) cloned in a Yeast Artificial Chromosome (YAC) and a
plasmid containing adenovirus sequences to target a specific region
in the YAC clone, an expression cassette for the gene of interest
and a positive and negative selectable marker. YACs are of
particular interest because they permit incorporation of larger
genes. This approach can be used for construction of
adenovirus-based vectors bearing nucleic acids encoding any of the
described modified protease polypeptides for gene transfer to
mammalian cells or whole animals.
[0515] The nucleic acids can be encapsulated in a vehicle, such as
a liposome, or introduced into a 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.
[0516] 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. Polynucleotides and
expression vectors provided herein can be made by any suitable
method. Further provided are nucleic acid vectors containing
nucleic acid molecules as described above. Further provided are
nucleic acid vectors containing nucleic acid molecules as described
above and cells containing these vectors.
[0517] For ex vivo and in vivo methods, nucleic acid molecules
encoding the protease polypeptide are 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.
[0518] For ex vivo treatment, cells from a donor compatible with
the subject to be treated or cells from a 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, 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 protease polypeptides 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.
[0519] In vivo expression of a protease polypeptide can be linked
to expression of additional molecules. For example, expression of a
protease polypeptide 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 protease
polypeptide can be used to enhance the cytotoxicity of the
virus.
[0520] In vivo expression of a protease polypeptide can include
operatively linking a protease polypeptide encoding nucleic acid
molecule to specific regulatory sequences such as a cell-specific
or tissue-specific promoter. Protease polypeptides also can be
expressed from vectors that specifically infect and/or replicate in
target cell types and/or tissues. Inducible promoters can be use to
selectively regulate protease polypeptide expression.
[0521] 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.
[0522] 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 can be targeted cells for in
vivo expression of protease polypeptides. Cells used for in vivo
expression of a protease polypeptide also include cells autologous
to the patient. Such cells can be removed from a patient, nucleic
acids for expression of a protease polypeptide introduced, and then
administered to a patient such as by injection or engraftment.
H. Therapeutic Uses
[0523] Therapeutic proteases have many potential advantages over
traditional therapeutic approaches. Chief among them is the ability
to inactivate disease targets in a catalytic manner (i.e. a one to
many stoichiometry). Additional differentiating advantages include
(1) irreversible inactivation; (2) low dosing; (3) small molecular
size; (4) the ability to target post-translational modifications;
(5) the ability to neutralize high target concentrations; and (6)
the ability to target away from the active site. As a therapeutic,
a protease must still exhibit the following characteristics: (1)
access to the molecular target (extracellular), and (2) possess
sufficiently stringent specificity for a target critical to a
disease state. The protease polypeptides provided herein can be
used in the treatment of diseases.
[0524] 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.
[0525] 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.
[0526] 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.
[0527] 1. Immune-Mediated Inflammatory Diseases
[0528] Proteases and modified proteases described herein, including
but not limited to modified MT-SP1 proteases, 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.
[0529] 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.
[0530] Complement activation modulates inflammation in diseases
such as rheumatoid arthritis (RA) (Wang et al., (1995) PNAS
92:8955). Proteases and modified proteases, including modified
MT-SP1 polypeptides, 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.
[0531] Proteases or modified proteases, including but not limited
to MT-SP1 polypeptides, 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.
[0532] Therapeutic proteases, such as those described herein
including MT-SP1 polypeptides, 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.
[0533] 2. Neurodegenerative Disease
[0534] 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 modified MT-SP1 polypeptides, 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.
[0535] 3. Cardiovascular Disease
[0536] Proteases and modified proteases described herein, including
but not limited to modified MT-SP1 proteases, 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.
[0537] 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 modified MT-SP1
polypeptide, 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.
[0538] 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 modified proteases including MT-SP1
polypeptides. The perfused hearts can be assayed for deposition of
human C3 and C5b-9, coronary artery perfusion pressure,
end-diastolic pressure, and heart rate.
[0539] Proteases and modified proteases, such as for example MT-SP1
polypeptides, 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.
I. Combination Therapies
[0540] Wildtype and modified proteases can be used in combination
with each other and with other existing drugs and therapeutic
agents to treat diseases and conditions. For example, as described
herein a number of proteases can be used to treat acute and chronic
inflammatory conditions and diseases. Such treatments can be
performed in conjunction with other anti-inflammatory drugs and/or
therapeutic agents. Examples of anti-inflammatory drugs and agents
useful for combination therapies include non steroidal
anti-inflammatory drugs (NSAIDs) including salicylates, such as
aspirin, traditional NSAIDs such as ibuprofen, naproxen,
ketroprofen, nabumetone, piroxicam, diclofenac, or indomethacin,
and Cox-2 selective inhibitors such as celecoxib (Celebrex.RTM.) or
Rotecoxin (Vioxx.RTM.). Other compounds useful in combination
therapies include antimetabolites such as methotrexate and
leflunomide, corticosteroids or other steroids such as cortisone,
dexamethasone, or prednisone, analgesics such as acetaminophen,
aminosalicylates such as mesalamine, and cytotoxic agents such as
azathioprine (Imuran.RTM.), cyclophosphamide (Cytoxan.RTM.), and
cyclosporine A. Additional agents that can be used in combination
therapies include biological response modifiers. Biological
response modifiers can include pro-inflammatory cytokine inhibitors
including inhibitors of TNF-alpha such as etanercept (Enbrel.RTM.),
infliximab (Remicade.RTM.), or adalimumad (Humira.RTM.), and
inhibitors of IL-1 such as anakinra (Kineret.RTM.). Biological
response modifiers also can include anti-inflammatory cytokines
such as IL-10, B cell targeting agents such as anti-CD20 antibodies
(Rituxmab.RTM.), compounds targeting T antigens, adhesion molecule
blockers, chemokines receptor antagonists, kinase inhibitors such
as inhibitors to MAP Kinase, JNK, or NF.kappa.B, and PPAR-.gamma.
ligands.
[0541] Wildtype and modified proteases also can be used in
combination with agents that are administered to treat
cardiovascular disease and/or administered during procedures to
treat cardiovascular disease such as for example those described
herein that contribute to inflammatory conditions associated with
complement-mediated ischemia-reperfusion injury. For example,
proteases provided herein such as scaffold proteases or modified
proteases can be administered in combination with anti-coagulants.
Examples of exemplary anti-coagulants include, but are not limited
to, heparin, warfarin, acenocoumarol, phenindione, EDTA, citrate,
oxalate, and direct thrombin inhibitors such as argatroban,
lepirudin, bivalirudin, ximelagatran.
[0542] Additional agents, such as other complement inhibitors, can
be used as anti-inflammatory drugs in combination therapy with
proteases and modified proteases as described herein. Examples of
such other complement inhibitors include cobra venom factor (CVF),
polyanionic molecules such as heparin, dextran sulphate, polyvinyl
sulphate, polylysine, or suramin, natural molecules such as
K-76COOH, Rosmarinic acid, or extract of the Chinese medicinal herb
Ephedra, synthetic molecules such as afamastat mesilate (FUT-175),
a synthetic inhibitor of C1s (C1s-INH-248), or an inhibitor against
C1s and fD (BCX-1470), peptide inhibitors such as compstatin,
antibody inhibitors of complement such as anti-C5 (N19-8), a
humanized anti-C5 (h5G1.1), anti-C6, or anti-C8 antibodies, and
soluble forms of membrane complement regulators such as soluble CR1
(sCR1), soluble DAF (sDAF), soluble MCP (sMCF), or soluble CD59
(sCD59) (Morgan et al., (2003) Mol Immunol. 40:159).
[0543] Pharmaceutical compositions containing a protease or
modified protease described herein can be used to treat any one or
more inflammatory diseases or conditions mediated by complement
activation. Also provided are combinations of a protease or
modified protease and another treatment or compound for treatment
of an inflammatory disease or condition. The protease or modified
protease and the anti-inflammatory agent can be packaged as
separate compositions for administration together or sequentially
or intermittently. Alternatively, they can provided as a single
composition for administration or as two compositions for
administration as a single composition. The combinations can be
packaged as kits, optionally with additional reagents, instructions
for use, vials and other containers, syringes and other items for
use of the modified protease.
J. EXAMPLES
[0544] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
embodiments provided herein.
Example 1
Cloning and Mutagenesis of MT-SP1
[0545] Wildtype MT-SP1 was cloned into the pQE32 bacterial
expression vector (Qiagen, SEQ ID NO:345) C-terminal to the 6
histidine tag using the BamH1 and HindIII restriction sites. The
construct included pro-region, activation sequence, and protease
domain, and contains residues 598 to the C-terminus of the sequence
published by Takeuchi et al. (1999) PNAS 96:11054 and SEQ ID NO:2
(i.e. corresponding to residues 598 to 855 of the sequence of amino
acids set forth in SEQ ID NO:2). Mutants were generated by the
Quikchange site directed mutagenesis (Stratagene). Briefly, a PCR
sample reaction was set up containing the wildtype MT-SP1 as a
template and oligonucleotide primers designed to contain the
desired mutation (see Table 21). The PCR reaction was as follows in
a 50 .mu.l total reaction volume: 5 .mu.l 10.times. Reaction
Buffer, 1 .mu.l MT-SP1 DNA template (100 ng/.mu.l), 0.5 .mu.l 50
.mu.M forward Primer, 0.5 .mu.l 50 .mu.M reverse Primer, 1.0 .mu.l
dNTPs, 41.0 .mu.l H.sub.2O, and 1.0 .mu.l Pfu Ultra (2.5
units/.mu.l). A control reaction also was performed in the absence
of forward or reverse Primers. The PCR reaction conditions were as
follows: 95.degree. C. for 30 sec, followed by 18 cycles at
95.degree. C. for 30 s, 55.degree. C. for 60 s, and 72.degree. C.
for 8 min, 24 s. The reaction was terminated with an elongation
step at 72.degree. C. for 10 min followed by incubation at
4.degree. C. Each reaction product was digested with DpnI for 1-2
hours at 37.degree. C. 1.0 ml of the products were transformed into
XL-1 Blue Supercompetent cells and plated at 2.0 .mu.l and 20.0
.mu.l on selective agar containing 50 .mu.g/ml carbenicillin.
TABLE-US-00021 TABLE 21 Mutagenesis Primers SEQ ID Primer Sequence
NO F97Dforward 5'-CACCCCTTCTTCAATGACGACACCTTCGACT 346 ATGACATCG-3'
F97Dreverse 5'-CGATGTCATAGTCGAAGGTGTCGTCATTGAA 347 GAAGGGGTG-3'
F97Eforward 5'-CCACCCCTTCTTCAATGACGAGACCTTCGAC 348 TATGACATCGC-3'
F97Ereverse 5'-GCGATGTCATAGTCGAAGGTCTCGTCATTGA 349 AGAAGGGGTGG-3'
F97Aforward 5'-CACCCCTTCTTCAATGACGCCACCTTCGACT 350 ATGACATC-3'
F97Areverse 5'-GATGTCATAGTCGAAGGTGGCGTCATTGAAG 351 AAGGGGTG-3'
F97Wforward 5'-CACCCCTTCTTCAATGACTGGACCTTCGACT 352 ATGACATC-3'
F97Wreverse 5'-GATGTCATAGTCGAAGGTCCAGTCATTGAAG 353 AAGGGGTG-3'
Y146Nforward 5'-GGACACACCCAGAACGGAGGCACTGGC-3' 354 Y146Nreverse
5'-GCCAGTGCCTCCGTTCTGGGTGTGTCC-3' 355 Y146Dforward
5'-GGACACACCCAGGACGGAGGCACTGGC-3' 356 Y146Dreverse
5'-GCCAGTGCCTCCGTCCTGGGTGTGTCC-3' 357 Y146Eforward
5'-GGGACACACCCAGGAGGGAGGCACTGGCG- 358 3' Y146Ereverse
5'-CGCCAGTGCCTCCCTCCTGGGTGTGTCCC- 359 3' Y146Aforward
5'-GGGACACACCCAGGCCGGAGGCACTGGCG- 360 3' Y146Areverse
5'-CGCCAGTGCCTCCGGCCTGGGTGTGTCCC- 361 3' Y146Wforward
5'-GGGACACACCCAGTGGGGAGGCACTGGCG- 362 3' Y146Wreverse
5'-CGCCAGTGCCTCCCCACTGGGTGTGTCCC- 363 3' Y146Rforward
5'-GGGGACACACCCAGAGGGGAGGCACTGGCG 364 C-3' Y146Rreverse
5'-GCGCCAGTGCCTCCCCTCTGGGTGTGTCCC 365 C-3' Q192Rforward
5'-TGGACTCCTGCCGGGGTGATTCCGG-3' 366 Q192Rreverse
5'-CCGGAATCACCCCGGCAGGAGTCCA-3' 367 K224Aforward
5'-CGCTCAGAGGAACGCGCCAGGCGTGTACA- 368 3' K224Areverse
5'-TGTACACGCCTGGCGCGTTCCTCTGAGCG- 369 3' K224Fforward
5'-GCTGCGCTCAGAGGAACTTCCCAGGCGTGTA 370 CACAAG-3' K224Freverse
5'-CTTGTGTACACGCCTGGGAAGTTCCTCTGAG 371 CGCAGC-3'
[0546] The sequences of each of the cloned protease domain MT-SP1
mutants designated by CB numbering is set forth in any of SEQ ID
NOS: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 552-605, or 672-680.
Example 2
Expression and Purification of MT-SP1
[0547] Wild-type and modified MT-SP1 were cloned into the pQE32
bacterial expression vector (Qiagen, SEQ ID NO:345) containing an
N-terminal 6 histidine tag, prodomain, and protease domain as
discussed in Example 1 above and the resulting constructs
transformed into BL-21 E. coli cells. Cells were grown in 100 mL
cultures to an OD of 0.6, and expression of the protease in
inclusion bodies was induced by adding IPTG to a final
concentration of 1 mM. After 4-6 hours, the bacteria were pelleted
by centrifugation and the pellet resuspended in 50 mM Tris pH 8,
500 mM KCl, and 10% glycerol (buffer A). Cells were lysed by
sonication and pelleted by centrifugation at 6000.times.g. Pellets
were resuspended in 50 mM Tris pH 8, 6 M urea, 100 mM NaCl and 1%
2-mercaptoethanol (buffer B). Membrane and organelles were pelleted
by centrifugation at 10,000.times.g and the supernatant was passed
over a nickel NTA column (Qiagen). The column was washed with 50 mM
Tris pH8, 6 M urea, 100 mM NaCl, 20 mM imidazole, 1%
2-mercaptoethanol and 0.01% Tween 20 (buffer D). The column was
washed again with buffer D without Tween 20. The protease was then
eluted from the column with 50 mM Tris pH 8, 6 M urea, 100 mM NaCl,
1% 2-mercaptoethanol and 250 mM imidazole (buffer E). The protease
was then concentrated to a volume of .about.1 mL and then dialyzed
at 4.degree. C. overnight in 1 L of 50 mM Tris pH8, 3 M urea, 100
mM NaCl, 1% 2-mercaptoethanol, and 10% glycerol. Finally, the
protease was dialyzed into 50 mM Tris pH 8, 100 mM NaCl, and 10%
glycerol at 4.degree. C. overnight. During the last dialysis step,
the protease becomes autoactivated by self-cleavage at the juncture
between the prodomain and the protease domain at the sequence
RQAR/VVGG, resulting in the removal of the 6 histidine tag and
prodomain.
Example 3
Expression and Purification of Modified MT-SP1 CB155 in Shake
Flasks
[0548] CB155 and related recombinant human serine protease mutants
as well as the wild-type MT-SP1 protease were cloned and expressed
in E. coli as inclusion bodies as described in Examples 1 and 2
above. The production of the MT-SP1 or mutants was adapted for
laboratory scale by pooling up to 30.times.1 L shake flasks for
subsequent isolation of the inclusion body pellets for
solubilization and refolding. Briefly, the MT-SP1 mutant CB155
plasmid construct was transformed into XL-1 Blue Supercompetent
cells and a single fresh colony was picked and grown in 25 ml of
luria broth (LB; Difco LB Broth Lennox, approximate formulation per
liter: 10.0 g Tryptone, 10.0 g Yeast Extract, 5.0 g Sodium
Chloride) containing 50 .mu.g/ml carbenecillin at 37.degree. C.
overnight. Ten milliliters of the overnight culture was diluted
into 1 L of LB in an Ultra Yield Flask (2.5L) and was shaken at
37.degree. C. to an OD600 of about 0.6 to about 0.7 (i.e. shaken at
37.degree. C. for about 2 hours). IPTG (Dioxane Free; Calbiochem)
at 1.0 M was added to the culture at a final concentration of 1 mM
to induce expression of the protease in inclusion bodies and the
culture was shaken for an additional 4 hours at 37.degree. C. The
culture was harvested by centrifugation at 6000 rpm in a Sorvall
rotor # SLC4000 for 20 minutes.
[0549] The cell pellet from the 1 L culture was resuspended in 50
ml of Wash Buffer II (300 mM Sodium Chloride, 50 mM Potassium
Phosphate pH 7.4) and was transferred to a rosette cell for
sonication. Cells were lysed by sonication as follows: 20-50 ml
solution for 2 minutes, 30% duty cycle, output=5-6, on ice,
repeated three times; 100+ ml solution for 2 minutes, 60% duty
cycle, output=8, on ice, repeated six times. The sonicated lysate
was centrifuged at 7000 rpm for 20 minutes at 4.degree. C.
Supernatant was discarded and the pellets were resuspended in 40 ml
Wash Buffer I (300 mM Sodium Chloride, 50 mM Potassium Phosphate pH
7.4, 0.5% LDAO) per about 2.0 gram of inclusion bodies using a
spatula and vortexing. The inclusion bodies were centrifuged at
9000 rpm for 15 minutes at 4.degree. C., and washed a total of
three times in Wash Buffer I. The centrifugation and washing steps
were repeated two additional times in Wash Buffer II. The washed
inclusion bodies were suspended in 20 ml of denaturing buffer (6 M
Guanadine Hydrochloride, 100 mM Tris HCL pH 8.0, 20 mM
Dithiothreitol) per about 2.0 grams of inclusion bodies using a
spatula to break up the pellet and rocked at room temperature for
30 minutes until the pellet was dissolved or mostly dissolved. Any
insoluble material was removed by centrifugation at 9000 rpm in a
Sorvall SLA600TC rotor followed by resuspension in 20 ml buffer.
The sample was slowly dripped into a beaker containing 100.times.
volume of refolding buffer (100 mM Tris HCL pH 8.0, 150 mM NaCl, 5
mM reduced Glutathione, 0.05M oxidized glutathione, 1.5 M
L-Arginine mono hydrochloride) and stirred slowly at 4.degree. C.
for 72 hours. The sample was diluted to 1 M final concentration of
Arginine-HCL in 50 mM Tris pH 8.0/50 mM NaCl and then concentrated
to about 700 ml using cross-flow filtration. The sample was then
transferred to the VivaFlow (Sartorius, Edgewood N.Y.) and further
concentrated to a final volume of about 300 ml. The sample was
dialyzed into 50 mM Tris pH 8.0/50 mM NaCl (8 L) overnight. Some
precipitation of the sample occurred. The sample was centrifuged at
9000 rpm to remove the precipitation.
[0550] The sample was incubated at room temperature until
auto-activation of the protease occurred by cleavage of the
proregion to release the mature enzyme. The autoactivation occurs
during the purification process. Activity was monitored by SDS-PAGE
(a 3 kilodalton shift). Activity also was monitored by enzyme
activity as assessed by cleavage of a fluorogenic substrate. To
measure enzyme activity, protease was diluted from 1:20 fold to
1:100 fold in assay buffer containing 50 mM Tris pH 8.0, 50 mM NaCl
and 0.01% Tween-20. Five .mu.l of the diluted protease was mixed
with 50 .mu.l of 100 .mu.M Ac-RQAR-AMC substrate, and the
fluorescence was measured in a fluorescence spectrophotometer
(Moleculare Devices Gemini XPS) at an excitation wavelength of 380
nm, an emission wavelength of 450 nm and using a cut-off filter set
at 435 nm. Activity was assessed over time as the sample was
allowed to incubate at room temperature for about 24 to 72 hours
until the activity stabilized. For more dilute samples, more time
may be needed. Typically, the protease was allowed to achieve
>75% activation before purification by anion exchange.
[0551] Once activity stabilized, the sample was dialyzed into 50 mM
Hepes pH 6.5 (8 L) overnight. The sample was filtered and loaded
onto a SourceQ column. After loading onto the SourceQ column, the
sample was washed with 3 column volumes of buffer A (50 mM Hepes pH
6.5) and eluted with a gradient from 0-20% buffer B (50 mM Hepes pH
6.5/1 M NaCl) over 10 column volumes. The activity of each fraction
was measured, and active fractions were combined and dialyzed into
PBS overnight. The protein sample was concentrated to about 5 ml
and benzamidine was added to a final concentration of 20 mM to
inhibit the autolysis of CB155. The overall yield of CB155 is
typically about 15 to 20 mg CB155/1 L cell culture. A yield of 22
mg/liter cell culture has been achieved by production of CB155 at
the shake flask scale. The overall yield from inclusion body to
native protein was typically less than 10%. Titers in the
fermentation up to 3 g/L have been achieved in shake flasks.
[0552] The purified protein was assayed for specific activity,
purity, and endotoxin levels. The specific activity or amount of
active protease in each prep was determined by active site
titration by incubation with an inhibitor that binds in the active
site of the functional protease at varying concentrations followed
by addition to a fluorogenic substrate for measurement of substrate
proteolysis. The amount of active enzyme was calculated from a plot
of the residual protease activity against the inhibitor
concentration where the intercept corresponds to the concentration
of active protease. Briefly, the purified protein was titrated with
ecotin M84R using the method of Harris et al. (JBC, 1988,
273:27354-73) by titrating a stock of trypsin with
4-methylumbelliferyl p-guandinobenzoate (MUGB), then a stock of
M84R ecotin was titrated with the trypsin. Finally, the purified
protease was titrated with the M84R ecotin stock. In each case, the
protease was incubated for 30 minutes at 30.degree. C. with
concentrations of the inhibitor between 0.1 and 2 times the
expected protease concentration. Residual activities between 30%
and 90% of the uninhibited protease activity were used to plot the
data. Enzyme activity was monitored at 30.degree. C. in the
presence of 40 .mu.M Ac-RQAR-AMC in assay buffer containing 50 mM
Tris pH 8.0, 50 mM NaCl, and 0.01% Tween-20 (for trypsin activity,
10 mM CaCl2 was added) on a Gemini XPS spectrafluorimeter
(Excitation: 380 nm; Emission: 450 nm; Cut-off: 435 mn). Purity of
the protein preparation was assessed by resolution of the protein
product on SDS-PAGE followed by staining with Coomassie Blue. The
mature protease runs as a single band at 25 kD. The level of
endotoxin in each prep was determined by Liumlus Amebocyte Lysate
Chromo-LAL assay (Associated of Cape Cod) according to the
manufacturer's protocol with the following modifications. The
protease was diluted 100 fold into LAL Reagent Water (LRW), and
split into two tubes. A challenge of 100 endotoxin unit (EU)/mL was
added to one tube and LRW to the other. Then, the sample was
diluted 2 fold into stock solutions such that the reaction
contained a final concentration of 5 mM
tosyl-lysyl-chloromethylketone (TLCK) and 10 mM Tris pH 8.0. Each
sample was incubated 2 hours at 37.degree. C. to inactivate the
protease activity. Samples were further diluted 50 fold and assayed
according to the manufacturer's instructions. Results were
considered valid if the recovery of the endotoxin challenge was
between 50 and 150% of theory. The final purified CB155 typically
had >95% purity by SDS-PAGE, >90% specific activity, and
contained approximately 50 to 500 EU/ml of endotoxin.
Example 4
Large Scale Fermentation of MT-SP1 and Mutants
[0553] Large scale fermentation of MT-SP1 mutants based on the
protocol described in Example 3 has been performed. A 2 liter
culture of a selected E. coli colony transformed with mutant MT-SP1
was grown overnight in LB media (Difco). The overnight culture was
seeded into 50 L fermentation and grown for about 5 hours with
shaking at 37.degree. C. until absorbance at OD600 reached log
phase. Expression of the protease in inclusion bodies was induced
with the addition of IPTG to a final concentration of 700 .mu.M and
fermentation was continued for an additional 4 hours in fed-batch
mode. The culture was harvested by centrifugation to yield a wet
weight cell pellet of about 750 grams to 1680 grams. The cell
pellet was resuspended and sonicated in 50 mM Tris, pH 8.0, 0.5 M
KCl, 10% glycerol, 1 mM beta-mercaptoethanol at 10 ml buffer per 1
gram cell pellet. The sonication conditions were pulse on: 4 s,
pulse off: 6 s, 700 W, 30 minutes. The sonicated lysate was
centrifuged at 7000 rpm for 30 minutes at 4.degree. C. Supernatant
was discarded and the pellets were resuspended in Wash Buffer I (50
mM Tris pH 8.0, 0.5 M KCl, 10% glycerol, 1 mM beta-mercaptoethanol,
0.01% Tween 20) at 10 ml buffer per 1 g of inclusion bodies using a
spatula and vortexing. The inclusion bodies were centrifuged and
washed two times. The inclusion body yield was about 176 to 506
grams.
Example 5
Production of MT-SP1 in Pichia pastoris
[0554] The production of multi-milligram amounts of the protease
domain of MT-SP1 was carried out by fermentation in a BioFlo 3000
fermentor (New Brunswick Scientific, NJ) equipped with a 3.3 L
capacity bioreactor using a SMD1168/pPIC9K:MTSP1 Sac SCI clone
(Friedrich et al. (2002) J. Biol. Chem., 277:2160-2168). ZA001
complex media (10 g/L yeast extract, 20 g/L peptone, 40 g/L
glycerol, 5 g/L ammonium sulfate, 0.2 g/L calcium sulfate
dehydrate, 2 g/L Magnesium sulfate hepahydrate, 2 g/L potassium
sulfate, 25 g/L sodium hexametaposhpate, 4.35 ml/L PTM1) was
inoculated with 100 ml of an overnight culture of the P. pastoris
transformant. The culture was supplemented with 50% glycerol by
fed-batch phase and induced for 18-24 hours with methanol
controlled at 0.025%.
[0555] To purify recombinant MT-SP1 secreted into the culture
media, cell and cell debris was removed by centrifugation at 5000 g
for 30 minutes. The resulting supernatant was decanted, adjusted to
pH 8.0 with 10 N NaOH, and filtered through a SartoBran 300
0.45+0.2 .mu.M capsule (Sartorius). The supernatant was
concentrated to 1 L by ultrafiltration using a 10 kDa
ultrafiltration cartridge (NC SRT UF system with AG/Technologies
UFP-10-C-5A filter), and the buffer was exchanged by cross-flow
filtration into buffer A (50 mM tris-HCl, 50 mM NaCl, 0.05%
tween-80, pH 8.0). The filtration unit was rinsed once with 1 L
buffer A, which was combined with the concentrate.
[0556] The concentrated MT-SP1-containing solution was applied onto
a 150 ml benzamidine column, that had been equilibrated with buffer
A, at a flow rate of 8 ml/min. The column was washed with 3 column
volumes of buffer B (50 mM tris-HCl, 1.0 M NaCl, 0.05% tween-80, pH
8.0) and eluted with 3 column volumes of buffer C (50 mM tris-HCl,
1.0M L-arginine, 0.05% tween-80, pH 8.0). Fractions containing
MT-SP1 activity were pooled and concentrated to 10 ml using a
JumboSep concentrator (Pall Gelman) and a 10 kDa cutoff membrane.
Once concentrated to 10 ml, the buffer was exchanged into buffer D
(50 mM Na.sub.2HPO.sub.4, 125 mM NaCl, pH 5.5), and the volume was
adjusted to 5-10 ml. The retentate was removed and the concentrator
washed with buffer D, which was added to the concentrate. The total
sample volume was adjusted to 15 ml.
[0557] The partially purified MT-SP1 from the benazmidine column
was passed through a 5 ml Q-sepharose Fast Flow HiTrap Column
(Amersham-Pharmacia Biotech) pre-equilibrated with 15 ml of buffer
D. The flow through was collected, and the protein concentration
was determined by measurement of OD280 (using an extinction
coefficient of 2.012 mg/OD280). Purified MT-SP1 was then
deglycosylated by the addition of 0.1 .mu.l of Endoglycosidase H
(ProZyme, 5 U/ml) per mg of protein and incubation overnight at
4.degree. C. with gentle swirling followed by a subsequent anion
exchange purification step (not required for CB155 MT-SP1
mutant).
[0558] The conductivity of the deglycosylated pool was adjusted to
2.0-3.0 mS/cm with Nanopure H.sub.2O and the pH was adjusted to 6.5
(about 200 to 300 ml final volume). MT-SP1 was then further
purified by anion exchange chromatography by loading directly onto
a Pharmacia Akta Explorer system using a 7 ml Source 15Q anion
exchange column (Amersham-Pharmacia Biotech). The protein was
eluted in a buffer containing 50 mM HEPES, pH 6.5 with a 0-0.33M
NaCl gradient over 10 column volumes at a flow rate of 6 ml/min.
Fractions containing protein were pooled, and benzamidine was added
to a final concentration determined by measurement of OD280 and use
of a theoretical extinction coefficient of 2.012 mg/OD280.
Example 6
Assessment of Plasma Activity
[0559] The activity in plasma of wildtype or modified proteases was
determined by cleavage of the peptide substrate Ac-RQAR-AMC, which
includes the MT-SP1 auto-activation site. One microliter of a 10
.mu.M protease stock was diluted into 9 .mu.l PBST or 9 .mu.l
pooled human citrated plasma (Innovative Research; Sarasota, Fla.)
(1 .mu.M final). The reaction was incubated for 5 minutes at
37.degree. C. At the end of the incubation, 1 .mu.l of the
incubated protease mixture was diluted into 250 .mu.l assay buffer
(50 mM Tris, pH 8.0, 50 mM NaCl, 0.01% Tween-20). 1 .mu.l of the
Ac-RQAR-AMC substrate (6.25 mM in DMSO) was added to each well of a
Nunc black microtiter plate and 50 .mu.l of the diluted, incubated
protease mixture was added to the well. Cleavage of the substrate
was monitored using a Spectromax fluorescence plate reader by
taking kinetic measurements with the excitation wavelength at 380
nm and the emission wavelength at 450 nm. Fractional activity of
the protease was calculated as a ratio of the activity of the
protease in plasma divided by the activity in PBST.
[0560] The plasma activity of wildtype MT-SP1 or a panel of MT-SP1
mutants was determined in parallel with assessment of the proteases
in hemolytic assays (see Example 7 below). The results are set
forth in Table 23 below.
Example 7
Hemolytic Assays for Screening and Titrating Protease Activity by
Detection of Complement-Induced Hemolysis of Red Blood Cells
[0561] The functional activity of the complement system can be
assessed using traditional hemolytic assays, which screen for
function of the total complement pathway by determining the ability
of the sample to lyse erythrocytes. Serial dilutions of the sample
to be analyzed are incubated with antibody-sensitized sheep
erythrocytes at a defined temperature. The number of red cells
lysed is determined by spectrophotometric absorbance of released
hemoglobin, which has a linear relationship to complement protein
levels in the 50% lysis range. The results are usually expressed as
reciprocal dilutions of the sample required to produce 50% lysis.
Thus, when evaluating the activity of the components of the
classical pathway, a CH.sub.50 value can be determined. Tests
evaluating the functional activity of the alternative pathway to
determine the AH.sub.50 (the titer at which 50% hemolysis occurs)
use guinea pig, rabbit or chicken erythrocytes as target cells.
Activation of the classical pathway is blocked by the addition of
EGTA and Mg to the alternative pathway hemolytic assay.
[0562] Hemolytic assays can be modified to determine the effect of
a given protease on complement activation. Protease is incubated
with the plasma prior to co-incubation with the erythrocytes.
Cleavage of complement products by the proteases will result in
decreased complement activity. By incubating the plasma with serial
dilutions of the proteases, an IC.sub.50 can be determined, which
is the concentration of protease at which 50% inhibition of
complement activity is achieved.
[0563] A. Classical Hemolytic Assays
[0564] 1. Classical Hemolytic Assay: Preincubation with 20%
Plasma
[0565] a. To assess complement activation following treatment with
proteases, human plasma with sodium citrate as an anticoagulant
(Innovative Research, Inc.) was diluted into PBST to a final
concentration of 20% (10 .mu.l plasma in 40 .mu.l PBST) before
addition of wildtype MT-SP1, CB155, or CB42 diluted to a final
concentration of 0-1 .mu.M. The reaction was incubated at
37.degree. C. for 1 hour. The plasma solution was further diluted
to a final concentration of 0.5% in a solution of sheep's red blood
cells activated with IgG (6.25 .mu.l plasma solution in 250 .mu.l
sheep's red blood cell solution, Diamedix (Miami, Fla.)). The
solution was incubated with shaking at room temperature for 45
minutes. The cells were spun down at 2000 rpm for 2 minutes and 100
.mu.l of the supernatant was removed and placed in a clear 96-well
microtiter plate. Release of hemoglobin from the lysed red blood
cells was monitored by reading the optical density (OD) at 415 nm.
The IC.sub.50 (nM) of hemolysis by wildtype MT-SP1, CB155, and CB42
was 131 nM, 94 nM, and 67 nM, respectively.
[0566] A panel of MT-SP1 modified proteases were tested for
inhibition of hemolysis. Diluted human plasma containing sodium
citrate as an anticoagulant was incubated with 200 nM of wildtype
MT-SP1, CB12, CB13, CB31, CB32, CB40, CB41, CB42, CB43, CB44, CB45,
CB64, CB66, CB67, and CB155. The reaction was incubated at
37.degree. C. for 1 hour. The plasma solution was further diluted
to a final concentration of 0.5% in a solution of sheep's red blood
cells activated with IgG and hemolysis was assayed as described
above. The CH50 value was determined. The fraction of hemolysis was
determined by comparing the CH50 value of the wildtype or modified
proteases compared to a sample containing no added protease
(positive control) where the fraction of hemolysis of the positive
control was set at 1.00. Table 22 depicts the raw fraction of
hemolysis values. The data shows that all proteases tested
inhibited hemolysis by at least 50% compared to the positive
control with hemolysis completely inhibited in the presence of
CB42. TABLE-US-00022 TABLE 22 Inhibition of Hemolysis by a Panel of
MT-SP1 Variants Fraction of Protease Hemolysis Wildtype 0.14 CB12
0.22 CB13 0.50 CB31 0.51 CB32 0.38 CB40 0.48 CB41 0.32 CB42 0.00
CB43 0.23 CB44 0.48 CB45 0.27 CB64 0.34 CB66 0.44 CB67 0.33 CB155
0.09 Negative control 0.00 Positive control 1.00
[0567] b. In another experiment, to assess classical complement
activation following treatment with proteases, proteases were
initially diluted in PBST to a concentration of 5.0 .mu.M for
screening protocols, while serial dilutions of the proteases from
50 .mu.M to 0.390 .mu.M were used for protocols to determine the
IC.sub.50. MT-SP1 or modified proteases were preincubated with a
final concentration of 20% plasma in an 0.2 ml tube by combining 2
.mu.l of the diluted protease solution, 10 .mu.l of human plasma
(with sodium citrate as an anticoagulant; Innovative Research,
Inc.) and 38 .mu.l of PBST. This resulted in a further dilution of
the protease to give a final concentration of 200 nM protease for
the screening protocol, and 2.0 .mu.M to 0.0156 .mu.M protease for
the IC.sub.50 protocol. A no-protease control (10 .mu.l plasma and
40 .mu.l PBST) and a background control (50 .mu.l PBST only) also
were included in the assays. The reaction was incubated at
37.degree. C. for 1 hour. Sensitized sheep erythrocytes (Diamdex,
Miami, Fla.) were added to polypropylene 96-well plates at a volume
of 120 .mu.l per well, and 3 .mu.l of the plasma/protease solution
was mixed into each well giving a final plasma concentration of
0.5%. The solution was incubated with shaking at room temperature
for 45 minutes. The cells were spun down at 2000 rpm for 5 minutes
in a Sorvall table top centrifuge to pellet the unbroken cells, and
100 .mu.l of the supernatant was removed and placed in a clear
96-well microtiter plate. Release of hemoglobin from the lysed red
blood cells was monitored by reading the optical density (OD) at
415 nm. The fraction hemolysis was calculated by subtracting the
background control from all of the wells, then dividing the
experimental samples by the no-protease control (positive control),
where the fraction of hemolysis of the positive control was set at
1.00. The IC.sub.50 (nM) of hemolysis by the proteases were
measured by plotting the fraction hemolysis versus protease
concentration on a 4 parameter logistic curve fit (SoftMax Pro
software, Molecular Devices, Calif.).
[0568] A panel of MT-SP1 modified proteases were tested for
inhibition of hemolysis through cleavage of one or more components
of the classical complement pathway. Diluted human plasma
containing sodium citrate as an anticoagulant was incubated with
either 200 nM, or serial dilutions from 2.0 .mu.M to 0.0156 .mu.M,
of wildtype MT-SP1 (CB200), CB12, CB16, CB17, CB20, CB21, CB42,
CB43, CB44, CB45, CB66, CB80, CB82, CB155, CB212, CB213, CB214,
CB216, CB218, CB219, CB232, CB235, CB238, CB244, CB245, CB251,
CB252, CB255, CB257, CB268, CB274, CB331, CB332, CB349, CB350,
CB351, CB353, CB357, CB367, CB373, CB377, CB381, CB383, CB385,
CB387, CB388, CB403, CB409, CB412, CB413, CB421, CB422, CB423,
CB450, CB451, CB458, CB464, CB486, CB487, CB488, CB489 and CB490.
The reaction was incubated at 37.degree. C. for 1 hour. The
protease/plasma solution was further diluted in sensitized sheep
erythrocytes and hemolysis was assayed as described above. The
fraction hemolysis and ID.sub.50 was determined. Table 23 depicts
the fraction hemolysis at 200 nM (Classical 200 nM hemolysis) and
the IC.sub.50 for each protease (20% plasma pre-incubation).
[0569] 2. Classical Hemolytic Assay: Preincubation with 90%
Plasma
[0570] The modified classical hemolytic assay can be adapted
further to measure the inhibitory activity of proteases under more
physiological conditions by adjusting the plasma and protease
concentrations. Proteases were initially diluted in PBST to a
concentration of 50 .mu.M for screening protocols, while serial
dilutions of the proteases from 200 .mu.M to 1.56 .mu.M were used
for protocols to determine the IC.sub.50. MT-SP1 or modified
proteases were preincubated with a final concentration of 90%
plasma in an 0.2 ml tube by combining 2 .mu.l of the diluted
protease solution, 18 .mu.l of human plasma (with sodium citrate as
an anticoagulant; Innovative Research, Inc.). This resulted in a
further dilution of the protease to give a final concentration of
5.0 .mu.M protease for the screening protocol, and 20 .mu.M to
0.156 .mu.M protease for the IC.sub.50 protocol. A no-protease
control (18 .mu.l plasma and 2 .mu.l PBST) and a background control
(20 .mu.l PBST only) also were included in the assays. The reaction
was incubated at 37.degree. C. for 1 hour. The reaction mixtures
were further diluted to 20% plasma with the addition of 70 .mu.l
PBST. Sensitized sheep erythrocytes (Diamdex, Miami, Fla.) were
concentrated to 10.times. by pelleting a 3.0 ml aliquot, removing
2.7 ml of buffer and resuspending the cell pellet in the remaining
0.3 ml buffer. The concentrated sensitized erythrocytes were added
to polypropylene 96-well plates at a volume of 12 .mu.l per well.
Preincubated protease/plasma mixtures at 6 .mu.l or 60 .mu.l were
added to the erythrocytes to give a final concentration of 1%
plasma or 10% plasma, respectively, in a final volume of 120 .mu.l
(PBST added to final volume). The solution was incubated with
shaking at room temperature for 45 minutes. The cells were spun
down at 2000 rpm for 5 minutes to pellet the unbroken cells, and
100 .mu.l of the supernatant was removed and placed in a clear
96-well microtiter plate. Release of hemoglobin from the lysed red
blood cells was monitored by reading the optical density (OD) at
415 nm. The fraction hemolysis was calculated by subtracting the
background control from all of the wells, then dividing the
experimental samples by the no-protease control (positive control),
where the fraction of hemolysis of the positive control was set at
1.00. The IC.sub.50 (nM) of hemolysis by the proteases were
measured by plotting the fraction hemolysis vs protease
concentration on a 4 parameter logistic curve fit (SoftMax Pro
software, Molecular Devices, Calif.).
[0571] Wildtype MT-SP1 (CB200), and MT-SP1 mutants CB252 and CB377
were tested for inhibition of hemolysis following preincubation
with 90% plasma. Preincubated protease/plasma mixtures (at a final
concentration of 20 nM to 2000 nM protease) were incubated with
sensitized erythrocytes at a final plasma concentration of 10% and
hemolysis was assessed as described above. The IC.sub.50 of
hemolysis was determined as described above. The IC.sub.50 of
CB200, CB252 and CB377 was 967 nM, 379 nM, and 205 nM,
respectively.
[0572] Modified MT-SP1 CB450 also was assessed for in vitro
classicial pathway-induced hemolysis following preincubation with
90% plasma. Preincubated protease/plasma mixtures (at a final
concentration of 20 nM to 2000 nM protease) were incubated with
sensitized erythrocytes at a final plasma concentration of 1% and
10% and hemolysis was assessed as described above. The results show
that CB450 dose-dependently decreased hemolysis induced by 1% or
10% plasma, with slightly greater inhibition of hemolysis observed
upon induction by 1% plasma. For example, there was no detectable
hemolysis observed upon induction in 1% plasma in the presence of
100 nM or greater CB450, whereas induction in 10% plasma required a
higher yield of protease to achieve complete inhibition of
hemolysis (i.e. about 750 nM or greater CB450 protease).
[0573] B. Alternative Hemolytic Assay
[0574] 1. Alternative Hemolytic Assay: Preincubation with 20%
Plasma
[0575] To assess alternative complement activation following
treatment with proteases, proteases were initially diluted in
GVB/Mg/EGTA buffer containing gelatin veronal buffer (GVB;
Comptech) with 1 mM MgCl.sub.2 and 8 mM EGTA. The proteases were
diluted to a concentration of 7.5 .mu.M for screening protocols,
while serial dilutions of the proteases from 30 .mu.M to 0.2344
.mu.M were used for protocols to determine the IC.sub.50. MT-SP1 or
modified proteases were preincubated with a final concentration of
20% plasma in a well of a 96-well polypropylene plate by combining
5 .mu.l of the diluted protease solution, 15 .mu.l of human plasma
(with sodium citrate as an anticoagulant; Innovative Research,
Inc.) and 55 .mu.l of GVB/Mg/EGTA buffer. This resulted in a
further dilution of the protease to give a final concentration of
500 nM protease for the screening protocol, and 2.0 .mu.M to 0.0156
.mu.M protease for the IC.sub.50 protocol. A no-protease control
(15 .mu.l plasma and 60 .mu.l GVB/Mg/EGTA) and a background control
(75 .mu.l GVB/Mg/EGTA only) also were included in the assays. The
reaction was incubated at room temperature for 1 hour. Following
the incubation, 5 .mu.l GVB/Mg/EGTA was added to the incubated
mixture, followed by 20 .mu.l of washed chicken Alsevyrs (50% mix
of whole blood from chickens and alsevers solution, which contains
anti-coagulants; Colorado Serum Company, CO) giving a final plasma
concentration of 15%. Prior to the addition, the Chicken Alsevyr
were sensitized as described in Example 19 below and the cells were
centrifuged and washed 3 times in cold PBS and resuspended in 10 ml
GVB/Mg/EGTA, and stored on ice until use. The plates were shaken at
37.degree. C. for 1 hour. The cells were spun down at 2000 rpm for
5 minutes to pellet the unbroken cells, and 100 .mu.l of the
supernatant was removed and placed in a clear 96-well microtiter
plate. Release of hemoglobin from the lysed red blood cells was
monitored by reading the optical density (OD) at 415 nm. The
fraction hemolysis was calculated by subtracting the background
control from all of the wells, then dividing the experimental
samples by the no-protease control (positive control), where the
fraction of hemolysis of the positive control was set at 1.00. The
IC.sub.50 (nM) of hemolysis by the proteases were measured by
plotting the fraction hemolysis vs protease concentration on a 4
parameter logistic curve fit (SoftMax Pro software, Molecular
Devices, CA).
[0576] A panel of MT-SP1 modified proteases were tested for
inhibition of hemolysis through cleavage of one or more components
of the alternative complement pathway. Human plasma containing
sodium citrate as an anticoagulant was incubated with either 500
nM, or serial dilutions from 2.0 .mu.M to 0.0156 .mu.M, of wildtype
MT-SP1 (CB200), CB12, CB16, CB17, CB20, CB21, CB42, CB43, CB44,
CB45, CB66, CB80, CB82, CB155, CB212, CB213, CB214, CB216, CB218,
CB219, CB232, CB235, CB238, CB244, CB245, CB251, CB252, CB255,
CB257, CB268, CB274, CB331, CB332, CB349, CB350, CB351, CB353,
CB0357, CB367, CB373, CB377, CB381, CB383, CB385, CB387, CB388,
CB403, CB409, CB412, CB413, CB421, CB422, CB423, CB450, CB451,
CB458, CB464, CB486, CB487, CB488, CB489 and CB490. The reaction
was incubated at room temperature for 1 hour. The protease/plasma
solution was further diluted in chicken erythrocytes and hemolysis
was assayed as described above. The fraction hemolysis and
ID.sub.50 was determined. Table 23 depicts the fraction hemolysis
at 500 nM (Alternative 500 nM hemolysis) and the IC.sub.50 for each
protease (20% plasma pre-incubation). TABLE-US-00023 TABLE 23
Assessment of Hemolysis and Plasma Activity by a Panel of MT-SP1
mutants Classical Alternative Classical Alternative IC.sub.50
IC.sub.50 200 nM 500 nM Hemolysis Hemolysis Plasma CB# Mutations
Hemolysis Hemolysis (nM) (nM) Activity CB200 wt 0.14 0.14 131 545
0.2 CB12 F97D 0.109 0.229 127.15 0.2 CB16 Y146F 0.058 0.049 182.6
204.95 0.23 CB17 L172N 0.198 0.044 211.7 180 0.19 CB20 Q175D 0.106
0.071 125.4 138.4 0.13 CB21 Q175E 0.156 0.047 123.7 81.7 0.16 CB42
Y146E 0.072 0.088 91.6 117.5 0.19 CB43 Y146A 0.195 0.226 160.2 0.07
CB44 Y146W 0.229 0.114 157.33 0.14 CB45 Y146R 0.195 0.297 170.42
0.07 CB66 K224A 0.236 0.097 166.3 102.2 0.24 CB80 R60cD 0.085 0.463
82 367 0.23 CB82 R60cW 0.147 0.48 74.8 283 0.29 CB155 Y146D/K224F
0.09 0.29 94 221 0.36 CB212 Y146N/K224F 0.477 0.538 371.5 0.47
CB213 Y146E/K224F 0.188 0.21 168.5 0.29 CB214 Y146A/K224F 0.197
0.32 171.25 0.42 CB216 Q192V/K224F 0.892 0.866 1200 0.47 CB0218
Q192F/K224F 0.975 0.947 1000 0.09 CB219 Y146D/Q192A/K224F 0.929
0.887 1100 0.63 CB232 Y146E/K224L 0.024 0.082 74.8 94.2 0.23 CB235
Y146E/K224A 0.035 0.126 96.7 89.8 0.26 CB238 Y146D/K224L 0.023
0.105 91.4 149.3 0.53 CB244 Y146D/K224R 0.046 0.562 66.08 617.5
0.18 CB245 Y146D/K224N 0.05 0.51 127.84 421.5 0.32 CB251
Y146E/K224R 0.052 0.47 57.57 625 0.19 CB252 Y146E/K224N 0.025 0.405
38.98 451.5 0.27 CB255 Y146E/K224T 0.115 0.84 179.46 580.1 0.55
CB257 Y146E/K224Y 0.032 0.548 118.65 158 0.44 CB268 Q221aD 0.049
0.359 55.3 512 0.2 CB274 G147E 0.102 0.441 80.7 311.5 0.23 CB331
I41D/Y146D/K224L 0.8 0.19 325.15 76.4 0.34 CB332 I41E/Y146D/K224L
0.254 0.124 150.05 52.3 0.53 CB349 I41D/Y146D/K224F 0.775 0.188
359.28 110.17 0.61 CB350 I41E/Y146D/K224F 0.729 0.24 284.14 177.07
0.69 CB351 I41T/Y146D/K224F 0.144 0.245 50.31 71.25 0.71 CB353
H143V/Y146D/K224F 0.278 0.193 160.85 461.4 0.26 CB357
I41T/Y146D/K224L 0.068 0.203 88.58 126.26 0.41 CB367
Y146D/Q175D/K224R 0.1 0.219 86 146.92 0.31 CB373 Y146E/Q175D/K224R
0.093 0.268 73.48 123.2 0.34 CB377 Y146E/Q175D/K224N 0.052 0.284
58.7 102.87 0.61 CB381 Y146D/Q175H/K224L 0.169 0.219 111.55 291.86
0.25 CB383 Y146D/Q175L/K224L 0.153 0.137 88.03 266.25 0.14 CB385
Y146D/Q175F/K224L 0.184 0.207 123.13 472.11 0.18 CB387
Y146D/Q175W/K224L 0.147 0.115 91.2 224.69 0.25 CB388
Y146D/Q175Y/K224L 0.272 0.194 114.83 317.21 0.23 CB403
Y146D/D217F/K224L 0.262 0.406 152.37 221.09 0.054 CB409
I41A/Y146D/K224F 0.22 0.174 281.2 0.498 CB412 I41L/Y146D/K224F
0.165 0.247 262.5 0.547 CB413 I41F/Y146D/K224F 0.222 0.215 251.11
0.446 CB421 I41T/Y146D/Q175D/K224F 0.714 0.271 478.9 0.585 CB422
I41T/Y146E/Q175D/K224N 0.05 0.15 50.35 0.511 CB423 I41T/Y146E/K224L
0.061 0.176 180.19 0.221 CB450 I41T/Y146D/G151L/K224F 0.255 0.223
269.14 0.287 CB451 Y146D/Q221aL/K224S 0.147 0.416 130.46 173.07
0.12 CB458 Y146E/Q221aE/K224R 0.136 0.58 93.25 332.95 0.21 CB464
Y146E/Q221aE/K224F 0.02 0.124 52.3 91.15 0.54 CB486
I41T/Y146E/Q175D/K224R 0.014 0.128 43.36 CB487
I41T/Y146E/G151L/Q175D/K224N 0.026 0.173 52.87 0 CB488
I41T/Y146E/G151L/Q175D/K224F 0.086 0.195 72.61 CB489
I41T/Y146E/G151L/Q175D/K224L 0.038 0.143 50.56 CB490
I41T/Y146E/G151L/Q175D/K224R 0.031 0.125 52.63
[0577] 2. Alternative Hemolytic Assay: Preincubation with 90%
Plasma
[0578] The modified alternative hemolytic assay can be adapted
further to measure the inhibitory activity of proteases, such as
for example those with low activity, by adjusting the plasma and
protease concentrations. Proteases were initially diluted in
GVB/Mg/EGTA to a concentration of 50 .mu.M for screening protocols,
while serial dilutions of the proteases from 200 .mu.M to 1.56
.mu.M were used for protocols to determine the IC.sub.50. MT-SP1 or
modified proteases were preincubated with a final concentration of
90% plasma in a well of a 96-well polypropylene plate by combining
2 .mu.l of the diluted protease solution with 18 .mu.l of human
plasma (with sodium citrate as an anticoagulant; Innovative
Research, Inc.). This resulted in a further dilution of the
protease to give a final concentration of 5.0 .mu.M protease for
the screening protocol, and 20 .mu.M to 0.156 .mu.M protease for
the IC.sub.50 protocol. A no-protease control (18 .mu.l plasma and
2 .mu.l GVB/Mg/EGTA) and a background control (20 .mu.l GVB/Mg/EGTA
only) also were included in the assays. The reaction was incubated
at room temperature for 1 hour. After the incubation, 80 .mu.l of
GVB/Mg/EGTA was added to the incubated plasma/protease mix,
followed by the addition of 20 .mu.l of washed chicken cells
(described above), giving a final plasma concentration of 15%. The
plates were shaken at 37.degree. C. for 1 hour. The cells were spun
down at 2000 rpm for 5 minutes to pellet the unbroken cells, and
100 .mu.l of the supernatant was removed and placed in a clear
96-well microtiter plate. Release of hemoglobin from the lysed red
blood cells was monitored by reading the optical density (OD) at
415 nm. The fraction hemolysis was calculated by subtracting the
background control from all of the wells, then dividing the
experimental samples by the no-protease control where the fraction
of hemolysis of the positive control was set at 1.00. The IC.sub.50
(nM) of hemolysis by the proteases were measured by plotting the
fraction hemolysis vs protease concentration on a 4 parameter
logistic curve fit (SoftMax Pro software, Molecular Devices,
CA).
[0579] Modified MT-SP1 CB450 was assessed for in vitro alternative
pathway-induced hemolysis following preincubation with 90% plasma.
Preincubated protease/plasma mixtures (at a final concentration of
20 nM to 2000 nM protease) were incubated with chicken cells at a
final plasma concentration of 15% and hemolysis was assessed as
described above. The results show that CB450 dose-dependently
decreased hemolysis induced by 15% plasma, with no detectable
hemolysis of the red blood cells observed at about 500 nM or
greater CB450 protease.
Example 8
Detection of Complement-Induced Hemolysis of Red Blood Cells Using
Complement-Depleted Sera
[0580] a. To assess complement activation by purified complement
factors treated in the presence or absence of wildtype MT-SP1,
CB155, or CB42 protease, purified complement factors C2, C3, C4,
and C5 and C2-, C3-, C4- and C5-complement depleted media were
purchased from Quidel. Purified complement proteins (C2, C3, C4, or
C5) were incubated with 100 nM wildtype MT-SP1, CB155 or CB42
protease at 37.degree. C. for 3 hours. The concentration of the
purified protein used in the reaction was 5 .mu.M. One microliter
of the reaction along with 1 .mu.l of the appropriate complement
depleted sera was added to 100 .mu.l of sensitized sheep red blood
cells and assayed for hemolytic activity as described in Example 7
above. The CH50 values of hemolysis were determined. The fraction
of hemolysis of the samples was determined by comparing the CH50
value of the sample to the C2-sera containing sample containing no
added protease which was set at 1.00 Table 24 depicts the raw
fraction of hemolysis values. Wildtype MT-SP1, CB155, and CB42
proteases inactivate C2 and C3, but not C4 and C5 as determined by
an inhibition of hemolysis. TABLE-US-00024 TABLE 24 Hemolysis by
Protease-incubated Complement Proteins Added Back to
Complement-depleted Sera Fraction of Protease Hemolysis C2 1 C2 +
WT 0 C2 + CB155 -0.00104 C2 + CB42 0.071429 C3 1.032325 C3 + WT
0.008342 C3 + CB155 0.07195 C3 + CB42 0.122449 C4 0.957766 C4 + WT
0.970027 C4 + CB155 0.944142 C4 + CB42 1.040816 C5 0.983651 C5 + WT
0.888283 C5 + CB155 0.942779 CB + CB42 1.020408
[0581] b. In another experiment, a panel of complement proteins
were treated in the presence or absence of MT-SP1 (CB200), CB252,
and CB377 to assess consequences on inactivation of purified
complement proteins by the proteases. Purified complement proteins
(all from Quidel; San Diego, Calif.) were incubated at their
physiological serum concentrations (C1q: 180 .mu.g/ml; C2: 25
.mu.g/ml; C3: 1000 .mu.g/ml; C4: 500 .mu.g/ml; C5: 75 .mu.g/ml; C6:
70 .mu.g/ml; C8: 80 .mu.g/ml; C9: 60 .mu.g/ml) in PBST with 25 nM
CB200, CB252, or CB377 protease. One .mu.l of the reaction was
added to 100 .mu.l of sensitized sheep red blood cells in the
presence of 1 .mu.l of sera that was depleted for the corresponding
complement factor (all depleted sera purchased from Quidel). As
controls, a no protease control, depleted sera only, and normal
sera only were included in the reactions. The samples were
incubated in wells of a 96-well plate for 1 hour at room
temperature with shaking. The cells were spun down by
centrifugation to pellet the unbroken cells, and 85 .mu.l of the
supernatant was removed and transferred to a clear 96-well
microtiter plate. Release of hemoglobin from the lysed red blood
cells was monitored by reading the optical density (OD) at 415 nm.
The results showed that the control depleted sera in the absence of
the added corresponding purified complement protein showed little
to no hemolysis as assessed by the OD415 reading, whereas the
control normal sera exhibited hemolysis as assess by an OD415 of
about 0.4. For each of the reactions where the corresponding
purified complement protein was added back to the depleted sera (no
protease control), the OD415 reading was comparable to that
observed with the normal sera. Preincubation of the complement
proteins C1q, C3, C4, C5, C6, C7, C8, or C9 with CB200, CB252, or
CB377 showed no decreased hemolysis as compared to normal sera or
the no protease control. In contrast, preincubation of C2 with
CB200, CB252, or CB377 resulted in an inhibition of hemolysis as
assessed by a reduced OD415 absorbance to levels comparable to the
depleted sera control.
Example 9
Visualization of the Proteolytic Cleavage of Complement Component
C2 in Human Plasma by Western Blotting
[0582] a. To visualize the proteolytic cleavage products, human
plasma was diluted to 5% in 40 .mu.l PBST and purified CB155,
CB200, or CB42 was added to a final concentration between 0 and 500
nM. The samples were incubated for 1 hour at 37.degree. C. Twenty
microliters of the reaction, or purified complement C2 as a control
(CompTech), was separated by SDS-PAGE on a 4-12% Tris-Glycine gel
at 200 V for 35 minutes using a Novex Mini-Cell Surelock apparatus
(Invitrogen), then transfered to PVDF membrane (Invitrogen) using a
Biorad Transblot SD semi-dry transfer cell. The membrane was
blocked 1 hour with 5% dry milk in TBST (tris-buffered saline with
1% Tween 20) followed by washing and incubation with 1:3000
dilution of goat anti-human C2 antisera (Quidel) in 5% dry
milk/TBST overnight at 4.degree. C. The membrane was washed again
three times in TBST for 10 minutes each wash, and incubated with
1:10,000 HRP conjugated rabbit anti-goat antibody (Invitrogen,
Carlsbad, Calif.) in 5% dry milk/TBST for one hour at room
temperature. The filter was washed three times in TBST for 10
minutes each wash, and the C2 in the plasma, and its cleavage
products, were developed with ECL Plus Western Blotting Detection
System (Amersham Biosciences) according to the manufacturer's
instructions. Densitometry was performed on a FluorChem imaging
system (Alpha Innotech Corp.) using AlphaEase FC Fluorchem 8800
software, version 3.1.2 (Alpha Innotech Corp.). Densitometry was
used to determine the ratio of uncleaved product to cleaved
product. The IC.sub.50 of the proteases for cleavage of C2 was
determined to be as follows: CB200: 15.7 nM; CB155: 18.4 nM; and
CB42:11.9 nM.
[0583] b. In another experiment, the cleavage of C2 and C3 by
wildtype MT-SP1 (CB200) and mutant MT-SP1 CB252 and CB377 was
compared. Nine microliters of human citrated plasma (Innovative
Research) was incubated with 1 .mu.l of protease to give a final
concentration of protease ranging from 0 to 2000 nM. Incubations
were performed for 1 hour at 37.degree. C. One microliter of the
preincubated protease/plasma reaction was combined with 10 .mu.l
(for assessment of C2) or 1000 .mu.l (for assessment of C3) NuPAGE
LDS same buffer and sample reducing reagent (Invitrogen, Carlsbad,
Calif.) and boiled for 5 minutes. In parallel, 10 .mu.l of the
boiled samples were loaded onto 4-12% NuPAGE Bis-Tris gradient gels
(Invitrogen) and run at 200 V for 35 minutes using the Novex
Mini-Cell Surelock apparatus (Invitrogen) for protein separation,
followed by transfer to a PVDF membrane filter (Invitrogen) using a
Biorad Transblot SD semi-dry transfer cell. The membranes were
blocked with 5% dry milk (Biorad, Hercules, Calif.) in TBST
(tris-buffered saline with 1% Tween 20) for 1 hour. The membranes
were then incubated with goat anti-human C2 (Quidel) or goat
anti-human C3 at 1:2000 in 5% dry milk/TBST overnight at 4 C. The
membranes were washed three times in TBST for 10 minutes, and then
incubated with HRP-conjugated anti-goat secondary antibody (Zymed;
San Francisco, Calif.) at 1:2000 in 5% dry mil/TBST for one hour at
room temperature. The membranes were washed three times in TBST for
10 minutes, and developed with ECL Plus Western Blotting Detection
System (Amersham Biosciences; Piscataway, N.J.). Densitomery was
performed with a FluorChem imaging system (Alpha Innotech Corp; San
Leandro, Calif.) using Alpha Ease FC Fluorchem 8800 software,
version 3.1.2 (Alpha Innotech Corp). The results show that each of
CB200, CB252, and CB377 cleaved C2 in human plasma in a
dose-dependent manner whereas none of the tested proteases cleaved
C3 in human plasma as compared to the no protease control even at
the highest concentration of protease (2000 nM). Degradation of C2
was noticeable by Western blot at 500 nM of each of the tested
proteases with little to no detectable C2 observed at 1000 nM and
complete degradation of C2 evident at 2000 nM of tested
proteases.
Example 10
Assessment of Alternative and Classical Hemolysis and Correlation
with Proteolytic Cleavage of Complement Component C3 in Human
Plasma
[0584] A panel of proteases were screened at a final concentration
of 200 nM (classical) or 500 nM (alternative) for their effects on
classical hemolysis or alternative hemolysis, respectively,
following preincubation with human plasma as set forth in Example
7, A.1 and B.1 as described above. The panel of proteases that were
tested included wildtype (CB200), CB238, CB331, CB349, CB357,
CB367, CB377, and CB387. CB200, CB357, CB367, and CB387 inhibiting
both the classical and alternative hemolysis to varying degrees.
CB238 and CB377 showed little alternative hemolysis inhibition, but
did show substantial inhibition of classical hemolysis. CB331 and
CB349 showed little classical hemolysis inhibition, but did exhibit
inhibition of alternative hemolysis. The results showed that CB331,
CB349, and CB387 were selective for cleaving the alternative
pathway as compared to the classical pathway.
[0585] The hemolysis results were correlated to cleavage of C3 in
plasma as assessed by the visualization of C3 in the presence or
absence of protease. Samples from the alternative hemolysis assay
also were examined by Western blot for C3 as described in Example 9
above. Consistent with the alternative hemolysis results, the
results show that CB331, CB349, and CB387 cleave C3 as assessed by
decreased C3 product as compared to the no protease treated plasma
sample.
Example 11
Cleavage of Purified Complement Components and Identification of
Cleavage Sites
[0586] To determine increased specificity of modified proteases for
a target protein compared to a scaffold or wildtype protease,
purified complement factors C2, iC3, and iC4 were purchased from
Quidel Corporation (San Diego, Calif.). 5 .mu.g of each protein was
diluted to a final concentration of 5 .mu.M in PBST. MT-SP1, CB155,
or Factor I were added to a final concentration of 100 nM, and the
reaction was incubated at 37.degree. C. for 5 hours. N-linked
glycosylation was removed by denaturing the protein and treating
with PNGaseF according to the manufacturer's protocol (New England
BioLabs, Ipswich, Mass.). The target proteins were separated by
SDS-PAGE on a 4-12% Tris-Glycine gradient gel (Invitrogen,
Carlsbad, Calif.) followed by transfer to a PVDF membrane. The
resulting membrane was stained with Coomassie Brilliant Blue R-250
stain (TekNova, Hollister, Calif.), rinsed with 50% methanol until
the protein band resolved, and air dried. Proteolytic fragments
were sequenced according to the Edmans' protocol by the UC Davis
Molecular Structure Facility to determine the cleavage sequences.
Table 25 below depicts the protease cleavage sequences of human C2,
C3, and C4. Where cleavage occurred, the respective cleavage sites
on C2, C3 and C4, as identified from sequencing of the cleavage
products, are shown for the natural protease Factor I, cathepsin K,
MT-SP1, and modified MT-SP1 (CB 155). The respective SEQ ID NOS are
indicated in parentheses next to the sequence. TABLE-US-00025 TABLE
25 Protease Cleavage Sequences MT-SP1 CB155 Cathepsin K Factor I
Human C2 GATR (391) GATR (391) SLGR (392) SLGR (392) VFAK (393)
Human C3 Beta chain REFK (394) Alpha chain GLAR (395) QHAR (398)
LGLA (399) LPSR (388) RLGR (396) LSVV (400) SLLR (389) AEGK (397)
Human C4 Beta chain Alpha chain HRGR (390) HRGR (390) Gamma
chain
Example 12
Correlation of C2 Cleavage with Complement Inactivation: Assessment
of the Formation of C3 Convertase and Hemolysis
[0587] C3 convertase is formed by the interaction of C1 complex
with C4 and C2. Activation of the C1r protease in the C1 complex
cleaves C1s yielding an active C1s protease. C4 is a sensitive
substrate for C1s, thereby resulting in cleavage of C4 into C4a and
C4b. The generation of an active C4b provides a binding site for
C2, a second substrate for C1s. Cleavage of C2 results in the
formation of fragments C2a and C2b. Upon cleavage by C1s, the C4b
and C2b fragments become associated, which together form the C3
convertase of the classical pathway. The SLGR cleavage site present
in C2 is the natural activation site for C2 cleavage yielding C2a
and C2b, whereas the VFAK cleavage sequence in C2 is present within
the protease domain of C2 in the C2a portion of the molecule. To
assess whether cleavage of cleavage sequences in C2 by wildtype or
modified MT-SP1 affects activation of C2 and the formation of C3
convertase, cleavage was assessed and the functional consequences
of cleavage was determined in an in vitro reconstituted cell
surface hemolysis model.
A. C2 Cleavage
[0588] The presence of C2a and C2b cleavage products was assessed
after cleavage of purified C2 by wildtype MT-SP1 (CB200), CB252, or
CB377. To assess cleavage products upon incubation of purified C2
with proteases, 5 .mu.g of purified C2 (Quidel) was incubated alone
or with a final concentration of 100 nM CB200, CB252, or CB377
protease for 1 hour at 37.degree. C. The entire reaction was
separated by SDS-PAGE on a 4-12% Tris-Glycine gradient gel
(Invitrogen, Carlsbad, Calif.) followed by transfer to a PVDF
membrane. The resulting membrane was stained with Coomassie
Brilliant Blue R-250 stain (TekNova, Hollister, Calif.), rinsed
with 50% methanol until the protein band resolved, and air dried.
The results show that in the presence of 100 nM CB200, CB252 or
CB377, C2 was almost completely degraded to yield cleavage products
of about 70 kD and 23 kD corresponding to C2a and C2b,
respectively. In addition, in the presence of CB200 a third
cleavage product of about 35 kD was observed.
B. Cell Surface Hemolysis Model
[0589] The activity of MT-SP1 or modified protease on the C2
protein specifically was assayed by isolating cells in the
intermediate stages of complement activation and exposing them to
plasma or protein treated with the protease. Briefly, activated
erythrocytes were stopped at the C1/C4b complex stage using the
method of Nagaki et al (1974, A New Method for the Preparation of
EAC14 cell with Human or Guinea-Pig Serum. Journal of Immunological
Methods 5:307-317.) Purchased activated erythrocytes (Diamedix
Corp) were washed three times by pelleting the cells at 2000 rpm
followed by resuspension into GGVB-TTHA (50% GVB.sup.0 (Diamedix
Corp.)+5% glucose+5 mM Ca-TTHA (equal parts of 100 mM CaCl.sub.2
with 100 mM TTHA (Sigma)), and after the final wash were
resuspended at 5.times.10.sup.8 cells/mL and stored on ice. The
cells were mixed with 2.5 volumes of 10% normal human serum (NHS;
made in GGVB-TTHA and incubated 15 minutes at 30.degree. C. to
ensure the chelation of Mg.sup.2+) and incubated for 5 minutes at
30.degree. C. The mixture was washed two times with GGVB-TTHA,
washed two times with GGVB-Ca (50% GVB.sup.0+5% glucose+0.3 mM
CaCl.sub.2), and washed two times with GGVB++ (50% GVB.sup.0+5%
glucose+1 mM MgCl.sub.2+0.15 mM CaCl.sub.2). After the final wash,
the cell mixture was incubated 2 hours at 37.degree. C. with mixing
every 30 minutes to avoid excess settling of the cells. The
incubated cell mixture was washed one time in GGVB++, and
resuspended at 1.5.times.10.sup.8 cells/mL. The suspension was
stored up to 1 week at 4.degree. C.
[0590] During complement activation, the C1/C4b complex is required
to cleave C2 generating C3 convertase and the resultant activation
of the remainder of the complement cascade resulting in the
formation of the membrane attack complex (MAC) and cell lysis. To
assess the consequences of C2 cleavage by protease, a 2.times.
protease solution of any of CB200, CB252, CB377 was serially
diluted by diluting a stock solution of the protease to 2 .mu.M in
GGVB++ and serially diluting this stock 1:2 across 11 wells of an
opaque 96 well assay plate (Nunc) for final protease concentrations
of 0.2 nM to 2000 nM. GGVB++ buffer alone was added to the 12th
well as a background control. A 40% solution of C3 depleted sera
(Sigma) was made by dilution with GGVB++. Five microliters of
serially diluted 2.times. protease (from above) was mixed with 5
.mu.L diluted C3-depleted sera and incubated for 1 hour at
37.degree. C. The reaction was diluted to 50 .mu.L with GGVB++ and
50 .mu.L EAC14b cells (at 1.5.times.10.sup.8 cells/mL) were added.
The mixture was incubated for 6 minutes at 30.degree. C. to
pre-form the C2 complex. 150 .mu.L C2 depleted sera, diluted 1:100
in GGVB-EDTA (50% GVB.sup.0+5% glucose+10 mM EDTA), was added to
each well. Samples were incubated 1 hour at 37.degree. C. with
gentle shaking. The plate was spun at 2000 rpm to pellet the cells
and 100 .mu.l of the supernatant was transferred into a clear 96
well assay plate. Release of hemoglobin from the lysed red blood
cells was monitored by reading the optical density (OD) at 415
nm.
[0591] The results show that C3 depleted sera pre-incubated with
increasing concentrations of proteases (CB200, CB252, or CB377)
conferred reduced hemolysis of erythrocytes containing preformed
C1/C4b when added to sera depleted for C2. The affects of each of
the tested proteases was dose-dependent with little to no
inhibition evident at concentrations ranging from 0.2 nM to 10 nM
of protease, with successive inhibition occurring at increasing
protease concentrations with little to no hemolysis observed at
protease concentrations of 800 nM or more. These results suggest
that preincubation of sera containing C2 in the presence of
proteases does not mimic cleavage of C2 by C1/C4b required for
complement activation and cell lysis.
Example 13
Determination of the Inhibitory Cleavage Site by Paired SDS-PAGE
and Hemolytic Assays
[0592] To correlate protease cleavage of complement components with
inhibition of complement activation, purified complement factors
were purchased from Quidel and CompTech. 5 .mu.g of each protein
was diluted to a final concentration of 5 .mu.M in PBST. A scaffold
or modified protease was diluted to a final concentration between 0
and 500 nM. Samples were incubated for 1 hour at 37.degree. C. 0.5
to 1 .mu.g of the treated complement component was removed and
diluted to a total volume of 10 .mu.l with PBST. This reaction was
mixed with 250 .mu.l IgG-activated sheep's red blood cells and 5
.mu.l media depleted of the corresponding complement factor to be
assayed. The solution was allowed to incubate at room temperature
for 45 minutes. The cells were spun down at 5000 rpm for 2 minutes
and 200 .mu.l of the supernatant was transferred to a 96-well
microtiter plate and absorbance at 415 nm was measured to determine
release of hemoglobin from the lysed red blood cells.
[0593] The remaining 4 to 4.5 .mu.g of the reaction sample was
deglycosylated according to the manufacturer's protocol for PNGaseF
(New England BioLabs). The samples were separated by SDS-PAGE on a
4-12% Tris-Glycine gradient gel followed by staining with Coomassie
Brilliant blue stain. Using the densitometry feature on an Alpha
Innotech Imager, the area of each band was determined and used to
calculate the percentage of the full length complement component
cleaved throughout the assay and the appearance of all major
degradation products.
Example 14
Correlation of C2 Cleavage with Complement Inactivation as Assessed
by Hemolysis in C2 Depleted Sera
[0594] The functional consequence of C2 cleavage of
complement-mediated hemolysis was assessed in a paired SDS-PAGE and
hemolytic assay as described above in Example 13. To assess
complement activation by purified complement factors, purified C2
and C2-depleted media were purchased from Quidel. 0.5 to 1 .mu.g of
purified C2 was incubated with 10 to 500 nM CB155 protease in a
total of 10 .mu.l PBST for 1 hour at 37.degree. C. The entire
reaction was added to 250 .mu.l of IgG-activated sheep's red blood
cells along with 5 .mu.l of plasma depleted of the C2 complement
factor being assayed. The solution was allowed to sit at room
temperature for 45 minutes. The cells were spun down at 5000 rpm
for 2 minutes and 200 .mu.l of the supernatant was transferred to a
96-well microtiter plate and absorbance at 415 nm was measured to
determine release of hemoglobin from the lysed red blood cells.
[0595] To visualize the proteolytic cleavage products, twenty
microliters of the reaction, or purified complement C2 as a control
(CompTech), was separated by SDS-PAGE on a 4-12% Tris-Glycine gel
and total protein was visualized by staining the gel with Coomassie
Blue according to the manufacturer's protocol. The cleavage of C2
was compared using gel densitometry. Cleavage of C2 occurred in a
dose-dependent manner with almost complete cleavage of C2 occurring
at 500 nM of protease. Further, the percent hemolysis decreased
with increasing concentrations of the protease. The IC.sub.50 of
CB155 based on gel densitometry of C2 cleavage products was 2.2 nM.
The IC.sub.50 of CB155 for hemolysis in C2 depleted serum
supplemented with protease-treated C2 was 17 nM. In vitro, complete
degradation of purified C2 is required for the functional decrease
in hemolysis.
Example 15
Detection of C5b-9 (Membrane Attack Complex) by ELISA and Effects
of MT-SP1 or Mutants on Complement Activation
A. C5b-9 ELISA
[0596] ELISAs for C5b-9 were performed according to the
manufacturer's protocol (Quidel). Briefly, microtiter plates coated
with the capture antibody were rehydrated with SuperBlock (Pierce,
Rockford, Ill.) for 30 minutes at room temperature. The plates were
washed in PBST and 20 .mu.l of the protease-treated, complement
stimulated serum solution from above diluted with 80 .mu.l
SuperBlock was added to wells of a microtiter plate. The plates
were incubated at room temperature for 1 hour and then washed in
PBST. 100 .mu.l of the detection antibody-HRP conjugate solution
was added and the plates were incubated for 1 hour and then washed
in PBST. Finally, 100 .mu.l of the substrate solution was added and
the plates were incubated about 15 minutes at room temperature to
develop. To stop the developing reaction, 100 .mu.l of stop
solution was added to each well and absorbance at 450/650 nm was
measured according to the manufacturer's protocol.
B. Effects of wildtype MT-SP1 (CB200), CB155 or CB42 on Complement
Activation
[0597] To detect complement activation, human plasma with sodium
citrate as an anticoagulant (Innovative Research, Inc., Southfield,
Mich.) was diluted into PBS with 0.05% Tween 20 (PBST) to a final
concentration of 20% (10 .mu.l serum in 40 .mu.l PBST), to which
wildtype MT-SP1, CB155, or CB42 were added to a final concentration
of 0-5 .mu.M. The solution was incubated at 37.degree. C. for 15
minutes. Activation of the classical pathway and activation of the
alternative pathway was initiated by the addition of
lipopolysaccharide (IgG or LPS, respectively at 1 mg/ml final
concentration, Sigma). The reaction was incubated at 37.degree. C.
for 30 minutes. The reaction was quenched by adding Pefabloc
(Roche) to a final concentration of 1 mg/ml and EDTA to a final
concentration of 50 mM. 20 .mu.l of the final solution was used for
the subsequent ELISA. The ELISA was performed as described in part
A above.
[0598] The IC50 of C5b-9 generation for each of the proteases was
determined. The IC.sub.50 following activation of the classical
pathway for wildtype MT-SP1, CB155, and CB42 was determined to be
103 nm, 47 nm, and 23 nm, respectively. The IC.sub.50 following
activation of the alternative pathway for wildtype MT-SP1, CB155,
and CB42 was determined to be 195 nm, 84 nm, and 41 nm,
respectively.
Example 16
Effects of wildtype MT-SP1 (CB200), CB252 or CB377 on Complement
Activation in a Total Complement System Screen
[0599] To assess the affect of MT-SP1 or mutant MT-SP1 proteases on
the classical, MBL, or alternative complement pathways, 9 .mu.l of
human citrated plasma (Innovative Research) was incubated with 1
.mu.l of CB200, CB252, or CB377 protease to give a final
concentration of 1 .mu.M of protease for 1 hour at 37.degree. C.
Each of the reactions were assessed for complement activation using
the Total Complement System Screen Classical, Lectin, Alternative
Pathways, according to the protocol from the manufacturer (WiesLab;
Sweden). In this assay, the wells of the microtiter strips provided
by the manufacturer are coated with specific activators of the
classical, alternative, or MBL (lectin) pathways. The reaction was
diluted into the appropriate buffer provided by the manufacturer to
give the concentration of plasma for each pathway and the reaction
was incubated as defined by the manufacturer. For the classical
pathway, the plasma sample was diluted in Dilutent CP and left at
room temperature for a maximum of 60 minutes before analysis. For
the lectin pathway, the plasma sample was diluted in Diluent LP and
incubated at room temperature for greater than 15 minutes but less
than 60 minutes before analysis. For the alternative pathway, the
plasma sample was incubated in Diluent AP and left at room
temperature for a maximum of 60 minutes before analysis. To the
microtiter plate, samples were added at 100 .mu.l/well in
duplicate. The plate was incubated for 60-70 minutes at 37.degree.
C. After the serum incubation, the wells of the microtiter plate
were washed three times with 300 .mu.l washing solution. After the
final wash, excess wash buffer was removed by tapping the plate on
an absorbent tissue. Complement activation was assessed in each
sample by detection of C5b-9. 100 .mu.l of the conjugate contain
alkaline phosphatase-labelled antibodies to C5b-9 was added to each
well and the plate incubated for 30 minutes at room temperature. To
develop the reaction, 100 .mu.l substrate solution was added to
each well and the plate incubated at room temperature for 30
minutes. The reaction was stopped by adding 5 mM EDTA at 100
.mu.l/well. The absorbance was read at 405 nm using a microplate
readed. The fraction of sC5b-9 generated was determined by
comparing the OD405 value of the sample to a no protease control
sample.
[0600] The results show that the fraction C5b-9 generated upon
complement activation induced by the classical and MBL pathways was
almost completely inhibited in the presence of each of the tested
proteases (CB200, CB252, or CB377). In contrast, little to no
inhibition of C5b-9 generation was observed by any of the tested
proteases when complement activation was induced by the alternative
pathway. Since the classical and MBL pathways require C2, but the
alternative pathway does not, these results suggest that inhibition
of the classical and MBL pathways is due to cleavage of C2. This
result is consistent with the observation that each of the tested
proteases (CB200, CB252, and CB377) when preincubated with C2
inhibit hemolysis (see Example 8.b above).
Example 17
Screening for Preferential Cleavage of SLGR or GLAR versus RQAR
Substrates
[0601] Modified proteases that match the desired specificity
profiles, as determined by substrate libraries, were assayed using
individual peptide substrates corresponding a desired cleavage
sequence to determine the magnitude change in specificity. One
native target substrate was designed: Ac-RQAR-AMC, which includes
the MT-SP1 auto-activation site; and two desired substrate cleavage
sequences were designed: Ac-SLGR-AMC (C2 cleavage site) and
Ac-GLAR-AMC (C3 cleavage site).
[0602] The substrates were diluted in a series of 12 concentrations
between 1 mM and 2 .mu.M in 50 .mu.l total volume of MT-SP1
activity buffer in the wells of a Costar 96 well black half-area
assay plate. The solution was warmed to 30.degree. C. for five
minutes, and 50 .mu.L of a protease solution (wildtype MT-SP1,
CB42, or CB155) was added to the wells of the assay. The
fluorescence was measured in a fluorescence spectrophotometer
(Molecular Devices Gemini XPS) at an excitation wavelength of 380
nm, an emission wavelength of 450 nm and using a cut-off filter set
at 435 nm. 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). The proteases wildtype MT-SP1 (CB200),
CB42, and CB155 cut the Ac-RQAR-AMC substrate at
1.9.times.10.sup.6, 1.8.times.10.sup.6, and 9.1.times.10.sup.4
M.sup.-1s.sup.-1, respectively. The proteases wildtype MT-SP1
(CB200), CB42, and CB155 cut the Ac-SLGR-AMC substrate at
2.0.times.10.sup.4, 5.9.times.10.sup.4, 3.7.times.10.sup.3
M.sup.-1s.sup.-1, respectively. The proteases wildtype MT-SP1
(CB200), CB42, and CB155 cut the Ac-GLAR-AMC substrate at
6.4.times.10.sup.4, 6.3.times.10.sup.4, and 3.5.times.10.sup.3
M.sup.-1s.sup.-1, respectively.
Example 18
Screening for Cleavage of Individual Substrates versus a
Full-Length Protein
[0603] The specificity of a wildtype or modified protease to a
substrate cleavage sequence versus a full length complement protein
was determined by comparing the specificity constant
(k.sub.cat/k.sub.m) to measure how well a substrate is cut by a
particular protein. A peptide substrate cleavage sequence was
designed containing a C2 cleavage sequence: Ac-SLGR-AMC. The
specificity constant (k.sub.cat/K.sub.m) of cleavage was determined
as described above in Example 17 by incubating the substrate
cleavage sequence with a protease (wildtype MT-SP1 (CB200), CB42,
or CB155) and the rate of increase in fluorescence was
determined.
[0604] In gel kinetics was used to determine the specificity
constant of the target complement protein C2. The kinetics of
cleavage of the C2 target protein was assayed by following by
SDS-PAGE the depletion of the target in the presence of a small
amount of protease over a time course. For this assay, 5 mM C2 was
incubated with 10 nM protease (wildtype MT-SP1 (CB200), CB42, or
CB155) in MTSP activity buffer at 37.degree. C. for 5 hours.
Aliquots were removed at 0, 10, 20, 40, 60, 100, 200 and 300
minutes, and immediately diluted and boiled in reducing agent. The
samples were treated with PNGase F (New England BioLabs), separated
by SDS-PAGE, and stained with Coomassie Brilliant Blue. The density
of the full length protein band was determined using the Alpha
Innotech Gel Imager. The specificity constant, k.sub.cat/K.sub.m
was determined by non-linear fitting of the curve produced by
plotting the integrated density value versus time with the
equation: density=exp(-1*time*[enzyme]*k.sub.cat/K.sub.m).
[0605] The results show that the specificity constant of cleavage
of a substrate peptide sequence versus a full length protein by the
proteases followed a similar pattern. The CB200 wildtype MT-SP1
showed almost identical specificity constants for cleavage of the
substrate peptide sequence versus the C2 full length protein,
whereas CB42 and CB155 showed slight variation in their specificity
constants. The specificity constant of cleavage of the Ac-SLGR-AMC
substrate sequence by CB200, CB42, and CB155 was
2.0.times.10.sup.4, 5.9.times.10.sup.4, and 3.7.times.10.sup.3
M.sup.-1s.sup.-1, respectively. The specificity constant of
cleavage of the C2 protein by CB200, CB42, and CB155 was
1.95.times.10.sup.4, 3.60.times.10.sup.4, and 7.20.times.10.sup.4
M.sup.-1s.sup.-1, respectively.
Example 19
Cynomolgus Hemolysis Protocol
[0606] The functional activity of the Cynomolgus monkey complement
system following protease treatment also can be assessed using
modified hemolytic assays.
[0607] A. Sensitization of Chicken Red Blood Cells
[0608] Chicken red blood cells were isolated from Chicken Alsevers
(50% mix of whole blood from chickens and alsevers solution, which
contains anti-coagulants; Colorado Serum Company, CO). The cells
were resuspended by gently pipetting up and down until no cell
pellet was visible and 50 .mu.l of cells were diluted into 1 ml
GVB++ buffer. The volume of cells was scaled as necessary for each
experiment assuming that 10 .mu.l per well of the final 1 ml
suspension would be added to each well so that a dilution of 50
.mu.l cells was sufficient for one plate. The cells were washed by
pelleting the cells at 2500 rpm in a tabletop centrifuge at
4.degree. C. for 1 minute, the supernatant discarded, and cells
resuspended in 1 ml GVB++ by gently pipetting up and down. The
washing steps were repeated twice more or until the supernatant was
completely clear upon the last spinning. One .mu.l of anti-chicken
erythrocyte antibody (Fitzgerald industries) was added to sensitize
the cells. After the antibody--cell suspension was mixed, the
solution was incubated on ice for a minimum of 15 minutes, then
centrifuged at 2000 rpm in a tabletop centrifuge at 4.degree. C.
for 2 minutes. The supernatant was discarded and the sensitized
cells were resuspended in 1 mL GVB++ and washed a further 2 times
by centrifugation at 2000 rpm for 1 minute until the supernatant
was clear. After the final wash, the cell pellet was resuspended in
a final volume of 1 ml GVB++ and the cells were diluted 10:50 in
GVB++ (i.e. 1 ml of resuspended sensitized cells 5 mL GVB++ for a
total volume of 6 ml). Sensitization of the chicken cells with
antibody was performed fresh the day of the experiment. The
sensitized cells were not kept overnight.
[0609] B. Hemolysis Assay From in vivo Pharmacodynamic (PD)
Experiments
[0610] Hemolysis reactions were set up at a final concentration of
1%, 2.5%, and 10% plasma for each plasma sample obtained from
protease treated animals or vehicle control (no protease) treated
animals in the presence of sensitized erythrocytes. Absorbance
controls containing no added concentrated sensitized erythrocytes
also were set up in parallel for each sample. All reactions were
set up in opaque plates with point divots. For the 1% plasma
samples, the hemolysis samples (in duplicate) were set up with 10
.mu.l sensitized erythrocytes, 89 .mu.l GVB++, and 1 .mu.l plasma
from protease or no-protease treated animals; and the corresponding
absorbance controls were set up with 99 .mu.l GVB++ and 1 .mu.l of
plasma from protease treated animals. For the 2.5% plasma samples,
the hemolysis samples (in duplicate) were set up with 10 .mu.l
sensitized erythrocytes, 87 .mu.l GVB++ and 2.5 .mu.l plasma from
protease or no protease treated animals; and the corresponding
absorbance controls were set up with 97 .mu.l GVB++ and 2.5 .mu.l
plasma from protease treated animals. For the 10% plasma samples,
the hemolysis samples (in duplicate) were set up with 10 .mu.l
sensitized erythrocytes, 80 .mu.l GVB++, and 10 .mu.l plasma from
protease or no protease treated animals; and the corresponding
absorbance controls were set up with 90 .mu.l GVB++ and 10 .mu.l
plasma from protease treated animals. The plates were incubated
with shaking at 37.degree. C. for 30 minutes. The cells were
centrifuged at 2000 rpm for 5 minutes to pellet the unbroken cells,
and 80 .mu.l of the supernatant was removed and placed in a clear
96-well round-bottom microtiter plate. This was done carefully
since the samples were sometimes gelatinous. The samples that were
gelatinous were noted. The supernatant-transferred plates were
centrifuged at 2500 rpm for 5 minutes to remove bubbles.
Centrifugation was repeated until no bubbles persisted or,
alternatively, remaining bubbles were popped with an 18 G needle.
Release of hemoglobin from the lysed red blood cells was monitored
photometrically by reading absorbance at 415 nm on a Biorad
Microplate Reader Model 680. If the absorbance was greater than 1,
the samples were diluted 1:3 in GVB++ and read again (i.e. 20 .mu.l
sample into 40 .mu.l GVB++). The fraction hemolysis was calculated
by subtracting the absorbance from the absorbance control samples
from the corresponding hemolysis well, then dividing the
experimental samples by the no-protease vehicle control sample.
ED50 values were determined by graphing the OD415 nm value as a
function of protease concentration.
[0611] C. In Vitro Titration Hemolysis Assay
[0612] For an in vitro titration of proteases using cynomolgus
monkey plasma, proteases at 10% of the final reaction volume were
incubated with purchased cynomolgus monkey plasma (i.e. in
polypropylene 96-well plates, 2 .mu.l of the protease solution was
added to 18 .mu.l cynomolgus monkey plasma) to give final protease
concentrations of protease of ranging from 20 .mu.M to 0.156 .mu.M
for the IC.sub.50 titration protocol, and a plasma concentration of
90%. A no-protease (18 .mu.l plasma and 2 .mu.l GVB/Mg/EGTA) and
background (20 .mu.l GVB/Mg/EGTA only) controls also were included
in the assays. The reaction was incubated at room temperature for 1
hour. After preincubation of the protease with 90% plasma,
hemolysis was performed as described in part B above. No absorbance
controls were included in the in vitro hemolysis titration.
Example 20
Mouse Hemolysis Protocol
[0613] A. Hemolysis Assay From in vivo Pharmacodynamic (PD)
Experiments
[0614] The functional activity of the mouse complement system
following protease treatment also can be assessed using modified
hemolytic assays. Erythrocytes used in the mouse hemolysis
protocols also were chicken red blood cells which were sensitized
as described in Example 19, part A above. Hemolysis reactions were
set up at a final concentration of 40% plasma for each plasma
sample obtained from protease treated animals or vehicle control
(no protease) treated animals in the presence of sensitized
erythrocytes. Absorbance controls containing no added concentrated
sensitized erythrocytes also were set up in parallel for each
sample at half the plasma concentration (20% plasma), such that the
absorbance control values were subtracted twice from the hemolysis
values during analysis as discussed below. The hemolysis samples
were set up in duplicate (if enough plasma allowed) in opaque
plates with divots by adding 60 .mu.l of concentrated sensitized
erythrocytes and 40 .mu.l of plasma from protease or no protease
treated animal to each well. The corresponding absorbance control
was set up by adding 80 .mu.l GVB++ and 20 .mu.l mouse plasma from
protease or no protease treated animals to each well. The plates
were incubated with shaking at 37.degree. C. for 1 hour. The cells
were centrifuged at 2000 rpm for 5 minutes to pellet the unbroken
cells, and 50 .mu.l of the supernatant was removed and placed in a
clear 96-well round-bottom microtiter plate. This was done
carefully since the samples were sometimes gelatinous. The samples
that were gelatinous were noted. The supernatant-transferred plates
were centrifuged at 2500 rpm for 5 minutes to remove bubbles.
Centrifugation was repeated until no bubbles persisted or,
alternatively, remaining bubbles were popped with an 18 G needle.
Release of hemoglobin from the lysed red blood cells was monitored
by reading at 415 nm. If the absorbance was greater than 1, the
samples were diluted 1:3 in GVB++ and read again (i.e. 20 .mu.l
sample into 40 .mu.l GVB++). The fraction hemolysis was calculated
by subtracting the 2.times. absorbance absorbance control samples
from the corresponding hemolysis well, then dividing the
experimental samples by the no-protease vehicle control. ED50
values were determined by graphing the OD415 nm value as a function
of protease concentration.
[0615] B. In Vitro Titration Hemolysis Assay
[0616] For an in vitro titration of proteases using mouse plasma,
proteases at 10% of the final reaction volume were incubated with
purchased mouse plasma or in-house control plasma (i.e. in
polypropylene 96-well plates, 2 .mu.l of the protease solution was
added to 18 .mu.l plasma) to give final protease concentrations of
protease of ranging from 20 .mu.M to 0.156 .mu.M for the IC.sub.50
titration protocol, and a plasma concentration of 90%. A
no-protease (18 .mu.l plasma and 2 .mu.l GVB/Mg/EGTA) and
background (20 .mu.l GVB/Mg/EGTA only) controls also were included
in the assays. The reaction was incubated at room temperature for 1
hour. After preincubation of the protease with 90% plasma,
hemolysis was performed as described in part A above. No absorbance
controls were included in the in vitro hemolysis titration.
Example 21
Classical C3b Deposition ELISA
[0617] To detect and quantitate C3b deposition, 96 well Maxisorp
plates (Nunc) were coated with 100 .mu.l/well of 0.5% ovalbumin for
2 hrs at 37.degree. C. or overnight at 4.degree. C. The plates were
washed 3 times with 250 .mu.l PBST using a Molecular Devices
SkanWasher 300 Version B. The plates were coated with 100
.mu.l/well of rabbit anti-chicken egg albumin antibody (MP
Biomedicals) dilted 1:1000 in PBS. The plates were incubated for 1
hr at room temperature or overnight at 4.degree. C. before being
washed 3 times with PBST. The plates were blocked with 200 .mu.l
Blocking Buffer (30% BSA Solution; Serologicals), and the plates
were shaken at room temperature for 1 hour. After washing 3 times
with PBST, 100 .mu.l of plasma sample, diluted to the desired
percentage (i.e. 1%, 10%) in GVB++ (Veronal (barbital)-buffered
saline, pH 7.4, containing 142 mM NaCl, 4.9 mM sodium Veronal, 0.1%
gelatin, 0.15 mM CaCl.sub.2, and 1 mM MgCl.sub.2; Comptech), was
added to each well, and the plates were shaken at room temperature
for 30 minutes. The wells were washed 3 times with PBST. Goat
anti-human C3b antibody (Quidel) diluted 1:4000 in blocking buffer
was added to the wells at a volume of 100 .mu.l, and the plates
were shaken at room temperature for 1 hour. After washing with
PBST, 100 .mu.l HRP-rabbit anti-goat conjugated antibody (Zymed)
diluted 1:8000 in blocking buffer was added to the wells and
allowed to incubate for 1 hour with shaking at room temperature.
The wells were washed with PBST and the ELISA was developed
according to the manufacturers instructions by the addition of 100
.mu.l TMB substrate (Pierce). The reaction was stopped by the
addition of 100 .mu.l 2M H.sub.2SO.sub.4 and the absorbance at 405
nm was read on a SpectraMax M5 plate reader (Molecular
Devices).
Example 22
Mouse Pharmacodynamic (PD) Analysis of Protease
[0618] A. Pharmacodynamics of CB450
[0619] Mice (n=6 for each dose) were injected intravenously with a
bolus of CB450 at varying dosages ranging (0 mg/kg, 2.5 mg/kg, 5
mg/kg, 10 mg/kg, and 15 mg/kg). Plasma was collected from the
treated mice at 5 minutes post-injection by cardiac puncture.
Complement activity of the plasma samples from the different
treatment groups were tested by hemolysis assay as described in
Example 20 or by C3b deposition as determined by a C3b ELISA
described in Example 21.
[0620] The results of the hemolysis experiment showed that there
was a dose-dependent decreased hemolysis of erythrocytes induced by
mouse plasma from CB450 treated mice as assessed by absorbance
levels at 415 nm. Plasma samples from mice treated with no protease
induced hemolysis in this assay as assessed by an absorbance at 415
nm of about 0.23, which decreased to about 0.1 in the 2.5 mg/kg
CB450 treatment group with little to no detectable absorbance
signal measured in samples from mice treated with 5, 20, or 15
mg/kg of CB450.
[0621] The C3b ELISA was performed using 1% or 10% plasma from each
of the treatment groups. The fraction hemolysis from the no
protease treated sample was set at 1.0 and the fraction hemolysis
of all experimental samples was accordingly determined. Thus, for
both the 1% and 10% plasma samples from mice treated with no
protease, the fraction of C3b deposition was measured to be 1. The
results of the C3b ELISA on the 10% plasma samples showed that the
plasma from mice treated with increasing doses of CB450 showed no
measurable difference in C3b levels at doses of CB450 of 2.5, 5, or
10 mg/kg as compared to the no protease treated sample, however,
mice treated with 15 mg/kg exhibited a decreased fraction of C3b
deposition which was measured to be about 0.40. The results of the
C3b ELISA on the 1% plasma samples showed that plasma from mice
treated with CB450 had a dose-dependent decrease in the fraction
C3b deposition as compared to the no protease treated plasma
sample, which was consistent with the results observed in the
hemolysis experiment. Plasma samples from mice treated with 2.5
mg/kg CB450 exhibited a decreased C3b deposition which was measured
to be about 0.40, while plasma from mice treated with 5, 20, or 15
mg/kg CB450 showed little to no detectable C3b.
[0622] B. Pharmacodynamics of a Panel of MT-SP1 Protease
Mutants
[0623] Mice were injected intravenously with a bolus of increasing
concentrations of a panel of MT-SP1 mutants including CB200
(wild-type), CB238, CB245, CB252, CB255, CB257, CB268, CB351,
CB377, CB409, CB422, CB450, CB464, and CB473. Plasma was collected
from the treated mice at 5 minutes post-injection by cardiac
puncture. Complement activity of the plasma samples from the
different treatment groups were tested by hemolysis assay as
described in Example 19 or by C3b deposition as determined by a C3b
ELISA (assayed in 1% and 10% plasma) as described in Example 20.
The results were graphed as a function of protease concentration to
determine the ED50 values. A summary of the results are depicted in
Table 26 below. The Table also sets forth the maximum tolerated
dose (MTD) of protease and the therapeutic index (TI) calculated as
the ratio of MTD to ED50. The results show differences in the in
vivo efficacy of some of the tested proteases. TABLE-US-00026 TABLE
26 Mouse Pharmacodynamics C3b in 1% C3b in 10% C3b TI MTD Hemolysis
Hemolysis plasma (ED50; C3b TI (1% plasma (ED50; (10% PROTEASE
(mg/kg) (ED50; mg/kg) TI mg/kg) plasma) mg/kg) plasma) CB200 10 6.4
1.56 2.7 3.7 13.5 0.74 CB238 15 9.4 1.6 6.1 2.46 16.2 0.92 CB245 10
2.4 4.1 2.63 3.8 >10 0 CB252 12.5 5.7 2.19 3.8 3.29 9.9 1.26
CB255 7 5 1.4 2.78 2.52 >7 0 CB257 5 3.1 1.6 4.86 1.03 >5 0
CB268 10 9.9 1 5.45 1.8 >10 0 CB351 10 6.6 1.52 5.8 1.72 10.8
0.93 CB377 15 8.6 1.75 2.6 5.77 6.2 2.42 CB409 15 8.2 1.8 3.2 4.65
17.99 0.83 CB422 12.5 10.25 1.22 3.82 3.27 20.36 0.61 CB450 15 1.8
8.5 3.5 4.3 15.9 0.9 CB464 15 7.72 1.94 5.07 2.96 33.1 0.45 CB473
12.5 14.25 0.86 1.95 6.41 8.01 1.56
Example 23
Rat Pharmacodynamic (PD) Analysis of Protease
A. CB252 and CB377
[0624] Rats were injected intravenously with a bolus of CB252 (23
mg/kg) followed by infusion for 1 hour at 3.3 mg/kg/hr or with a
bolus of CB377 (18 mg/kg) followed by infusion for 1 hour at 1.8
mg/kg/hr. Rats treated with a vehicle control also were included in
the study. Plasma was collected at various time points after
injection (where t=0 is pre-injection; i.e. 0, 5, 15, 30, 60, or
120 minutes) and analyzed for complement activity by assaying for
C2 cleavage by Western Blot as set forth in Example 9 (except that
only 1.5 .mu.l of plasma was used) and for hemolysis using the
Cynomolgus hemolysis protocol as set forth in Example 19 using
either 1% or 10% rat plasma.
[0625] The results showed increased cleavage of C2 in plasma from
CB252 and CB377 treated rats. CB252 showed a greater cleavage of C2
as there was little detectable C2 present in the plasma samples as
assessed by Western Blot even after only 5 minutes following
injection, with no detectable C2 present at 60 or 120 minutes after
injection. CB377 also showed diminished C2 levels as compared to
vehicle control at early time points, however, by 60 minutes and
120 minutes the levels of C2 were comparable to those from vehicle
control samples.
[0626] The results of hemolysis induced by 10% plasma from the
treated rats showed that plasma from CB377 had no effect on the
inhibition of hemolysis as compared to vehicle control, while
plasma from CB252 showed a marked inhibition of hemolysis. Plasma
samples from rats treated with CB252 collected at 5, 15, 30, and 60
minutes after injection showed little to no detectable hemolysis.
Hemolysis was increased to levels comparable to vehicle control by
plasma from CB252 treated mice at later time points (i.e. by 90 and
120 minutes). The effects of CB252 and CB377 was more pronounced
when hemolysis was induced by 1% plasma from each of the treated
rats. The fraction hemolysis (set at 1.0 for the t=0 time point
vehicle control sample) induced by 1% plasma from vehicle control
rats did not change among the tested time points and was always
around about 1.0. Plasma from rats treated with CB252 induced no
detectable hemolysis at any of the collected time points. Plasma
from CB377 treated rats also showed reduced hemolysis as compared
to plasma from vehicle control treated animals at all time points,
although to a lesser extent than plasma from CB252 treated rats.
Hemolysis was reduced to the greatest extent in plasma collected 15
minutes after injection of CB377 with a reported fraction of
hemolysis of about 0.2 as compared to plasma control treated mice,
and steadily increased at longer time points after injection to
about 0.6 at 120 minutes after injection.
[0627] B. Comparison of CB200, CB155, and CB42
[0628] Rats were injected intravenously with a bolus of CB200
(wildtype), CB155, and CB42 at 2 mg/kg, 10 mg/kg, and 25 mg/kg.
Plasma was collected at various time points after injection (where
t=0 is pre-injection) up to about 1380 minutes after injection and
analyzed for complement activity by assaying for hemolysis using
the Cynomolgus hemolysis protocol as set forth in Example 19 using
1% plasma. The results show that plasma from rats treated with
CB200 or CB42 at 2 mg/kg and 10 mg/kg exhibited levels of hemolysis
comparable to levels observed at t=0 before pre-injection of the
protease. Plasma from rats treated with 25 mg/kg of CB200 or CB42
induced reduced hemolysis of erythrocytes at early time points,
with little to no hemolysis observed at time points up to about 60
minutes after injection of the protease. Hemolysis was increased to
levels comparable to hemolysis at t=0 before pre-injection of the
protease from plasma samples collected by 1380 minutes after
injection of CB200 or CB42. Plasma from CB155 treated rats,
however, showed decreased hemolysis at all doses tested. Treatment
of rats with 2 mg/kg or 10 mg/kg showed slight but reproducibly
decreased hemolysis induced by plasma collected at early time
points as compared to t=0 before pre-injection of the protease.
Plasma samples collected at about 30 minutes after rats receiving a
dosage of 2 mg/kg or 10 mg/kg of CB155 resulted in an observed
OD415 of hemolysis of about 0.3 or about 0.25, respectively, as
compared to about 0.45 for t=0 (no protease) plasma samples. Plasma
collected at 240 minutes or longer after preinjection of CB155 at 2
mg/kg and 10 mg/kg induced hemolysis to levels comparable to that
observed from t=0 treated animals. Plasma from rats treated with 25
mg/kg of CB155 induced reduced hemolysis of erythrocytes at early
time points, with little to no hemolysis observed at timepoints up
to about 240 minutes after injection of the protease. Hemolysis was
increased to levels comparable to hemolysis at t=0 before
pre-injection of the protease from plasma samples collected by 1380
minutes after injection of CB155. These results show that CB155 has
a greater in vivo pharmacodynamic efficacy on complement
inactivation than do CB200 and CB42 as assessed in this
experiment.
Example 24
[0629] Cynomologus Monkey Pharmacodynamic (PD) Analysis of
Protease
A. CB252 Cynomolgus ex vivo Complement Inhibition
[0630] Two naive male and two naive female cynomolgus monkeys
(approximately 2.2-4.4 kg, and 2-4 years of age at initiation of
treatment) were assigned to a single treatment group. Each animal
was permanently tattooed with a unique identification number and
assigned to a 14-day acclimation period prior to dosing. Study
animals were intravenously administered 1 and 3 mg/ml doses of
CB252 at volumes of 5 mg/kg. Blood samples for ex vivo
pharmacodynamic analyses were collected at scheduled time points
(Pre-injection, i.e. t=0; 5 minutes, 30 minutes, and 60 minutes
post-injection). Blood was collected by venipuncture from a
peripheral vein of restrained, conscious animals. Blood samples
were collected from spare animals to be used as baseline values.
Approximately 1 ml of blood was transferred to a tube treated with
lithium heparin, placed on ice, and then centrifuged at 2000 g for
15 minutes at 4.degree. C. within 30 minutes of collection. Plasma
obtained was divided into two approximately equal aliquots and then
transferred to cyrovials which were frozen on dry ice. Samples were
stored at approximately -60.degree. C. or colder prior to thawing
and analysis. Plasma samples were tested for effects on complement
activation by assaying for C2 cleavage by Western Blot, C3b
deposition by ELISA at 1% and 10% plasma concentration, and through
hemolysis of sensitized chicken red blood cells at 1%, 2.5%, and
10% plasma.
[0631] 1. C2 Cleavage
[0632] C2 cleavage in the plasma samples was assessed by Western
Blot as described in Example 9 with the following modifications: 1
.mu.l plasma, boiled with NuPAGE LDS sample buffer and sample
reducing agent (Invitrogen) for 5 minutes, was used in the
analysis; goat anti-human C2 was diluted to 1:2000 in 5% dry
milk/TBST; and HRP-conjugated anti-goat secondary was diluted to
1:4000 in 5% dry milk/TBST. The results showed that ex vivo plasma
from cynomolgus monkeys dosed with bolus IV injection of 1 or 3
mg/kg CB252 demonstrated partial cleavage of C2 at 3 mg/kg only.
Plasma from monkeys treated with 1 mg/kg CB252 had no discernable
C2 cleavage. Plasma collected from monkeys treated with 3 mg/kg
CB252, there was a significant C2 cleavage observed for all three
animals for which plasma samples were available. The average extent
of degradation of C2 as determined by densitometry of C2 Western
blots was 60% degraded at 5 minutes, 50% degraded at 30 minutes,
and 40% degraded at 60 minutes. The percent inhibition of
complement as assessed by C2 cleavage in plasma from all animals
treated with 3 mg/kg CB252 are summarized in Table 27 below.
TABLE-US-00027 TABLE 27 CB252 Ex Vivo Complement Inhibition: C2
cleavage Animal Time point 2 4 5 5 minutes 62% 55% 65% 30 minutes
55% 45% 50% 60 minutes 50% 37% 38%
[0633] 2. C3b Deposition
[0634] C3b deposition in the plasma samples was assessed by ELISA
as described in Example 21. In the C3b deposition assay no
significant inhibition of complement was observed in plasma samples
from monkeys administered with 1 mg/kg CB252 administered for any
animal at any time point assayed. At the 3 mg/kg dose of CB252, at
10% plasma, C3b deposition was inhibited by an average of
approximately 50% at the 5 minute time point, 30% at the 30 minute
time point, and 15% at 60 minutes. The level of inhibition by CB252
was observed to be greater when measured in 1% plasma. For the 3
mg/kg dose of CB252, at 1% plasma, C3b deposition was inhibited by
an average of approximately 70% at 5 minutes, 55% at 30 minutes,
and 50% at 60 minutes. The percent inhibition of complement as
assessed by C3b deposition in plasma from all animals treated with
3 mg/kg CB252 are summarized in Table 28 below. TABLE-US-00028
TABLE 28 CB252 Ex Vivo Complement Inhibition: C3b deposition 10%
plasma 1% plasma Animal Animal Time point 2 4 5 2 4 5 5 minutes 52%
45% 50% 50% 74% 77% 30 minutes 37% 38% 13% 25% 60% 40% 60 minutes
40% 10% 3% 82% 16% 78%
[0635] 3. Hemolysis
[0636] Hemolysis was assessed in the sensitized chicken red blood
cell (RBC) hemolysis assay as described in Example 19. The results
showed that plasma from monkeys treated with 1 mg/kg dose of CB252
exhibited no observable effect on hemolysis of sensitized chicken
RBC for any animal at any time point assayed. Plasma samples from
monkeys treated with 3 mg/kg CB252 showed significant inhibition of
hemolysis at the various time points assayed, and inhibition was
observed at 1%, 2.5% and 10% plasma. In 10% plasma, plasma from
monkeys treated with 3 mg/kg CB252 showed an average of 80%
inhibition of hemolysis at 5 minutes, 45% inhibition at 30 minutes,
and 25% inhibition at 60 minutes. In 2.5% plasma, plasma from
monkeys treated with 3 mg/kg CB252 showed an average of 92%
inhibition of hemolysis at 5 minutes, 80% inhibition at 30 minutes,
and 65% inhibition at 60 minutes. In 1% plasma, plasma from monkeys
treated with 3 mg/kg CB252 showed an average of 99% inhibition of
hemolysis at 5 minutes, 98% inhibition at 30 minutes, and 90%
inhibition at 60 minutes. The percent inhibition of complement as
assessed by hemolysis of chicken RBCs by plasma from all animals
treated with 3 mg/kg CB252 are summarized in Table 29 below.
TABLE-US-00029 TABLE 29 CB252 Ex Vivo Complement Inhibition:
Hemolysis 10% Plasma 2.5% Plasma 1% Plasma Time point Animals
Animals Animals (minutes) 2 4 5 2 4 5 2 4 5 5 55% 90% 80% 90% 95%
91% 96% 95% 97% 30 43% 65% 30% 85% 91% 62% 96% 95% 91% 60 ND 40%
10% 98% 78% 21% 96% 91% 79% ND: not determined
[0637] In summary, cynomolgus plasma from monkeys treated with a
single bolus intravenous injection of 1 mg/kg CB252 did not show
significant inhibition of complement as measured by C2 degradation,
C3b deposition ELISA, or hemolysis of sensitized chicken red blood
cells. Cynomolgus plasma from monkeys treated with a single bolus
intravenous injection of 3 mg/kg CB252 did show significant
inhibition of all ex vivo complement assays at the 5 minute and 30
minute time points. At the least stringent assay levels, 1% plasma
in C3b deposition ELISA and 1% hemolysis, significant inhibition
persists to the 60 minute time point.
B. Pharmacodynamic Efficacy of CB252 Compared to Other MT-SP1
Mutants in Cynomolgus
[0638] Monkeys were injected intravenously with a bolus of CB252 or
CB377 at 1 mg/kg and 3 mg/kg protease. Plasma was collected at
various time points after injection (where t=0 is pre-injection;
i.e. 0, 5, 30, and 60 minutes) and analyzed for complement activity
by assaying for C2 cleavage by Western Blot as set forth in Example
9 and for hemolysis using the Cynomologous hemolysis protocol as
set forth in Example 19 using either 1%, 2.5% or 10% monkey
plasma.
[0639] The results showed increased cleavage of C2 in plasma from
CB252 and CB377 treated monkeys after treatment with 3 mg/kg
protease but not after treatment with 1 mg/kg protease. Plasma
collected from monkeys treated with 3 mg/kg CB252 and CB377
protease showed a time-dependent cleavage of C2, with greatest C2
cleavage occurring in plasma collected from monkeys at 5 minutes
after protease treatment and decreased cleavage occurring at
increased timepoints. The results also showed that a greater
cleavage of C2 occurred in plasma collected form CB252 treated
monkeys as compared to CB377 treated monkeys at all time points
tested.
[0640] The results of the hemolysis experiment correlated with the
C2 cleavage results since there was no observed difference in
hemolysis induced by either 2.5% plasma or 10% plasma from monkeys
treated with 1 mg/kg CB252 or CB377 protease at any of the
collected time points as compared to hemolysis induced by plasma
from monkeys not treated with protease (i.e. t=0). The results also
showed that the hemolysis observed using either 2.5% or 10% plasma
from monkeys treated with 3 mg/kg CB252 or CB377 was similar. Under
both assay conditions, plasma from monkeys treated with 3 mg/kg
CB377 showed only a slight decrease in hemolysis of erythrocytes as
compared to plasma from t=0. The fraction of hemolysis at t=0 was
set at 1.0 and the fraction hemolysis observed from plasma from
CB377 treated monkeys was about 0.7 for all time points tested.
CB252 was markedly more potent than CB377 in this experiment.
Plasma from monkeys treated with 3 mg/kg CB252 collected 5 minutes
after injection induced no detectable hemolysis of erythrocytes
with an observed fraction hemolysis of at or close to 0. The
effects of CB252 on hemolysis was time-dependent since the fraction
hemolysis induced from plasma from monkeys treated with 3 mg/kg
CB252 increased to about 0.4 and about 0.7 in plasma collected 30
minutes and 60 minutes, respectively, from CB252 treated
monkeys.
[0641] In another experiment, the pharmacodynamic efficacy of
proteases CB238, CB252, and CB377 was compared upon administration
to cynomologous. Monkeys were injected intravenously with a bolus
of CB238, CB252 or CB377 at the maximum tolerated dose (MTD) for
each protease (i.e. 2 mg/kg, 3 mg/kg, and 3 mg/kg, respectively).
Plasma was collected at various time points after injection (where
t=0 is pre-injection; i.e. 0, 5, 30, and 60 minutes) and analyzed
for complement activity by assaying for the ability to support
hemolysis of chicken red blood cells using the Cynomologous
hemolysis protocol as set forth in Example 19 using either 2.5% or
10% monkey plasma. The % inhibition of hemolysis was determined as
compared to hemolysis induced by 2.5% or 10% plasma collected from
t=0 monkeys. The results are summarized in Table 30 below.
TABLE-US-00030 TABLE 30 Cynomologous Pharmacodynamics % inhib %
inhib % inhib % inhib hemol hemol hemol hemol (2.5% (2.5% (10% (10%
Monkey MTD plasma) plasma) plasma) plasma) Protease (mg/kg) @ 5 min
@ 30 min @ 5 min @ 30 min CB238 2 24 15 20 11 CB252 3 92 79 75 46
CB377 3 18 8 14 7
Example 25
Examination of the Complement-Mediated Cardiovascular Effects of
Proteases Ex Vivo in Rabbit Hearts
[0642] The effects of proteases on complement-mediated injury was
assessed ex vivo using the Langendorff Assay to examine cardiac
damage in rabbit hearts. Studies on the isolated heart allows for
simultaneous observations of a compound's hemodynamic,
electrocardiographic, and electrophysiologic effects. New Zealand
White rabbits were used in this study because the amino acid
sequence of the rabbit I.kappa..sub.r channel shares 99% homology
with the human I.kappa..sub.r channel sequence (Wymore et al.
(1997) Circ Res., 80: 261-268). The rabbit has been used
extensively for cardiovascular studies and is an appropriate
species to model potential effects on the human heart, since rabbit
cardiac action potentials (similar to human cardiac action
potentials) appear to be strongly driven by I.kappa..sub.r (Weirich
et al. (1998) Basic Res Cardiol., 93:125-132; Carmeliet et al.
(1992) J Pharmacol Exp Ther., 262:809-817). Also, the interaction
between human plasma and rabbit heart tissue has been previously
characterized and has been shown to be primarily complement
mediated (Kilgore et al. (1998) J Pharmacol Exp. Ther., 285:
987-94). For example, contact of human plasma and the foreign
surface of the rabbit heart activates complement, which then
mediates damage to the myocardium ultimately resulting in asystole.
Therefore, this model is appropriate to determine the efficacy of
complement inhibitors, such as proteases or modified proteases that
target one or more complement components.
A. Experimental Design and Methods
[0643] Rabbits were euthanized via stunning followed by
cardiectomy. Hearts were rapidly removed, mounted on a Langendorff
apparatus, and perfused with modified, oxygenated, Krebs-Henseleit
buffer (37.degree. C.; Krebs-Henseleit buffer: 118.1 mM NaCl, 4.7
mM KCl, 1.17 mM MgSO.sub.4, 1.18 mM KH.sub.2PO.sub.4, 11.1 mM
d-glucose, 2.5 mM CaCl.sub.2, 24.8 mM NaHCO.sub.3, and 2.0 mM
pyruvate; modified with the addition of 2.5 g of bovine serum
albumin/1000 ml of perfusion medium; and oxygenated via pressurized
oxygen/carbon dioxide (95%/5%)). A ventricular drain and
fluid-filled latex balloon was secured in the left ventricle with a
purse string suture at the atrial appendage. A pulmonary artery
drain was secured. Hearts were paced via pacing electrodes placed
onto the right atrium. Hearts were deemed acceptable for the study
if they exhibited acceptable hemodynamic parameters (e.g.,
dP/dT>1000 mm Hg/sec) throughout the equilibration period.
[0644] The protease test compound (at a final concentration of 1
.mu.M) was incubated with incubation media (containing human plasma
diluted to 50% in perfusion buffer; i.e. 12 ml human serum diluted
into 12 ml perfusion medium) for 1 hour at 37.degree. C. Following
the incubation, the test compound mixture was added to the
experimental perfusion medium, reciruculating, to give a final
serum concentration of about 4-6% in 300 ml total volume. Isolated
rabbit hearts that were previously equilibrated with perfusion
medium for 10-15 minutes followed by collection of baseline
measurements for 10 minutes, were exposed to perfusion medium
containing the incubated test compound mixture for approximately 1
hour with measurements collected continuously as described below.
The experiment was terminated if irreversible ventricular
fibrillation occurred. Ventricular fibrillation was deemed
irreversible if the heart did not spontaneously revert within 90
seconds of initiation. After exposure to the test compounds, the
hearts were fixed in O.C.T., frozen on dry ice, and then stored in
a freezer set at -80.degree. C. for immunohistochemistry
evaluation.
[0645] 1. Hemodynamic Measurements
[0646] The latex balloon in the left ventricle (LV) was expanded
with water to achieve an LV end-diastolic pressure (LVEDP) of
approximately 5 mmHg. The balloon was connected with tubing to a
pressure transducer to measure LEPD, LV diastolic pressure (LVDP)
and LV systolic pressure (LVSP). Coronary perfusion was measured
with a pressure transducer connected to a side-arm port of the
aortic cannula. Hemodynamic measurements were continuously
monitored with the Notocord HEM (Kalamazoo, Mich.) v3.5 data
capture system. Digital markers were used to indicate test compound
exposure periods. LVDP was defined as the difference between LVEDP
and LVSP. Both maximal rate of increase in LV pressure (+dP/dt) and
minimal rate of decrease in LV pressure (-dP/dt) were measured, as
the first derivative of the time from LVEDP to LVSP and LVSP to
LVEDP, respectively. Coronary perfusion pressure (CPP) also was
measured.
[0647] Hemodynamic measurements from the final minute of the
equilibration period (0 min) and during the last minute of each 15
minute period (i.e. 15 min, 30 min, 45 min, 60 min) within the hour
of the test compound exposure period were evaluated and used to
determine effects of the test compound. Average values taken from
five consecutive cardiac cycles uninterrupted by interference of
ectopic beats were used for analysis of hemodynamic parameters.
Values from each individual heart were pooled to determine an
average for each variable at individual concentrations. Average
percent change of each variable between baseline and each
concentration also was determined. The effect of each test compound
on hemodynamic parameters was examined for statistical significance
using repeated measures analysis of variance (ANOVA) followed by a
post-hoc test for group comparisons when warranted. A value of
p<0.05 was considered statistically significant. Data was
presented as mean.+-.SEM or percent change from baseline when
appropriate.
[0648] 2. Creatine Kinase Concentration Analysis
[0649] Approximately 2.0 ml of perfusion medium was collected just
prior to the end of each 15 minute test period from the pulmonary
artery drain. Prior to an early termination of the experiment
(e.g., due to ventricular fibrillation), a sample was taken for
analysis. The samples were frozen on dry ice, and ten stored in a
freezer set at -80.degree. C. for analysis.
B. Experimental Results
[0650] One micromolar of CB200, CB155, or CB42 was preincubated
with human plasma diluted to 50% in perfusion medium for 1 hour at
37.degree. C. The protease test compound mixture was then diluted
to a final concentration of 6% plasma and perfused over isolated
rabbit hearts to induce complement activation and the effects of
the proteases on complement activation was determined based on
hemodynamic measurements. Perfusion of hearts with heat-inactivated
plasma was used as a negative control. Maximal rate of increase in
LV pressure (+dP/dt) was determined at baseline (0 min), and 15,
30, 45, and 60 minutes after perfusion with the test compound
proteases. The results show that plasma alone induced reduced rate
of increase in LV pressure indicating damage to the mycocardium.
The +dP/dt value was decreased about 5-fold from the baseline value
and was similar between all time points tested. In contrast, plasma
that was first heat-inactivated showed no change in the +dP/dt
value as compared to that observed at baseline indicating no
complement activation. Perfusion of rabbit hearts with protease
test compounds protected the hearts from complement-mediated
injury. Both CB42 and CB155 gave full protection of heart function
as indicated by +dP/dt values comparable to baseline levels at all
measured time points. CB200 (wildtype), however, only gave partial
protection of heart function in this model. At 15 minutes after
perfusion with CB200, the heart function observed indicated almost
complete protection with +dP/dt value comparable to baseline
levels. By 30 minutes, CB200 showed little to no protection of
heart function with greater than 3-fold decreased values of +dP/dt
observed, approaching the levels observed by treatment of rabbit
hearts with plasma alone. The rate of increase in LV pressure
levels in rabbits perfused in the presence of CB200 remained low at
45 and 60 minutes indicating cardiac damage at these time
points.
Example 26
Expression and Purification of Modified MT-SP1 CB238 in Shake
Flasks
[0651] CB238 and related recombinant MT-SP1 mutants or wild-type
MT-SP1 were cloned and expressed in E. coli as inclusion bodies as
described in Example 1 and 2 above. The production of the MT-SP1 or
mutants was adapted for laboratory scale by optimizing production
of the MT-SP1 mutant CB238 by pooling up to about 44.times.1 L
shake flasks for subsequent isolation of the inclusion body pellets
for solubilization and refolding. Briefly, 1 .mu.l of plasmid DNA
(from DNA miniprep purification) was mixed with 50 .mu.l of BL-21
cells. The cells were incubated the plasmid DNA on ice for 30
minutes, and then heat shocked at 42.degree. C. for 45 seconds. The
cells were then incubated on ice for 2 minutes for recovery. 500
.mu.l of LB (LB; Difco LB Broth Lennox, approximate formulation per
liter: 10.0 g Tryptone, 10.0 g Yeast Extract, 5.0 g Sodium
Chloride) was added to the cells, and the culture was incubated at
37.degree. C. with shaking for 1 hour. 50 .mu.l of the cells was
then plated out on agar plates containing 50 .mu.g/ml carbenicillin
for selection. The plate was incubated at 37.degree. C. for 16-18
hours.
[0652] 25 ml of LB containing 50 .mu.g/ml carbenicillin was
inoculated from a single colony and grown until fully confluent.
0.5 ml of the seed culture was added to 800 ml of 2XYT containing
10 .mu.g/ml of carbenicillin and grown overnight (.about.12-16
hours; approximately 44 flasks). The cells were harvested by
centrifugation at 6,000.times.g in a Sorvall rotor # SLC4000. The
cell pellets were pooled and weighed. From 35.2 L of E. coli
culture, 320 g of wet cell pellet was obtained. 600 ml of a buffer
containing 50 mM Potassium Phosphate (KPO.sub.4) pH 7.4 and 300 mM
Sodium Chloride (NaCl) was added to the cell pellet. After the
cells were completely resuspended, the batch was split into two and
each part was sonicated in a glass vessel on ice. The sonicator was
set at 60% duty cycle, output level 8, for 4 minutes. The
sonication procedure was repeated two times for each sample. The
resulting sonicated sample was centrifuged at 16,900.times.g for 20
minutes at 4.degree. C. The supernatant was poured out and replaced
with .about.300 ml of fresh buffer containing 50 mM KPO.sub.4 pH
7.4, 300 mM NaCl, and 0.5% lauryldimethylamine oxide (LDAO)
volume/volume. The inclusion body was resuspended using a spatula
and the solution was stirred until homogenous. The stirred sample
was then centrifuged again and the supernatant decanted. The LDAO
wash was performed a total of three times followed by three rounds
of washing with buffer containing 50 mM KPO.sub.4 pH 7.4, 300 mM
NaCl that does not contain LDAO.
[0653] To the 70 g of purified wet inclusion body, 700 ml of
denaturing buffer (6 M Guanidine HCl in 100 mM Tris pH 8.0, 20 mM
dithiothreitol (DTT)) was added, and the protein was solubilized.
The sample was then centrifuged at 20,400.times.g for 30 minutes at
22.degree. C., and the supernatant was collected. The protein
solution was then slowly dripped into 35 L of refolding solution
(100 mM Tris pH 8.0, 150 mM NaCl, 1.5 M Arginine, 5 mM reduced
glutathione, 0.05 mM oxidized glutathione) while vigorously
stirring. The protein solution was left at 4.degree. C. for 72
hrs.
[0654] The resulting protein solution was concentrated by hollow
filtration to .about.1-2L then dialyzed into 16 L of buffer
containing 50 mM Tris pH 8.0, 50 mM NaCl at 4.degree. C. overnight.
The buffer was exchanged for fresh buffer the following morning,
and the sample was dialyzed for an additional 8 hours. The protease
sample was then removed from the dialysis tubing and incubated at
room temperature until auto-activation of the protease occurred by
cleavage of the proregion to release the mature enzyme. Activity
was monitored as described in Example 3 above using a fluorogenic
RQAR-AMC substrate and SDS-PAGE. Upon complete activation, the
sample was then dialyzed into buffer containing 50 mM HEPES pH 6.5
at 4.degree. C. overnight.
[0655] The protein solution was then loaded onto a Source 15S
column (Amersham) and eluted using a buffer gradient from 50 mM
HEPES pH 6.5 to 50 mM HEPES pH 6.5 containing 0.15 M NaCl. Prior to
all chromatography steps, each column is washed in reverse with 0.5
N NaOH then rinsed with water. The active fractions were pooled. An
equal volume of buffer containing 2 M (NH.sub.4).sub.2SO.sub.4 in
50 mM PO.sub.4 pH 7.0 was added, and the resulting solution was
loaded onto a Phenyl Sepharose HP column pre-equilibrated with
buffer containing 50 mM PO.sub.4 pH 7.0, 1 M
(NH.sub.4).sub.2SO.sub.4. The active protein was eluted with a
buffer gradient from 50 mM PO.sub.4 pH 7.0, 1 M
(NH.sub.4).sub.2SO.sub.4 to 50 mM PO.sub.4 pH 7.0. The active
fractions were pooled and buffer exchanged into 50 mM HEPES pH 6.5.
The sample was then reloaded and purified on Source 15Q as in the
first chromatography step. Active fractions were then pooled,
buffer exchanged into PBS using a stirred cell, and concentrated to
.about.10 mg/ml. A sample was removed to measure protein
concentration, A280. Benzamidine was then added to a final
concentration of 20 mM to the remaining sample prior to filtration
of the protein sample through a 0.2 uM syringe filter. The protein
solution was frozen in liquid nitrogen and stored at -80.degree. C.
The final yield was .about.800 mg of pure protein (.about.20 mg of
protease/L of culture). The purified protein was assayed for
specific activity, purity, and endotoxin levels as described in
Example 3 above.
[0656] A similar strategy was employed for other MT-SP1 mutants or
wildtype MT-SP1. The protocol is altered depending on the specific
mutant. For the mutants that don't purify well over Phenyl
Sepharose, Benzamidine Sepharose was used instead. For example, the
MT-SP1 mutant CB450 is purified over a Benzamidine column.
Example 27
Assessment of Hemolysis and Plasma Activity by a Panel of MT-SP1
Mutants
[0657] A panel of proteases were tested for their ability to
support classical hemolysis or alternative hemolysis following
preincubation with 20% plasma as described in Example 7, part A.1.b
and Example 7, part B.1 above. In addition, the proteases were
tested for Plasma Activity as described in Example 6. Table 31
depicts the fraction classical hemolysis at 200 nM, the fraction
alternative hemolysis at 500 nM, and the IC50 for each protease for
both Classical and Alternative hemolysis.
[0658] In addition, the percent protease unbound by alpha-2
macroglobulin (a2M) also was determined. Inactivation of a protease
by alpha-2 macroglobulin traps the protease in a complex where it
is still able to turn over small fluorescent substrates, but unable
to access large protein substrates. This property of alpha-2
macroglobulin complicates the assessment of a proteases activity in
plasma. To determine the actual activity of the free, uncomplexed
protease in plasma, a two step measurement is required. First, the
sample's activity on fluorescent substrates is measured. Second, a
macromolecular inhibitor is added to bind all of the free protease
(ATIII or M84R ecotin), and the protease activity trapped (and
hence protected from inhibition) in alpha-2 macroglobulin is
measured. The percent unbound by alpha-2 macroglobulin activity is
the percentage of the plasma residual activity that is inhibited by
the addition of the ecotin. Briefly, in a 0.2 mL PCR tube, 1 .mu.L
10.times. protease was mixed with 9 .mu.L human plasma in citrate
(Innovative Research). A uninhibted control also was prepeared
containing 1 .mu.L 10.times. protease mixed with 9 .mu.L PBST. The
mixtures were incubated for 5 minutes at 37.degree. C. The samples
were diluted 250 fold in PBST and stored on ice. Two 50 .mu.L
aliquots for each sample were transferred to an opaque assay plate
(Costar #3694) containing 2 .mu.L PBST or 2 .mu.L 520 nM M84R
ecotin. The plates were incubated 10 minutes at room temperature.
Five microliters 0.4 mM Ac-RQAR-AMC substrate was added and the
fluorescence was measured over time with a SpectraMax M5
spectrafluorometer (Molecular Devices) set to read every 20 seconds
for 30 minutes at 30.degree. C. (Ex: 380 nm, EM: 450 nm, Cut-off:
435 nm). The percent unbound by alpha-2 macroglobulin was
calculated with the following formula: (1-([(Protease in
plasma/Protease in PBST)-(Protease in plasma+ecotin/Protease in
PBST)]/(Protease in plasma/Protease in PBST)))*100. The results of
% unbound alpha-2 macroglobulin for the panel of proteases tested
is set forth in Table 31 below. TABLE-US-00031 TABLE 31 Assessment
of Hemolysis and Activity of a Panel of Proteases Classical
Alternative Classical Alternative IC.sub.50 IC.sub.50 % 200 nM 500
nM Hemolysis Hemolysis Plasma Unbound CB# Mutations Hemolysis
Hemolysis (nM) (nM) Activity by a2M CB421 I41T/Y146D/Q175D/ 0.714
478.90 0.271 0.585 84% K224F CB422 I41T/Y146E/Q175D/ 0.050 50.35
0.150 111.8 0.511 83% K224N CB450 I41T/146D/G151L/ 0.255 269.14
0.223 262.4 0.287 K224F CB476 I41T/Y146D/Q175D/ 0.111 87.26 0.217
162.3 0.595 89% K224L CB477 I41T/Y146D/Q175D/ 0.016 53.43 0.140
116.2 0.346 67% K224R CB478 I41T/Y146D/Q175D/ 0.254 86.48 0.316
230.3 0.550 89% K224N CB480 I41T/Y146D/G151L/ 0.272 131.01 0.367
268.1 0.670 88% Q175D/K224F CB481 I41T/Y146D/G151L/ 0.050 143.19
0.169 154.3 0.687 72% Q175D/K224L CB482 I41T/Y146D/G151L/ 0.042
57.39 0.255 296.2 0.306 10% Q175D/K224R CB483 I41T/Y146D/G151L/
0.076 57.39 0.425 257.3 0.642 73% Q175D/K224N CB484
I41T/Y146E/Q175D/ 0.235 67.21 0.365 268.5 0.649 94% K224F CB485
I41T/Y146E/Q175D/ 0.072 103.78 0.184 160.4 0.593 82% K224L CB486
I41T/Y146E/Q175D/ 0.014 43.36 0.128 125.4 0.326 44% K224R CB487
I41T/Y146E/G151L/ 0.026 52.87 0.173 169.2 0.548 53% Q175D/K224N
CB488 I41T/Y146E/G151L/ 0.086 72.61 0.195 179.3 0.658 85%
Q175D/K224F CB489 141T/Y146E/G151L/ 0.038 50.56 0.143 140.8 0.526
58% Q175D/K224L CB490 I41T/Y146E/G151L/ 0.031 52.63 0.125 193.8
0.288 0% Q175D/K224R
Example 28
Additional Mutants
[0659] Additional mutants are prepared as described herein. Such
mutants include, but are not limited to, those set forth in Table
32 below. TABLE-US-00032 TABLE 32 SEQ SEQ Additional Mutants ID ID
I41T/Y146D/G151L/K224N 681 696 Y146D/Q175D/K224N 682 697
I41T/Y146D/K224N 683 698 Y146D/G151L/K224N 684 699
Y146D/Q175R/K224N 685 700 Y146D/Q175K/K224N 686 701
Y146D/Q175H/K224N 687 702 I41T/Y146D/G151L/Q175K/K224F 688 703
I41T/Y146D/G151L/Q175R/K224F 689 704 I41T/Y146D/G151L/Q175H/K224F
690 705 I41T/Y146D/G151L/Q175Y/K224F 691 706
I41T/Y146D/G151L/Q175K/K224N 692 707 I41T/Y146D/G151L/Q175R/K224N
693 708 I41T/Y146D/G151L/Q175H/K224N 694 709
I41T/Y146D/G151L/Q175Y/K224N 695 710
[0660]
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=US20070093443A1).
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=US20070093443A1).
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