U.S. patent application number 16/734256 was filed with the patent office on 2021-07-22 for modified urokinase-type plasminogen activator polypeptides and methods of use.
The applicant listed for this patent is Catalyst Biosciences, Inc.. Invention is credited to Eric Steven Furfine, Edwin L. Madison, Mikhail Popkov, Vanessa Soros, Christopher Thanos, Kimberly Tipton, Matthew John Traylor, Jeffrey Charles Way.
Application Number | 20210222143 16/734256 |
Document ID | / |
Family ID | 1000005693003 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210222143 |
Kind Code |
A9 |
Madison; Edwin L. ; et
al. |
July 22, 2021 |
MODIFIED UROKINASE-TYPE PLASMINOGEN ACTIVATOR POLYPEPTIDES AND
METHODS OF USE
Abstract
Provided are u-PA polypeptides and fusion proteins containing
the u-PA polypeptides. The u-PA polypeptides are modified to have
altered activity and/or specificity so that they cleave a
complement protein, such as complement protein C3, to thereby
inhibit complement activation. The modified u-PA polypeptides and
fusion proteins that inhibit complement activation can be used for
treatment of diseases and conditions that are mediated by
complement activation, or in which complement activation plays a
role. These disorders include ischemic and reperfusion disorders,
including myocardial infarction and stroke, sepsis, autoimmune
diseases, diabetic retinopathies, age-related macular degeneration,
transplanted organ rejection, inflammatory diseases and diseases
with an inflammatory component.
Inventors: |
Madison; Edwin L.; (San
Francisco, CA) ; Thanos; Christopher; (Tiburon,
CA) ; Soros; Vanessa; (San Francisco, CA) ;
Popkov; Mikhail; (San Diego, CA) ; Tipton;
Kimberly; (Boston, MA) ; Traylor; Matthew John;
(Boulder, CO) ; Furfine; Eric Steven; (Lincoln,
MA) ; Way; Jeffrey Charles; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Catalyst Biosciences, Inc. |
South San Francisco |
CA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200208133 A1 |
July 2, 2020 |
|
|
Family ID: |
1000005693003 |
Appl. No.: |
16/734256 |
Filed: |
January 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US19/68839 |
Dec 27, 2019 |
|
|
|
16734256 |
|
|
|
|
62786302 |
Dec 28, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 304/21073 20130101;
C12N 9/6462 20130101; A61K 38/00 20130101; C07K 2319/50 20130101;
C07K 2319/02 20130101; C07K 14/765 20130101 |
International
Class: |
C12N 9/72 20060101
C12N009/72; C07K 14/765 20060101 C07K014/765 |
Claims
1. A modified urokinase-type plasminogen activator (u-PA)
polypeptide, comprising one or more amino acid modifications
selected from among replacements corresponding to R35Q, H37Y, V41R,
V41L, Y40Q, D60aP, L97bA, T97aI and H99Q, and conservative amino
acid modifications therefor, whereby the modified u-PA polypeptide
has increased activity/specificity for a complement protein
compared to the unmodified active form of the u-PA polypeptide,
wherein: the amino acid modifications are selected from among
replacements, insertions and deletions in the primary sequence of
the modified u-PA polypeptide; the modified u-PA polypeptide
cleaves a complement protein to thereby inhibit or reduce
complement activation compared to the unmodified u-PA polypeptide
that does not contain the amino acid modifications; the complement
protein is C3; the modified u-PA polypeptide has reduced activity
or specificity for cleavage of a substrate sequence in plasminogen
compared to the unmodified u-PA polypeptide; the modified u-PA
polypeptide has at least 90% sequence identity with the
polypeptides of any of SEQ ID NOs: 1-6; residues are numbered by
chymotrypsin numbering; the unmodified u-PA polypeptide comprises
the sequence set forth in any of SEQ ID NOs: 1-6, which sets forth
wild-type (WT) full-length u-PA, WT protease domain u-PA, WT mature
u-PA, full-length u-PA with a C122S, by chymotrypsin numbering,
protease domain u-PA with C122S, mature u-PA with C122S, or a
catalytically active fragment thereof that includes the amino acid
replacement(s); and the conservative modifications are selected
from among R35Y, W, F or N; H37R, Q, E, W or F; V41K; D60aS; T97aD,
L or V; L97bG or S, and H99N.
2. The modified u-PA polypeptide of claim 1 that cleaves within
residues QHARASHLG (residues 737-745) of human C3 (SEQ ID
NO:47).
3. The modified u-PA polypeptide of claim 1 that has increased
activity for cleavage of C3 that is least 3-fold greater than the
unmodified u-PA polypeptide comprising the protease domain of SEQ
ID NO:5, or a corresponding form of u-PA set forth in any of SEQ ID
NOs: 1-4 and 6.
4. The modified u-PA polypeptide of any claim 1, wherein: the
modified u-PA polypeptide has ED.sub.50 for inactivation cleavage
of C3 of less than or 100 nM, or 50 nM or 30 nM or 25 nM in an in
vitro assay; and the modified u-PA polypeptide has stability of
greater than 50% or 80% after incubation in PBS, or a body fluid
for 7 days.
5. The modified u-PA polypeptide of claim 1, wherein the unmodified
u-PA polypeptide consists of the sequence of amino acids set forth
in any of SEQ ID NOs: 1-6.
6. The modified u-PA polypeptide of claim 1 that has 1 or up to 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
amino acid replacements, insertions or deletions, compared to the
unmodified u-PA polypeptide of any of SEQ ID NOs: 1-6 or a
catalytically active portion thereof.
7. The modified u-PA polypeptide of claim 1, comprising one or more
amino acid modifications selected from among replacements
corresponding to R35Q, H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI,
and H99Q.
8. The modified u-PA polypeptide of claim 7, comprising V41R or
V41L and one or more of the replacements L97bA, R35Q, H99Q, D60aP,
and T97aI.
9. The modified u-PA polypeptide of claim 1, comprising the
replacement V41R or V41L, and optionally C122S.
10. The modified u-PA polypeptide of claim 1, further comprising
the replacement V38E.
11. The modified u-PA polypeptide of claim 7, comprising the
replacement H37Y.
12. The modified u-PA polypeptide of claim 1, comprising the
modifications V38E/V41R.
13. The modified u-PA polypeptide of claim 1, comprising the
replacements R35 Y/H37S/V38E/V41R or R35 Y/H37Y/V38E/V41R.
14. The modified u-PA polypeptide of claim 1, comprising the
replacements H37Y/V38E, R35Y/H37K, R35Q/H37K, R35Q/H37Y, V38E/V41R,
V38E/V41R/Y149R, T39Y/V41R/D60aP/L97bA/H99Q/C122S,
T39Y/V41R/D60aP/L97bA/H99Q, T39Y/V41R/Y60bQ/L97bA/H99Q or
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S.
15. The modified u-PA polypeptide of claim 1, comprising the amino
acid modifications R35Q/H37Y/T39Y/V41R, R35Q/H37Y/T39Y/V41R/C122S,
R35Q/H37Y/T39Y/V41R/L97bA/H99Q/C122S, or
R35Q/H37Y/T39Y/V41R/L97bA/H99Q.
16. The modified u-PA polypeptide of claim 1, comprising the
modifications selected from:
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;
R35W/R36Q/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151L;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82R/T97aI/L97bA/H99Q/K110aR/C122-
S/Y149R/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K/K179R;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92R/T97aI/L97bA/H99Q/C12-
2S/Y149R/M157K;
F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bW/T97aI/L97bA/H99Q/C122S/Y149-
E/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92S/T97aI/L97bA/H99Q/C12-
2S/Y149R/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61R/K62R/T97aI/L97bA/H99Q/C122S/-
Y149R/M 157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K/K179S;
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S-
;
F30Y/R35W/R36T/H37S/V38S/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y14-
9R/Y151L/M157R/Q192Y;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61
S/K62S/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
R35A/H37E/R37aG/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;
R35W/R36Q/H37S/V38T/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y15-
1P/M157R;
F30Y/R35W/H37Y/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/-
Y149R; V38E/T39W/V41R/D60aW/Y60bP/L97bG/H99L/C122S;
R35W/R36K/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y15-
1L/M157S/Q192H;
R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aQ/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R;
I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/T158-
A;
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L-
/Q192H;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y14-
9R/M157K;
R35W/R36N/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/-
Y149R/M157S;
R35Y/H37D/V38E/T39W/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82S/T97aI/L97bA/H99Q/K110aS/C122-
S/Y149R/M157K;
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/H99L/C12-
2S;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/Y149R/M157K;
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S;
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R/M15-
7K;
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/M157K;
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158A;
R35Q/R36H/H37Y/V38E/T39Y/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
R35W/H37P/R37aG/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
V38D/V41Q/D60aH/Y60bS/T97aW/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R;
F30Y/R35W/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bQ/T97aE/L97bA/H99Q/C122-
S/Y149R/M157K;
F30Y/R35W/R36Q/H37S/V38P/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/M157R;
F30H/R35W/R36T/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y-
149R/Y151L/M157 S;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bD/T97aI/L97bA/H99Q/C122S/M157-
K;
F30Y/R35Y/R36H/H37N/V38E/T39F/Y40F/V41R/K61E/R72H/T97aI/L97bA/H99Q/C122-
S/Y149R/M157K/Q169K;
R35W/R36Q/H37S/V38S/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151L/M1-
57S/Q192H;
R35W/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C12-
2S/Y151L/Q192T;
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S;
F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/Y149-
R/M157K;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C1-
22S/Y149R;
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S-
/Y149R;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C12-
2S/M157K;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C-
122S;
F30Y/R35W/R36S/H37S/V38Q/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S-
/Y149R/Y151L/M157S/Q192N;
F30Y/R35W/R36H/H37P/R37aD/V38E/T39Y/Y40F/V41R/D60aE/Y60bS/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K;
R35Q/H37Y/R37aS/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
R37aS/V38E/Y40V/V41R/H99L/C122S/Y151L/R217V;
V38D/V41R/L97bG/H99Q/C122S/Y151L/R217E;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S;
F30Y/R35V/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bP/T97aI/L97bA/H99Q/C122S/Y149-
R/M157K;
I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M1-
57K;
F30Y/R35V/R36H/H37S/V38E/T39F/Y40H/V41R/Y60bS/T97aM/L97bA/H99Q/C122S/-
Y149W/M157K;
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;
N26D/F30Y/R35Y/R36H/H37E/V38E/T39F/Y40F/V41R/K61E/T97aI/L97bA/H99Q/R110dS-
/P114S/C122S/Y149R/M157K;
F30Y/R35W/R36H/H37P/R37aE/V38E/T39Y/Y40F/V41R/Y60bA/T97aE/L97bA/H99Q/C122-
S/Y149R/M157K;
R35L/H37D/R37aN/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/Y149-
K/M157K;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R-
; R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S/E167K;
R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35Y/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bE/T97aI/L97bA/H99Q/C122S/Y149-
R/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y1-
49R/M157K;
F30Y/R35Y/R36H/H37E/V38E/T39F/Y40F/V41R/K61E/T97aI/L97bA/H99Q/C-
122S/Y149R/M157K/T 242A;
F30Y/R35L/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A;
F30Y/R35Y/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bH/T97aE/L97bA/H99Q/C122-
S/Y149R/M157K; V38D/V41R/L97bR/H99E/C122S/Y151L/R217E;
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/-
R217E/K224R;
R35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y151L/Q-
192T;
F30Y/R35M/R36H/H37G/V38E/T39F/Y40H/V41R/Y60bP/T97aF/L97bA/H99Q/C122S-
/Y149R/M157K;
F30Y/R35W/R36Q/H37S/V38T/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/Y151L/M157K/Q192T;
R35W/H37D/R37aP/V38E/T39W/V41R/D60aR/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;
I17V/F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S-
/Y149K/M157K;
I17V/F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/M157K/T15-
8A; R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/I138V/E167K;
F30Y/R35W/R36Q/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/Y151L/M157T/Q192H;
R35H/G37bD/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122 S/T158S;
R35H/H37P/R37aG/V38E/T39F/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122-
S/Y149R;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C1-
22S/Y149R/M157K; V38D/T39Y/Y40L/V41R/L97bI/H99E/C122S/R217E;
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/T158A;
F30H/R35W/R36H/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/M157K;
R35V/R36H/H37D/V38E/T39W/Y40M/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M-
157K;
R35W/R36K/H37S/V38A/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y15-
1L/M157R/Q192T;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/Y149-
R/M157K;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C1-
22S/N145 S/S146V/T147M/D148G/Y149Q/L150F/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/Y149R/M157K;
F30Y/R35I/R36H/H37D/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K; R35V/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R;
R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aN/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R;
V38E/Y40Q/V41L/L97bG/H99Q/C122S/R217T;
R35H/V38E/T39Y/V41R/T56S/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S;
F30H/R35Q/H37W/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K;
R35Q/H37Y/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R;
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q-
192A; V38D/V41R/Y60bR/T97aW/L97bR/H99E/C122S/E175D/R217E/K224R;
F30Y/R35W/R36H/H37S/V38E/Y40H/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R;
V38D/V41L/Y60bP/T97aM/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R;
F30Y/R35W/R36H/H37D/V38E/T39F/Y40H/V41R/Y60bE/T97aI/L97bA/H99Q/C122S/Y149-
R/M157K;
F30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C1-
22S/Y149K/M157K;
F30Y/R35W/R36K/H37S/V38D/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/Y151L/M157R/Q192T; V41R/H99Q/C122S/Y151L/R217V;
V38E/Y40P/V41L/L97bG/H99L/C122S/Y151Q/R217E;
V38E/Y40L/V41R/H99L/C122S/Y151L/R217S;
V38E/Y40Q/V41L/L97bG/H99Q/C122S/Y151P/R217T;
V38E/T39Y/V41R/D60aW/Y60bP/L97bR/H99I/C122S;
R35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R36H/H37F/V38E/T39Y/Y40H/V41R/Y60bD/T97aV/L97bA/H99Q/C122S/Y149L/M15-
7K;
F30Y/R35W/R36H/H37E/S37dP/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/C-
122S/Y149K/M157K;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aA/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R;
R36H/V38D/V41R/A96D/D97E/A98G/T97adel/H99L/L97bdel/C122S/T178
S/R217D; V38D/V41R/L97bG/H99Q/C122S/Y151L/R217A;
F30H/R35Q/R36H/H37Y/V38E/T39Y/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K;
F30Y/R35W/R36K/H37E/V38E/T39W/Y40H/V41R/Y60bQ/K61E/I65T/T97aE/L97bA/H99-
Q/C122S/Y149K/M157K;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bS/T97aL/L97bA/H99Q/C122S/Y149-
L/M157K;
F30Y/R35Q/R36H/H37Y/R37aE/V38E/T39Y/Y40F/V41R/D60aS/Y60bP/T97aE/L-
97bA/H99Q/C122S/Y149R/M157K;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aG/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;
I24N/F30Y/R35W/R36H/H37E/V38E/T39W/Y40L/V41R/Y60bQ/N87D/T97aE/L97bA/H99Q/-
C122S/Y149K/M157K; V38E/Y40Q/V41L/L97bA/H99Q/C122S;
F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/M157K/T158A;
F30Y/R35W/R36A/H37E/V38E/T39W/Y40H/V41R/Y60bQ/K61D/I65R/T97aE/L97bA/H99Q/-
C122S/Y149K/M157K;
F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K/K187R/K223R/K224R;
R35W/R36Q/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y15-
1L/M157S/Q192T;
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/P60cS/L97bA/H99Q/C122S/I138V/E167K;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;
I17V/F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K;
R35Y/R36H/H37K/V38E/T39F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
R35H/V38E/T39Y/V41R/T56A/D60aP/Y60bQ/L97bA/H99Q/C122S;
F30Y/V38D/Y40F/V41L/L97bA/H99Q/C122S/Y151L/M157R;
V38E/Y40A/V41L/L97bG/H99Q/C122S/R217T;
I24T/F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S-
/Y149K/M157K;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/M157K; I17V/F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A;
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q-
192T; F30H/R35L/H37D/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K/R217E;
F30Y/R35W/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/D60aE/Y60bF/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K;
F30Y/R35L/R36H/H37G/V38E/T39Y/Y40H/V41R/Y60bP/T97aE/L97bA/H99Q/C122S/Y149-
M/M157K; I17V/F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K;
F30Y/R35V/R36H/H37K/V38E/T39F/Y40H/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/Y149-
R/M157K;
R35W/R36H/H37N/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M-
157K/Q192H; R35V/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R;
F30Y/R35M/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K; R35Q/V38D/V41R/L97bG/H99Q/C122S/Y151;
R37aS/V38E/Y40P/V41L/L97bG/H99Q/C122S/Y151Q/R217T;
R35V/R37aE/V38E/Y40Q/V41L/T97aE/L97bA/H99Q/C122S/Y149R;
F30H/V38D/V41R/A96G/L97bA/H99Q/C122S/Y151L/M157K;
T39L/Y40L/V41R/T97aI/L97bA/H99Q/C122S;
F30Y/R35W/R36H/H37E/V38E/T39Y/Y40F/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/Y149-
R/M157K Y40Q/V41L/Y60bL/L97bA/H99Q/C122S;
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/S146F/M157K/Q192H/K243Q
Y40Q/V41L/L97bA/H99Q/C122S/Y149R;
F30Y/R35W/R36Q/H37E/V38E/T39W/Y40H/V41R/Y60bQ/K61L/I65V/T97aE/L97bA/H99Q/-
C122S/Y149K/M157K;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
R35Q/V38D/V41R/T97aS/L97bA/H99Q/C122S/Y151L;
V41R/L97bR/H99Q/C122S/Y151L/R217V;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/Y149R;
F30Y/R35V/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bP/T97aE/L97bA/H99Q/C122S/Y149-
E/M157K;
R35A/H37T/R37aD/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S-
/Y151L/Q192S; R35S/V38D/V41R/L97bA/H99Q/C122S/Y151L;
R35S/V38D/V41L/L97bG/H99Q/C122S/Y151L/R217Q;
F30Y/R35H/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158S;
R35Q/H37S/R37aE/V38E/T39Y/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;
H37G/R37aD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/-
K224R;
H37G/R37aD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/-
R217E;
R35W/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bS/T97aI/L97bA/H99Q/C122S/Y-
149R;
R35Q/H37G/R37aD/V38E/T39Y/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/Y1-
49R;
R35Q/H37D/R37aK/V38E/T39F/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y14-
9R;
R35Y/R36H/H37S/V38D/T39Y/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R36H/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/M157K;
R37aS/V38D/V41Q/L97bG/H99Q/C122S/Y151L/R217T;
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K;
F30Y/R35K/R36H/H37E/R37aK/V38E/T39F/Y40F/V41R/D60aP/Y60bS/T97aI/L97bA/H99-
Q/C122S/Y149R/M157K;
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bG/T97aI/L97bA/H99-
Q/C122S/Y149R/M157K;
F30Y/R35W/R36Q/H37E/V38A/T39W/Y40H/V41R/Y60bQ/K61D/I65V/T97aE/L97bA/H99Q/-
C122S/Y149K/M157K;
F30Y/R35H/V38D/Y40H/V41R/T56A/L97bA/H99Q/C122S/M157K;
R35N/H37T/R37aY/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
R37aH/V38E/T39Y/V41R/T56A/D60aP/Y60bQ/L97bA/H99Q/C122S/T158A;
F30H/R35Q/H37T/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K;
F30H/R36L/V38E/V41R/K82R/L97bA/H99Q/C122S/Y151L/M157K;
V38D/V41R/H99Q/C122S/Y151L/R217V;
R35Q/H37G/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35Q/R36H/H37Y/R37aE/V38E/T39Y/Y40F/V41R/D60aE/Y60bA/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K;
R35Q/H37Y/R37aD/V38E/T39L/V41R/D60aE/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35L/R36H/H37E/V38E/T39N/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K; R36S/V38E/Y40L/V41N/L97bG/H99Q/C122S/Y151L/R217T;
T39W/V41R/L97bG/H99Q/C122S;
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/Y149-
K/M157K;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R-
; R35S/R37aA/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149V;
F30Y/R35W/R36H/H37Q/V38E/T39H/Y40H/V41R/T97aE/L97bA/H99Q/C122S/Y149L/M157-
K;
F30Y/R35Q/R36H/H37Y/R37aD/V38E/T39Y/Y40F/V41R/Y60bV/T97aE/L97bA/H99Q/C1-
22S/Y149R/M157K; F30H/V38D/V41R/L97bA/H99Q/Y151L/M157K;
F30H/R35H/H371/V38D/V41R/L97bA/H99Q/C122S/Y149W/Y151L/M157K/R217S;
V38D/T39Y/Y40H/V41R/T97aI/L97bA/H99Q/C122S;
R35F/H37D/R37aN/V38E/T39Y/V41R/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;
T39Y/V41R/Y60bQ/L97bG/H99Q/C122S; T39Y/V41R/D60aP/Y60bQ/L97bA/H99
Q/C122S; V38D/V41R/L97bR/H99Q/C122S/Y151L/R217E; R36
S/V38D/T39L/Y40L/V41R/L97bI/H99E/C122S/R217T;
R35S/R37aD/V38E/Y40Q/V41L/Y60b V/T97aL/L97bA/H99Q/C122S/Y149L;
Y40Q/V41L/Y60bT/T97aE/L97bA/H99Q/C122S/Y149R;
F30Y/V38E/Y40H/V41R/T56A/L97bA/H99Q/C122S/M157K/K243M;
F30Y/R36H/R37aH/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/M157K;
F30H/R35Q/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K;
V38D/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R;
H37G/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/-
R217E/K224R;
R35S/R37aD/V38E/Y40Q/V41L/T97aE/L97bA/H99Q/C122S/Y149R;
R35V/R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S;
Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R;
F30Y/R35H/V38D/Y40H/V41R/L97bA/H99Q/C122S/I138V/M157K;
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S;
F30Y/R35H/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bD/T97aI/L97bA/H99-
Q/C122S/Y149R/M157K;
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A;
V38E/T39W/V41R/D60aP/Y60bD/L97bA/H99L/C122S;
F30Y/R36H/V38E/Y40H/V41R/I65T/T97aI/L97bA/H99Q/C122S/M157K;
V38D/V41R/L97bR/H99Q/C122S/Y151L/R217V;
R35Q/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
R35W/R36H/H37S/V38E/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157K;
R36S/V38E/Y40Q/V41R/L97bG/H99L/C122S/Y151P/R217E;
V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122S;
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/-
R217E;
H37G/R37aD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/-
Q192T/R217E;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157-
K/K187S/K223S/K224Y; Y40Q/V41L/L97bA/H99Q/C122S;
F30H/R35H/V38D/V41R/K61E/L97bA/H99Q/C122S/Y151L/M157K/R206H;
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K;
F30Y/R36H/V38E/Y40H/V41R/T97aE/L97bA/H99Q/C122S/Y149R/M157K;
R35A/R37aE/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149R;
V38D/V41L/L97bG/H99Q/C122S/Y151L/R217Q;
F30H/R35Q/H37W/V38D/V41R/D60aE/L97bA/H99Q/C122S/Y149L/Y151L/M157K/R217D;
F30Y/R35F/R36H/H37G/V38E/T39Y/Y40H/V41R/Y60bS/T97aD/L97bA/H99Q/C122S/Y149-
R/M157K; T39Y/V41R/L97bG/H99Q/C122S;
F30Y/R35I/R36H/H37E/V38E/T39Y/Y40H/V41R/Y60bS/T97aV/L97bA/H99Q/C122S/Y149-
L/M157K; R35S/R37aD/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149R; Y40H/V41
Q/L97bG/H99Q/C122S/R217T;
R35W/H37D/V38D/T39Y/V41R/Y60bS/L97bA/H99Q/C122S/Y149R;
V38D/T39F/Y40L/V41R/T97aW/L97bA/H99Q/C122S;
V38D/T39Y/Y40L/V41R/T97aE/L97bA/H99Q/C122S;
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39F/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K; V38D/T39L/Y40L/V41R/T97aI/L97bA/H99Q/C122S;
V38D/T39Y/Y40L/V41R/T97aW/L97bA/H99Q/C122S;
F30Y/R36H/V38D/Y40H/V41R/L97bA/H99L/C122S/F141L/M157K/T158A;
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aA/Y60bS/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K;
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K; T39Y/V41R/Y60bP/L97bG/H99Q/C122S;
F30H/R36H/V38D/V41R/T56A/L97bA/H99Q/C122S/Y151L/M157K;
F30Y/R35E/R36H/H37D/R37aN/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122-
S/Y149R/M157K; V38E/Y40Q/V41L/D60aP/Y60bL/L97bA/H99Q/C122S/Y149W;
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K;
F30H/R35Q/H37
W/V38D/V41R/D60aE/Y60bS/L97bA/H99Q/C122S/Y149L/Y151L/M157K;
R35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149-
R/Y151P/M157K/Q192H;
F30Y/R35M/R36H/H37D/R37aD/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/H99-
Q/C122S/Y149R/M157K;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bT/T97aD/L97bA/H99Q/C122S/Y149-
R/M157K; V38D/T39L/Y40L/V41R/T97aV/L97bA/H99Q/C122S;
V38D/V41R/Y60bS/T97aI/L97bR/H99E/C122S/Y151L/E175D/Q192F/R217E/K224R;
T39Y/V41R/Y60bP/L97bA/H99Q/C122S;
R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192E/R217-
D;
R35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R-
; F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/Y151L/M157K/Q192H;
F30H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151L/M157F;
H37M/R37aD/V38E/T39A/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;
F30H/V38D/V41R/L97bA/H99Q/Y151L/M157K;
T22I/F30Y/R35S/V38D/Y40H/V41R/L97bA/H99Q/C122S/I138V/M157K;
R35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35L/V38D/Y40H/V41R/N76S/L97bA/H99Q/C122S/M157K/K187E;
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157S;
R35W/H37D/V38D/T39Y/V41R/Y60bH/L97bA/H99Q/C122S/Y149R;
F30Y/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bA/T97aE/L97bA/H99Q/C122S/Y149Q/M15-
7K;
R35Q/H37G/R37aE/V38W/T39Y/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175-
D/Q192T/R217E/K224R;
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/-
R217E/K224R; V38D/T39Y/Y40M/V41R/T97aE/L97bA/H99Q/C122S;
R35Q/H37N/V38D/T39Y/V41R/Y60bP/L97bA/H99Q/C122S;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/Y149-
K/M157K;
R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149-
R; V38E/T39L/V41R/D60aN/Y60bP/L97bG/H99Q/C122S;
F30Y/R36H/H37A/V38E/T39Y/Y40H/V41R/Y60bQ/T97aV/L97bA/H99Q/C122S/Y149R/M15-
7K;
F30Y/R35W/R36H/H37E/R37aP/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C-
122S/Y149Q/M157K;
H37T/R37aL/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192R;
H37G/R37aD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/-
R217E/K224R;
F30Y/R35W/R36H/H37S/V38E/Y40H/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157K;
V38D/T39W/Y40L/V41R/T97aL/L97bA/H99Q/C122S;
H37G/R37aD/G37bD/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T-
/R217E/K224R; T39Y/V41R/L97bA/H99Q/C122S;
V38D/T39L/Y40L/V41R/T97aW/L97bA/H99Q/C122S;
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/Y149N/L150V/M157K;
R35 S/V38D/L97bA/H99Q/C122S/Y151L/M157Y;
R37aS/V38D/T39Y/Y40F/V41R/H99L/C122S/R217T;
Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R;
Y40H/V41T/L97bG/H99Q/C122S/R217T; and any of these polypeptides in
which C122S is C122C, by chymotrypsin numbering.
17. The modified u-PA polypeptide of claim 1, comprising the amino
acid modifications:
H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;
or
R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aA/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R;
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149-
R; or
R35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y1-
49R; or
R35Q/H37G/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/-
Y149R; or
R35A/H37G/R37aE/V38E/T39F/V41R/D60aE/Y60bP/T97aI/L97bA/H99Q/C122-
S/Y149R; or
R35Q/H37S/R37aE/V38E/T39Y/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37T/R37aP/V38E/T39Y/V41R/D60aE/Y60bD/T97aI/L97bA/H99Q/C122S/Y149-
R; or
R35Q/H37G/R37aE/V38E/T39H/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/Y1-
49R; or
R35W/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bS/T97aI/L97bA/H99Q/C122S/-
Y149R; or
R35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122-
S/Y149R; or
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/H99L/C12-
2S; or
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y-
151L/Q192A; or
R35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y151L/Q-
192T; or
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S-
/Y151L; or
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C12-
2S/Y151L/Q192T; or each of the foregoing with no replacement at
C122.
18. The modified u-PA polypeptide of claim 1, comprising the amino
acid modifications corresponding to Y40Q/V41L/L97bA/C122S or
Y40Q/V41R/L97bA/C122S or Y40Q/V41L/L97bA or Y40Q/V41R/L97bA.
19. The modified u-PA polypeptide of claim 1, comprising the amino
acid modifications corresponding to R37aS/V41R/L97bG/H99Q or
R37aS/V41R/L97bG/H99Q/C122S.
20. The modified u-PA polypeptide of claim 1, comprising the amino
acid modifications corresponding to T39Y/V41L/L97bA/H99Q/C122S or
T39Y/V41R/L97bA/H99Q/C122S or T39Y/V41L/L97bA/H99Q or
T39Y/V41R/L97bA/H99Q.
21. The modified u-PA polypeptide of claim 18, further comprising
the replacement corresponding to H99Q.
22. The modified u-PA polypeptide of claim 1, comprising the amino
acid replacements
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149-
R; or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/Y149R.
23. The modified u-PA polypeptide of claim 1, comprising the amino
acid replacements
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R,
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/Y149R.
24. The modified u-PA polypeptide of claim 1 comprising the amino
acid modifications corresponding to
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R,
wherein the unmodified u-PA polypeptide comprises the protease
domain set forth in SEQ ID NO:2 or SEQ ID NO:5.
25. The modified u-PA polypeptide of claim 23, wherein the
unmodified polypeptide consists of the mature u-PA of SEQ ID NO:3
or SEQ ID NO:6.
26. The modified u-PA polypeptide of claim 1, wherein the modified
u-PA polypeptide comprises the sequence of amino acid residues set
forth in any of SEQ ID NOs: 8-44.
27. The modified u-PA polypeptide of claim 1, wherein the modified
u-PA polypeptide comprises the sequence of amino acid residues set
forth in SEQ ID NO:21 or SEQ ID NO: 18 or SEQ ID NO:987.
28. The modified u-PA polypeptide of claim 1 that comprises two
chains and is activated, wherein the modified u-PA polypeptide
contains the residue C122 that forms a disulfide bind with another
free cysteine in the polypeptide.
29. The modified u-PA polypeptide of claim 1 that is conjugated to
another moiety or polymer either directly or via a linker.
30. The modified u-PA polypeptide of claim 29, wherein the moiety
or polymer increases serum half-life and/or to reduces
immunogenicity or both.
31. The modified u-PA polypeptide of claim 1 that is PEGylated.
32. The modified u-PA polypeptide of claim 29 that is a fusion
protein.
33. The modified u-PA polypeptide of claim 29 that is linked
directly or indirectly to serum albumin.
34. The modified u-PA polypeptide of claim 33, wherein the serum
albumin is a human serum albumin (HSA) that comprises the sequence
of amino acids set for in SEQ ID NO: 991, or a form that has at
least 90% or at least 95% sequence identity thereto.
35. The modified u-PA polypeptide of claim 29, that is conjugated
to a polymer that is a polypeptide, wherein the polypeptide is
multimerization domain.
36. The modified u-PA polypeptide of claim 35, wherein the
multimerization domain is an Fc domain that comprises the sequence
set forth in SEQ ID NO: 50 or SEQ ID NO:992 or a form that has at
least 90% or at least 95% sequence identity thereto.
37. The modified u-PA polypeptide of claim 29, wherein the moiety
or polymer is linked via a peptide linker to the modified u-PA
polypeptide.
38. The modified u-PA polypeptide of claim 37, wherein the linker
comprises Gly and/or Ser.
39. The modified u-PA polypeptide of claim 29, comprising the
sequence of amino acid residues set forth in any of SEQ ID Nos:
1001-1003, 1024-1029, multimers thereof, and sequences having at
least 99% sequence identity thereto.
40. A fusion protein, comprising a modified u-PA polypeptide or a
catalytically active portion of a modified u-PA polypeptide of
claim 1 that is fused to a non-protease polypeptide or a portion
thereof.
41. The fusion protein of claim 40 that comprises a heterologous
activation sequence or a u-PA activation sequence.
42. The fusion protein of claim 41, wherein the activation sequence
comprises a cysteine, and the modified u-PA polypeptide comprises a
free cysteine, whereby, upon activation, the resulting activated
polypeptide comprises two chains.
43. The fusion protein of claim 41, wherein the activation sequence
is a u-PA activation sequence or a furin activation sequence.
44. The fusion protein of claim 43, wherein the activation sequence
is an activation sequence set forth in any of SEQ ID NOs:995-998,
1041, and 1044, or a sequence having at least 95% sequence identity
to the sequence set forth in any of SEQ ID NOs:995-998, 1041, and
1044.
45. The fusion protein of claim 40 that comprises a signal
sequence, wherein the signal sequence effects secretion of the
fusion protein and is removed from the fusion protein.
46. The fusion protein of claim 40, comprising a fusion
partner.
47. The fusion protein of claim 46, wherein the fusion partner is
albumin, or an F.sub.c domain, or a single chain antibody or other
antigen binding fragment of an antibody, or a hyaluronic acid
binding domain (HABD), or an antibody or antigen binding fragment
thereof that is an anti-type II collagen antibody scFv fragment or
an anti-VEGFR antibody or fragment thereof.
48. The fusion protein of claim 40, comprising an activation
sequence, a modified u-PA polypeptide, and HSA.
49. The fusion protein of claim 40, comprising the sequence of
amino acids set forth in: a) any of SEQ ID Nos: 1004-1019 and
1034-1040, or b) a sequence having at least 95% sequence identity
to the sequence of amino acids of any of SEQ ID Nos: 1004-1019 and
1034-1040, or c) a sequence of amino acids of a) or b) from which
the signal sequence has been removed upon expression or is not
included.
50. The fusion protein of claim 49, wherein the sequence of amino
acids in a), b), and c) is the sequence set forth in any of SEQ ID
NOs: 1006, 1007, 1009, and 1010.
51. The fusion protein of claim 49, wherein the sequence of amino
acids in a), b), and c) is the sequence set forth in SEQ ID NO:1015
or 1019.
52. The fusion of claim 49 that is a two-chain activated form
containing an A chain and a B chain.
53. The fusion protein of claim 52, wherein the B chain starts at
residues IIGG of the modified u-PA polypeptide and ends at the
C-terminus of the fusion protein.
54. The fusion protein of claim 52, comprising the sequence of
amino acids set forth in any of SEQ ID NOs: 1005, 1011, 1014, 1015,
1016, 1019, and 1036, but lacking the signal sequence.
55. The fusion protein of claim 54, comprising an A chain of
residues 21-178, and a B chain of residues 179- to the C-terminus
of the protein with a disulfide linkage between residues
168-299.
56. The fusion protein of claim 54 that is a two chain activated
fusion protein, comprising an A chain and a B chain, wherein the A
chain consists of residues 21-178 of SEQ ID NO: 1015, and the B
chain consists of residues 179-1022; and the A and B chains are
linked via a disulfide bridge between C168 and C299 of SEQ ID
NO:1015.
57. The fusion protein of claim 40 that comprises a multimerization
domain, and that is a dimer via interaction of complementary
multimerization domains.
58. The fusion protein of claim 57, wherein the multimerization
domain is an F.sub.c domain.
59. The fusion protein of claim 57, wherein the modified u-PA
polypeptide comprises the replacement C122S.
60. A nucleic acid molecule, comprising a sequence of nucleotides
encoding a modified u-PA polypeptide of claim 1 or a fusion protein
comprising the modified u-PA polypeptide of claim 1.
61. A vector, comprising the nucleic acid molecule of claim 60.
62. A method of treating a disease or condition mediated by or
involving complement activation or reducing the risk of developing
the disease or condition, comprising administering the modified
u-PA polypeptide of claim 1, or a fusion protein comprising the
modified u-PA polypeptide, or nucleic acid encoding the modified
u-PA polypeptide or fusion protein, to a subject with the condition
or disease.
63. The method of claim 62, wherein the modified u-PA polypeptide
or fusion protein comprises a protease domain having the sequence
of amino acids set forth in SEQ ID NO:21 or SEQ ID NO:987, or the
nucleic acid encodes a modified u-PA polypeptide or fusion protein
comprising the sequence of amino acids set forth in SEQ ID NO:21 or
SEQ ID NO:987.
64. The method of claim 62, wherein the complement-mediated disease
or condition is selected from among inflammatory diseases and
conditions.
65. The method of claim 62, wherein the complement-mediated disease
or condition is selected from among: Complement 3 Glomerulopathy
(C3G), atypical hemolytic uremic syndrome (aHUS), sepsis,
rheumatoid arthritis (RA), a cardiovascular disease,
membranoproliferative diseases and conditions, ophthalmic or ocular
diseases or disorders, 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), transplanted
organ rejection, and ischemia-reperfusion injury.
66. The method of claim 65, wherein the disease or condition is an
ocular or ophthalmic disease or is rejection or inflammation due to
a transplanted organ.
67. The method of claim 65, wherein the disease or condition is a
diabetic retinopathy or age-related macular degeneration (AMD).
68. An isolated cell or a cell culture, comprising the nucleic acid
of claim 60, wherein the isolated cell is not a human zygote.
69. A method of producing a modified u-PA polypeptide or fusion
protein comprising the modified u-PA polypeptide, comprising
culturing the cell or cell culture of claim 68 under conditions for
expression of the encoded modified u-PA polypeptide or fusion
protein.
70. A pharmaceutical composition, comprising a modified u-PA
polypeptide of claim 1, or a fusion protein comprising the a
modified u-PA polypeptide of claim 1, or nucleic acid encoding the
modified u-PA polypeptide or fusion protein.
71. The pharmaceutical composition of claim 70, wherein the
modified u-PA polypeptide or fusion protein is in a two-chain
activated form.
72. The pharmaceutical composition of claim 71, wherein the
modified u-PA polypeptide or fusion protein comprises a protease
domain having the sequence of amino acids set forth in SEQ ID NO:21
or SEQ ID NO:987, or the nucleic acid encodes a modified u-PA
polypeptide or fusion protein comprising the sequence of amino
acids set forth in SEQ ID NO:21 or SEQ ID NO:987.
73. The pharmaceutical composition of claim 70, wherein the
modified u-PA polypeptide or fusion protein has the sequence set
forth in SEQ ID NO: 1015 or 1019.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of International PCT
application No. PCT/US2019/068839, entitled "MODIFIED
UROKINASE-TYPE PLASMINOGEN ACTIVATOR POLYPEPTIDES AND METHODS OF
USE," filed Dec. 27, 2019, to inventors Edwin L. Madison,
Christopher Thanos, Vanessa Soros, Mikhail Popkov, Kimberly Tipton,
Matthew John Traylor, Eric Steven Furfine, and Jeffrey Charles Way,
and applicant Catalyst Biosciences, Inc. International PCT
application No. PCT/US2019/068839 claims priority, and benefit of
priority to U.S. provisional application Ser. No. 62/786,302.
[0002] Benefit of priority is claimed to U.S. provisional
application Ser. No. 62/786,302, entitled "MODIFIED UROKINASE-TYPE
PLASMINOGEN ACTIVATOR POLYPEPTIDES AND METHODS OF USE," filed Dec.
28, 2018, to inventors Edwin L. Madison, Christopher Thanos,
Vanessa Soros, Mikhail Popkov, and Kimberly Tipton, and applicant
Catalyst Biosciences, Inc.
[0003] Where permitted, the subject matter of each of these
applications is incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED
ELECTRONICALLY
[0004] An electronic version of the Sequence Listing is filed
herewith, the contents of which are incorporated by reference in
their entirety. The electronic file was created on Dec. 27, 2019,
is 2,294 kilobytes in size, and is titled 4940seq001.txt. A
substitute Sequence Listing is filed electronically herewith, the
contents of which are incorporated by reference in their entirety.
The electronic file was created on Jan. 17, 2020, is 2,294
kilobytes in size, and is titled 4940SEQ002.txt.
FIELD OF THE INVENTION
[0005] Provided are modified u-PA polypeptides and fusion proteins
that cleave a complement protein, thereby, inhibiting complement
activation. By virtue of this inhibition the modified u-PA
polypeptides and fusion proteins can be used for treatment of
diseases and conditions mediated by complement or in which
complement activation plays a role. These diseases and conditions,
include, but are not limited to, ophthalmic indications, including
macular degeneration, such as age-related macular degeneration
(AMD) and Stargardt disease, renal delayed graft function (DGF),
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).
Complement typically is activated or triggered by 1 of these 3
pathways, which, as shown in FIG. 1, converge at C3 activation. In
a fourth complement-activation mechanism, referred to as the
intrinsic pathway, serine proteases associated with the
coagulation/fibrinolytic cascade activate the complement system
directly through cleavage of C3 or C5, independently of the
classical, alternate, and lectin pathways.
[0008] 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.
[0009] 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 can be a facet
of, or can occur in a variety of disorders that involve
inappropriate inflammatory and immune responses, such as those
observed in acute and chronic inflammatory diseases and other
conditions involving an inappropriate immune response. 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.
[0010] 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 can 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 a 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 objectives herein, it
is an objective to provide such therapeutics to target the
activation of the complement cascade and to provide therapeutics
and methods of treatment of diseases.
SUMMARY
[0011] Provided are modified urokinase-type plasminogen activator
(u-PA) polypeptides that include insertions, deletions and/or
replacements of amino acids in the protease domain that result in
increased cleavage activity on the complement protein C3 compared
to wild-type u-PA protease domain (where the protease domain can
include the replacement of the free Cys with Ser to
reduce/eliminate aggregation). The modified u-PA polypeptides and
fusion proteins are any that comprise the protease domain, such as
full length activated protease, zymogen forms thereof, and fusion
proteins the contain a modified u-PA polypeptide and a fusion
partner that confers pharmacological property or activity. The
modified u-PA polypeptides and fusion proteins containing the
modified u-PA polypeptides, when in active form, inhibit complement
activation. In particular these polypeptides and fusion proteins
cleave C3 whereby C3 activity is inhibited or eliminated.
[0012] Modifications, including amino acid deletions, replacements
and insertions, provided herein are in the protease domain. The
modified u-PA polypeptides (and fusion proteins) include or are the
protease domains. The modified u-PA polypeptides further can
include post-translational and other modifications to other than
the primary amino acid sequence, such as conjugation or linkage to
other polypeptides and moieties that alter properties, such as
serum half-life, and resistance to endogenous protease. Such
modifications include, but are not limited to, linkage to albumin,
linkage to multimerization domain(s), and PEGylation. Thus,
modified u-PA polypeptides, can be modified by PEGylation,
albumination, farnysylation, carboxylation, hydroxylation,
phosphorylation, and other polypeptide modifications known in the
art. Among the modifications is the replacement of a free cysteine,
in the zymogen, such as C122, by chymotrypsin numbering, with
serine or alanine, to reduce aggregation, particularly upon
expression in vitro. This replacement is optional, and not
necessarily included in polypeptides that to be pegylated or
expressed in vivo.
[0013] The modified u-PA polypeptides and fusion proteins
inactivate complement protein C3 by cleavage, thereby reducing,
inhibiting or preventing complement activation. The modified u-PA
polypeptides (and fusion proteins) cleave C3 to thereby inhibit
complement activation. They cleave C3 at a site, such as in the
active site of C3, that inactivates or inhibits C3 activity to
thereby inhibit complement activation. The modified u-PA
polypeptides provided herein were selected and designed to cleave
within QHARASHLG, and in particular where P1-P1' is RA
(QHAR.dwnarw.ASHL; see SEQ ID NO:47 residues 737-744, where
cleavage is between residues 740 and 741). As a result, these
modified u-PA polypeptides can be used as therapeutics for treating
disorders, diseases and/or conditions in which complement
activation plays a role such that inhibition thereof can treat the
disorders, diseases and/or conditions. The modified u-PA
polypeptides also can have reduced activity for a native substrate,
such as plasminogen, compared to a wild-type u-PA or compared to
one that just has the replacement corresponding to C122S, by
chymotrypsin numbering.
[0014] Among the diseases and conditions for which the modified
u-PA polypeptides and fusion proteins are used for treatment are
any C3-mediated or complement mediated or involved disease and
conditions. These include ophthalmic disorders, such as age-related
macular degeneration (AMD) and diabetic retinopathies, and organ
rejection, such as renal Delayed Graft Function (DGF) as well as
other diseases, disorders and conditions that can be treated by
inhibiting complement activation. AMD is treated by administration
to the vitreous humor, such as by intravitreal injection or
intraretinal or subretinal injection, and DGF is treated by
intravenous or other systemic administration. The modified u-PA
polypeptides and fusion proteins further can be modified, such as
by PEGylation, to enhance or improve or impart desirable
pharmacological properties, including increased half-life and/or
decreased immunogenicity. Other diseases and conditions include,
for example, Rheumatoid arthritis (RA), ocular diseases,
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, atypical
hemolytic uremic syndrome (aHUS), and Complement 3 Glomerulopathy
(C3G).
[0015] The unmodified u-PA polypeptides include precursor forms,
mature forms, the catalytic domain, and catalytically active forms
thereof, and also fusion proteins, such as those described in
Examples 14-16. Exemplary of the unmodified u-PA polypeptides are
those whose sequences are set forth in SEQ ID NOs: 1-6. Included
among the unmodified u-PA polypeptides are those in which the free
cysteine in the catalytic domain (corresponding to C122 by
chymotrypsin numbering) is replaced by another amino acid, such as
S or A, particularly S, which does not alter catalytic activity,
but decreases aggregation of the polypeptides. It is understood
that all modified u-PA polypeptides can include a replacement,
generally S, at the residue corresponding to C122 by chymotrypsin
numbering.
[0016] Among the modified urokinase-type plasminogen activator
(u-PA) polypeptides provided herein are those that contain one or
more amino acid modifications selected from among replacements
corresponding to R35Q, H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI,
and H99Q, and conservative amino acid modifications therefor,
whereby the modified u-PA polypeptide has increased
activity/specificity for a complement protein compared to the
unmodified active form of the u-PA polypeptide, where: the amino
acid modifications are selected from among replacements, insertions
and deletions; corresponding residues can be determined by
alignment with the mature form of u-PA; the modified u-PA
polypeptide cleaves a complement protein to thereby inhibit or
reduce complement activation compared to the unmodified u-PA
polypeptide that does not contain the amino acid modifications;
residues are numbered by chymotrypsin numbering; the unmodified
u-PA polypeptide comprises the sequence set forth in any of SEQ ID
NOs: 1-6 (wild-type human full-length u-PA, wild-type protease
(catalytic) domain u-PA, wild-type mature u-PA, full-length u-PA
with the replacement corresponding to C122S, protease domain u-PA
with the replacement corresponding to C122S, and mature u-PA with
the replacement corresponding to C122S) and catalytically active
fragment thereof that includes the amino acid replacement(s). The
conservative modifications are selected from among R35Y, W, F or N;
H37 R, Q, E, W or F, V41K, D60aS, T97aD, L or V, L97bG or S and
H99N, by chymotrypsin numbering.
[0017] In particular, among these modified urokinase-type
plasminogen activator (uPA) polypeptides are those containing one
or more amino acid modifications selected from among replacements
corresponding to R35Q, H37Y, V41R, V41L, Y40Q, D60aP, L97bA, T97aI,
and H99Q.
[0018] The modified u-PA polypeptides have reduced activity and/or
specificity for cleavage of a substrate sequence in plasminogen.
The complement protein for which the polypeptides have increased
specificity/activity is C3; cleavage inactivates C3. Exemplary of
cleavage sites is within the active site of C3. Among the modified
u-PA polypeptides are those that have increased activity for
cleavage of C3 that is least 3-fold greater than the unmodified
u-PA polypeptide of SEQ ID NO:5 (protease domain with the C122S
replacement).
[0019] The modified u-PA polypeptides include those that contain
the replacement H37Y, such as the replacements H37Y/V38E. The
modified u-PA polypeptides include those that contain the
replacements R35Y/H37K or R35Q/H37K, such as those that comprise
the replacements R35Y/H37K/V38E or R35Q/H37K/V38E.
[0020] Also provided are the modified u-PA polypeptides, including
those described above, that also contain the replacement L97bA
and/or R35Q, and or H99Q, and/or D60aP, and/or T97aI.
[0021] The modified u-PA polypeptide can further include the amino
acid replacement corresponding to T39Y, T39W, T39F, such as T39Y,
or conservative replacements selected from T39M or T39L. Others of
the modified u-PA polypeptides include or further include the amino
acid replacements R35Q/H37Y and/or V38E/V41R/Y149R.
[0022] Others of the modified u-PA polypeptides are those that
comprise the modification V41R, such as modified u-PA polypeptides
comprising the modifications V38E/V41R, including those that
further comprise a replacement at one or more of positions R35, H37
and V38. These include modified u-PA polypeptide in which the
replacement at V38 is E, such as for example, modified u-PA
polypeptides comprising R35Y/H37S/V38E/V41R, H37Y/V38E, and other
combinations of residues that contribute to cleavage of C3 and/or
stability, such as in a body fluid.
[0023] Among the modified u-PA polypeptides provided herein are
that have an ED.sub.50 for inactivation cleavage of C3 of less than
or 100 nM, or 50 nM or 30 nM or 25 nM in an in vitro assay.
Exemplary of these are those set forth in Table 14, where the
ED.sub.50 is 100 nM or less, or those set forth in Table 14, where
the ED.sub.50 is less than 50 nM, or those set forth in Table 14,
where the ED.sub.50 is less than 30 nM, or those set forth in Table
14, where the ED.sub.50 less than 25 nM. Exemplar of an assay to
assess ED.sub.50 is one that comprises incubation of the substrate
complement protein human C3 with various concentrations of each
modified protease for 1 hour at 37.degree. C. to determine the
ED.sub.50. In particular, the modified u-PA polypeptides are any
that cleave C3 with an ED.sub.50 of 50 nM or less.
[0024] The unmodified u-PA polypeptides can consist of the sequence
of amino acids set forth in any of SEQ ID NOs: 1-6 or can include
additional modifications, including additional insertions, and
deletions. Any of the replacements, insertions or deletions herein
can be included in the unmodified u-PA polypeptides, such as the
protease domain, particularly the protease domain of SEQ ID NO:5.
The modified u-PA polypeptide can have at least 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with
the polypeptides of any of SEQ ID NOs: 1-6 or a catalytically
active portion thereof. The modified u-PA polypeptides can contain
1 or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or
17 amino acid replacements, insertions or deletions, compared to
the unmodified u-PA polypeptide of any of SEQ ID NOs: 1-6 or a
catalytically active portion thereof.
[0025] Hence, provided are modified u-PA polypeptides that contain
the modification V41R, or H37Y, or L97bA, or R35Q, or H99Q, or
D60aP, or T97aI or combinations thereof. Any of the modified u-PA
polypeptides can further contain the amino acid replacement
corresponding to T39Y, T39W, T39F or conservative replacements
thereof selected from T39M or T39L. In particular, the modified
u-PA polypeptides can further contain the amino acid replacement
T39Y, such as the combination T39Y/V41R, and up to 12 or 13
additional modifications as well as the optional C122S. Any of the
modified u-PA polypeptides further can contain the amino acid
replacement V38E, and can further contain one or more of the amino
acid modifications R35Q, Y60bQ and/or Y149R. Any of the modified
u-PA polypeptides can further contain the amino acid modification
R37aE or R37aS. Hence, modified u-PA polypeptides provided herein
can contain the replacements R35Q/H37Y/T39Y/V41R or
R35Q/H37Y/T39Y/V41R/C122S. Any of the modified u-PA polypeptides
can contain the replacement corresponding to H99Q.
[0026] Among the modified u-PA polypeptides provided herein are
those that contain the amino acid modifications
R35Q/H37Y/T39Y/V41R/L97bA/H99Q/C122S or
R35Q/H37Y/T39Y/V41R/L97bA/H99Q, or T39Y/V41R/Y60bQ/L97bA/H99Q or
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S or
T39Y/V41R/D60aP/L97bA/H99Q/C122S or
T39Y/V41R/D60aP/L97bA/H99Q/C122S. Also among the modified u-PA
polypeptides provided herein are those that contain the amino acid
modifications corresponding to Y40Q/V41L/L97bA/C122S or
Y40Q/V41R/L97bA/C122S or Y40Q/V41L/L97bA or Y40Q/V41R/L97bA or
R37aS/V41R/L97bG/H99Q or R37aS/V41R/L97bG/H99Q/C122S or
T39Y/V41L/L97bA/H99Q/C122S or T39Y/V41R/L97bA/H99Q/C122S.
[0027] Included among the modified u-PA polypeptides are those that
contain the modifications:
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R.
[0028] Provided are modified u-PA polypeptides that contain the
amino acid modifications, included are polypeptides with the
modifications:
H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/H99Q/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/C122S/Y149R;
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aA/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R
or
R35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R
or
R35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37G/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R
or
R35A/H37G/R37aE/V38E/T39F/V41R/D60aE/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37S/R37aE/V38E/T39Y/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37T/R37aP/V38E/T39Y/V41R/D60aE/Y60bD/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37G/R37aE/V38E/T39H/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/Y149R
or
R35W/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122S/Y149R
or
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/H99L/C122-
S or
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q1-
92A or
R35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y151L/Q1-
92T or
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q1-
92T or
[0029] each with no replacement at C122. Exemplary of these
modified u-PA polypeptides are those that contain the
modifications
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R.
[0030] Exemplary of these polypeptides are those whose sequences
are set forth in any of SEQ ID NOs:8-44 and 987, such as 21 and
39-44 as well as precursor and full-length modified u-PA
polypeptides that contain the polypeptides whose sequences are set
forth in SEQ ID NOs:8-44 and catalytically active portions thereof.
It also is understood that in any of the modified u-PA polypeptides
provided herein the Cys at residue 122, by chymotrypsin numbering,
can be substituted with Ser, or can remain Cys. The Cys is retained
for embodiments in which the polypeptide, including fusion
proteins, containing the modified u-PA protease domain is intended
for use as a two chain form in which the free C122 forms a
disulfide bond with another free Cys in the polypeptide, or the Cys
is modified, such as by PEGylation. In all embodiments described
herein, position 122 can be Cys or Ser. The skilled person can
select the appropriate residue depending upon the intended use.
[0031] The unmodified u-PA polypeptide comprises the protease
domain of any of SEQ ID NOs: 1-6, or a catalytically active portion
thereof, including or containing only the protease domain of SEQ ID
NO:2 or SEQ ID NO:5.
[0032] The modified u-PA polypeptide can contain additional
modifications, including post-translational modifications,
modifications that introduce or remove a glycosylation site,
modification, such as linkage or conjugation to a polymer, such as
a PEG to increase serum half-life and/or to reduce immunogenicity
or both. In particular, any and all of the modified u-PA
polypeptides described and provided herein can be PEGylated. Fusion
proteins containing the modified u-PA polypeptides provided herein,
such as fusion with an Fc domain, or a targeting agent specific for
a targeted cell or antigen also are provided.
[0033] Among the modified u-PA polypeptides and fusion proteins
provided herein are those that have stability of greater than 50%
or 80% after incubation in PBS, or in a body fluid, such as aqueous
humor or serum for 7 days. Also among the modified u-PA
polypeptides are those that, when in active form, have at least
100-fold decreased activity on plasmin compared to a corresponding
form of unmodified u-PA polypeptide.
[0034] Also among the modified u-PA polypeptides and fusion
proteins provided herein are those that have an ED.sub.50 for
inactivation cleavage of C3 of less than or 100 nM, or 50 nM or 30
nM or 25 nM or 15 nM or 10 nM in an in vitro assay, such as any
exemplified in the Examples herein. These include polypeptides that
contain or are the protease domains set forth in Table 14, which
lists numerous mutation strings and the ED.sub.50 for modified u-PA
polypeptide protease domains that exhibit the ED.sub.50 assessed as
described in Example 2. Modified u-PA polypeptides and fusion
polypeptides that have an ED.sub.50 of 100 nM or less, less than 50
nM, less than 30 nM, less than 25 nM, less than 15 nM, and less
than 10 nM, are among those that can be used as protease domains,
or in longer u-PA forms and/or in fusion proteins as described
herein.
[0035] Provided are conjugated proteins, including fusion proteins
containing a modified u-PA polypeptide or a catalytically active
portion of any of the modified u-PA polypeptides fused to a
non-protease polypeptide or a portion thereof. Non-protease
polypeptides such as those that include a multimerization domain,
such as an Fc domain, a polypeptide, such as albumin, that
increases serum stability, or a protein transduction domain (PTD)
are provided.
[0036] As discussed above, all of the modifications can be in the
unmodified polypeptides whose sequences are set forth in any of SEQ
ID NOs: 1-6 and catalytically active portions thereof. Included
among the polypeptides are those in which the unmodified
polypeptide has the sequence set forth in SEQ ID NO:5 (the protease
domain with the C122S replacement).
[0037] Also provided are fusion proteins that contain the modified
u-PA polypeptides provided herein and additional polypeptides, such
as serum albumin, multimerization domains, signal sequences and
other trafficking sequences and tags to facilitate expression and
isolation. The fusion proteins also can include activation
sequences to activate the u-PA portions. Active forms of the fusion
proteins are produced upon expression, and removal of signal
sequences, and any other processing and trafficking signals to
result in active fusion proteins that cleave C3. The active forms
of the fusion proteins include 2 chain activated forms and also
dimers, such as the those resulting from inclusion of a
multimerization domain.
[0038] Among the fusion proteins are those that contain a modified
u-PA polypeptide or a catalytically active portion of a modified
u-PA polypeptide, such as those in Table 14, that is fused to a
non-protease polypeptide or a portion thereof. The fusion proteins
also can include activation sequences, and, before processing,
signal sequences and other trafficking signals. Non protease
polypeptides, include, but are not limited to, any known to those
of skill in the art to confer a desirable pharmaceutical activity
or property, a multimerization domain, such as an Fc, a protein
transduction domain (PTD), a hyaluronic acid binding domain (HABD),
an antibody to target to a particular antigen. The fusion proteins
also can include activation sequences, such as a native u-PA
activation sequence or a furin activation sequence. Exemplary of
furin activation sequences are those that are or comprise
QSGQKTLRRRKR (SEQ ID NO:996) or QCGQKTLRRRKR (SEQ ID NO:995) or
QSGQKTLRRKR (SEQ ID NO: 1044) or a furin activation sequence having
at least 98% sequence identity thereto.
[0039] For example, fusion proteins that comprise any of the
modified u-PA polypeptides as described or provided herein, and
also include, prior to processing or activation, a signal sequence
and the modified u-PA polypeptide or catalytically active portion
thereof. Signal sequences to encode for secretion of the fusion
proteins include, for example, a signal sequence from 11-2, u-PA,
or IgG.kappa..
[0040] The fusion proteins can include a fusion partner, such as a
multimerization domain, or a polypeptide that increases serum
half-life, or one that confers another desirable pharmacological
property or activity. Exemplary of these are an albumin, or an Fc
domain, or a single chain antibody or other antigen binding
fragment of an antibody, or a hyaluronic acid binding domain
(HABD). Exemplary fusion partners include, but are not limited to,
Tumor Necrosis factor-Stimulated Gene-6 (TSG-6); HSA, IgG Fc, an
antibody or antigen binding fragment thereof, such as an anti-type
II collagen antibody scFv fragment or an anti-VEGFR antibody or
fragment thereof.
[0041] The fusion proteins also can include an activation sequence
so that the resulting fusion protein containing u-PA is in an
active form, such as a two chain form. Activation sequences can
contain or be modified to contain a cysteine, which can form a
disulfide bond with a free Cys, such as C122, in the modified u-PA
polypeptide, whereby, upon activation, the resulting activated
polypeptide comprises two chains. Exemplary activation sequences
are a u-PA activation sequence and a furin activation sequence, and
modified forms thereof, such an activation sequence that has the
sequence set forth in any of SEQ ID NOs:995-998, 1041, and 1044 or
a sequence having at least 98% or 99% sequence identity
thereto.
[0042] Exemplary fusion proteins are those that contain an
activation sequence, a modified u-PA polypeptide, and HSA, such as
any comprising the sequence of amino acids set forth in any of SEQ
ID NOs: 1014, 1015, 1016, 1019 and 1040 or a modified form thereof
having at least 95%, 96%, 97%, 98%, 99% sequence identity (and
containing the modifications in the sequence of the u-PA portion).
For use in methods of treatment, the fusion proteins generally do
not contain the signal sequence. For use in gene therapy methods,
the nucleic acid can encode the signal sequence.
[0043] Provided are such fusion proteins, such as those containing
the sequence of amino acids set forth in any of SEQ ID Nos:
1004-1019 and 1034-1040 or any having at least 95%, 96%, 97%, 98%,
99% sequence identity (and containing the modifications in the
sequence of the u-PA portion). Exemplary of fusion proteins are
those having the sequence of amino acids set forth in SEQ ID
NO:1015 or 1019. In particular, the signal sequence is removed
prior to use or upon expression in vivo or when produced in vitro.
These include those that are in two-chain activated form containing
an A chain and a B chain. For example, fusion proteins, where the B
chain starts at residues IIGG of the modified u-PA polypeptide and
ends at the C-terminus of the fusion protein, such as those
containing a modified u-PA polypeptide and HSA, those containing
the sequence of amino acids set forth in any of SEQ ID NOs:1005,
1011, 1014, 1015, and 1036, but lacking the signal sequence.
Exemplary of fusion proteins in activated form is a fusion protein
that contains an A chain of residues 21-178, and a B chain of
residues 179- to the C-terminus of the protein with a disulfide
linkage between residues 168-299. It is understood that these also
include fusion proteins having at least 95%, 96%, 97%, 98%, 99%
sequence identity (and containing the modifications in the sequence
of the u-PA portion). For example, provided is a fusion protein
containing an A chain and a B chain, where the A chain consists of
residues 21-178 of SEQ ID NO:1015, and B chain consists of residues
179-1022; and the A and B chains are linked via a disulfide bridge
between C168 and C299 of SEQ ID NO: 1015.
[0044] Other fusion proteins provided herein contain
multimerization domains such that, upon processing, they form
multimers, such as dimers that form via interaction of
complementary multimerization domains, such as Fc domains.
[0045] Also provided are combinations, which can be packaged as a
kit, that contain a first composition containing a modified u-PA
polypeptide, including, as in all embodiments, fusion proteins,
particularly those in activated form, or plurality thereof, and a
second composition containing a second agent or agents for treating
a complement-mediated disease or disorder. The second agent or
agents, for example, can be an anti-inflammatory agent(s) or
anticoagulant(s). Exemplary of such agents are an anti-inflammatory
agent(s) selected from among any one or more of a nonsteroidal
anti-inflammatory drug (NSAID), antimetabolite, corticosteroid,
analgesic, cytotoxic agent, pro-inflammatory cytokine inhibitor,
anti-inflammatory cytokines, B cell targeting agents, compounds
targeting T antigens, adhesion molecule blockers, chemokine
receptor antagonists, kinase inhibitors, PPAR-.gamma. (gamma)
ligands, complement inhibitors, heparin, warfarin, acenocoumarol,
phenindione, EDTA, citrate, oxalate, argatroban, lepirudin,
bivalirudin, and ximelagatran.
[0046] Provided are nucleic acid molecules that encode any of the
modified u-PA polypeptides and fusion proteins provided herein.
Also provided are vectors containing such nucleic acid molecules
and encoding the modified u-PA polypeptides. Vectors include
prokaryotic vectors, and eukaryotic vectors, including mammalian
and insect vectors, such as a baculovirus vector, yeast vectors,
such as Pichia and Saccharomyces, and viral vectors, such as a
herpes virus simplex vector, or a vaccinia virus vector, an AAV
vector, an adenoviral vector or a retroviral vector. The vectors
can be expression vectors for production of the modified u-PA
polypeptides and/or vectors, such as adenoviruses and AAV viruses,
particularly those with tropism for the tissue of interest, such as
liver or the eye, for gene therapy.
[0047] Provided are methods of producing the modified u-PA
polypeptides by growing a cell containing a vector or nucleic acid
encoding a modified u-PA polypeptide or fusion protein under
conditions in which the vector is expressed, and, optionally,
isolating or recovering the expressed modified u-PA
polypeptide.
[0048] Also provided are isolated cells and cell cultures that
contain the nucleic acid molecules or the vectors. The cells can be
non-human cells, or human cell cultures, but do not include any
zygotes or cells that develop into a human. Cells include mammalian
cells and bacterial cells, including, but not limited to, bacterial
cells, such as E. coli, CHO, Balb/3T3, HeLa, MT2, mouse NS0, BHK,
insect cells, yeast cells and other cells routinely used for
recombinant expression of polypeptides. Methods for producing the
modified u-PA polypeptide include growing the cells under
conditions whereby the encoded modified u-PA polypeptide is
expressed and optionally isolating or purifying the modified u-PA
polypeptide. Generally, the modified u-PA polypeptides and
conjugates thereof, such as fusion proteins, are produced in cells
that glycosylate the proteins. The isolated modified u-PA
polypeptides can be further modified, such as by PEGylation.
[0049] Also provided are pharmaceutical compositions containing the
modified u-PA polypeptides and fusion proteins and/or the nucleic
acids and/or the vectors. Provided are uses of the pharmaceutical
compositions, nucleic acids or modified u-PA polypeptides for
inhibiting complement activation to thereby treat a disease or
disorder mediated by complement activation or in which complement
activation plays a role in the etiology or underlying etiology of
the disease or disorder. In particular, provided are uses of the
nucleic acid molecules and/or vectors for gene therapy for treating
such diseases, disorders and conditions, mediated by or involving
complement activation, where inhibition of complement activation
effects treatment or amelioration of the disease or condition. Also
provided are methods of treating a disease or condition mediated by
or involving complement activation by administering the vectors or
administering the nucleic acid molecules. In particular, the
diseases, disorders and conditions are those in which inactivation
of C3 to thereby inhibit or reduce complement activation effects
treatment.
[0050] Complement mediated diseases, disorders or conditions or
diseases, disorders and conditions in which complement activation
plays a role in the etiology or underlying etiology, include, but
are not limited to, any inflammatory disorder, sepsis, rheumatoid
arthritis (RA), ocular or ophthalmic disease, cardiovascular
disorders, 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, atypical hemolytic uremic syndrome
(aHUS), Complement 3 Glomerulopathy (C3G), and organ transplant
rejection, particularly delayed organ transplant rejection.
Particular diseases and disorders include ocular or ophthalmic
disorders, such as a macular degeneration or a diabetic
retinopathy, or inflammation due to a transplanted organ. Included
among the diseases, disorders and conditions are age-related
macular degeneration (AMD) and delayed renal graft function
(DGF).
[0051] Methods of inhibiting complement activation are provided.
The methods are effected by contacting a modified u-PA polypeptide
with complement protein C3, whereby complement protein C3 is
cleaved such that complement activation is reduced or inhibited.
Contacting can be effected in vitro, but generally is in vivo, by
administering the modified u-PA polypeptide to a subject in whom
complement inactivation or reduction is desired. Administration can
be systemic, such as parenterally, including intravenously, or
locally, such as by contacting an affected tissue, such as the eye.
Administration to the eye includes by drops, by linking the
modified u-PA polypeptide to a protein transduction domain, or by
intravitreal injection, intraretinal, or subretinal injection, or
other such method. For diseases and conditions, such as DGF,
administration can be effected by intravenous administration. Other
methods include subcutaneous and transdermal administration.
[0052] The methods and uses include treatment of any disease,
disorder or condition where inhibition of complement activation
leads to a reduction of inflammatory symptoms associated with a
complement-mediated disease or disorder selected from among an
inflammatory disorder, a neurodegenerative disorder, an ophthalmic
disorder and a cardiovascular disorder. These include, but are not
limited to, inflammatory diseases, conditions and disorders,
sepsis, rheumatoid arthritis (RA), ocular disorders,
membranoproliferative glomerulonephritis (MPGN), multiple sclerosis
(MS), myasthenia gravis (MG), asthma, inflammatory bowel disease,
immune complex (IC)-mediated acute inflammatory tissue injury,
atypical hemolytic uremic syndrome (aHUS), complement 3
glomerulopathy (C3G), Alzheimer's Disease (AD), ophthalmic
disorders, such as AMD and diabetic retinopathies, and
ischemia-reperfusion injury. The ischemia-reperfusion injury can
involve or be caused by an event or treatment selected from among
myocardial infarct (MI), stroke, angioplasty, coronary artery
bypass graft, cardiopulmonary bypass (CPB), and hemodialysis or a
treatment of a subject. The treatment with the modified u-PA
polypeptide is effected prior to treatment of a subject. Treatments
include organ transplantation. The disease, disorder or condition
include ophthalmic conditions or is an ocular disease or is
rejection or inflammation due to a transplanted organ, such as a
diabetic retinopathy or a macular degeneration. In particular,
methods of treatment of age-related macular degeneration (AMD) are
provided, as are methods of treatment of delayed renal graft
function (DGF). Treatment can be effected intravenously or
subcutaneously or locally, such as by injection of the modified
u-PA polypeptide into the eye. Included is intravitreal or
intraretinal, subretinal, injection or linking the modified u-PA
polypeptide to a protein transduction domain to facilitate
transduction into the vitreous humor. The modified u-PA polypeptide
can be linked to or conjugated to moieties that effect targeting of
the polypeptide to a particular organ or tissue, or that increase
serum half-life or reduce immunogenicity, such as PEGylation and/or
linkage to an Fc domain or to an antibody or antigen-binding
portion thereof.
[0053] Hence, provided are methods for treating a subject with a
complement-mediated disorder or condition or one in which
complement activation plays a role in such disorder or condition,
by administering a modified u-PA polypeptide provided herein. Such
uses of the modified u-PA polypeptides and fusion proteins provided
herein also are provided. The modified u-PA polypeptides and fusion
proteins effect treatment or can be used for such treatment because
they cleave complement protein C3 to thereby inhibit or reduce
complement activation. Inhibition of complement activation leads to
a reduction of inflammatory symptoms associated with a
complement-mediated disorder, disease or condition that involves an
inflammatory response, leading to a reduction of inflammatory
symptoms associated with a complement-mediated disease, condition
or disorder selected from among an inflammatory disorder, a
neurodegenerative disorder and a cardiovascular disorder. These
include ophthalmic conditions, such as diabetic retinopathy and
macular degeneration, and also delayed organ rejection, such as
DGF.
[0054] Dosages for the uses and methods and single dosage
formulations are provided herein. A single dosage can be
empirically determined by the skilled medical practitioner, and
includes, for example, single dosages that are in the range from
0.1 mg to 1 mg for local administration, and 0.1 mg to 10, 15, 20,
30 mg or more for systemic, such as intravenous administration. The
particular dosage depends upon the particular disorder or disease
or condition, the subject treated, the stage of the disease, the
disorder or condition, the route of administration, the regimen and
other such parameters. Dosages can be repeated daily, every two,
three, four, five, six, or seven days, at least bi-weekly, at least
every two weeks, three weeks, four weeks or longer intervals. The
particular regimen and dosage depend, for example, upon the
disorder treated, the mode of administration, and particulars, such
as weight, of the subject. Determination thereof is within the
skill of the skilled medical practitioner.
[0055] Also provided are the methods, uses and combinations and
modified u-PA polypeptides and fusion proteins, where the modified
u-PA polypeptide comprises the modification V41R or V41L,
particularly V41R, such as V41I or R and V38E, and those containing
H37Y/V38E. Exemplary of such modified u-PA polypeptide are modified
u-PA polypeptides that contain the modifications Y40Q/V41R/L97bA or
Y40Q/V41L/L97BA or R37aS/V41R/L97bG/H99Q, or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R.
The modifications are in any unmodified u-PA polypeptide, including
those set forth in any of SEQ ID NOs: 1-6, and catalytically active
portions thereof that include the residue corresponding to V41.
Exemplary of such modified u-PA polypeptides are the modified u-PA
polypeptides that comprise the sequence of amino acid residues set
forth in in SEQ ID NO: 21 or 987 or in any of SEQ ID NOs:40-44, or
40-44 without the modification at C122, by chymotrypsin numbering,
and catalytically active portions thereof, and modified forms
thereof, such as PEGylated forms, and fusion proteins and modified
forms thereof.
[0056] Also provided are methods of treating disorders, such as
DGF, by intravenously administering a modified u-PA polypeptide or
fusion protein (in activated form) as described and provided
herein, including the modified u-PA polypeptides that comprises the
sequence of amino acid residues set forth in any of SEQ ID NOs:21
and 40-44, and modified forms thereof, such as PEGylated forms. A
single dosage can be empirically determined by the skilled medical
practitioner, and includes single dosages that are in the range
from 0.1 mg to 1 mg. The dosage depends upon the subject, the
severity or stage of the disease or disorder, such as DGF.
Treatment can be repeated a plurality of times, such as two, three
or four times a day, once a day, repeated every 1 day, 2 days, 3
days, 4 days, 5 days, 6 days, weekly, bi-monthly or monthly. The
modified u-PA polypeptide can be one that comprises the
replacements/insertions, by chymotrypsin numbering,
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R;
and by mature numbering R20Q/H22Y/R23E/V27E/T28Y/V30R/D50P/Y51Q/T91
I/L92A/H94Q/C121S/Y148R. Exemplary thereof is the modified u-PA
polypeptide that contains the protease domain set forth in SEQ ID
NO:21 or a catalytically active portion thereof, or the full-length
or precursor forms that contain the protease domain, and modified
forms thereof, such as PEGylated forms and fusion proteins.
Administration can be effected by any suitable method, including
intravenous, subcutaneous, transdermal, local, intramuscular, oral,
and other systemic administration routes. Generally the
administered form of the modified u-PA polypeptides provided herein
is an activated form, which generally, depending upon the
components of the protein (see, e.g., Example 15), is a two chain
form.
[0057] The methods as described herein as described above and
below, include methods of treating an ophthalmic disorder or ocular
disorder by administering any of the modified u-PA polypeptides,
and modified forms thereof, such as PEGylated forms and fusion
proteins, such as those containing a protein transduction domain,
provided herein to the eye. Ophthalmic disorders, diseases or
conditions, involving complement activation include diabetic
retinopathies and macular degeneration, such as AMD. The dosage is
as described above, and includes single dosages of 0.1 mg to 1 mg.
Modified u-PA polypeptides include those that contain the
replacements
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R,
Y40Q/V41L/L97bA/C122S or Y40Q/V41R/L97bA/C122S or Y40Q/V41L/L97bA
or Y40Q/V41R/L97bA, and those that contain the sequence of amino
acid residues set forth in any of SEQ ID NOs:21 and 40-44 and
catalytically active portions thereof, as well as modified forms
thereof. Treatment can be repeated a plurality of times, such as
once a day. Uses of the modified u-PA polypeptides and modified
forms thereof for treating AMD or DGF are provided. The modified
u-PA polypeptides include any described herein, including those
that contain the replacements
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R
or Y40Q/V41L/L97bA/C122S or Y40Q/V41R/L97bA/C122S or
Y40Q/V41L/L97bA or Y40Q/V41R/L97bA, and modified forms thereof that
are PEGylated or that are fusion proteins as described herein.
[0058] Also provided are combinations containing any of the
modified u-PA polypeptides or fusion protein comprising the
modified u-PA polypeptides or nucleic acid, including vectors,
encoding the modified u-PA polypeptides or fusion proteins; and a
second agent or agents for treating a complement-mediated disease
or disorder. For example, the second agent or agents can be an
anti-inflammatory agent(s) or anticoagulant(s), such as, but not
limited to, an anti-inflammatory agent(s) selected from among any
one or more of a nonsteroidal anti-inflammatory drug (NSAID),
antimetabolite, corticosteroid, analgesic, cytotoxic agent,
pro-inflammatory cytokine inhibitor, anti-inflammatory cytokine, B
cell targeting agent, compound targeting T antigens, adhesion
molecule blocker, chemokine receptor antagonist, kinase inhibitor,
PPAR-.gamma. (gamma) ligand, complement inhibitor, heparin,
warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate,
argatroban, lepirudin, bivalirudin, and ximelagatran.
[0059] Methods of treatment or prevention (reduction of the risk)
of a complement mediated disease or disorder by administering the
modified u-PA polypeptide, fusion protein, or nucleic acid,
pharmaceutical compositions, or combinations, using the
polypeptides, fusion proteins and nucleic acids for treatment or
prevention are provided.
[0060] Exemplary of the modified u-PA polypeptides for the
combinations, pharmaceutical compositions, methods, and uses are
those that comprise the modification(s) V41R or V41L, or those that
comprise the modifications V38E/V41R, or the modifications
Y40Q/V41R/L97bA or Y40Q/V41L/L97bA or R37aS/V41R/L97bG/H99Q, or the
modifications:
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R,
and optionally C122S. The unmodified u-PA polypeptide can be the
unmodified u-PA polypeptide comprises the sequence of amino acid
residues set forth in SEQ ID NO:2 or SEQ ID NO:5, SEQ ID NO:3, or
SEQ ID NO:6.
[0061] Provided are modified u-PA polypeptides and fusion proteins
that comprise the sequence of amino acid residues set forth in SEQ
ID NO: 21 or 987 or in any of SEQ ID Nos: 40-44, or 40-44,
including those without the modification at C122, by chymotrypsin
numbering, and nucleic acids encoding modified u-PA polypeptides
and fusion proteins that have these sequences, and polypeptides and
proteins that have at least 95% sequence identity thereto.
[0062] The methods of treatment include methods of treating delayed
graft function (DGF), atypical hemolytic uremic syndrome (aHUS),
Complement 3 Glomerulopathy (C3G), and age-related macular
degeneration (AMD). Dosage depends upon the particular disorder.
Administration can be systemic or local, such as, for treatment of
ophthalmic disorders, intravitreal or subretinal. Dosage for
ophthalmic diseases and disorders, can be, for example, 0.1 to 3
mg, or 0.1 to 2 mg, or 1 to 3 mg, or 1 to 10 mg. Treatment can be
repeated a plurality of times, such as at least every 2 days, 3
days, 4 days, 5 days, 6 days, weekly, bi-monthly, monthly, every
two months, every three months, or every four months, every 6
months, or longer intervals. The modified u-PA polypeptides and
fusion proteins and nucleic acids include any described or provided
or suggested herein that cleave C3. These include modified u-PA
polypeptides, and fusion proteins that comprise or encode the
modifications
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/Y149R,
or
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R,
such as any that comprise or encode the protease domain set forth
in SEQ ID NO:21 or 987 or a catalytically active portion
thereof.
[0063] Methods of making or producing the modified u-PA
polypeptides or fusions proteins are provided. The methods are
effected by culturing cells, such as mammalian cells and cell
cultures (not including human zygotes) under conditions, whereby
the encoded polypeptide or fusion protein is expressed, and
optionally isolating the polypeptide or fusion protein. The
polypeptide or fusion protein as isolated generally does not
include a signal protein or other trafficking signal, which is
removed by the cell. The modified u-PA polypeptide or fusion
protein can be in activated two chain form, or can be further
treated to produce a two chain activated form. Alternatively, the
fusion protein can be one that contains a multimerization domain so
that the fusion protein is a multimer, such as a dimer.
BRIEF DESCRIPTION OF THE FIGURES
[0064] 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). 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.
[0065] FIGS. 2A-2B are schematics of N-terminal u-PA fusion
proteins. FIG. 2A is a schematic of N-terminal u-PA fusion proteins
which contain the fusion partner (i.e., Fc)N-terminal to the u-PA
catalytic domain. An exemplary N-terminal fusion protein is set
forth in SEQ ID NO:1004, which contains human immunoglobulin light
chain kappa (.kappa.) signal sequence, Fc (Fusion partner), AGS
(linker), the u-PA activation sequence, and a modified u-PA
catalytic domain. FIG. 2B is a schematic of N-terminal wild-type
protein which does not contain a fusion partner. An exemplary
N-terminal wild-type protein is set forth in SEQ ID NO:1005, which
contains human immunoglobulin light chain kappa (.kappa.) signal
sequence, the N-terminus of u-PA, u-PA activation sequence, and a
modified u-PA catalytic domain.
[0066] FIGS. 3A-3C are schematics of C-terminal u-PA fusion
proteins. FIG. 3A is a schematic of C-terminal u-PA fusion proteins
which contain the fusion partner C-terminal to the u-PA catalytic
domain where the fusion protein lacks an activation sequence
N-terminal to the u-PA catalytic domain. An exemplary C-terminal
fusion protein is set forth in SEQ ID NO: 1006, which contains a
human IL2 Signal sequence (hIL2SP), a modified u-PA catalytic
domain, a linker, and Fc (Fusion partner). Another exemplary
C-terminal fusion protein is set forth in SEQ ID NO: 1007, which
contains a human IL2 Signal sequence (hIL2SP), a modified u-PA
catalytic domain, a linker, and HSA (human serum albumin as a
fusion partner). Another exemplary C-terminal fusion protein is set
forth in SEQ ID NO: 1008, which contains a human IL2 Signal
sequence (hIL2SP), a modified u-PA catalytic domain, a linker, and
a scFv that binds Collagen II (C2scFv) (Fusion partner). Another
exemplary C-terminal fusion protein is set forth in SEQ ID NO:
1009, which contains a human IL2 Signal sequence (hIL2SP), a
modified u-PA catalytic domain, a linker, and a HABD (hyaluronic
acid binding domain (Fusion partner). Another exemplary C-terminal
fusion protein is set forth in SEQ ID NO:1012, which contains a
human IL2 Signal sequence (hIL2SP), the wild-type u-PA catalytic
domain, a linker, and Fc (Fusion partner). Another exemplary
C-terminal fusion protein is set forth in SEQ ID NO:1013, which
contains a human IL2 Signal sequence (hIL2SP), the wild-type u-PA
catalytic domain, a linker, and HSA (Fusion partner). FIG. 3B is a
schematic of C-terminal u-PA fusion proteins which contain the
fusion partner (i.e., Fc or HSA)C-terminal to the u-PA catalytic
domain. An exemplary C-terminal fusion protein is set forth in SEQ
ID NO:1010, which contains a human immunoglobulin light chain kappa
(i) signal sequence, a furin activation sequence, a modified u-PA
catalytic domain, a linker, and Fc (Fusion partner). Another
exemplary C-terminal fusion protein is set forth in SEQ ID NO:
1016, which contains a human immunoglobulin light chain kappa
(.kappa.) signal sequence, a furin activation sequence, a modified
u-PA catalytic domain, a linker, and HSA (Fusion partner). FIG. 3C
is a schematic of u-PA fusion proteins which contain a fusion
partner (i.e., Fc or HSA)C-terminal to the u-PA catalytic domain
and a fusion partner (i.e., the wild-type N-terminus of
u-PA)N-terminal to the u-PA catalytic domain. An exemplary fusion
protein is set forth in SEQ ID NO:1011, which contains a human
immunoglobulin light chain kappa (.kappa.) signal sequence, the
u-PA N-terminal domain, a modified u-PA catalytic domain, a linker,
and Fc (Fusion partner). Another exemplary C-terminal fusion
protein is set forth in SEQ ID NO:1014, which contains a human
immunoglobulin light chain kappa (.kappa.) signal sequence, the
N-terminal region of u-PA, a furin activation sequence, a modified
u-PA catalytic domain, a linker, and HSA (Fusion partner). Another
exemplary C-terminal fusion protein is set forth in SEQ ID NO:1015,
which contains a human immunoglobulin light chain kappa (.kappa.)
signal sequence, the N-terminal region of u-PA, the u-PA activation
sequence, a modified u-PA catalytic domain, a linker, and HSA
(Fusion partner).
[0067] FIGS. 4A-4H are schematics of the activated forms of the
fusion proteins, where SPD refers to the Serine protease domain
(the modified u-PA polypeptide protease domains provided herein;
the u-PA N-terminus refers generally to residues 1-178 of u-PA or
any modified forms thereof. FIG. 4A is a schematic of the fusion
protein of SEQ ID NO: 1010, which contains an Fc domain at the
C-terminus of the u-PA protease domain (SEQ ID NO: 21) and a furin
activation sequence, where disulfide linkage between the Fc domains
to form a dimer. FIG. 4B is a schematic of the fusion protein of
SEQ ID NO: 1011, which contains an Fc domain at the C-terminus of
the u-PA protease domain (SEQ ID NO: 987), and the N-terminus of
u-PA and the u-PA activation sequence at the N-terminus of the
protein, where disulfide linkage between the Fc domains to form a
dimer. FIG. 4C is a schematic of the fusion protein set forth in
SEQ ID NO: 1036, which contains an Fc domain at the C-terminus of
the u-PA protease domain (SEQ ID NO: 987), and the N-terminus of
u-PA and a furin activation sequence at the N-terminus of the
fusion protein, where disulfide linkage between the Fc domains form
a dimer. FIG. 4D is a schematic of the fusion protein set forth in
SEQ ID NO: 1014, which contains HSA at the C-terminus of the u-PA
protease domain (SEQ ID NO: 987), and the N-terminus of u-PA and a
furin activation sequence at the N-terminus of the fusion protein.
FIG. 4E is a schematic of the fusion protein set forth in SEQ ID
NO: 1015, which contains HSA at the C-terminus of the u-PA protease
domain (SEQ ID NO: 987), and the N-terminus of u-PA and the u-PA
activation sequence at the N-terminus of the fusion protein. FIG.
4F is a schematic of the fusion protein set forth in SEQ ID NO:
1016, which contains HSA at the C-terminus of the u-PA protease
domain (SEQ ID NO: 21) and a furin activation sequence N-terminal
to the protease domain. FIG. 4G is a schematic of the fusion
protein set forth in SEQ ID NO: 1017, which contains HSA at the
C-terminus of the u-PA protease domain (SEQ ID NO: 21) and a SUMO
activation sequence N-terminal to the protease domain. FIG. 4H is a
schematic of the fusion protein set forth in SEQ ID NO: 1018, which
contains an Fc domain at the C-terminus of the u-PA protease domain
(SEQ ID NO: 21) and the N-terminus of u-PA and a SUMO activation
sequence N-terminal to the protease domain, where a disulfide
linkage between the Fc domains form a dimer.
DETAILED DESCRIPTION
[0068] Outline [0069] A. DEFINITIONS [0070] B. u-PA STRUCTURE AND
FUNCTION [0071] 1. Serine proteases [0072] 2. Structure [0073] 3.
Function/activity [0074] C. COMPLEMENT INHIBITION BY TARGETING C3
[0075] 1. Complement Protein C3 and its Role in Initiating
Complement [0076] a. Classical Pathway [0077] b. Alternative
Pathway [0078] c. Lectin Pathway [0079] d. Complement-mediated
effector functions [0080] i. Complement-mediated lysis: Membrane
[0081] Attack Complex [0082] ii. Inflammation [0083] iii.
Chemotaxis [0084] iv. Opsonization [0085] v. Activation of the
Humoral Immune Response [0086] 2. C3 Structure and Function [0087]
a. C3a [0088] b. C3b [0089] c. Inhibitors of C3b [0090] D. MODIFIED
U-PA POLYPEPTIDES THAT CLEAVE C3 [0091] 1. Exemplary modified u-PA
polypeptides [0092] 2. Additional Modifications [0093] a. Decreased
immunogenicity [0094] b. Fc domain [0095] c. Conjugation to
polymers [0096] d. Protein transduction domain [0097] E. ASSAYS TO
ASSESS OR MONITOR u-PA ACTIVITY ON COMPLEMENT-MEDIATED FUNCTIONS
[0098] 1. Methods for assessing effects of u-PA on complement
protein C3 activity [0099] a. Protein Detection [0100] i. SDS-PAGE
analysis [0101] ii. Enzyme Immunoassay [0102] iii. Radial
Immunodiffusion (RID) [0103] b. Hemolytic assays [0104] c. Methods
for determining cleavage sites [0105] 2. Methods for assessing wild
type u-PA activity [0106] a. Cleavage of plasminogen [0107] b.
Plasminogen Activation Assays [0108] c. u-PA-uPAR Binding Assays
[0109] d. C3 cleavage [0110] ACC-AGR+ELISA [0111] Assessing
specificity using peptide libraries [0112] 3. Specificity [0113] 4.
Disease Models [0114] F. METHODS OF PRODUCING NUCLEIC ACIDS
ENCODING MODIFIED U-PA POLYPEPTIDES THEREOF [0115] 1. Isolation or
Preparation of Nucleic Acids Encoding u-PA Polypeptides [0116] 2.
Generation of Mutant or Modified Nucleic Acids and Encoding
Polypeptides [0117] 3. Vectors and Cells [0118] 4. Expression
[0119] a. Prokaryotic Cells [0120] b. Yeast Cells [0121] c. Insects
and Insect Cells [0122] d. Mammalian Expression [0123] e. Plants
[0124] 5. Purification [0125] 6. Additional Modifications [0126] a.
PEGylation [0127] b. Fusion Proteins and other conjugates [0128] 7.
Nucleic acid molecules [0129] G. COMPOSITIONS, FORMULATIONS AND
DOSAGES [0130] 1. Administration of modified u-PA polypeptides
[0131] 2. Administration of nucleic acids encoding modified u-PA
polypeptides (gene therapy) [0132] H. THERAPEUTIC USES AND METHODS
OF TREATMENT [0133] 1. Disease mediated by Complement activation
[0134] a. Rheumatoid Arthritis [0135] b. Sepsis [0136] c. Multiple
Sclerosis [0137] d. Alzheimer's Disease [0138] e.
Ischemia-Reperfusion Injury [0139] f. Ocular disorders [0140]
Age-Related Macular Degeneration (AMD) [0141] g. Organ
transplantation and Delayed Graft Function (DGF) [0142] 2.
Therapeutic Uses [0143] a. Immune-mediated Inflammatory Disease
[0144] b. Neurodegenerative Disease [0145] c. Cardiovascular
Disease [0146] d. Age-Related Macular Degeneration (AMD) [0147] e.
Organ transplant [0148] Delayed Graft Function (DGF) [0149] 3.
Combination Therapies [0150] I. EXAMPLES
A. DEFINITIONS
[0151] 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, 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 is 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 is understood that such identifiers can
change and particular information on the internet can come and go,
but equivalent information is known and can be readily accessed,
such as by searching the internet and/or appropriate databases.
Reference thereto evidences the availability and public
dissemination of such information.
[0152] 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). In human C3,
cleavage by a C3 convertase occurs between residues R and S (see
residues 746-751 of SEQ ID NO: 47, cleavage between residues 748
and 749 in human C3) of C3:
TABLE-US-00001 P3 P2 P1 P1' P2' P3' Leu Ala Arg .dwnarw. Ser Asn
Leu
[0153] Typically, cleavage of a substrate in a biochemical pathway
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. 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 a C3 convertase
into C3a and C3b is an activation cleavage. In contrast, the
modified u-PA polypeptides provided herein effect inhibitory
cleavage of C3, such as by cleavage in the active site.
[0154] As used herein, an inhibitory cleavage or inactivation
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 or at least about 1%, 2%,
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% 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.
[0155] As used herein, "complement activation" refers to the
activation of complement pathways, for example complement
activation refers to an increase in the functions or activities of
any one or more of the complement pathways by a protease or an
increase in the activity of any of the proteins in the complement
pathway. Complement activation can lead to complement-mediated cell
lysis or can lead to cell or tissue destruction. Inappropriate
complement activation on host tissue plays an important role in the
pathology of many autoimmune and inflammatory diseases, and also is
responsible for or associated with many disease states associated
with bioincompatibility. It is understood that activation can mean
an increase in existing activity as well as the induction of a new
activity. A complement activation 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. In some embodiments, complement activation is
inhibited by a protease, such as a protease described herein, by
40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% or more compared to
the activity of complement in the absence of a protease.
[0156] 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 1%, 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% or more compared to the activity of complement
in the absence of a protease.
[0157] As used herein, a "complement protein" or a "complement
component" is a protein of the complement system that functions in
the host's defense against infections and in the inflammatory
process. Complement proteins include those that function in the
classical pathway, those that function in the alternative pathway,
and those that function in the lectin pathway. Among the complement
proteins are proteases that participate in the complement
pathways.
[0158] 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 iC3b and C3a-desArg. 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, C4 bp, MCP, DAF, CD59 (MIRL), clusterin and
HRF and allelic and species variants of any complement protein.
[0159] 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.
[0160] 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 C3 are
proteases which are themselves activated by protease cleavage such
that cleavage of C3 by the C3 convertase C4b2b generates the active
fragments C3a and C3b. 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 contains 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 is biologically active.
Activation of C3 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.
[0161] 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.
[0162] 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 termed iC3. In another example, anaphylatoxins including C3a,
C5a, and C4a can be desarginated by carboxypeptidase N into more
stable, less active forms.
[0163] As used herein, a "fragment" or "cleavage product" of a
complement protein is a region or segment 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 a complement cascade.
Generally, a fragment results from the proteolytic cleavage of a
native complement protein. For example, complement protein C3 is
enzymatically cleaved by a C3 convertase, resulting in two
fragments: C3a which constitutes the N-terminal portion of C3; and
C3b 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 of a complement protein.
[0164] 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 a 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.
[0165] As used herein, "anaphylatoxins" are cleavage effector
proteins that trigger degranulation of, or 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 include, for example, C3a, C4a
and C5a. Anaphylatoxins also indirectly mediate spasms of smooth
muscle cells (such as bronchospasms), increases in permeability of
blood capillaries, and chemotaxis.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] As used herein, "complement protein C3" or "C3" refers to
complement protein C3 of the complement system that functions in
the host defense against infections and in the inflammatory
process. Human complement protein C3 is a 1663 amino acid
single-chain pre-proprotein or zymogen set forth in SEQ ID NO:47
that that contains a 22 amino acid signal peptide (amino acids 1-22
of SEQ ID NO:47) and a tetra-arginine sequence (amino acids 678-671
of SEQ ID NO:47) that is removed by a furin-like enzyme resulting
in a mature two chain protein containing a beta chain (amino acids
23-667 of SEQ ID NO:47) and an alpha chain (amino acids 672-1663 of
SEQ ID NO:47) linked by a disulfide bond between residues C559 and
C816. Complement protein C3 is further activated by proteolytic
cleavage by a C3 convertase (C4b2b or C3bBb) between amino acids
748 and 749 of SEQ ID NO:47 generating the anaphylatoxin C3a and
the opsonin C3b.
[0170] As used herein, a "zymogen" refers to a protein 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 protein. Such precursors are generally larger, although not
necessarily larger, than the active form. With reference to u-PA or
complement protein C3, 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.
[0171] As used herein, a "proregion," "propeptide," or "pro
sequence," refers to a region or a segment of a protein 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.
[0172] 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. 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.
[0173] 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. An exemplary polypeptide domain
is a part of the polypeptide that can form an independently folded
structure within a polypeptide made up of one or more structural
motifs (e.g., combinations of alpha helices and/or beta strands
connected by loop regions) and/or that is recognized by a
particular functional activity, such as enzymatic activity,
dimerization or substrate-binding. A polypeptide can have one or
more, typically more than one, distinct domains. For example, the
polypeptide can have one or more structural domains and one or more
functional domains. A single polypeptide domain can be
distinguished based on structure and function. A domain can
encompass a contiguous linear sequence of amino acids.
Alternatively, a domain can encompass a plurality of non-contiguous
amino acid portions, which are non-contiguous along the linear
sequence of amino acids of the polypeptide. Typically, a
polypeptide contains a plurality of domains. For example, serine
proteases can be characterized based on the sequence of protease
domain(s). Those of skill in the art are familiar with polypeptide
domains and can identify them by virtue of structural and/or
functional homology with other such domains. For exemplification
herein, definitions are provided, but it is understood that it is
well within the skill in the art to recognize particular domains by
name. If needed, appropriate software can be employed to identify
domains.
[0174] As used herein, a "structural region" of a polypeptide is a
region of the polypeptide that contains at least one structural
domain.
[0175] 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.
[0176] As used herein, a "catalytically active portion" or
"catalytically active domain" of a protease, for example a u-PA
polypeptide, refers to the protease domain, or any fragment or
portion thereof that retains protease activity. For example, a
catalytically active portion of a u-PA polypeptide can be a u-PA
protease domain including an isolated single chain form of the
protease domain or an activated two-chain form. 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.
[0177] 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.
[0178] 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 optionally can, and generally
include additional non-protease-derived sequences of amino
acids.
[0179] As used herein, an "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, such as amino acids Gln His Ala Arg
Ala Ser His Leu (active site of C3; residues 737-744 of SEQ ID
NO:47) as well as amino acids that contribute to substrate sequence
recognition, such as amino acids that contribute to extended
substrate binding specificity. For example, cleavage in the active
site of C3 can inhibit its activity, such as:
TABLE-US-00002 Q H A R .dwnarw. A S H L (residues 737-744 of SEQ ID
NO: 47) P4 P3 P2 P1 .dwnarw.P1' P2' P3' P4'.
[0180] 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, 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. 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.
[0181] 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 complement protein C3, or any portion or
fragment thereof containing a cleavage sequence recognized by a
u-PA polypeptide. 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.
[0182] As used herein, "u-PA" or "uPA" or "u-PA polypeptide" refers
to any u-PA polypeptide including, but not limited to, a
recombinantly produced polypeptide, a synthetically produced
polypeptide and a u-PA polypeptide extracted or isolated from cells
or tissues including, but not limited to, liver and blood.
Alternative names that are used interchangeably for u-PA include
urokinase and urinary plasminogen activator and urokinase
plasminogen activator and urinary-type plasminogen activator and
urokinase-type plasminogen activator. u-PA includes related
polypeptides from different species including, but not limited to
animals of human and non-human origin. Human u-PA includes u-PA,
allelic variants, isoforms, synthetic molecules from nucleic acids,
protein isolated from human tissue and cells, and modified forms
thereof. Exemplary unmodified human u-PA polypeptides include, but
are not limited to, unmodified and wild-type native mature u-PA
polypeptides (SEQ ID NO:3), the unmodified and wild-type precursor
u-PA polypeptide that includes a propeptide and/or signal peptides
(such as the u-PA polypeptide set forth in SEQ ID NO:1) and the
protease domain (such as the u-PA protease domain set forth in SEQ
ID NO: 2). One of skill in the art would recognize that the
referenced positions of the mature u-PA polypeptide (SEQ ID NO:3)
differ by 20 amino acid residues when compared to the precursor
u-PA polypeptide (SEQ ID NO:1), which is the u-PA polypeptide
containing the signal peptide sequence. Thus, the first amino acid
residue of SEQ ID NO:3 "corresponds to" the twenty-first (21st)
amino acid residue of SEQ ID NO:1.
[0183] Recitation of "u-PA" encompasses the activated or two-chain
form of the u-PA polypeptide containing the N-terminal A chain
(amino acids 1-158 of SEQ ID NO:3) and the C-terminal B chain
(amino acids 159-411 of SEQ ID NO:3) linked by a disulfide bond
between residues 148C and 279C (corresponding to the mature u-PA
polypeptide set forth in SEQ ID NO:3). The two-chain form, or high
molecular weight (HMW) u-PA, is formed from a mature u-PA
polypeptide (e.g., that set forth in SEQ ID NO:3) by proteolytic
cleavage after amino acid residue Lys158 before residue Ile159.
Proteolytic cleavage can be carried out, for example, by plasmin,
kallikrein, cathepsin B, matriptase and nerve growth factor-.gamma.
(gamma). The u-PA polypeptides provided herein can be further
modified, such as by chemical modification or post-translational
modification. Such modifications include, but are not limited to,
glycosylation, pegylation, albumination, farnysylation,
carboxylation, hydroxylation, phosphorylation, and other
polypeptide modifications known in the art.
[0184] u-PA includes u-PA from any species, including human and
non-human species. u-PA polypeptides of non-human origin include,
but are not limited to, murine, canine, leporine, avian, bovine,
ovine, porcine and other primate u-PA polypeptides. Exemplary u-PA
polypeptides of non-human origin include, for example, mouse (Mus
musculus, SEQ ID NO:52), rat (Rattus norvegicus, SEQ ID NO:53), cow
(Bos taurus, SEQ ID NO:54), pig (Sus scrofa, SEQ ID NO:55), rabbit
(Oryctolagus cuniculus, SEQ ID NO:56), chicken (Gallus gallus, SEQ
ID NO:57), yellow baboon (Papio cynocephalus, SEQ ID NO:58),
Sumatran orangutan (Pongo abelii, SEQ ID NO:59), dog (Canis lupus,
SEQ ID NO:60), sheep (Ovis aries, SEQ ID NO:61), marmoset
(Callithrix jacchus, SEQ ID NO:62), rhesus monkey (Macaca mulatta,
SEQ ID NO:63), northern white-cheeked gibbon (Nomascus leucogenys,
SEQ ID NO:64) and chimpanzee (Pan troglodytes, SEQ ID NO:65).
[0185] Reference to u-PA polypeptides also includes precursor
polypeptides and mature u-PA polypeptides in single-chain or
two-chain forms, truncated forms thereof that have activity, the
isolated protease domain and includes allelic variants and species
variants, variants encoded by splice variants, and other variants,
including polypeptides that have at least or at least about 40%,
45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or more sequence identity to the precursor polypeptide set
forth in SEQ ID NO: 1 or the mature form thereof (SEQ ID NO:3) or
the protease domain thereof (SEQ ID NO: 2). u-PA polypeptides
include, but are not limited to, tissue-specific isoforms and
allelic variants thereof, synthetic molecules prepared by
translation of nucleic acids, proteins generated by chemical
synthesis, such as syntheses that include ligation of shorter
polypeptides, through recombinant methods, proteins isolated from
human and non-human tissue and cells, chimeric u-PA polypeptides
and modified forms thereof. u-PA polypeptides also include
fragments or portions of u-PA that are of sufficient length or
include appropriate regions to retain at least one activity (upon
activation if needed) of a full-length mature polypeptide. In one
example the portion of u-PA is the protease domain, such as, for
example, the protease domain set forth in SEQ ID NO: 2 which
corresponds to amino acids 179-431 of the u-PA sequence set forth
in SEQ ID NO: 1. u-PA polypeptides also include those that contain
chemical or posttranslational modifications and those that do not
contain chemical or posttranslational modifications. Such
modifications include, but are not limited to, pegylation,
albumination, glycosylation, farnysylation, carboxylation,
hydroxylation, phosphorylation, HESylation (half-life extension by
on coupling drug molecules to the biodegradable hydroxyethyl starch
(HES)), PASylation (conjugation via genetic fusion or chemical
coupling of pharmacologically active compounds, such as proteins,
peptides and low molecular weight drugs, with natively disordered
biosynthetic polymers made of the small L-amino acids Pro, Ala
and/or Ser), and other polypeptide modifications known in the
art.
[0186] As used herein, "u-PA protease" or "u-PA protease domain"
refers to any u-PA polypeptide including, but not limited to, a
recombinantly produced polypeptide, a synthetically produced
polypeptide and a u-PA polypeptide extracted or isolated from cells
or tissues including, but not limited to, liver and blood. u-PA
protease includes related polypeptides from different species
including, but not limited to animals of human and non-human
origin. A human u-PA protease or u-PA protease domain includes
u-PA, allelic variants, isoforms, synthetic molecules from nucleic
acids, protein isolated from human tissue and cells, and modified
forms thereof. Exemplary reference human u-PA protease domains
include, but are not limited to, unmodified and wild-type u-PA
protease domain (SEQ ID NO:2) and an alternate protease domain
(such as the u-PA protease domain set forth in SEQ ID NO: 5). One
of skill in the art would recognize that the referenced positions
of the u-PA protease domain (SEQ ID NO:2) differ by 178 amino acid
residues when compared to the mature u-PA polypeptide (SEQ ID
NO:1), which is the u-PA polypeptide containing the full length WT
sequence. Thus, the first amino acid residue of SEQ ID NO:2
"corresponds to" the one hundred seventy-ninth (179th) amino acid
residue of SEQ ID NO: 1.
[0187] As used herein, a "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 or
nucleotides, respectively. Methods of modifying a polypeptide are
routine to those of skill in the art, such as by using recombinant
DNA methodologies. There is a distinction between modifications to
the sequence of amino acids of polypeptide and modification of the
polypeptide. The former refers to insertions, deletions, and
replacements or substitutions of amino acids; the latter to
modifications of the polypeptide, such as post-translational
modifications, PEGylation, and other such modifications of proteins
to alter properties and/or activities.
[0188] As used herein, "substitution" or "replacement" refers to
the replacing of one or more nucleotides or amino acids in a
native, target, wild-type or other nucleic acid or polypeptide
sequence with an alternative nucleotide or amino acid, without
changing the length (as described in numbers of residues) of the
molecule. Thus, one or more substitutions in a molecule does not
change the number of amino acid residues or nucleotides of the
molecule. Amino acid replacements compared to a particular
polypeptide can be expressed in terms of the number of the amino
acid residue along the length of the polypeptide sequence. For
example, a modified polypeptide having a modification in the amino
acid at the 35.sup.th position of the amino acid sequence that is a
substitution/replacement of Arginine (Arg; R) with glutamine (Gln;
Q) can be expressed as R35Q, Arg35Gln, or 35Q. Simply R35 can be
used to indicate that the amino acid at the modified 35.sup.th
position is an arginine.
[0189] As used herein, a "modified u-PA" or "modified u-PA
polypeptide" refers to a u-PA protease that exhibits altered
activity, such as altered substrate specificity, compared to the
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 wild type u-PA such
that an activity, such as substrate specificity or selectivity, of
the u-PA protease for cleaving complement protein C3 is altered. A
modified u-PA can be a full-length u-PA protease, or can be a
portion thereof of a full length protease, such as the protease
domain of u-PA, as long as the modified u-PA protease contains
modifications in regions that alter the activity or substrate
specificity of the protease and the protease is proteolytically
active. A modified u-PA protease, or a modified u-PA protease
domain, also can include other modifications in regions that do not
impact on substrate specificity of the protease. Hence, a modified
u-PA polypeptide 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 wild type u-PA
polypeptide. A modified full-length u-PA polypeptide or a
catalytically active portion thereof or a protease domain thereof
of a modified u-PA polypeptide can include polypeptides that are
fusion proteins as long as the fusion protein possesses the target
specificity.
[0190] As used herein, chymotrypsin numbering refers to the amino
acid numbering of a mature chymotrypsin polypeptide of SEQ ID
NO:76. Alignment of a protease domain of another protease, such as,
for example, the protease domain of u-PA, can be made with
chymotrypsin. In such an instance, the amino acids of u-PA
polypeptide 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. The
corresponding chymotrypsin numbers of amino acid positions 159-411
of the u-PA polypeptide set forth in SEQ ID NO:3 are provided in
Table 1. The amino acid positions relative to the sequence set
forth in SEQ ID NO:3 are in normal font, the amino acid residues at
those positions are in bold, and the corresponding chymotrypsin
numbers are in italics. For example, upon alignment of the serine
protease domain of u-PA (SEQ ID NO:2) with mature chymotrypsin, the
isoleucine (I) at position 159 in u-PA is given the chymotrypsin
numbering of 116. Subsequent amino acids are numbered accordingly.
In one example, a phenylalanine (F) at amino acid position 173 of
mature u-PA (SEQ ID NO:3) corresponds to amino acid position F30
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 u-PA sequence
compared to chymotrypsin, are referred to for example as D60a, Y60b
or P60c. These residues correspond to D208, Y209 and P210,
respectively, by numbering relative to the mature u-PA sequence set
forth in SEQ ID NO:3.
TABLE-US-00003 TABLE 1 Chymotrypsin numbering of u-PA 159 160 161
162 163 164 165 166 167 168 169 170 171 172 173 I I G G E F T T I E
N Q P W F 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 174 175 176
177 178 179 180 181 182 183 184 185 186 187 188 A A I Y R R H R G G
S V T Y V 31 32 33 34 35 36 37 37A 37B 37C 37D 38 39 40 41 189 190
191 192 193 194 195 196 197 198 199 200 201 202 203 C G G S L I S P
C W V I S A T 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 204 205
206 207 208 209 210 211 212 213 214 215 216 217 218 H C F I D Y P K
K E D Y I V Y 57 58 59 60 60A 60B 60C 61 62 62A 63 64 65 66 67 219
220 221 222 223 224 225 226 227 228 229 230 231 232 233 L G R S R L
N S N T Q G E M K 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 234
235 236 237 238 239 240 241 242 243 244 245 246 247 248 F E V E N L
I L H K D Y S A D 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 249
250 251 252 253 254 255 256 257 258 259 260 261 262 263 T L A H H N
D I A L L K I R S 97A 97B 98 99 100 101 102 103 104 105 106 107 108
109 110 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278
K E G R C A Q P S R T I Q T I 110A 110B 110C 110D 111 112 113 114
115 116 117 118 119 120 121 279 280 281 282 283 284 285 286 287 288
289 290 291 292 293 C L P S M Y N D P Q F G T S C 122 123 124 125
126 127 128 129 130 131 132 133 134 135 136 294 295 296 297 298 299
300 301 302 303 304 305 306 307 308 E I T G F G K E N S T D Y L Y
137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 309 310
311 312 313 314 315 316 317 318 319 320 321 322 323 P E Q L K M T V
V K L I S H R 152 153 154 155 156 157 158 159 160 161 162 163 164
165 166 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338
E C Q Q P H Y Y G S E V T T K 167 168 169 170 170A 1708 171 172 173
174 175 176 177 178 179 339 340 341 342 343 344 345 346 347 348 349
350 351 352 353 M L C A A D P Q W K T D S C Q 180 181 182 183 184
185 185A 1858 186 187 188 189 190 191 192 354 355 356 357 358 359
360 361 362 363 364 365 366 367 368 G D S G G P L V CS L Q G R M
193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 369 370
371 372 373 374 375 376 377 378 379 380 381 382 383 T L T G I V S W
G R G C A L K 208 209 210 211 212 213 214 215 216 217 218 220 221
222 223 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398
D K P G V Y T R V S H F L P W 223A 224 225 226 227 228 229 230 231
232 233 234 235 236 237 399 400 401 402 403 404 405 406 407 408 409
410 411 I R S H T K E E N G L A L 238 239 240 241 242 243 244 245
246 247 248 249 250
[0191] As used herein, k.sub.cat measures the catalytic activity of
an enzyme; the units of k.sub.cat are seconds.sup.-1. The
reciprocal of k.sub.cat is the time required by an enzyme molecule
to "turn over" one substrate molecule; k.sub.cat measures the
number of substrate molecules turned over per enzyme molecule per
second. k.sub.cat is sometimes called the turnover number. In
enzymology, k.sub.cat (also referred to as turnover number) is the
maximum number of chemical conversions of substrate molecules per
second that a single catalytic site executes for a given enzyme. It
is the maximum rate of reaction (V.sub.max) when all the enzyme
catalytic sites are saturated with substrate.
[0192] As used herein, specificity for a target substrate refers to
a preference for cleavage of a target substrate by a protease
compared to another substrate, referred to as a non-target
substrate. Specificity is reflected in the 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.
k.sub.cat/K.sub.m is a measure of enzyme efficiency; a large value
of k.sub.cat (rapid turnover) or a small value of K.sub.m (high
affinity for substrate) makes k.sub.cat/K.sub.m large.
[0193] As used herein, a specificity constant for cleavage is
(k.sub.cat/K.sub.m), where 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
preference of a protease for one target substrate over another
substrate.
[0194] 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 ratio of specificity
constants.
[0195] 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 or more substrates.
For example, substrate specificity of a protease for competing
substrates or of competing proteases for a substrate can be
compared by comparing k.sub.cat/K.sub.m. For example, a protease
that has a specificity constant of 2.times.10.sup.6 M.sup.-1
sec.sup.-1 for a target substrate and 2.times.10.sup.4 M.sup.-1
sec.sup.-1 for a non-target substrate is more specific for the
target substrate. Using the specificity constants from above, the
protease has a substrate specificity ratio of 100 for the target
substrate.
[0196] 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.
[0197] As used herein, altered specificity refers to a change in
substrate specificity of a modified protease compared to a starting
wild type 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 wild type substrate of the protease
(herein referred to as a non-target substrate). Typically, modified
u-PA proteases provided herein exhibit increased substrate
specificity for complement protein C3 compared to the substrate
specificity of the wild type u-PA 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.
[0198] As used herein, "selectivity" can be used interchangeably
with specificity when referring to the ability of a protease to
choose and cleave one target substrate from among a mixture of
competing substrates. Increased selectivity of a protease for a
target substrate compared to any other one or more target
substrates can be determined, for example, by comparing the
specificity constants of cleavage of the target substrates by a
protease. For example, if a protease has a specificity constant of
cleavage of 2.times.10.sup.6 M.sup.-1 sec.sup.-1 for a target
substrate and 2.times.10.sup.4 M.sup.-1 sec.sup.-1 for any other
one of more substrates, the protease is more selective for the
target substrate.
[0199] As used herein, an "activity" or a "functional activity" of
a polypeptide, such as a protease, refers to any activity exhibited
by the polypeptide. Such activities can be empirically determined.
Exemplary activities include, but are not limited to, ability to
interact with a biomolecule, for example, through
substrate-binding, DNA binding, or dimerization, enzymatic
activity, for example, kinase activity or proteolytic activity. For
a protease (including protease fragments), activities include, but
are not limited to, the ability to specifically bind a particular
substrate, affinity and/or specificity of substrate-binding (e.g.,
high or low affinity and/or specificity), effector functions, such
as the ability to promote substrate (e.g. protein, i.e. C3)
inhibition, neutralization, cleavage or clearance, and in vivo
activities, such as the ability to promote protein cleavage or
clearance. Activity can be assessed in vitro or in vivo using
recognized assays, such as ELISA, flow cytometry, surface plasmon
resonance or equivalent assays to measure on- or off-rate,
immunohistochemistry and immunofluorescence histology and
microscopy, cell-based assays, and binding assays. For example, for
a protease, e.g. a modified u-PA protease, activities can be
assessed by measuring substrate protein cleavage, turnover,
residual activity, stability and/or levels in vitro and/or in vivo.
The results of such in vitro assays that indicate that a
polypeptide exhibits an activity can be correlated to activity of
the polypeptide in vivo, in which in vivo activity can be referred
to as therapeutic activity, or biological activity. Activity of a
modified polypeptide can be any level of percentage of activity of
the unmodified polypeptide, including, but not limited to, at or
about 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
100%, 200%, 300%, 400%, 500%, or more of activity compared to the
unmodified polypeptide. Assays to determine functionality or
activity of modified (or variant) proteases are well-known in the
art.
[0200] 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.
[0201] 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. Exemplary of assays for testing
activities of complement activity include hemolysis of red blood
cells, and detection of complement effector molecules such as by
ELISA or SDS-PAGE.
[0202] 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.
[0203] 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.
[0204] As used herein, "increased activity" with reference to a
modified u-PA polypeptide means that, when tested under the same
conditions, the modified u-PA polypeptide exhibits greater activity
compared to an unmodified u-PA polypeptide not containing the amino
acid replacement(s). For example, a modified u-PA polypeptide
exhibits at least or about at least 110%, 120%, 130%, 140%, 150%,
160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%,
800%, 900%, 1000% or more of the activity of the unmodified or
reference u-PA polypeptide.
[0205] As used herein, the term "the same," when used in reference
to antibody binding affinity, means that the EC.sub.50, association
constant (Ka) or dissociation constant (Kd) is within about 1 to
100 fold or 1 to 10 fold of that of the reference antibody (1-100
fold greater affinity or 1-100 fold less affinity, or any numerical
value or range or value within such ranges, than the reference
antibody).
[0206] As used herein, "binding activity" refers to characteristics
of a molecule, e.g., a polypeptide, relating to whether or not, and
how, it binds one or more binding partners. Binding activities
include the ability to bind the binding partner(s), the affinity
with which it binds to the binding partner (e.g., high affinity),
the strength of the bond with the binding partner and/or
specificity for binding with the binding partner.
[0207] As used herein, EC.sub.50, also called the apparent Kd, is
the concentration (e.g., nM) of protease, where 50% of the maximal
activity is observed on a fixed amount of substrate (e.g., the
concentration of modified u-PA polypeptide required to cleave
through 50% of the available hC3). Typically, EC.sub.50 values are
determined from sigmoidal dose-response curves, where the EC.sub.50
is the concentration at the inflection point. A high protease
affinity for its substrate correlates with a low EC.sub.50 value
and a low affinity corresponds to a high EC.sub.50 value. Affinity
constants can be determined by standard kinetic methodology for
protease reactions, for example, immunoassays, such as ELISA,
followed by curve-fitting analysis.
[0208] As used herein, "affinity constant" refers to an association
constant (Ka) used to measure the affinity or molecular binding
strength between a protease and a substrate. The higher the
affinity constant the greater the affinity of the protease for the
substrate. Affinity constants are expressed in units of reciprocal
molarity (i.e., M.sup.-1 and can be calculated from the rate
constant for the association-dissociation reaction as measured by
standard kinetic methodology for protease-substrate reactions
(e.g., immunoassays, surface plasmon resonance, or other kinetic
interaction assays known in the art). The binding affinity of a
protease also can be expressed as a dissociation constant, or Kd.
The dissociation constant is the reciprocal of the association
constant, Kd=1/Ka. Hence, an affinity constant also can be
represented by the Kd. Affinity constants can be determined by
standard kinetic methodology for protease reactions, for example,
immunoassays, surface plasmon resonance (SPR) (Rich and Myszka
(2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst.
123:1599), isothermal titration calorimetry (ITC) or other kinetic
interaction assays known in the art (see, e.g., Paul, ed.,
Fundamental Immunology, 2nd ed., Raven Press, New York, pages
332-336 (1989)). Instrumentation and methods for real time
detection and monitoring of binding rates are known and are
commercially available (e.g., BIAcore 2000, BIAcore AB, Uppsala,
Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem.
Soc. Trans. 27:335).
[0209] Methods for calculating affinity are well-known, such as
methods for determining EC.sub.50 values or methods for determining
association/dissociation constants, including those exemplified
herein. For example, with respect to EC.sub.50, high binding
affinity means that the protease specifically binds to a target
protein with an EC.sub.50 that is less than about 10 ng/mL, 9
ng/mL, 8 ng/mL, 7 ng/mL, 6 ng/mL, 5 ng/mL, 3 ng/mL, 2 ng/mL, 1
ng/mL or less. High binding affinity also can be characterized by
an equilibrium dissociation constant (Kd) of 10.sup.-6 M or lower,
such as 10.sup.-7 M, 10.sup.-8 M, 10.sup.-10 M, 10.sup.-11 M or
10.sup.-12 M or lower. In terms of equilibrium association constant
(Ka), high binding affinity is generally associated with Ka values
of greater than or equal to about 10.sup.6 M.sup.-1, greater than
or equal to about 10.sup.7 M.sup.-1, greater than or equal to about
10.sup.8 M.sup.-1, or greater than or equal to about 10.sup.9
M.sup.-1, 10.sup.10 M.sup.-1, 10.sup.11 M.sup.-1 or 10.sup.12
M.sup.-1. Affinity can be estimated empirically or affinities can
be determined comparatively, e.g., by comparing the affinity of two
or more antibodies for a particular antigen, for example, by
calculating pairwise ratios of the affinities of the antibodies
tested. For example, such affinities can be readily determined
using conventional techniques, such as by ELISA; equilibrium
dialysis; surface plasmon resonance; by radioimmunoassay using
radiolabeled target antigen; or by another method known to the
skilled artisan. The affinity data can be analyzed, for example, by
the method of Scatchard et al., Ann N. Y. Acad. Sci., 51:660 (1949)
or by curve fitting analysis, for example, using a 4 Parameter
Logistic nonlinear regression model using the equation:
y=((A-D)/(1+((x/C){circumflex over ( )}B)))+D, where A is the
minimum asymptote, B is the slope factor, C is the inflection point
(EC.sub.50), and D is the maximum asymptote.
[0210] As used herein, "ED.sub.50" is the dose (e.g., mg/kg or nM)
of a protease (e.g., a modified u-PA) that produces a specified
result (e.g., cleavage of the complement protein C3) in 50% of the
total population (e.g., total amount of C3 present in the
sample).
[0211] As used herein, the term "surface plasmon resonance" refers
to an optical phenomenon that allows for the analysis of real-time
interactions by detection of alterations in protein concentrations
within a biosensor matrix, for example, using the BIAcore system
(GE Healthcare Life Sciences).
[0212] 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 wild type or prominent sequence of a
human protein.
[0213] As used herein, the residues of naturally occurring a-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.
[0214] As used herein, non-naturally occurring amino acids refer to
amino acids that are not genetically encoded.
[0215] As used herein, "nucleic acid" refers to at least two linked
nucleotides or nucleotide derivatives, including a deoxyribonucleic
acid (DNA) and a ribonucleic acid (RNA) and analogs thereof, joined
together, typically by phosphodiester linkages. Also included in
the term "nucleic acid" are analogs of nucleic acids such as
peptide nucleic acid (PNA), phosphorothioate DNA, and other such
analogs and derivatives or combinations thereof. Nucleic acids also
include DNA and RNA derivatives containing, for example, a
nucleotide analog or a "backbone" bond other than a phosphodiester
bond, for example, a phosphotriester bond, a phosphoramidate bond,
a phosphorothioate bond, a thioester bond, or a peptide bond
(peptide nucleic acid). The term also includes, as equivalents,
derivatives, variants and analogs of either RNA or DNA made from
nucleotide analogs, single (sense or antisense) and double-stranded
nucleic acids. Deoxyribonucleotides include deoxyadenosine,
deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the
uracil base is uridine. 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 nucleotides long.
[0216] As used herein, an "isolated nucleic acid molecule" is one
which is separated from other nucleic acid molecules which are
present in the natural source of the nucleic acid molecule. An
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
Exemplary isolated nucleic acid molecules provided herein include
isolated nucleic acid molecules encoding a u-PA protease
provided.
[0217] As used herein, "synthetic," with reference to, for example,
a synthetic nucleic acid molecule or a synthetic gene or a
synthetic peptide refers to a nucleic acid molecule or polypeptide
molecule that is produced by recombinant methods and/or by chemical
synthesis methods.
[0218] As used herein, "polypeptide" refers to two or more amino
acids covalently joined. The terms "polypeptide" and "protein" are
used interchangeably herein.
[0219] As used herein, a "peptide" refers to a polypeptide that is
from 2 to about or 40 amino acids in length.
[0220] 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 2).
The nucleotides which occur in the various nucleic acid fragments
are designated with the standard single-letter designations used
routinely in the art.
[0221] 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
(Table 2), non-natural amino acids and amino acid analogs (i.e.,
amino acids where the .alpha.-carbon has a side chain). As used
herein, the amino acids, which occur in the various amino acid
sequences of polypeptides appearing herein, are identified
according to their well-known, three-letter or one-letter
abbreviations (see Table 2). The nucleotides, which occur in the
various nucleic acid molecules and fragments, are designated with
the standard single-letter designations used routinely in the
art.
[0222] As used herein, "amino acid residue" refers to an amino acid
formed upon chemical digestion (hydrolysis) of a polypeptide at its
peptide linkages. The amino acid residues described herein are
presumed to be in the "L" isomeric form. Residues in the "D"
isomeric form, which are so designated, can be substituted for any
L-amino acid residue as long as the desired functional property is
retained by the polypeptide. NH.sub.2 refers to the free amino
group present at the amino terminus of a polypeptide. COOH refers
to the free carboxy group present at the carboxyl terminus of a
polypeptide. In keeping with standard polypeptide nomenclature
described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted in
37 C.F.R. .sctn..sctn. 1.821-1.822, abbreviations for amino acid
residues are shown in Table 2:
TABLE-US-00004 TABLE 2 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 Asparagine B Asx Asn and/or Asp C Cys Cysteine X Xaa
Unknown or other
[0223] All sequences of amino acid residues represented herein by a
formula have a left to right orientation in the conventional
direction of amino-terminus to carboxyl-terminus. The phrase "amino
acid residue" includes the amino acids listed in the Table of
Correspondence (Table 2), modified, non-natural and unusual amino
acids. Furthermore, 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 or to an amino-terminal group such
as NH.sub.2 or to a carboxyl-terminal group such as COOH.
[0224] As used herein, "naturally occurring amino acids" refer to
the 20 L-amino acids that occur in polypeptides. 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.
[0225] 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-stereoisomers of amino
acids. Exemplary non-natural amino acids are known to those of
skill in the art, and include, but are not limited to, para-acetyl
Phenylalanine, para-azido Phenylalanine, 2-Aminoadipic acid (Aad),
3-Aminoadipic acid (bAad), .beta.-alanine/.beta.-Amino-propionic
acid (Bala), 2-Aminobutyric acid (Abu), 4-Aminobutyric
acid/piperidinic acid (4Abu), 6-Aminocaproic acid (Acp),
2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib),
3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm),
2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2'-Diaminopimelic
acid (Dpm), 2,3-Diaminopropionic acid (Dpr), N-Ethylglycine
(EtGly), N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl),
allo-Hydroxylysine (Ahyl), 3-Hydroxyproline (3Hyp),
4-Hydroxyproline (4Hyp), Isodesmosine (Ide), allo-Isoleucine
(Aile), N-Methylglycine, sarcosine (MeGly), N-Methylisoleucine
(Melle), 6-N-Methyllysine (MeLys), N-Methylvaline (MeVal),
Norvaline (Nva), Norleucine (Nle), and Ornithine (Orn). Exemplary
non-natural amino acids are described herein and are known to those
of skill in the art.
[0226] 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).
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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 is understood to be equivalent to the term base
pairs. Those skilled in the art understand 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 cannot be paired. Such
unpaired ends generally do not exceed 20 nucleotides in length.
[0231] As used herein, alignment of a sequence refers to the use of
homology to align two or more sequences 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 sequences. Related or variant
polypeptides or nucleic acid molecules can be aligned by any method
known to those of skill in the art. Such methods typically maximize
matches, and include methods, such as using manual alignments and
by using the numerous alignment programs available (e.g., BLASTP)
and others, known to those of skill in the art. By aligning the
sequences of polypeptides or nucleic acids, one skilled in the art
can identify analogous portions or positions, using conserved and
identical amino acid residues as guides. Further, one skilled in
the art also can employ conserved amino acid or nucleotide residues
as guides to find corresponding amino acid or nucleotide residues
between and among human and non-human sequences. Corresponding
positions also can be based on structural alignments, for example
by using computer simulated alignments of protein structure. In
other instances, corresponding regions can be identified. One
skilled in the art also can employ conserved amino acid residues as
guides to find corresponding amino acid residues between and among
human and non-human sequences.
[0232] As used herein, "sequence identity" refers to the number of
identical or similar amino acids or nucleotide bases in a
comparison between a test and a reference polypeptide or
polynucleotide. Sequence identity can be determined by sequence
alignment of nucleic acid or protein sequences to identify regions
of similarity or identity. For purposes herein, sequence identity
is generally determined by alignment to identify identical
residues. The alignment can be local or global. Matches, mismatches
and gaps can be identified between compared sequences. Gaps are
null amino acids or nucleotides inserted between the residues of
aligned sequences so that identical or similar characters are
aligned. Generally, there can be internal and terminal gaps.
Sequence identity can be determined by taking into account gaps as
the number of identical residues/length of the shortest
sequence.times.100. When using gap penalties, sequence identity can
be determined with no penalty for end gaps (e.g terminal gaps are
not penalized). Alternatively, sequence identity can be determined
without taking into account gaps as the number of identical
positions/length of the total aligned sequence.times.100.
[0233] As used herein, "at a position corresponding to," or
recitation that nucleotides or amino acid positions "correspond to"
nucleotides or amino acid positions in a disclosed sequence, such
as set forth in the Sequence listing, refers to nucleotides or
amino acid positions identified upon alignment with the disclosed
sequence to maximize identity using a standard alignment algorithm,
such as the GAP algorithm. For purposes herein, alignment of a u-PA
sequence is to the amino acid sequence of the protease domain of
human u-PA set forth in SEQ ID NO: 2 or 5, particularly a reference
human u-PA of SEQ ID NO:5. By aligning the sequences, one skilled
in the art can identify corresponding residues, for example, using
conserved and identical amino acid residues as guides. In general,
to identify corresponding positions, the sequences of amino acids
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; and Carillo et al. (1988)
SIAM J Applied Math 48:1073). Alternatively, the skilled person can
number the residues by chymotrypsin number, thereby identify
corresponding residues. For closely related sequences, a computer
algorithm is not needed; alignment can be done visually.
[0234] As used herein, a "global alignment" is an alignment that
aligns two sequences from beginning to end, aligning each letter in
each sequence only once. An alignment is produced, regardless of
whether or not there is similarity or identity between the
sequences. For example, 50% sequence identity based on "global
alignment" means that in an alignment of the full sequence of two
compared sequences each of 100 nucleotides in length, 50% of the
residues are the same. It is understood that global alignment also
can be used in determining sequence identity even when the length
of the aligned sequences is not the same. The differences in the
terminal ends of the sequences are taken into account in
determining sequence identity, unless the "no penalty for end gaps"
is selected. Generally, a global alignment is used on sequences
that share significant similarity over most of their length.
Exemplary algorithms for performing global alignment include the
Needleman-Wunsch algorithm (Needleman et al. (1970) J. Mol. Biol.
48: 443). Exemplary programs for performing global alignment are
publicly available and include the Global Sequence Alignment Tool
available at the National Center for Biotechnology Information
(NCBI) website (ncbi.nlm.nih.gov/), and the program available at
deepc2.psi.iastate.edu/aat/align/align.html.
[0235] As used herein, a "local alignment" is an alignment that
aligns two sequences, but only aligns those portions of the
sequences that share similarity or identity. Hence, a local
alignment determines if sub-segments of one sequence are present in
another sequence. If there is no similarity, no alignment is
returned. Local alignment algorithms include BLAST and
Smith-Waterman algorithm (Adv. Appl. Math. 2: 482 (1981)). For
example, 50% sequence identity based on "local alignment" means
that in an alignment of the full sequence of two compared sequences
of any length, a region of similarity or identity of 100
nucleotides in length has 50% of the residues that are the same in
the region of similarity or identity.
[0236] For purposes herein, sequence identity can be determined by
standard alignment algorithm programs used with default gap
penalties established by each supplier. 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. Whether any two nucleic acid molecules
have nucleotide sequences or any two polypeptides have amino acid
sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% "identical," or other similar variations reciting a percent
identity, can be determined using known computer algorithms based
on local or global alignment (see, e.g.,
wikipedia.org/wiki/Sequence_alignment software, providing links to
dozens of known and publicly available alignment databases and
programs). Generally, for purposes herein sequence identity is
determined using computer algorithms based on global alignment,
such as the Needleman-Wunsch Global Sequence Alignment tool
available from NCBI/BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi?
CMD=Web&Page_TYPE=BlastHome); LAlign (William Pearson
implementing the Huang and Miller algorithm (Adv. Appl. Math.
(1991) 12:337-357)); and program from Xiaoqui Huang available at
deepc2.psi.iastate. edu/aat/align/align.html. Generally, when
comparing nucleotide sequences herein, an alignment with penalty
for end gaps is used. Local alignment also can be used when the
sequences being compared are substantially the same length.
[0237] As used herein, the term "identity" represents a comparison
or alignment between a test and a reference polypeptide or
polynucleotide. In one non-limiting example, "at least 90%
identical to" refers to percent identities from 90% to 100%
relative to the reference polypeptide or polynucleotide. Identity
at a level of 90% or more is indicative of the fact that, assuming
for exemplification purposes a test and reference polypeptide or
polynucleotide length of 100 amino acids or nucleotides are
compared, no more than 10% (i.e., 10 out of 100) of amino acids or
nucleotides in the test polypeptide or polynucleotide differs from
that of the reference polypeptides. Similar comparisons can be made
between a test and reference polynucleotides. Such differences can
be represented as point mutations randomly distributed over the
entire length of an amino acid sequence 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 also can be due to deletions or truncations
of amino acid residues. Differences are defined as nucleic acid or
amino acid substitutions, insertions or deletions. Depending on the
length of the compared sequences, at the level of homologies or
identities above about 85-90%, the result can be independent of the
program and gap parameters set; such high levels of identity can be
assessed readily, often without relying on software.
[0238] As used herein, a disulfide bond (also called an S--S bond
or a disulfide bridge) is a single covalent bond derived from the
coupling of thiol groups. Disulfide bonds in proteins are formed
between the thiol groups of cysteine residues, and stabilize
interactions between polypeptide domains.
[0239] As used herein, "coupled" or "conjugated" means attached via
a covalent or noncovalent interaction. Conjugates provided herein,
contain a modified u-PA polypeptide protease domain (referred to as
a "SPD," see, e.g., FIG. 4), and all or portion of the remaining
u-PA polypeptide, linked directly or vial a linker to another
moiety, such as a polypeptide that confers a property, such as
increased serum half life (i.e., human serum albumin HSA), or
facilitates expression or purification (i.e., SUMO, his-SUMO,
TSG-6), or targets the protein to receptor, such as an antibody
that binds to a receptor. The polypeptide can be linked directly or
via a polypeptide linker, generally a short, about 4-20, amino
acids, such as combinations of Ser and Gly residues. Conjugates
that contain a polypeptide generally are fusion proteins.
Conjugates also include modified u-PA polypeptides in which amino
acid residues are linked to moieties, such as PEG moieties,
glycosylation moieties and other such moieties.
[0240] 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. The skilled
person understands 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.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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.
[0246] As used herein, the wild-type form of a polypeptide or
nucleic acid molecule is a form encoded by a gene or by a coding
sequence encoded by the gene. Typically, a wild-type form of a
gene, or molecule encoded thereby, does not contain mutations or
other modifications that alter function or structure. The term
wild-type also encompasses forms with allelic variation as occurs
among and between species. As used herein, a predominant form of a
polypeptide or nucleic acid molecule refers to a form of the
molecule that is the major form produced from a gene. A
"predominant form" varies from source to source. For example,
different cells or tissue types can produce different forms of
polypeptides, for example, by alternative splicing and/or by
alternative protein processing. In each cell or tissue type, a
different polypeptide can be a "predominant form."
[0247] 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 wild type 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 wild-type 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 or at least
about 80%, 85%, 90% or 95% identity or greater with a wild type
and/or predominant form, including at least or at least about 96%,
97%, 98%, 99% or greater identity with a wild-type and/or
predominant form of a polypeptide.
[0248] 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.
[0249] As used herein, species variants refer to variants in
polypeptides among different species, including different mammalian
species, such as mouse and human. Generally, species variants have
about or 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% or more sequence identity. Corresponding residues
between and among species variants can be determined by comparing
and aligning sequences to maximize the number of matching
nucleotides or residues, for example, such that identity between
the sequences is equal to or greater than 95%, equal to or greater
than 96%, equal to or greater than 97%, equal to or greater than
98% or equal to greater than 99%. The position of interest is then
given the number assigned in the reference nucleic acid molecule.
Alignment can be effected manually or by eye, particularly, where
sequence identity is greater than 80%.
[0250] 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.
[0251] As used herein, modification in reference to modification of
the primary 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. This in contrast to modifications of the polypeptide
itself, which include post-translational modifications, such as
glycosylation, farnysylation, pegylation, and fusions, such as
fusions with other polypeptides to change a property, such as serum
half-life, such as by albumination, fusion with albumin, such as
human serum albumin, and other such modifications to the
polypeptide. Thus reference to modifications of the sequence of
amino acids refers to insertions, deletions,
substitutions/replacements, and combinations thereof. Modification
of the polypeptide refers to modifications that are added to the
polypeptide that do not change the sequence thereof.
[0252] For purposes herein, amino acid substitutions, deletions
and/or insertions, can be made in any of u-PA polypeptide or
catalytically active fragment 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.
[0253] Amino acid substitutions contemplated include conservative
substitutions, such as those set forth in Table 3, 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.
[0254] As used herein, 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 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). Such substitutions
can be made in accordance with those set forth in Table 3 as
follows:
TABLE-US-00005 TABLE 3 Original residue Exemplary conservative
substitution Ala (A) Gly; Ser Arg (R) Lys 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 Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr
Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0255] Other substitutions also are permissible and can be
determined empirically or in accord with known conservative
substitutions.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] As used herein, "expression" refers to the process by which
polypeptides are produced by transcription and translation of
polynucleotides. The level of expression of a polypeptide can be
assessed using any method known in art, including, for example,
methods of determining the amount of the polypeptide produced from
the host cell. Such methods can include, but are not limited to,
quantitation of the polypeptide in the cell lysate by ELISA,
Coomassie blue staining following gel electrophoresis, Lowry
protein assay and Bradford protein assay.
[0262] As used herein, a "host cell" is a cell that is used to
receive, maintain, reproduce and/or amplify a vector. Host cells
also can be used to express the polypeptide encoded by the vector.
The nucleic acid contained in the vector is replicated when the
host cell divides, thereby amplifying the nucleic acids.
[0263] As used herein, a "vector" or "plasmid" is a replicable
nucleic acid from which one or more heterologous proteins can be
expressed when the vector is transformed into an appropriate host
cell. Reference to a vector includes discrete elements that are
used to introduce heterologous nucleic acid into cells for either
expression or replication thereof. Reference to a vector also
includes those vectors into which a nucleic acid encoding a
polypeptide or fragment thereof can be introduced, typically by
restriction digest and ligation. Reference to a vector also
includes those vectors that contain nucleic acid encoding a
protease, such as a modified u-PA. The vector is used to introduce
the nucleic acid encoding the polypeptide into the host cell for
amplification of the nucleic acid or for expression/display of the
polypeptide encoded by the nucleic acid. 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. A 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] As used herein, "operably linked" with reference to nucleic
acid sequences, regions, elements or domains means that the nucleic
acid regions are functionally related to each other. For example,
nucleic acid encoding a leader peptide can be operably linked to
nucleic acid encoding a polypeptide, whereby the nucleic acids can
be transcribed and translated to express a functional fusion
protein, where the leader peptide effects secretion of the fusion
polypeptide. In some instances, the nucleic acid encoding a first
polypeptide (e.g., a leader peptide) is operably linked to nucleic
acid encoding a second polypeptide and the nucleic acids are
transcribed as a single mRNA transcript, but translation of the
mRNA transcript can result in one of two polypeptides being
expressed. For example, an amber stop codon can be located between
the nucleic acid encoding the first polypeptide and the nucleic
acid encoding the second polypeptide, such that, when introduced
into a partial amber suppressor cell, the resulting single mRNA
transcript can be translated to produce either a fusion protein
containing the first and second polypeptides, or can be translated
to produce only the first polypeptide. In another example, a
promoter can be operably linked to nucleic acid encoding a
polypeptide, whereby the promoter regulates or mediates the
transcription of the nucleic acid.
[0268] As used herein, "primary sequence" refers to the sequence of
amino acid residues in a polypeptide or the sequence of nucleotides
in a nucleic acid molecule.
[0269] 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.
[0270] As used herein, a "tag" or an "epitope tag" refers to a
sequence of amino acids, typically added to the N- or C-terminus of
a polypeptide, such as a u-PA provided herein. The inclusion of
tags fused to a polypeptide can facilitate polypeptide purification
and/or detection. Typically, a tag or tag polypeptide refers to a
polypeptide that has enough residues to provide an epitope
recognized by an antibody or can serve for detection or
purification, yet is short enough such that it does not interfere
with activity of the polypeptide to which it is linked. The tag
polypeptide typically is sufficiently unique so that an antibody
that specifically binds thereto does not substantially cross-react
with epitopes in the polypeptide to which it is linked. Epitope
tagged proteins can be affinity purified using highly specific
antibodies raised against the tags.
[0271] Suitable tag polypeptides generally have at least 5 or 6
amino acid residues and usually between about 8-50 amino acid
residues, typically between 9-30 residues. The tags can be linked
to one or more proteins and permit detection of the protein or its
recovery from a sample or mixture. Such tags are well-known and can
be readily synthesized and designed. Exemplary tag polypeptides
include those used for affinity purification and include, Small
Ubiquitin-like Modifier (SUMO) tags, FLAG tags, His tags, the
influenza hemagglutinin (HA) tag polypeptide and its antibody
12CA5, (Field et al. (1988) Mol. Cell. Biol. 8:2159-2165); the
c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies
thereto (see, e.g., Evan et al. (1985) Molecular and Cellular
Biology 5:3610-3616); and the Herpes Simplex virus glycoprotein D
(gD) tag and its antibody (Paborsky et al. (1990) Protein
Engineering 3:547-553). An antibody used to detect an
epitope-tagged antibody is typically referred to herein as a
secondary antibody.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] 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 3, 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] As used herein, "linker" refers to short sequences of amino
acids that join two polypeptides (or nucleic acid encoding such
polypeptides). "Peptide linker" refers to the short sequence of
amino acids joining the two polypeptide sequences. Exemplary of
polypeptide linkers are linkers joining two antibody chains in a
synthetic antibody fragment such as an scFv fragment. Linkers are
well-known and any known linkers can be used in the provided
methods. Exemplary of polypeptide linkers are (Gly-Ser).sub.n amino
acid sequences, with some Glu or Lys residues dispersed throughout
to increase solubility. Other exemplary linkers are described
herein; any of these and other known linkers can be used with the
provided compositions and methods.
[0281] As used herein, derivative or analog of a molecule refers to
a portion derived from or a modified version of the molecule.
[0282] 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, conditions related to environmental
exposures and human behaviors, and conditions characterized by
identifiable symptoms. Diseases or disorders include clinically
diagnosed disease as well as disruptions in the normal state of the
organism that have not been diagnosed as clinical disease. 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 of interest herein include
those characterized by complement activation (e.g., age-related
macular degeneration and renal delayed graft function).
[0283] As used herein, macular degeneration occurs when the small
central portion of the retina, known as the macula, deteriorates.
There are two types of AMD: dry (atrophic) and wet (neovascular or
exudative). Most AMD starts as the dry type and in 10-20% of
individuals, it progresses to the wet type. Age-related macular
degeneration is always bilateral (i.e., occurs in both eyes), but
does not necessarily progress at the same pace in both eyes.
[0284] As used herein, age-related macular degeneration (AMD) is an
inflammatory disease that causes visual impairment and blindness in
older people. The proteins of the complement system are central to
the development of this disease. Local and systemic inflammation in
AMD are mediated by the deregulated action of the alternative
pathway of the complement system.
[0285] As used herein, delayed graft function (DGF) is a
manifestation of acute kidney injury (AKI) with attributes unique
to the transplant process. It occurs post-transplant surgery.
Delayed graft function (DGF) is a common complication frequently
defined as the need for dialysis during the first post transplant
week. Intrinsic renal synthesis of the third complement component
C3 (C3) contributes to acute rejection by priming a T-cell-mediated
response. For example, in brain dead donors, local renal C3 levels
are higher at procurement and inversely related to renal function
14 days after transplant.
[0286] As used herein, a complement-mediated disease or 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 a complement protein or complement-related protein or activation
or inactivation of a complement or complement-related protein. In
some embodiments, a complement-mediated disorder is one that is due
to a deficiency in a complement protein(s). In other embodiments as
described herein a complement-mediated disorder is one that is due
to activation or over-activation of a complement protein(s). A
complement-mediated disorder also is one that is due to the
presence of any one or more of the complement proteins and/or the
continued activation of the complement pathway.
[0287] As used herein, "macular degeneration-related disorder"
refers to any of a number of conditions in which the retinal macula
degenerates or becomes dysfunctional (e.g., as a consequence of
decreased growth of cells of the macula, increased death or
rearrangement of the cells of the macula (e.g., RPE cells), loss of
normal biological function, or a combination of these events).
Macular degeneration results in the loss of integrity of the
histoarchitecture of the cells and/or extracellular matrix of the
normal macula and/or the loss of function of the cells of the
macula. Examples of macular degeneration-related disorder include
age-related macular degeneration (AMD), geographic atrophy (GA),
North Carolina macular dystrophy, Sorsby's fundus dystrophy,
Stargardt's disease, pattern dystrophy, Best disease, dominant
drusen, and malattia leventinese (radial drusen). Macular
degeneration-related disorder also encompasses extramacular changes
that occur prior to, or following dysfunction and/or degeneration
of the macula. Thus, the term "macular degeneration-related
disorder" also broadly includes any condition which alters or
damages the integrity or function of the macula (e.g., damage to
the RPE or Bruch's membrane). For example, the term encompasses
retinal detachment, chorioretinal degenerations, retinal
degenerations, photoreceptor degenerations, RPE degenerations,
mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies
and cone degenerations.
[0288] A macular degeneration-related disorder described herein
includes macular degeneration, such as, for example, AMD macular
degeneration. A macular degeneration-related disorder includes
disorders treated by anti-VEGF treatment, such as, for example,
anti-VEGF antibodies, or laser treatment or an implantable
telescope.
[0289] 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 u-PA polypeptide
and compositions provided herein.
[0290] As used herein, "prevention" or "prophylaxis" refers to
methods in which the risk or probability of developing a disease or
condition is reduced.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] As used herein, a "pharmaceutically effective agent"
includes any therapeutic agent or bioactive agents, including, but
not limited to, for example, anesthetics, vasoconstrictors,
dispersing agents, and conventional therapeutic drugs, including
small molecule drugs and therapeutic proteins.
[0295] 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.
[0296] As used herein, a "therapeutically effective amount" or a
"therapeutically effective dose" refers to the quantity of an
agent, compound, material, or composition containing a compound
that is at least sufficient to produce a therapeutic effect
following administration to a subject. Hence, it is the quantity
necessary for preventing, curing, ameliorating, arresting or
partially arresting a symptom of a disease or disorder.
[0297] As used herein, a "therapeutic effect" means an effect
resulting from treatment of a subject that alters, typically
improves or ameliorates, the symptoms of a disease or condition or
that cures a disease or condition.
[0298] As used herein, a "prophylactically effective amount" or a
"prophylactically effective dose" refers to the quantity of an
agent, compound, material, or composition containing a compound
that when administered to a subject, have the intended prophylactic
effect, e.g., preventing or delaying the onset, or reoccurrence, of
disease or symptoms, reducing the likelihood of the onset, or
reoccurrence, of disease or symptoms, or reducing the incidence of
viral infection. The full prophylactic effect does not necessarily
occur by administration of one dose, and can occur only after
administration of a series of doses. Thus, a prophylactically
effective amount can be administered in one or more
administrations.
[0299] As used herein, "administration of a non-complement
protease", such as a modified u-PA 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, such as a modified u-PA
protease. For in vivo administration, the modified non-complement
protease, such as a modified u-PA 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.
[0300] 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.
[0301] 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 and
cows; and rodents such as mice, rats, hamsters and gerbils.
[0302] As used herein, a "combination" refers to any association
between or among two or more items. The association can be spatial
or refer to the use of the two or more items for a common purpose.
The combination can be two or more separate items, such as two
compositions or two collections, a mixture thereof, such as a
single mixture of the two or more items, or any variation thereof.
The elements of a combination are generally functionally associated
or related.
[0303] 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.
[0304] As used herein, a stabilizing agent refers to compound added
to the formulation to protect either the antibody or conjugate,
such as under the conditions (e.g. temperature) at which the
formulations herein are stored or used. Thus, included are agents
that prevent proteins from degradation from other components in the
compositions. Exemplary of such agents are amino acids, amino acid
derivatives, amines, sugars, polyols, salts and buffers,
surfactants, inhibitors or substrates and other agents as described
herein.
[0305] 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.
[0306] 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 a therapeutic agent with a modified
u-PA polypeptide or nucleic acid molecule contained in the same or
separate articles of packaging.
[0307] 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, such as a
modified u-PA protease provided herein, 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.
[0308] As used herein, a "cellular extract" refers to a preparation
or fraction which is made from a lysed or disrupted cell.
[0309] 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; porcine, 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.
[0310] As used herein, a "single dosage" formulation refers to a
formulation containing a single dose of therapeutic agent for
direct administration. Single dosage formulations generally do not
contain any preservatives.
[0311] As used herein, a multi-dose formulation refers to a
formulation that contains multiple doses of a therapeutic agent and
that can be directly administered to provide several single doses
of the therapeutic agent. The doses can be administered over the
course of minutes, hours, weeks, days or months. Multi-dose
formulations can allow dose adjustment, dose-pooling and/or
dose-splitting. Because multi-dose formulations are used over time,
they generally contain one or more preservatives to prevent
microbial growth.
[0312] As used herein, a "control" or "standard" 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. For
example, a control can be a sample, such as a virus, that has a
known property or activity.
[0313] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "an" agent includes one
or more agents.
[0314] As used herein, the term "or" is used to mean "and/or"
unless explicitly indicated to refer to alternatives only or if the
alternatives are mutually exclusive.
[0315] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 bases" means "about 5 bases" and also "5
bases."
[0316] As used herein, "optional" or "optionally" means that the
subsequently described event or circumstance does or does not
occur, and that the description includes instances where said event
or circumstance occurs and instances where it does not. For
example, an optionally substituted group means that the group is
unsubstituted or is substituted.
[0317] 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).
[0318] For clarity of disclosure, and not by way of limitation, the
detailed description is divided into the subsections that
follow.
B. U-PA STRUCTURE AND FUNCTION
[0319] Urokinase-type plasminogen activator (u-PA, also called
urokinase or urinary plasminogen activator) is a serine protease
that catalyzes the hydrolysis of plasminogen into plasmin. u-PA is
found in urine, blood, seminal fluids, and in many cancer tissues.
It is involved in a variety of biological processes, which are
linked to its conversion of plasminogen to plasmin, which itself is
a serine protease. Plasmin has roles in a variety of normal and
pathological processes including, for example, cell migration and
tissue destruction through its cleavage of a variety of molecules
including fibrin, fibronectin, proteoglycans, and laminin. u-PA is
involved in tissue remodeling during wound healing, inflammatory
cell migration, neovascularization and tumor cell invasion. u-PA
also cleaves and activates other substrates, including, but not
limited to, hepatocyte growth factor/scatter factor (HGF/SF), the
latent form of membrane type 1 matrix metalloprotease (MT-SP1),
platelet derived growth factors, and others.
[0320] Provided herein are modified Urokinase-type plasminogen
activator (u-PA) polypeptides that are modified so that they cleave
inhibitory sequences in C3, such that activation of C3 into C3a and
C3b fragments is inhibited. The activity/specificity of the
modified u-PA polypeptides provided herein is such that they cleave
C3 with greater activity and/or specificity or k.sub.cat/k.sub.m
compared to the unmodified u-PA polypeptide, particularly of any of
SEQ ID NOs: 1-6. The modified u-PA polypeptides also can have
reduced activity or specificity or both for a native physiological
substrate plasminogen of the unmodified u-PA polypeptide. Thus, the
modified u-PA polypeptides provided herein inhibit complement
activation in a complement pathway. The modified u-PA polypeptides
also exhibit increased selectivity for cleaving C3 compared to
other u-PA substrates, such as plasminogen. Therefore, the modified
u-PA polypeptides provided herein do not exhibit undesired cleavage
activities against physiological native u-PA substrates so that
they do no exhibit undesirable side effects. In some embodiments,
the modified u-PA polypeptide is a protease domain or a single
chain form; in such instances, the free cysteine (residue position
122 by chymotrypsin numbering) is replaced with a serine, to
decrease or eliminate aggregation upon preparation of the protein.
In embodiments in which the modified u-PA polypeptide is full
length or other form in which it is activated by cleavage, the
residue at position 122 (by chymotrypsin numbering) generally is
not replaced with S so that the disulfide bond can form to produce
the two chain activated polypeptide.
[0321] 1. Serine Proteases
[0322] Serine proteases (SPs), which include secreted enzymes and
enzymes sequestered in cytoplasmic storage organelles, have a
variety of physiological roles, including in blood coagulation,
wound healing, digestion, immune responses and tumor invasion and
metastasis. For example, chymotrypsin, trypsin, and elastase
function in the digestive tract; Factor 10, Factor 11, Thrombin,
and Plasmin are involved in clotting and wound healing; and C1r,
C1s, and the C3 convertases play a role in complement
activation.
[0323] 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.
[0324] 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.
[0325] More than 20 families (denoted S1-S27) of serine protease
have been identified, and they are 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).
[0326] Examples of serine proteases of the chymotrypsin superfamily
include tissue-type plasminogen activator (tPA), trypsin,
trypsin-like protease, chymotrypsin, plasmin, elastase, urokinase
(or urinary-type plasminogen activator, u-PA), acrosin, activated
protein C, C1 esterase, cathepsin G, chymase, and proteases of the
blood coagulation cascade including kallikrein, thrombin, and
Factors VIIa, IXa, Xa, XIa, and XIIa (Barret, A. J., In: Proteinase
Inhibitors, Ed. Barrett, A. J., et al., Elsevier, Amsterdam, Pages
3-22 (1986); Strassburger, W. et al., (1983) FEBS Lett.,
157:219-223; Dayhoff, M. O., Atlas of Protein Sequence and
Structure, Vol 5, National Biomedical Research Foundation, Silver
Spring, Md. (1972); and Rosenberg, R. D. et al. (1986) Hosp. Prac.,
21: 131-137).
[0327] 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 (Ser195) in all three.
Despite their similarities, they have different substrate
specificities; they cleave different peptide bonds during protein
digestion. For example, chymotrypsin prefers an aromatic side chain
on the residue whose carbonyl carbon is part of the peptide bond to
be cleaved. Trypsin prefers a positively charged Lys or Arg residue
at this position. Serine proteases differ markedly in their
substrate recognition properties: some are highly specific (i. e.
the proteases involved in blood coagulation and the immune
complement system); some are only partially specific (i.e. the
mammalian digestive proteases trypsin and chymotrypsin); and
others, like subtilisin, a bacterial protease, are completely
non-specific. Despite these differences in specificity, the
catalytic mechanism of serine proteases is well conserved.
[0328] The mechanism of cleavage of a target protein by a serine
protease is based on nucleophilic attack of the targeted peptidic
bond by a serine. Cysteine, threonine or water molecules associated
with aspartate or metals also can play this role. In many cases the
nucleophilic property of the group is improved by the presence of a
histidine, held in a "proton acceptor state" by an aspartate.
Aligned side chains of serine, histidine and aspartate build the
catalytic triad common to most serine proteases. For example, the
active site residues of chymotrypsin, and serine proteases that are
members of the same family as chymotrypsin, such as for example
MTSP-1, are Asp102, His57, and Ser195.
[0329] The catalytic domains of all serine proteases of the
chymotrypsin superfamily have sequence homology and structural
homology. The sequence homology includes the conservation of: 1)
the characteristic active site residues (e.g., Ser195, His57, and
Asp102 in the case of trypsin); 2) the oxyanion hole (e.g., Gly193,
Asp194 in the case of trypsin); and 3) the cysteine residues that
form disulfide bridges in the structure (Hartley, B. S., (1974)
Symp. Soc. Gen. Microbiol., 24: 152-182). The structural homology
includes 1) a common fold characterized by two Greek key structures
(Richardson, J. (1981) Adv. Prot. Chem., 34:167-339); 2) a common
disposition of catalytic residues; and 3) detailed preservation of
the structure within the core of the molecule (Stroud, R. M. (1974)
Sci. Am., 231: 24-88).
[0330] 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 u-PA are listed in Table 4, 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.
[0331] 2. Structure
[0332] u-PA cDNA has been cloned from numerous mammalian species.
Exemplary u-PA precursor polypeptides, or prepro-urokinase
polypeptides include, but are not limited to, human (SEQ ID NO:1
and encoded by SEQ ID NO:7), mouse (SEQ ID NO:52), rat (SEQ ID
NO:53), bovine (SEQ ID NO:54), pig (SEQ ID NO:55), rabbit (SEQ ID
NO:56), chicken (SEQ ID NO:57), yellow baboon (SEQ ID NO:58),
Sumatran orangutan (SEQ ID NO:59), dog (SEQ ID NO:60), ovine (SEQ
ID NO:61), marmoset (SEQ ID NO:62), rhesus monkey (SEQ ID NO:63),
northern white-cheeked gibbon (SEQ ID NO:64) and chimpanzee (SEQ ID
NO:65) u-PA polypeptides. The mRNA transcript is typically
translated to generate a precursor protein containing a 20 amino
acid signal sequence at the N-terminus. Following transport to the
ER, the signal peptide is removed to yield a prourokinase
polypeptide. Exemplary prourokinase polypeptides include, but are
not limited to, human (SEQ ID NO:3), mouse (SEQ ID NO:66), rat (SEQ
ID NO:67), bovine (SEQ ID NO:68), pig (SEQ ID NO:69), rabbit (SEQ
ID NO:70), chicken (SEQ ID NO:71), yellow baboon (SEQ ID NO:72),
Sumatran orangutan (SEQ ID NO:73), dog (SEQ ID NO:74), and ovine
(SEQ ID NO:75) u-PA polypeptides. For example, the human u-PA mRNA
transcript is normally translated to form a 431 amino acid
precursor protein (SEQ ID NO: 1) containing a 20 amino acid signal
sequence at the N-terminus Met Arg Ala Leu Leu Ala Arg Leu Leu Leu
Cys Val Leu Val Val Ser Asp Ser Lys Gly (amino acid residues 1-20
of SEQ ID NO:1). Thus, following transport to the ER and removal of
the signal peptide, a 411 amino acid prourokinase polypeptide with
a sequence of amino acids set forth in SEQ ID NO:3 is produced. As
described in further detail below, prourokinase is a zymogen or
proenzyme that is further processed by proteolytic cleavage to
generate a two chain mature u-PA polypeptide. Thus, for example,
with reference to mature u-PA (SEQ ID NO:3), the wild type chain
activated u-PA contains a first chain (A chain), residues 1-158
linked by disulfide to residues 159-411 (B chain) via a disulfide
bond between Cys148 (C97a chymotrypsin numbering) and Cys279 (C122
chymotrypsin numbering). Hence, in the modified u-PA polypeptides
provided herein, when the protease domain is produced, it contains
the replacement C122S, but when an activated form is produced that
is a 2 chain form, the residue at 122 (chymotrypsin numbering) is C
so that it forms a disulfide bond with another C, generally in the
activation sequence (see discussion below and Example 15).
[0333] Human precursor u-PA has a sequence of amino acids set forth
in SEQ ID NO: 1 and encoded by a sequence of nucleotides set forth
in SEQ ID NO:7. Human pro-u-PA, also termed mature u-PA, lacking
the signal sequence is set forth in SEQ ID NO:3. Two isoforms of
human u-PA exist, as produced by alternative splicing. Isoform 1 of
human u-PA is the canonical form described above set forth in SEQ
ID NO:1. In isoform 2 of human u-PA, amino acids 1-29 of SEQ ID
NO:1 are replaced with amino acids 1-12 of SEQ ID NO: 51, with the
resulting protein containing 414 amino acids (set forth in SEQ ID
NO:51). Allelic variants and other variants of human u-PA are
known. For example, a uPA variant is known containing the amino
acid modification V15L in the sequence of amino acids set forth in
SEQ ID NO: 1. In another example, a modified u-PA polypeptide is
known containing the amino acid modification C299S (C122S by
chymotrypsin numbering) in the sequence of amino acids set forth in
SEQ ID NO: 1 (corresponding to the sequence of amino acids set
forth in SEQ ID NO: 4). Additional variants include those
containing amino acid modifications P121L, D130G, C131W, I194M,
K211Q, G366c and A410V in mature u-PA set forth in SEQ ID NO:3
(corresponding to amino acid modifications P141L, D150G, C151W,
I214M, K231Q, G386C and A430V in SEQ ID NO:1).
[0334] u-PA polypeptides are synthesized and secreted as a
single-chain zymogen molecules (also called prourokinases or
single-chain urokinases), which are converted into active two-chain
u-PAs by a variety of proteases including, for example, plasmin,
kallikrein, cathepsin B, matriptase and nerve growth factor 7.
Cleavage to generate the two chain form occurs between residues 158
and 159 (SEQ ID NO:3) in the human prourokinase sequence
(corresponding to amino acid residues 178 and 179 in SEQ ID NO: 1).
The two resulting chains are linked by a disulfide bond between
Cys148 and Cys279, thereby forming the two-chain form of u-PA. The
two chain form of u-PA also is called high molecular weight u-PA
(HMW-u-PA). HMW-u-PA can be further processed into low molecular
weight u-PA (LMW-u-PA) by cleavage of the A chain into a short
chain A (A1, amino acids 136-157 of SEQ ID NO:3) and an amino
terminal fragment. 21-178 linked disulfide to 179-411 linked via
Cys corresponding to Cys148 and Cys279 (SEQ ID NO:3).
[0335] Urokinase-type plasminogen activator, u-PA, is a classical
serine protease, containing a His-Asp-Ser catalytic triad, that
cleaves a specific Arg-Val bond in plasminogen to form plasmin.
Plasmin in turn can cleave u-PA at Lys158-Ile159 of SEQ ID NO:3
(corresponding to Lys15-Ile16 by chymotrypsin numbering) forming
the two-chain form described above. The catalytic triad of human
u-PA includes amino acids His204, Asp255 and Ser356 of SEQ ID NO:3
(corresponding to His57, Asp102 and Ser195 by chymotrypsin
numbering). Residues Ser138 and Ser303 of the human uPA set forth
in SEQ ID NO:3 are phosphorylated (Franco et al. (1997) J Cell Biol
137:779-791). Human u-PA contains O-linked glycosylation, e.g.
fucosylation, at amino acid residue Thr18 of SEQ ID NO:3 (Buko et
al. (1991) Proc Natl Acad Sci USA 88:3992-3996) and N-linked
glycosylation at amino acid residue Asn302 of SEQ ID NO:3. Mature
human u-PA contains intrachain disulfide bonds between residues
C11-C19, C13-C31, C33-C42, C50-C131, C71-C113, C102-C126,
C189-C205, C197-C268, C293-C362, C325-C341 and C352-C380 of SEQ ID
NO:3 and an interchain disulfide bond between residues C148-C279 of
SEQ ID NO:3.
[0336] The mature form of u-PA is a 411 residue protein
(corresponding to amino acid residues 21 to 431 in the sequence of
amino acids set forth in SEQ ID NO: 1 which is the precursor form
containing a 20 amino acid signal peptide). u-PA contains three
domains: the serine protease domain, the kringle domain and the
growth factor domain. In the mature form of human u-PA, amino acids
1-158 represent the N-terminal A chain including a growth factor
domain (amino acids 1-49), a kringle domain (amino acids 50-131),
and an interdomain linker region (amino acids 132-158). Amino acids
159-411 represent the C-terminal serine protease domain or B chain.
u-PA is synthesized and secreted as a single-chain zymogen
molecule, which is converted into an active two-chain u-PA by a
variety of proteases including, for example, plasmin, kallikrein,
cathepsin B, and nerve growth factor-.gamma. (gamma). Cleavage into
the two chain form occurs between residues 158 and 159 in a mature
u-PA sequence (corresponding to amino acid residues 178 and 179 in
SEQ ID NO:3). The two resulting chains are kept together by a
disulfide bond, thereby forming the two-chain form of u-PA.
[0337] Urokinase-type plasminogen activators contain three domains:
a serine protease domain, a kringle domain and a growth factor
domain. In the zymogen or proenzyme form of human u-PA, amino acids
1-158 of SEQ ID NO:3 represent the N-terminal A chain (or long
chain A) including an epidermal growth factor domain (amino acids
1-49), a kringle domain (amino acids 50-131) and an interdomain
linker region (amino acids 132-158) and amino acids 159-411
represent the catalytically active C-terminal serine protease
domain or B chain. The epidermal growth factor domain is
responsible for binding of u-PA to the cell surface-anchored u-PA
receptor (uPAR). In the extracellular matrix, u-PA is tethered to
the cell membrane by binding to the u-PA receptor. LMW-u-PA is
proteolytically active but does not bind the u-PA receptor. The
serine protease domain contains surface-exposed loops around
residues 37, 60, 96, 110, 170 and 185, by chymotrypsin numbering.
Upon activation or cleavage, the amino terminus inserts into a
hydrophobic binding cleft of the catalytic protease domain forming
hydrophobic interactions and a salt bridge to the side pocket of
Asp194 which stabilizes the substrate binding pocket and oxyanion
hole in a catalytically productive conformation. Asp194, according
to chymotrypsin numbering, participates in hydrogen bonding to the
main chain amino group of Gly142 and the main chain carbonyl group
of Lys143 (Blouse et al. (2009) J Biol Chem 284:4647-4657).
Conformational changes after cleavage involves four disordered
regions of the activation domain, including the activation loop
(residues 16-21), the autolysis loop (residues 142-152), the
oxyanion stabilizing loop (residues 184-194) and the Si entrance
frame (residues 216-223), all numbering according to chymotrypsin
numbering (see, Blouse et al. (2009) J Biol Chem 284:4647-4657;
Hedstrom (2002) Chem Rev 102:4501-4524; Huber and Bode (1978) Acc
Chem Res 11:114-122; Madison et al. (1993) Science
262:419-421).
[0338] Structural determinants for u-PA are set forth in Table 4
below with numbering based on the numbering of mature chymotrypsin.
The number underneath the Cys168-Cys182 and 60's loop column
headings indicates the number of amino acids in the loop between
the two amino acids and in the loop. The yes designation under the
Cys191-Cys220 column headings indicates a disulfide bridge is
present. These regions are variable within the family of
chymotrypsin-like serine proteases and represent structural
determinants in themselves. Modification of a u-PA polypeptide to
alter any one or more of the amino acids in the S1-S4 pocket
affects the specificity or selectivity of the u-PA polypeptide for
a target substrate. The extended substrate specificity (P1-P4)
reveals that u-PA has a high specificity for cleavage after P1 Arg,
a preference for small amino acids at the P2 position, a preference
for small polar amino acids (Thr and Ser) at the P3 position and no
preference at the P4 position (Ke et al. (1997) J. Biol. Chem.,
272:16603-16609; Harris et al. (2000) Proc Natl Acad Sci USA,
97:7754-7759).
TABLE-US-00006 TABLE 4 Structural Determinants for u-PA substrate
cleavage (chymotrypsin numbering) Residues that Determine
Specificity S4 S1 Cys Cys 168 S2 191 Cys S3 60's Cys 171 174 180
215 182 192 218 99 57 loop 189 190 226 220 H S M W 15 Q R H H 11 D
S G yes
[0339] 3. Function/activity
[0340] Urokinase-type plasminogen activator is a serine protease
that catalyzes the hydrolysis of plasminogen into plasmin. Plasmin
acts directly on the degradation of extracellular matrix proteins
(Andreasen et al. (2000) Cell. Mol. Life Sci. 57:25-40). u-PA plays
an important role in cell adhesion, migration and invasion, tissue
remodeling and cancer (Blasi et al. (2002) Rev Mol Cell Biol 3:932;
Andreasen et al. (2000) Cell. Mol. Life Sci. 57:25-40; Mondino and
Blasi (2004) Trends Immunol 25:450; Ploug (2003) Curr Pharm Des
9:1499). Abnormal expression of u-PA has been associated with
rheumatoid arthritis, allergic vasculitis, xeroderma pigmentosum
and the invasive capacity of malignant tumors.
[0341] u-PA is regulated by the binding to the high affinity cell
surface receptor uPAR. Binding of u-PA to uPAR increases the rate
of plasminogen activation and enhances extracellular matrix
degradation and cell invasion. The binary complex formed between
uPAR and u-PA interacts with membrane-associated plasminogen to
form higher order activation complexes that reduce the Km (i. e.
kinetic rate constant of the approximate affinity for a substrate)
for plasminogen activation (Bass et al. (2002) Biochem. Soc.
Trans., 30: 189-194). Binding of u-PA to uPAR protects the protease
from inhibition by the cognate inhibitor, i.e. PAI-1. This is
because single chain u-PA normally present in plasma is not
susceptible to inhibition by PAI-1, and any active u-PA in the
plasma will be inhibited by PAI-1. Active u-PA that is receptor
bound is fully available for inhibition by PAI-1, however, PAI-1 is
unable to access the bound active molecule (Bass et al. (2002)
Biochem. Soc. Trans., 30: 189-194). As a result, u-PA primarily
functions on the cell surface and its functions are correlated with
the activation of plasmin-dependent pericellular proteolysis.
[0342] u-PA also cleaves hepatocyte growth factor/scatter factor
(HGF/SF), the latent form of membrane type 1 matrix metalloprotease
(MT-SP1; matriptase), platelet derived growth factor C (PDGF-C),
platelet derived growth factor D (PDGF-D), platelet derived growth
factor DD (PDGF-DD) and other proteins (see, e.g., Hurst et al.
(2012) Biochem J 441:909-918; Ustach and Kim (2005) Mol Cell Biol
5:6279-6288; Ehnman et al. (2009) Oncogene 28(4):534-544). Plasmin
degrades fibrin clots, cleaves fibrin, fibronectin, thrombospondin,
laminin and von Willebrand factor, proteolyzes mediators of
complement system and activates collagenases. As such, plasmin
participates in thrombolysis or extracellular matrix degradation,
linking to plasmin to vascular diseases and cancer. For example,
components of the plasminogen activation system have been observed
to be highly expressed in malignant tumors. Hepatocyte growth
factor/scatter factor regulates cell growth, cell motility and
morphogenesis by binding of activated HGF to the HGF-receptor c-Met
and its ability to stimulate mitogenesis, cell motility and matrix
invasion link it to angiogenesis, tumorogenesis and tissue
regeneration. Platelet derived growth factors regulate cell growth
and division, and play a significant role in angiogenesis, which,
when uncontrolled, is a characteristic of cancer. Once activated by
proteolytic cleavage, PDGFs bind PGDF receptor tyrosine kinases
leading to phosphorylation and a number of downstream signaling
pathways involved in cancer. Due to the role of u-PA and the above
mentioned proteins in vascular diseases the u-PA polypeptides
provided herein are altered such that they reduced selectivity
towards these proteins. By virtue of the changes in their
specificity and activity the modified u-PA polypeptides provided
herein exhibit reduced or no activity or no substantial activity on
native substrates, and high activity, compared to unmodified u-PA
on complement protein C3. As a result, at therapeutic dosages, the
modified u-PA polypeptides provided herein specifically inhibit
complement activation but have none or few side effects from
cleavage of natural u-PA targets.
C. COMPLEMENT INHIBITION BY TARGETING C3
[0343] The modified u-PA polypeptides provided herein exhibit
increased specificity and/or activity for an inhibitory cleavage
sequence in complement protein C3 compared to u-PA not containing
the amino acid modifications (e.g. wild type human u-PA (see, SEQ
ID NO: 1 or 3)) or the catalytic domain or protease domain thereof
(see, SEQ ID NO:2) or corresponding unmodified u-PA polypeptides
that include the replacement C122S, by chymotrypsin numbering.
Replacement with S at residue 122 does not alter specificity or
activity on C3, but reduces aggregation. Since C3 is involved in
the 3 initiation pathways of complement (see, e.g., FIG. 1),
targeting C3 by proteolytic inhibition provides a general and broad
therapeutic target for inactivation of the complement cascade.
Inactivation cleavage of C3 blocks terminal activity of complement
as well as the alternative pathway amplification loop. All three
pathways converge at C3 (see, e.g., FIG. 1). By virtue of the
ability to inhibit complement activation, such modified u-PA
polypeptides can be used to treat various diseases, conditions and
pathologies associated with complement activation, such as
inflammatory responses and autoimmune diseases. Complement
activation is associated with the development of diseases and
conditions by promoting local inflammation and damage to tissues
caused in part by the generation of effector molecules and a
membrane attack complex. 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 not adequately
controlled. The role of C3 in complement activation is discussed in
further detail below.
[0344] 1. Complement Protein C3 and its Role in Initiating
Complement
[0345] The complement system involves over 30 soluble and
cell-membrane bound proteins that 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 activation is
initiated on pathogen surfaces through three distinct pathways: 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 (e.g., C3 convertases)
which cleave complement protein C3 to trigger the formation of the
membrane attack complex (MAC) (see, e.g., FIG. 1). Thus, complement
protein C3 is an attractive target for a therapeutic since
modulation of C3 results in modulation of various opsonins,
anaphylatoxins and the MAC. Further, naturally occurring complement
inhibitor proteins including factor H (FH), CR1, complement
receptor Ig (CR1g), DAF and MCP inhibit at the C3 level.
[0346] There are three (3) pathways of complement activation (See,
FIG. 1, which depicts these pathways). The pathways of complement
are distinct; each relies on different molecules and mechanisms for
initiation. The pathways are similar in that they converge to
generate the same set of effector molecules, i.e., C3 convertases.
In the classical and lectin pathways C4b2b acts as a C3 convertase;
in the alternative pathway, C3bBb is a C3 convertase (see Table 5).
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 that generates 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. Alternatively, C3 can be cleaved and activated
by extrinsic proteases, such as lysosomal enzymes and elastase
(Markiewski and Lambris (2007) Am J Pathology 171:715-727; Ricklin
and Lambris (2007) Nat Biotechnol 25:1265-1275).
TABLE-US-00007 TABLE 5 Complement Cascades Alternative Pathway
Classical Pathway Lectin Pathway Activators Pathogen surface
antigen-bound IgM Pathogens via molecules and IgG; non- recognition
of LPS, teichoic immune molecules carbohydrates on acid, zymosan
surface C3 convertase C3bBb C4b2b C4b2b C5 convertase C3bBb3b
C4b2b3b C4b2b3b MAC C5678poly9 C5678poly9 C5678poly9 anaphylatoxins
C3a, C5a C3a, C4a, C5a C3a, C4a, C5a
[0347] a. Classical Pathway
[0348] 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 et al., (1994) Clin Exp Immunol.
97(2):4-9; Holmskov et al. (1994) Immunol Today 15(2):67-74).
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. (2003) J. Biol.
Chem. 278(47):46974-46982).
[0349] 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. 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 initiation by an
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.
[0350] C1q is part of the C1 complex which contains a single C1q
molecule bound to two molecules each of the zymogens C1r and Cis.
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 C1 s 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 6 summarizes the proteins involved in the classical pathway
of complement.
TABLE-US-00008 TABLE 6 Proteins of the Classical Pathway Native
Active Component Form Function of the Active Form C1 C1q Binds
directly to pathogen surfaces or (C1q:(C1r:C1s).sub.2) 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
[0351] b. Alternative Pathway
[0352] The alternative pathway is initiated by foreign pathogens in
the absence of antibody. 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 sialic acid which inactivate C3b if
it binds, but bacteria contain low external sialic acid levels 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
complement 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 and additionally initiates the alternative pathway, 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 7
summarizes the proteins involved in the alternative pathway of
complement.
TABLE-US-00009 TABLE 7 Proteins of the Alternative Pathway Native
Active Component 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
[0353] c. Lectin Pathway
[0354] 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). 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. MBL also acts as an opsonin to
facilitate the phagocytosis of bacterial, viral, and fungal
pathogens by phagocytic cells. Additional 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. 175:3150-3156).
Similar to MBL, ficolins recognize carbohydrate moieties such as,
for example, N-acetyl glucosamine and mannose structures.
[0355] 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 C s 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 8
summarizes the proteins involved in the lectin pathway of
complement.
TABLE-US-00010 TABLE 8 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
[0356] d. Complement-Mediated Effector functions
[0357] 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), which act as effector molecules to mediate
diverse effector functions. The recognition of complement effector
molecules by cells for the initiation of effector functions (e.g.
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.
[0358] Thus, through complement receptors, these complement
effector molecule 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 9.
TABLE-US-00011 TABLE 9 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 chemotactic factor) vascular permeability;
smooth 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
[0359] i. Complement-Mediated Lysis: Membrane Attack Complex
[0360] 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.
[0361] ii. Inflammation
[0362] 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. C5a, the
most potent anaphylatoxin, primarily acts on white blood cells,
particularly 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.
[0363] 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.
[0364] 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. 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.
[0365] iii. Chemotaxis
[0366] 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 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.-10 M.
[0367] iv. Opsonization
[0368] 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.
[0369] C3b and, to a lesser extent, C4b help to remove harmful
immune complexes from the body. 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.
[0370] v. Activation of the Humoral Immune Response
[0371] 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
bind 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.
[0372] 2. C3 Structure and Function
[0373] The variant u-PA polypeptides provided herein cleave
complement protein C3 or its proteolytic fragments thereby
inhibiting complement. Human complement protein C3 (Uniprot
Accession No. P01024) is a 1663 amino acid single chain
pre-proprotein having an amino acid sequence set forth in SEQ ID
NO:47. The protein is encoded by a 41 kb gene located on chromosome
19 (nucleotide sequence set forth in SEQ ID NO:46). The
pre-proprotein contains a 22 amino acid signal peptide (amino acids
1-22 of SEQ ID NO:47) and a tetra-arginine sequence (amino acids
678-681 of SEQ ID NO:47) that is removed by a furin-like enzyme
resulting in formation of a mature two chain protein containing a
beta chain (amino acids 23-667 of SEQ ID NO:47) and an alpha chain
(amino acids 672-1663 of SEQ ID NO:47), that are linked by an
interchain disulfide bond between amino acid residues Cys559 and
Cys816. The mature 2 chain protein has a sequence of amino acids
set forth in SEQ ID NO:77.
[0374] During the complement cascade, complement protein C3 is
further processed by proteolytic cleavage to form various C3
proteolytic fragments. As described above, all three complement
initiation pathways converge on the C3 convertases C4b2b and C3bBb.
C3 convertases cleave C3 between residues 748 and 749 of SEQ ID
NO:47 (see Table 10 below) generating the anaphylatoxin C3a (amino
acids 672-748 of SEQ ID NO:47) and the opsonin C3b (C3b alpha'
chain; amino acids 749-1663 of SEQ ID NO:47). C3a is involved in
inflammation and C3b forms the C5 convertases ultimately leading to
C5a anaphylatoxin and the MAC. The variant u-PA polypeptides
provided herein inhibit complement, and as such, do not cleave C3
at this GLAR cleavage site.
[0375] C3b has binding sites for various complement components
including C5, properdin (P), factors H, B and I, complement
receptor 1 (CR1) and the membrane co-factor protein (MCP) (see Sahu
and Lambris (2001) Immunological Reviews 180:35-48). Binding of
Factor I, a plasma protease, in the presence of cofactors H, CR1
and MCP results in inactivation of C3b whereas binding of factors B
and P in the presence of factor D results in amplification of C3
convertase and initiation of MAC. Factor I cleaves C3b in the
presence of cofactors between residues 1303-1304, 1320-1321 and
954-955 of SEQ ID NO:47 (see Table 10 below) generating fragments
iC3b (amino acids 749-1303 of SEQ ID NO:47) and C3f (amino acids
1304-1320 of SEQ ID NO:47). Factor I subsequently cleaves iC3b
generating C3c (C3c alpha' chain Fragment 1; amino acids 749-954 of
SEQ ID NO:47) and C3dg (amino acids 955-1303 of SEQ ID NO:47). The
end result is that C3b is permanently inactivated (see Sahu and
Lambris (2001) Immunological Reviews 180:35-48). Since Factor I
inactivates C3b, the Factor I cleavage sites are candidates for
cleavage by the variant u-PA polypeptides provided herein.
Additional C3b proteolytic fragments include C3g (amino acids
955-1001 of SEQ ID NO:47), C3d (amino acids 1002-1303 of SEQ ID
NO:47), and C3c alpha' chain Fragment 2 (amino acids 1321-1663 of
SEQ ID NO:47). Cleavage sequences in complement protein C3 are set
forth in Table 10 below, which lists the P4-P1 residues, the amino
acid residues of the cleavage site (P1-P1' site) and the protease
responsible for cleavage. The modified u-PA polypeptides provided
herein do not cleave at these sites.
TABLE-US-00012 TABLE 10 Complement Protein C3 Cleavage Sequences
Cleavage Site (in SEQ ID NO: 47) P4-P1 Residues Between residues
Protease SEQ ID NO. GLAR 748-749 C3 convertase 78 RLGR 954-955
Factor I 79 LPSR 1303-1304 Factor I 80 SLLR 1320-1321 Factor I
81
[0376] a. C3a
[0377] C3a (amino acids 672-748 of SEQ ID NO:47) is an
anaphylatoxin that is involved in inflammation, basophil and mast
cell degranulation, enhanced vascular permeability, smooth muscle
contraction and induction of suppressor T cells.
[0378] b. C3b
[0379] C3b (amino acids 749-1663 of SEQ ID NO:47) has various roles
in the complement cascade. C3b is an opsonin that facilitates the
uptake and destruction of pathogens by phagocytic cells.
Additionally, C3b combines with the C3 convertases to generate the
C5 convertases which activate complement protein C5 thereby
generating the C5a anaphylatoxin and C5b, which combines with C6,
C7, C8 and C9 to form the membrane attack complex. Furthermore, as
described in section 1b above, C3b is involved in the alternative
pathway of complement initiation. C3b is regulated by complement
regulatory protein Factor I, a plasma protease which degrades C3b
into various fragments, including iC3b, C3c, C3d, C3f and C3dg,
thereby permanently inactivating C3b.
[0380] C3b plays a critical role in complement-mediated effector
functions by virtue of its ability to bind to the C3 convertases
C4b2b and C3bBb thereby generating the C5 convertases C4b2b3b and
C3bBb3b. The C5 convertases cleave the zymogen C5 into its active
fragments, namely the C5a anaphylatoxin and C5b. C5a is involved in
chemotaxis and inflammation and C5b is involved in formation of
MAC.
[0381] c. Inhibitors of C3b
[0382] C3b has binding sites for various complement components
including C5, properdin (P), factors H, B and I, complement
receptor 1 (CR1) and the membrane co-factor protein (MCP) (see Sahu
and Lambris (2001) Immunological Reviews 180:35-48). Binding of
factor I, a plasma protease, in the presence of cofactors H, CR1
and MCP results in inactivation of C3b whereas binding of factors B
and P in the presence of factor D results in amplification of C3
convertase and initiation of MAC. Factor I cleaves C3b in the
presence of cofactors between residues 1303-1304, 1320-1321 and
954-955 of SEQ ID NO:47 generating fragments iC3b (amino acids
749-1303 of SEQ ID NO:47) and C3f (amino acids 1304-1320 of SEQ ID
NO:47). 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. The protein iC3b functions as an opsonin.
Factor I subsequently cleaves iC3b generating fragments C3c (C3c
alpha' chain Fragment 1: amino acids 749-954 of SEQ ID NO:47 and
C3c alpha' chain Fragment 2: amino acids 1321-1663 of SEQ ID NO:47)
and C3dg (amino acids 955-1303 of SEQ ID NO:47). The end result is
that C3b is permanently inactivated (see Sahu and Lambris (2001)
Immunological Reviews 180:35-48). C3dg can be further cleaved to
generate fragments C3g (amino acids 955-1001 of SEQ ID NO: 47) and
C3d (amino acids 1002-1303 of SEQ ID NO:47).
D. MODIFIED U-PA POLYPEPTIDES THAT CLEAVE C3
[0383] Provided herein are modified or variant urokinase-type
plasminogen activator (u-PA) polypeptides. Also provided are
conjugates, such as fusion proteins, that contain modified u-PA
polypeptides, so that resulting activated forms thereof cleave C3.
The modified u-PA polypeptides provided herein exhibit altered
activities or properties compared to a wild-type, native or
reference u-PA polypeptide. For example, the u-PA polypeptides
provided herein contain modifications compared to a wild-type,
native or reference u-PA polypeptide set forth in any of SEQ ID
NOS: 1-6, or in a polypeptide that has at least 65%, 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, particularly at least 95% sequence identity to any of
SEQ ID NOS:1-6, such as the reference u-PA protease domain set
forth in SEQ ID NO:5. Included among the modified u-PA polypeptides
provided herein are u-PA polypeptides that alter (inhibit)
complement activation by effecting inhibitory cleavage of
complement protein C3. Among the modified u-PA polypeptides
provided herein are those that effect inhibitory cleavage of
complement protein C3. Included are those that effect inhibitory
cleavage of C3 with greater activity or specificity,
K.sub.cat/K.sub.m, compared to a corresponding form of the u-PA
that does not contain the modification (the replacement, deletion
and/or insertion) or compared to the corresponding form of
unmodified u-PA whose sequences are set forth in any of SEQ ID NOs:
1-6. The modified u-PA polypeptides also can have decreased
specificity and/or and selectivity for substrates and targets
cleaved or recognized by unmodified u-PA, including cleavage of
plasminogen and/or binding to uPAR, compared to the corresponding
u-PA polypeptide not containing the amino acid modification(s).
[0384] The modified u-PA polypeptides provided herein inhibit or
inactivate complement through inhibitory or inactivation cleavage
of complement protein C3. The modified u-PA polypeptides provided
herein inhibit or inactivate complement by cleaving complement
protein C3 at a cleavage site that results in inhibition or
inactivation of C3. Inactivation or inhibition cleavage of
complement protein C3 can be at any sequence in C3 so long as the
resulting cleavage of C3 results in inactivation or inhibition of
activation of complement. Since the modified u-PA polypeptides
provided herein inhibit complement activation, the modified u-PA
polypeptides do not effect cleavage of the zymogen form of C3 to
generate the C3a and C3b activated fragments. Thus, modified u-PA
polypeptides provided herein do not cleave C3 between residues
748-749 of SEQ ID NO: 47, which would result in generation of C3a
and C3b. Inhibition or inactivation cleavage sites of complement
protein C3 can be empirically determined or identified. If
necessary, a modified u-PA polypeptide can be tested for its
ability to inhibit complement as described in section E below and
as exemplified in the Examples.
[0385] The modified u-PA polypeptides provided herein catalyze
inhibitory or inactivation cleavage of complement protein C3. The
modified u-PA polypeptides provided herein cleave complement
protein C3 at any cleavage sequence as long as the resulting C3
fragments are inactive, or unable to activate a complement-mediated
effector function. The modified u-PA polypeptides provided herein
have altered (i. e., decreased) specificity and/or selectivity for
natural targets of u-PA, including plasminogen and uPAR. In one
example, the modified u-PA polypeptides provided herein have
reduced specificity for cleavage of plasminogen. In another
example, the modified u-PA polypeptides provided herein have
reduced selectivity for binding to uPAR. In some examples, the
modified u-PA polypeptides provided herein have reduced specificity
for cleavage of plasminogen and reduced selectivity for binding to
uPAR. In other examples, the modified u-PA polypeptides provided
herein have increased specificity for cleavage of complement
protein C3 and decreased specificity for cleavage of plasminogen.
In other examples, the modified u-PA polypeptides provided herein
have increased selectivity for complement protein C3 and decreased
selectivity for plasminogen and/or uPAR.
[0386] The modified u-PA polypeptides provided herein and described
in the examples are, for example, isolated protease domains of
u-PA. Smaller portions thereof that retain protease activity also
are contemplated. The modified u-PA polypeptides provided herein
are mutants of the protease domain of u-PA, particularly modified
u-PA polypeptides in which the Cys residue in the protease domain
that is free (i.e., does not form disulfide linkages with any other
Cys residue in the protein) is substituted with another amino acid
substitution, preferably with a conservative amino acid
substitution or a substitution that does not eliminate the
activity, such as, for example, substitution with Serine, and
modified u-PA polypeptides in which a glycosylation site(s) is
eliminated. Modified u-PA polypeptides in which other conservative
amino acid substitutions in which catalytic activity is retained
are also contemplated (see e.g., Table 3, for exemplary amino acid
substitutions).
[0387] The modified u-PA polypeptides provided herein contain one
or more amino acid modifications such that they cleave complement
protein C3 in a manner that results in inactivation or inhibition
of complement. The modifications can be a single amino acid
modification, such as single amino acid replacements
(substitutions), insertions or deletions, or multiple amino acid
modifications, such as multiple amino acid replacements, insertions
or deletions. Exemplary modifications are amino acid replacements,
including single or multiple amino acid replacements. The amino
acid replacement can be a conservative substitution, such as set
forth in Table 3, or a non-conservative substitution, such as any
described herein. Modified u-PA polypeptides provided herein can
contain at least or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, or more modified positions compared to the
u-PA polypeptide not containing the modification.
[0388] The modifications described herein can be made in any u-PA
polypeptide. For example, the modifications are made in a human
u-PA polypeptide having a sequence of amino acids including or set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO: 5 or SEQ ID NO:6, or allelic variants thereof; a mouse u-PA
polypeptide having a sequence of amino acids including or set forth
in SEQ ID NOS:52 or 66; a rat u-PA polypeptide having a sequence of
amino acids including or set forth in SEQ ID NOS:53 or 67; a cow
u-PA polypeptide having a sequence of amino acids including or set
forth in SEQ ID NOS:54 or 68; a porcine u-PA polypeptide having a
sequence of amino acids including or set forth in SEQ ID NOS:55 or
69; a rabbit u-PA polypeptide having a sequence of amino acids
including or set forth in SEQ ID NOS:56 or 70; a chicken u-PA
polypeptide having a sequence of amino acids including or set forth
in SEQ ID NOS:57 or 71; a yellow baboon u-PA polypeptide having a
sequence of amino acids including or set forth in SEQ ID NOS:58 or
72; a Sumatran orangutan u-PA polypeptide having a sequence of
amino acids including or set forth in SEQ ID NOS:59 or 73; a dog
u-PA polypeptide having a sequence of amino acids including or set
forth in SEQ ID NOS:60 or 74; a ovine u-PA polypeptide having a
sequence of amino acids including or set forth in SEQ ID NOS:61 or
75; a marmoset u-PA polypeptide having a sequence of amino acids
including or set forth in SEQ ID NO:62; a rhesus monkey u-PA
polypeptide having a sequence of amino acids including or set forth
in SEQ ID NO:63; a northern white-cheeked gibbon u-PA polypeptide
having a sequence of amino acids including or set forth in SEQ ID
NO:64; and a chimpanzee u-PA polypeptide having a sequence of amino
acids including or set forth in SEQ ID NOS:65; or in sequence
variants or catalytically active fragments that exhibit at least
65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of
SEQ ID NOS:1-6 and 52-75.
[0389] The modified u-PA polypeptides provided herein can be
modified in any region or domain of a u-PA polypeptide provided
herein, as long as the modified u-PA polypeptide retains its
ability to effect inactivation or inhibitory cleavage of complement
protein C3. The modified u-PA polypeptides provided herein can be
single-chain or two chain polypeptides, species variants, allelic
variants, isoforms, or catalytically active fragments thereof, such
as, for example, the protease domain thereof. The u-PA polypeptides
provided herein can be full length or truncated u-PA polypeptides.
The modified u-PA polypeptides provided herein can be the protease
domain of u-PA or a modified form of the protease domain of u-PA.
Also contemplated for use herein are zymogen, precursor or mature
forms of modified u-PA polypeptides, provided the u-PA polypeptides
retain their ability to effect inhibitory or inactivation cleavage
of complement protein C3. Modifications in a u-PA polypeptide also
can be made to a u-PA polypeptide that also contains other
modifications, including modifications of the primary sequence and
modifications not in the primary sequence of the polypeptide. For
example, a modification described herein can be in a u-PA
polypeptide that is a fusion polypeptide or chimeric polypeptide.
The modified u-PA polypeptides provided herein also include
polypeptides that are conjugated to a polymer, such as a PEG
reagent.
[0390] For purposes herein, reference to positions and amino acids
for modification, including amino acid replacement or replacements,
herein are with reference to the u-PA polypeptide set forth in any
of SEQ ID NOs: 1-6. It is within the level of one of skill in the
art to make any of the modifications provided herein in another
u-PA polypeptide by identifying the corresponding amino acid
residue in another u-PA polypeptide, such as the u-PA polypeptide
set forth in any of SEQ ID NOs: 1-6 or a variant thereof that
exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to
a u-PA polypeptide set forth in any of SEQ ID NOs: 1-6.
Corresponding positions in another u-PA polypeptide can be
identified by alignment of the u-PA polypeptide with the reference
a u-PA polypeptide set forth in any of SEQ ID NOs: 1-6. For
purposes of modification (e.g. amino acid replacement), the
corresponding amino acid residue can be any amino acid residue, and
need not be identical to the residue set forth in any of SEQ ID
NOs: 1-6. Typically, the corresponding amino acid residue
identified by alignment with, for example, residues in SEQ ID NO:5
is an amino acid residue that is identical to SEQ ID NO:5, or is a
conservative or semi-conservative amino acid residue thereto. It
also is understood that the exemplary replacements provided herein
can be made at the corresponding residue in a u-PA polypeptide,
such as the protease domain of u-PA, so long as the replacement is
different than exists in the unmodified form of the u-PA
polypeptide, such as the protease domain of u-PA. Based on this
description and the description elsewhere herein, it is within the
level of one of skill in the art to generate a modified u-PA
polypeptide containing any one or more of the described mutations,
and test each for a property or activity as described herein.
[0391] The modified u-PA polypeptides provided herein alter
complement activity by proteolysis-mediated inhibition or
inactivation of complement protein C3. Further, the modified u-PA
polypeptides provided herein have decreased specificity for
cleavage of plasminogen and/or binding to uPAR. For example, the
modified u-PA polypeptides provided herein exhibit less than 100%
of the wild type activity of a u-PA polypeptide for cleavage of
plasminogen, such as less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,
20%, 10% or less of the activity for cleavage of plasminogen of a
wild type or reference u-PA polypeptide, such as the corresponding
polypeptide not containing the amino acid modification. In another
example, the modified u-PA polypeptides provided herein exhibit
less than 100% of the wild type binding activity of a u-PA
polypeptide for uPAR, such as less than 90%, 80%, 70%, 60%, 50%,
40%, 30%, 20%, 10% or less of the activity for binding to uPAR of a
wild type or reference u-PA polypeptide, such as the corresponding
polypeptide not containing the amino acid modification.
[0392] Also provided herein are nucleic acid molecules that encode
any of the modified u-PA polypeptides provided herein. In some
examples, the encoding nucleic acid molecules also can be modified
to contain a heterologous signal sequence to alter (e.g. increased)
expression and secretion of the polypeptide. The modified u-PA
polypeptides and encoding nucleic acid molecules provided herein
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. The modified polypeptides and encoding nucleic acid
molecules provided herein can be produced by standard recombinant
DNA techniques known to one of skill in the art. 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 or random mutagenesis of encoding nucleic acid
molecules, or solid phase polypeptide synthesis methods. For
example, nucleic acid molecules encoding a u-PA polypeptide can be
subjected to mutagenesis, such as random mutagenesis of the
encoding nucleic acid, error-prone PCR, site-directed mutagenesis,
overlap PCR, gene shuffling, or other recombinant methods. The
nucleic acid encoding the polypeptides can then be introduced into
a host cell to be expressed heterologously. Hence, also provided
herein are nucleic acid molecules encoding any of the modified
polypeptides provided herein. In some examples, the modified u-PA
polypeptides are produced synthetically, such as using solid phase
or solutions phase peptide synthesis. The nucleic acid molecules
can be provided in gene therapy vectors, such as AAV or adenovirus
vectors, for expression of the encoded modified u-PA polypeptide in
vivo, such as in the eye or for systemic administration. The
encoded u-PA polypeptide can be a full-length polypeptide or a
protease domain or other form that is active or that can be
activated.
[0393] The u-PA polypeptides provided herein have been modified to
have increased specificity and/or selectivity for cleavage of an
inhibitory or inactivation cleavage sequence of complement protein
C3. u-PA polypeptides 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 u-PA polypeptide to determine if the modified u-PA
polypeptide targets complement protein C3 for cleavage and
inactivation. Exemplary methods to identify a u-PA polypeptide and
the modified u-PA polypeptides are provided herein.
[0394] 1. Exemplary Modified u-PA Polypeptides
[0395] Provided herein are modified u-PA polypeptides that contain
one or more, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 and more amino acid modifications in a u-PA polypeptide and that
cleave complement protein C3 such that complement is inhibited or
inactivated. Modifications are in the primary amino acid sequence,
and include replacements, deletions and insertions of amino acid
residues. The modification alter the specificity/activity of the
u-PA polypeptide, when in an active form. The modified u-PA
polypeptides herein are selected to recognize and cleave a target
site in a complement protein, particularly C3 to inactivate it.
They also can be further modified and screened to have reduced
specificity/activity on in vivo substrates, such as plasminogen.
They can be selected and identified by any suitable protease screen
method. The modified u-PA polypeptides herein initially were
identified using the screening method described in U.S. Pat. No.
8,211,428, in which a library of modified proteases are reacted
with a cognate or other inhibitory serpin that is modified to
include a target sequence in the reactive site loop to capture
modified proteases that would cleave such target.
[0396] Modified u-PA polypeptides provided herein display increased
activity or specificity or K.sub.cat/K.sub.m for complement protein
C3 at a site that inactivates C3, and also can have reduced
activity or specificity for plasminogen and/or display increased
selectivity, specificity and/or activity for a target site
complement protein C3, whereby the modified u-PA polypeptide
inactivates C3. The modified u-PA polypeptides exhibit increased
activity for cleaving and inactivating C3 compared to the
corresponding form of wild-type or wild-type with the replacement
C122S (by chymotrypsin numbering). In particular, the protease
domain of the modified u-PA polypeptide exhibits increased
inactivation cleavage activity of C3 compared to the u-PA protease
domain of SEQ ID NO:5 (u-PA protease domain with C122S). The
increase in activity can be 10%, 20%, 50%, 100%, 1-fold, 2-fold,
3-fold, 4, 5, 6, 7, 8, 9, 10-fold and more compared to the
unmodified u-PA.
[0397] The modified u-PA polypeptide can have reduced activity for
a native substrate, such as plasminogen. For example, the modified
u-PA polypeptides can exhibit 0 to 99% of the u-PA activity of a
wild type or reference u-PA polypeptide, such as the u-PA
polypeptide set forth in SEQ ID NO:5, for plasminogen and at least
0.5-fold, 1-fold, 2-fold, 3-fold or more for cleaving C3 to
inactivate it. For example, modified u-PA polypeptides provided
herein exhibit less than or less than about 99%, 95%, 90%, 80%,
70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the u-PA activity of a
wild type or reference u-PA polypeptide, such as the corresponding
polypeptide not containing the amino acid modification (e.g. amino
acid replacement), for example, a u-PA protease domain set forth in
SEQ ID NO:5.
[0398] For example, exemplary positions that can be modified, for
example by amino acid replacement or substitution, include, but are
not limited to, any of positions corresponding to position 173,
178, 179, 180, 181, 185, 186, 187, 188, 208, 209, 249, 250, 252,
306, 314 or 353 with reference to the sequence of amino acids set
forth in SEQ ID NO:3 (corresponding to positions 30, 35, 36, 37,
37a, 38, 39, 40, 41, 60a, 60b, 97a, 97b, 99, 149, 157 or 192
according to chymotrypsin numbering). For example, the amino acid
positions can be replacements at positions corresponding to
replacement of phenylalanine (F) at one or more of positions 173,
R178, R179, H180, R181, V185, T186, Y187, V188, D208, Y209, T249,
L250, H252, Y306, M314 or Q353 with reference to amino acid
positions set forth in SEQ ID NO:3 (corresponding to F30, R35, R36,
H37, R37a, V38, T39, Y40, V41, D60a, Y60b, T97a, L97b, H99, Y149,
M157 and Q192, respectively according to chymotrypsin
numbering).
[0399] Exemplary amino acid replacements at any of the above
positions are set forth in Table 11. Reference to corresponding
position in Table 11 is with reference to positions set forth in
SEQ ID NO:3. (See, also the Examples, below). It is understood that
the replacements can be made in the corresponding position in
another u-PA polypeptide by alignment with the sequence set forth
in SEQ ID NO:3, whereby the corresponding position is the aligned
position. For example, the replacement can be made in the u-PA
protease domain with the sequence set forth in SEQ ID NO: 2 or a
reference u-PA protease domain with the sequence set forth in SEQ
ID NO: 5. In some examples, the amino acid replacement(s) can be at
the corresponding position in a u-PA polypeptide as set forth in
SEQ ID NO: 5 or a variant thereof having at least or at least about
75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, particularly 95%, or more
sequence identity thereto, so long as the resulting modified u-PA
polypeptide exhibits altered (i. e., enhanced) specificity towards
complement protein C3 compared to a u-PA activity towards
plasminogen and/or an altered selectivity for complement protein
C3. In one example, any one or more of the replacements are in any
of SEQ ID NOs: 1-6, so long as the resulting modified u-PA
polypeptide exhibits altered (i.e., enhanced) specificity towards
complement protein C3 compared to a u-PA activity towards
plasminogen and/or an altered selectivity for complement protein
C3.
TABLE-US-00013 TABLE 11 Exemplary mutations that result in
increased activity for cleavage of C3 Corresponding Position
Corresponding Position (in SEQ ID NO: 3) (chymotrypsin numbering)
Replacement 173 30 Y 178 35 W Y Q 179 36 H 180 37 E P D N G K Y 181
37a Q P E N S 185 38 D E 186 39 W Y F 187 40 H F Q 188 41 R L 208
60a P T 209 60b Q H S A T L 249 97a E I 250 97b A G 252 99 Q 279
122 S 306 149 K R 314 157 K 353 192 H
[0400] Exemplary of amino acid modifications in the modified u-PA
polypeptides provided herein include, but are not limited to,
replacement with tyrosine (Y) at a position corresponding to
position 173 (30 by chymotrypsin numbering); W at a position
corresponding to position 178 (35 by chymotrypsin numbering); Y at
a position corresponding to position 178; Q at a position
corresponding to position 178; H at a position corresponding to
position 179 (36 by chymotrypsin numbering); E at a position
corresponding to position 180 (37 by chymotrypsin numbering); P at
a position corresponding to position 180; D at a position
corresponding to position 180; N at a position corresponding to
position 180; G at a position corresponding to position 180; K at a
position corresponding to position 180; Y at a position
corresponding to position 180; Q at a position corresponding to
position 181 (37a by chymotrypsin numbering); P at a position
corresponding to position 181; E at a position corresponding to
position 181; N at a position corresponding to position 181; S at a
position corresponding to position 181; D at a position
corresponding to position 185 (38 by chymotrypsin numbering); E at
a position corresponding to position 185; W at a position
corresponding to position 186 (39 by chymotrypsin numbering); Y at
a position corresponding to position 186; F at a position
corresponding to position 186; H at a position corresponding to
position 187 (40 by chymotrypsin numbering); F at a position
corresponding to position 187; Q at a position corresponding to
position 187; R at a position corresponding to position 188 (41 by
chymotrypsin numbering); L at a position corresponding to position
188; P at a position corresponding to position 208; T at a position
corresponding to position 208 (60a by chymotrypsin numbering); Q at
a position corresponding to position 209 (60b by chymotrypsin
numbering); H at a position corresponding to position 209; S at a
position corresponding to position 209; A at a position
corresponding to position 209; T at a position corresponding to
position 209; L at a position corresponding to position 209; E at a
position corresponding to position 249 (97a by chymotrypsin
numbering); I at a position corresponding to position 249; A at a
position corresponding to position 250 (97b by chymotrypsin
numbering); G at a position corresponding to position 250; Q at a
position corresponding to position 252 (99 by chymotrypsin
numbering); K at a position corresponding to position 306 (149 by
chymotrypsin numbering); R at a position corresponding to position
306; K at a position corresponding to position 314 (157 by
chymotrypsin numbering); or H at a position corresponding to
position 353 (192 by chymotrypsin numbering); each with reference
to the amino acid positions set forth in SEQ ID NO:3. S at a
position corresponding to position 279 (122S) by chymotrypsin
numbering) replaces a free Cys to thereby reduce a tendency for
aggregation.
[0401] Exemplary modified u-PA polypeptides containing 2 or more
amino acid modifications are set forth in Table 12 below, and their
activity for cleaving C3 described in Table 14. The Sequence ID NO.
references an exemplary u-PA protease domain that contains the
recited replacements, which include the replacement at C122S to
reduce or eliminate aggregation. C122 is a free cysteine, which can
result in cross-linking among the protease polypeptides. It is
understood that the protease domain is exemplary, and full-length
and precursor molecules, as well as other catalytically active
portions of the protease domain, full-length and precursor
polypeptide can include the recited replacements, to form
full-length activated modified u-PA polypeptides and other
forms.
TABLE-US-00014 TABLE 12 modified u-PA polypeptides Exemplary SEQ ID
Mature u-PA numbering Chymotrypsin numbering NO
F173Y/V185D/Y187H/V188R/L250A/ F30Y/V38D/Y40H/V41R/L97bA/ 8
H252Q/C279S/M314K H99Q/C122S/M157K F173Y/R178W/R179H/H180E/V185E/
F30Y/R35W/R36H/H37E/V38E/T39W/ 9 T186W/Y187H/V188R/Y209Q/T249E/
Y40H/V41R/Y60bQ/T97aE/L97bA/ L250A/H252Q/C279S/Y306K/M314K
H99Q/C122S/Y149K/M157K F173Y/R178W/R179H/H180D/V185E/
F30Y/R35W/R36H/H37D/V38E/T39Y/ 10 T186Y/Y187F/V188R/T249I/L250A/
Y40F/V41R/T97aI/L97bA/H99Q/ H252Q/C279S/Y306R/M314K
C122S/Y149R/M157K R178W/R179H/H180N/V185E/T186F/
R35W/R36H/H37N/V38E/T39F/Y40F/ 11 Y187F/V188R/T249I/L250A/H252Q/
V41R/T97aI/L97bA/H99Q/C122S/ C279S/Y306R/M314K/Q353H
Y149R/M157K/Q192H F173Y/R178Y/R179H/H180K/V185E/
F30Y/R35Y/R36H/H37K/V38E/T39F/ 12 T186F/Y187F/V188R/T249I/L250A/
Y40F/V41R/T97aI/L97bA/H99Q/ H252Q/C279S/Y306R/M314K
C122S/Y149R/M157K F173Y/R178W/R179H/H180N/V185E/
F30Y/R35W/R36H/H37N/V38E/T39Y/ 13 T186Y/Y187F/V188R/Y209S/T249E/
Y40F/V41R/Y60bS/T97aE/L97bA/ L250A/H252Q/C279S/Y306K/M314K
H99Q/C122S/Y149K/M157K F173Y/R178W/R179H/H180P/V185E/
F30Y/R35W/R36H/H37P/V38E/T39Y/ 14 T186Y/Y187F/V188R/Y209S/T249E/
Y40F/V41R/Y60bS/T97aE/L97bA/ L250A/H252Q/C279S/Y306K/M314K
H99Q/C122S/Y149K/M157K V185E/Y187Q/V188L/Y209L/L250A/
V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/ 15 H252Q/C279S C122S
F173Y/R178Q/R179H/H180G/R181E/V185E/
F30Y/R35Q/R36H/H37G/R37aE/V38E/ 16 T186F/Y187F/V188R/D208P/Y209S/
T39F/Y40F/V41R/D60aP/Y60bS/ T249I/L250A/H252Q/C279S/Y306R/M314K
T97aI/L97bA/H99Q/C122S/Y149R/ M157K
F173Y/R178Y/R179H/H180P/R181Q/V185E/
F30Y/R35Y/R36H/H37P/R37aQ/V38E/ 17 T186Y/Y187F/V188R/Y209H/T249I/
T39Y/Y40F/V41R/Y60bH/T97aI/ L250A/H252Q/C279S/Y306R/M314K
L97bA/H99Q/C122S/Y149R/M157K R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 18 D208T/Y209T/T249I/L250A/H252Q/
D60aT/Y60bT/T97aI/L97bA/H99Q/ C279S/Y306R C122S/Y149R
R178W/H180P/R181N/V185E/T186Y/V188R/
R35W/H37P/R37aN/V38E/T39Y/V41R/ 19 D208P/Y209L/T249I/L250A/H252Q/
D60aP/Y60bL/T97aI/L97bA/H99Q/ C279S/Y306R C122S/Y149R
R178W/H180D/R181P/V185E/T186W/V188R/
R35W/H37D/R37aP/V38E/T39W/V41R/ 20 Y209A/T249I/L250A/H252Q/C279S/
Y60bA/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 21 D208P/Y209Q/T249I/L250A/H252Q/
D60aP/Y60bQ/T97aI/L97bA/H99Q/ C279S/Y306R C122S/Y149R
H180Y/R181E/V185E/T186Y/V188R/D208P/
H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 22 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/R181E/V185E/T186Y/V188R/D208P/
R35Q/R37aE/V38E/T39Y/V41R/D60aP/ 23 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/V185E/T186Y/V188R/D208P/
R35Q/H37Y/V38E/T39Y/V41R/D60aP/ 24 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/T186Y/V188R/D208P/
R35Q/H37Y/R37aE/T39Y/V41R/D60aP/ 25 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/V188R/D208P/
R35Q/H37Y/R37aE/V38E/V41R/D60aP/ 26 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/D208P/
R35Q/H37Y/R37aE/V38E/T39Y/D60aP/ 27 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 28 Y209Q/T249I/L250A/H252Q/C279S/
Y60bQ/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 29 D208P/T249I/L250A/H252Q/C279S/
D60aP/T97aI/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 30 D208P/Y209Q/L250A/H252Q/C279S/
D60aP/Y60bQ/L97bA/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 31 D208P/Y209Q/T249I/H252Q/C279S/
D60aP/Y60bQ/T97aI/H99Q/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 32 D208P/Y209Q/T249I/L250A/C279S/
D60aP/Y60bQ/T97aI/L97bA/C122S/ Y306R Y149R
R178Q/H180Y/R181E/V185E/T186Y/V188R/
R35Q/H37Y/R37aE/V38E/T39Y/V41R/ 33 D208P/Y209Q/T249I/L250A/H252Q/
D60aP/Y60bQ/T97aI/L97bA/H99Q/ C279S C122S
Y187Q/V188L/Y209L/L250A/H252Q/ Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 34
C279S V185E/Y187Q/Y209L/L250A/H252Q/
V38E/Y40Q/Y60bL/L97bA/H99Q/C122S 35 C279S
V185E/Y187Q/V188L/L250A/H252Q/ V38E/Y40Q/V41L/L97bA/H99Q/C122S 36
C279S V185E/Y187Q/V188L/Y209L/H252Q/
V38E/Y40Q/V41L/Y60bL/H99Q/C122S 37 C279S
V185E/Y187Q/V188L/Y209L/L250A/ V38E/Y40Q/V41L/Y60bL/L97bA/C122S 38
C279S Y187Q/V188L/L250A/H252Q/C279S Y40Q/V41L/L97bA/H99Q/C122S 39
Y187Q/V188L/L250A/C279S Y40Q/V41L/L97bA/C122S 40
R181S/V188R/L250G/H252Q/C279S R37aS/V41R/L97bG/H99Q/C122S 41
T186Y/V188R/L250A/H252Q/C279S T39Y/V41R/L97bA/H99Q/C122S 42
T186Y/V188R/Y209Q/L250A/H252Q/C279S
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S 43
T186Y/V188R/D208P/L250A/H252Q/C279S
T39Y/V41R/D60aP/L97bA/H99Q/C122S 44
[0402] 2. Additional Modifications
[0403] Any of the modified u-PA polypeptides provided herein can
contain any one or more additional modifications. The additional
modifications can include, for example, any amino acid
substitution, deletion or insertion known in the art, typically any
that increase specificity towards complement protein C3 compared to
u-PA activity towards plasminogen and/or alter selectivity for
complement protein C3. Also, contemplated are modifications that
alter any other activity of interest. It is long known in the art
that amino acid modifications of the primary sequence are additive
(see, e.g., Wells (1990) Biochem 29:8509-8517). Any modified u-PA
polypeptide provided herein can contain 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino
acid modifications to provide additional activities or alter
activities.
[0404] Examples of additional modifications that can be included in
the modified u-PA polypeptides provided herein include, but are not
limited to, those described in U.S. Pat. Nos. 4,997,766; 5,126,134;
5,129,569; 5,275,946; 5,571,708; 5,580,559; 5,648,253; 5,728,564;
5,759,542; 5,811,252; 5,891,664; 5,932,213; 5,980,886; 6,248,712;
6,423,685; 7,070,925; 7,074,401; 7,807,457; 7,811,771; and
8,211,428; U.S. Patent Publication Nos. 2002/0106775; 2004/0265298;
2004/0146938; 2009/0010916; 2011/0055940; 2008/0020416; and
2006/0142195; International Patent Publication Nos. WO1988/008451;
WO1989/010401; WO1990/004635; WO1996/013160; and WO 2002/40503;
Petersen et al. (2001) Eur J Biochem 268:4430-4439; Skeldal et al.
(2006) FEBS J 273:5143-5149; Sun et al. (1997) J Biol Chem
272:23818-23823; Blouse et al. (2009) J Biol Chem 284:4647-4657;
Nelles et al. (1987) JBC 262:5682-5689; Crowley et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:5021-5025; Zeslawska et al. (2000) J Mol
Biol 301:465-475; Zeslawska et al. (2003) J Mol Biol 328:109-118;
Quax et al. (1998) Arterioscler Thromb Vasc Biol 18:693-701;
Homandberg and Wai (1990) Thrombin Res 58:403-412; Zaitsev et al.
(2010) Blood 115:5241-5248; Yang et al. (1994) Biochemistry
33:606-612; Davidow et al. (1991) Protein Eng 4:923-928; Boutad and
Castellino (1993) Arch Biochem Biophys 303:222-230; Tsujikawa et
al. (1996) Yeast 12:541-553; Carriero et al. (2002) Biol Chem
383:107-113; Stopelli et al. (1985) Proc. Natl. Acad. Sci. U.S.A.
82:4939-4943; Stoppelli et al. (1987) J Biol Chem 262:4437-4440;
Franco et al. (1998)J Biol Chem 273:27734-27740; Franco et al.
(1997)J Cell Biol 137:779-791; Li et al. (1995) J Biol Chem
270:30282-30285; Botkjaer et al. (2009) Biochemistry 48:9606-9617;
Bdeir et al. (2003) Blood 102:3600-3608; Eguchi et al. (1990) J
Biochem 108:72-79; Miyake et al. (1988) J Biochem 104:643-647;
Bergstrom et al. (2003) Biochem 42:5395-5402; Sun and Liu (2005)
Proteins 61:870-877; Sun et al. (1998) Biochemistry 37:2935-2940;
Anderson et al. (2008) Biochem J 412:447-457; Li et al. (1992)
Biochim Biophys Acta 1159:37-43; Lijnen et al. (1988) Eur J Biochem
177:575-582; Lijnen et al. (1988) Eur J Biochem 172:185-188; Lijnen
et al. (1992) Eur J Biochem 205:701-709; Lijnen et al. (1994) Eur J
Biochem 224:567-574; Lijnen et al. (1990) J Biol Chem
265:5232-5236; Yoshimoto et al. (1996) Biochim Biophys Acta
1293:83-89; Magdolen et al. (1996) Eur J Biochem 237:743-751;
Nienaber et al. (2000) J Biol Chem 275:7239-7248; Gurewich et al.
(1988) J Clin Invest 1956-1962; Liu et al. (1996) Biochemistry
35:14070-14076; Liu et al. (2002) Circ Res 90:757-763; Mukhina et
al. (2000) J Biol Chem 275:16450-16458; Peng et al. (1997) Biochem
Mol Biol Int 41:887-894; Turkmen et al. (1997) Electrophoresis
18:686-689; Peng et al. (1999) Biotechnol Lett 21:979-985; Ueshima
et al. (1994) Thromb Haemost 71:134-140; and Melnick et al. (1990)
J Biol Chem 265:801-807. Non-limiting examples of exemplary amino
acid modifications described in the art include any one or more of
S9A, C13A, T18A, C19A, V20A, S21A, N22Y, N22A, N22Q, N22R, K23A,
K23H, K23Q, K23E, Y24A, F25A, S26A, S26F, N27A, N27R, I28A, H29A,
H29R, W30A, W30R, W30F, N32S, K35A, G38R, E43A, I44A, D45A, K46A,
S47A, S47G, K48A, K48P, T49A, Y51A, N54A, L80H, Q81R, Q82P, T83R,
H99Y, P105A, D106A, N107A, R108A, R108D, R109A, R110A, G118N,
L119R, K120R, K120A, P121L, L122T, L122R, V123Y, V123W, Q124A,
E125A, H129A, D130G, C131W, K135G, K135S, K135Y, K135Q, K136P,
S138E, C148S, C148A, K151E, T152A, R154G, R154P, R154A, P155R,
P155L, P155A, P155N, P155S, P155G, P155Q, R156P, R156A, R156H,
R156S, R156Y, R156E, R156G, R156L, F157L, F157T, F157G, F157Q,
F157D, F157E, K158R, K158E, K158A, K158H, K158S, K158Y, K158G,
K158W, K158V, K158M, I159R, I159A, I159P, I159G, I160A, I160K,
G162R, E163A, F164V, F164A, F164V, I167L, P171L, F173I, F173V,
F173L, F173T, F173G, F173M, A175S, Y177A, R178A, R179A, H180A,
R181A, S184A, T186A, T186E, T186D, Y187A, Y187H, V188A, S192N,
I194M, S195A, H204A, H204Q, F206A, D208A, Y209A, P210A, K211A,
K211Q, K212A, E213A, D214A, Y215A, I216A, Y218A, R221A, S222L,
R223G, R223A, L224A, L224P, N225A, S226P, N227A, Q229A, E231G,
K233E, K233A, F234A, E235K, E235A, E237A, I240V, K243E, K243A,
D244A, Y245A, D255A, R262A, K264A, E265A, R267A, C268Y, C279S,
C279A, F289L, G290D, E294G, I295T, G297D, F298A, G299A, G299H,
K300A, K300H, K300W, E301D, E301A, E301H, N302A, N302Q, N302V,
N302L, N302I, N302S, N302T, S303E, S303A, S303E, T304A, T304V,
T304M, D305A, Y306A, Y306G, Y306V, Y306H, L307A, Y308A, P309A,
P309S, P309T, P309V, P309G, P309N, P309L, P309D, P309R, P309H,
P309F, P309W, E310A, Q311A, L312P, L312V, L312M, K313Y, K313T,
K313A, K313H, T315A, T315I, V316A, V317A, Y330H, A343T, D344A,
Q346A, W347A, K348A, K348E, T349I, D350A, S351A, Q353A, G354R,
D355A, S356A, G357E, G366C, R378C, R378A, K383A, K385A, R400A,
H402A, K404A, E405A, E406A G408A, or A410V, according to the
sequence of amino acids set forth in SEQ ID NO:3. Additional
modifications include amino acid replacements that introduce a
glycosylation site.
[0405] The modified u-PA polypeptides include those that contain
chemical or post-translational modifications. In some examples,
modified u-PA polypeptides provided herein do not contain chemical
or post-translational modifications. Chemical and
post-translational modifications include, but are not limited to,
pegylation, sialation, albumination, glycosylation, farnysylation,
carboxylation, hydroxylation, PASylation, HESylation,
phosphorylation, linkage to a multimerization domain(s), such as
Fc, and other polypeptide modifications known in the art. In
addition to any one or more amino acid modifications, such as amino
acid replacements, insertions, deletions, and combinations thereof,
provided herein, modified u-PA polypeptides provided herein can be
conjugated or fused to any moiety using any method known in the
art, including chemical and recombinant methods, providing the
resulting polypeptide, when in active form, retains the ability to
effect inhibitory or inactivation cleavage of complement protein
C3.
[0406] For example, in addition to any one or more amino acid
modifications, such as amino acid replacements, provided herein,
modified u-PA polypeptides provided herein also can contain other
modifications that are or are not in the primary sequence of the
polypeptide, including, but not limited to, modification with a
carbohydrate moiety, a polyethylene glycol (PEG) moiety, a silation
moiety, an Fc domain from immunoglobulin G, or any other domain or
moiety. For example, such additional modifications can be made to
increase the stability or serum half-life of the protein.
[0407] a. Decreased Immunogenicity
[0408] The modified u-PA polypeptides provided herein can be
modified to have decreased immunogenicity. Decreased immunogenicity
can be effected by sequence changes that eliminate antigenic
epitopes from the polypeptide or by altering post-translational
modifications. One of skill in the art is familiar with methods of
identifying antigenic epitopes in a polypeptide (see e.g. Liang et
al. (2009) BMC Bioinformatics, 10:302; Yang et al. (2009) Rev. Med.
Virol., 19:77-96). In some examples, one or more amino acids can be
modified in order to remove or alter an antigenic epitope. In
another example, altering the glycosylation of a protein also can
affect immunogenicity. For example, altering the glycosylation of
the peptide is contemplated, so long as the polypeptides retain the
ability to effect inhibitory or inactivation cleavage of complement
protein C3. Glycosylation sites can be removed by single mutations.
Glycosylation sites can be added by introducing a canonical
sequence, such as by insertion or single or a plurality of
mutations, such as NXS(T), where X is not a proline. Glycosylation
sites also can increase serum half-life.
[0409] b. Fc Domain
[0410] The modified u-PA polypeptides can be linked to the Fc
region of an immunoglobulin polypeptide. Typically, such a fusion
retains at least a functionally active hinge, C.sub.H2 and C.sub.H3
domains of the constant region of an immunoglobulin heavy chain.
For example, a full-length Fc sequence of IgG1 includes amino acids
99-330 of the sequence set forth in the SEQ ID NO: 45 below.
TABLE-US-00015 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230
235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys. 325 330 An exemplary Fc sequence for hIgG1
is set forth in SEQ ID NO: 50: Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 1 5 10 15 Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45 Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60 Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 65 70
75 80 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 85 90 95 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 100 105 110 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 115 120 125 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys 130 135 140 Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 145 150 155 160 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195
200 205 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 210 215 220 Leu Ser Leu Ser Pro Gly Lys 225 230
It contains almost all of the hinge sequence corresponding to amino
acids 100-110 of SEQ ID NO:45; the complete sequence for the
C.sub.H2 and C.sub.H3 domain as set forth in SEQ ID NO:45.
[0411] Another exemplary Fc polypeptide is set forth in PCT
application Publication No. WO 93/10151, and is a single chain
polypeptide extending from the N-terminal hinge region to the
native C-terminus of the Fc region of a human IgG1 antibody (SEQ ID
NO:50). The precise site at which the linkage is made is not
critical: particular sites are well known and can be selected in
order to optimize the biological activity, secretion, or binding
characteristics of the HABP polypeptide. For example, other
exemplary Fc polypeptide sequences begin at amino acid C109 or P113
of the sequence set forth in SEQ ID NO: 45 (see e.g., U.S. Pub. No.
2006/0024298).
[0412] In addition to hIgG1 Fc, other Fc regions and other
multimerization domains also can be used. For example, where
effector functions mediated by Fc/Fc.gamma.R interactions are to be
minimized, fusion with IgG isotypes that poorly recruit complement
or effector cells, such as for example, the Fc of IgG2 or IgG4, is
contemplated. Additionally, the Fc fusions can contain
immunoglobulin sequences that are substantially encoded by
immunoglobulin genes belonging to any of the antibody classes,
including, but not limited to IgG (including human subclasses IgG1,
IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and
IgA2), IgD, IgE, and IgM classes of antibodies. Linkers can be used
to covalently link Fc to another polypeptide to generate an Fc
chimera.
[0413] Modified Fc domains also are well known. In some examples,
the Fc region is modified such that it exhibits altered binding to
an FcR to result in altered (i.e. more or less) effector function
than the effector function of an Fc region of a wild-type
immunoglobulin heavy chain. Thus, a modified Fc domain can have
altered affinity, including but not limited to, increased or low or
no affinity for the Fc receptor. For example, the different IgG
subclasses have different affinities for the Fc.gamma.Rs, with IgG1
and IgG3 typically binding substantially better to the receptors
than IgG2 and IgG4. Different Fc.gamma.Rs mediate different
effector functions. Fc.gamma.R1, Fc.gamma.RIIa/c, and
Fc.gamma.RIIIa are positive regulators of immune complex triggered
activation, characterized by having an intracellular domain that
has an immunoreceptor tyrosine-based activation motif (ITAM).
Fc.gamma.RIIb, however, has an immunoreceptor tyrosine-based
inhibition motif (ITIM) and is therefore inhibitory. Altering the
affinity of an Fc region for a receptor can modulate the effector
functions and/or pharmacokinetic properties associated by the Fc
domain. Modified Fc domains are known to one of skill in the art
and described in the literature, see e.g. U.S. Pat. No. 5,457,035;
U.S. Patent Publication No. US 2006/0024298; and International
Patent Publication No. WO 2005/063816 for exemplary
modifications.
[0414] The resulting chimeric polypeptides containing Fc moieties,
and multimers formed therefrom, can be easily purified by affinity
chromatography over Protein A or Protein G columns.
[0415] In another example, the modified u-PA polypeptide can be
linked to human serum albumin (HSA), such as residues 25-608 of
HSA, or the full length, or portion thereof:
TABLE-US-00016 10 20 30 40 MKWVTFISLL FLFSSAYSRG VFRRDAHKSE
VAHRFKDLGE 50 60 70 80 ENFKALVLIA FAQYLQQCPF EDHVKLVNEV TEFAKTCVAD
90 100 110 120 ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP 130 140
150 160 ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK 170 180 190 200
KYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA 210 220 230 240
CLLPKLDELR DEGKASSAKQ GLKCASLQKF GERAFKAWAV 250 260 270 280
ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD 290 300 310 320
RADLAKYICE NQDSISSKLK ECCEKPLLEK SHCIAEVEND 330 340 350 360
EMPADLPSLA ADFVGSKDVC KNYAEAKDVF LGMFLYEYAR 370 380 390 400
RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE 410 420 430 440
FKPLVEEPQN LIKQNCELFE QLGEYKFQNA LLVRYTKKVP 450 460 470 480
QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDCLSVF 490 500 510 520
LNQLCVLHEK TPVSDRVTKC CTESLVNGRP CFSALEVDET 530 540 550 560
YVPKEFNAET FTFHADICTL SEKERQIKKQ TALVELVKHK 570 580 590 600
PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV AASQAALGL
[0416] c. Conjugation to Polymers
[0417] In some examples, the modified u-PA polypeptides provided
herein are conjugated to other polymers. Polymers can increase the
size of the polypeptide to reduce kidney clearance and thereby
increase half-life or to modify the structure of the polypeptide to
increase half-life or reduce immunogenicity. Exemplary polymers
that can be conjugated to the u-PA polypeptides include natural and
synthetic homopolymers, such as polyols (i.e. poly-OH), polyamines
(i.e. poly-NH2) and polycarboxylic acids (i. e. poly-COOH), and
other heteropolymers i.e. polymers comprising one or more different
coupling groups e.g. a hydroxyl group and amine groups. Examples of
suitable polymeric molecules include polymeric molecules selected
from among polyalkylene oxides (PAO), such as polyalkylene glycols
(PAG), including polyethylene glycols (PEG), methoxypolyethylene
glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers
(Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched
polyethylene glycols (PEGs), polyvinyl alcohol (PVA),
polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids,
polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid
anhydride, dextrans including carboxymethyl-dextrans, heparin,
homologous albumin, celluloses, including methylcellulose,
carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,
carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of
chitosan, starches such as hydroxyethyl-starches and
hydroxypropyl-starches, glycogen, agaroses and derivatives thereof,
guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin,
alginic acid hydrolysates and biopolymers.
[0418] Typically, the polymers are polyalkylene oxides (PAO), such
as polyethylene oxides, such as PEG, typically mPEG, which have few
reactive groups capable of cross-linking. Typically, the polymers
are non-toxic polymeric molecules such as (methoxy)polyethylene
glycol (mPEG) which can be covalently conjugated to the u-PA
polypeptides (e.g., to attachment groups on the protein surface)
using a relatively simple chemistry.
[0419] Suitable polymeric molecules for attachment to the u-PA
polypeptides include, but are not limited to, polyethylene glycol
(PEG) and PEG derivatives such as methoxy-polyethylene glycols
(mPEG), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole
(CDI-PEG), branched PEGs, and polyethylene oxide (PEO) (see, e.g.,
Roberts et al., Advanced Drug Delivery Review 2002, 54: 459-476;
Harris and Zalipsky (eds.) "Poly(ethylene glycol), Chemistry and
Biological Applications" ACS Symposium Series 680, 1997; Mehvar et
al., J. Pharm. Pharmaceut. Sci., 3(1): 125-136, 2000; Harris and
Chess (2003) Nat Rev Drug Discov. 2(3):214-21; and Tsubery, J Biol.
Chem 279(37):38118-24, 2004). The polymeric molecule can be of a
molecular weight typically ranging from about 3 kDa to about 60
kDa. In some embodiments the polymeric molecule that is conjugated
to a U-PA polypeptide provided herein has a molecular weight of 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 kDa.
[0420] Methods of modifying polypeptides by covalently attaching
(conjugating) a PEG or PEG derivative (i.e. "PEGylation") are well
known in the art (see, e.g., U.S. 2006/0104968; U.S. Pat. Nos.
5,672,662; 6,737,505; and U.S. 2004/0235734). Techniques for
PEGylation include, but are not limited to, specialized linkers and
coupling chemistries (see, e.g., Harris, Adv. Drug Deliv. Rev.
54:459-476, 2002), attachment of multiple PEG moieties to a single
conjugation site (such as via use of branched PEGs; see, e.g.,
Veronese et al., Bioorg. Med. Chem. Lett. 12:177-180, 2002),
site-specific PEGylation and/or mono-PEGylation (see, e.g., Chapman
et al., Nature Biotech. 17:780-783, 1999), and site-directed
enzymatic PEGylation (see, e.g, Sato, Adv. Drug Deliv. Rev.,
54:487-504, 2002) (see, also, for example, Lu and Felix (1994) Int.
J. Peptide Protein Res. 43:127-138; Lu and Felix (1993) Peptide
Res. 6:142-6, 1993; Felix et al. (1995) Int. J. Peptide Res.
46:253-64; Benhar et al. (1994) J. Biol. Chem. 269:13398-404;
Brumeanu et al. (1995)J Immunol. 154:3088-95; see also, Caliceti et
al. (2003) Adv. Drug Deliv. Rev. 55(10):1261-77 and Molineux (2003)
Pharmacotherapy 23 (8 Pt 2):3S-8S). Methods and techniques
described in the art can produce proteins having 1, 2, 3, 4, 5, 6,
7, 8, 9, 10 or more than 10 PEG or PEG derivatives attached to a
single protein molecule (see, e.g, U.S. 2006/0104968).
[0421] Numerous reagents for PEGylation have been described in the
art. Such reagents include, but are not limited to,
N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG,
mPEG2-N-hydroxysuccinimide, mPEG succinimidyl
alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG
succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid
succinimidyl ester, homobifunctional PEG-succinimidyl propionate,
homobifunctional PEG propionaldehyde, homobifunctional PEG
butyraldehyde, PEG maleimide, PEG hydrazide,
p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate,
propionaldehyde PEG, mPEG butryaldehyde, branched mPEG2
butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone,
mPEG "linkerless" maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG
orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS,
Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluorescein
PEG-NHS, and biotin PEG-NHS (see, e.g, Monfardini et al.,
Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. Bioactive
Compatible Polymers 12:197-207, 1997; U.S. Pat. Nos. 5,672,662;
5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614;
5,183,550; 5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581;
5,795,569; 5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237;
6,113,906; 6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339;
6,437,025; 6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S.
2001/0021763; U.S. 2001/0044526; U.S. 2001/0046481; U.S.
2002/0052430; U.S. 2002/0072573; U.S. 2002/0156047; U.S.
2003/0114647; U.S. 2003/0143596; U.S. 2003/0158333; U.S.
2003/0220447; U.S. 2004/0013637; US 2004/0235734; U.S. 2005/000360;
U.S. 2005/0114037; U.S. 2005/0171328; U.S. 2005/0209416; EP
01064951; EP 0822199; WO 00176640; WO 0002017; WO 0249673; WO
9428024; and WO 0187925).
[0422] d. Protein Transduction Domain
[0423] The modified u-PA polypeptides provided herein can be
linked, such as a fusion protein containing an antibody, or antigen
binding fragment thereof, conjugated to a protein transduction
domain (PTD) that increases the retention of the antibody at a
target site for therapy, such as a mucosal site, such as the eye.
Any PTD can be employed so long as the PTD promotes the binding to
target cell surfaces at the therapeutic site (e.g. mucosal site)
and/or uptake of the modified u-PA polypeptide by target cells at
the therapeutic site (e.g. mucosal site, such as the eye).
[0424] Generally, PTDs include short cationic peptides that can
bind to the cell surface through electrostatic attachment to the
cell membrane and can be uptaken by the cell by membrane
translocation (Kabouridis (2003) TRENDS Biotech 21(11) 498-503).
The PTDs provided generally interact with a target cell via binding
to glycosaminoglycans (GAGs), such as for example, hyaluronic acid,
heparin, heparan sulfate, dermatan sulfate, keratin sulfate or
chondroitin sulfate and their derivatives.
[0425] The protein transduction domain can be of any length.
Generally the length of the PTD ranges from 5 or about 5 to 100 or
about 100 amino acids in length. For example, the length of the PTD
can range from 5 or about 5 to 25 or about 25 amino acids in
length. In some examples, the PTD is 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in
length.
[0426] A single PTD or a plurality thereof can be conjugated to a
modified u-PA polypeptide. These are advantageously employed for
treatment of ocular or ophthalmic disorders, such as diabetic
retinopathies or macular degeneration, including AMD. For example,
multiple copies of the same PTD (e.g., dimer, trimer, tetramer,
pentamer, hexamer, heptamer, octamer, nonamer, decamer or larger
multimer) or different PTDs can be conjugated to the modified u-PA
polypeptide.
[0427] Several proteins and their peptide derivatives possess cell
internalization properties. Exemplary PTDs are known in the art and
include, but are not limited to, PTDs listed in the Table below,
including, for example, PTDs derived from human immunodeficiency
virus 1 (HIV-1) TAT (SEQ ID NOS: 125-135; Ruben et al. (1989) J.
Virol. 63:1-8), the herpes virus tegument protein VP22 (SEQ ID NO:
140; Elliott and O'Hare (1997) Cell 88:223-233), the homeotic
protein of Drosophila melanogaster Antennapedia (Antp) protein
(Penetratin PTD; SEQ ID NO: 112; Derossi et al. (1996)J. Biol.
Chem. 271:18188-18193), the protegrin 1 (PG-1) anti-microbial
peptide SynB (e.g., SynB1 (SEQ ID NO: 121), SynB3 (SEQ ID NO: 122),
and SynB4 (SEQ ID NO: 123); Kokryakov et al. (1993) FEBS Lett.
327:231-236) and the Kaposi fibroblast growth factor (SEQ ID NO:
105; Lin et al., (1995) J. Biol. Chem. 270-14255-14258).
[0428] Other proteins and their peptide derivatives have been found
to possess similar cell internalization properties. The carrier
peptides that have been derived from these proteins show little
sequence homology with each other, but are all highly cationic and
arginine or lysine rich. Indeed, synthetic poly-arginine peptides
have been shown to be internalized with a high level of efficiency
and can be selected for conjugation to can antibody provided
(Futaki et al. (2003) J. Mol. Recognit. 16:260-264; Suzuki et al.
(2001) J. Biol. Chem. 276:5836-5840). The PTD also can be selected
from among one or more synthetic PTDs, including but not limited
to, transportan (SEQ ID NO: 136; Pooga et al. (1988) FASEB J.
12:67-77; Pooga et al. (2001) FASEB J. 15:1451-1453), MAP (SEQ ID
NO: 103; Oehlke et al. (1998) Biochim. Biophys. Acta.
1414:127-139), KALA (SEQ ID NO: 101; Wyman et al. (1997)
Biochemistry 36:3008-3017) and other cationic peptides, such as,
for example, various .beta.-cationic peptides (Akkarawongsa et al.
(2008) Antimicrob. Agents and Chemother. 52(6):2120-2129).
Additional PTD peptides and variant PTDs also are provided in, for
example, U.S. Patent Publication Nos. US 2005/0260756, US
2006/0178297, US 2006/0100134, US 2006/0222657, US 2007/0161595, US
2007/0129305, European Patent Publication No. EP 1867661, PCT
Publication Nos. WO 2000/062067, WO 2003/035892, WO 2007/097561, WO
2007/053512 and Table 13 herein (below). Any such PTDs provided
herein or known in the art can be conjugated to a provided
therapeutic antibody.
TABLE-US-00017 TABLE 13 Known Protein Transduction Domains Protein
Transduction SEQ Domain (PTD) Source Protein ID NO
TRSSRAGLQFPVGRVHRLLRK Buforin II 82 RKKRRRESRKKRRRES DPV3 83
GRPRESGKKRKRKRLKP DPV6 84 GKRKKKGKLGKKRDP DPV7 85 GKRKKKGKLGKKRPRSR
DPV7b 86 RKKRRRESRRARRSPRHL DPV3/10 87 SRRARRSPRESGKKRKRKR DPV10/6
88 VKRGLKLRHVRPRVTRMDV DPV1047 89 VKRGLKLRHVRPRVTRDV DPV1048 90
SRRARRSPRHLGSG DPV10 91 LRRERQSRLRRERQSR DPV15 92
GAYDLRRRERQSRLRRRERQS DPV15b 93 R WEAALAEALAEALAEHLAEAL GALA 94
AEALEALAA KGSWYSMRKMSMKIRPFFPQQ Fibrinogen beta 95 chain
KTRYYSMKKTTMKIIPFNRL Fibrinogen gamma 96 chain precursor
RGADYSLRAVRMKIRPLVTQ Fibrinogen alpha 97 chain
LGTYTQDFNKFHTFPQTAIGV hCT(9-32) 98 GAP TSPLNIHNGQKL HN-1 99
NSAAFEDLRVLS Influenza virus 100 nucleoprotein (NLS)
WEAKLAKALAKALAKHLAKAL KALA 101 AKALKACEA VPMLKPMLKE Ku70 102
KLALKLALKALKAALKLA MAP 103 GALFLGFLGAAGSTMGAWSQP MPG 104 KKKRKV
AAVALLPAVLLALLAP Human Fibroblast 105 growth factor 4 (Kaposi
Fibroblast growth factor) VQRKRQKLM N50 (NLS of NF-kB 106 P50)
KETWWETWWTEWSQPKKKRKV Pep-1 107 SDLWEMMMVSLACQY Pep-7 108
RQIKIWFQNRRMKWKK Penetratin 109 GRQIKIWFQNRRMKWKK Penetratin
variant 110 RRMKWKK Short Penetratin 111 ERQIKIWFQNRRMKWKK
Penetratin 42-58 112 RRRRRRR Poly Arginine-R7 113 RRRRRRRRR Poly
Arginine-R9 114 RVIRVWFQNKRCKDKK pISL 115 MANLGYWLLALFVTMWTDVGL
Prion mouse PrPc1- 116 CKKRPKP 28 LLIILRRRIRKQAHAHSK pVEC 117
LLIILRRRIRKQAHAH pVEC variant 118 VRLPPPVRLPPPVRLPPP SAP 119
PKKKRKV SV-40 (NLS) 120 RGGRLSYSRRRFSTSTGR SynB1 121 RRLSYSRRRF
SynB3 122 AWSFRVSYRGISYRRSR SynB4 123 YGRKKRRQRRRPPQ Tat 47-60 124
YGRKKRRQRRR Tat 47-57 125 YGRKKRRQRR Tat 47-56 126 GRKKRRQRR Tat
48-56 127 GRKKRRQRRR Tat 48-57 128 RKKRRQRRR Tat 49-57 129 RKKRRQRR
Tat 49-56 130 GRKKRRQRRRPPQ Tat 48-60 131 GRKKR Tat 48-52 132
CFITKALGISYGRKKRRQRRR Tat 37-72 133 PPQFSQTHQVSLSKQ
FITKALGISYGRKKRRQRRRP Tat 38-72 134 QFSQTHQVSLSKQ YGRKKRRQRRRPP Tat
47-59 135 GWTLNSAGYLLGKINLKALAA Transportan 136 LAKKIL
AGYLLGKINLKALAALAKKIL Transportan 10 137 GWTLNSAGYLLG Transportan
138 derivative INLKALAALAKKIL Transportan 139 derivative
DAATATRGRSAASRPTERPRA VP22 140 PARSASRPRRPVD DPKGDPKGVTVTVTVTVTGKG
VT5 141 DPKPD GALFLGWLGAAGSTMGAWSQP Signal Sequence- 142 KKKRKV
based peptide KLALKLALKALKAALKLA Amphiphilic 143 model peptide
KFFKFFKFFK Bacterial cell 144 wall permeating LLGDFFRKSKEKIGKEFKRIV
LL-37 145 QRIKDFLRNLVPRTES SWLSKTAKKLENSAKKRISEG Cecropin P1 146
IAIAIQGGPR ACYCRIPACIAGERRYGTCIY alpha defensin 147 QGRLWAFCC
DHYNCVSSGGQCLYSACPIFT beta defensin 148 KIQGTCYRGKAKCCK RKCRIWIRVCR
Bactenecin 149 RRRPRPPYLPRPRPPPFFPPR PR-39 150
LPPRIPPGFPPRFPPRFPGKR ILPWKWPWWPWRR Indolicidin 151
GALFLGWLGAAGSTMGAWSQP MPS 152 KKKRKV PVIRRVWFQNKRCKDKK pIs1 153
[0429] In some examples, the PTDs can be modified by replacement of
a lysine or arginine with another basic amino acid, such as
replacement of a lysine with an arginine or by replacement of an
arginine with a lysine.
E. ASSAYS TO ASSESS OR MONITOR U-PA ACTIVITY ON COMPLEMENT-MEDIATED
FUNCTIONS
[0430] The modified u-PA polypeptides provided herein exhibit
altered specificity and/or selectivity for complement protein C3.
Exemplary modified u-PA polypeptides specifically cleave complement
protein C3 and thereby alter complement activation. Further,
exemplary modified u-PA polypeptides provided herein have altered,
or reduced, specificity and/or selectivity for cleavage of natural
substrates of u-PA, such as plasminogen, and binding to uPAR.
[0431] Various in vitro and in vivo assays can be used to monitor
or screen u-PA polypeptides for their ability to cleave complement
protein C3 and for their effects on complement activation and
complement-mediated diseases and disorders. Such assays are well
known to those of skill in the art. One of skill in the art can
test a particular u-PA polypeptide for cleavage of complement
protein C3 and/or test to assess any change in the effects of a
u-PA on a complement-mediated activity compared to the absence of a
protease. Some such assays are exemplified herein.
[0432] Exemplary in vitro and in vivo assays are provided herein
for comparison of an activity of a modified u-PA polypeptide on the
function of complement protein C3. As discussed below, numerous
assays, such as assays for measuring complement activation, are
known to one of skill in the art. Also provided herein are
exemplary assays for determining the activity of the modified u-PA
polypeptides for wild type u-PA activities, such as cleavage of
plasminogen or binding to uPAR. Also provided are assays for
determining the specificity of the modified u-PA polypeptides for
complement protein C3. Exemplary assays are described below.
[0433] 1. Methods for Assessing Effects of u-PA on Complement
Protein C3 Activity
[0434] A modified u-PA 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, such as, for example, C3, compared to the corresponding
full-length, scaffold or wild-type form of the modified u-PA
protease. Modified u-PA proteases retain their protease activity,
but can exhibit an increased specificity and/or selectivity to any
one or more complement proteins. Exemplary modified u-PA proteases
specifically cleave any one or more complement protein, such as,
for example, C3, and thereby alter the activity of a complement
protein. All such modified u-PA proteases with increased
specificity and/or selectivity to any one or more complement
protein are candidate therapeutics.
[0435] Where the modified u-PA 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 modified u-PA protease for cleavage of any one
or more complement protein, such as, for example, C3, and/or test
to assess any change in the effects of a modified u-PA protease on
a complement-mediated activity compared to the absence of a
modified u-PA protease. Some such assays are exemplified
herein.
[0436] Exemplary in vitro and in vivo assays are provided herein
for comparison of an activity of a modified u-PA protease on the
function of any one or more targeted complement proteins. Many of
the assays are applicable to other proteases and modified
proteases. As discussed above, 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.
[0437] In one example, in vitro assays can be performed using
purified complement protein C3, as exemplified in Example 2-4. In
another example, in vitro assays can be conducted in
physiologically relevant solutions (i.e., vitreous humor), as
exemplified in Example 5. In another example, in vitro assays can
be performed using peptide libraries to assess cleavage
specificity. In another example, assays can be conducted to assess
the normal functions of the modified u-PA polypeptides, i.e.,
activity towards normal substrates. Various disease models known to
one of skill in the art can be used to test the efficacy of u-PA
polypeptides provided herein on various complement-mediated
diseases and disorders.
[0438] a. Protein Detection
[0439] 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 u-PA polypeptide on
cleavage of complement protein C3, or alternatively, complement
proteins can be measured as a means to assess complement
activation. Complement protein C3, treated in the presence or
absence of a u-PA polypeptide, 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.
[0440] i. SDS-PAGE Analysis
[0441] Analysis of complement proteins in the presence or absence
of increasing concentrations of a u-PA polypeptide 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 Coomassie 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, complement protein C3,
can be incubated in the presence or absence of a u-PA polypeptide.
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.
[0442] In another embodiment, a sample, such as for example human
serum or plasma, can be treated in the presence or absence of a
u-PA polypeptide or can be collected after treatment of an animal
or a human with or without a u-PA polypeptide. The u-PA-treated
sample can be analyzed on SDS-PAGE and a specific complement
protein can be detected, such as for example C3, 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 u-PA polypeptide.
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 u-PA polypeptide. 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, Bb, and C5-b9. For
example, decreased expression in a sample of Bb can indicate that a
u-PA polypeptide 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 u-PA polypeptide on the cleavage of
complement effector molecules themselves.
[0443] ii. Enzyme Immunoassay
[0444] 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 u-PA polypeptides 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 u-PA polypeptide 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 u-PA polypeptide. 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.
[0445] 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, C5b-9, C3a,
C3a-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.
[0446] 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 percent cleavage can be
determined.
[0447] iii. Radial Immunodiffusion (RID)
[0448] 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 u-PA polypeptide. 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, C3, 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.
[0449] b. Hemolytic Assays
[0450] 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 u-PA polypeptide, or can be
collected following treatment of an animal or human in the presence
or absence of a u-PA polypeptide. 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.
[0451] 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 C3, can be incubated in the presence
or absence of increasing concentrations of a u-PA polypeptide. 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.
[0452] 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.
[0453] c. Methods for Determining Cleavage Sites
[0454] Cleavage sequences in complement protein C3 can be
identified by any method known in the art (see e.g., published U.S.
Publication No. US 2004/0146938). In one example, a cleavage
sequence is determined by incubating complement protein C3 with any
modified u-PA polypeptide provided herein. Following incubation
with the u-PA polypeptide, the C3 protein can be separated by
SDS-PAGE and degradative products can be identified by staining
with a protein dye such as Coomassie Brilliant Blue. Proteolytic
fragments can be sequenced to determine the identity of the
cleavage sequences. After identification, fluorogenic peptide
substrates designed based on the cleavage sequence of a desired
target substrate can be used to assess activity, as described
below.
[0455] 2. Methods for Assessing Wild Type u-PA Activity
[0456] The modified u-PA polypeptides provided herein have altered,
or reduced, specificity for plasminogen and uPAR. u-PA polypeptides
can be tested to determine whether they retain catalytic efficiency
and/or substrate specificity for their native substrate
plasminogen. For example, cleavage of plasminogen can be assessed
by incubation of a u-PA polypeptide with plasminogen and detecting
protein cleavage products. In another example, cleavage of
plasminogen can be determined in vitro by measuring cleavage of a
fluorogenically tagged tetrapeptide of the peptide substrate, for
example, a fluorogenic substrate, such as fluorophores ACC
(7-amino-4-carbamoylmethylcoumarin) or AMC
(7-amino-4-methylcoumarin) linked to a tetrapeptide substrate. In
some examples, plasminogen activation assays are used to determine
the specificity of the u-PA polypeptides provided herein. In other
examples, the ability of the u-PA polypeptides provided herein to
bind to the u-PA receptor (uPAR) is determined.
[0457] a. Cleavage of Plasminogen
[0458] In one example, modified u-PA polypeptides can be assayed
using individual fluorogenic peptide substrates corresponding to
the desired cleavage sequence. For example, a method of assaying
for a modified u-PA protease that can cleave any one or more of the
desired cleavage sequences includes: (a) contacting a peptide
fluorogenic sample (containing a desired target cleavage sequence)
with a protease, in such a manner whereby a fluorogenic moiety is
released from a peptide substrate sequence upon action of the
protease, thereby producing a fluorescent moiety; and (b) observing
whether the sample undergoes a detectable change in fluorescence,
the detectable change being an indication of the presence of the
enzymatically active protease in the sample. In such an example,
the desired cleavage sequence is made into a fluorogenic peptide by
methods known in the art. In one embodiment, the individual peptide
cleavage sequences can be attached to a fluorogenically tagged
substrate, such as for example an ACC or AMC fluorogenic leaving
group, and the release of the fluorogenic moiety can be determined
as a measure of specificity of a protease for a peptide cleavage
sequence. The rate of increase in fluorescence of the target
cleavage sequence can be measured such as by using a fluorescence
spectrophotometer. The rate of increase in fluorescence can be
measured over time. Michaelis-Menton kinetic constants can be
determined by the standard kinetic methods. The kinetic constants
k.sub.cat, K.sub.m and k.sub.cat/K.sub.m can be calculated by
graphing the inverse of the substrate concentration versus the
inverse of the velocity of substrate cleavage, and fitting to the
Lineweaver-Burk equation
(1/velocity=(K.sub.m/V.sub.max)(1/[S])+1/V.sub.max; where
V.sub.max=[E.sub.T]k.sub.cat). The second order rate constant or
specificity constant (k.sub.cat/K.sub.m) is a measure of how well a
substrate is cut by a particular protease. For example, an ACC- or
AMC-tetrapeptide such as Ac-CPGR-AMC can be made and incubated with
a modified u-PA polypeptide provided herein and activity of the
u-PA polypeptide can be assessed by assaying for release of the
fluorogenic moiety. The choice of the tetrapeptide depends on the
desired cleavage sequence to target and can be empirically
determined.
[0459] In other embodiments, u-PA polypeptides also can be assayed
to ascertain that, when in an active form, they cleave the desired
sequence when presented in the context of the full-length protein.
In one example, a purified target protein, i.e. plasminogen, can be
incubated in the presence or absence of a selected u-PA polypeptide
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.
[0460] b. Plasminogen Activation Assays Any assay known to one of
skill in the art can be used to determine if the u-PA polypeptides
activate plasminogen. In one example, activation of plasminogen can
be determined by incubating the polypeptides in the presence of
plasminogen and a detectable plasmin substrate, such as, for
example, the chromogenic substrate H-D-Val-Leu-Lys-p-nitroanalide
(Chromogenix S-2251) or the fluorogenic substrate
H-D-Val-Leu-Lys-7-amido-4-methylcoumarin. Hydrolysis is then
monitored by measuring absorbance at 405 nm or by detecting
fluorescence using a fluorescence plate reader with an excitation
wavelength of 390 nm and an emission wavelength of 480 nm. In
another example, activation of plasminogen is assessed while the
u-PA polypeptides are bound to uPAR. In such example, the u-PA
polypeptides are first bound to uPAR on a cell surface, such as a
U397 cell, followed by addition of plasminogen and a detectible
plasmin substrate and hydrolysis is measured as described
above.
[0461] c. u-PA-uPAR Binding Assays
[0462] Binding of the u-PA polypeptides to uPAR can be assessed by
any assay known to one of skill in the art to detect
protein-protein binding interactions, including, but not limited
to, solid phase binding assays, ELISA, surface plasmon resonance
and FACS. In one example, ELISA can be used. The recombinant uPAR
is immobilized on a microtiter plate and u-PA polypeptide binding
is assessed by addition of a reagent that specifically binds to
u-PA, such as, for example, a u-PA binding antibody. In another
example, binding can be determined in a cell based assay using a
cell line, such as, for example, U397 cells, that expresses the
u-PA receptor. The u-PA polypeptides can be labeled, for example,
with a chromogenic, fluorogenic or radioactive substrate to effect
detection of binding.
[0463] d. C3 Cleavage
[0464] The activity of the modified uPA polypeptides can be
assessed by cleavage of the substrate complement protein human C3
by measuring the amount of intact human C3 remaining after
incubation with various concentrations of the modified uPA
protease. In accord with this assay, signal is generated in the
presence of intact human C3, and is lost as the C3 is cleaved. In
other examples, C3 activation assays are used to determine the
specificity of the modified uPA polypeptides provided herein.
[0465] Purified C3 protein can be incubated with the modified u-PA
polypeptides and the residual levels of undigested human C3 can be
quantified by any assay known in the art to assess protein
concentration, such as, for example using an Amplified Luminescent
Proximity Homogeneous Assay Screen (AlphaScreen.RTM.; Perkin
Elmer). The C3/uPA polypeptide mixture is incubated with a-mouse
IgG-coated acceptor beads, and following incubation the a-hC3
mAb/acceptor beads mixture is incubated with a biotinylated a-hC3
pAb. Streptavidin-coated donor beads are added to the mixture and
the `alphascreen` signal (Excitation=680 nm, Emission=570 nm) is
then measured. This signal corresponds to the concentration of
remaining C3 protein. The concentration of uPA polypeptide required
to cleave through 50% of the available hC3 (EC.sub.50) can be
calculated.
[0466] ACC-AGR+ELISA
[0467] Provided herein are methods of assessing substrate
specificity of the modified u-PA polypeptides. The use of a
fluorogenic peptide substrate, 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 the activity of a modified protease
whereby a fluorogenic moiety is released from a peptide substrate
upon action of the protease, 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=[E.sub.T]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.
[0468] In one example, 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 C3 cleavage
sequences. In another example, a sequence corresponding to a
substrate of the wild-type protease can be used to assay residual
protease activity.
[0469] 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 C3 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 C3 by a modified
protease can be compared to cleavage of another full-length
substrate, or the cleavage can be compared to a fluorogenic
tetrapeptide cleavage sequence of C3. 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.
[0470] In an additional embodiment, the activity of a modified u-PA
polypeptide can be assessed after prolonged incubation in
cynomolgus plasma or vitreous humor. In one example, the residual
protease activity is assayed with fluorogenic substrate AGR-ACC
(7-amino-4-carbamoylmethyl-coumarin) after incubation in 80%
Cynomolgus vitreous humor. For example, the modified u-PA
polypeptide of SEQ ID NO:21 exhibits comparable ability to cleave
the fluorogenic substrate AGR-ACC after 7 days incubation in
vitreous and PBS. In another example, the modified u-PA polypeptide
of SEQ ID NO:21 cleaves the fluorogenic substrate AGR-ACC at a
similar levels before and after 7 day incubation in vitreous
humor.
[0471] Assessing Specificity Using Peptide Libraries
[0472] Provided herein are methods of assessing substrate
specificity of the resulting modified u-PA polypeptides using
peptide libraries coupled to fluorogenic peptides. A modified u-PA
polypeptide can be verified for P1-P4 substrate specificity at any
given sub-site using a peptide library coupled to a fluorogenic
substrate (Harris et al., (2000) Proc. Natl. Acad. Sci. U.S.A.
97:7754; US 2004/0175777; US 2004/0146938). Use of a peptide
library or peptide libraries allows for the rapid and facile
determination of proteolytic substrate. This strategy involves the
use of libraries of peptides whereby one position in the library is
held constant (i.e., the P1 position), while the remaining
positions (i.e., P4-P2 and/or P1' and/or P2') 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 15
amino acids. 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.
[0473] Those of skill in the art 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) Proc. Natl. Acad. Sci. U.S.A. 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.
[0474] 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) de-protecting, 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) de-protecting, 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) de-protecting, thereby
forming a fourth conjugate having a reactive amine moiety.
[0475] 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.
[0476] 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, 2nd ed. Pierce Chem. Co.,
Rockford, Ill. Solid phase synthesis is most easily accomplished
with commercially available peptide synthesizers utilizing Fmoc or
t-BOC chemistry.
[0477] 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-butyl, t-boc, and
S-trityl groups is accomplished using, for example,
TFA:phenol:water:thio-aniso:ethanedithio (85:5:5:2.5:2.5), or
TFA:phenol:water (95:5:5).
[0478] 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). Modified proteases,
such as for example a modified u-PA protease described herein, can
be assayed using individual fluorogenic peptide substrates
corresponding to a desired cleavage sequence. A method of assaying
for a modified protease that can cleave any one or more of the C3
cleavage sequences includes: (a) contacting a peptide fluorogenic
sample (containing a C3 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-AGR-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.
[0479] 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.
[0480] 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.
[0481] 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) or in a
test condition (e.g., vitreous humor or serum), 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.
[0482] 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
C3 cleavage sites are in the C3 activation cleavage or active
sites. 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, for example C3, 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.
[0483] 3. Specificity
[0484] The specificity constant of cleavage of target substrate,
e.g., complement protein C3 or plasminogen, by a modified u-PA
polypeptide can be determined by using gel densitometry to assess
changes in densitometry over time of a full-length target substrate
incubated in the presence of a u-PA polypeptide. In specific
embodiments, comparison of the specificities of a modified u-PA
polypeptide can be used to determine if the modified u-PA
polypeptide exhibits altered, for example, increased, specificity
for C3 compared to the wild-type u-PA polypeptide. The specificity
of a u-PA polypeptide for a target substrate, e.g. complement
protein C3, can be determined from the specificity constant of
cleavage of a target substrate compared to a non-target substrate
(e.g. the native wild-type substrate of u-PA). A ratio of the
specificity constants of a modified u-PA polypeptide for the target
substrate C3 versus a non-target substrate, such as plasminogen,
can be made to determine a ratio of the efficiency of cleavage of
the modified u-PA polypeptide. Comparison of the ratio of the
efficiency of cleavage between a modified u-PA polypeptide and a
wild-type u-PA polypeptide can be used to assess the fold change in
specificity for a target substrate. Specificity can be at least
2-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,
400, 500, 600, 700, 800, 900, or 1000 times or more when compared
to the specificity of a wild-type u-PA polypeptide for a target
substrate versus a non-target substrate.
[0485] Kinetic analysis of cleavage of native substrates of a u-PA
polypeptide can be compared to analysis of cleavage of desired
target substrates in complement protein C3 to assess specificity of
the modified u-PA polypeptide for complement protein C3. Second
order rate constants of inhibition (ki) can be assessed to monitor
the efficiency and reactivity of a modified u-PA polypeptide for
complement protein C3. For purposes herein, the modified u-PA
polypeptides cleave C3 so that complement activation is inhibited,
and, as shown in the Examples, they do so with significantly
greater activity, such as at least 5-fold more activity, than the
unmodified u-PA polypeptide (or u-PA polypeptide modified with the
C122S replacement, which eliminates a free cysteine to thereby
reduce aggregation). For example, the modified u-PA polypeptide of
SEQ ID NO:21 cleaves human C3 in the assay described herein with a
an EC.sub.50 of 19 nM, compared to 3380 nM for the wild-type
protease domain of SEQ ID NO:5.
[0486] 4. Disease Models
[0487] The modified u-PA polypeptides provided herein can be used
in any clinically relevant disease model known to one of skill in
the art to determine their effects on complement-mediated diseases
or disorders. Exemplary assays include, but are not limited to,
assays for transplantation, including in vitro assays with human
islet cells (Tjernberg et al. (2008) Transplantation 85:1193-1199)
and ex vivo assays with pig kidneys (Fiane et al. (1999)
Xenotransplantation 6:52-65); bioincompatibility, including in
vitro artificial surface-induced inflammation (Lappegard et al.
(2008) J BiomedMater Res A 87:129-135; Lappegard et al. (2005) Ann
Thorac Surg 79:917-923; Nilsson et al. (1998) Blood 92:1661-1667;
Schmidt et al. (2003) JBiomedMater Res A 66:491-499); inflammation,
including in vitro E. coli-induced inflammation (Mollnes et al.
(2002) Blood 100:1867-1877) and heparin/protamine complex-induced
inflammation in baboons (Soulika et al. (2000) Clin Immunol
96:212-221); age-related macular degeneration in rabbits and
monkeys and rodents (Chi et al. (2010) Adv Exp Med Biol
703:127-135; Pennesi et al. (2012) Mol. Aspects Med. 33(4):487-509;
Fletcher et al. (2014) Optm. Vis. Sci. 91(8):878-886; Forest et
al., (2015) Disease Models and Mechanisms 8:421-427); and delayed
graft function in pigs (Hanto et al., (2010) Am J Transplant
10(11):2421-2430) and dogs (Petrinec et al., (1996) Surgery
61:1331-1337).
F. METHODS OF PRODUCING NUCLEIC ACIDS ENCODING MODIFIED U-PA
POLYPEPTIDES THEREOF
[0488] Polypeptides of a modified u-PA polypeptide set forth herein
can be obtained by methods well known in the art for protein
purification and recombinant protein expression. Polypeptides also
can be synthesized chemically. Modified or variant, including
truncated forms, can be engineered from a wild type polypeptide
using standard recombinant DNA methods. For example, modified u-PA
polypeptides can be engineered from a wild type polypeptide, such
as by site-directed mutagenesis.
[0489] 1. Isolation or Preparation of Nucleic Acids Encoding u-PA
Polypeptides
[0490] Polypeptides can be cloned or isolated using any available
methods known in the art for cloning and isolating nucleic acid
molecules. Such methods include PCR amplification of nucleic acids
and screening of libraries, including nucleic acid hybridization
screening, antibody-based screening and activity-based screening.
For example, when the polypeptides are produced by recombinant
means, 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 or partial (i.e., encompassing the entire coding region)
cDNA or genomic DNA clone encoding a u-PA, such as from a cell or
tissue source.
[0491] Methods for amplification of nucleic acids can be used to
isolate nucleic acid molecules encoding a desired polypeptide,
including for example, polymerase chain reaction (PCR) methods.
Exemplary of such methods include use of a Perkin-Elmer Cetus
thermal cycler and Taq polymerase (Gene Amp). A nucleic acid
containing material can be used as a starting material from which a
desired polypeptide-encoding nucleic acid molecule can be isolated.
For example, DNA and mRNA preparations, cell extracts, tissue
extracts, fluid samples (e.g. blood, serum, saliva), and samples
from healthy and/or diseased subjects can be used in amplification
methods. The source can be from any eukaryotic species including,
but not limited to, vertebrate, mammalian, human, porcine, bovine,
feline, avian, equine, canine, and other primate sources. Nucleic
acid libraries also can be used as a source of starting material.
Primers can be designed to amplify a desired polypeptide. For
example, primers can be designed based on expressed sequences from
which a desired polypeptide is generated. Primers can be designed
based on back-translation of a polypeptide amino acid sequence. If
desired, degenerate primers can be used for amplification.
Oligonucleotide primers that hybridize to sequences at the 3' and
5' termini of the desired sequence can be used as primers to
amplify by PCR sequences from a nucleic acid sample. Primers can be
used to amplify the entire full-length u-PA, or a truncated
sequence thereof, such as a nucleic acid encoding any of the
soluble u-PA polypeptides provided herein. Nucleic acid molecules
generated by amplification can be sequenced and confirmed to encode
a desired polypeptide.
[0492] Additional nucleotide sequences can be joined to a
polypeptide-encoding nucleic acid molecule, including linker
sequences containing restriction endonuclease sites for the purpose
of cloning the synthetic gene into a vector, for example, a protein
expression vector or a vector designed for the amplification of the
core protein coding DNA sequences. Furthermore, additional
nucleotide sequences specifying functional DNA elements can be
operatively linked to a polypeptide-encoding nucleic acid molecule.
Examples of such sequences include, but are not limited to,
promoter sequences designed to facilitate intracellular protein
expression, and secretion sequences, for example heterologous
signal sequences, designed to facilitate protein secretion. Such
sequences are known to those of skill in the art. Additional
nucleotide residue sequences such as sequences of bases specifying
protein binding regions also can be linked to enzyme-encoding
nucleic acid molecules. Such regions include, but are not limited
to, sequences of residues that facilitate or encode proteins that
facilitate uptake of an enzyme into specific target cells, or
otherwise alter pharmacokinetics of a product of a synthetic
gene.
[0493] Tags and/or other moieties can be added, for example, to aid
in detection or affinity purification of the polypeptide. For
example, additional nucleotide residue sequences such as sequences
of bases specifying an epitope tag or other detectable marker also
can be linked to enzyme-encoding nucleic acid molecules. Exemplary
of such sequences include nucleic acid sequences encoding a SUMO
tag or His tag or Flag Tag.
[0494] The identified and isolated nucleic acids then can be
inserted into an appropriate cloning vector. A large number of
vector-host systems known in the art can be used. Possible vectors
include, but are not limited to, plasmids or modified viruses, but
the vector system must be compatible with the host cell used. Such
vectors include, but are not limited to, bacteriophages such as
lambda derivatives, or plasmids such as pCMV4, pBR322 or pUC
plasmid derivatives or the Bluescript vector (Stratagene, La Jolla,
Calif.). The insertion into a cloning vector can, for example, be
accomplished by ligating the DNA fragment into a cloning vector
which has complementary cohesive termini. Insertion can be effected
using TOPO cloning vectors (Invitrogen, Carlsbad, Calif.).
[0495] If the complementary restriction sites used to fragment the
DNA are not present in the cloning vector, the ends of the DNA
molecules can be enzymatically modified. Alternatively, any site
desired can be produced by ligating nucleotide sequences (linkers)
onto the DNA termini; these ligated linkers can contain specific
chemically synthesized oligonucleotides encoding restriction
endonuclease recognition sequences. In an alternative method, the
cleaved vector and protein gene can be modified by homopolymeric
tailing.
[0496] Recombinant molecules can be introduced into host cells via,
for example, transformation, transfection, infection,
electroporation and sonoporation, so that many copies of the gene
sequence are generated. In specific embodiments, transformation of
host cells with recombinant DNA molecules that incorporate the
isolated protein gene, cDNA, or synthesized DNA sequence enables
generation of multiple copies of the gene. Thus, the gene can be
obtained in large quantities by growing transformants, isolating
the recombinant DNA molecules from the transformants and, when
necessary, retrieving the inserted gene from the isolated
recombinant DNA.
[0497] In addition to recombinant production, modified u-PA
polypeptides provided herein, can be produced by direct peptide
synthesis using solid-phase techniques (see e.g., Stewart et al.
(1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San
Francisco; Merrifield J (1963) J Am Chem Soc., 85:2149-2154). In
vitro protein synthesis can be performed using manual techniques or
by automation. Automated synthesis can be achieved, for example,
using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer,
Foster City Calif.) in accordance with the instructions provided by
the manufacturer. Various fragments of a polypeptide can be
chemically synthesized separately and combined using chemical
methods.
[0498] Also provided herein, are methods of expression of active or
activated or activatable forms of the modified u-PA polypeptides,
such as two chain activated forms and dimers. As discussed and
described herein, and exemplified in Examples 14-16, the nucleic
acid encoding modified u-PA polypeptide fusion proteins can be
prepared. The nucleic acids encode the modified u-PA protease
domains, linked to nucleic acid encoding other sequences,
including, but are limited to, secretion signals, such as, for
example, the u-PA signal sequence, an IgG kapp chain signal
sequence, and an IL-2 signal sequence, the N-terminal portion of
u-PA (to produce full-length u-PA), activation sequences, such as
for example, the u-PA activation sequence or a furin sequence, and
fusion partners, such as an albumin, to alter a property of the
u-PA, such as serum half-life, and/or a sequence, such as a His Tag
and/or SUMO to increase expression and/or facilitate isolation.
These nucleic acid molecules can be expressed in suitable host
cells, well known to those of skill in the art, for production of
the modified u-PA and/or fusion protein. Generally the nucleic
acids encode a signal sequence or other trafficking sequence for
secretion or trafficking to an locus for purification. Including
nucleic acid encoding an activation sequence can be used to produce
an activated form of the modified u-PA polypeptide.
[0499] 2. Generation of Mutant or Modified Nucleic Acid and
Encoding Polypeptides
[0500] The modifications provided herein can be made by standard
recombinant DNA techniques such as are routine to one of skill in
the art. 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 QuikChange available from Stratagene) of encoding nucleic acid
molecules, or by solid phase polypeptide synthesis methods.
[0501] 3. Vectors and Cells
[0502] For recombinant expression of one or more of the desired
proteins, such as any modified u-PA polypeptide described herein,
the nucleic acid containing all or a portion of the nucleotide
sequence encoding the protein can be inserted into an appropriate
expression vector, i. e., a vector that contains the necessary
elements for the transcription and translation of the inserted
protein coding sequence. The necessary transcriptional and
translational signals also can be supplied by the native promoter
for enzyme genes, and/or their flanking regions.
[0503] Also provided are vectors that contain a nucleic acid
encoding the enzyme. Cells containing the vectors also are
provided. The cells include eukaryotic and prokaryotic cells, and
the vectors are any suitable for use therein. Generally, the cell
is a cell that is capable of effecting glycosylation of the encoded
protein.
[0504] Prokaryotic and eukaryotic cells containing the vectors are
provided. Such cells include bacterial cells, yeast cells, fungal
cells, Archea, plant cells, insect cells and animal cells. The
cells are used to produce a protein thereof by growing the
above-described cells under conditions whereby the encoded protein
is expressed by the cell, and recovering the expressed protein. For
purposes herein, for example, the enzyme can be secreted into the
medium.
[0505] A host cell strain can be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation and
acylation. Post-translational processing can impact the folding
and/or function of the polypeptide. Different host cells, such as,
but not limited to, CHO (DG44, DXB11, CHO-K1), HeLa, MCDK, 293 and
WI38 have specific cellular machinery and characteristic mechanisms
for such post-translational activities and can be chosen to ensure
the correct modification and processing of the introduced protein.
Generally, the choice of cell is one that is capable of introducing
N-linked glycosylation into the expressed polypeptide. Hence,
eukaryotic cells containing the vectors are provided. Exemplary of
eukaryotic cells are mammalian Chinese Hamster Ovary (CHO) cells.
For example, CHO cells deficient in dihydrofolate reductase (e.g.
DG44 cells) are used to produce polypeptides provided herein.
[0506] Provided are vectors that contain a sequence of nucleotides
that encodes the modified u-PA polypeptide, coupled to the native
or heterologous signal sequence, as well as multiple copies
thereof. The vectors can be selected for expression of the enzyme
protein in the cell or such that the enzyme protein is expressed as
a secreted protein.
[0507] 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 a-mating factor signal sequence or a portion thereof, or
the native signal sequence.
[0508] 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.
[0509] Any methods known to those of skill in the art for the
insertion of DNA fragments into a vector can be used to construct
expression vectors containing a chimeric gene containing
appropriate transcriptional/translational control signals and
protein coding sequences. These methods can include in vitro
recombinant DNA and synthetic techniques and in vivo recombinants
(genetic recombination). Expression of nucleic acid sequences
encoding protein, or domains, derivatives, fragments or homologs
thereof, can be regulated by a second nucleic acid sequence so that
the genes or fragments thereof are expressed in a host transformed
with the recombinant DNA molecule(s). For example, expression of
the proteins can be controlled by any promoter/enhancer known in
the art. In a specific embodiment, the promoter is not native to
the genes for a desired protein. Promoters which can be used
include, but are not limited to, the SV40 early promoter (Bernoist
and Chambon, Nature 290:304-310 (1981)), the promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al.
Cell 22:787-797 (1980)), the herpes thymidine kinase promoter
(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)),
the regulatory sequences of the metallothionein gene (Brinster et
al., Nature 296:39-42 (1982)); prokaryotic expression vectors such
as the .beta.-lactamase promoter (Jay et al., (1981) Proc. Natl.
Acad. Sci. USA 78:5543) or the tac promoter (DeBoer et al., Proc.
Natl. Acad. Sci. USA 80:21-25 (1983); see also "Useful Proteins
from Recombinant Bacteria": in Scientific American 242:79-94
(1980)); plant expression vectors containing the nopaline
synthetase promoter (Herrara-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 (Magram 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 (Shani,
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)).
[0510] In a specific embodiment, a vector is used that contains a
promoter operably linked to nucleic acids encoding a desired
protein, or a domain, fragment, derivative or homolog thereof, one
or more origins of replication, and optionally, one or more
selectable markers (e.g., an antibiotic resistance gene). Depending
on the expression system, specific initiation signals also are
required for efficient translation of a u-PA sequence. These
signals include the ATG initiation codon and adjacent sequences. In
cases where the initiation codon and upstream sequences of u-PA or
catalytically active fragments thereof are inserted into the
appropriate expression vector, no additional translational control
signals are needed. In cases where only coding sequence, or a
portion thereof, is inserted, exogenous transcriptional control
signals including the ATG initiation codon must be provided.
Furthermore, the initiation codon must be in the correct reading
frame to ensure transcription of the entire insert. Exogenous
transcriptional elements and initiation codons can be of various
origins, natural and synthetic. The efficiency of expression can be
enhanced by the inclusion of enhancers appropriate to the cell
system in use (Scharf et al. (1994) Results Probl Cell Differ
20:125-62; Bittner et al. (1987) Methods in Enzymol,
153:516-544).
[0511] Exemplary plasmid vectors for transformation of E. coli
cells, include, for example, the pQE expression vectors (available
from Qiagen.RTM., Valencia, Calif.; see also literature published
by Qiagen.RTM. 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, to and Ti transcriptional
terminators, ColE1 origin of replication, and a beta-lactamase gene
for conferring ampicillin resistance. The pQE vectors enable
placement of a 6.times.His tag at either the N- or C-terminus of
the recombinant protein. Such plasmids include pQE 32, pQE 30, and
pQE 31 which provide multiple cloning sites for all three reading
frames and provide for the expression of N-terminally
6.times.His-tagged proteins. Other exemplary plasmid vectors for
transformation of E. coli cells, include, for example, the pET
expression vectors (see, U.S. Pat. No. 4,952,496; available from
Novagen.RTM., Madison, Wis.; see, also literature published by
Novagen.RTM. describing the system). Such plasmids include pET 11a,
which contains the T71ac 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.RTM., 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.
[0512] Typically, vectors can be plasmid, viral, or others known in
the art, used for expression of the modified u-PA polypeptide in
vivo or in vitro. For example, the modified u-PA polypeptide is
expressed in mammalian cells, including, for example, Chinese
Hamster Ovary (CHO) cells.
[0513] Viral vectors, such as adenovirus, retrovirus or vaccinia
virus vectors, can be employed. In some examples, the vector is a
defective or attenuated retroviral or other viral vector (see U.S.
Pat. No. 4,980,286). For example, a retroviral vector can be used
(see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These
retroviral vectors have been modified to delete retroviral
sequences that are not necessary for packaging of the viral genome
and integration into host cell DNA. In some examples, viruses armed
with a nucleic acid encoding a modified u-PA polypeptide can
facilitate their replication and spread within a target tissue. The
virus also can be a lytic virus or a non-lytic virus where the
virus selectively replicates under a tissue specific promoter. As
the viruses replicate, the coexpression of the u-PA polypeptide
with viral genes will facilitate the spread of the virus in
vivo.
[0514] 4. Expression
[0515] Modified u-PA polypeptides can be produced by any method
known to those of skill in the art including in vivo and in vitro
methods. Desired proteins can be expressed in any organism suitable
to produce the required amounts and forms of the proteins, such as
for example, needed for administration and treatment. Expression
hosts include prokaryotic and eukaryotic organisms such as E. coli,
yeast, plants, insect cells, mammalian cells, including human cell
lines and transgenic animals. Expression hosts can differ in their
protein production levels as well as the types of
post-translational modifications that are present on the expressed
proteins. The choice of expression host can be made based on these
and other factors, such as regulatory and safety considerations,
production costs and the need and methods for purification.
[0516] Many expression vectors are available and known to those of
skill in the art and can be used for expression of proteins. The
choice of expression vector will be influenced by the choice of
host expression system. In general, expression vectors can include
transcriptional promoters and optionally enhancers, translational
signals, and transcriptional and translational termination signals.
Expression vectors that are used for stable transformation
typically have a selectable marker which allows selection and
maintenance of the transformed cells. In some cases, an origin of
replication can be used to amplify the copy number of the
vector.
[0517] Modified u-PA polypeptides also can be utilized or expressed
as protein fusions. For example, an enzyme fusion can be generated
to add additional functionality to an enzyme. Examples of enzyme
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.
[0518] For example, a modified u-PA polypeptide described herein is
one that is generated by expression of a nucleic acid molecule
encoding the protease domain set forth in any one of SEQ ID NOS:
1-6, 8-44 and 52-75 or a sequence of amino acids that exhibits at
least 65%, 70%, 75%, 80%, 84%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to a sequence set forth in any
of SEQ ID NOS: 1-6, 8-44 and 52-75.
[0519] For long-term, high-yield production of recombinant
proteins, stable expression is desired. For example, cell lines
that stably express a modified u-PA polypeptide can be transformed
using expression vectors that contain viral origins of replication
or endogenous expression elements and a selectable marker gene.
Following the introduction of the vector, cells can be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells that successfully express the introduced
sequences. Resistant cells of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the
cell types.
[0520] Any number of selection systems can be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler et al., (1977) Cell
11:223-232) and adenine phosphoribosyltransferase (Lowy I et al.
(1980) Cell, 22:817-23) genes, which can be employed in TK- or
APRT-cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection. For
example, DHFR, which confers resistance to methotrexate (Wigler M
et al. (1980) Proc. Natl. Acad. Sci, 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin F et al. (1981) J. Mol. Biol., 150:1-14); and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively, can be used.
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of typtophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman S C and R C Mulligan (1988) Proc. Natl. Acad. Sci,
85:8047-8051). Visible markers, such as but not limited to,
anthocyanins, beta glucuronidase and its substrate, GUS, and
luciferase and its substrate luciferin, also can be used to
identify transformants and also to quantify the amount of transient
or stable protein expression attributable to a particular vector
system (Rhodes C A et al. (1995) Methods Mol. Biol.
55:121-131).
[0521] The presence and expression of u-PA polypeptides can be
monitored. For example, detection of a functional polypeptide can
be determined by testing the conditioned media for hyaluronidase
enzyme activity under appropriate conditions. Exemplary assays to
assess the solubility and activity of expressed proteins are
provided herein.
[0522] a. Prokaryotic Cells
[0523] Prokaryotes, especially E. coli, provide a system for
producing large amounts of proteins. Transformation of E. coli is a
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.
[0524] Proteins, such as any provided herein, can be expressed in
the cytoplasmic environment of E. coli. The cytoplasm is a reducing
environment and for some molecules, this can result in the
formation of insoluble inclusion bodies. Reducing agents such as
dithiothreotol and .beta.-mercaptoethanol and denaturants, such as
guanidine-HCl and urea can be used to resolubilize the proteins. An
alternative approach is the expression of proteins in the
periplasmic space of bacteria which provides an oxidizing
environment and chaperonin-like and disulfide isomerases and can
lead to the production of soluble protein. Typically, a leader
sequence is fused to the protein to be expressed which directs the
protein to the periplasm. The leader is then removed by signal
peptidases inside the periplasm. Examples of periplasmic-targeting
leader sequences include the pelB leader from the pectate lyase
gene and the leader derived from the alkaline phosphatase gene. In
some cases, periplasmic expression allows leakage of the expressed
protein into the culture medium. The secretion of proteins allows
quick and simple purification from the culture supernatant.
Proteins that are not secreted can be obtained from the periplasm
by osmotic lysis. Similar to cytoplasmic expression, in some cases
proteins can become insoluble and denaturants and reducing agents
can be used to facilitate solubilization and refolding. Temperature
of induction and growth also can influence expression levels and
solubility, typically temperatures between 25.degree. C. and
37.degree. C. are used. Typically, bacteria produce aglycosylated
proteins. Thus, if proteins require glycosylation for function,
glycosylation can be added in vitro after purification from host
cells.
[0525] b. Yeast Cells
[0526] Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces
pombe, Yarrowia lipolytica, Kluyveromyces lactis and Pichia
pastoris are well known yeast expression hosts that can be used for
production of proteins, such as any described herein. Yeast can be
transformed with episomal replicating vectors or by stable
chromosomal integration by homologous recombination. Typically,
inducible promoters are used to regulate gene expression. Examples
of such promoters include GAL1, GAL7 and GALS and metallothionein
promoters, such as CUP1, AOX1 or other Pichia or other yeast
promoter. Expression vectors often include a selectable marker such
as LEU2, TRP 1, 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.
[0527] c. Insects and Insect Cells
[0528] Insect cells, particularly using baculovirus expression, are
useful for expressing polypeptides such as u-PA polypeptides.
Insect cells express high levels of protein and are capable of most
of the post-translational modifications used by higher eukaryotes.
Baculovirus have a restrictive host range which improves the safety
and reduces regulatory concerns of eukaryotic expression. Typical
expression vectors use a promoter for high level expression such as
the polyhedrin promoter of baculovirus. Commonly used baculovirus
systems include the baculoviruses such as Autographa 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. The cell lines Pseudaletia unipuncta (A7S) and
Danaus plexippus (DpN1) produce proteins with glycosylation
patterns similar to mammalian cell systems. Exemplary insect cells
are those that have been altered to reduce immunogenicity,
including those with "mammalianized" baculovirus expression vectors
and those lacking the enzyme FT3.
[0529] 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.
[0530] d. Mammalian Expression
[0531] Mammalian expression systems can be used to express proteins
including U-PA polypeptides. 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. IRES elements also can be added to permit
bicistronic expression with another gene, such as a selectable
marker. 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 (DHFR) and thymidine kinase. For example,
expression can be performed in the presence of methotrexate to
select for only those cells expressing the DHFR gene. Fusion with
cell surface signaling molecules such as TCR-.zeta. and
FcERI-.gamma. can direct expression of the proteins in an active
state on the cell surface.
[0532] 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.
Examples include CHO-S cells (Invitrogen.RTM., Carlsbad, Calif.,
cat #11619-012) and the serum free EBNA-1 cell line (Pham et al.,
(2003) Biotechnol. Bioeng. 84:332-42.). Cell lines also are
available that are adapted to grow in special mediums optimized for
maximal expression. For example, DG44 CHO cells are adapted to grow
in suspension culture in a chemically defined, animal product-free
medium.
[0533] e. Plants
[0534] Transgenic plant cells and plants can be used to express
proteins such as any described herein. Expression constructs are
typically transferred to plants using direct DNA transfer such as
microprojectile bombardment and PEG-mediated transfer into
protoplasts, and with agrobacterium-mediated transformation.
Expression vectors can include promoter and enhancer sequences,
transcriptional termination elements and translational control
elements. Expression vectors and transformation techniques are
usually divided between dicot hosts, such as Arabidopsis and
tobacco, and monocot hosts, such as corn and rice. Examples of
plant promoters used for expression include the cauliflower mosaic
virus promoter, the nopaline syntase promoter, the ribose
bisphosphate carboxylase promoter and the ubiquitin and UBQ3
promoters. Selectable markers such as hygromycin, phosphomannose
isomerase and neomycin phosphotransferase are often used to
facilitate selection and maintenance of transformed cells.
Transformed plant cells can be maintained in culture as cells,
aggregates (callus tissue) or regenerated into whole plants.
Transgenic plant cells also can include algae engineered to produce
hyaluronidase polypeptides. Because plants have different
glycosylation patterns than mammalian cells, this can influence the
choice of protein produced in these hosts.
[0535] 5. Purification
[0536] Host cells transformed with a nucleic acid sequence encoding
a modified u-PA polypeptide can be cultured under conditions
suitable for the expression and recovery of the encoded protein
from cell culture. The protein produced by a recombinant cell is
generally secreted, but may be contained intracellularly depending
on the sequence and/or the vector used. As understood by those of
skill in the art, expression vectors containing nucleic acid
encoding u-PA can be designed with signal sequences that facilitate
direct secretion of u-PA through prokaryotic or eukaryotic cell
membrane.
[0537] Thus, methods for purification of polypeptides from host
cells 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.
[0538] Proteins, such as modified u-PA polypeptides, can be
purified using standard protein purification techniques known in
the art including but not limited to, SDS-PAGE, size fractionation
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 u-PA proteins can be used
in affinity purification.
[0539] Expression constructs also can be engineered to add an
affinity tag to a protein such as a Small Ubiquitin-like Modifier
(SUMO) tag, myc epitope, GST fusion or His6 and affinity purified
with SUMO or myc antibody, glutathione resin and Ni-resin,
respectively. Such tags can be joined to the nucleotide sequence
encoding a u-PA as described elsewhere herein, which can facilitate
purification of soluble proteins. For example, a modified u-PA
polypeptide can be expressed as a recombinant protein with one or
more additional polypeptide domains added to facilitate protein
purification. Such purification facilitating domains include, but
are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle
Wash.). The inclusion of a cleavable linker sequence such as Factor
XA or enterokinase (Invitrogen.RTM., San Diego, Calif.) between the
purification domain and the expressed u-PA polypeptide is useful to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing a u-PA polypeptide in and
an enterokinase cleavage site. The Small Ubiquitin-like Modifier
(SUMO) tag facilitates purification on IMIAC (immobilized metal ion
affinity chromatography), while the enterokinase cleavage site
provides a means for purifying the polypeptide from the fusion
protein.
[0540] Purity can be assessed by any method known in the art
including gel electrophoresis, orthogonal HPLC methods, staining
and spectrophotometric techniques. The expressed and purified
protein can be analyzed using any assay or method known to one of
skill in the art, for example, any described in Section 3. These
include assays based on the physical and/or functional properties
of the protein, including, but not limited to, analysis by gel
electrophoresis, immunoassay and assays of u-PA activity.
[0541] 6. Additional Modifications
[0542] The modified u-PA polypeptides provided herein can be
modified to improve or alter pharmacokinetic and pharmacological
properties. In particular, the modified u-PA polypeptides can be
conjugated to a polymer, such as a PEG moiety or dextran or
sialiation to reduce immungeniciaty and/or increase half-life in
serum and other body fluids including vitreous humor.
[0543] a. PEGylation
[0544] Polyethylene glycol (PEG) is used in biomaterials,
biotechnology and medicine primarily because PEG is a
biocompatible, nontoxic, water-soluble polymer that is typically
nonimmunogenic (Zhao and Harris, ACS Symposium Series 680: 458-72,
1997). In the area of drug delivery, PEG derivatives have been
widely used in covalent attachment (i. e., "PEGylation") to
proteins to reduce immunogenicity, proteolysis and kidney clearance
to increase serum half-life, and to enhance solubility (Zalipsky,
Adv. Drug Del. Rev. 16:157-82, 1995). Similarly, PEG has been
attached to low molecular weight, relatively hydrophobic drugs to
enhance solubility, reduce toxicity and alter biodistribution.
Typically, PEGylated drugs are injected as solutions.
[0545] A related application is synthesis of crosslinked degradable
PEG networks or formulations for use in drug delivery since much of
the same chemistry used in design of degradable, soluble drug
carriers also can be used in design of degradable gels (Sawhney et
al., Macromolecules 26: 581-87, 1993). It also is known that
intermacromolecular complexes can be formed by mixing solutions of
two complementary polymers. Such complexes are generally stabilized
by electrostatic interactions (polyanion-polycation) and/or
hydrogen bonds (polyacid-polybase) between the polymers involved,
and/or by hydrophobic interactions between the polymers in an
aqueous surrounding (Krupers et al., Eur. Polym J. 32:785-790,
1996). For example, mixing solutions of polyacrylic acid (PAAc) and
polyethylene oxide (PEO) under the proper conditions results in the
formation of complexes based mostly on hydrogen bonding.
Dissociation of these complexes at physiologic conditions has been
used for delivery of free drugs (i.e., non-PEGylated). Complexes of
complementary polymers have been formed from homopolymers and
copolymers.
[0546] Numerous reagents for PEGylation are known as are PEG moiety
(PEGylated) therapeutic proteins. Such reagents include, but are
not limited to, reaction of the polypeptide with
N-hydroxysuccinimidyl (NHS) activated PEG, succinimidyl mPEG,
mPEG.sub.2-N-hydroxysuccinimide, mPEG succinimidyl
alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG
succinimidyl butanoate, mPEG carboxymethyl 3-hydroxybutanoic acid
succinimidyl ester, homobifunctional PEG-succinimidyl propionate,
homobifunctional PEG propionaldehyde, homobifunctional PEG
butyraldehyde, PEG maleimide, PEG hydrazide,
p-nitrophenyl-carbonate PEG, mPEG-benzotriazole carbonate,
propionaldehyde PEG, mPEG butryaldehyde, branched mPEG.sub.2
butyraldehyde, mPEG acetyl, mPEG piperidone, mPEG methylketone,
mPEG "linkerless" maleimide, mPEG vinyl sulfone, mPEG thiol, mPEG
orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS,
Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluorescein
PEG-NHS, and biotin PEG-NHS (see, e.g., Monfardini et al.,
Bioconjugate Chem. 6:62-69, 1995; Veronese et al., J. Bioactive
Compatible Polymers 12:197-207, 1997; U.S. Pat. Nos. 5,672,662;
5,932,462; 6,495,659; 6,737,505; 4,002,531; 4,179,337; 5,122,614;
5,324,844; 5,446,090; 5,612,460; 5,643,575; 5,766,581; 5,795,569;
5,808,096; 5,900,461; 5,919,455; 5,985,263; 5,990,237; 6,113,906;
6,214,966; 6,258,351; 6,340,742; 6,413,507; 6,420,339; 6,437,025;
6,448,369; 6,461,802; 6,828,401; 6,858,736; U.S. 2001/0021763; U.S.
2001/0044526; U.S. 2001/0046481; U.S. 2002/0052430; U.S.
2002/0072573; U.S. 2002/0156047; U.S. 2003/0114647; U.S.
2003/0143596; U.S. 2003/0158333; U.S. 2003/0220447; U.S.
2004/0013637; US 2004/0235734; WO0500360; U.S. 2005/0114037; U.S.
2005/0171328; U.S. 2005/0209416; EP 01064951; EP 0822199; WO
00176640; WO 0002017; WO 0249673; WO 9428024; and WO 0187925).
[0547] In one example, the polyethylene glycol has a molecular
weight ranging from about 3 kD to about 50 kD, and typically from
about 5 kD to about 30 kD. Covalent attachment of the PEG to the
drug (known as "PEGylation") can be accomplished by known chemical
synthesis techniques. For example, the PEGylation of protein can be
accomplished by reacting NHS-activated PEG with the protein under
suitable reaction conditions.
[0548] While numerous reactions have been described for PEGylation,
those that are most generally applicable confer directionality, use
mild reaction conditions, and do not necessitate extensive
downstream processing to remove toxic catalysts or bi-products. For
instance, monomethoxy PEG (mPEG) has only one reactive terminal
hydroxyl, and thus its use limits some of the heterogeneity of the
resulting PEG-protein product mixture. Activation of the hydroxyl
group at the end of the polymer opposite to the terminal methoxy
group is generally necessary to accomplish efficient protein
PEGylation, with the aim being to make the derivatised PEG more
susceptible to nucleophilic attack. The attacking nucleophile is
usually the epsilon-amino group of a lysyl residue, but other
amines also can react (e.g. the N-terminal alpha-amine or the ring
amines of histidine) if local conditions are favorable. A more
directed attachment is possible in proteins containing a single
lysine or cysteine. The latter residue can be targeted by
PEG-maleimide for thiol-specific modification. Alternatively, PEG
hydrazide can be reacted with a periodate oxidized
hyaluronan-degrading enzyme and reduced in the presence of
NaCNBH.sub.3. More specifically, PEGylated CMP sugars can be
reacted with a hyaluronan-degrading enzyme in the presence of
appropriate glycosyl-transferases. One technique is the
"PEGylation" technique where a number of polymeric molecules are
coupled to the polypeptide in question. When using this technique
the immune system has difficulties in recognizing the epitopes on
the polypeptide's surface responsible for the formation of
antibodies, thereby reducing the immune response. For polypeptides
introduced directly into the circulatory system of the human body
to give a particular physiological effect (i. e. pharmaceuticals)
the typical potential immune response is an IgG and/or IgM
response, while polypeptides which are inhaled through the
respiratory system (i.e. industrial polypeptide) potentially can
cause an IgE response (i.e. allergic response). One of the theories
explaining the reduced immune response is that the polymeric
molecule(s) shield(s) epitope(s) on the surface of the polypeptide
responsible for the immune response leading to antibody formation.
Another theory or at least a partial factor is that the heavier the
conjugate is, the more reduced immune response is obtained.
[0549] Typically, to make the PEGylated modified u-PA polypeptide
provided herein, PEG moieties are conjugated, via covalent
attachment, to the polypeptides. The Modified u-PA polypeptides for
PEGylation can be prepared without the C122S replacement; instead
the C122 can serve as a site for conjugate to a PEG moiety and/or
for forming a desired disulfide bond, such as for a two chain
activated form or an dimer.
[0550] Techniques for PEGylation include, but are not limited to,
specialized linkers and coupling chemistries (see, e.g, Harris,
Adv. Drug Deliv. Rev. 54:459-476, 2002), attachment of multiple PEG
moieties to a single conjugation site (such as via use of branched
PEGs; see, e.g., Veronese et al., Bioorg. Med. Chem. Lett.
12:177-180, 2002), site-specific PEGylation and/or mono-PEGylation
(see, e.g, Chapman et al., Nature Biotech. 17:780-783, 1999), and
site-directed enzymatic PEGylation (see, e.g, Sato, Adv. Drug
Deliv. Rev., 54:487-504, 2002). Methods and techniques described in
the art can produce proteins having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more than 10 PEG or PEG derivatives attached to a single protein
molecule (see, e.g., U.S. Patent Publication No. 2006/0104968).
[0551] b. Fusion Proteins and Other Conjugates
[0552] Provided herein are conjugates of u-PA and the modified u-PA
polypeptides provided herein. Exemplary such conjugates are the
fusion proteins exemplified in Examples 14-16. As described herein,
some of the conjugates when activated by cleavage of an included
activation polypeptide forms a two-chain activated u-PA
polypeptide; others, such as those that contain Fc domains can form
tow chains via linkage of the Fc domains. Others contain sequences,
such as SUMO and HIS-SUMO that facilitate expression and
isolation/purification. Examples 14 and 15, and also FIGS. 1-4,
describe and depict resulting conjugates. For use as
pharmaceuticals, the modified u-PA polypeptides generally are
provided in activated form, such as a two chain activated form. It
is understood that the following discussion describes the fusion
polypeptides that can include signal sequences and other regulatory
sequences that will not appear in the product as produced. In
particular, the fusion polypeptides can include activation
sequences, whereby upon cleavage, the resulting polypeptide is a
two chain activated polypeptide. It is the activated forms of the
polypeptides that, in general, will be the pharmaceutical product
administered to a subject.
[0553] i. Exemplary Fusion Proteins and Other Protein
Conjugates
[0554] The modified u-PA polypeptides provided herein can be fused
to other polypeptides and portions thereof and to moieties to
confer desired properties, such as increased serum half-life,
and/or reduced immunogenicity, and/or other properties. These
include, for example, fusion to albumin, fusion to targeting
moieties, such as antibodies and antigen binding fragments thereof,
fusion to immunoglobulins, Fc fusions, modification of
glycosylation patterns, farsnylation and other such modifications
(see, Strohl (2015) BioDrugs 29:215-239 for a review of a variety
of fusion proteins for improving pharmacokinetic properties of
therapeutic proteins). Any such modalities for altering
pharmacological properties of therapeutics can be applied to the
modified u-PA polypeptides provided herein. Generally, where the
modification is a polypeptide or portion thereof, the modified u-PA
is produced as a fusion protein. For non-polypeptidic
modifications, such as pegylation, modification is effected on
isolated protein. The modified u-PA polypeptides include those that
have Cys at residue 122 (by chymotrypsin number), to provide sites
for podt-translational or post-purification modification. The
modified u-PA polypeptides include those that are full-length and
catalytically active portions thereof, such as the protease domain,
or the mature polypeptide or the activated two-chain
polypeptide.
[0555] Fusion proteins containing a modified u-PA polypeptide
provided herein and one or more other polypeptides also are
provided. Pharmaceutical compositions containing such fusion
proteins formulated for administration by a suitable route are
provided. Fusion proteins are formed by linking in any order a
modified u-PA polypeptide 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
modified u-PA polypeptide by directing the u-PA polypeptide to a
targeted cell or tissue, and/or increasing the expression or
secretion of a u-PA polypeptide. Within a u-PA polypeptide fusion
protein, the u-PA polypeptide can be all or a catalytically active
portion thereof of a u-PA polypeptide or the catalytically active
portion of a u-PA polypeptide and a further portion of u-PA that is
not full-length u-PA. Fusion proteins provided herein retain
substantially all of their specificity and/or selectivity for
complement protein C3. Generally, u-PA 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
u-PA polypeptide, including 96%, 97%, 98%, 99% or greater substrate
specificity compared with a non-fusion u-PA polypeptide.
[0556] ii. Construct Generation
[0557] A u-PA fusion protein can be produced by standard
recombinant techniques. For example, DNA fragments encoding 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). Many expression vectors are commercially
available that encode a fusion moiety (e.g., a his tag, SUMO
polypeptide, or GST polypeptide). A u-PA-encoding nucleic acid can
be cloned into such an expression vector such that the fusion
moiety is linked in-frame to the u-PA polypeptide.
[0558] Exemplary expression vectors include any mammalian
expression vector such as, for example, pCMV. For bacterial
expression, such vectors include pBR322, pUC, pSKF, pET23D, and
fusion vectors such as MBP, GST and LacZ. Other eukaryotic vectors,
for example any containing regulatory elements from eukaryotic
viruses, can be used as eukaryotic expression vectors. These
include, for example, SV40 vectors, papilloma virus vectors, and
vectors derived from Epstein-Bar virus. Exemplary eukaryotic
vectors include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus
pDSCE, and any other vector allowing expression of proteins under
the direction of the CMV promoter, SV40 early promoter, SV40 late
promoter, metallothionein promoter, murine mammary tumor virus
promoter, Rous sarcoma virus promoter, polyhedron promoter, or
other promoters shown effective for expression in eukaryotes.
[0559] iii. Signal Sequence
[0560] u-PA fusion proteins can contain a signal peptide (SP or
signal sequence or localization signal or leader peptide) for
directing transport of the protease. Signal peptides are sequence
motifs found at the N-terminus of nascent proteins that target
proteins for translocation across the endoplasmic reticulum
membrane to their specific destination within the cell, or outside
the cell if the proteins are to be secreted. Thus, SP selection and
modifying the SP influences protein targeting (Zhang et al. (2005)
J Gene Med 7:354-365). Optimized SPs have been developed for more
efficient activity. Computational models and algorithms have been
developed to predict SP efficacy and define SP consensus sequences
(Burdukiewicz et al. (2018) Int J Mol Sci 19(12): 3709; Peason et
al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448).
[0561] Various proteins are known to have SPs, including but not
limited to: receptors (nuclear, 4 transmembrane, G protein-coupled
and tyrosine kinase), cytokines (chemokines), hormones (growth and
differentiation factors), neuropeptides and vasomediators, protein
kinases, phosphatases, phospholipases, phosphodiesterases,
nucleotide cyclases, matrix molecules (adhesion, cadherin,
extracellular matrix molecules, integrin, and selectin), G
proteins, ion channels (calcium, chloride, potassium, and sodium),
proteases, transporter/pumps (amino acid, protein, sugar, metal and
vitamin; calcium, phosphate, potassium, and sodium) and regulatory
proteins. In some examples the original signal peptide is optimized
for the secretion of the protein in the desired host cell selected
for production. A u-PA polypeptide, such as a modified u-PA
protease domain provided herein, can be fused, directly or
indirectly, to a non-uPA signal peptide for u-PA targeting.
[0562] The signal peptides may be signal peptides of antibodies
such as the signal peptides of the heavy chains of antibodies and
the light chain of antibodies. The isotype of the antibody may
comprise, but is not limited, to IgG, IgM, IgD, IgA and IgE. Thus,
the heavy chain may comprise gamma, mu, delta, alpha and epsilon
heavy chains, and the light chain may comprise a kappa or a lambda
light chain. The u-PA fusion proteins set forth herein can be
prepared with an antibody signal peptide such as the human
immunoglobulin light chain kappa (.kappa.) leader signal peptide
sequence, such as the signal sequence set forth in SEQ ID NO:
999.
[0563] Other exemplary signal peptides are those derived from human
interleukin-2 (IL-2) which are used extensively for research and
protein production (Bamford et al. (1998) J Immunol 160:4418;
Komada et al. (1999) Biol Pharm Bull 22:846). Modified IL-2 SPs
with increased basicity and hydrophobicity have been developed that
increased secretion of fused proteins by up to 3.5 fold (Zhang et
al. (2005) J. Gene Med. 7:354). The u-PA fusion proteins herein can
be prepared, for example, with an IL-2 signal peptide, such as the
human IL2 Signal Peptide (hIL2SP), such as, for example, the signal
sequence set forth in SEQ ID NO: 1000.
[0564] Exemplary u-PA fusion proteins set forth herein can contain
a signal peptide for directing transport of the protease. For
example, the u-PA fusion polypeptides set forth as SEQ ID NOs:1004,
1005, 1010, 1011, 1014-1018, 1036 and 1040 contain a human
immunoglobulin light chain kappa (.kappa.) leader signal peptide
sequence (SEQ ID NO: 999). In another example, the u-PA fusion
polypeptides set forth as SEQ ID NOs:1006-1009, 1012, 1013, 1034
and 1035 contain a human IL2 Signal Peptide (hIL2SP) sequence (SEQ
ID NO: 1000).
[0565] iii. Exemplary Fusion Proteins and Peptide Linkers
[0566] Linkage of a modified u-PA 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 u-PA polypeptide to another polypeptide can be to the
N- or C-terminus of the modified u-PA polypeptide, such as the
modified u-PA protease domain. Non-limiting examples of
polypeptides that can be used in fusion proteins with a u-PA
polypeptide provided herein include, for example, a Fc domain from
immunoglobulin G, serum albumin (i. e., human serum albumin)), scFv
that binds to Collagen IIm (C2scFv), Hyaluronic Acid Binding Dmain
(HABD), GST (glutathione S-transferase) polypeptide, a his tag
(i.e., HHHHHH), a Small Ubiquitin-like Modifier (SUMO) tag, the
influenza hemagglutinin (HA) tag polypeptide and its antibody
12CA5, and/or a heterologous signal sequence (e.g., from thrombin
or a mouse Ig kappy chain V-III region (IgG.kappa.) or human
Interleukin-2 (hIL2)). 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 Publication No. WO 01/32711).
[0567] Peptide linkers can be included in u-PA fusion proteins. In
one example, peptide linkers can be fused to the C-terminal end of
a first polypeptide and the N-terminal of a second polypeptide.
This structure can be repeated a plurality times such that at least
one, and optionally 2, 3, 4 or more polypeptides are linked to one
another via peptide linkers at their respective termini. For
example, a fusion protein can include a sequence X-Y-Z, where X is
the wild-type or modified u-PA catalytic domain, Y is a peptide
linker, and Z is all or part of fusion partner (e.g., HSA, Fc,
HABD, or C2 scFv). In some instances, X is all of a modified u-PA
including the N-terminus of u-PA, and the protease domain of u-PA.
In other instances, X is part of a modified u-PA including the 12
amino acids directly upstream of the u-PA protease domain, and the
u-PA protease domain. In another example, the polypeptide can
include the sequence A-X-Y-Z, where "A" is another fusion partner,
such as a polypeptide, such as SUMO or HIS-SUMO, that facilitates
expression and/or isolation of the resulting polypeptide.
[0568] Peptide linkers generally include Gly, Ser, and combinations
thereof, or Ala and Proline. Linkers generally contain from two up
to 20 or 25 residues. Examples of peptide linkers include, but are
not limited to: -Gly-Gly-, GSG, AGS (SEQ ID NO: 1003), GGGGS (SEQ
ID NO:1001), GGSSGG (SEQ ID NO:1002), SSSSG (SEQ ID NO:1024),
GKSSGSGSESKS (SEQ ID NO:1025), GGSTSGSGKSSEGKG (SEQ ID NO: 1026),
GSTSGSGKSSSEGSGSTKG (SEQ ID NO: 1027), GSTSGSGKPGSGEGSTKG (SEQ ID
NO: 1028), EGKSSGSGSESKEF (SEQ ID NO: 1029), or AlaAlaProAla or
(AlaAlaProAla)n (SEQ ID NO: 1030), where n is 1 to 6, such as 1, 2,
3, 4, 5 or 6.
[0569] Linking moieties are described, for example, in Huston et
al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, Whitlow et
al. (1993) Protein Engineering 6:989-995, and Newton et al., (1996)
Biochemistry 35:545-553. Other suitable peptide linkers include any
of those described in U.S. Pat. No. 4,751,180 or 4,935,233. A
polynucleotide encoding a desired peptide linker can be inserted
between, and in the same reading frame as a polynucleotide encoding
all or part of a u-PA including the u-PA protease domain, using any
suitable conventional technique. In one example, the fusion protein
contains a u-PA polypeptide, for example a u-PA protease domain,
and a fusion partner, such as HSA, Fc, HABD, or C2 scFv, separated
by a peptide linker(s).
[0570] Exemplary u-PA fusion polypeptides include a linker at the
C-terminus of the u-PA protease domain which links the u-PA
protease domain to a C-terminal fusion partner, such as HSA or Fc.
u-PA-linker-Fc and u-PA-linker-HSA molecules optionally can contain
an epitope tag and/or a signal for expression and secretion. An
exemplary u-PA-linker-Fc fusion protein is set forth in SEQ ID NO:
1018, which contains human immunoglobulin light chain kappa ( )
leader signal peptide sequence (SEQ ID NO: 999), HIS-SUMO (SEQ ID
NO: 990), a u-PA protease domain (SEQ ID NO: 21), a linker (SEQ ID
NO: 1002), and an Fc fragment of the human IgG1 heavy chain (SEQ ID
NO:992).
[0571] In other examples, the exemplary u-PA fusion proteins are
u-PA-linker-HSA fusion polypeptides, such as the fusion proteins
set forth as SEQ ID NOs: 1015-1017. For example, the fusion
polypeptide set forth in SEQ ID NO:1015 contains human
immunoglobulin light chain kappa (.kappa.) leader signal peptide
sequence (SEQ ID NO: 999), the N-terminal domain of u-PA (SEQ ID
NO: 1042), the wild-type u-PA activation sequence (SEQ ID NO: 997),
a u-PA protease domain (SEQ ID NO: 987), a linker (SEQ ID NO:
1002), and HSA (SEQ ID NO:991). In another example, the fusion
polypeptide set forth in SEQ ID NO: 1016 contains human
immunoglobulin light chain kappa (.kappa.) leader signal peptide
sequence (SEQ ID NO: 999), a furin activation sequence in the u-PA
activation sequence (SEQ ID NO:996), a u-PA protease domain (SEQ ID
NO: 21), a linker (SEQ ID NO: 1002), and HSA (SEQ ID NO:991). In
another example, the fusion polypeptide set forth in SEQ ID NO:1017
contains human immunoglobulin light chain kappa (.kappa.) leader
signal peptide sequence (SEQ ID NO: 999), HIS-SUMO (SEQ ID NO:
990), a u-PA protease domain (SEQ ID NO: 21), a linker (SEQ ID NO:
1002), and HSA (SEQ ID NO:991).
[0572] In other examples the linker is at the N-terminus of the
u-PA protease domain and links the protease domain to an N-terminal
fusion partner. For example, the fusion protein may contain an
N-terminal Fc linked to u-PA. An exemplary FC-linker-u-PA fusion
polypeptide is set forth in SEQ ID NO: 1004, which contains human
immunoglobulin light chain kappa (.kappa.) leader signal peptide
sequence (SEQ ID NO: 999), an Fc fragment of the human IgG1 heavy
chain (SEQ ID NO:992), a linker (SEQ ID NO: 1003), the wild-type
u-PA activation sequence (SEQ ID NO: 997), and a u-PA protease
domain (SEQ ID NO:987).
[0573] iv. Fusion Partners
[0574] Fusion proteins, such as fusion proteins containing fusion
to Fc, fusion to human serum albumin (HSA), fusion to a
single-chain fragment variable (scFv) antibody, such as scFv that
binds Collagen II (C2scFv), fusion to HABD, and fusion to other
polypeptides, are known modifications for improving
pharmacokinetics of peptide or biologic drugs. Also among these is
conjugation to either linear or branched-chain monomethoxy
poly-ethylene glycol (PEG), resulting in increases in the molecular
mass and hydrodynamic radius, and a decrease in the rate of
glomerular filtration by the kidney. Another approach to for
improving pharmacokinetic parameters includes modification of
glycosylation patterns, resulting in reduced clearance and
extension of half-life.
[0575] Exemplary u-PA fusion polypeptides include placement of the
fusion partner (i.e., HSA, HABD, C2 scFv or Fc)N-terminal to the
u-PA protease domain or C-terminal to the u-PA protease domain. An
exemplary u-PA fusion protein where the fusion partner is
N-terminal to the u-PA protease domain is set forth in SEQ ID NO:
1004. Exemplary u-PA fusion proteins where the fusion partner is
C-terminal to the u-PA protease domain are set forth in SEQ ID NOs:
1006-1018.
[0576] (a) Fc Domain
[0577] Some examples of u-PA fusion proteins include the heavy
chain of an immunoglobulin polypeptide, most usually the constant
domains of the heavy chain. Exemplary sequences of heavy chain
constant regions for human IgG sub-types are set forth in SEQ ID
NO: 45 (IgG1), SEQ ID NO: 1020 (IgG2), SEQ ID NO: 1021 (IgG3), and
SEQ ID NO: 1022 (IgG4). For example, for the exemplary heavy chain
constant region set forth in SEQ ID NO: 45, the CH1 domain
corresponds to amino acids 1-98, the hinge region corresponds to
amino acids 99-110, the C.sub.H2 domain corresponds to amino acids
111-223, and the CH3 domain corresponds to amino acids 224-330.
[0578] In one example, a u-PA fusion protein can include the Fc
region of an immunoglobulin polypeptide, such as human
immunoglobulin. Typically, such a fusion retains at least a
functionally active hinge, C.sub.H2 and C.sub.H3 domains of the
constant region of an immunoglobulin heavy chain. For example, a
full-length Fc sequence of IgG1 includes amino acids 105-330 of the
sequence set forth in SEQ ID NO:45. Exemplary Fc sequences for
hIgG1 are set forth in SEQ ID NO: 992 and 1023, and contain almost
all of the hinge sequence corresponding to amino acids 100-110 of
SEQ ID NO:45, and the complete sequence for the C.sub.H2 and
C.sub.H3 domain as set forth in SEQ ID NO:45. Another exemplary Fc
polypeptide is set forth in PCT application WO 93/10151, and is a
single chain polypeptide extending from the N-terminal hinge region
to the native C-terminus of the Fc region of a human IgG1 antibody
(SEQ ID NO:50). The precise site at which the linkage is made is
not critical: particular sites are well known and can be selected
in order to optimize the biological activity, or stability of the
u-PA polypeptide. For example, other exemplary Fc polypeptide
sequences begin at amino acid C109 or P113 of the sequence set
forth in SEQ ID NO: 45 (see e.g., U.S. Pub. No. 2006/0024298).
[0579] In addition to hIgG1 Fc, other Fc regions also can be
included in the u-PA fusion proteins provided herein. For example,
where effector functions mediated by Fc/Fc.gamma.R interactions are
to be minimized, fusion with IgG isotypes that poorly recruit
complement or effector cells, such as for example, the Fc of IgG2
or IgG4, is contemplated. Additionally, the Fc fusions can contain
immunoglobulin sequences that are substantially encoded by
immunoglobulin genes belonging to any of the antibody classes,
including, but not limited to IgG (including human subclasses IgG1,
IgG2, IgG3, or IgG4), IgA (including human subclasses IgA1 and
IgA2), IgD, IgE, and IgM classes of antibodies. Further, linkers
can be used to covalently link Fc to another polypeptide to
generate a Fc chimera.
[0580] Modified Fc domains also are contemplated herein for use in
chimeras with u-PA fusion polypeptides. In some examples, the Fc
region is modified such that it exhibits altered binding to an FcR
so has to result altered (i. e. more or less) effector function
than the effector function of an Fc region of a wild-type
immunoglobulin heavy chain. Thus, a modified Fc domain can have
altered affinity, including but not limited to, increased or low or
no affinity for the Fc receptor. For example, the different IgG
subclasses have different affinities for the Fc.gamma.Rs, with IgG1
and IgG3 typically binding substantially better to the receptors
than IgG2 and IgG4. Different Fc.gamma.Rs mediate different
effector functions. Fc.gamma.R1, Fc.gamma.RIIa/c, and
Fc.gamma.RIIIa are positive regulators of immune complex triggered
activation, characterized by having an intracellular domain that
has an immunoreceptor tyrosine-based activation motif (ITAM).
Fc.gamma.RIIb, however, has an immunoreceptor tyrosine-based
inhibition motif (ITIM) and is therefore inhibitory. In some
instances, an u-PA polypeptide-Fc fusion protein provided herein
can be modified to enhance binding to the complement protein C1q.
Further, an Fc can be modified to alter its binding to FcRn,
thereby improving the pharmacokinetics of an u-PA-Fc fusion
polypeptide. Thus, altering the affinity of an Fc region for a
receptor can modulate the effector functions and/or pharmacokinetic
properties associated by the Fc domain. Modified Fc domains are
known to one of skill in the art and described in the literature,
see e.g. U.S. Pat. No. 5,457,035; U.S. Patent Publication No. US
2006/0024298; and International Patent Publication No. WO
2005/063816 for exemplary modifications.
[0581] In some examples, a u-PA polypeptide multimer is formed.
Typically, a polypeptide multimer is a dimer of two chimeric
proteins created by linking, directly or indirectly, two of the
same or different u-PA polypeptides, such as a u-PA protease
domain, to an Fc polypeptide. In some examples, a gene fusion
encoding the u-PA-Fc fusion protein is inserted into an appropriate
expression vector. The resulting u-PA-Fc fusion proteins can be
expressed in host cells transformed with the recombinant expression
vector, and allowed to assemble much like antibody molecules, where
interchain disulfide bonds form between the Fc moieties to yield
divalent u-PA polypeptides.
[0582] u-PA fusion polypeptides containing Fc regions also can be
engineered to include a tag with metal chelates or other epitope.
The tagged domain can be used for rapid purification by
metal-chelate chromatography, and/or by antibodies, to allow for
detection of western blots, immunoprecipitation, or activity
depletion/blocking in bioassays.
[0583] Exemplary u-PA-Fc fusion polypeptides include fusion of the
u-PA protease domain and Fc. Exemplary u-PA-Fc fusion proteins are
set forth in SEQ ID NOs: 1004, 1006, 1010, 1011, 1012 and 1018. The
u-PA-Fc molecules optionally can contain an epitope tag or a signal
for expression and secretion. For example, the exemplary u-PA-Fc
fusion polypeptides set forth as SEQ ID NOs: 1004, 1010, and 1011
contain human immunoglobulin light chain kappa (.kappa.) leader
signal peptide sequence (SEQ ID NO: 999), an Fc fragment of the
human IgG1 heavy chain (SEQ ID NO:992) and a u-PA protease domain
(SEQ ID NO: 21 or 987) either N-terminal (SEQ ID NO:1004) or
C-terminal (SEQ ID NOs:1010 and 1011) to the Fc. In another
example, the exemplary u-PA-Fc fusion polypeptides set forth as SEQ
ID NOs: 1006 and 1012 contain human IL2 Signal Peptide (hIL2SP)
sequence (SEQ ID NO: 1000), a u-PA protease domain (SEQ ID NO: 5 or
21), and an Fc fragment of the human IgG1 heavy chain (SEQ ID
NO:992)N-terminal to the u-PA protease domain.
[0584] (b) Serum Albumin
[0585] u-PA fusion proteins can be generated with albumin as a
fusion partner in order to increase the half-life, stability,
bioavailability, distribution and/or improve the pharmacokinetics
of u-PA. Numerous products linked to human serum albumin (HSA) are
approved for use as therapeutics, including use as cancer
therapeutics and for treatment of type 2 diabetes (AlQahtani et al.
(2019) Biomed and Pharmacotherapy 113:108750; Roscoe et al., (2018)
Mol. Pharmaceutics 151:15046-5047; Strohl, W. R. (2015) BioDrugs
4:215-239). In some examples, the mature HSA protein, lacking the
signal sequence and activation sequence is fused to a protein of
interest. In some examples of a u-PA fusion protein, serum albumin,
such as human serum albumin (HSA), is conjugated to the u-PA, such
as the u-PA protease domain. An exemplary HSA is set forth in SEQ
ID NO: 991.
[0586] u-PA-HSA fusion polypeptides include fusion of the u-PA
protease domain and HSA. Exemplary u-PA-HSA fusion proteins are set
forth in SEQ ID NOs: 1007 and 1013-1017. u-PA-HSA molecules
optionally can contain an epitope tag and/or a signal for
expression and secretion. For example, the exemplary u-PA-HSA
fusion polypeptides set forth as SEQ ID NOs: 1014-1017 contain
human immunoglobulin light chain kappa (.kappa.) leader signal
peptide sequence (SEQ ID NO: 999), a u-PA protease domain (SEQ ID
NO: 987 or 21), and a C-terminal HSA (SEQ ID NO:991). In another
example, the exemplary u-PA-HSA fusion polypeptide set forth as SEQ
ID NO:1013 contains human IL2 Signal Peptide (hIL2SP) sequence (SEQ
ID NO: 1000), the u-PA protease domain (SEQ ID NO:5), and a
C-terminal HSA (SEQ ID NO:991).
[0587] (c) scFv that binds Collagen II (C2scFv)
[0588] Recombinant antibody fragments in the form of single-chain
fragment variable (scFv) antibodies, such as a scFv that binds
Collagen II (C2scFv), can be used as a fusion partner with u-PA.
scFv antibodies produced from phage display can be fused to
markers, or active or therapeutic proteins (Ahmad et al. (2012)
Clin Dev Immunol 2012:980250). Fusion of scFvs can be used to
increase yield and activity of conjugated proteins (Martin et al.,
(2006) BMC Biotech 6:46).
[0589] Single-chain fragment variable antibodies comprise heavy
(V.sub.H) and light (V.sub.L) chain variable regions joined by a
peptide linker or disulfide bond (Glockshuber et al. (1990)
Biochemistry 29(6): 1362-1367). The peptide linker plays a critical
role in folding of the polypeptide chain. Commonly utilized linkers
comprise Gly and Ser residues for flexibility or Glu and Lys to
enhance solubility (Whitlow et al. (1993) Protein Engineering
6(8):989-995).
[0590] scFvs can be fused to proteins for specific delivery to
antigen-presenting cells (Ahmad et al. (2012) Clin Dev Immunol
2012:980250). For example, the scFv can be generated to target
collagen II, such as for uses as research agents, and as a delivery
agent of therapeutic molecules to sites expressing human collagen
II. For example, the scFv is an isolated monoclonal antibody or
fragment thereof that binds human collagen II, comprising a VH
region and a VL region, where the C2scFv comprises an amino acid
sequence having a sequence shown in SEQ ID NO: 993.
[0591] Exemplary u-PA-C2scFv fusion polypeptides include fusion of
the u-PA protease domain and C2scFv. An exemplary u-PA-C2scFv
fusion protein is set forth in SEQ ID NO: 1008. u-PA-C2scFv
molecules optionally can contain an epitope tag or a signal for
expression and secretion. For example, the exemplary u-PA-C2scFv
fusion polypeptide set forth as SEQ ID NO:1008 contains a human IL2
Signal Peptide (hIL2SP) sequence (SEQ ID NO: 1000), a u-PA protease
domain (SEQ ID NO:21), and a C-terminal C2scFv (SEQ ID NO:993).
[0592] (d) Hyaluronic Acid Binding Domain (HABD)
[0593] In some examples, the u-PA fusion proteins contain a HABD
fusion partner, such as Tumor Necrosis factor-Stimulated Gene-6
(TSG-6), such as the TSG-6 set forth as SEQ ID NO: 994
(corresponding to amino acids 32-134 of human TSG-6; NCBI No.
NP_009046.2). u-PA fusion proteins can be generated with a HABD,
such as TSG-6, as a fusion partner in order to increase the
half-life, stability, bioavailability, distribution and/or improve
the pharmacokinetics of u-PA.
[0594] Tumor necrosis factor-Stimulated Gene-6 (TSG-6, tumor
necrosis factor alpha-induced protein 6, TNFAIP6; NCBI No.
NP_009046.2) is a .about.35 kDa secreted glycoprotein composed of a
single N-terminal link module and C-terminal CUB domain. Expression
of TSG-6 is induced in many cell types by inflammatory mediators,
including cytokines and growths factors. Via its link module, which
has been reported to contain approximately amino acids 35-132,
TSG-6 is a potent inhibitor of polymorphonuclear leukocyte
migration. TSG-6 forms a stable complex with the serine protease
inhibitor Inter-alpha-Inhibitor (I.alpha.I) and potentiates the
anti-plasmin activity of I.alpha.I. TSG-6 also is important for the
formation and remodeling of HA-rich pericellular coats and
extracellular matrices.
[0595] Exemplary u-PA-HABD fusion polypeptides include fusion of
the u-PA protease domain and HABD. An exemplary u-PA-HABD fusion
protein is set forth in SEQ ID NO: 1009. u-PA-HABD molecules can,
optionally, contain an epitope tag or a signal for expression and
secretion. For example, the exemplary u-PA-HABD fusion polypeptide
set forth as SEQ ID NO: 1009 contains human IL2 Signal Peptide
(hIL2SP) sequence (SEQ ID NO: 1000), a u-PA protease domain (SEQ ID
NO:21), and a C-terminal HABD (SEQ ID NO:994).
[0596] v. Activation Sequences (sites)
[0597] Exemplary u-PA fusion proteins contain a site (sequence) for
u-PA activation. For example, u-PA fusion proteins comprise
wild-type u-PA sequence for auto-activation; contain furin sequence
for activation during protein expression; or are activated after
secretion signal cleavage, all generating the activated u-PA
protease.
[0598] (a) Furin
[0599] Furin proteins have been implicated in the endoproteolytic
maturation processing of inactive precursor proteins at single,
paired or multiple basic consensus sites within the secretory
pathway (Nakayama(1997) Biochem. J. 327:625-635; Seidah and
Chretien, Current Opinions in Biotechnology (1997) 8:602-607). Upon
transit of a newly synthesized precursor protein from the
endoplasmic reticulum to the Golgi compartment, the propeptide is
autocatalytically removed in a two-step processing event at a furin
cleavage motif (Leduc et al. (1992) J. Biol. Chem 267:14304-14308;
Anderson et al. (1997) EMBO 1508-1518). Furin requires a R-X-X-R
site for cleavage, and optimum processing occurs at a R-X-K/R-R
motif (Molloy et al. (1992) J. Biol Chem 267:16396-16402).
Exemplary u-PA activation sequences, containing the furin RRKR
cleavage sites, are set forth in SEQ ID NOs: 995 and 996.
[0600] u-PA fusion proteins may include a furin activation sequence
(site)N-terminal to the u-PA protease domain, so that u-PA protein
is activated during expression. u-PA activation during expression,
such as by inclusion of a furin activation sequence in the u-PA
activation sequence, is intended to remove the need for an
activation step during downstream processing.
[0601] u-PA fusion polypeptides including a furin activation
sequence and the u-PA protease domain were generated. Exemplary
furin-u-PA proteins are set forth in SEQ ID NOs: 1010, 1014 and
1016. Furin activated u-PA molecules optionally contain a fusion
partner, and/or a signal for expression and secretion. For example,
the exemplary u-PA fusion proteins set forth as SEQ ID NOs: 1014
and 1016 contain human immunoglobulin light chain kappa (.kappa.)
leader signal peptide sequence (SEQ ID NO: 999), a furin activation
sequence in the u-PA activation sequence (SEQ ID NO: 995 or 996),
the u-PA protease domain (SEQ ID NO: 21 or 987), and HSA (SEQ ID
NO:991). The u-PA fusion protein set forth as SEQ ID NO: 1014
further contains the N-terminus of u-PA (set forth as amino acids
21-178 of SEQ ID NO:1 or SEQ ID NO: 1042), N-terminal to the
furin-u-PA protease domain with the u-PA protease domain set forth
in SEQ ID NO: 987. In another example, u-PA fusion protein set
forth as SEQ ID NO:1010 contains human immunoglobulin light chain
kappa (.kappa.) leader signal peptide sequence (SEQ ID NO: 999), a
furin activation sequence in the u-PA activation sequence (SEQ ID
NO: 995), the u-PA protease domain (SEQ ID NO: 21), and Fc (SEQ ID
NO:992).
[0602] (b) u-Pa
[0603] u-PA zymogen activation occurs by cleavage of a single
peptide bond N-terminal to the u-PA catalytic domain, initiating a
conformational change in the protein. u-PA constructs generated
herein can contain the 12 amino acid u-PA activation sequence (SEQ
ID NO: 997) or a modified form thereof (SEQ ID NO: 998) or can
contain an extended portion of the u-PA N-terminus including the
activation sequence, such that the u-PA comprises the full-length
mature polypeptide, such as the polypeptide set forth in SEQ ID NO:
3. In other examples, the u-PA comprises the N-terminus, such as
the N-terminal region of u-PA set forth as amino acids 21-178 of
SEQ ID NO: 1 or SEQ ID NO: 1042, and the 12 amino acid u-PA
activation sequence (SEQ ID NO: 997) or a modified form of the u-PA
activation sequence (SEQ ID NO: 998).
[0604] Fusion proteins containing the modified u-PA polypeptides
provided herein have been prepared. u-PA fusion polypeptides
including the wild-type or a modified u-PA activation sequence and
the u-PA protease domain were generated. Exemplary u-PA proteins
containing the wild-type u-PA activation sequence for activation
are set forth in SEQ ID NOs: 1004, 1005, 1011, and 1015. The fusion
peptides optionally can contain a fusion partner, and/or a signal
for expression and/or secretion. For example, the exemplary u-PA
fusion protein set forth as SEQ ID NO:1004 contains human
immunoglobulin light chain kappa (.kappa.) leader signal peptide
sequence (SEQ ID NO: 999), Fc (SEQ ID NO:992), the u-PA activation
sequence (SEQ ID NO:995), and the u-PA protease domain (SEQ ID NO:
987). In a further example, the u-PA fusion protein set forth as
SEQ ID NO: 1005 contains the full-length mature u-PA sequence (SEQ
ID NO: 3 with the modified protease domain set forth in SEQ ID NO:
987) and an N-terminal human immunoglobulin light chain kappa
(.kappa.) leader signal peptide sequence (SEQ ID NO: 999). In a
further example, the u-PA fusion protein set forth as SEQ ID NO:
1011 contains an N-terminal human immunoglobulin light chain kappa
(.kappa.) leader signal peptide sequence (SEQ ID NO: 999), the
full-length mature u-PA sequence (SEQ ID NO: 3 with the modified
protease domain set forth in SEQ ID NO: 987), and Fc (SEQ ID NO:
992). In another example, the u-PA fusion protein set forth as SEQ
ID NO:1015 contains human immunoglobulin light chain kappa
(.kappa.) leader signal peptide sequence (SEQ ID NO: 999), the
N-terminus of u-PA (set forth as amino acids 21-178 of SEQ ID NO:1)
including the u-PA activation sequence, the u-PA protease domain
(SEQ ID NO: 987), and HSA (SEQ ID NO:991). Modified u-PA
polypeptides, such as those of SEQ ID NOs: 1006, 1007, 1009 and
1010, upon expression, demonstrated u-PA protease activity.
Modified u-PA with a furin activation sequence N-terminal to u-PA
with an Ig FC fusion at the C-terminus (such as set forth in SEQ ID
NO: 1010) showed the highest activity.
[0605] vi. Purification Tags
[0606] Exemplary u-PA fusion proteins contain a tag for
purification of the u-PA or u-PA fusion protein. Exemplary tags for
purification of u-PA fusion proteins are set forth in Section F,
above. Exemplary u-PA fusion proteins can comprise a SUMO or His
sequence for purification.
[0607] (a) His Tag
[0608] u-PA fusion proteins may include a His tag, such as the
6.times.His set forth in SEQ ID NO: 989, and the u-PA protease
domain.
[0609] u-PA fusion polypeptides including a His purification tag
and the u-PA protease domain were generated. Exemplary HIS-u-PA
fusion proteins are set forth in SEQ ID NOs: 1017 and 1018. His
tagged u-PA molecules optionally can contain a fusion partner,
and/or a signal for expression and secretion. For example, the
exemplary His-u-PA fusion protein set forth as SEQ ID NO: 1017
contains human immunoglobulin light chain kappa (.kappa.) leader
signal peptide sequence (SEQ ID NO: 999), 6.times.His (SEQ ID
NO:989), SUMO (SEQ ID NO:1031), the u-PA protease domain (SEQ ID
NO: 21), and HSA (SEQ ID NO:991). In another example, the exemplary
His tagged-u-PA fusion protein set forth as SEQ ID NO: 1018
contains human immunoglobulin light chain kappa (.kappa.) leader
signal peptide sequence (SEQ ID NO: 999), 6.times.His (SEQ ID
NO:989), SUMO (SEQ ID NO:1031), the u-PA protease domain (SEQ ID
NO: 21), and Fc (SEQ ID NO:992).
[0610] (b) SUMO
[0611] u-PA fusion proteins can include a His tag and/or SUMO
sequences for accumulation in inclusion bodies can be include. For
example, the HIS-SUMO sequence set forth in SEQ ID NO: 990, and the
u-PA protease domain, can be linked to the full-length modified
u-PA polypeptide, or to a catalytically active portion thereof,
such to the protease domain, or to a larger portion of the modified
u-PA polypeptide. u-PA fusion polypeptides including His-SUMO tags
and the u-PA protease domain were generated. Exemplary
HIS-SUMO-u-PA proteins are set forth in SEQ ID NOs: 1017 and 1018.
HIS-SUMO tagged u-PA molecules optionally can contain a fusion
partner, and/or a signal for expression and secretion. For example,
the His-SUMO-u-PA fusion protein set forth as SEQ ID NO: 1017
contains human immunoglobulin light chain kappa (.kappa.) leader
signal peptide sequence (SEQ ID NO: 999), 6.times.His (SEQ ID
NO:989), SUMO (SEQ ID NO:1031), the u-PA protease domain (SEQ ID
NO: 21), and HSA (SEQ ID NO:991). In another example, the exemplary
His-SUMO-u-PA fusion protein set forth as SEQ ID NO: 1018 contains
human immunoglobulin light chain kappa (.kappa.) leader signal
peptide sequence (SEQ ID NO: 999), 6.times.His (SEQ ID NO:989),
SUMO (SEQ ID NO:1031), the u-PA protease domain (SEQ ID NO: 21),
and Fc (SEQ ID NO:992).
[0612] 7. Nucleic Acid Molecules
[0613] Nucleic acid molecules encoding u-PA polypeptides are
provided herein. Nucleic acid molecules include allelic variants or
splice variants of any encoded u-PA polypeptide, or catalytically
active portion thereof. In one embodiment, nucleic acid molecules
provided herein have at least 50, 55, 60, 65, 70, 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any
nucleic acid encoded u-PA polypeptide or catalytically active
portion thereof. In another embodiment, a nucleic acid molecule can
include those with degenerate codon sequences of any of the u-PA
polypeptides or catalytically active portions thereof such as those
provided herein.
[0614] 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.
[0615] A u-PA protease 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 or AMD or DGF, 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 transplant site for
therapy.
G. COMPOSITIONS, FORMULATIONS AND DOSAGES
[0616] Pharmaceutical compositions containing modified u-PA
polypeptides, modified u-PA 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. In most
embodiments, the modified u-PA polypeptide or fusion protein will
be in an activated form in the composition for administration.
Thus, for example, the polypeptides will be two chain activated
forms or, where the fusion protein contains a multimerization
domain, the protein can be a multimer, such as a dimer.
[0617] Selection of the carrier or excipient is within the skill of
the administering professional 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.
[0618] 1. Administration of Modified u-PA Polypeptides
[0619] For purposes of this section, modified u-PA polypeptides
refer to u-PA polypeptides that contain modifications, such as the
modified protease domains, and include the conjugates, such as
fusion proteins. 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).
[0620] 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.
[0621] The u-PA 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 u-PA polypeptide provided herein, including any of the
modified u-PA polypeptides provided herein. For example, when
administering a compound ex vivo, a body fluid, such as the
vitreous, or tissue sample from a subject can be contacted with the
u-PA polypeptides that are coated on a tube or filter, such as for
example, a true 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, intravitreally,
intraretinally, subretinally, periocularly, subcutaneously, or
topically, in liquid, semi-liquid or solid form and are formulated
in a manner suitable for each route of administration.
Determination of dosage is within the skill of the physician, and
can be a function of the particular disorder, route of
administration and subject. Exemplary dosages, include for example
0.1-1 mg.
[0622] The modified u-PA polypeptide 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 u-PA polypeptide 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.
[0623] For pulmonary administration to the lungs, the modified u-PA
polypeptide 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.
[0624] 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 sulfate). 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.
[0625] 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.
[0626] The modified u-PA 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.
[0627] The modified u-PA polypeptide 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.
[0628] The modified u-PA polypeptides can be formulated for ocular
or opthalmic delivery. Ocular drug delivery may be, for example,
topical, oral or systemic, and/or injected. For example, a modified
u-PA polypeptide(s) or pharmaceutical composition containing a
modified u-PA polypeptide(s) may be administered topically, such as
in the form of eye drops. In another example, a modified u-PA
polypeptide(s) or pharmaceutical composition containing a modified
u-PA polypeptide(s) can be administered by periocular and/or
intravitreal or intraretinal or subretinal administration, such as,
for example, by periocular, or intraretinal, or intravitreal
injection(s).
[0629] The modified u-PA polypeptides or pharmaceutical composition
containing modified u-PA polypeptides or nucleic acids encoding
modified u-PA polypeptides can be formulated for systemic
administration for treatment of DGF. In another example, the
modified u-PA polypeptides or pharmaceutical composition containing
modified u-PA polypeptides or nucleic acids encoding modified u-PA
polypeptides are directly infused or injected into the kidney or
into the tissues or organs adjacent or surrounding the transplanted
kidney. The modified u-PA polypeptides or pharmaceutical
composition containing modified u-PA polypeptides can be
administered before the time of allograft transplantation or at the
time of transplantation with administration continuing in a chronic
fashion, and/or can be administered during a rejection episode in
the event such an episode does occur.
[0630] 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).
[0631] 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 u-PA polypeptide
compared to the unmodified and/or wild type u-PA polypeptide.
[0632] 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.
[0633] Also provided are compositions containing nucleic acid
molecules, including expression vectors, encoding the u-PA
polypeptides. In some embodiments, the compositions of nucleic acid
molecules encoding the u-PA polypeptides and expression vectors
encoding them 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 acid
therein, and reintroduction into the host or a compatible
recipient.
[0634] 2. Administration of Nucleic Acids Encoding Modified u-PA
Polypeptides (Gene Therapy)
[0635] The modified u-PA polypeptides can be delivered to cells and
tissues by expression of nucleic acid molecules. The modified u-PA
polypeptides can be administered as nucleic acid molecules encoding
the modified u-PA 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. Methods for administering u-PA 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.
[0636] u-PA polypeptides also can be used in ex vivo gene
expression therapy using vectors. Suitable gene therapy vectors and
methods of delivery are known to those of skill in the art. For
example, cells can be engineered to express a modified u-PA
polypeptide, such as by integrating u-PA 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.
[0637] Viral vectors including, for example, adenoviruses, herpes
viruses, adeno-associated viruses (AAV), retroviruses, such as
lentiviruses, EBV, SV40, cytomegalovirus vectors, vaccinia virus
vectors, and others designed for gene therapy can be employed. The
vectors can be those that remain episomal or those that can
integrate into chromosomes of the treated subject. A modified u-PA
polypeptide can encoded in a viral vector, such as AAV, which is
administered to a subject in need of treatment.
[0638] Virus vectors suitable for gene therapy include adenovirus,
adeno-associated virus, retrovirus, lentivirus, 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.RTM.,
Rockville, Md.). Adenovirus can be used ex vivo, for example, cells
are isolated from a patient in need of treatment, and transduced
with a modified u-PA polypeptide-expressing adenovirus vector.
After a suitable culturing period, the transduced cells are
administered to a subject, locally and/or systemically.
Alternatively, u-PA 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 10.sup.14 particles per kilogram subject
weight, generally between 10.sup.6 or 10.sup.8 particles to
10.sup.12 particles per kilogram subject weight.
[0639] 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)
32(21):e172) can be engineered to encode and express the u-PA
polypeptide. 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.
[0640] Another method for introducing nucleic acids encoding the
modified u-PA 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 u-PA polypeptides for
gene transfer to mammalian cells or whole animals.
[0641] The nucleic acids can be encapsulated in a vehicle, such as
a liposome, or introduced into a cell, 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.
[0642] In some embodiments, 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.
[0643] For ex vivo and in vivo methods, nucleic acid molecules
encoding the u-PA 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, including 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.
[0644] 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 u-PA 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.
[0645] In vivo expression of a modified u-PA polypeptide can be
linked to expression of additional molecules. For example,
expression of a u-PA 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 u-PA
polypeptide can be used to enhance the cytotoxicity of the
virus.
[0646] In vivo expression of a u-PA polypeptide can include
operatively linking a u-PA polypeptide encoding nucleic acid
molecule to specific regulatory sequences such as a cell-specific
or tissue-specific promoter. u-PA polypeptides also can be
expressed from vectors that specifically infect and/or replicate in
target cell types and/or tissues. Inducible promoters can be used
to selectively regulate u-PA polypeptide expression.
[0647] 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.
[0648] Administration also can be direct, such as by administration
of a vector or cells that typically targets a cell or tissue. For
example, tumor cells and proliferating cells can be targeted cells
for in vivo expression of u-PA polypeptides. Cells used for in vivo
expression of a u-PA polypeptide also include cells autologous to
the patient. Such cells can be removed from a patient, nucleic
acids for expression of a u-PA polypeptide introduced, and then
administered to a patient such as by injection or engraftment.
[0649] Administration for Treatment of AMD and Other Ocular
Diseases
[0650] Nucleic acids encoding the modified u-PA polypeptides
provides can be administered for treatment of diseases or
conditions involving complement activation in their etiology, in
which inhibition thereof can ameliorate a symptom of the disease or
condition or otherwise treat the disease or condition. Nucleic
acids, such as vectors, such as viral vectors, designed for
delivery of nucleic acids that encode the modified u-PA
polypeptides described herein can be administered to subjects by
any suitable route or a combination of different routes, depending
upon the disease or condition. Nucleic acid delivery can be
effected via direct delivery to the eye (such as via ocular
delivery, subretinal injection, intravitreal (IVT) injection,
intraretinal injection, or topical (e.g., eye drops) delivery), or
delivery via systemic routes, e.g., intraarterial, intraocular,
intravenous, intramuscular, subcutaneous, intradermal, and other
parental routes of administration.
[0651] One skilled in the art can select any mode of administration
compatible with the subject and virus for administration, and that
also is likely to result in the virus reaching and entering the
target cell-type or tissue, e.g., eye, such as retinal pigment
epithelial (RPE) cells and/or photoreceptor cells. The route of
administration can be selected by one skilled in the art according
to any of a variety of factors, including the nature of the
disease, the properties of the target cell or tissue (e.g., cell
type), and the particular virus to be administered. Administration
can be selected where cells or tissue of interest are targeted,
such as the eye, e.g., the retinal pigment epithelial (RPE) cells
and/or photoreceptor cells or the subretinal space.
[0652] Nucleic acid encoding the modified u-PA polypeptides, such
as viral expression vectors, can be delivered, for example, to the
target cell which is characterized by the disease, such as an
ocular disease, such as AMD. For example, the composition
containing the virus can be delivered by subretinal injection, such
as subretinal injection to the retinal pigment epithelium (RPE),
photoreceptor cells or other ocular cells (e.g., retinal ganglion
cells). In some examples, subretinal administration of a virus,
such as any virus containing the nucleic acids described herein,
requires the skilled physician to perform a vitrectomy (i.e., where
a needle hole is created in the retina (retinomy) and fluid is
injected, such as fluid containing a virus, such as any virus
described herein). Subretinal injections can be effected via a
transcomeal route, through the pupil and then passing through the
lens, vitreous and retina. In other examples, subretinal injection
can be performed by passing a needle or any other administration
device through the sclera, entering the pars plana or limbus area,
though the mid- or posterior vitreous and to the opposite side of
the retina, into the subretinal space. In other examples,
subretinal injection can be performed by passing a needle or any
other administration device through the sclera and through the
choroid and Bruch's membrane, avoiding the retina to achieve
delivery to the RPE. Other appropriate routes for subretinal
administration can be determined by the skilled artisan or
physician or surgeon. In some examples, bleb formation signals
successful administration.
[0653] In other examples, the composition containing nucleic acid
encoding the modified u-PA polypeptide for expression in the eye
can be delivered by intravitreal injection to ocular cells, such as
administration to target vitreal cells and cells in the inner
retina. In some examples, intravitreal injection is performed by
passing a needle or any other administration device through the
pars plana, though the mid- or posterior vitreous and to the
opposite side of the retina. After intravitreal injection, the
composition containing the virus can be delivered to infect
ganglion cells. In other examples, the composition containing the
virus delivered by intravitreal injection target inner nuclear
layer cells. Efficacy of delivery depends on virus titer and
serotype. In some examples, treatment comprises direct intravitreal
injection combined with an intravitreal implantable device (i.e.,
bioerodible and nonbioerodible intravitreal implantable devices) to
increase concentration of the administered agent to the back of the
eye (Hwang et al. (2012) J Korean Med Sci 27:1580-85).
[0654] In other examples, the composition containing the nucleic
acid encoding the modified u-PA polypeptide is injected via the
palpebral vein to target ocular cells. In other examples, the virus
is applied ex vivo (e.g., applied to excised RPE choroid or fetal
retinal cells or retinal cells) for transplantation into the eye,
such as, for example, as a retinal graft. In other examples, one of
skill in the art, such as the skilled physician can select the
appropriate route for administration of any virus containing the
nucleic acids described herein. If desired, routes of
administration can be combined.
[0655] In one example, the virus is administered locally, at the
site where the target cells, e.g., diseased cells, are present,
i.e., in the eye or the retina. Topical administration often is
used in eye diseases of the anterior segment of the eye (Patel et
al. (2013) World J Pharmacol 2:47-64).
[0656] In one example, a virus to be delivered intravitreally can
be administered with a thin needle (27 to 30 G) through the pars
plana inside the vitreous body. The skilled artisan will determine
how far the needle shaft is inserted into the eye (e.g., insertion
depth), the speed of administration (e.g., the pressure applied to
the plunger), the angle of orientation of the bevel, and the angle
between the shaft and the pars plana.
H. THERAPEUTIC USES AND METHODS OF TREATMENT
[0657] The modified u-PA polypeptides provided herein target
complement protein C3 and permit modulation of complement-mediated
diseases and disorders. Therapeutic proteases, such as the modified
u-PA polypeptides provided herein, 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). Thus, proteases can maintain effective
regulation at concentrations significantly below the target
concentration. Additional differentiating advantages include (1)
irreversible inactivation; (2) low dosing; (3) decreased dosing
frequency (4) small molecular size; (5) the ability to target
post-translational modifications; (6) the ability to neutralize
high target concentrations; and (7) 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 modified u-PA
polypeptides provided herein can be used in the treatment of
complement-mediated diseases and disorders.
[0658] The skilled artisan understands the role of the complement
system in disease processes and is aware of a variety of such
diseases. Provided is a brief discussion of exemplary diseases and
the role of the complement protein C3 in their etiology and
pathology. The modified u-PA 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, modified u-PA polypeptides provided
herein. These described therapies are exemplary and do not limit
the applications of the modified u-PA polypeptides provided herein.
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 age-related macular degeneration (AMD), geographic atrophy (GA),
paroxysmal nocturnal hemoglobinuria (PNH), renal delayed graft
function (DGF), 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, chronic obstructive
pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF)
and/or Guillain Barre syndrome.
[0659] Treatment of diseases and conditions with modified u-PA
polypeptides provided herein can be effected by any suitable route
of administration using suitable formulations as described herein
including, but not limited to, subcutaneous injection, oral,
intravitreal, intraretinal, subretinal, periocular and transdermal
administration. If necessary, a particular dosage and duration and
treatment protocol can be empirically determined or extrapolated.
For example, exemplary doses of wild type u-PA polypeptides can be
used as a starting point to determine appropriate dosages. Modified
u-PA polypeptides that have more specificity and/or selectivity
compared to a wild type u-PA polypeptide 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 will vary depending upon the host treated and the
particular mode of administration.
[0660] 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.
[0661] 1. Disease Mediated by Complement Activation
[0662] 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.
[0663] 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),
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), age-related macular
degeneration (Bradley et al. Eye 25: 683-693 (2011); Gemenetzi et
al. Eye 30: 1-14 (2016)) and renal delayed graft function
(Danobeitia et al. [abstract]. Am J Transplant. 2013; 13 (suppl 5);
Yu et al. (2016) Am J Transplant 16(9):2589-2597; Castallano et al.
(2010) Am J Pathol 176(4): 1648-1659). Some exemplary examples of
complement-mediated diseases are described below.
[0664] a. Rheumatoid Arthritis
[0665] 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.
[0666] b. Sepsis
[0667] 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.
[0668] 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 plays 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.
[0669] c. Multiple Sclerosis
[0670] 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.
[0671] d. Alzheimer's Disease
[0672] 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 the precise cause of AD is not entirely clear, chronic
neuroinflammation in affected regions of AD brains suggests 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 an 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 amyloid-beta
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 progression and likely
contributes to neurite destruction.
[0673] e. Ischemia-Reperfusion Injury
[0674] 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.
Ischemia-reperfusion injury 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.
[0675] 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.
[0676] 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.
[0677] f. Ocular Disorders
[0678] In the normal eye, the complement system is continuously
activated at low levels; membrane-bound and soluble intraocular
complement regulatory proteins tightly regulate this spontaneous
complement activation. Low level complement activation protects
against pathogens without causing any damage to self-tissue and
vision loss. The complement system and complement regulatory
proteins control the intraocular inflammation in autoimmune uveitis
and play an important role in the development of corneal
inflammation, age-related macular degeneration and diabetic
retinopathy. The complement system plays an important role in the
pathogenesis of diabetic retinopathy (see, e.g., Ghosh et al.
(2015) Endocr Rev 36:272-288) as well as diabetic neuropathy and
diabetic cardiovascular disease. Spontaneous complement activation
can cause damage to the corneal tissue after the infection.
Complement inhibition is a relevant therapeutic target in the
treatment of various ocular diseases (see, e.g., Purushottam et al.
(2007) Mol Immunol. 44:3901-3908).
[0679] Age-Related Macular Degeneration (AMD)
[0680] Age-related macular degeneration is a clinical term that
describes a variety of diseases that are characterized by the
progressive loss of central vision. AMD is the leading cause of
vision loss in aged individuals in many industrialized countries
(Jager et al. (2008) N Engl J Med 358:2606-2617). Vision loss
occurs due to the progressive degeneration of the macula, the
region at the back of the eye comprising a high density of cone
photoreceptors, which is specialized for high-acuity, central
vision.
[0681] AMD can manifest as Dry (non-neovascular) AMD and/or Wet
AMD. Dry AMD is the more common (85-90% of cases) and milder form
of AMD, and is characterized by small, round, white-yellow lesions
(drusen) in and under the macula. Advanced dry AMD, or geographic
atrophy, leads to thinning of the retina due to loss of PRE
photoreceptors, deterioration of the macula and eventual blindness.
Although rarer, vision loss associated with wet AMD is generally
more dramatic than in dry AMD. Wet AMD includes the formation of
pathogenic blood vessels, termed choroidal neovascularization
(CNV), in which abnormal blood vessels develop beneath the retinal
pigment epithelium (RPE) layer of the retina. CNV invasion of the
retina from the underlying choroid through fractures in Bruch
membrane, the extracellular matrix between the choroid and the
retinal pigment epithelium (RPE), or their breakage can cause
vision loss in AMD (e.g., due to subretinal hemorrhage and/or
scarring).
[0682] Early clinical hallmarks of AMD include thickening of the
Bruch membrane and the appearance of drusen (Gass, J. D. (1972)
Trans. Am. Ophthalmol. Soc. 70: 409-36), which are extracellular
lipoproteinaceous deposits consisting of aggregated proteins (i.e.,
albumin, apolipoprotein E (APOE)), components of the complement
pathway (e.g., complement factor H (CFH), C1q, C3, C5, C5b, C6, C7,
C8, C9, and vitronectin (Hageman et al., (2001) Prog. Retin. Eye.
Res 29:95-112; Hageman et al. (2005) Proc. Nat. Acad. Sci. 102:
7227-7232; Mullins et al. (2000) FASEB H 14:835-846; Anderson et
al., (2010) Pro. Retin. Eye Res. 29:95-112)), immunoglobulins
and/or amyloid-.beta. (Crabb et al., (2002) Proc Natl Acad Sci 99:
14682-14687; Johnson et al., (2002) 99: 11830-11835)) and lipids
and cellular components that are localized between the RPE and the
Bruch membrane.
[0683] Inflammation in AMD is mediated by the deregulation of the
alternative complement pathway. Complement components C3 and C5 are
principal constituents of drusen in patients with AMD (Mullins et
al., (2000) FASEB J 14, 835-46; Johnson et al., (2000) Exp Eye Res
70, 441-9; Anderson et al., (2002) Am J Ophthalmol 134, 411-31; and
Leitner et al., (2001) Exp Eye Res 73, 887-96). It is hypothesized
that drusen biogenesis involves chronic inflammatory processes that
either can trigger complement activation and formation of MAC,
which may lyse RPE cells or disturb physiological homeostasis in
RPE cells, leading to inflammation characteristic of AMD (Johnson
et al. (2001) Exp Eye Res 73, 887-896). Complement proteins (e.g.,
C3d) were also detected in blood in AMD patients (Scholl et al.,
(2008) PLoS One 3: e2593), indicating that AMD-induced inflammation
may be systemic. There is genetic evidence for a role in complement
in the pathogenesis of dry AMD (Klein et al. Science
308(5720):385-389 (2005); Yates et al., NEJM 357:553-561 (2007)),
compstatin (and compstatin derivatives APL-1 and APL-2) and POT-4
(Potentia Pharmaceuticals), small peptide inhibitors of C3, may
slow the progression of geographic atrophy (Ricklin et al. (2008)
Adv. Exp. Med. Biol. 632: 273-292) in AMD, indicating that C3
(i.e., C3 inhibition) may be a viable target for AMD treatment.
[0684] g. Organ Transplantation and Delayed Graft Function
(DGF)
[0685] Complement plays a role in the pathogenesis of
ischemia-reperfusion injury. The mechanism of renal reperfusion
injury depends on the generation of C5a and C5b-9, both of which
have direct toxicity on the renal tubules contributing to acute
tubular necrosis and apoptosis, and leading to post-ischemic acute
renal failure and tissue fibrosis. In turn, the generation of these
terminal pathway components depends on intra-renal synthesis of C3
and availability of other complement components that are essential
for complement activation. The level of expression of C3 in the
donor organ is strongly dependent on the cold ischemic time (Elham
et al. (2010) Curr Opin Organ Transplant. 15:486-491).
[0686] Rejection in solid organ transplantation is influenced by
the initial inflammatory response and subsequent adaptive
alloimmune response, both of which are affected by various
complement components. Complement proteins play a significant part
in organ damage following transplantation in the process of
ischemia reperfusion and in modulating the activation of the
adaptive immune response. Inhibiting complement or modulating the
function of complement protein molecules can reduce transplant
organ damage and increase the organ lifespan (see, e.g., Elham et
al. (2010) Curr Opin Organ Transplant. 15:486-491). Targeting
complement components for therapeutic intervention can reduce organ
damage at the time of organ recovery, transfer and after
transplantation. Exemplary of such organs is the kidney. The
modified u-PA polypeptides provided herein can be administered to
mitigate and/or treat organ damage following transplantation.
[0687] Delayed graft function (DGF), such as renal delayed graft
function, is a condition occurring in a subset of kidney transplant
patients in which the transplanted organ fails to function normally
immediately following transplant. Other possible transplants
include, but are not limited to, heart, lung, vascular tissue, eye,
cornea, lens, skin, bone marrow, muscle, connective tissue,
gastrointestinal tissue, nervous tissue, bone, stem cells, islets,
cartilage, hepatocytes, and hematopoietic cells. Renal DGF is
characterized by acute necrosis of the renal allograft and is
clinically defined by the need for dialysis shortly following
transplantation. Acute kidney injury during the transplant process
frequently manifests as DGF. The pathology underlying DGF is
complex with contributions from donor-derived factors such as donor
age and duration of ischemia, and recipient factors such as
reperfusion injury, immunological responses and treatment with
immunosuppressant medications.
[0688] Components of the complement cascade and complement
activation play a critical role as mediators of transplant
rejection and ischemia-reperfusion injury leading to DGF. Animal
studies have established a key role for complement in ischemic
reperfusion injury. For example, Eculizumab, a humanized monoclonal
antibody directed against C5, blocks complement activation and was
shown to prevent delayed graft function in a subset of high-risk
kidney transplant patients (see, e.g., Horizon Scanning Research
and Intelligence Centre brief, 2016 September; Johnson et al.
(2015) Curr Opin Organ Transplant 20(6):643-651; Yu et al. (2016)
Am J Transplant 16(9):2589-2597). Granular C4d deposition was
associated with DGF in human renal allograft recipients (Kikid et
al. (2014) Transpl Int 27(3):312-321). Increased C3 production is
associated with kidney transplant rejection (Pratt et al. (2002)
Nat Med 8(6):582-587; Damman et al. (2011) Nephrol Dial Transplant
26(7):2345-2354). Hence, the modified u-PA polypeptides provided
herein, can be used as a therapeutic for preventing or ameliorating
or eliminating transplant rejection and DGF.
[0689] 2. Therapeutic Uses
[0690] a. Immune-Mediated Inflammatory Diseases
[0691] Modified u-PA polypeptides described herein can be used to
treat inflammatory diseases. Inflammatory diseases that can be
treated with proteases include acute and chronic inflammatory
diseases. Exemplary inflammatory diseases include central nervous
system diseases (CNS), autoimmune diseases, airway
hyper-responsiveness conditions such as in asthma, rheumatoid
arthritis, inflammatory bowel disease, and immune complex
(IC)-mediated acute inflammatory tissue injury.
[0692] 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. Modified u-PA polypeptides can be
assessed in EAE animal models. Modified u-PA polypeptides 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.
[0693] Complement activation modulates inflammation in diseases
such as rheumatoid arthritis (RA) (Wang et al., (1995) Proc. Natl.
Acad. Sci. U.S.A. 92:8955). Modified u-PA polypeptides can be used
to treat RA. For example, u-PA polypeptides can be injected locally
or systemically. Modified u-PA polypeptides can be dosed daily or
weekly. PEGylated u-PA polypeptides 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
re-immunized using the identical protocol. To examine the effects
of a u-PA polypeptide, 3 weeks following the initial challenge with
B-CII, a u-PA polypeptide 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 effect of a u-PA polypeptide on
established disease, a u-PA polypeptide 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 u-PA polypeptides and
controls to assess u-PA polypeptide treatment.
[0694] Modified u-PA polypeptide 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, neutrophilia, 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 u-PA polypeptide, 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 u-PA polypeptide 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 u-PA polypeptide 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.
[0695] Therapeutic u-PA polypeptides, such as those described
herein, can be used to treat sepsis and severe sepsis that can
result in lethal shock. A model of complement-mediated lethal shock
can be used to test the effects of a u-PA polypeptide 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 et al., (2002) Int Arch
Allergy Immunol 127:55-62). A u-PA polypeptide 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.
[0696] b. Neurodegenerative Disease
[0697] Complement activation exacerbates the progression of
Alzheimer's disease (AD) and contributes to neurite loss in AD
brains. Modified u-PA polypeptides described herein can be used to
treat AD. Mouse models that mimic some of the neuropathological and
behavioral features of AD can be used to assess the therapeutic
effects of u-PA polypeptides. 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. u-PA polypeptides can be administered,
such as by daily intraperitoneal or intravenous injections, and the
course and progression of symptoms is monitored compared to control
animals.
[0698] c. Cardiovascular Disease
[0699] Modified u-PA polypeptides provided herein can be used to
treat cardiovascular disease. u-PA polypeptides 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. u-PA polypeptides also can be used in the
treatment of the inflammatory response associated with
cardiopulmonary bypass that can contribute to tissue injury.
Generally, a u-PA polypeptide 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 u-PA polypeptide 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.
[0700] Effects of a u-PA polypeptide 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 u-PA polypeptide, such as for example
a modified u-PA 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 u-PA polypeptide 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.
[0701] 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
modified u-PA polypeptides 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 modified u-PA
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.
[0702] Modified u-PA polypeptides provided herein can be used as
therapeutics prior to or following Cardiopulmonary Bypass (CPB) or
coronary artery bypass graft to inhibit the inflammatory immune
response that often follows bypass and that can contribute to
tissue injury. An in vitro recirculation of whole blood in an
extracorporeal bypass circuit can be used to stimulate platelet and
leukocyte changes and complement activation induced by CPB (Rinder
et al. (1995) J. Clin. Invest. 96:1564). In such a model, addition
of a u-PA polypeptide 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.
[0703] d. Age-Related Macular Degeneration (AMD)
[0704] Modified u-PA polypeptides described herein can be used to
treat Age-Related Macular Degeneration (AMD). Age-Related Macular
Degeneration (AMD) that can be treated with proteases include wet
AMD, dry AMD and geographic atrophy.
[0705] Numerous animal models of AMD are available that mimic many
of the characteristics of the human disorder (Pennesi et al. (2012)
Mol. Aspects Med. 33(4):487-509)). Mutations in complement pathway
genes were shown to increase or decrease susceptibility to AMD
(Edwards et al. (2005) Science 308(5720):421-424; Hageman et al.
(2005) Proc. Nat. Acad. Sci 102(20): 7227-7232; Klein et al. (2005)
Science 308(5720):385-389). For example, in complement factor H
(CFH), which normally interacts with C3b, the single nucleotide
polymorphism Y402H prevented binding of C3b with factor B, leading
to inhibition of C3 formation. Y402H is associated with an
increased risk of AMD in people and the mutation was previously
identified in 43-59% of AMD patients (Haines et al. (2005) Science
308(5720): 419-421; Thakkinstian et. al. (2006) Hum. Mol. Genet.
15(18): 2784-2790; Zareparsi et al. (2005) Am. J. Hum. Genet.
77(1): 149-153).
[0706] Genetically modified mice that lack the ability to make CFH
develop characteristics of AMD, including retinal abnormalities,
decreased visual acuity and complement deposition (Coffey et al.
(2007) Proc. Nat. Acad. Sci. 104:16651-16656). Mutations in
complement proteins Factor B (Montes et al. (2009) Proc. Nat. Acad.
Sci. 106(11): 4366-4371), C2 (Gold et al. (2006) Nat. Genet. 38(4):
458-462), and C3 (Maller et al. (2007) Nat. Genet. 39(10):
1200-1201; Yates et al. (2007) New Engl. J. Med. 357(6): 553-561)
are associated with increased or decreased risk of developing AMD
based on their impact on expression and/or activity of the various
complement proteins (Reynolds et al. (2009) Invest. Ophthalmol.
Vis. Sci. 50(12): 5818-5827).
[0707] Modified u-PA proteases, such as modified u-PA proteases
provided herein, where an activity, such as substrate specificity
or selectivity, of the u-PA protease for cleaving complement
protein C3 is altered can be can be used as therapeutics. The
modified u-PA polypeptides provided herein are administered, for
example, by bi-monthly intravitreal or subretinally, or
intraretinal injection, and the course and progression of symptoms
is monitored compared to control animals or subjects. The levels of
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.
[0708] Complement activation plays a role in disease progress in
Age-Related Macular Degeneration (AMD) (see, e.g., Bradley et al.,
(2011) Eye 25:683-693; Gemenetzi et al. (2016) 30:1-14). Modified
u-PA polypeptides can be used to treat AMD. For example, u-PA
polypeptides or a pharmaceutical composition containing u-PA
polypeptides, such as the modified u-PA polypeptides described
herein, can be injected intravitreally, or intraretinally, or
subretinally, or periocularly. Modified u-PA polypeptides can be
dosed daily or weekly or less frequently, such as for example,
monthly or less frequently, such as bi-monthly. For AMD, modified
uPA polypeptides that are not further "modified" for extended
duration in the eye (e.g., fusion proteins, PEGylation, etc.)
monthly dosing is likely (bi-monthly dosing also is contemplated).
After appropriate "modification", every 3 months (or less
frequently) may be possible. The modified u-PA polypeptides can be
modified, such as by PEGylation to reduce potential immunogenicity
and/or to increase serum half-life. For AMD, modified u-PA
polypeptides that are not further modified for extended duration in
the eye (e.g., fusion proteins, PEGylation) monthly dosing or
bi-monthly dosing is used. If modified, such as by PEGylation,
dosing can be effected every 3 months or more.
[0709] e. Organ Transplant
[0710] Delayed Graft Function (DGF)
[0711] Modified u-PA polypeptides described herein can be used to
treat Delayed Graft Function (DGF), including, such as, for
example, DGF as a result of Ischemia-Reperfusion Injury in kidney
transplant recipients. u-PA polypeptides also can be used in the
treatment of the inflammatory response associated with organ
transplant that can contribute to tissue injury. Generally, a u-PA
polypeptide 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
u-PA polypeptide 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 kidney transplant or kidney
allograft. Effects of a u-PA polypeptide on treatment of delayed
graft function, for example delayed graft function as a result of
ischemia-reperfusion injury, can be assessed in animal models of
the injury, which mimic characteristics displayed in human kidney
allografts or transplants.
[0712] The presence of early biomarkers of early graft dysfunction
leading to DGF, including biomarkers for tubular epithelial cell
injury, may indicate the need for therapeutics. Biomarkers of DGF
(i.e., serum creatine) have been identified (Malyszko et al. (2015)
Nature Scientific Reports 5:11684; Wanga et al. (2015) PLoS One
10(9):e0136276). Early detection of biomarkers for DGF and
therapeutic intervention, such as, for example, therapeutic
treatment with a modified u-PA polypeptide, may improve clinical
outcomes.
[0713] Complement activation modulates disease progress in
disorders such as delayed graft function after organ transplant,
for example kidney transplant (Yu et al. (2016) Am J of
Transplantation 16(9):2589-2597). Modified u-PA polypeptides can be
used to treat DGF. For example, u-PA polypeptides can be
administered for systemic delivery or can be injected directly into
the graft or the surrounding tissues. Modified u-PA polypeptides
can be administered prior to, during or after transplant. Modified
u-PA polypeptides can be dosed daily or weekly or less frequently,
such as, for example, monthly or less frequently, such as
bi-monthly. In some instances a single systemic dose of the
modified u-PA polypeptide is administered. Multiple infusions of
the modified u-PA polypeptide over several hours are also
considered.
[0714] Modified u-PA polypeptides can be delivered chronically, if
needed, for example, the modified u-PA polypeptides, such as the
modified u-PA polypeptides described herein, can be delivered on a
daily basis or on another schedule to maintain an effective amount
in the allograft recipient. Modified u-PA polypeptides can be used
to prolong allograft survival in a recipient, in particular,
chronic survival of the allograft. PEGylated u-PA polypeptides can
be used to reduce immunogenicity.
[0715] 3. Combination Therapies
[0716] u-PA polypeptides provided herein can be used in combination
with other existing drugs and therapeutic agents to treat diseases
and conditions. 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 (sold under the trademark Celebrex.RTM.) or Rotecoxin
(sold under the trademark 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 (sold under the trademark Imuran.RTM.),
cyclophosphamide (sold under the trademark 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 (sold under
the trademark Enbrel.RTM.), infliximab (sold under the trademark
Remicade.RTM.), or adalimumad (sold under the trademark
Humira.RTM.), and inhibitors of IL-1 such as anakinra (sold under
the trademark Kineret.RTM.). Biological response modifiers also can
include anti-inflammatory cytokines such as IL-10, B cell targeting
agents such as anti-CD20 antibodies (sold under the trademark
Rituximab.RTM.), compounds targeting T antigens, adhesion molecule
blockers, chemokines receptor antagonists, kinase inhibitors such
as inhibitors to mitogen-activated protein (MAP) Kinase, c-Jun
N-terminal Kinase (JNK), or nuclear factor (NF) KB (NF.kappa.B),
and peroxisome proliferator-activated receptor-gamma (PPAR-.gamma.)
ligands. Additional agents that can be used in combination
therapies include immunosuppressants. Immunosuppressants can
include tacrolimus or FK-506; mycophenolic acid; calcineurin
inhibitors (CNIs); CsA; sirolimus or other agents known to suppress
the immune system.
[0717] u-PA polypeptides provided herein 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, u-PA
polypeptides provided 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, and ximelagatran.
[0718] u-PA polypeptides provided herein also can be used in
combination with agents that are administered to treat DGF. u-PA
polypeptides provided herein can, for example, be administered in
combination with an immunosuppressive agent. Such combination is
useful in prolonging allograft survival in a recipient, in
particular, chronic survival of the allograft. In preferred
embodiments, the combination is formulated and prepared such that
it is suitable for chronic administration to the recipient of the
allograft, for example, stable formulations are employed. In
certain embodiments, the combination is formulated and prepared
such that it is suitable for concurrent administration of the
modified u-PA polypeptides and the immunosuppressive drug to the
recipient of the allograft. In certain embodiments, the combination
is formulated and prepared such that it is suitable for sequential
(in either order) administration of the modified u-PA polypeptides
and the immunosuppressive drug to the recipient of the
allograft.
[0719] u-PA polypeptides provided herein also can be used in
combination with other agents that are administered to treat
macular degeneration. For example, modified u-PA polypeptides can
be administered with any one or more of ranibizumab (sold under the
trade name Lucentis.TM.); bevacizumab (sold under the trade name
Avastin.TM.); pegaptanib sodium (sold under the trade name
Macugen.TM.); aflibercept (sold under the trade name Eylea.TM.);
and verteporfin (sold under the trade name Visudyne.TM.). U-PA
polypeptides and fusion proteins provided herein also can be used
in combination with an implantable telescope, laser treatment or
laser photocoagulation, surgery, and/or photodynamic therapy, alone
or in combination with the therapeutic verteporfin, to treat
macular degeneration.
[0720] Additional agents, such as other complement inhibitors, can
be used as anti-inflammatory drugs in combination therapy with
modified u-PA polypeptides 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
C1 s 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
(sCRi), soluble DAF (sDAF), soluble MCP (sMCF), or soluble CD59
(sCD59) (Morgan et al., (2003) Mol Immunol. 40:159).
[0721] Pharmaceutical compositions containing u-PA polypeptides
described herein can be used to treat any one or more inflammatory
diseases or conditions mediated by complement activation. Also
provided are combinations of u-PA polypeptides and another
treatment or compound for treatment of an inflammatory disease or
condition. The u-PA polypeptides 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 u-PA
polypeptides.
I. EXAMPLES
[0722] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
Example 1
Cloning and Expression of Modified u-PA Polypeptides and Screening
for Modified u-PA Polypeptides that Cleave C3 at the QHAR/AS
Site
[0723] A. Cloning of the u-PA
[0724] Nucleic acid encoding amino acids 179-431 with the C122S
mutation by chymotrypsin numbering (set forth in SEQ ID NO:5) of
the human u-PA polypeptide (Uniprot P00749; set forth in SEQ ID NO:
1) was cloned into the pE-SUMO-AMP expression vector C-terminal to
the small ubiquitin-like modifier (SUMO) tag. The construct
included the signal peptide (amino acids 1-20) and the protease
domain (amino acids 179-431).
B. Generation of Modified u-PA Polypeptides
[0725] Modified u-PA polypeptides were generated by Quikchange site
directed mutagenesis (Stratagene) according to the manufacturer's
instructions with specifically designed oligonucleotides that
served as primers to incorporate designed mutations into the newly
synthesized DNA. A PCR reaction was set up containing the wild type
u-PA DNA as a template and oligonucleotide primers designed to
contain the desired mutation(s). Following PCR, each reaction
product was digested with DpnI to remove dam methylated parental
strands of DNA. The DNA then was transformed into E. coli XL-1 Blue
Supercompetent cells (Stratagene) and plated on selective agar
containing 50 .mu.g/ml carbenicillin. Plasmid DNA was isolated from
selected clones, and sequenced to verify incorporation of the
intended mutation(s) at the selected location(s) within the u-PA
encoding DNA and the absence of any additional, undesired
mutations.
C. Preparation of u-PA Polypeptides
[0726] 1. Transformation
[0727] DNA encoding wild-type and each of the variant u-PA
polypeptides was cloned into the pE-SUMO-AMP expression vector
C-terminal to the small ubiquitin-like modifier (SUMO) tag and
prodomain as detailed in Section A. above, and the resulting
constructs were transformed into BL21 Gold (DE3) E. coli cells
(Agilent Technologies, Catalog number: 230132). Approximately 50
.mu.L of chemically competent BL21 Gold (DE3) cells were
transformed with 0.5 .mu.L of the appropriate plasmid DNA
(typically containing 1 pg-50 ng of total DNA). Cells and DNA were
incubated on ice for 30 minutes, cells were then heat shocked at
42.degree. C. for 45 sec and further incubated on ice for 2
minutes. 450 .mu.L of room temperature Terrific Broth (TB) media
(VWR International, Catalog number 100219-866) was added to the
mixture, and cells were incubated in the TB media for 1 hour with
shaking at 240 rpm at 37.degree. C. 20 .mu.L of this transformation
mixture was spread on a 2.times. YT medium+100 .mu.g/mL
carbenicillin plate from Teknova (Cat #: Y4420) and incubated
overnight at 37.degree. C.
[0728] 2. Expression of u-PA Polypeptides
[0729] Cells containing DNA encoding a desired u-PA polypeptide
(typically obtained from a single, "confirmed" colony from the
transformation process described above) were grown in approximately
50 mL of medium prepared by combining 50 .mu.L of Carbenicillin,
0.3 mL of 20% Lactose solution, 5 mL of phosphate buffer, and 45mls
of base Terrific Broth (TB) media (Teknova, Catalog number L0350).
The cells and growth medium were rotated at 400 rpm in an Infors
Multitron Shaker at 37.degree. C. After 18 to 22 hours of growth,
bacteria were pelleted by centrifugation at 7,000 rpm in a 50 ml
Falcon centrifuge tube in a Beckman Sorval RC6 Plus Centrifuge with
Fiberlite F13-14.times.50cs centrifugation rotor (Thermo-Fisher)
for 10 minutes at 4.degree. C. After centrifugation, the
supernatant was decanted.
[0730] The cell pellet from the 50 mL culture was resuspended in 10
ml of cell lysis buffer A (50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM
EDTA, 0.1 mg/mL Lysozyme). The cell pellet was resuspended in
buffer A by shaking at 240 rpm for 1 hr at 37.degree. C. The
resulting mixture was subjected to centrifugation at 7,000 rpm for
15 minutes, and the supernatant was decanted. The resulting pellets
were resuspended in 10 ml BugBuster.RTM. extraction reagent (Merck
Millipore, NC9591474) containing 20 .mu.L Benzonase.TM. (Millipore
Sigma). Cells were resuspended by vortexing and shaking at 240 rpm
for 1 hr at 37.degree. C. Following shaking, the remaining
insoluble material was pelleted by centrifugation at 10,000 rpm for
15 minutes at 4.degree. C., and the supernatant was decanted. The
resulting pellet was resuspended by homogenizing in 10 ml of Wash
Buffer A [50 mM Tris (pH 8.0), 300 mM NaCl, and 1% Triton X-100]
using a Power Gen 500 homogenizer (Fisher Scientific, 14-261-04P).
This mixture, containing resuspended u-PA polypeptide inclusion
bodies (IBs) was centrifuged at 10,000 rpm for 15 minutes at
4.degree. C., and the supernatant was discarded. The new pellets
were resuspended in 10 mL of Wash Buffer B (50 mM Tris (pH 8.0))
and homogenized repeatedly until the pellet was well dispersed. The
resulting mixture was again centrifuged at 10,000 rpm for 15
minutes at 4.degree. C., the supernatant was decanted, and the
pellet was allowed to air dry for 10 to 15 minutes. This pellet of
u-PA polypeptide inclusion bodies (IB) can be stored at -20.degree.
C. or used immediately for the unfolding and refolding described
below.
[0731] 3. Unfolding of uPA
[0732] The insoluble SUMO-u-PA polypeptide fusion protein inclusion
bodies were dissolved and denatured in 5 mL of unfolding buffer [6M
GuHCl, 50 mM Tris pH 8, (Teknova, Catalog number: G0380)]. Freshly
prepared DTT was added to a final concentration of 10 mM on the day
of the re-folding procedure. This IB solution was agitated at 240
rpm at 37.degree. C. for at least 1 hour (typically 2 hours), or
until the inclusion bodies were fully dissolved. The
fully-dissolved IB solution is clear but can exhibit a brownish
tint.
[0733] 4. Refolding of u-PA
[0734] The 5 ml solution of unfolded u-PA polypeptide described
above is split into two aliquots of 2.5 ml, and each aliquot is
added to 200 ml of refolding buffer [1.5 M Arginine, 50 mM Tris pH
8.0, 150 mM NaCl, 5 mM GSH (L-Glutathione Reduced, Sigma-Aldrich),
and 4.0 mM GSSG (L-Glutathione Oxidized, Sigma-Aldrich)]. This
solution containing u-PA polypeptides in Refolding Buffer was
incubated on a shaker at 150 rpm for 24 hours at room temperature
to allow folding to take place.
[0735] The resulting protein solution was transferred to
12,000-14,000 Dalton molecular weight cutoff (MWCO)
Spectra/Por.RTM. regenerated cellulose dialysis tubing (VWR) that
was approximately 35 cm in length, and dialyzed in 25 mM Bis-Tris,
pH 6.1. Samples were dialyzed at least overnight, and, more
typically, for several days. Samples dialyzed for only one day were
incubated at room temperature, and samples dialyzed for more than
one day were incubated at 4.degree. C. The optimal ratio of total
dialysis buffer volume to total sample volume was at least 100.
Lower ratios typically produced lower yields of properly folded
u-PA polypeptide. Following dialysis, the protease samples were
removed from the dialysis tubing and filtered using a 500 mL 0.22 m
flask (Millipore).
[0736] 4. Column Purification of Zymogen
[0737] The protein solution was then purified using Sulfopropyl
Sepharose Fast Flow (SPFF) system. The column was prepared by
adding of 6 mL of SPFF Superflow slurry (GE Lifesciences) (with
approximately 3 mL of resin) to each Econo Column (BioRad), and the
storage solution was allowed to drain from the resin. 10 mL of 25
mM Bis-Tris pH 6.1, 1 M NaCl was then added to the column
containing the resin, and the solution was allowed to flow through
the column. Then, 10 mL of 25 mM Bis-Tris pH 6.1 was added to the
column containing the resin, and the solution was allowed to pass
through. The bottom of the column is capped and stored with the
addition of 10 mL 25 mM Bis-Tris pH 6.1 buffer to equilibrate the
resin.
[0738] The refolded and dialysed u-PA polypeptide sample solution
was applied to the equilibrated SPFF column, followed by 10 ml of
25 mM Bis-Tris pH 6.1, 50 mM NaCl. The unactivated, u-PA
polypeptide zymogen was then eluted with 4 ml of 25 mM Tris pH 7.5,
500 mM NaCl that was collected into a 50 mL Falcon tube. The sample
was then diluted with 25 mM Tris pH 7.5 to a total volume of 12
ml.
[0739] 5. Activation of u-PA Zymogen
[0740] The 12 ml sample containing the purified u-PA polypeptide
zymogen was diluted by addition of 12 ml of activation buffer (25
mM Tris pH 7.5, 20 mM Benzamidine). The u-PA polypeptide zymogen
was then converted into the corresponding active u-PA protease with
the SUMO Protease ubiquitin-like specific protease-1 (ULP-1) from
Saccharomyces cerevisiae. "Activation" of the u-PA polypeptide
zymogen was accomplished by adding 120 .mu.g of ULP-1 to the
purified zymogen, briefly swirling the solution and incubating the
sample overnight at room temperature.
[0741] 6. Purification of Activated u-PA Polypeptides
[0742] Active u-PA polypeptides were purified using ion exchange
chromatography. Prior to chromatography, the sample was filtered
with a 50 ml filter unit. A Vivapure Q spin column was
pre-conditioned with 5 ml of 25 mM Tris pH 7.5, 1M NaCl and 10 ml
of 25 mM Tris pH 7.5 followed by centrifugation at 500g in a
Sorvall Legend RT centrifuge for 5 minutes.
[0743] Each sample containing an activated u-PA polypeptide was
loaded onto an individual Q-spin column in 19 mL batches and
centrifuged at 500g for 5 minutes for each run. The flow-through
containing activated u-PA without SUMO tag and Zymogen was
collected. The pH of the resultant u-PA sample was adjusted by
adding 12 ml of 25 mM Citric Acid, pH=5.0 and 60 .mu.L of 1M Citric
Acid. The resulting protein solution was then loaded onto a
pre-conditioned Vivapure S spin column. This column was
preconditioned with 5 ml of 25 mM Tris pH 7.5, 1M NaCl and 10 mL of
25 mM Tris pH 7.5, followed by centrifuging the column at 500 g for
5 mins. Samples were loaded onto the S-column (HiTrap SP HP; GE
Healthcare) in 19 ml batches and the column was centrifuged at 500
g for 5 mins. The flow through from this process was discarded. The
column is then "washed" with 10 ml of 25 mM Sodium Citrate pH 5.0,
20 mM NaCl. After washing the column, the collection tube is
replaced with a new tube that contains 7 ml of the dilution buffer
50 mM Sodium Citrate pH 5.0. u-PA polypeptide is then eluted from
the column with 7 ml of 25 mM Sodium Phosphate pH 7.0, 250 mM NaCl.
The elute, containing a u-PA polypeptide, is then concentrated and
"buffer-exchanged" into citrate buffered saline (CBS; 20 mM Sodium
Citrate pH 5.0, 50 mM NaCl) using an Amicon Ultra-15 Centrifugal
Filter Unit to achieve a final concentration of .gtoreq.60 .mu.M
(A280 of .gtoreq.2.6). Optical density of the solutions was
measured using a Nanodrop device. The quality of the preparation
was initially assessed by SDS-PAGE. Two g of u-PA polypeptide
sample in 1.times. Sample Buffer containing Bond-Breaker TCEP was
loaded on each "lane" of a 12-well 4-12% PAGE NovexBis-Tris gel,
and run in 1.times.MES Running Buffer at 200 V for 40 min. Proteins
were "visualized" by staining the gel with Comassie Blue followed
by destaining. Fractions containing single bands migrating at
approximately 25 kDa were snap-frozen in liquid nitrogen and stored
at -80.degree. C. until use. The quality of individual u-PA
polypeptide samples were further assessed by activity assays and
mass spectroscopy.
[0744] D. Selection and Identification of Modified u-PA
Polypeptides that Cleave C3 to Inactivate it
[0745] Modified u-PA polypeptides were identified by screening a
library of modified u-PA polypeptides against a modified serpin
(PAI-1) as described, for example in detail in U.S. Pat. No.
8,211,428 (see, also published US application Publication No.
US-2014-0242062-A1). An inhibitory serpin, or fragment thereof,
capable of forming a covalent acyl enzyme intermediate between the
serpin and protease is used for screening. Generally, the serpin
used is one that in vivo normally targets the protease. In the
assay a serpin modified by replacement of its reactive site loop
(RSL) to include the target sequence (i.e. the active site in C3)
captures modified proteases that will cleave the target site to
form stable complexes. The captured modified protease is then
isolated/identified. For u-PA, PAI-1, PAI-2, PAI-3, particularly
PAI-1 which is an inhibitor thereof, are cognate serpins. The
serpin PAI-1 was modified by replacing the residues indicated below
with QHARASHLG (residues 737-745 of C3, SEQ ID NO: 47), which is
the active site of human C3. All modified u-PA polypeptides were
selected so that they cleave within the active site of C3. In
particular they cleave between R and A.
TABLE-US-00018 Q H A R .dwnarw. A S H L
[0746] ATIII "bait" (BELOW) with inserted sequence QHARASHLG
(corresponding to an inactivating cleavage site in C3 (residues
737-745 of SEQ ID NO: 47)):
TABLE-US-00019 10 20 30 40 CHHPPSYVAH LASDFGVRVF QQVAQASKDR
NVVFSPYGVA 50 60 70 80 SVLAMLQLTT GGETQQQIQA AMGFKIDDKG MAPALRHLYK
90 100 110 120 ELMGPWNKDE ISTTDAIFVQ RDLKLVQGFM PHFFRLFRST 130 140
150 160 VKQVDFSEVE RARFIINDWV KTHTKGMISH LLGTGAVDQL 170 180 190 200
TRLVLVNALY FNGQWKTPFP DSSTHRRLFH KSDGSTVSVP 210 220 230 240
MMAQTNKFNY TEFTTPDGHY YDILELPYHG DTLSMFIAAP 250 260 270 280
YEKEVPLSAL TNILSAQLIS HWKGNMTRLP RLLVLPKFSL 290 300 310 320
ETEVDLRKPL ENLGMTDMFR QFQADFTSLS DQEPLHVALA 330 340 350 360
LQKVKIEVNE SGTVASSSTL RRQHARASHL EIIIDRPFLF 370 VVRHNPTGTV
LFMGQVMEP
[0747] The mutations in habove modified PAT-1 are as follows:
TABLE-US-00020 RCL LRRQHARASRL 341 350 Mutation V1C 1 1 Mutation
N150H 150 150 Mutation K154T 154 154 Mutation Q319L 319 319
Mutation A340L 340 340 Mutation V341R 341 341 Mutation I342R 342
342 Mutation V343Q 343 343 Mutation S344H 344 344 Mutation M347A
347 347 Mutation A348S 348 348 Mutation P349H 349 349 Mutation
E350L 350 350 Mutation M354I 354 354
[0748] Table 14, in Example 2 below, sets forth mutations, and
provides SEQ IDs for exemplary protease domains of modified u-PA
polypeptides that contain the mutations. Numbering is chymotrypsin
numbering. The modified u-PA polypeptides were generated and
selected to inactivate C3, with the mutations indicated. While the
SEQ ID NOs. reference protease domains, it is understood that the
mutations can be included in precursor, full-length and mature
modified u-PA polypeptides. The C122S replacement, or other
conserved replacement for S, is included to reduce aggregation;
while advantageous, it is optional. For modified u-PA for use for
gene therapy or for PEGylation, the C122S replacement is not
included in the modified u-PA or in the encoding nucleic acid. C122
can serve as a site for conjugate of a pegylation moiety or other
modification. When expressed in vivo, aggregation generally is not
a concern. Also for which the active form is a two chain form
linked by a disulfide bond, the free Cys at residue 122 generally
is not modified to Ser so that it is available to form the
disulfide bond.
Example 2
In Vitro Cleavage of Complement Protein C3
[0749] The activity of the modified u-PA polypeptides for
inactivation cleavage of C3 was determined by measuring the amount
of intact human C3 remaining after incubation of the substrate
complement protein human C3 with various concentrations of each
modified protease for 1 hour at 37.degree. C. In accord with this
assay, signal is generated in the presence of intact human C3, and
is lost as the C3 is cleaved.
[0750] 2 .mu.M plasma purified human C3 (Complement Technologies;
Tyler, Tex.) was incubated with the modified u-PA polypeptides
(0-250 nM) for 1 hour at 37.degree. C. in buffer containing 50 mM
Tris, pH 8.0, 50 mM NaCl, and 0.01% Tween-20. The activity of the
modified u-PA polypeptides was quenched by the addition of EGR-CMK
(Haematologic Technologies, EGRCK-01) to a final concentration of
10 .mu.M and the hC3/modified u-PA polypeptide mixture was allowed
to stand for 30 minutes at ambient temperature.
[0751] Residual levels of undigested human C3 were quantified using
an Amplified Luminescent Proximity Homogeneous Assay Screen (sold
under the trademark AlphaScreen.RTM.; Perkin Elmer). a-mouse
IgG-coated acceptor beads at 100 .mu.g/mL (Perkin Elmer #6760606)
were incubated with 5 nM mouse a-hC3a mAb (Abcam # ab11872-50) in
50 mM Tris, pH 8.0, 50 mM NaCl, 0.01% Tween-20 and 0.2% BSA to form
the acceptor bead mixture. The acceptor bead mixture was shielded
from light and placed on a rotating shaker for 30-60 minutes. The
hC3/modified u-PA polypeptide reaction mixtures (prepared above)
were diluted 1600-fold into 50 mM Tris, pH 8.0, 50 mM NaCl, 0.01%
Tween-20, 0.2% BSA and 4 .mu.L aliquots were placed in duplicate
wells of a 384-well Optiplate (Perkin Elmer #6007299). 8 .mu.L of a
a-hC3 mAb/acceptor beads mixture was incubated with 8 .mu.L of 25
nM biotinylated goat a-hC3 pAb (prepared using EZ-Link
Sulfo-NHS-LC-Biotin kit from Thermo Scientific #21327 from the
unbiotinylated version from Complement Technologies #A213). The
plate was then shielded from light and incubated for 30 minutes at
ambient temperature. After this time, 4 .mu.L of 100 .mu.g/mL
streptavidin-coated donor beads (Perkin Elmer #6760606) were added
to each well and incubated for 60 minutes, shielded from light. The
alphascreen signal (Excitation=680 nm, Emission=570 nm) was then
measured using an Envision 2104 Multilabel plate reader (Perkin
Elmer). This signal (corresponding to the concentration of
remaining hC3 ([hC3])) was plotted as a function of Alterase.RTM.
concentration ([Alterase]) and the data were fitted to the four
parameter equation below to determine the concentration of modified
u-PA polypeptide (the Alterase.RTM. concentration) required to
cleave through 50% of the available hC3 (EC.sub.50), the Hill slope
(Hill) as well as the maximum (Max) and minimum (Min) signals in
the assay.
[ h C 3 ] = Min + Max - Min 1 + ( [ Alterase ] E C 50 ) Hill
##EQU00001##
[0752] Cleavage of hC3 by the u-PA variant containing the mutations
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
(see, SEQ ID NO:21) was measured independently a total of 13 times,
using 9 different lots of the protease. The average EC.sub.50 value
for this modified u-PA polypeptide was determined to be 19 nM
(n=13, SD=2.2); in the experiment for which the results are
reported in the Table 14 below, it was 24.5 nM.
[0753] About 600 modified u-PA polypeptides comprising a protease
domain with the mutations set forth in Table 14. Results are set
forth in Table 14 below. The majority of the tested modified u-PA
polypeptides cleaved human complement protein C3 significantly more
efficiently (i.e., lower ED.sub.50) than the wild type u-PA
protease domain containing the C122S replacement; many with an
ED.sub.50 below 100 nM. The polypeptides include the C122S
replacement to prevent aggregation upon expression, for example, of
the protease domain. In some embodiments, the modified u-PA
polypeptide is full length that is activated to form a two chain
activated polypeptide. For such embodiments, the modified u-PA
protease domain does not include the C122S replacement (279 by
mature numbering); the cysteine forms a disulfide bond (between
148C and 279C by mature numbering, with reference to SEQ ID
NO:3)
[0754] Among the tested u-PA variants are those that were less
potent than wild type u-PA, particularly those variants for which
no cleavage (i.e., indicated as NA in Table 14 below) of hC3 was
observed during the one hour assay. Other low activity variants
cleaved less than 25% of the hC3 during the assay and were
therefore not assigned an ED.sub.50. Based on these results, the
skilled person can select mutations that increase cleavage activity
for C3.
TABLE-US-00021 TABLE 14 SEQ ID ED50 NO* Mutation String (nM) 749
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
0.92 Y151L 678
R35W/R36Q/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/
2.38 Y151L 277
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82R/T97aI/L97bA/H99Q/K110aR/
2.47 C122S/Y149R/M157K 280
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
2.49 M157K/K179R 278
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92R/T97aI/L97bA/H99Q/C122S/
3.04 Y149R/M157K 614
F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bW/T97aI/L97bA/H99Q/
3.47 C122S/Y149E/M157K 279
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92S/T97aI/L97bA/H99Q/C122S/
3.48 Y149R/M157K 276
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61R/K62R/T97aI/L97bA/H99Q/
3.51 C122S/Y149R/M157K 281
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
3.87 M157K/K179S 291
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S
4.38 690
F30Y/R35W/R36T/H37S/V38S/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
4.47 Y149R/Y151L/M157R/Q192Y 272
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/M157K
4.68 287
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61S/K62S/T97aI/L97bA/H99Q/
4.75 C122S/Y149R/M157K 751
R35A/H37E/R37aG/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
4.76 Y151L 676
R35W/R36Q/H37S/V38T/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
4.78 Y149R/Y151P/M157R 668
F30Y/R35W/H37Y/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
4.92 Y149R 977 V38E/T39W/V41R/D60aW/Y60bP/L97bG/H99L/C122S 4.94 682
R35W/R36K/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
5.01 Y149R/Y151L/M157S/Q192H 792
R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aQ/Y60bP/T97aI/L97bA/H99Q/C122S/
5.14 Y149R 225
I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/
5.21 T158A 750
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
5.26 Y151L/Q192H 10
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
5.33 M157K 675
R35W/R36N/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
5.52 Y149R/M157S 802
R35Y/H37D/V38E/T39W/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R
5.57 288
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82S/T97aI/L97bA/H99Q/K110aS/
5.82 C122S/Y149R/M157K 744
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/
6.17 H99L/C122S 275
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/Y149R/M157K
6.24 753
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S
6.24 669
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/
6.35 Y149R/M157K 286
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
6.38 M157K 981
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158A 7.23 656
R35Q/R36H/H37Y/V38E/T39Y/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
7.33 M157K 705
R35W/H37P/R37aG/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
7.4 Y149R 824
V38D/V41Q/D60aH/Y60bS/T97aW/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R
7.48 731
F30Y/R35W/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bQ/T97aE/L97bA/
7.52 H99Q/C122S/Y149R/M157K 677
F30Y/R35W/R36Q/H37S/V38P/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
7.55 Y149R/M157R 679
F30H/R35W/R36T/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
7.59 Y149R/Y151L/M157S 613
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bD/T97aI/L97bA/H99Q/C122S/
7.64 M157K 707
F30Y/R35Y/R36H/H37N/V38E/T39F/Y40F/V41R/K61E/R72H/T97aI/L97bA/H99Q/
7.64 C122S/Y149R/M157K/Q169K 688
R35W/R36Q/H37S/V38S/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/
8.04 Y151L/M157S/Q192H 752
R35W/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
8.07 Y151L/Q192T 19
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S/
8.13 Y149R 290
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S
8.23 616
F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/
8.3 Y149R/M157K 670
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
8.44 Y149R 285
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
8.44 Y149R 283
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
8.6 M157K 284
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S
8.78 683
F30Y/R35W/R36S/H37S/V38Q/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
8.86 Y149R/Y151L/M157S/Q192N 727
F30Y/R35W/R36H/H37P/R37aD/V38E/T39Y/Y40F/V41R/D60aE/Y60bS/T97aE/L97bA/
8.9 H99Q/C122S/Y149R/M157K 799
R35Q/H37Y/R37aS/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
8.95 Y149R 949 R37aS/V38E/Y40V/V41R/H99L/C122S/Y151L/R217V 9.04 831
V38D/V41R/L97bG/H99Q/C122S/Y151L/R217E 9.09 33
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S
9.27 624
F30Y/R35V/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bP/T97aI/L97bA/H99Q/C122S/
9.54 Y149R/M157K 223
I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K
9.65 617
F30Y/R35V/R36H/H37S/V38E/T39F/Y40H/V41R/Y60bS/T97aM/L97bA/H99Q/C122S/
10.2 Y149W/M157K 703
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
10.3 Y149R 706
N26D/F30Y/R35Y/R36H/H37E/V38E/T39F/Y40F/V41R/K61E/T97aI/L97bA/H99Q/
10.3 R110dS/P114S/C122S/Y149R/M157K 732
F30Y/R35W/R36H/H37P/R37aE/V38E/T39Y/Y40F/V41R/Y60bA/T97aE/L97bA/
10.3 H99Q/C122S/Y149R/M157K 796
R35L/H37D/R37aN/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R
10.4 14
F30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/
10.6 Y149K/M157K 29
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R
10.6 979
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S/E167K 10.6
24
R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
10.7 633
F30Y/R35Y/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bE/T97aI/L97bA/H99Q/C122S/
10.8 Y149R/M157K 12
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
10.8 M157K 708
F30Y/R35Y/R36H/H37E/V38E/T39F/Y40F/V41R/K61E/T97aI/L97bA/H99Q/C122S/
11.1 Y149R/M157K/T242A 557
F30Y/R35L/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A 11.3 724
F30Y/R35Y/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bH/T97aE/L97bA/H99Q/
11.3 C122S/Y149R/M157K 882 V38D/V41R/L97bR/H99E/C122S/Y151L/R217E
11.4 335
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
11.5 E175D/R217E/K224R 746
R35Y/H37V/R37aW/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
11.6 Y151L/Q192T 615
F30Y/R35M/R36H/H37G/V38E/T39F/Y40H/V41R/Y60bP/T97aF/L97bA/H99Q/C122S/
11.7 Y149R/M157K 684
F30Y/R35W/R36Q/H37S/V38T/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
11.8 Y149R/Y151L/M157K/Q192T 701
R35W/H37D/R37aP/V38E/T39W/V41R/D60aR/Y60bS/T97aI/L97bA/H99Q/C122S/
11.8 Y149R 260
I17V/F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/
11.9 C122S/Y149K/M157K 218
I17V/F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/ 12
M157K/T158A 978
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/I138V/E167K 12.1
680
F30Y/R35W/R36Q/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
12.4 Y149R/Y151L/M157T/Q192H 980
R35H/G37bD/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S 12.4
794
R35H/H37P/R37aG/V38E/T39F/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
12.6 Y149R 611
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
12.7 Y149R/M157K 926 V38D/T39Y/Y40L/V41R/L97bI/H99E/C122S/R217E
12.8 224
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/T158A 12.9
689 F30H/R35W/R36H/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/
13 C122S/Y149R/M157K 650
R35V/R36H/H37D/V38E/T39W/Y40M/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
13.1 M157K 685
R35W/R36K/H37S/V38A/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/
13.3 Y151L/M157R/Q192T 628
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/
13.5 Y149R/M157K 717
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
13.6 N145S/S146V/T147M/D148G/Y149Q/L150F/M157K 275
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/Y149R/M157K
13.7 658
F30Y/R35I/R36H/H37D/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
14.1 M157K 373
R35V/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 14.4 800
R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aN/Y60bN/T97aI/L97bA/H99Q/C122S/
14.5 Y149R 957 V38E/Y40Q/V41L/L97bG/H99Q/C122S/R217T 14.5 983
R35H/V38E/T39Y/V41R/T56S/D60aP/Y60bQ/L97bA/H99Q/C122S/T158S 14.6
513 F30H/R35Q/H37W/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 14.7 300
R35Q/H37Y/V38E/T39Y/V41R/D60aP/T97aI/L97bA/H99Q/C122S/Y149R 14.7
745
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
14.8 Y151L/Q192A 821
V38D/V41R/Y60bR/T97aW/L97bR/H99E/C122S/E175D/R217E/K224R 14.8 674
F30Y/R35W/R36H/H37S/V38E/Y40H/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R
15.1 823
V38D/V41L/Y60bP/T97aM/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R 15.3
627
F30Y/R35W/R36H/H37D/V38E/T39F/Y40H/V41R/Y60bE/T97aI/L97bA/H99Q/C122S/
15.4 Y149R/M157K 13
F30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/
15.7 Y149K/M157K 681
F30Y/R35W/R36K/H37S/V38D/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
15.9 Y149R/Y151L/M157R/Q192T
382 V41R/H99Q/C122S/Y151L/R217V 16 959
V38E/Y40P/V41L/L97bG/H99L/C122S/Y151Q/R217E 16 950
V38E/Y40L/V41R/H99L/C122S/Y151L/R217S 16.1 956
V38E/Y40Q/V41L/L97bG/H99Q/C122S/Y151P/R217T 16.4 973
V38E/T39Y/V41R/D60aW/Y60bP/L97bR/H99I/C122S 16.4 20
R35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/T97aI/L97bA/H99Q/C122S/Y149R
16.6 597
F30Y/R36H/H37F/V38E/T39Y/Y40H/V41R/Y60bD/T97aV/L97bA/H99Q/C122S/
16.8 Y149L/M157K 664
F30Y/R35W/R36H/H37E/S37dP/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/
16.8 H99Q/C122S/Y149K/M157K 733
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aA/Y60bP/T97aI/L97bA/H99Q/C122S/
17.4 Y149R 469
R36H/V38D/V41R/A96D/D97E/A98G/T97adel/H99L/L97bdel/C122S/T178S/R217D
17.5 830 V38D/V41R/L97bG/H99Q/C122S/Y151L/R217A 17.7 653
F30H/R35Q/R36H/H37Y/V38E/T39Y/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
17.8 M157K 264
F30Y/R35W/R36K/H37E/V38E/T39W/Y40H/V41R/Y60bQ/K61E/I65T/T97aE/L97bA/
17.9 H99Q/C122S/Y149K/M157K 604
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bS/T97aL/L97bA/H99Q/C122S/
18 Y149L/M157K 714
F30Y/R35Q/R36H/H37Y/R37aE/V38E/T39Y/Y40F/V41R/D60aS/Y60bP/T97aE/L97bA/
18.1 H99Q/C122S/Y149R/M157K 743
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aG/Y60bS/T97aI/L97bA/H99Q/C122S/
18.1 Y149R 666
I24N/F30Y/R35W/R36H/H37E/V38E/T39W/Y40L/V41R/Y60bQ/N87D/T97aE/L97bA/
18.2 H99Q/C122S/Y149K/M157K 36 V38E/Y40Q/V41L/L97bA/H99Q/C122S 18.2
217
F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/M157K/
18.3 T158A 263
F30Y/R35W/R36A/H37E/V38E/T39W/Y40H/V41R/Y60bQ/K61D/I65R/T97aE/L97bA/
18.4 H99Q/C122S/Y149K/M157K 221
F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K
18.6 289
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
18.9 M157K/K187R/K223R/K224R 687
R35W/R36Q/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/ 19
Y149R/Y151L/M157S/Q192T 986
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/P60cS/L97bA/H99Q/C122S/I138V/E167K
19.4 18
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/
19.5 Y149R 222
I17V/F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K 19.6 271
R35Y/R36H/H37K/V38E/T39F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K
19.9 982 R35H/V38E/T39Y/V41R/T56A/D60aP/Y60bQ/L97bA/H99Q/C122S 19.9
538 F30Y/V38D/Y40F/V41L/L97bA/H99Q/C122S/Y151L/M157R 20.2 953
V38E/Y40A/V41L/L97bG/H99Q/C122S/R217T 20.4 665
I24T/F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/
20.6 C122S/Y149K/M157K 292
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
20.7 Y149R/M157K 219
I17V/F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A 21.6 747
R35W/H37P/R37aN/V38E/T39Y/V41K/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
21.7 Y151L/Q192T 514
F30H/R35L/H37D/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K/R217E 21.8
713
F30Y/R35W/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/D60aE/Y60bF/T97aE/L97bA/
21.8 H99Q/C122S/Y149R/M157K 619
F30Y/R35L/R36H/H37G/V38E/T39Y/Y40H/V41R/Y60bP/T97aE/L97bA/H99Q/C122S/
22.1 Y149M/M157K 505
I17V/F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 22.2 623
F30Y/R35V/R36H/H37K/V38E/T39F/Y40H/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/
22.6 Y149R/M157K 11
R35W/R36H/H37N/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
22.8 M157K/Q192H 360
R35V/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 22.8 729
F30Y/R35M/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/
22.9 H99Q/C122S/Y149R/M157K 834
R35Q/V38D/V41R/L97bG/H99Q/C122S/Y151L 22.9 961
R37aS/V38E/Y40P/V41L/L97bG/H99Q/C122S/Y151Q/R217T 23.4 364
R35V/R37aE/V38E/Y40Q/V41L/T97aE/L97bA/H99Q/C122S/Y149R 23.5 486
F30H/V38D/V41R/A96G/L97bA/H99Q/C122S/Y151L/M157K 23.7 311
T39L/Y40L/V41R/T97aI/L97bA/H99Q/C122S 23.7 265
F30Y/R35W/R36H/H37E/V38E/T39Y/Y40F/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/
23.8 Y149R/M157K 34 Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 23.9 586
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/S146F/M157K/Q192H/
24.1 K243Q 346 Y40Q/V41L/L97bA/H99Q/C122S/Y149R 24.1 262
F30Y/R35W/R36Q/H37E/V38E/T39W/Y40H/V41R/Y60bQ/K61L/I65V/T97aE/L97bA/
24.4 H99Q/C122S/Y149K/M157K 21
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/
24.5 Y149R 832 R35Q/V38D/V41R/T97aS/L97bA/H99Q/C122S/Y151L 24.8 369
V41R/L97bR/H99Q/C122S/Y151L/R217V 25.1 30
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/Y149R
25.3 621
F30Y/R35V/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bP/T97aE/L97bA/H99Q/C122S/
25.4 Y149E/M157K 754
R35A/H37T/R37aD/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
25.5 Y151L/Q192S 835 R35S/V38D/V41R/L97bA/H99Q/C122S/Y151L 25.7 836
R35S/V38D/V41L/L97bG/H99Q/C122S/Y151L/R217Q 26.1 562
F30Y/R35H/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158S 26.3 738
R35Q/H37S/R37aE/V38E/T39Y/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/
26.3 Y149R 338
H37G/R37aD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/
26.7 R217E/K224R 341
H37G/R37aD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/Q192T/
26.8 R217E 741
R35W/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bS/T97aI/L97bA/H99Q/C122S/
26.9 Y149R 795
R35Q/H37G/R37aD/V38E/T39Y/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/
27.1 Y149R 798
R35Q/H37D/R37aK/V38E/T39F/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/
27.1 Y149R 671
R35Y/R36H/H37S/V38D/T39Y/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R
27.3 230 F30Y/R36H/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/M157K
27.9 829 R37aS/V38D/V41Q/L97bG/H99Q/C122S/Y151L/R217T 27.9 484
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 28.1 693
F30Y/R35K/R36H/H37E/R37aK/V38E/T39F/Y40F/V41R/D60aP/Y60bS/T97aI/L97bA/
28.1 H99Q/C122S/Y149R/M157K 694
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bG/T97aI/L97bA/
28.3 H99Q/C122S/Y149R/M157K 261
F30Y/R35W/R36Q/H37E/V38A/T39W/Y40H/V41R/Y60bQ/K61D/I65V/T97aE/L97bA/
28.4 H99Q/C122S/Y149K/M157K 494
F30Y/R35H/V38D/Y40H/V41R/T56A/L97bA/H99Q/C122S/M157K 28.5 797
R35N/H37T/R37aY/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
28.7 Y149R 984
R37aH/V38E/T39Y/V41R/T56A/D60aP/Y60bQ/L97bA/H99Q/C122S/T158A 28.8
515 F30H/R35Q/H37T/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 28.9 503
F30H/R36L/V38E/V41R/K82R/L97bA/H99Q/C122S/Y151L/M157K 29 370
V38D/V41R/H99Q/C122S/Y151L/R217V 29.1 736
R35Q/H37G/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
29.2 Y149R 726
F30Y/R35Q/R36H/H37Y/R37aE/V38E/T39Y/Y40F/V41R/D60aE/Y60bA/T97aE/L97bA/
29.3 H99Q/C122S/Y149R/M157K 704
R35Q/H37Y/R37aD/V38E/T39L/V41R/D60aE/Y60bT/T97aI/L97bA/H99Q/C122S/
29.4 Y149R 657
F30Y/R35L/R36H/H37E/V38E/T39N/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
29.6 M157K 963 R36S/V38E/Y40L/V41N/L97bG/H99Q/C122S/Y151L/R217T
29.6 401 T39W/V41R/L97bG/H99Q/C122S 29.7 9
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/
29.8 C122S/Y149K/M157K 28
R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
29.8 940 R35S/R37aA/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149V 30.2 612
F30Y/R35W/R36H/H37Q/V38E/T39H/Y40H/V41R/T97aE/L97bA/H99Q/C122S/Y149L/
30.3 M157K 715
F30Y/R35Q/R36H/H37Y/R37aD/V38E/T39Y/Y40F/V41R/Y60bV/T97aE/L97bA/
30.4 H99Q/C122S/Y149R/M157K 171
F30H/V38D/V41R/L97bA/H99Q/Y151L/M157K 30.6 511
F30H/R35H/H37I/V38D/V41R/L97bA/H99Q/C122S/Y149W/Y151L/M157K/R217S
30.6 875 V38D/T39Y/Y40H/V41R/T97aI/L97bA/H99Q/C122S 30.7 702
R35F/H37D/R37aN/V38E/T39Y/V41R/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R
30.9 402 T39Y/V41R/Y60bQ/L97bG/H99Q/C122S 31.1 398
T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S 31.4 881
V38D/V41R/L97bR/H99Q/C122S/Y151L/R217E 31.5 924
R36S/V38D/T39L/Y40L/V41R/L97bI/H99E/C122S/R217T 31.5 936
R35S/R37aD/V38E/Y40Q/V41L/Y60bV/T97aL/L97bA/H99Q/C122S/Y149L 31.5
350 Y40Q/V41L/Y60bT/T97aE/L97bA/H99Q/C122S/Y149R 31.6 496
F30Y/V38E/Y40H/V41R/T56A/L97bA/H99Q/C122S/M157K/K243M 31.9 231
F30Y/R36H/R37aH/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/M157K
31.9 516 F30H/R35Q/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 32 818
V38D/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R
32 324
H37G/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
32.1 Q192T/R217E/K224R 948
R35S/R37aD/V38E/Y40Q/V41L/T97aE/L97bA/H99Q/C122S/Y149R 32.2 368
R35V/R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S 32.2 351
Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 32.5 551
F30Y/R35H/V38D/Y40H/V41R/L97bA/H99Q/C122S/I138V/M157K 33.1 43
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S 33.2 696
F30Y/R35H/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bD/T97aI/L97bA/
33.4 H99Q/C122S/Y149R/M157K 495
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T158A 33.5 972
V38E/T39W/V41R/D60aP/Y60bD/L97bA/H99L/C122S 33.5 242
F30Y/R36H/V38E/Y40H/V41R/I65T/T97aI/L97bA/H99Q/C122S/M157K 33.6 884
V38D/V41R/L97bR/H99Q/C122S/Y151L/R217V 33.7 801
R35Q/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
33.8 Y149R 673
R35W/R36H/H37S/V38E/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/
34 M157K 958 R36S/V38E/Y40Q/V41R/L97bG/H99L/C122S/Y151P/R217E 34.3
15 V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 34.5 340
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/
34.5 Q192T/R217E 339
H37G/R37aD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
34.8 Q192T/R217E 282
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
34.9 M157K/K187S/K223S/K224Y 39 Y40Q/V41L/L97bA/H99Q/C122S 35.2 504
F30H/R35H/V38D/V41R/K61E/L97bA/H99Q/C122S/Y151L/M157K/R206H 35.4 8
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 35.7 273
F30Y/R36H/V38E/Y40H/V41R/T97aE/L97bA/H99Q/C122S/Y149R/M157K 35.7
943 R35A/R37aE/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149R 35.9 827
V38D/V41L/L97bG/H99Q/C122S/Y151L/R217Q 36.2 517
F30H/R35Q/H37W/V38D/V41R/D60aE/L97bA/H99Q/C122S/Y149L/Y151L/M157K/
36.4 R217D 620
F30Y/R35F/R36H/H37G/V38E/T39Y/Y40H/V41R/Y60bS/T97aD/L97bA/H99Q/C122S/
36.7 Y149R/M157K 399 T39Y/V41R/L97bG/H99Q/C122S 36.8 608
F30Y/R35I/R36H/H37E/V38E/T39Y/Y40H/V41R/Y60bS/T97aV/L97bA/H99Q/C122S/
36.9 Y149L/M157K 934
R35S/R37aD/V38E/Y40Q/V41L/L97bA/H99Q/C122S/Y149R 37.2 387
Y40H/V41Q/L97bG/H99Q/C122S/R217T 37.2 645
R35W/H37D/V38D/T39Y/V41R/Y60bS/L97bA/H99Q/C122S/Y149R 37.4 869
V38D/T39F/Y40L/V41R/T97aW/L97bA/H99Q/C122S 37.8 878
V38D/T39Y/Y40L/V41R/T97aE/L97bA/H99Q/C122S 37.8 718
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39F/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/
38.2 H99Q/C122S/Y149R/M157K 873
V38D/T39L/Y40L/V41R/T97aI/L97bA/H99Q/C122S 38.2 876
V38D/T39Y/Y40L/V41R/T97aW/L97bA/H99Q/C122S 38.4
550 F30Y/R36H/V38D/Y40H/V41R/L97bA/H99L/C122S/F141L/M157K/T158A
38.5 728
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aA/Y60bS/T97aE/L97bA/
38.5 H99Q/C122S/Y149R/M157K 712
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/
38.6 H99Q/C122S/Y149R/M157K 404 T39Y/V41R/Y60bP/L97bG/H99Q/C122S
39.2 507 F30H/R36H/V38D/V41R/T56A/L97bA/H99Q/C122S/Y151L/M157K 40
720
F30Y/R35E/R36H/H37D/R37aN/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/ 40
H99Q/C122S/Y149R/M157K 268
V38E/Y40Q/V41L/D60aP/Y60bL/L97bA/H99Q/C122S/Y149W 40.4 498
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K 40.5 509
F30H/R35Q/H37W/V38D/V41R/D60aE/Y60bS/L97bA/H99Q/C122S/Y149L/Y151L/
40.8 M157K 742
R35Q/H37G/R37aE/V38E/T39Y/V41R/D60aP/Y60bT/T97aI/L97bA/H99Q/C122S/
40.9 Y149R 686
F30Y/R35W/R36H/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/
41 Y149R/Y151P/M157K/Q192H 710
F30Y/R35M/R36H/H37D/R37aD/V38E/T39Y/Y40F/V41R/D60aP/Y60bS/T97aE/L97bA/
41.2 H99Q/C122S/Y149R/M157K 618
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bT/T97aD/L97bA/H99Q/
41.3 C122S/Y149R/M157K 872
V38D/T39L/Y40L/V41R/T97aV/L97bA/H99Q/C122S 41.6 822
V38D/V41R/Y60bS/T97aI/L97bR/H99E/C122S/Y151L/E175D/Q192F/R217E/K224R
42.1 403 T39Y/V41R/Y60bP/L97bA/H99Q/C122S 42.1 446
R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192E/
42.2 R217D 735
R35M/H37G/R37aD/V38E/T39W/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
42.3 Y149R 543
F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/Y151L/M157K/Q192H 42.4 475
F30H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151L/M157F 42.5 697
H37M/R37aD/V38E/T39A/V41R/D60aP/Y60bS/T97aI/L97bA/H99Q/C122S/Y149R
42.7 171 F30H/V38D/V41R/L97bA/H99Q/Y151L/M157K 42.8 552
T22I/F30Y/R35S/V38D/Y40H/V41R/L97bA/H99Q/C122S/I138V/M157K 42.9 734
R35L/H37D/R37aS/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
42.9 Y149R 561
F30Y/R35L/V38D/Y40H/V41R/N76S/L97bA/H99Q/C122S/M157K/K187E 43.6 481
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157S 43.8 648
R35W/H37D/V38D/T39Y/V41R/Y60bH/L97bA/H99Q/C122S/Y149R 44.2 595
F30Y/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bA/T97aE/L97bA/H99Q/C122S/
44.6 Y149Q/M157K 904
R35Q/H37G/R37aE/V38W/T39Y/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
44.6 E175D/Q192T/R217E/K224R 330
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/L97bR/H99E/C122S/Y151L/E175D/
44.6 Q192T/R217E/K224R 879
V38D/T39Y/Y40M/V41R/T97aE/L97bA/H99Q/C122S 45 647
R35Q/H37N/V38D/T39Y/V41R/Y60bP/L97bA/H99Q/C122S 45.4 663
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/Y60bS/T97aE/L97bA/H99Q/C122S/
45.8 Y149K/M157K 23
R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
46.1 975 V38E/T39L1V41R/D60aN/Y60bP/L97bG/H99Q/C122S 46.8 601
F30Y/R36H/H37A/V38E/T39Y/Y40H/V41R/Y60bQ/T97aV/L97bA/H99Q/C122S/
46.9 Y149R/M157K 722
F30Y/R35W/R36H/H37E/R37aP/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/
47.7 H99Q/C122S/Y149Q/M157K 748
H37T/R37aL/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/
47.9 Q192R 325
H37G/R37aD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
47.9 Q192T/R217E/K224R 667
F30Y/R35W/R36H/H37S/V38E/Y40H/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/
48.6 M157K 868 V38D/T39W/Y40L/V41R/T97aL/L97bA/H99Q/C122S 48.9 326
H37G/R37aD/G37bD/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
49 Q192T/R217E/K224R 42 T39Y/V41R/L97bA/H99Q/C122S 49 874
V38D/T39L/Y40L/V41R/T97aW/L97bA/H99Q/C122S 49.1 584
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/Y149N/L150V/M157K
49.3 534 R35S/V38D/L97bA/H99Q/C122S/Y151L/M157Y 49.8 925
R37aS/V38D/T39Y/Y40F/V41R/H99L/C122S/R217T 49.9 349
Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R 49.9 390
Y40H/V41T/L97bG/H99Q/C122S/R217T 50 506
F30H/R35S/V38E/V41R/Y60bH/L97bA/H99Q/C122S/Y151L/M157K 50.1 877
V38D/T39Y/Y40M/V41R/T97aW/L97bA/H99Q/C122S 50.1 900
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
50.9 E175D/Q192T/R217E/K224R 737
R35A/H37G/R37aE/V38E/T39F/V41R/D60aE/Y60bP/T97aI/L97bA/H99Q/C122S/
51.1 Y149R 22
H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
51.1 598
F30Y/R36H/H37N/V38E/T39Y/Y40H/V41R/Y60bQ/T97aV/L97bA/H99Q/C122S/
51.4 Y149L/M157K 545
F30Y/V38D/Y40L//V41R/L97bA/H99Q/C122S/Y151L/M157A/Q192Y 51.5 954
V38E/Y40H/V41Q/L97bG/H99Q/C122S/R217T 51.6 870
V38D/T39Y/Y40Q/V41L/T97aY/L97bA/H99Q/C122S 51.7 942
R35V/R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 51.7
358 R35S/Y40Q/V41L/Y60bT/T97aE/L97bA/H99Q/C122S/Y149R 52.4 535
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157T 52.9 356
R35K/Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R 53.2 692
F30Y/R35V/R36H/H37T/V38E/T39A/Y40L/V41Q/T97aI/L97bA/H99Q/C122S/Y149R/
53.7 M157K 184 F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K/R217D 54
249
F30Y/R36H/V38E/Y40H/V41R/I65T/T97aI/L97bA/H99Q/C122S/Y149R/M157K
54.1 672
R36H/H37S/V38E/T39W/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R 54.1
371 V38D/V41R/L97bR/C122S/Y151L/R217V 54.3 553
F30Y/R36S/V38D/T39I/Y40H/V41R/L97bA/H99Q/C122S/S146P/M157K/T158S
54.6 365 R35V/R37aE/V38E/Y40Q/V41L/Y60bS/L97bA/H99Q/C122S/Y149R
54.6 37 V38E/Y40Q/V41L/Y60bL/H99Q/C122S 54.9 347
R37aE/Y40Q/V41L/L97bA/H99Q/C122S 54.9 709
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39F/Y40F/V41R/D60aS/Y60bP/T97aE/L97bA/
55.3 H99Q/C122S/Y149R/M157K 487
F30H/G37bD/V38D/T39H/V41R/L97bA/H99Q/R110dH/C122S/Y151L/M157K/S240I
55.5 902
R35K/H37A/R37aE/V38H/T39Y/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
55.9 E175D/Q192T/R217E/K224R 603
F30Y/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bN/T97aV/L97bA/H99Q/C122S/ 56
Y149R/M157K 25
R35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
56.2 436
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192E/R217D
56.3 606
F30Y/R36H/H37E/V38E/T39F/Y40H/V41R/Y60bN/T97aV/L97bA/H99Q/C122S/
56.4 Y149R/M157K 337
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
56.4 E175D/Q192T/R217E 587
F30Y/R36H/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/Y149H/M157K/
57.7 K187R 232
F30Y/R36H/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/Y149R/M157K 58
44 T39Y/V41R/D60aP/L97bA/H99Q/C122S 58.2 329
H37G/R37aD/G37bD/V38F/T39H/V41R/T97aS/L97bR/H99E/C122S/Y151L/E175D/
58.5 Q192T/R217E/K224R 216
I17V/F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/
58.6 M157K 519
F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149W/M157K/R217D 58.8
270 F30Y/R36H/V38E/Y40H/V41R/T97aE/L97bA/H99Q/C122S/M157K 59.3 348
R37aE/Y40Q/V41L/L97bA/H99Q/C122S/Y149R 59.3 793
R35N/H37S/R37aE/V38E/T39F/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
59.5 Y149R 226
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K 59.9
719
F30Y/R35Q/R36H/H37G/R37aL/V38E/T39N/Y40F/V41R/D60aP/Y60bT/T97aE/L97bA/
59.9 H99Q/C122S/Y149R/M157K 625
F30Y/R35V/R36H/H37G/V38E/T39L/Y40H/V41R/Y60bT/T97aI/L97bA/H99Q/C122S/
60.1 Y149R/M157K/Q192T/R239H 903
R35Q/H37G/R37aE/V38Y/T39F/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
60.1 E175D/Q192T/R217E/K224R 35 V38E/Y40Q/Y60bL/L97bA/H99Q/C122S
60.2 176 F30H/V38D/V41R/L97bA/H99Q/C122S/Y149N/Y151L/M157K 60.3 381
H99Q/C122S/Y151L/R217V 60.4 841
R35S/V38D/Y40H/V41L/Y60bD/L97bG/H99Q/C122S 61.5 698
R35F/H37D/R37aS/V38E/T39Y/V41R/D60aE/Y60bD/T97aI/L97bA/H99Q/C122S/
63.4 Y149R 935
R35V/R37aD/V38E/Y40Q/V41L/Y60bG/T97aE/L97bA/H99Q/C122S/Y149R 63.5
951 R36L/V38E/Y40Q/V41R/L97bG/H99Q/C122S/Y151P/R217S 63.7 600
F30Y/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bV/T97aE/L97bA/H99Q/C122S/
63.9 Y149R/M157K 607
F30Y/R36H/H37G/V38E/T39Y/Y40H/V41R/Y60bN/T97aA/L97bA/H99Q/C122S/
64.2 Y149R/M157K 947
R37aD/V38E/Y40Q/V41L/Y60bP/T97aV/L97bA/H99Q/C122S/Y149K 64.8 555
F30Y/R35L/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 65.1 357
R37aD/Y40Q/V41L/Y60bT/T97aE/L97bA/H99Q/C122S/Y149R 65.3 699
R35A/H37D/R37aN/V38E/T39F/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/
65.8 Y149R 198 I17V/F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 66
267 V38E/Y40Q/V41Q/D60aP/Y60bL/L97bA/H99Q/C122S/Y149W 66 711
F30Y/R35Q/R36H/H37G/R37aN/V38E/T39Y/Y40F/V41R/Y60bA/T97aE/L97bA/
66.2 H99Q/C122S/Y149R/M157K 849
V38D/V41R/Y60bR/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R 66.2
518 F30H/R35Q/V38D/V41R/Y60bE/L97bA/H99Q/C122S/Y149N/Y151L/M157K
66.3 313 V38D/T39L/V41R/T97aI/L97bA/H99Q/C122S 66.3 937
R37aP/V38E/Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R 67.2 591
F30Y/R36H/H37S/V38E/T39L/Y40H/V41R/Y60bH/T97aI/L97bA/H99Q/C122S/Y149R/
67.5 M157K 901
R35Q/H37G/R37aE/V38Y/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
67.6 E175D/Q192T/R217E/K224R 955
V38E/Y40H/V41T/L97bG/H99Q/C122S/R217T 67.8 833
V38D/V41Q/L97bG/H99Q/C122S/Y151L/R217T 68.6 417
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/R217D 68.8 695
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39I/Y40F/V41R/D60aP/Y60bD/T97aI/L97bA/
69.2 H99Q/C122S/Y149M/M157K 893
R35G/H37T/R37aE/V38T/T39Y/V41Q/Y60bR/T97aF/L97bR/H99E/C122S/Y151L/
69.7 E175D/Q192T/R217E/K224R 932
R37aS/V38D/T39Y/V41R/L97bI/H99E/C122S/R217T 70.1 233
F30Y/R36H/R37aH/V38E/Y40H/V41R/K61E/T97aI/L97bA/H99Q/C122S/Y149R/
70.3 M157K 661
F30H/R35L/R36H/H37E/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
70.4 M157K 629
F30Y/R35W/R36H/H37E/V38E/T39Y/Y40H/V41R/Y60bD/T97aI/L97bA/H99Q/C122S/
70.8 Y149R/M157K/Q192M 807
V38E/T39L/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y149V/Y151L/Q192T
71.1 199 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149W/M157K 71.3 725
F30Y/R35F/R36H/H37D/R37aD/V38E/T39H/Y40F/V41R/D60aP/Y60bA/T97aE/L97bA/
71.3 H99Q/C122S/Y149R/M157K 565
F30H/R35Q/H37Y/V38D/V41R/D60aE/Y60bN/L97bA/H99Q/C122S/Y149R/Y151L/
71.4 M157K 376 L97bR/H99Q/C122S/Y151L/R217V 72.3 960
R37aS/V38E/Y40H/V41T/L97bG/H99Q/C122S/R217T 72.6 307
Y40Q/Y60bL/L97bA/H99Q/C122S 73 466
R36H/G37cD/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/M157T/
75 R217D 839 R35K/V38D/Y40H/V41R/Y60bS/L97bG/H99Q/C122S 75.6 299
R35Q/H37Y/R37aE/V38D/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/
77.3 Y149R 592
F30Y/R35L/R36H/H37D/V38E/T39W/Y40H/V41R/Y60bA/T97aD/L97bA/H99Q/
77.9 C122S/Y149R/M157K 520
F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149H/M157K
78.3 389 Y40H/V41Q/L97bG/H99Q/C122S 78.4 400
T39H/V41R/L97bG/H99Q/C122S 78.4 315
V38D/T39L/Y40L/V41R/L97bA/H99Q/C122S 79 740
R35Q/H37G/R37aE/V38E/T39H/V41R/D60aP/Y60bA/T97aI/L97bA/H99Q/C122S/
80.1 Y149R 716
F30Y/R35A/R36H/H37T/R37aD/V38E/T39Y/Y40L/V41R/D60aP/Y60bE/T97aE/L97bA/
80.7 H99Q/C122S/Y149R/M157K 700
R35Q/H37D/R37aA/V38E/T39F/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/
80.7 Y149R
312 V38D/Y40L/V41R/T97aI/L97bA/H99Q/C122S 80.7 651
F30Y/R35L/R36H/H37D/V38E/T39Y/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
80.9 M157K/Q192N 871
R37aS/V38D/T39Y/Y40M/V41R/T97aL/L97bA/H99Q/C122S 81.2 374
R35V/R37aE/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 81.4 359
R37aE/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 81.5 938
R35M/R37aQ/V38E/Y40Q/V41L/Y60bE/T97aE/L97bA/H99Q/C122S/Y149R 81.6
38 V38E/Y40Q/V41L/Y60bL/L97bA/C122S 82.9 243
F30Y/R36H/V38E/Y40L/V41R/T97aI/L97bA/H99Q/C122S/M157K 83.5 500
F30Y/R35H/V38D/T39S/Y40L/V41R/L97bA/H99Q/C122S/T147S/M157K 84.7 564
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K/Q192H/R239H 85.7 721
F30Y/R36H/H37M/R37aD/V38E/T39G/Y40F/V41R/D60aP/Y60bE/T97aE/L97bA/
85.9 H99Q/C122S/Y149R/M157K 523
F30Y/R35H/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 87.3 544
F30Y/V38D/Y40L/V41Q/L97bA/H99Q/C122S/Y151L/M157R/Q192Y 88.1 228
F30Y/R35Q/H37W/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149R/M157K
88.8 826
V38D/V41Q/Y60bR/T97aW/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R
90 985 R37aH/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S 90.7 547
F30H/V38D/V41Q/L97bA/H99Q/C122S/Y151L/M157R 91.3 886
V38D/T39Y/Y40L/V41K/L97bR/H99L/C122S/E175D 91.8 944
R37aD/V38E/Y40Q/V41L/Y60bP/T97aE/L97bA/H99Q/C122S/Y149R 91.9 32
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/C122S/Y149R
92.3 542 N26D/F30Y/V38D/Y40H/V41R/I65T/L97bA/H99Q/C122S/M126I/M157K
93.2 946
R35A/R37aD/V38E/Y40Q/V41L/Y60bV/T97aE/L97bA/H99Q/C122S/Y149K 93.4
939 R35S/R37aD/V38E/Y40Q/V41L/Y60bT/T97aE/L97bA/H99Q/C122S/Y149R
93.5 508
F30H/R35Q/H37T/V38D/V41R/L97bA/H99Q/C122S/Y149I/Y151L/M157K/R217D
93.6 541 F30H/V38D/L97bA/H99Q/C122S/Y151L/M157K 94.2 377
V38D/H99Q/C122S/Y151L/R217V 94.4 576
F30Y/R35T/H37T/V38D/Y40H/V41R/D60aP/Y60bD/L97bA/H99Q/C122S/Y149T/
94.7 M157K 227
I17V/F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149R/M157K 94.8 855
V38D/V41R/Y60bR/L97bR/H99L/C122S/Y151L/E175D/Q192Y/R217E/K224R 94.9
372 R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 95 594
F30Y/R36H/H37E/V38E/T39Y/Y40H/V41R/Y60bN/T97aV/L97bA/H99Q/C122S/
96.3 Y149K/M157K 820
V38D/V41Q/D60aP/Y60bS/T97aS/L97bR/H99E/C122S/Y151L/E175D/Q192Y/R217E/
96.4 K224R 510
F30H/R35Q/H37Q/V38D/V41R/A96D/L97bA/H99Q/C122S/Y149L/Y151L/M157K/
96.8 R217D 240
F30Y/R35Q/H37Y/V38D/Y40H/V41R/Y60bE/L97bA/H99Q/C122S/Y149R/M157K
98.7 891
H37A/R37aE/V38F/T39L/V41Q/Y60bR/T97aW/L97bR/H99E/C122S/Y151L/E175D/
99.6 Q192T/R217E/K224R 605
F30Y/R36H/V38E/T39V/Y40H/V41R/Y60bH/T97aE/L97bA/H99Q/C122S/Y149S/
99.7 M157K 485 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S 99.9 622
F30Y/R35V/R36H/H37S/V38E/T39F/Y40H/V41R/Y60bS/T97aI/L97bA/H99Q/C122S/
100 Y149R/M157K/Q192T 585
F30Y/R36H/V38E/Y40H/V41R/Y60bN/N87D/A96T/T97aI/L97bA/H99Q/C122S/N145S/
101 M157K/M207K 309 V38E/Y40Q/V41L/L97bA/C122S 101 952
R37aS/V38E/V41R/L97bG/H99Q/C122S/R217V 101 445
R36H/V38D/Y40L/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192T/
102 R217D 649
F30Y/R35Q/R36H/H37G/V38E/T39H/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
102 M157K/Q192M 363
R35V/R37aE/Y40Q/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 103 314
V38D/T39L/Y40L/T97aI/L97bA/H99Q/C122S 104 183
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/R217E 105 655
F30L/R35L/R36H/H37D/V38E/T39Y/V41K/T97aI/L97bA/H99Q/C122S/Y149R/M157K
105 921 R36S/V38D/T39A/Y40F/V41R/L97bA/H99E/C122S/R217T 105 568
F30Y/R35K/H37E/V38D/Y40H/V41R/D60aP/Y60bD/L97bA/H99Q/C122S/Y149N/
106 M157K 691
F30Y/R35L/R36H/H37E/V38E/T39S/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
106 M157K 739
R35Q/H37T/R37aP/V38E/T39Y/V41R/D60aE/Y60bD/T97aI/L97bA/H99Q/C122S/
106 Y149R 448
R36H/V38D/Y40I/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Q192A/R217D
107 540 F30Y/R36H/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/T210S 107
397 T39Y/V41R/Y60bQ/L97bA/C122S 108 208
F30Y/R35Q/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149W/M157K 109 211
F30Y/V38D/Y40H/V41R/T56A/L97bA/H99Q/C122S/M157K 109 546
F30Y/R36S/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 109 850
V38D/V41R/D60aP/Y60bD/T97aW/L97bR/H99Q/C122S/Y151L/E175D/Q192A/R217E/
109 K224R 842
V38D/V41R/Y60bN/T97aL/L97bR/H99E/C122S/Y151L/E175D/Q192T/R217E/K224R
109 883 R35H/V38D/V41Q/L97bR/H99Q/C122S/Y151L/R217V 109 967
R35H/V38E/L97bR/C122S/Y151L/R217V 109 447
R36H/V38D/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192G/R217D
110 254
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bH/T97aI/L97bA/H99Q/C122S/
111 Y149R/M157K/Q192A 385 R37aS/V41R/L97bG/H99Q/C122S/R217V 111 887
R35Q/H37G/R37aN/V38F/T39H/V41Q/Y60bR/T97aL/L97bR/H99E/C122S/Y151L/
112 E175D/Q192T/R217E/K224R 164
R36S/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/M157K/R217D
113 210 F30Y/R35Q/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 113
838 R35S/V38D/Y40Q/V41R/Y60bS/L97bG/H99Q/C122S 113 898
H37S/R37aD/V38H/T39L/V41Q/Y60bR/T97aW/L97bR/H99E/C122S/Y151L/E175D/
113 Q192T/R217E/K224R 196
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K 114 310
Y40Q/L97bA/H99Q/C122S 114 355
R37aE/Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R 114 945
R35S/R37aD/V38E/Y40Q/V41L/T97aD/L97bA/H99Q/C122S/Y149K 115 643
R35Q/H37M/V38D/T39V/V41R/Y60bP/L97bA/H99Q/C122S/Y149R 116 644
R35L/H37E/V38D/T39Y/V41R/L97bA/H99Q/C122S/Y149R 116 450
R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192D/
118 R217D 497
F30Y/Y34N/V38D/Y40H/V41R/Y94F/S95R/L97bA/H99Q/C122S/M157K/T242I 118
323
R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
118 Q192T/R217E/K224R 334
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
118 Q192T/R217E/K224R 392 Y40H/V41T/L97bG/H99Q/C122S 118 8
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 119 434
R37aH/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192G/
119 R217D 419
R36H/V38D/V41M/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217D 120 317
V38D/L97bR/H99Q/C122S/Y151L/R217V 120 201
F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149W/M157K 121 549
F30Y/R37aH/V38D/Y40H/V41R/K61E/L97bA/H99Q/C122S/M157K 121 767
V38D/L97bR/H99E/C122S/E175D/K224S 121 888
H37N/R37aD/V38Y/T39F/V41Q/Y60bR/T97aW/L97bR/H99E/C122S/Y151L/E175D/
121 Q192T/R217E/K224R 452
R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192R/
122 R217D 560 F30Y/R37aS/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 122
837 V38D/V41R/L97bG/H99Q/C122S/Y151L/Q192S/R217A 122 361
R37aD/Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R 122 880
R35S/V38D/V41T/L97bR/H99Q/C122S/Y151L/R217E 124 40
Y40Q/V41L/L97bA/C122S 124 173
F30H/V38D/V41R/L97bV/H99Q/C122S/Y151L/M157K 125 229
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149R/M157K 125 322
V38D/V41R/L97bR/H99Q/C122S/Y151L 125 895
H37A/R37aE/V38Y/T39Y/V41Q/Y60bR/T97aV/L97bR/H99E/C122S/Y151L/E175D/
126 Q192T/R217E/K224R 892
H37N/R37aE/V38F/T39L/V41Q/Y60bR/T97aW/L97bR/H99E/C122S/Y151L/E175D/
127 Q192T/R217E/K224R 652
F30Y/R35L/R36H/H37E/V38E/T39L/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
129 M157K/Q192H 451
R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151M/Q192T/
130 R217D 854
V38D/V41R/Y60bR/L97bR/H99L/C122S/Y151L/E175D/Q192T/R217E/K224R 130
308 V38E/Y40Q/L97bA/H99Q/C122S 133 968
V38E/T56I/H99Q/C122S/Y151L/S190G/R217V 134 556
F30Y/S37dP/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 135 964
R35S/V38E/L97bR/C122S/Y151L/R217V/H241N 135 396
T39Y/V41R/L97bA/C122S 135 384 L97bR/C122S/Y151L/R217V 136 599
F30Y/R35L/R36H/H37E/V38E/T39L/Y40H/V41R/Y60bS/T97aD/L97bA/H99Q/C122S/
137 Y149R/M157K 596
F30Y/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bD/T97aD/L97bA/H99Q/C122S/
137 Y149R/M157K 894
R35K/H37S/R37aE/V38F/T39Y/V41Q/Y60bR/T97aL/L97bR/H99E/C122S/Y151L/
141 E175D/Q192T/R217E/K224R 269
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99L/ 142
C122S/Y149K/M157K 840 R35Q/V38D/Y40L/V41L/Y60bP/L97bG/H99Q/C122S
142 471 F30H/V38D/V41R/L97bA/H99Q/C122S/M157E 146 429
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192D/R217D
146 501 F30Y/R36S/V38H/Y40L/V41R/N76K/L97bA/H99Q/C122S/M157K 146
195 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K 147 539
F30H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151L/M157R/Q192H 149 927
R37aP/V38D/T39L/Y40I/V41R/H99L/C122S/R217Q 150 293
V38E/V41R/T97aI/L97bA/H99Q/C122S 152 375
R35V/R37aE/V38E/Y40Q/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 153 214
V38D/V41R/L97bA/H99Q/C122S/M157K/T158A 154 189
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K/R217E 155 31
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/H99Q/C122S/Y149R
155 393 V41R/L97bG/H99Q/C122S 155 890
R35K/H37G/R37aE/G37bS/V38F/T39H/V41Q/Y60bR/T97aW/L97bR/H99E/C122S/
156 Y151L/E175D/Q192T/R217E/K224R 571
F30Y/R35S/H37G/V38D/Y40H/V41R/D60aP/Y60bE/L97bA/H99Q/C122S/Y149K/
158 M157K 602
F30Y/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bM/T97aD/L97bA/H99Q/C122S/
159 Y149R/M157K 203 F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K
160 843
V38D/V41Q/Y60bR/T97aF/L97bR/H99Q/C122S/Y151L/E175D/Q192H/R217E/K224R
160 897
H37A/R37aD/V38Y/T39Y/V41Q/Y60bR/T97aD/L97bR/H99E/C122S/Y151L/E175D/
160 Q192T/R217E/K224R 896
H37D/R37aD/V38Y/T39Y/V41Q/Y60bR/T97aL/L97bR/H99E/C122S/Y151L/E175D/
162 Q192T/R217E/K224R 353 Y40Q/V41L/L97bA/C122S/Y149R 162 488
F30H/V38D/V41R/L97bA/H99Q/C122S/Y149N/Y151L/M157K/R239H 163 930
R36S/V38D/T39S/V41R/L97bI/H99E/C122S/R217T 163 906
V38D/T39V/V41R/L97bV/H99E/C122S/R217E 165 962
R37aS/V38E/Y40Q/V41L/L97bG/C122S/Y151P/R217T 166 484
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 167 378
V38D/L97bR/C122S/Y151L/R217V 170 593
F30Y/R36H/H37P/V38E/T39Y/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/Y149R/
171 M157K 825
V38D/V41K/D60aN/Y60bN/L97bR/H99E/C122S/Y151L/E175D/Q192H/R217E/K224R
172 889
H37A/R37aE/V38F/T39L/V41Q/Y60bR/T97aF/L97bR/H99E/C122S/Y151L/E175D/
174 Q192T/R217E/K224R 345 Y40Q/V41L/H99Q/C122S 174 574
F30Y/R35Q/V38D/Y40H/V41R/Y60bS/L97bA/H99Q/C122S/Y149R/M157K/R217E
175 730
F30Y/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/Y60bN/T97aE/L97bA/H99Q/
176 C122S/Y149R/M157K 941
R35K/R37aD/V38E/Y40Q/V41L/Y60bE/L97bA/H99Q/C122S/Y149R 178 209
F30Y/R35Q/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K 179 321
V38D/T39L/Y40L/V41R/T97aI/L97bA/C122S 179 245
N26D/F30Y/R36H/R37aH/V38E/Y40H/V41R/K61E/I65T/L97bA/H99Q/C122S/M126I/
185 M157K 367
R35V/R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/L97bA/C122S/Y149R 185 483
F30Y/V38D/Y40M/V41R/L97bA/H99Q/C122S 188 768
V38D/L97bV/H99E/C122S/K224S 192 861
V38D/V41R/D60aQ/Y60bP/L97bG/H99Q/C122S 192 202
F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K 194 931
R37aS/V38D/T39G/V41R/L97bM/H99E/C122S/R217S 194 521
F30Y/R35Q/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149I/M157K 196 473
F30Y/V38D/Y40H/V41A/L97bA/H99Q/C122S/M157R 197 191
F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217E 199 206
F30Y/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 199 327
H37G/R37aD/G37bD/V38F/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
199 Q192T/R217E/K224R 41 R37aS/V41R/L97bG/H99Q/C122S 199 244
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/Q192H 201
248
N26D/F30Y/V38D/Y40H/V41R/I65T/L97bA/H99Q/C122S/M126I/Y149R/M157K
201 864 R35K/V38D/Y40H/Y60bP/L97bG/H99Q/C122S 201 525
F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/E153D/M157K 202 526
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/D148N/M157K/T188S 202 660
R35E/R36H/H37D/V38E/T39W/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
202 M157K/Q192T 558
F30Y/R37aS/V38D/Y40H/V41R/E86D/L97bA/H99Q/C122S/M157K/F234Y 203 723
F30Y/R35Q/R36H/H37D/R37aE/V38E/T39Y/Y40F/V41R/D60aE/Y60bG/T97aE/L97bA/
203 H99Q/C122S/Y149R/M157K 563
F30Y/R37aS/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/K187Q/T208S 205
465
R36H/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/Q192R/R217F
208 170 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/R217D 208 583
F30Y/R35T/H37T/V38D/Y40H/V41R/D60aT/Y60bS/L97bA/H99Q/C122S/Y149K/
208 M157K 609
F30Y/R35L/H37D/V38D/Y40H/V41R/D60aE/Y60bT/L97bA/H99Q/C122S/Y149R/
212 M157K 352 R37aE/Y40Q/V41L/L97bA/C122S 212 920
R36S/V38D/T39K/Y40F/V41R/L97bI/H99E/C122S/R217S 213 320
V38D/T39L/Y40L/V41K/L97bR/H99L/C122S/E175D 215 169
V38D/V41R/A96E/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151L/T178S/Q192E/
217 R217D 588
F30Y/R37aP/V38D/Y40H/V41R/Y60bN/E84D/L97bA/H99Q/C122S/M157K/T242I
219 190 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K/R217D 222
566
F30Y/R35T/H37Q/V38D/Y40H/V41R/D60aE/Y60bN/L97bA/H99Q/C122S/Y149R/
230 M157K 536 R37aL/V38D/L97bA/H99Q/C122S/Y151L/M157S 231 760
V38D/L97bR/H99E/C122S/E175D/R217E/K224R 234 478
F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/M157R 235 238
F30Y/R35Q/V38D/Y40H/V41R/D60aE/Y60bS/L97bA/H99Q/C122S/Y149T/M157K
235 234
F30Y/R35Q/H37D/V38D/Y40H/V41R/D60aE/Y60bN/L97bA/H99Q/C122S/Y149S/
236 M157K 207
F30Y/R35Q/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K 238 344
Y40Q/V41L/C122S 238 554
F30Y/Y34S/R37aH/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K 239 354
R37aE/Y40Q/V41L/L97bA/C122S/Y149R 245 247
N26D/F30Y/V38D/Y40L/V41R/I65T/L97bA/H99Q/C122S/M126I/M157K 247 476
F30Y/V38D/Y40H/V41N/L97bA/H99Q/C122S 250 524
F30Y/R35H/V38D/Y40H/V41R/T56A/L97bV/H99Q/R116S/C122S/M157K/T210N
250 212 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y151F/M157K 252 388
Y40H/V41Q/L97bG/C122S/R217T 252 974
V38E/T39H/D60aP/Y60bK/L97bG/H99Q/C122S 252 205
F30Y/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K 255 167
V38D/Y40P/V41K/L97bA/H99Q/C122S 257 573
F30Y/R35T/H37A/V38D/Y40H/V41R/D60aP/Y60bE/L97bA/H99Q/C122S/Y149K/
263 M157K/R217Y 559
F30Y/R37aH/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/S174P 267 569
F30Y/R35L/H37D/V38D/Y40H/V41R/Y60bD/L97bA/H99Q/C122S/Y149R/M157K
267 578
F30Y/R35S/V38D/Y40H/V41R/D60aE/Y60bS/L97bA/H99Q/C122S/Y149R/M157K
270 383 L97bR/H99Q/C122S/Y151L 271 572
F30Y/R35Q/V38D/Y40H/V41R/D60aE/Y60bE/L97bA/H99Q/C122S/Y149N/M157K
275 200 F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K 276
250 F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/M157K 277 567
F30Y/R35T/V38D/Y40H/V41R/D60aE/Y60bQ/L97bA/H99Q/C122S/Y149R/M157K/
277 R217K 477 F30S/V38D/V41R/L97bA/H99Q/C122S/Y151L/M157K 279 819
V38D/V41Q/D60aK/Y60bD/T97aA/L97bR/H99E/C122S/Y151L/E175D/Q192Y/R217E/
279 K224R 186
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217E 280 976
V38E/T39H/D60aP/Y60bW/L97bG/H99Q/C122S 281 662
F30H/R35L/R36H/H37D/V38E/T39Y/Y40F/V41Q/T97aI/L97bA/H99Q/C122S/Y149R/
284 M157K 316 V38D/T39L/Y40L/V41R/T97aI/H99Q/C122S 284 757
V38D/L97bR/H99Q/C122S/E175D/R217E/K224R 292 923
R37aS/V38D/T39L/Y40L/V41R/L97bM/H99L/C122S/R217S 292 654
F30Y/R36H/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K/Q192Y
297 637 R35E/H37D/V38D/T39N/V41R/Y60bN/L97bA/H99Q/C122S/Y149R 306
766 V38D/L97bR/H99E/C122S/E175D/K224A 306 763
V38D/L97bV/H99L/C122S/E175D/K224S 310 813
V38D/L97bR/H99L/C122S/E175D/K224A 310 912
R37aP/V38D/T39Y/V41R/L97bV/H99Q/C122S/R217T 316 175
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151M/M157K 321 502
F30H/V38D/V41R/L73Q/L97bA/H99Q/E110bD/C122S/Y149N/Y151L/M157K 321
582
F30Y/R35Q/V38D/Y40H/V41R/D60aE/Y60bN/L97bA/H99Q/C122S/Y149R/M157K
324 817 V38D/L97bT/H99E/C122S/E175D/K224S 326 204
F30Y/H37T/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K 330 522
F30Y/R35Q/H37T/V38D/Y40H/V41R/D60aE/Y60bN/L97bA/H99Q/C122S/Y149H/
331 M157K 589
F30Y/R37aP/G37bD/V38D/Y40H/V41R/Y60bN/L97bA/H99Q/C122S/M157K 331
237
F30Y/R35Q/V38Y/Y40H/V41R/D60aE/Y60bE/L97bA/H99Q/C122S/Y149V/M157K/
332 R217M 764 V38D/L97bR/H99L/C122S/E175D/R217E 333 235
F30Y/R35Q/H37T/V38D/Y40H/V41R/D60aE/Y60bN/L97bA/H99Q/C122S/Y149L/
338 M157K 773 V38D/L97bG/H99Q/C122S/R217Q 338 187
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217D 341 933
R35E/R37aD/V38E/Y40Q/V41L/Y60bN/L97bA/H99Q/C122S/Y149R 342 610
F30Y/R35Q/V38D/Y40H/V41R/D60aK/Y60bV/L97bA/H99Q/C122S/Y149R/M157K
344 581
F30Y/R35Q/V38D/Y40H/V41R/D60aP/Y60bE/L97bA/H99Q/C122S/Y149R/M157K/
345 R217K 865 R35Q/V38D/Y40M/V41R/Y60bA/L97bG/H99Q/C122S 345 252
F30Y/R36H/V38E/Y40L/V41R/T97aI/L97bA/H99Q/C122S/M157K/Q192H 346 331
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/H99E/C122S/Y151L/E175D/
347 Q192T/R217E/K224R 26
R35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
348 866 R35K/V38D/Y40F/V41R/Y60bT/L97bG/H99Q/C122S 348 579
F30Y/R35H/H37T/V38D/Y40H/V41R/D60aR/Y60bD/L97bA/H99Q/C122S/Y149R/
349 M157K 774 F30H/V38D/L97bA/H99Q/C122S/Y151L/M157T/Q192H 349 380
L97bR/H99Q/C122S/Y151L/R217V 351 860
R35S/V38D/V41L/D60aP/Y60bW/L97bG/H99Q/C122S 353 391
Y40H/V41T/L97bG/C122S/R217T 357 301
V38E/V41L/Y60bL/L97bA/H99Q/C122S 364 815
V38D/L97bK/H99L/C122S/E175D/K224A 365 302 L97bG/H99Q/C122S/R217Q
367 646 R35G/H37G/V38D/T39W/V41A/Y60bN/L97bA/H99Q/C122S 370 636
R35F/H37G/V38D/T39H/V41A/Y60bD/L97bA/H99Q/C122S/Y149R 371 332
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/C122S/Y151L/E175D/
371 Q192T/R217E/K224R 193
F30Y/H37D/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217E 378 862
R35Q/V38D/Y40H/V41L/Y60bD/L97bG/H99Q/C122S 380 828
V38D/V41Q/L97bG/H99Q/C122S/Y151L/Q192S/R217A 385 965
R35S/V38E/H99Q/C122S/S146F/Y151L/V159L/K179N/Q192R/R217V/T242S 408
590
F30Y/R35L/R36H/H37P/V38E/Y40H/V41R/Y60bG/T97aD/L97bA/H99Q/C122S/
409 Y149R/M157K 971 V38E/T39R/D60aP/Y60bD/L97bG/H99Q/C122S 412 846
R37aS/V38D/V41R/Y60bT/T97aD/L97bR/H99Q/C122S/Y151L/E175D/Q192A/R217E/
413 K224R 318 V38D/V41R/L97bR/H99Q/C122S/R217V 418 438
R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151L/Q192D/
421 R217D 537 F30N/V38D/V41L/L97bA/H99Q/C122S/Y151L/M157R 428 899
H37G/R37aD/V38Y/T39H/V41Q/Y60bR/T97aD/L97bR/H99E/C122S/Y151L/E175D/
433 Q192T/R217E/K224R 659
F30Y/R35W/R36H/H37E/V38E/T39S/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/
434 M157K/Q192A 844
V38D/V41R/D60aP/Y60bD/T97aP/L97bR/H99Q/C122S/Y151L/E175D/Q192R/R217E/
437 K224R 460
F30Y/R36H/V38D/V41K/D97E/T97adel/A98G/L97bdel/H99L/C122S/R217D 445
274 L97bA/H99Q/C122S 449 966 F30Y/R37aH/V38E/H99Q/C122S/Y151L/R217V
449 246
N26D/F30Y/V38D/Y40H/V41R/I65T/L97bA/H99Q/C122S/M126I/M157K/Q192H
455 253
F30Y/R35W/R36H/H37E/V38E/T39S/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/ 463
C122S/Y149R/M157K/Q192Y 970 V38E/T39S/V41L/Y60bP/L97bG/H99Q/C122S
464 319 V38D/Y40L/V41K/L97bR/H99L/C122S/E175D 466 236
F30Y/R35Q/V38D/Y40H/V41R/Y60bE/L97bA/H99Q/C122S/Y149Q/M157K/R217S
470 297 V38D/L97bR/H99E/C122S/E175D/R217E 470 343
V41L/L97bA/H99Q/C122S 471 366
R35V/R37aE/V38E/Y40Q/V41L/Y60bS/T97aE/H99Q/C122S/Y149R 478 640
R35F/H37E/V38D/T39S/V41A/Y60bD/L97bA/H99Q/C122S 487 333
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/E175D/
499 Q192T/R217E/K224R 213 V38D/L97bA/H99Q/C122S/M157K/T158A 503 256
F30Y/R35W/R36H/H37E/V38E/T39R/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/ 511
C122S/Y149R/M157K/Q192Y 529
F30H/V38D/L97bA/H99Q/C122S/Y151L/M157L/Q192H 515 492
F30Y/R37aH/V38D/Y40L/V41R/L97bA/H99Q/C122S/M157K 516 239
F30Y/R35Q/V38D/Y40H/V41R/D60aE/Y60bE/L97bA/H99Q/C122S/Y149L/M157K/
530 R217S 814 V38D/L97bM/H99S/C122S/K224S 538 575
F30Y/R37aD/V38D/Y40L/V41R/L97bA/H99Q/C122S/M126K/M157K 539 394
V41Q/L97bG/H99Q/C122S 547 435
V38D/Y40P/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Q192L/R217D
557 258
F30Y/R35Y/R36H/H37E/V38E/T39R/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/C122S/
569 Y149R/M157K/Q192H 908
R36S/V38D/T39H/Y40L/V41R/L97bV/H99V/C122S/R217Q 574 638
R35Q/H37Y/V38D/T39A/V41R/Y60bP/L97bA/H99Q/C122S/Y149R/Q192S 575 845
V38D/V41Q/Y60bP/T97aW/L97bR/H99Q/C122S/Y151L/E175D/Q192K/R217E/K224R
578 907 R37aH/V38D/T39M/Y40A/V41K/L97bR/H99Q/C122S/R217E 582 328
H37G/R37aD/G37bD/V38F/T39H/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/E175D/
594 Q192T/R217E/K224R 811 V38D/L97bR/H99L/C122S/E175D/R217E/K224R
610 848
R37aS/V38D/V41R/D60aS/Y60bP/L97bR/H99Q/C122S/Y151L/E175D/Q192R/R217E/
614 K224R 570
F30Y/R35H/H37T/V38D/Y40H/V41R/D60aE/Y60bD/L97bA/H99Q/C122S/Y149K/
622 M157K/R217K 295 V38E/T97aI/L97bA/H99Q/C122S 630 499
F30Y/R35H/V38D/T39S/Y40H/V41R/S75P/L97bA/H99Q/C122S/M157K/I163M/
632 Q204H/K243E 528
F30Y/V38D/Y40M/V41R/L97bA/H99Q/C122S/Y151L/M157R/Q192M 641 395
V41T/L97bG/H99Q/C122S 646 463
R36H/V38D/V41M/A55S/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217D 647
928 V38D/T39V/Y40L/V41S/H99V/C122S/R217V 653 577
F30Y/R35H/H37D/V38D/Y40H/V41R/D60aK/Y60bS/L97bA/H99Q/C122S/Y149R/
659 M157K 759 V38D/L97bR/H99L/C122S/E175D/R217E/K224S 661 769
V38D/L97bK/H99L/C122S/R217E 673 174
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151F/M157K 688 867
R35Q/V38D/Y40F/V41T/Y60bP/L97bG/H99Q/C122S 698 462
R36H/V38D/V41M/F59I/D97E/T97adel/A98G/L97bdel/H99L/C122S/R217D 702
969 F30Y/V38E/L97bR/C122S/Y151L/R217V 702 914
V38D/T39H/Y40F/V41K/L97bM/H99T/C122S/R217E 717 386
R37aS/V41R/L97bG/C122S/R217V 726 379 V38D/L97bR/H99Q/C122S/R217V
731 885 R37aS/V38D/V41R/L97bR/H99Q/C122S/Y151L/Q192T/R217V 738 27
R35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
743 362 R35V/R37aE/V38E/V41L/Y60bS/T97aE/L97bA/H99Q/C122S/Y149R 765
241 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/Q192H 778 527
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/K223N/P236S 781 255
F30Y/R35W/R36H/H37E/V38E/T39Q/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/ 802
C122S/Y149R/M157K/Q192F 490
F30Y/R37aH/V38D/Y40L/V41R/L97bA/H99Q/R110dH/C122S/M157K/V160I 808
630
F30Y/R35W/R36H/H37E/V38E/T39V/Y40H/V41W/Y60bS/T97aI/L97bA/H99Q/C122S/
819 Y149R/M157K/Q192H 771 V38D/L97bV/H99L/C122S/K224A 824 444
R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151M/Q192D/
825 R217D 479
F30Y/R36H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151M/M157Y/Q192A 830 812
V38D/L97bT/H99L/C122S/E175D/R217K 851 294
V38E/T97aE/L97bA/H99Q/C122S 862 580
F30Y/R35Q/V38D/Y40H/V41R/D60aE/Y60bK/L97bA/H99Q/C122S/Y149R/M157K/
917 R217I 816 V38D/L97bR/H99V/C122S/E175D/R217E/K224R 930 853
V38D/V41Q/Y60bP/T97aY/L97bR/H99L/C122S/Y151L/E175D/Q192K/R217E/K224R
960 922 R35Q/H37Y/R37aE/V38E/T39Y/V41R/L97bG/H99S/C122S 989 852
V38D/V41Q/D60aN/Y60bP/T97aN/L97bR/H99I/C122S/Y151L/E175D/Q192R/R217E/
993 K224R 635
R35Q/H37D/V38D/T39H/V41R/Y60bP/L97bA/H99Q/C122S/Y149R/Q192T 998 548
F30H/V38D/L97bA/H99Q/C122S/Y151F/M157K 1000 259
F30Y/R35Y/R36H/H37E/V38E/T39R/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/C122S/
1030 Y149R/M157K/Q192F 847
V38D/V41K/D60aE/Y60bP/T97aA/L97bR/H99L/C122S/Y151L/E175D/Q192R/R217E/
1040 K224R 851
V38D/V41Q/D60aN/Y60bP/L97bR/H99L/C122S/Y151L/E175D/Q192R/R217E/K224R
1040 336
H37G/R37aD/G37bD/V38F/T39H/V41R/Y60bK/T97aS/L97bR/H99E/C122S/Y151L/
1040 E175D/Q192T/K224R 639
R35Q/H37Y/V38D/T39F/V41R/Y60bP/L97bA/H99Q/C122S/Y149R/Q192G 1130
215 F30Y/R37aH/V38D/Y40Q/V41I/L97bA/H99Q/C122S/Y151L/M157W/Q192Y
1140 631
F30Y/R35Y/R36H/H37E/V38E/T39A/Y40H/V41W/Y60bV/T97aI/L97bA/H99Q/C122S/
1170
Y149R/M157K/Q192H 859 R36H/V38D/T97aR/L97bG/H99Q/C122S/E175F 1180
177 F30H/V38D/V41R/L97bA/H99Q/C122S/Y149N/Y151F/M157K 1190 305
V38D/L97bG/H99Q/C122S 1190 909
R36S/V38D/T39K/Y40F/V41R/L97bN/H99E/C122S/R217S 1230 251
F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/M157K/Q192H 1240 423
V38D/L97bA/H99Q/C122S 1310 916
V38D/T39L/Y40L/V41K/L97bH/H99L/C122S/R217A 1320 642
R35Q/H37G/V38D/T39W/V41R/Y60bP/L97bA/H99Q/C122S/Y149R/Q192G 1340
929 R37aS/V38D/T39V/V41E/L97bG/H99Q/C122S/R217T 1390 626
F30Y/R35Y/R36H/H37E/V38E/T39L/Y40H/V41W/Y60bE/T97aI/L97bA/H99Q/C122S/
1450 Y149R/M157K/Q192F 863
R37aS/V38D/V41R/D60aT/Y60bN/L97bG/H99Q/C122S 1450 805
V38E/T39W/V41T/D60aP/Y60bW/T97aI/L97bA/H99Q/C122S/Y149L/Y151G/Q192T
1480 770 V38D/L97bR/H99L/C122S/E175D/R217T/K224S 1570 808
V38D/L97bR/H99M/C122S/E175D/R217E 1570 424
R36S/V38D/L97bR/H99E/C122S/R217D/K224R 1580 432
R36H/V38D/V41R/A96D/D97G/T97adel/A98G/L97bdel/H99L/C122S/Y151F/Q192D/
1600 R217D 809 V38D/L97bR/H99M/C122S/E175D/R217E/K224R 1660 634
F30Y/R35W/R36H/H37E/V38E/T39V/Y40H/V41W/Y60bA/T97aI/L97bA/H99Q/C122S/
1760 Y149R/M157K/Q192A 197
I17L/F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 1790 632
F30Y/R35Y/R36H/H37P/V38E/T39R/Y40H/V41W/Y60bE/T97aI/L97bA/H99Q/C122S/
1900 M157K/Q192F 810 V38D/L97bR/C122S/E175D/R217E/K224R 1990 761
V38D/L97bR/H99E/C122S/E175D/R217S/K224S 2040 532
F30H/V38D/Y40H/V41Q/L97bA/H99Q/C122S/Q192H 2110 482
F30Y/V38D/Y40H/V41K/L97bA/H99Q/C122S/Y151R/M157R 2190 491
F30Y/A32E/Y34N/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 2210 422
A32S/V38D/H99E/C122S/K224A 2230 493
F30Y/V38D/V41R/L97bA/H99Q/C122S/M157K 2240 762
V38D/L97bR/H99V/C122S/E175D/R217E/K224N 2240 533
F30Q/V38D/Y40L/V41R/L97bA/H99Q/C122S/Y151H/M157R/Q192Y 2300 179
F30H/V38D/V41R/L97bV/H99Q/C122S/Y151F/M157K 2390 772
V38D/L97bR/H99V/C122S/E175D/R217E/K224S 2490 756
V38D/L97bR/H99E/C122S/Y172F/E175D/R217E 2720 154
V38D/T97adel/L97bdel/C122S 2950 180
F30H/V38D/V41R/L97bV/H99Q/C122S/Y149N/Y151F/M157K 3230 765
V38D/L97bG/H99Q/C122S/E175D/R217K/K224A 3310 5 C122S 3380 192
F30Y/R35Q/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217G 3860
461 R36H/V38D/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/V213A/R217D
4030 182 F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K/R217G 4210 489
F30Y/A32E/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 4280 172
F30Y/V38D/V41R/L97bV/H99Q/C122S/Y151F/M157K 5890 178
F30Y/V38D/V41R/L97bV/H99Q/C122S/M157K 6030 181
F30Y/V38D/V41R/L97bV/H99Q/C122S/Y149N/Y151F/M157K 7110 530
F30H/V38D/L97bA/H99Q/C122S/Y151L/M157S/Q192L 7340 194
F30Y/H37D/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217G 7760
755 V38D/L97bR/H99E/C122S/E175D/R217A/K224S 7770 758
V38D/L97bR/H99E/C122S/E175D/R217A/K224Q 7770 775
F30Y/V38D/Y40H/V41K/L97bA/H99Q/C122S/Y151K/M157R/Q192E 7770 776
F30Y/V38D/Y40H/V41K/L97bA/H99Q/C122S/Y151K/M157S/Q192E 7770 777
F30Y/V38D/Y40H/V41K/L97bA/H99Q/C122S/Y151K/Q192E 7770 778
F30M/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151G/Q192T 7770 779
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151M/M157R/Q192T 7770 780
F30H/V38D/Y40P/V41K/L97bA/H99Q/C122S/Y151H/M157E/Q192A 7770 296
V38D/L97bR/H99E/C122S/E175D 7770 804
V38E/T39W/V41T/D60aP/Y60bQ/T97aE/L97bA/H99Q/C122S/Y149V/Y151G/Q192T
7770 303 V38D/H99Q/C122S/R217Q 7770 304 V38D/L97bG/C122S/R217Q 7770
856 V38D/V41R/L97bT/C122S/Y151F/R217E 7770 858
V38D/V41R/L97bN/H99E/C122S/E175D/Q192M/R217E 7770 911
R36H/V38D/T39R/Y40F/V41R/H99S/C122S/R217T 7770 918
R36S/V38D/T39A/V41R/L97bV/H99S/C122S/R217T 7770 905
R36L/V38D/T39R/Y40L/V41T/L97bD/H99P/C122S/R217S 7770 913
R36S/V38D/T39K/Y40M/V41K/L97bI/C122S/R217E 7770 919
R37aH/V38D/T39R/Y40F/L97bT/C122S/R217E 7770 910
V38D/T39K/Y40F/V41Q/L97bI/H99S/C122S/R217T 7770 917
V38D/T39F/Y40L/V41K/L97bT/H99S/C122S/R217T 7770 480
F30Y/V38D/Y40L/V41R/L97bA/H99Q/C122S/Y151L/M157T/Q192L 9990 474
F30H/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151R/M157K 9990 472
F30H/V38D/V41R/L97bA/H99Q/C122S/Y151F/M157K/Q192W 9990 185
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149L/M157K/R217G 9990 188
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/Y149F/M157K/R217G 9990 266
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/Y60bQ/T97aE/L97bA/H99Q/
9990 C122S/Y149K/M157K/Q192P 781
R37aH/V38E/T39S/D60aR/Y60bG/T97aE/L97bA/H99Q/C122S/Y149T/Y151S 9990
782
R37aH/V38E/T39A/V41R/D60aG/Y60bG/T97aI/L97bA/H99Q/C122S/Y149V/Y151F/
9990 Q192R 783
R37aH/V38E/T39H/V41R/D60aT/Y60bT/T97aI/L97bA/H99Q/C122S/Y149M/Y151N/
9990 Q192T 784
R37aH/V38E/T39G/V41A/D60aG/Y60bE/T97aE/L97bA/H99Q/C122S/Y149R/Y151K/
9990 Q192T 785
V38E/T39D/V41A/D60aA/Y60bR/T97aE/L97bA/H99Q/C122S/Y149W/Y151K/Q192T
9990 786
V38E/T39A/V41R/D60aH/Y60bH/T97aI/L97bA/H99Q/C122S/Y149M/Y151N/Q192T
9990 787
V38E/T39F/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149A/Y151G/Q192T 9990
789 R37aH/V38E/V41A/Y60bS/T97aE/L97bA/H99Q/C122S/Y149A/Y151M/Q192T
9990 790
R37aH/V38E/T39S/V41A/D60aG/Y60bE/T97aE/L97bA/H99Q/C122S/Y149S/Y151H
9990 791
R37aP/V38E/T39G/V41R/Y60bR/T97aE/L97bA/H99Q/C122S/Y149T/Y151P/Q192T
9990 788
V38E/T39D/V41A/D60aS/Y60bR/T97aE/L97bA/H99Q/C122S/Y149W/Y151K/Q192T
9990 298 V38D/L97bR/H99E/C122S/E175D/K224R 9990 806
V38E/T39W/V41Q/Y60bE/T97aE/L97bA/H99Q/C122S/Y149I/Y151G/Q192T 9990
803 V38E/T39W/V41T/Y60bK/T97aE/L97bA/H99Q/C122S/Y149I/Y151G/Q192T
9990 857 V38D/V41K/L97bA/H99L/C122S/Y151R/R217E 9990 915
R36S/V38D/T39K/Y40M/V41K/L97bH/H99T/C122S/R217S 9990 641
R35D/H37R/V38D/T39V/V41T/Y60bT/L97bA/H99Q/C122S/Q192A 16600 5 C122S
NA 154 V38D/T97a_L97bdel/C122S NA 155 L73R/L97bG/H99Q/C122S NA 409
V38D/V41T/A96E/D97E/T97aG/A98_H99del/C122S/E175K/R217H NA 407
V38D/V41S/A96E/D97E/T97aG/A98_H99del/C122S/E175K NA NA
V38D/V41S/D97E/A96_-nulldelinsVG/A98_H99del/C122S/E175K/R217H NA
408 V38D/V41A/A96_T97adelinsERG/A98_H99del/C122S/E175N NA NA
V38D/V41L/A96_A98del/-null_H100insRGL/C122S/Y172E/E175P NA 406
V38D/V41S/A96D/D97E/T97aG/A98_H99del/C122S/E175S NA NA
V38D/V41A/A96del/-nulldelinsRG/A98_H99del/C122S/E175N/R217delinsF
NA 405 V38D/V41S/A96_T97adelinsERG/A98_H99del/C122S/E175A NA 156
V38D/L73R/L97bG/H99Q/C122S NA NA
V38D/A96_-nulldelinsVG/D97E/A98_H99del/C122S NA NA
V38D/V41Q/A96_-nulldelinsAG/D97E/A98_H99del/C122S/R217K/K224R NA NA
R35S/V38_Y40delinsDRF/D60adelinsY/A96del/- NA
nulldelinsLK/A98_H99del/C122S/Y151L NA
R35T/V38_V41delinsDRF/V41M/D60aP/A96del/- NA
nulldelinsLK/A98_H99del/C122S/Y151L NA
R35K/V38_-nulldelinsDQHR/A96del/-nulldelinsLK/A98_H99del/C122S/Y151W
NA 410
R35K/V38D/T39S/Y40F/V41M/D60aN/A96D/D97L/A98K/T97adel/H99L/L97bdel/
NA C122S/Y151L NA R35K/V38D/T39S/Y40F/V41M/D60aN/A96del/- NA
nulldelinsLK/A98_H99del/C122S/Y151L NA
R35S/V38E/T39S/Y40H/V41R/A96del/-nulldelinsLK/A98_H99del/C122S/Y151W
NA NA
R35K/V38D/T39K/Y40F/A96del/-nulldelinsLK/A98_H99del/C122S/Y151delinsG
NA 411 V38D/V41R/A96E/D97G/T97adelinsSG/A98_H99del/C122S NA 412
V38D/L97bG/H99P/C122S/R217V/K224Q NA 412
V38D/L97bG/H99P/C122S/R217V/K224Q NA 413
V38D/A96G/D97R/A98G/T97adel/H99I/L97bdel/C122S NA NA
V38D/A96_-nulldelinsRGI/A98G/C122S NA 414
V38D/A96E/D97E/T97aG/A98_H99del/L97bM/C122S NA NA
V38D/V41R/A96del/-null_T97ainsG/-null_L97binsG/A98_H99del/C122S/R217A
NA 415 V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/R217F NA
NA V38D/V41R/-nulldelinsDG/D97N/T97aG/A98_H99del/C122S/R217delinsF
NA NA V38D/V41R/A96E/- NA
null_L97binsG/D97E/A98_H99del/C122S/E175K/R217E/K224S 416
V38D/V41Q/A96E/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217H NA 416
V38D/V41Q/A96E/D97E/T97aG/A98_H99del/C122S/R217H NA NA
V38D/V41S/A96del/-nulldelinsRG/A98_H99del/C122S/R217delinsY NA NA
V38D/V41R/A96del/-nulldelinsGG/A98_H99del/C122S/R217D NA 418
V38D/V41K/A96D/D97E/T97aG/A98_H99del/C122S/R217K/K224N NA 419
R36H/V38D/V41M/D97E/T97aG/A98_H99del/C122S/R217D NA 419
R36H/V38D/V41M/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217D NA 420
V38D/V41H/A96D/D97E/T97aG/A98_H99del/C122S/R217delinsF NA 421
V38D/D97E/T97aG/A98_H99del/C122S/E175G/R217H NA NA
V38D/A96P/D97_-nulldelinsVG/A98_H99del/C122S NA 157
L97bP/H99L/C122S NA NA -null_H99delinsRVG/L97bM/C122S NA NA
V41R/A96del/-null_T97ainsG/-null_L97binsG/A98_H99del/C122S NA NA
-null_H99delinsRG/D97G/L97bM/C122S/R217E/K224R NA 423
V38D/L97bA/H99Q/C122S NA 425
V38D/V41Q/A96N/D97G/T97aI/A98G/L97bdel/H99L/C122S NA 427
V38D/V41K/A96K/D97E/A98G/T97adel/H99L/L97bdel/C122S NA 426
R35S/V38D/V41T/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S NA 428
R36H/V38D/V41S/A96E/D97R/A98G/T97adel/H99L/L97bdel/C122S/K224R NA
158
R37aP/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/M157K/R217F
NA 159
R37aS/G37bD/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/R217F
NA 160
R36H/V38D/V41R/A96D/D97G/A98R/T97adel/H99L/L97bdel/C122S/M157K/R217D
NA 161
R37aH/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/S174P/R217D
NA 162
R37aH/V38D/V41G/A96G/D97E/T97aA/A98G/L97bdel/H99M/C122S/Y151N NA
163 R36S/V38D/Y40L/V41R/A96P/D97V/T97aR/A98G/L97bdel/H99L/C122S NA
165 V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Q192E/R217D
NA 166
F30Y/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/M157K/R217D
NA 431
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D
NA 433
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151W/Q192M/R217D
NA 437
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151N/Q192M/R217D
NA 439
R36H/V38D/Y40H/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192V/
NA R217D 440
R36H/V38D/Y40L/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151R/Q192L/
NA R217D 441
R36H/V38D/Y40V/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151F/Q192F/
NA R217D 442
R36H/V38D/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D NA
443
R36H/V38D/Y40L/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/
NA R217D 449
R36H/V38D/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D
NA 453
R36H/V38D/Y40F/V41K/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151R/Q192E/
NA R217D 454
R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151N/Q192V/
NA R217D 455
R36H/V38D/Y40L/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151N/Q192D/
NA R217D 456
R36H/V38D/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/R217D
NA 457
R36H/V38D/Y40F/V41R/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151K/Q192E/
NA R217D 458
R36H/V38D/Y40I/V41A/D97E/A98G/T97adel/H99L/L97bdel/C122S/Y151M/Q192L/
NA R217D 459
R36H/V38D/Y40H/V41M/D97E/A98G/T97adel/H99L/L97bdel/C122S/R217D NA
464
R36H/V38D/V41R/A96D/D97G/T97aN/A98G/L97bdel/H99L/C122S/G193R/R217F
NA 467
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Q192R/R217D NA
468
F21S/R36H/V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/E175D/
NA R217D 470
R36H/V38D/V41R/A96D/D97E/A98G/T97adel/H99L/L97bdel/C122S/G193R/R217D
NA 168 V38D/Y40P/V41K/L97bA/H99Q/C122S/M157K NA 430
V38D/V41R/A96D/D97G/A98G/T97adel/H99L/L97bdel/C122S/Y151M/Q192E/R217D
NA 512
F30H/R35T/H37M/V38D/V41R/D60aS/Y60bT/L97bA/H99Q/A112V/C122S/Y151L/
NA M157K/R217T 220 V38D/V41R/L97bA/H99Q/C122S NA 531
F30N/V38D/Y40F/V41R/L97bA/H99Q/C122S/Y151L/M157S/Q192H NA 257
F30Y/R35Y/R36H/H37E/V38E/T39S/Y40H/V41W/Y60bW/T97aI/L97bA/H99Q/C122S/
NA Y149R/M157K/Q192F 306 V38D/L97bG/H99Q/C122S/S195A/R217Q NA 342
V38D/C122S/S190H/G216A NA *SEQ ID the of an exemplary protease
domain containing the replacements
Example 3
Anti-C3 Activity of u-PA Variants in Cynomolgus Monkey Plasma
[0755] The ex vivo anti-C3 activity of some modified u-PA
polypeptides was measured in purchased cynomolgus monkey plasma
(BioChemed). The median effective dose (ED.sub.50) of modified u-PA
polypeptides for cleaving C3 was calculated using ELISA. Briefly,
exemplary modified u-PA polypeptides were serially diluted 1:1.5
fold from 1000 nM to 39.0 nM (9 point dilution). The hC3 standard
was serially diluted 1:1.5 fold from 450 to 39 (7 point dilution)
in 1% BSA in 1.times.PBST (Phosphate Buffered Saline Tween-20) A
recipe for 1.times.PBST includes:
[0756] 1. Dissolve the following in 800 ml of distilled H.sub.2O
[0757] 8 g of NaCl [0758] 0.2 g of KCl [0759] 1.44 g of
Na.sub.2HPO.sub.4 [0760] 0.24 g of KH.sub.2PO.sub.4 [0761] 2 ml of
tween-20
[0762] 2. Adjust pH to 7.2
[0763] 3. Adjust volume to 1 L with additional distilled
H.sub.2O
[0764] 4. Sterilize by autoclaving.
[0765] 80% Cynomolgus vitreous plasma (obtained from BioChemed) in
buffer containing 50 mM Tris, pH 8.0, 50 mM NaCl, and 0.01%
Tween-20, and dilutions of C3, was incubated with modified u-PA
polypeptides at a final concentration of 0.1 .mu.M at 37.degree. C.
for 10 minutes. The 80% plasma digests were diluted 1:15,625 in 1%
BSA in 1.times.PBST using the Biomek liquid handling system
(Beckman Coulter). Flat bottom EIA plates (Bio-Rad) coated with
purified A213 antibody (anti-C4; Quidel specialty products) at 2.0
.mu.g/mL, were incubated with 50 .mu.L/well of sample or standard
and detected with Anti-hC3a antibody (ab11872; Abcam) in 1% BSA in
1.times.PBST. The wells were further coated with HRP conjugate Goat
anti Mouse-HRP antibody (Jackson ImmunoResearch; catalog number:
115-035-003) at a 1:30,000 dilution in 1% BSA in 1.times.PBST and
developed with WesternBright ECL (chemiluminescent) HRP substrate
for detection.
[0766] The ED.sub.50 is defined as the concentration of protease
that produces a 50% loss of C3. The results show an increased loss
(i.e., cleavage) of C3 in the presence of the modified u-PA
polypeptides with the sequence set forth in SEQ ID NOs: 8-14 and
16-20. The u-PA polypeptide protease domain with the sequence set
forth in SEQ ID NO: 8, cleaved with an ED.sub.50 that was several
fold higher than the others. The results are set forth in Table 15
below.
TABLE-US-00022 TABLE 15 anti-C3 Activity in cynomolgus monkey
plasma ED.sub.50 80% SEQ cynomolgus ID plasma Chymotrypsin
numbering NO* (10 min, nM) F30Y/V38D/Y40H/V41R/L97bA/H99Q/ 8 1700
C122S/M157K F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/ 9 176
V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/ Y149K/M157KF30Y/R35W/R36H/ 10
114 H37D/V38E/T39Y/Y40F/ V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K
R35W/R36H/H37N/V38E/T39F/Y40F/V41R/T97aI/ 11 309
L97bA/H99Q/C122S/Y149R/M157K/Q192H
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/ 12 286
T97aI/L97bA/H99Q/C122S/Y149R/M157K
F30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/ 13 222
Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157K
F30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/ 14 145
Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157K
F30Y/R35Q/R36H/H37G/R37aE/V38E/ 16 481
T39F/Y40F/V41R/D60aP/Y60bS/T97aI/ L97bA/H99Q/C122S/Y149R/M157K
F30Y/R35Y/R36H/H37P/R37aQ/V38E/ 17 215
T39Y/Y40F/V41R/Y60bH/T97aI/L97bA/ H99Q/C122S/Y149R/M157K
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/ 18 244
Y60bT/T97aI/L97bA/H99Q/C122S/Y149R
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/ 19 214
Y60bL/T97aI/L97bA/H99Q/C122S/Y149R
R35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/ 20 175
T97aI/L97bA/H99Q/C122S/Y149R *SEQ ID the of exemplary protease
domain containing the replacements
Example 4
Anti-C3 Activity of Modified u-PA Polypeptides in Cynomolgus Monkey
Vitreous Humor
[0767] The activity of modified u-PA polypeptides was assessed by
cleavage of the substrate complement protein human C3. 2 .mu.M
purified human C3 (Complement Technologies; Tyler, Tex.) was
incubated with the modified u-PA polypeptides (0-250 nM) for 1 hour
at 37.degree. C. in purchased monkey vitreous humor (BioChemed).
The activity of the modified u-PA polypeptides was then quenched by
the addition of the urokinase inhibitor Glu-Gly-Arg Chlormethyl
Ketone (EGR-CMK; Haematologic Technologies, EGRCK-01) to a final
concentration of 10 .mu.M and the hC3/modified u-PA polypeptide
mixture was allowed to stand for 30 minutes at ambient temperature.
Residual levels of undigested human C3 were quantified using an
ELISA. All modified u-PA polypeptides, including those that contain
the replacements
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R,
Y40Q/V41L/L97bA/C122S and Y40Q/V41L/L97bA/C122S, and
Y40Q/V41R/L97bA/C122S, cleaved complement protein C3 with a higher
turnover number (per hour) than the reference u-PA polypeptide
containing the C122S replacement set forth in SEQ ID NO: 5. The
results are set forth in Table 16 below.
TABLE-US-00023 TABLE16 C3 cleavage in vitreous humor C3 SEQ
Turnover ID Number Chymotrypsin numbering NO* (hr.sup.-1) u-P wild
type u-PA with C122S 5 0.3
V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122SR35Q/ 15 29
H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/ T97aI/L97bA/H99Q/C122S/Y149R
21 48 Y40Q/V41L/L97bA/C122S 40 8 Y40Q/V41L/Y60bL/L97bA/H99Q/C122S
34 42 V38E/Y40Q/Y60bL/L97bA/H99Q/C122S 35 17
V38E/Y40Q/V41L/L97bA/H99Q/C122S 36 56
V38E/Y40Q/V41L/Y60bL/H99Q/C122S 37 18
V38E/Y40Q/V41L/Y60bL/L97bA/C122S 38 12 R37aS/V41R/L97bG/H99Q/C122S
41 5 T39Y/V41R/L97bA/H99Q/C122S 42 17
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S 43 26
T39Y/V41R/D60aP/L97bA/H99Q/C122S 44 17 *SEQ ID the of protease
domain containing the replacements
Example 5
Ex Vivo Stability of Modified u-PA Polypeptides in Cynomolgus
Monkey Vitreous Humor
[0768] The ex vivo stability of modified u-PA polypeptides was
assessed in purchased cynomolgus monkey vitreous humor or Phosphate
Buffered Saline (PBS) control. Modified u-PA polypeptides that
exhibit stability in vitreous humor can be used for treatment of
AMD.
[0769] 80% Cynomolgus vitreous humor (obtained from BioChemed;
Catalog Nos. BC7615-V1, BC60815-V1, BC33115-V6) in buffer
containing 50 mM Tris, pH 8.0, 50 mM NaCl, and 0.01% Tween-20 or
PBS control was incubated with modified u-PA polypeptides at a
final concentration of 0.1 .mu.M. These mixtures were incubated at
37.degree. C. for 7 days, and the residual protease activity was
assayed with 100 .mu.M fluorogenic substrate AGR-ACC
(7-amino-4-carbamoylmethyl-coumarin) in 50 mM Tris, pH 8.0, 50 mM
NaCl, 0.01% Tween-20 (assay results were assessed at excitation
wavelength=380 nm and emission wavelength=460 nm). The results show
that the modified u-PA polypeptides with the sequence set forth in
SEQ ID NOs: 21-33 exhibit comparable activity in cynomolgus plasma
and PBS. The results are set forth in Table 17 below.
TABLE-US-00024 TABLE17 Stability of Modified u-PA polypeptides in
vitreous humor SEQ Activity (%) ID on Day 7 Chymotrypsin numbering
NO* vitreous PBS wild type u-PA protease domain with C122S 5 102
111 R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 21 83 94
Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/ 22 73 79
T97aI/L97bA/H99Q/C122S/Y149R R35Q/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/
23 88 92 T97aI/L97bA/H99Q/C122S/Y149R
R35Q/H37Y/V38E/T39Y/V41R/D60aP/Y60bQ/ 24 87 99
T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/T39Y/V41R/D60aP/Y60bQ/
25 105 103 T97aI/L97bA/H99Q/C122S/Y149R
R35Q/H37Y/R37aE/V38E/V41R/D60aP/Y60bQ/ 26 93 108
T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/D60aP/Y60bQ/
27 88 100 T97aI/L97bA/H99Q/C122S/Y149R
R35Q/H37Y/R37aE/V38E/T39Y/V41R/Y60bQ/ 28 93 97
T97aI/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/
29 58 61 T97aI/L97bA/H99Q/C122S/Y149R
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 30 86 92
Y60bQ/L97bA/H99Q/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/
31 90 111 Y60bQ/T97aI/H99Q/C122S/Y149R
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 32 89 108
Y60bQ/T97aI/L97bA/C122S/Y149R R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/
33 74 99 Y60bQ/T97aI/L97bA/H99Q/C122S *SEQ ID the of exemplary
protease domain containing the replacements
[0770] The ex vivo stability of the anti-C3 u-PA variants in Table
18 below was assessed after incubation in purchased cynomolgus
monkey vitreous humor for both 7 and 28 days. The results show that
several of the variants maintain significant activity even after
the 28 day incubation. The results are set forth in Table 18
below.
TABLE-US-00025 Table 18 Stability of Modified u-PA polypeptides in
vitreous humor SEQ ID Activity (%) Chymotrypsin numbering NO* Day 7
Day 28 wild type u-PA with C122S 5 106 90
V38E/Y40Q/V41L/Y60bL/L97bA/H99Q/C122S 15 43 n/d
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/ 21 83 34
Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R Y40Q/V41L/Y60bL/L97bA/H99Q/C122S
34 42 n/d V38E/Y40Q/Y60bL/L97bA/H99Q/C122S 35 28 n/d
V38E/Y40Q/V41L/L97bA/H99Q/C122S 36 17 n/d
V38E/Y40Q/V41L/Y60bL/H99Q/C122S 37 73 n/d
V38E/Y40Q/V41L/Y60bL/L97bA/C122S 38 71 n/d Y40Q/V41L/L97bA/C122S 40
56 28 R37aS/V41R/L97bG/H99Q/C122S 41 100 74
T39Y/V41R/L97bA/H99Q/C122S 42 92 61
T39Y/V41R/Y60bQ/L97bA/H99Q/C122S 43 98 58
T39Y/V41R/D60aP/L97bA/H99Q/C122S 44 86 42 *SEQ ID the of protease
domain containing the replacements
Example 6
Ex Vivo Pharmacodynamic Assay in Human Plasma
[0771] Modified u-PA polypeptides (protease domains) were incubated
with 80% human plasma prior to addition of erythrocytes to assess
cleavage of complement protein C3 in a hemolytic assay of
complement activity. Performing functional assays in the presence
of human plasma tests the activity of the anti-C3 proteases in a
pharmaceutically relevant environment and, for example, examines
whether they are sensitive to inactivation by serpins or other
protease inhibitors present in human blood. The modified u-PA
polypeptides provided herein were not inhibited, in general, in the
presence of human plasma. This is of significance for treatment of
diseases and disorders and conditions, such as DGF, in which the
administered modified u-PA polypeptides are exposed to human
plasma, such as when administered intravenously. It is of lesser or
no importance for applications, such as treatment of AMD by
intravitreal or intraretinal or subretinal injection, where the
modified u-PA polypeptides are not exposed to plasma.
[0772] An ED.sub.50 value, which is the concentration of protease
at which 50% inhibition of complement activity is achieved, was
measured. The wild-type u-PA protease domain (SEQ ID NO:5, with
C122S), and various exemplary modified u-PA protease domains were
serially diluted from 3 .mu.M to 0.11 .mu.M (9 point serial
dilution 1:2) to measure the ED.sub.50. The wild-type u-PA (SEQ ID
NO:5) protease domain and modified u-PA protease domains were
preincubated with a final concentration of 80% plasma in an 0.2 mL
tube by combining 4 .mu.L of the diluted protease solution and 16
.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 0.6 .mu.M to 0.0022
.mu.M protease for the EC.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.
[0773] 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.
[0774] 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
ED.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).
[0775] The results are shown in Table 19 below, which sets forth
the ED.sub.50 (nM) for hemolysis in 80% human plasma by wild type
u-PA with the C122S mutation set forth in SEQ ID NO: 5 and the
modified u-PA polypeptides. As shown in Table 19, the ED.sub.50 for
wild type u-PA in 80% human plasma is greater than 6 .mu.M; whereas
exemplary modified u-PA protease domain polypeptides have
significantly increased ability to inhibit complement as indicated
by a lower ED.sub.50 (e.g., between 173 nM and 1.028 .mu.M).
TABLE-US-00026 TABLE 19 C3 inhibition in human plasma ED50 80% SEQ
human ID plasma Chymotrypsin numbering NO.* (60 min, nM) wild type
u-PA (protease domain) with C122S 5 >6000
F30Y/V38D/Y40H/V41R/L97bA/H99Q/ 8 1028 C122S/M157K
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/ 9 257
Y60bQ/T97aE/L97bA/H99Q/C122S/Y149K/M157K
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/ 10 195
T97aI/L97bA/H99Q/C122S/Y149R/M157K
R35W/R36H/H37N/V38E/T39F/Y40F/V41R/T97aI/ 11 208
L97bA/H99Q/C122S/Y149R/M157K/Q192H
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/ 12 227
T97aI/L97bA/H99Q/C122S/Y149R/M157K
F30Y/R35W/R36H/H37N/V38E/T39Y/Y40F/V41R/ 13 220
Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157K
F30Y/R35W/R36H/H37P/V38E/T39Y/Y40F/V41R/ 14 185
Y60bS/T97aE/L97bA/H99Q/C122S/Y149K/M157K
F30Y/R35Q/R36H/H37G/R37aE/V38E/T39F/ 16 220
Y40F/V41R/D60aP/Y60bS/T97aI/L97bA/ H99Q/C122S/Y149R/M157K
F30Y/R35Y/R36H/H37P/R37aQ/V38E/T39Y/ 17 173
Y40F/V41R/Y60bH/T97aI/L97bA/H99Q/ C122S/Y149R/M157K
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/ 18 253
Y60bT/T97aI/L97bA/H99Q/C122S/Y149R
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/ 19 318
Y60bL/T97aI/L97bA/H99Q/C122S/Y149R
R35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/ 20 196
T97aI/L97bA/H99Q/C122S/Y149R *SEQ ID the of exemplary protease
domain containing the rep acements
Example 7
Kinetic Analysis of Plasminogen Activation Using an Indirect
Chromogenic Assay
[0776] An indirect chromogenic assay was performed to determine the
activities of the wild-type and modified u-PA polypeptides produced
as purified protein preparations (see, Madison et al. (1989)
Nature, 339: 721-724; Madison et al. (1990) J Biol. Chem., 265:
21423-21426). In this assay, free p-nitroaniline is released from
the chromogenic substrate Spectrozyme PL
(H-D-norleucylhexahydrotyrosyl-lysine-p-nitroanilide diacetate
salt, American Diagnostics, Inc.) by the action of plasmin
generated by the action of u-PA on plasminogen. The release of free
p-nitroaniline was measured spectrophotometrically at OD.sub.405
nm.
[0777] For the assay, 100 .mu.L reaction mixtures containing 0.25-1
ng of the u-PA enzymes to be tested, 0.62 mM Spectrozyme PL, and
0.2 .mu.M Lys-plasminogen (American Diagnostics, Inc.), were
combined in a buffer containing 50 mM Tris-HCL (pH 7.5), 0.1 M
NaCl, 1.0 mM EDTA and 0.01% (v/v) Tween 80. The reaction was
incubated at 37.degree. C. in 96-well, flat-bottomed microtiter
plates (Costar, Inc.) and the optical density at 405 nm
(OD.sub.405) was read every 30 s for 1 hour in a Molecular Devices
Thermomax. The kinetic constants k.sub.cat, K.sub.m, and
k.sub.cat/K.sub.m (specificity constant) were calculated (see,
e.g., Madison, E. L (1989) Nature 339: 721-724).
[0778] The results are set forth in Table 20 below. The results
show that the modified u-PA polypeptides have significantly
decreased enzymatic activity for the substrate plasminogen. All of
modified u-PA polypeptides provided herein have reduced activity on
and specificity for plasminogen; and all have many-fold increases
in specificity and activity on C3, and for inhibiting complement
activation compared to the unmodified u-PA.
TABLE-US-00027 TABLE 20 Kinetic Analysis of Plasminogen Activation
SEQ ID k.sub.cat/K.sub.m Chymotrypsin numbering NO.*
(M.sup.-1s.sup.-1) wild type u-PA with C122S 5 1.54E+04
F30Y/V38D/Y40H/V41R/L97bA/H99Q/C122S/M157K 8 4.03E+02
F30Y/R35W/R36H/H37E/V38E/T39W/Y40H/V41R/ 9 <1.0E+01
Y60bQ/T97aE/L97bA/H99Q/C122S/Y149K/M157K
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/ 12 <1.0E+01
T97aI/L97bA/H99Q/C122S/Y149R/M157K
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aT/ 18 <1.0E+01
Y60bT/T97aI/L97bA/H99Q/C122S/Y149R
R35W/H37D/R37aP/V38E/T39W/V41R/Y60bA/ 20 <1.0E+01
T97aI/L97bA/H99Q/C122S/Y149R F30Y/R35Y/R36H/H37P/R37aQ/V38E/ 17
<1.0E+01 T39Y/Y40F/V41R/Y60bH/T97aI/L97bA/
H99Q/C122S/Y149R/M157K *SEQ ID the of exemplary protease domain
containing the replacements
Example 8
Anti-C3 Activity and Stability of Anti-C3 u-PA Variant in
Cynomolgus Monkey Vitreous Humor In Vivo
[0779] The in vivo activity and stability of the modified u-PA
polypeptide set forth in SEQ ID NO:21, which is the protease domain
that contains the replacements
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
was assessed. Stability in vitreous humor and ability to cleave C3
are parameters indicative of a candidate for treatment of AMD.
[0780] Twelve naive cynomolgus monkeys were assigned to a single
treatment group. Study animals were intravitreally administered a
single dose 125 .mu.g of modified u-PA polypeptide in one eye. The
isolated protease domain whose sequence is set forth in SEQ ID
NO:21, which has a molecular weight of approximately 25 kDa, was
administered. The right eye received the test article and the left
eye was injected with vehicle control. Four animals were sacrificed
at each of the following time points: 24 hours post-dose, day 2 and
on day 6. Vitreous humor samples were collected from the right and
left eyes and analyzed for modified u-PA polypeptide stability and
level of C3 after treatment with modified u-PA polypeptide or
vehicle control; C3 and modified u-PA polypeptide concentration
were determined by ELISA as detailed above.
[0781] The concentration of the modified u-PA polypeptide present
in vitreous humor samples obtained 24 hours post-dose, on day 2 and
on day 6 was determined by ELISA. The activity of the modified u-PA
polypeptides was then quenched by the addition of EGR-CMK
(Haematologic Technologies, EGRCK-01) to a final concentration of
10 .mu.M and the hC3/modified u-PA polypeptide mixture was allowed
to stand for 30 minutes at ambient temperature.
[0782] The half-life of modified u-PA polypeptide of SEQ ID NO:21
was determined to be approximately 2 days, which should correspond
to approximately 5 days in a human system (Deng et al. MAbs 3(1):
61-66 (2011)). In vivo recovery (i.e., the peak level of modified
u-PA polypeptide divided by the dose of modified u-PA polypeptide)
of the modified u-PA polypeptide (of SEQ ID NO:21) was calculated
by ELISA from the observed maximum level of modified u-PA
polypeptide. The theoretical predicted value for 100% in vivo
recovery was 2.5 .mu.M. The measured in vivo recovery of modified
u-PA protease domain (SEQ ID NO: 21) containing the replacements:
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
was calculated to be approximately 80% of the predicted value, or
approximately 2.0 .mu.M.
[0783] C3 levels in vitreous humor were assessed by ELISA as
detailed in Example 3. C3 levels in vehicle-injected negative
control eye ranged between 0.4 nM-50 nM (2 samples from
vehicle-injected eyes differed significantly from the other 10,
likely due to blood contamination of the vitreous). The baseline
level of C3 prior to u-PA administration was approximately 2.2 nM.
C3 was undetectable in variant-treated eye after 1 day and 7 days.
After 28 days, C3 concentration in the eye treated with the
modified u-PA polypeptide with the sequence set forth in SEQ ID NO:
21 was approximately 2.2 nM, which is equivalent to
before-treatment levels. Thus, modified u-PA polypeptides provided
herein are candidates for treatment of AMD.
Example 9
Exemplary Mutations in u-PA and Confirmation of Cleavage Sites
[0784] Exemplary positions and mutations in u-PA polypeptides,
including the full-length, precursor and protease domains and
catalytically active portions thereof are set forth in Table 22
(below).
TABLE-US-00028 TABLE 22 Exemplary mutations in u-PA Mutation Chymo
Mature in the poly- Conser- numb- numb- peptide of Exemplary vative
to ering ering wt SEQ ID NO: 21 mutations Mutations 30 173 F Y, W,
F 35 178 R Q Q, W, Y Y, W, F, N 36 179 R H N, Q 37 180 H Y Y, E, P,
D, E, Q, D, N, G, H, P, R, Q, K, Y E, W, F 37a 181 R E E, P, Q, N
D, Q, H 38 185 V E E 39 186 T Y W, Y, F M, L 40 187 Y Q, F M, L, Y,
N, Q 41 188 V R R, L K 60a 208 D P P S 60b 209 Y Q L, Q, S, N, T,
G, S, A, Y, T I, V, Q 97a 249 T I E, I D, L, V 97b 250 L A A, G G,
S 99 252 H Q Q N 149 306 Y R K, R Q, E 157 314 M K R, Q, E 192 353
Q H N, Q
[0785] The replacements are in any form of u-PA, including the
protease domain (SEQ ID NO: 2 or 5); the full length (SEQ ID NO: 1
or 4) and mature form (SEQ ID NO: 3 or 6). The replacements can be
combined, including as exemplified herein, including up to as many
as 15-18 or more replacements.
[0786] The data show that the modified u-pA polypeptides with these
mutations, cleave and inactivate C3 in multiple species such as
human and cynomolgus monkey. Cleavage of human C3 can be between
residues 740 and 741 (SEQ ID NO:47), and this cleavage inactivates
C3:
TABLE-US-00029 Q H A R .dwnarw. A S H L 737-744 P4 P3 P1 .dwnarw.
P1' P4'.
[0787] As demonstrated above, and throughout the disclosure, the
modified u-PA polypeptides cleave and inactivate C3. The modified
u-PA polypeptides were selected for cleavage in this region, and it
was confirmed by testing them. For example, C3 was incubated with
either modified u-PA protease domain (SEQ ID NO: 21) containing the
replacements:
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
or with the modified u-PA protease domain (SEQ ID NO:40) with the
replacements Y40Q/V41L/L97bA/C122S at enzyme to substrate ratios of
1:10 or 1:50 for a total of one hour. Samples were removed from
these reactions at 0, 5, 10, 20, 40, and 60 minutes, and the
cleavage reaction was terminated immediately in each sample by
addition of TFA and flash freezing in dry ice. Prior to further
analysis of these samples, cysteine side chains were reduced and
alkylated, the e-amino group of lysine side chains was blocked by
treatment with O-methylisourea, and peptide amino termini were then
labeled with NHS-SS-biotin. The resulting biotinylated C3 peptides
were captured and further digested with trypsin and GluC protease.
Following this second protease digestion, peptide products were
once again affinity captured. Biotin was then removed from the
captured peptides by reduction, and the peptide mixture was
analyzed by LC-MS/MS. At each time point after 0 minutes a fragment
of MW 8289 was observed, indicating cleavage at the arginine in the
QHAR site in C3. No additional C3 cleavage sites were observed in
these reactions. Hence the modified u-PA polypeptides provided
herein cleave at the QHAR.
TABLE-US-00030 Q H A R .dwnarw. A S H L 737-744 P4 P3 P2 P1.dwnarw.
P1' P4'.
Example 10
u-PA Toxicity in Cynomolgus Monkey Vitreous Humor
[0788] Safety and tolerability of modified u-PA polypeptides were
assessed in vivo in cynomolgus monkeys. Three naive cynomolgus
monkeys were assigned to each of three treatment groups. Study
animals were intravitreally administered either 12.5 .mu.g, 37.5
.mu.g or 125 .mu.g per eye, of each modified u-PA polypeptide. The
right eye received the test polypeptide and the left eye was
injected with vehicle control. Animals were clinically observed
(i.e., food consumption) and ophthalmic examinations were
conducted. Ophthalmic examination included slit-lamp biomicroscopy
and indirect ophthalmoscope observations, followed by color fundus
photography or optical coherence tomography (OCT) prior to dosing
(T=0) and on days 2, 8 and 15 post-dosing. All observations
continued for up to 4 weeks or until resolution.
[0789] The no-observed-adverse-effect-level (NOAEL) was assessed
for all animals. The NOAEL for animals administered a modified u-PA
polypeptide with the sequence set forth in SEQ ID NO: 42 was
.gtoreq.37.5 .mu.g. No adverse effects were noted for animals
administered a modified u-PA polypeptide with the sequence set
forth in SEQ ID NO: 21; therefore, the NOAEL for animals
administered a modified u-PA polypeptide with the sequence set
forth in SEQ ID NO: 21 was .gtoreq.125 .mu.g (equivalent to
.gtoreq.375 .mu.g/eye in man).
Example 11
[0790] Calculations were performed to identify candidate
immunogenic hotspots in the wild-type u-PA, a C122S variant of
wild-type u-PA (SEQ ID NO:5) protease domain and exemplary u-PA
variant protease domains to confirm that the mutations in the
exemplary variants did not introduce any immunogenic hotspots.
Calculations also were performed to compare the overall profile of
hotspots for the variants of interest to a panel of comparison
proteins.
[0791] Overview of Methods
[0792] An important step in the T-cell response to foreign proteins
is the binding and presentation of constituent peptides (derived
from the cleavage of the foreign protein) to any of a host of HLA
complexes expressed in the antigen presenting cells. The
identification of peptides, derived from a protein, that are
predicted to bind to known HLAs can be used as an indication of
possible hotspots in the sequence for eliciting a T-cell response.
When considering a variant of a protein with known immunogenic
properties, comparing the profile of predicted HLA-binders can help
to identify whether the changes introduced any new immunogenic
hotspots.
[0793] The profiles can be generated using publically available
databases. For example, the publically available binding prediction
service (NetMHCII 2.2server), Technical University of Denmark, was
used to predict binding to HLAs. The NetMHCII 2.3 server predicts
binding of peptides to HLA-DR, HLA-DQ, HLA-DP and mouse MHC class
II alleles using artificial neuron networks. Predictions can be
obtained for 25 HLA-DR alleles, 20 HLA-DQ, 9 HLA-DP, and 7 mouse H2
class II alleles. The prediction values are given in nM IC.sub.50
values, and as a %-Rank to a set of 1,000,000 random natural
peptides. Strong and weak binding peptides are indicated in the
output.
[0794] The service was used to predict the binding affinity of all
possible peptides of 15 contiguous amino acids in exemplary
variants (SEQ ID NO:40, and SEQ ID NO:21), the wild-type protease
domain (SEQ ID NO:2), and wild-type protease domain with C122S (SEQ
ID NO:5) against a panel of 14 HLA-DR variants (see Table B, below)
based on the primary sequence of the peptide. For each peptide/HLA
pair, the NetMHCII server provides a predicted binding affinity, as
well as a classification as a tight binder (K.sub.d.ltoreq.500),
weak binder K.sub.d.ltoreq.50 nM), or non-binder (K.sub.d>500
nM). For a protein sequence containing N amino acids, this results
in a total of T=(N-14).times.14 possible peptide-HLA binding
pairs.
[0795] Using the binding predictions from the NetMHCII server, the
results were used to identify possible hotspots in these protein
sequences, based on areas where a number of candidate binding pairs
were identified. These changes were compared with the known changes
in the corresponding protein sequences.
[0796] For each protein, an overall binding score based on the
number of predicted tight-binding peptide-HLA pairs (TB), the
number of predicted weak-binding peptide-HLA pairs (WB), and the
total number of peptide-HLA pairs considered (T) as:
Score=(TB+0.5*WB)/T
TABLE-US-00031 TABLE B HLA-DR molecules included as possible
binders HLA-DRB1*1101 HLA-DRB1*0101 HLA-DRB1*0301 HLA-DRB1*0401
HLA-DRB1*0404 HLA-DRB1*0405 HLA-DRB1*0701 HLA-DRB1*0802
HLA-DRB1*0901 HLA-DRB1*1302 HLA-DRB1*1501 HLA-DRB3*0101
HLA-DRB4*0101 HLA-DRB5*0101
[0797] This score has shown good agreement with the in silico
immunogenicity predictions generated by the company EpiVax
(Providence, R.I.), which uses immunoinformatics and in vitro
techniques to predict immunogenicity, when using a subset of 8
HLAs. This subset of 8 HLAs was used. The same calculations were
performed on a panel of standard proteins to provide a comparison
set against which to compare the u-PA variants.
[0798] Impact of Mutations on Predicted HLA Binding Profiles
[0799] The number of times a given sequence position for any of the
variant u-PA protease domains were mapped for each-HLA complex at a
K.sub.d cutoff of 50 nM and 500 nM. The scale in each column is
fixed, but differs between columns and compared with the wild-type
polypeptides to assess whether the mutations altered the
immunogenic profile. No significant differences among the
polypeptides were observed.
[0800] Aggregate Immunogenicity Scores
[0801] To compute an aggregate immunogenicity score for each
sequence, the total number of peptide-HLA binding pairs at each
binding threshold across a panel of 8 HLAs (Table C) that
previously have been validated by EpiVax (Providence, R.I.) to
provide good agreement with published in silico immunogenicity
scores. EpiVax is a company that uses immunoinformatics and in
vitro techniques to predict immunogenicity. These values then were
used to compute an overall score as described in the methods
summary above.
TABLE-US-00032 TABLE C HLA-DR subset used for overall
immunogenicity score HLA-DRB1*0101 HLA-DRB1*0301 HLA-DRB1*0401
HLA-DRB1*0701 HLA-DRB1*0802 HLA-DRB1*1101 HLA-DRB1*1302
HLA-DRB1*1501
[0802] The number of peptide-HLA binding pairs for the subset of 8
HLAs and the composite scores for uPA are shown below:
TABLE-US-00033 Sequence ID # tight (TB) # weak (WB) Total possible
(T) Score 2 (WT) 98 526 1888 .191 5 (WTS) 109 542 1888 .201 40 107
528 1912 .194 21 96 514 1912 .185
[0803] The results indicate that the immunogenicity of the variants
should not be different from the wild-type polypeptides. The
results of the same analysis for a panel of comparator proteins is
shown below:
TABLE-US-00034 Sequence # tight (TB) # weak (WB) Total possible (T)
Score Follitropin-Beta 13 120 920 .079 Fibrinogen-Alpha 125 582
5040 .083 Insulin 32 107 768 .111 Albumin 192 743 4760 .118 Amylase
210 711 3976 .142 Thrombopoietin 266 609 2712 .210 Interferon-Beta
175 353 1384 .254 Interleukin-11 233 316 1480 .264
Example 12
Activation of Plasminogen by Wt- and Variant u-PA Polypeptides
[0804] The catalytic efficiency for activation of Glu-plasminogen
by wt- and anti-C3 variants of u-PA polypeptides provided herein
was tested as described below. Data from these experiments
demonstrated that the anti-C3 u-PA variant proteins displayed
significantly reduced activity towards plasminogen, a
physiologically relevant substrate of wild type u-PA. In
combination with data from Example 7, these data demonstrate that
the anti-C3 u-PA polypeptides display not only substantially
greater activity towards a "new" substrate, C3, but also
substantially reduced activity towards the normal physiological
substrate of wt u-PA.
[0805] Mutations introduced into the anti-C3 variants described in
this application, therefore, have dramatically altered substrate
specificity of the anti-C3 variant polypeptides compared with that
of wt-u-PA.
Plasminogen Activation by Wt- and Anti-C3 Variant u-PA
Polypeptides
1) Analysis Using a Single Time Point Reaction (37 C for 30
Minutes)
[0806] a) Reaction Conditions
[0807] Plasminogen activation activity was measured for wild type
u-PA/C122S (SEQ ID NO:5) as well as anti-C3 u-PA polypeptide
variants containing the mutations Y40Q/V41L/L97bA/C122S (SEQ ID
NO:40) and
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
(SEQ ID NO:21). Protease concentrations in the reaction mixture
were 25 nM (wt-u=PA) or 250 nM (anti-C3 u-PA variant) and substrate
(i. e, Glu-plasminogen) concentration was 2.5 uM. The reaction
volume was 20 uL and the reaction proceeded for 30 minutes at
37.degree. C. Each reaction was terminated by adding of 2 uL of 1 M
DTT and 7 uL of 4.times.sample buffer, and heating to 80.degree. C.
for 45 s. Terminated reaction mixtures were loaded into individual
wells of a 4-12% BIS-TRIS gel and run in XT MES buffer at 200 V for
45 min and then stained with SimplyBlue SafeStain.
[0808] b) Measurement of Reaction Product (i.e., Plasmin)
[0809] Analysis of the stained SDS gel indicated that wt u-PA
cleaved all the plasminogen present in the reaction, converting it
to two-chain, active plasmin during the 30 minute incubation. By
contrast, the activity of u-PA variant Y40Q/V41L/L97bA/C122S (seq
ID 40) in this reaction was substantially lower than that of
wt-u-PA while no plasminogen activation was observed by u-PA
variant
R35Q/H37Y/R37aE/V38E/T39Y/V41R/D60aP/Y60bQ/T97aI/L97bA/H99Q/C122S/Y149R
in this reaction.
2) Kinetics of Glu-Plasminogen Activation by Anti-C3 u-PA
Polypeptides
[0810] Kinetic analysis of the activation of Glu-plasminogen by wt-
or anti-C3 uPA variant polypeptides was performed as described
below. Activation reactions were performed at 37.degree. C. in an
I=0.16 M Hepes/NaCl/EDTA buffer containing 0.1% BSA. U-PA
polypeptides were present at concentrations of 1 (wt) or 10
(variant) nM. Second-order rate constants (k.sub.cat/KM) were
derived from the fitted linear relationship of the initial reaction
velocities for hydrolysis (by the reaction product plasmin) of the
chromogenic substrate S-2251 (H-D-Val-Leu-Lys-pNA.2HCl) vs. the
Glu-plasminogen (i.e., u-PA substrate) concentration (0-4 VM). See
also Table 24.
TABLE-US-00035 SEQ ID k.sub.cat/K.sub.M Ratio NO. Mutations
(M.sup.-1s.sup.-1) .+-.S.D. % CV n= to WT 5 C122S (WT) 2.28E+04
2.62E+03 11.5% 4 1 423 V38D/L97bA/H99Q/C122S 1.38E+03 1.79E+02
12.9% 4 17 8 F30Y/V38D/Y40H/V41R/L97bA/ 4.59E+02 6.42E+01 14.0% 4
50 H99Q/C122S/M157K 254b* C[4]S/F30Y/R35W/R36H/H37E/ 9.91E-01
1.88E+00 190.0% 4 23034 V38E/T39W/Y40H/V41R/Y60bH/
T97aI/L97bA/H99Q/C122S/ Y149R/M157K/Q192A
C4S Mutation is not in the Protease Domain
[0811] Comparison among 15 variants showed the results in the Table
24 appended below labeled "Activation of Glu-Plasminogen by anti-C3
u-PA Variants."
[0812] For Table 24:
[0813] Glu-plasminogen was activated by C122S uPA or selected uPA
variants at protease concentrations of 1 nM or 10 nM protease
[0814] Reactions were carried out at 37.degree. C. in an I=0.16 M
Hepes/NaCl/EDTA buffer containing 0.1% BSA
[0815] Second-order rate constants (k.sub.cat/KM) were derived from
the fitted linear relationship of the initial reaction velocities
for hydrolysis of the chromogenic substrate S-2251
(H-D-Val-Leu-Lys-pNA 2HC1) vs. the Glu-plasminogen concentration
(0-4 .mu.M).
Example 13
Exemplary u-PA Protease Domain Mutants Cleavage of Complement
Protein C3 in Cynomolgus Monkey Vitreous Humor
[0816] Exemplary mutants are shown in Table 23 below with reference
to the WT u-PA protease domain set forth in SEQ ID NO: 5. Shaded
cells indicate that the modified u-PA polypeptides are mutated at
this residue when compared to the reference u-PA polypeptide set
forth in SEQ ID NO: 5. Unshaded cells indicate that the modified
u-PA polypeptides contain the same amino acid as the reference u-PA
polypeptide set forth in SEQ ID NO: 5. The activity of the modified
u-PA polypeptides was determined by cleavage of the substrate
complement protein C3 as detailed and presented in Example 2 and
are presented as the residual levels of C3 (nM) after u-PA
treatment. Cleavage of C3 by the modified u-PA polypeptides was
performed in purchased cynomolgus monkey vitreous humor or
Phosphate Buffered Saline (PBS) control as detailed in Example 3.
The results set forth in Table 23, below, show that the modified
u-PA polypeptides exhibit greater activity against C3 compared to
the reference u-PA protease domain, whose sequence is set forth in
SEQ ID NO:5. The activity % in vitreous humor and PBS show the
percentage of remaining activity after 7 days. The modified u-PA
polypeptides are relatively stable compared to the reference
wild-type protease domain of SEQ ID NO:5, with some showing more
stability than others. Therapeutic candidates, including those in
the table below, are those having high C3 cleavage activity, and
greater stability, particularly in vitreous humor.
Among other things, these data and the other data show that:
[0817] R35Q: this mutation increased the intrinsic anti-C3 activity
(i.e., in buffer) by approximately 2.7-fold and by approximately
4.3-fold in the presence of 80% human plasma
[0818] H37Y: this mutation increased anti-C3 activity by
approximately 2.4-2.5-fold in buffer and in the presence of 80%
human plasma
[0819] R37aE: this mutation decreased the intrinsic anti-C3
activity by approximately 3.2-fold; however, in the presence of 80%
human plasma it had no effect on anti-C3 activity
[0820] V38E: this mutation improved the stability of the protein in
buffer and vitreous humor and improved the anti-C3 activity in 80%
human plasma by .about.1.5-fold
[0821] T39Y: This mutation increased the intrinsic anti-C3 activity
and the anti-C3 activity in 80% human plasma by approximately
7.5-8.5-fold
[0822] V41R: This mutation increased the intrinsic anti-C3 activity
and the anti-C3 activity in 80% human plasma by approximately
25-27-fold
[0823] D60aP: This mutation increased the intrinsic anti-C3
activity and the anti-C3 activity in 80% human plasma by
approximately 1.2-1.6-fold
[0824] Y60bQ: This mutation decreased the intrinsic anti-C3
activity by approximately 1.7-fold and decreased the stability of
the protein in vitreous after incubation for 7 days at 37.degree.
C. by .about.1.4-fold; but in the presence of 80% human plasma, it
increased anti-C3 activity by approximately 1.1-fold
[0825] T97aI: This mutation increased the intrinsic anti-C3
activity and anti-C3 activity in the presence of 80% human plasma
by approximately 1.2-1.3-fold
[0826] L97bA: This mutation increased anti-C3 activity by
approximately 6.4-8.2-fold in buffer and in 80% human plasma
H99Q: This mutation increased anti-C3 activity by approximately
2.9-4.8-fold in buffer and 80% human plasma Y149R: This mutation
decreased anti-C3 activity by approximately 1.6-2.1-fold in buffer
and 80% human plasma.
[0827] Similar results were achieved for the replacements in lower
mutation load modified u-PA polypeptides that contain:
I41D/C122S/G151N/Q192T (see, e.g., the modified u-PA polypeptide
whose sequence is set forth in SEQ ID NO:40, and also full-length
and precursor and mature forms that contain these
replacements).
[0828] Data indicate that the modified u-PA provided herein cleave
and inactivate C3 in a variety of species. For example, cleavage of
human C3 to inactivate it can be between residues 740 and 741 (SEQ
ID NO:47):
TABLE-US-00036 Q H A R .dwnarw. A S H L 737-744 P4 P3 P1 .dwnarw.
P1' P4'.
Example 14
Cloning, Expression and Preparation of u-PA-Human Serum Albumin
(HSA) Fusion Proteins
[0829] A. Cloning of the u-PA-HSA Fusion Polypeptide
[0830] A construct for expression of a u-PA-HSA fusion protein was
generated (SEQ ID NO: 1015). The u-PA-linker-HSA fragment was
assembled from synthetic oligonucleotides and/or PCR products and
cloned into the pcDNA3.4-TOPO vector (Invitrogen; Cat. No. A14697)
for expression under control of the human cytomegalovirus (CMV)
immediate-early promoter/enhancer or a proprietary expression
vector (Lake Pharma). A secretion signal sequence (SEQ ID NO:999
(METDTLLLWVLLLWVPGSTG)) was cloned upstream of the u-PA N-terminal
domain (amino acids 1-158 of SEQ ID NO: 3), and an exemplary
modified u-PA protease domain (set forth in SEQ ID NO:987), and
linked via a linker (SEQ ID NO: 1002 (GGSSGG)) to the coding region
of human serum albumin (HSA; SEQ ID NO: 991):
TABLE-US-00037 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT
EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQ
EPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYE
IARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDE
GKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLV
TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKP
LLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGM
FLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDE
FKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTL
VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS
DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLS
EKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK
ETCFAEEGKKLVAASQAALGL.
[0831] The construct includes the signal peptide (amino acids
1-20), the u-PA N-terminal domain, the modified protease domain of
SEQ ID NO:21, except with C at position 122 (set forth in SEQ ID
NO: 987), the GS linker (GGSSGG, SEQ ID NO: 1002) followed by the
HSA coding region. The complete construct sequence, set forth in
SEQ ID NO: 1015, is:
TABLE-US-00038 METDTLLLWVLLLWVPGSTGSNELHQVPSNCDCLNGGTCVSNKYFSN
IHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPW
NSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKP
LVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIE
NQPWFAAIYQRYEGGSEYYRCGGSLISPCWVISATHCFIPQPKKEDY
IVYLGRSRLNSNTQGEMKFEVENLILHKDYSADIAAQHNDIALLKIR
SKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDRLYP
EQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDS
GGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT
KEENGLALGGSSGGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQ
CPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL
RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAF
HDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK
AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQ
RFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQ
DSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV
CKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCA
AADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVV
LNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFN
AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDD
FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.
B. Preparation of u-PA-HSA Fusion Polypeptides
[0832] 1. Transformation and Expression of u-PA-HSA Fusion
Polypeptides
[0833] DNA encoding the modified u-PA-HSA fusion polypeptide was
cloned into the pcDNA3_4 expression vector (Thermo Fisher) or a
proprietary vector (Lake Pharma)C-terminal to the secretion signal
sequence as detailed in Section A. Modified u-PA-HSA fusion
proteins were subsequently expressed in a 1 L volume of expression
media for 6 days in HEK expi293 or expiCHO expression cells at
ThermoFisher or the proprietary TunaCHO.TM. cell line at Lake
Pharma.
[0834] 2. Affinity Purification of u-PA-HSA Fusion Polypeptides
[0835] The zymogen form of the modified u-PA-HSA fusion proteins
were purified using the system sold as the CaptureSelect.TM. Human
Albumin Affinity Matrix system (ThermoFisher Scientific; Cat. No.
19129701L or 19127005) according to the manufacturer's
instructions. A column was prepared by adding approximately 10 mL
of CaptureSelect.TM. Affinity Matrices resin (ThermoFisher) to the
column. After the storage solution was allowed to flow through the
column, 10 column volumes (CVs) of PBS (137 mM NaCl, 2.7 mM KCl, 10
mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4, pH 7.4) was added to
wash the resin. Next, the expression harvests containing the
u-PA-HSA fusion proteins were applied to the column and
subsequently washed with 5-10 CVs of PBS. Bound u-PA-HSA fusion
proteins were eluted with 10 CVs of elution buffer (20 mM Tris, 2 M
MgCl.sub.2, pH 7.0). The column was later stripped with 10 CVs of
glycine (0.2 M, pH 3.0) and neutralized with 10% Tris-HCl (1.5 M,
pH 7.4) and re-equilibrated in PBS. Samples of flow through and
elution steps were collected and analyzed on reduced and
non-reduced SDS PAGE gel electrophoresis to evaluate sample purity.
The u-PA-HSA fusion proteins were dialyzed for 16 hours into PBS at
4.degree. C.
[0836] 3. Plasmin Activation of Modified u-PA-HSA Fusion
Polypeptides
[0837] Plasmin selectively cleaves a single bond in wild-type u-PA
and other u-PA fusion polypeptides having the wild-type u-PA
activation sequence to activate the u-PA-HSA zymogen and form a
two-chain u-PA-HSA fusion protein, linked via a disulfide with the
C-terminal active protease domain tethered to the N-terminal u-PA
domain by a disulfide linkage. For these experiments the construct
used has the sequence set forth in SEQ ID NO: 1015, which contains
the modified u-PA polypeptide of SEQ ID NO:21, except that the
C122S, by chymotrypsin numbering, is C122C to provide a free
cysteine, and the signal sequence (residues 1-20 of SEQ ID NO:1015)
is not included. Following activation the resulting product has two
chains (with reference to SEQ ID NO: 1015): an A chain that has
residues 21-178, and a B chain that has residues 179-1022, u-PA
(residues 179-431), linker (residues 432-437), and HSA (residues
438-1022). The A and B chains are linked by a disulfide bond
between C168 and C299 (corresponding to C122 by chymotrypsin
numbering).
[0838] The HSA domain remains conjugated through the GGSSGG linker
to the C-terminus of the protease domain. Activation followed the
following procedure: a plasmin-agarose resin slurry (Molecular
Innovations; Cat. No. HPL-1) was prepared by washing the resin with
1.times.PBS, 3 times. Subsequently, 200 .mu.L of resin slurry in
1.times.PBS per milligram of u-PA fusion polypeptide was added to a
solution containing the dialyzed u-PA-HSA fusion polypeptide in
PBS. "Activation" of the u-PA polypeptide zymogen was accomplished
by gently shaking the protein/resin solution for 3 hours at room
temperature. The modified u-PA-HSA polypeptide zymogen was
thenceforth fully converted into the corresponding active modified
u-PA-HSA protease.
[0839] The activated modified u-PA-HSA polypeptides were recovered
from the plasmin resin using 0.2 .mu.m spin filters (2.0 mL
capacity) and centrifuging to recover activated modified u-PA-HSA
from the plasmin resin as per the manufacturer's instructions. To
maximize recovery, additional filtration techniques may be
envisioned (as opposed to spinning down the resin and recovering
the supernatant by pipette extraction). Other low-protein binding
filtration apparatus also can be used for further filtration of the
resin. Activated u-PA-HSA fusion proteins were stored at 4.degree.
C. or frozen in aliquots. Confirmation of the complete activation
step was visualized by SDS-PAGE under reducing conditions to
separate the N-terminal domain from the protease domain HSA fusion
conjugate.
[0840] 4. Inhibition of Activated Modified u-PA-HSA Fusion
Proteins
[0841] For some experiments it was desired to use a catalytically
inactive form of the u-PA-HSA fusion polypeptide (set forth as
residues 21-1022 of SEQ ID NO:1015, as discussed above; see e.g.,
SEQ ID NO: 1019). To generate a protein designated as inhibited
modified u-PA-HSA (u-PA-HSAi), activated modified u-PA-HSA
(u-PA-HSAa) was incubated with an irreversible active site
inhibitor, Glu-Gly-Arg-Chloromethylketone (EGR-CMK), to prevent any
autocatalysis of the modified u-PA-HSA polypeptides or
inactivation/cleavage during in vivo experiments (see e.g.,
Examples 15 and 16). To prepare the u-PA-HSAi, lyophilized EGR-CMK
(Molecular Innovations; Cat. No. GGACK) was reconstituted to 100 mM
in 10 mM HCl. Concentrated EGR-CMK was added to the activated
modified u-PA-HSA sample (u-PA-HSAa) at the stock concentration to
reach a final inhibitor concentration of 1 mM EGR-CMK inhibitor in
1.times.PBS, and the mixture was allowed to incubate for 1 hour at
RT.
[0842] To assess whether the u-PA-HSAa was fully inhibited, the
degradation of modified u-PA-HSAi was compared to modified
u-PA-HSAa in a stability experiment. Briefly, modified u-PA-HSAi
and modified u-PA-HSAa were incubated for 0 hours, 1 day, or 7 days
at 37.degree. C. Modified u-PA-HSAi and u-PA-HSAa degradation was
visually monitored by reduced and non-reduced SDS-PAGE gel
electrophoresis under reduced and non-reduced conditions. Bands
were visualized by Comassie or other similar commercially available
stain. The results demonstrate that preincubation with the
inhibitor stabilizes the polypeptide, as no degradation products
were observed for the modified u-PA-HSAi at any time point up to 7
days, compared to the observed degradation of modified u-PA-HSAa
after incubation for 1 day or 7 days at 37.degree. C.
[0843] 5. Purification of u-PA-HSAa and u-PA-HSAi Polypeptides
after Activation
[0844] During the final purification round, active and inhibited
modified u-PA-HSA polypeptides were isolated from high and low
molecular weight impurities using size exclusion chromatography.
Purification excluded a high molecular weight species that eluted
as a discrete peak prior to the main u-PA-HSA peak. The high
molecular weight species was not further analyzed, however could
represent aggregates or multimers that formed during the expression
or activation steps in the process. A secondary, low molecular
weight species thought to be free from albumin generated during
expression or activation was also eliminated.
[0845] Purification proceeded as follows; a .about.300 .mu.L sample
of modified u-PA-HSAi (0.8 mg) or a .about.400 .mu.L sample of
modified u-PA-HSAa (0.8 mg) was loaded onto a size-exclusion
chromatography column (HiPrep 16/60 Sephacryl S-200 HR; GE
Heathcare, Cat. No. GE17-1166-01) in PBS at pH 7.0 at a flow rate
of 0.5 mL/min. Proteins were generally purified according to
manufacturer instructions for the resin (GE Healthcare) with the
main peak containing modified u-PA-HSAi or u-PA-HSAa retained.
[0846] The quality of the preparations and extent of separation was
further assessed by reducing and non-reducing SDS-PAGE gel
electrophoresis. Elution fractions of modified u-PA-HSAi or
u-PA-HSAa polypeptide sample were loaded in each "lane" of a
12-well non-reducing SDS-PAGE gel and run at 40V until the bands
were sufficiently distinguishable and the various sized protein
species were visualized by silver staining to improve sensitivity
over the standard Coomassie stain. Fractions containing single
bands migrating at the expected molecular weight of 75 kDa were
pooled and snap-frozen in liquid nitrogen at a final concentration
of approximately 2 mg/mL and stored at -80.degree. C. until use.
The final concentration was determined by absorption at 280 nM
using a NanoDrop spectrophotometer. Protein size and expression was
later confirmed by Comassie stain, and SDS-PAGE. The quality of
individual u-PA-HSA polypeptide samples was further assessed by
activity assays and mass spectroscopy.
Example 15
Modified u-PA Protease Domain and Modified u-PA-HSA Fusion Protein
Pharmacokinetic Evaluation Following Intravitreal Injection
[0847] The pharmacokinetics and overt ocular toxicity of a modified
u-PA-HSA fusion polypeptide was assessed. The fusion protein
contains ae modified u-PA polypeptide protease domain (SEQ ID
NO:987, which is SEQ ID NO:21, except that the C122S is C122C), as
described above in Example 14. The fusion protein has the sequence
set forth in SEQ ID NO:1019, which is residues 21-1022 of SEQ ID
NO: 1015. As described in Example 14, the protease domain of the
modified u-PA is set forth in SEQ ID NO:987. The pharmacokinetic
profile of activated and inhibited modified u-PA-HSA fusion
proteins (SEQ ID NO:1015) were assessed in vivo in Dutch Belted
rabbits and compared to that of the pharmacokinetic profile of the
modified u-PA protease domain only as set forth in SEQ ID NO:21.
Eight rabbits were assigned to each of three treatment groups
(group 1: modified u-PA-HSAa; group 2: modified u-PA-HSAi; group 3:
modified u-PA protease domain (SEQ ID NO: 21)). Study animals were
administered 50 .mu.L of either 2.1 mg/mL of modified u-PA-HSAi and
u-PA-HSAa, or 1.15 mg/mL of modified u-PA (SEQ ID NO: 21), per eye,
via intravitreal injection (IVT). For the group treated with the
modified u-PA containing the modified protease, the right eye
received the modified u-PA polypeptide, and the left eye was
injected with PBS as the vehicle control (PBS). The table below
summarizes the groups and conditions, and assessments
performed:
TABLE-US-00039 Irritation Fluids and Concen- scoring Terminal
Tissues tration Dose/ method/ Time Collected Group Formulation
(mg/mL) Route timing points (OS and OD).sup.* 1 (n = 8) modified u-
2.1 50 .mu.L/eye Draize/ 1, 3, 7, and Aqueous humor, PA-HSA mg/mL
intravitreal prior to 14 days post- vitreous humor, Activated
euthanasia dose retina, choroid, (u-PA-HSAa) (n = 2/time plasma
point) 2 (n = 8) modified u- 2.1 50 .mu.L/eye Draize/ 1, 3, 7, and
Aqueous humor, PA-HSA mg/mL intravitreal prior to 14 days post-
vitreous humor, Inhibited euthanasia dose retina, choroid,
(u-PA-HSAi) (n = 2/time plasma point) 3 (n = 8) Modified u- 1.15 50
.mu.L left Draize/ 1, 3, and Aqueous humor, PA Protease mg/mL
eye/intra- prior to 7 days vitreous humor, Domain vitreal
euthanasia postdose retina, choroid, (SEQ ID NO: Right eye/ (n =
2-3/ plasma 21) control timepoint) .sup.*OS and OD refer to the
right and left eyes, respectively
[0848] 1. Modified u-PA and Modified u-PA-HSA Fusion Protein
Occular Tolerability
[0849] During the dosing period and prior to euthanasia, clinical
and ocular observations were conducted and body weight were
recorded. Ocular exams were performed on both eyes at 1, 3, 7, and
14 days (Groups 1 and 2) or at 1, 3, and 7 days (Group 3) following
dosing, and ocular irritation was scored using the Draize scale.
The Draize scoring system (Draize et al., (1944) J Pharm Exper Ther
82 (3) 377-90) assesses eye irritation in the cornea, iris and
conjunctiva and provides criteria for scoring irritation on a 0-2
or 0-4 scale. A score of "0" indicates that the cornea, iris or
conjunctiva is normal. At all timepoints, ocular irritation was
scored a 0 as assessed using the Draize criteria for all animals
except one rabbit with 2 scores of "1" indicating "redness" where
the "vessels are definitely injected above normal" and "chemosis"
with "swelling above normal" of the conjunctiva. These "1" scores
were observed for one rabbit in Group 2 (u-PA-HSAi) within a few
hours following the intravitreal administration of u-PA-HSAi at day
1. Most importantly there was no toxicity observed after
intravitreal administration of the modified u-PA and modified
u-PA-HSA activated fusion proteins.
[0850] 2. Modified u-PA and Modified u-PA-HSA Fusion Protein
Pharmacokinetics
[0851] For the determination of modified u-PA and modified u-PA-HSA
polypeptide concentrations (nM) and pharmacokinetic parameters,
terminal samples of ocular tissues and fluids were collected after
enucleation of both eyes on days 1, 3, 7 and 14 using two animals
per time point. Following euthanasia, both eyes of each rabbit were
harvested and dissected for collection of ocular tissues and fluids
(aqueous humor (AH), vitreous humor (VH), retina, and choroid)) for
assessment of u-PA and u-PA-HSA expression and activity. Following
collection, weighed amounts of rabbit vitreous humor, retina, and
choroid were homogenized in impact resistant microtubes (USA
Scientific) containing 2.8 mm ceramic beads. For VH, retina, and
choroid tissues, a consistent aliquot of phosphate buffered saline
per milligram of tissue was added to each tube. Retina and choroid
samples were diluted 9:1 (diluent volume: tissue volume) and VH
samples were diluted 4:1 (diluent volume: VH volume) with phosphate
buffered saline. Samples were homogenized (Precellys.RTM.
homogenizer) at 0 to 10.degree. C., at 5500 rpm for 3.times.30
second cycles with 20 second pauses between cycles until thoroughly
homogenized.
[0852] The concentration as determined by ELISA (nM) and activity
(nM) of modified u-PA and modified u-PA-HSA polypeptides in VH was
determined using an ELISA and activity assay, respectively. For the
concentration determination, the anti-u-PA sandwich ELISA was
carried out as follows: the capture antibody PA1-36166 (Invitrogen)
was coated on ELISA plates overnight at 4.degree. C. or 2 hours at
RT at a concentration of 1.0 ug/mL in 100 mM carbonate buffer, pH
9.5. The plates were subsequently washed 3.times. with PBST
(1.times.PBS containing tween) followed by blocking with 1% BSA in
PBS-tween overnight at 4.degree. C. or 2 hours at RT. 50 .mu.L of
each sample were incubated for for 30 mins at RT with shaking
followed by washing 3.times. with PBS and incubation with the
detection antibody PA1-36015 (Invitrogen at 0.25 ug/mL for 30
mins). Wells were again washed and subsequently incubated with HRP
conjugated anti Goat antibody (Rockland) at 1:30,000 dilution for
30 min with shaking at RT. After washing 6.times. with PBST, bound
modified u-PA and modified u-PA-HSA polypeptides were visualized by
detection with 1-step TMB (34028, Thermo), quenching with 2N
sulfuric acid prior to reading the absorbance at 450 nm.
[0853] Quantification of the modified u-PA and modified u-PA-HSA
polypeptides in VH by activity was followed using an assay based on
the hydrolysis of a quenched-fluorescence peptide substrate (FRET)
and calibrated to a standard curve of modified u-PA polypeptides of
known active concentration. This assay uses a FRET peptide
substrate based on the cleavage sequence of human complement 3
(C3). The sequence of the peptide is RQHAR/ASHL, where the "/"
indicates the cleavage site. The N-terminal side of the peptide is
labeled with a DABCYL fluorophore, and the C-terminal side is
labeled with an EDANS fluorophore. Cleavage of the peptide
separates the EDANS/DABCYL FRET pair to generate a fluorescent
signal, which is measured in a multi-well fluorescence plate
reader.
[0854] The assay was conducted as follows: test samples are
typically diluted to a minimum required dilution of 1:20 in assay
buffer (100 mM Tris, 50 mM NaCl, 0.01% Tween-80, pH 7.4). The
diluted samples are further diluted 1:2 with 80 .mu.M FRET
substrate in a 96-well plate. Immediately upon the combination of
diluted test samples and substrate, the fluorescence signal
following FRET substrate hydrolysis was evaluated in a fluorescence
plate reader with measurements every 30 seconds for 2 hours.
Enzymatic hydrolysis of the FRET peptide substrate generates an
EDANS fluorescent product. The rate of generation of fluorescence
intensity is interpolated against an EDANS standard curve to yield
the EDANS product generation rate. The specific activity may be
calculated in two ways. First, the product generation rate is
multiplied by the dilution factor to yield a volumetric specific
activity in units of nmol product per minute of reaction per mL of
sample (nmol/min/mL). The volumetric specific activity indicates
the total amount of active enzyme in the sample. Secondly, the
specific activity is calculated by dividing the volumetric specific
activity by the sample enzyme concentration to yield an enzyme
specific activity in units of nmol product per minute of reaction
per mg of enzyme (nmol/min/mg). For testing to determine the
apparent protease concentration of unknown samples (e.g., in vivo
PK samples), the volumetric specific activity of the sample
(nmol/min/mL) is divided by the enzyme specific activity of the
control modified u-PA or modified u-PA-HSA polypeptide
(nmol/min/mg) to yield the apparent protease concentration in the
sample (mg/mL).
[0855] Data for each eye and animal are provided in the Table
below. The concentrations obtained for each eye were averaged per
animal. Then the drug concentrations for each animal was averaged
at each time point to compensate for the inter-animal variability.
The means at each time point were computed and presented in the
Table below. The resulting data then were subjected to the
following analytical methodology: First, a semi-parametric
piecewise robust regression approach developed by Lee et al., (see,
Lee et al., (1990) J. Lab Clin. Med. 115:745-748; and Lee et al.
(1997) "The use of robust regression techniques to obtain improved
coagulation factor half-life estimates. XVIth Congress of the
International Society for Thrombosis and Hemostasis," Florence,
Italy) was used for computing the half-life. It is a compartmental
model. The data were evaluated using the program Demitasse, which
has been validated and used for FDA submissions. For analyses of
area under the time curve (AUC) and the other PK parameters, a
non-compartmental model based on the trapezoidal rule was used. The
PK parameters were calculated and are set forth in the tables
below.
TABLE-US-00040 TABLE Drug Drug time Concentration Concentration
Condition point (d) by ELISA (nM) by Activity (nM) Modified u-PA- 0
842.90 n.d. HSAi 1 385.08 n.d. 1 122.79 n.d. 1 289.90 n.d. 1 270.36
n.d. 3 289.25 n.d. 3 257.84 n.d. 3 254.54 n.d. 3 265.59 n.d. 7
120.94 n.d. 7 138.35 n.d. 7 144.15 n.d. 7 113.22 n.d. 14 30.13 n.d.
14 27.53 n.d. 14 36.28 n.d. 14 25.37 n.d. Modified u-PA- 0 842.90
842.90 HSAa 1 368.35 749.14 1 264.23 344.78 1 311.69 460.72 1
<LOD 0.35 3 199.27 505.84 3 168.21 283.60 3 132.80 179.47 3
181.28 239.77 7 64.47 156.92 7 61.08 82.45 7 73.70 98.58 7 51.94
51.25 14 9.69 31.96 14 7.55 72.49 14 5.35 15.63 14 6.79 10.38
Modified u-PA 0 1838.69 1838.69 (SEQ ID NO. 21) 1 214.47 203.44 1
222.15 251.46 1 182.63 183.70 3 <LOD 6.48 3 130.01 110.85 3
98.91 75.91 7 21.55 25.24 7 31.49 19.44 *Cells in bold were outside
limits of detection and/or considered outliers or excluded based on
an outlier test
TABLE-US-00041 TABLE Study Modified u-PA- Modified u-PA- Modified
u-PA day HSAi (ELISA) HSAa (ELISA) (ELISA) 1 267.03 314.76 206.42 3
266.81 170.39 114.46 7 129.16 62.80 26.52 14 29.83 7.35 n/a Study
Modified u-PA- Modified u-PA- Modified u-PA day HSAi (ELISA) HSAa
(ELISA) (Activity) 1 n.d. 388.75 212.86 3 n.d. 302.17 64.41 7 n.d.
97.30 22.34 14 n.d. 32.61 n/a ELISA Results (t-half Activity
Results (t-half Test article terminal-days) terminal-days) Modified
u-PA-HSAi 3.42 (MRT = 5.56) n.d. Modified u-PA-HSAa 2.42 (MRT =
3.79) 3.27 (MRT = 4.71) Modified u-PA 2.01 (MRT = 3.29) 1.95 (MRT =
3.01) (SEQ ID NO: 21)
[0856] Based on ELISA and activity assays, the modified protease
domain of u-PA (containing the modified u-PA polypeptide of SEQ ID
NO:21) has a half-life of approximately 2 days. Fusion to HSA,
increases the half-life of the protein. The activated modified
u-PA-HSAa had a half-life of 2.42 days and 3.27 days, as measured
by ELISA and activity, respectively. Half-life was increased for
u-PA-HSA subjected to the inhibition protocol with an
ELISA-determined half-life of 3.42 days. Thus, fusion of u-PA to
HSA increases the protein half-life in vivo, and the fusion protein
retains protease activity in vivo.
Example 16
Cloning, Expression and Preparation of u-PA Fusion Proteins
[0857] A. Cloning of u-PA Fusion Polypeptides
[0858] u-PA fusion proteins were generated with fusion partners at
either the N-terminus or C-terminus.
[0859] 1. Exemplary Fusion Proteins
[0860] Several alternate constructs for expression of u-PA fusion
proteins were generated (described below). For example, N-terminal
fusion proteins were generated (see, e.g., FIG. 2A). The constructs
contained (from N-terminal to C-terminal): (1) a secretion signal
(e.g., mouse Ig kappa chain V-III region (IgG.kappa.) (SEQ ID NO:
999); (2) a fusion partner (e.g., IgG1 Fc (SEQ ID NO: 992)); (3)
linker, such as AGS (SEQ ID NO: 1003); to the (4) wild-type u-PA
activation sequence (SEQ ID NO: 997; amino acids 167-178 of SEQ ID
NO: 1); and (5) the modified u-PA protease domain (SEQ ID NO: 987).
An example of an N-terminal fusion protein is set forth in SEQ ID
NO: 1004.
[0861] C-terminal fusion proteins also were generated (see, e.g.,
FIG. 3). In some examples the constructs contained (from N-terminal
to C-terminal): (1) a secretion signal (mouse Ig kappa chain V-III
region (IgG.kappa.) (SEQ ID NO: 999); or human Interleukin-2 (hIL2)
(SEQ ID NO: 1000)); (2) the wild-type u-PA activation sequence (SEQ
ID NO: 997 or 998), furin activation sequence (SEQ ID NO: 995, 996,
or 1041), or no activation sequence; (3) the modified u-PA protease
domain (SEQ ID NO: 987 or 21) or the wild-type u-PA protease domain
(SEQ ID NO: 2 or 5); (4) a linker (SEQ ID NO: 1002 or 1003); and
(5) a fusion partner (i.e., IgG1 Fc (SEQ ID NO: 992); human serum
albumin (HSA) (SEQ ID NO: 991); cFv that binds to Collagen IIm
(C2scFv) (SEQ ID NO: 993); or an Hyaluronic acid binding domain
(HABD)(SEQ ID NO: 994)). Examples of C-terminal fusion proteins are
set forth in SEQ ID NOs: 1006-1010, 1012, 1013, 1016, and 1040 (see
e.g., FIGS. 3A and 3B).
[0862] C-terminal fusion proteins containing the u-PA N-terminal
region also were generated. The constructs contained (from
N-terminal to C-terminal): (1) a secretion signal (mouse Ig kappa
chain V-III region (IgG.kappa.); SEQ ID NO: 999); (2) the wild-type
u-PA N-terminal region (amino acids 21-178 of SEQ ID NO: 1 or SEQ
ID NO: 1042); (3) an activation sequence of u-PA (SEQ ID NO: 997 or
998) or a furin activation sequence (SEQ ID NO: 995, 996, or 1041);
(4) the modified u-PA catalytic domain (SEQ ID NO: 987 or SEQ ID
NO:5, except with C122, by chymotrypsin numbering, or SEQ ID NO:
21); (5) a linker (SEQ ID NO: 1002 or 1003); and (6) a fusion
partner (i.e., IgG1 Fc (SEQ ID NO: 992); human serum albumin (HSA)
(SEQ ID NO: 991)). Examples of C-terminal fusion proteins are set
forth in SEQ ID NOs: 1011, 1014, 1015 and 1036 (see e.g., FIG. 3C).
See, also SEQ ID NO:1010, which contains a furin activation
sequence in place of the u-PA
[0863] Fusion proteins containing SUMO at the N-terminus and the
fusion partner at the C-terminus also were generated. The
constructs contained (from N-terminal to C-terminal): (1) a
secretion signal (mouse Ig kappa chain V-III region (IgG) (SEQ ID
NO: 999)); (2) a HIS linker and SUMO sequence (SEQ ID NO: 990); (3)
the modified u-PA catalytic domain (SEQ ID NO: 987); (4) a linker
(SEQ ID NO: 1002); and (5) a fusion partner (i.e., IgG1 Fc (SEQ ID
NO: 992); or human serum albumin (HSA) (SEQ ID NO: 991)). Examples
of C-terminal fusion proteins are set forth in SEQ ID NOs: 1016 and
1017.
[0864] A full-length u-PA protein that was not fused to a fusion
partner was generated as a control (see, e.g., FIG. 2B). The
construct contained (from N-terminal to C-terminal): (1) secretion
signal (mouse Ig kappa chain V-III region (IgG.kappa.) (SEQ ID NO:
999)); (2) the N-terminal domain of u-PA (SEQ ID NO:1040; amino
acids 21-166 of SEQ ID NO: 1); (3) a u-PA activation sequence (SEQ
ID NO: 997); and (4) the modified u-PA protease domain (SEQ ID NO:
987). An example of a full-length u-PA protein is set forth in SEQ
ID NO: 1005. The following is a summary of the constructs that were
generated:
TABLE-US-00042 SEQ ID Signal Fusion Activation Fusion partner
Protease domain NO: Sequence partner Sequence location of u-PA 1004
IgG.kappa. IgG1 Fc u-PA with Cys N-terminus SEQ ID NO: 987 1005
IgG.kappa. No fusion Full-length uPA No fusion SEQ ID NO: 987
partner w/Cys partner 1006 hIL2 IgG1 Fc No Activation C-terminus
SEQ ID NO: 21 Sequence 1007 hIL2 HSA No Activation C-terminus SEQ
ID NO: 21 Sequence 1008 hIL2 C2 scFv No Activation C-terminus SEQ
ID NO: 21 Sequence 1009 hIL2 HABD No Activation C-terminus SEQ ID
NO: 21 Sequence 1010 IgG.kappa. IgGlFc u-PA furin C-terminus SEQ ID
NO: 21 w/o Cys 1011 IgG.kappa. IgGlFc Full-length wild C-terminus
SEQ ID NO: 987 type uPA w/Cys 1012 hIL2 IgG1 Fc No Activation
C-terminus SEQ ID NO: 5 Sequence 1013 hIL2 HSA No Activation
C-terminus SEQ ID NO: 5 Sequence 1014 IgG.kappa. HSA furin with Cys
C-terminus SEQ ID NO: 987 1015 IgG.kappa. HSA u-PA with Cys
C-terminus SEQ ID NO: 987 1016 IgG.kappa. HSA furin without Cys
C-terminus SEQ ID NO: 21 1017 IgG.kappa. HSA SUMO C-terminus SEQ ID
NO: 21 1018 IgG.kappa. IgG1 Fc SUMO C-terminus SEQ ID NO: 21
[0865] The control protein has the sequence set forth in SEQ ID NO:
1005. The control protein contains the u-PA N-terminus (residues
1-158 of SEQ ID NO:3), wild-type u-PA activation sequence, and u-PA
protease domain (SEQ ID NO:987), and no fusion partner. The fusion
protein set forth in SEQ ID NO: 1004 includes the fusion partner
(Fc) at the N-terminus. The fusion proteins set forth in SEQ ID
NOs: 1006-1009 have different fusion partners at the C-terminus and
lack an activation sequence N-terminal to the modified u-PA
protease domain. The fusion proteins set forth in SEQ ID NOs: 1010
and 1011 contain Fc at the C-terminus and are activated differently
from each other: the fusion protein set forth in SEQ ID NO: 1010
contains a furin activation sequence; and the fusion protein set
forth in SEQ ID NO: 1011 contains the n-terminal region of u-PA and
a wild-type u-PA activation sequence. The fusion proteins set forth
in SEQ ID NOs: 1012 and 1013 are the same as the fusion proteins
set forth in SEQ ID NOs: 1006 and 1007, respectively, but have the
wild-type u-PA protease domain in place of the modified u-PA
protease domain.
[0866] The fusion proteins set forth in SEQ ID NOs: 1014-1016
contain HSA at the C-terminus and are activated differently from
each other: the fusion protein set forth in SEQ ID NO: 1014
contains a furin activation sequence; the fusion protein set forth
in SEQ ID NO: 1015 contains a wild type u-PA activation sequence
for activation; and the fusion protein set forth in SEQ ID NO: 1016
contains a furin domain for activation. The fusion proteins set
forth in SEQ ID NOs: 1017 and 1018 contain SUMO as an activation
domain at the N-terminus, and HSA or IgG-Fc at the C-terminus,
respectively. Furin activation sequences were added to the fusion
proteins set forth in SEQ ID NOs: 1010, 1014 and 1016 so that the
protein can be activated during expression, thereby eliminating a
requirement for an activation step during downstream
processing.
[0867] 2. Construct Generation
[0868] (a) Preparation of u-PA Constructs
[0869] The constructs were assembled from synthetic
oligonucleotides and/or PCR products and cloned into the
pcDNA3.4-TOPO vector (Invitrogen; Cat. No. A14697) for expression
under control of the human cytomegalovirus (CMV) immediate-early
promoter/enhancer.
[0870] (b) Preparation of SUMO-u-PA Constructs
[0871] For expression of fusion proteins containing a SUMO tag (set
forth in SEQ ID NOs: 1017 and 1018), DNA encoding the modified u-PA
polypeptide with C122S (set forth in SEQ ID NO: 21) was cloned into
the codon optimized pE5 expression vector (Thermo Scientific;
sequence set forth in SEQ ID NO:988). The pE5 plasmid contains a
multiple cloning site C-terminal to a SUMO sequence for cloning the
fusion partner (HSA or Fc) and the modified u-PA protease domain.
The final fusion protein is (1) a fusion partner (HSA or FC); (2)
the modified u-PA protease domain with C122S (SEQ ID NO: 21); with
an (3)N-terminal 6.times.His purification tag; and (4) SUMO.
[0872] B. Preparation of u-PA Fusion Polypeptides
[0873] Expected molecular weights of the u-PA fusion proteins are
set forth below:
TABLE-US-00043 Expected MW Reduced Expected MW MW without Name
Non-Reduced MW with activation activation 1004 111,012 Modified
u-PA: 28,445.36 55506.07 Fc: 27,078.73 1005 46,389 Modified u-PA:
28,445.36 46388.7 uPA: 17,961.36 1006 108,467 54233.44 1007 95,286
95285.85 1008 55,226 55226 1009 40,297 40297 1010 111,146 54233.44
55573 1011 144,386 uPA: 17,961.36 72192.85 Modified u-PA-Fc:
54,249.50 1012 108,461 54230.5 1013 95,283 95282.91
[0874] 1. Transformation, Expression, Folding and Refolding of
u-PA
[0875] Fusion Proteins Fusion proteins with the sequences set forth
in SEQ ID NOs: 1004-1013 were transformed and expressed as detailed
in Example 14, above.
[0876] 2. Transformation, Expression, Folding and Refolding of
u-PA-SUMO (Small Ubiquitin-Like Modifier) Fusion Proteins
[0877] Fusion proteins with the sequences set forth in SEQ ID NOs:
1017 and 1018 were prepared as detailed below.
[0878] Cloning of the SUMO-Modified u-PA Fusion Polypeptide for E.
coli Expression
[0879] Competent BL21 Gold (DE3) E. coli cells are transformed with
u-PA fusion protein in an expression vector (SEQ ID NO: 988), which
was prepared by Thermo Scientific, using the standard heat shock
method. The plasmid DNA is resuspended in 50 .mu.L MQ water to
obtain a 100 ng/mL stock solution. The plasmid DNA and competent
BL21 Gold DE3 cells are thawed on ice. 0.5 .mu.L DNA, are added to
50 .mu.L cells in a sterile microfuge tube and incubated on ice for
30 minutes. The cell/DNA mixture are heat shocked by placing at
42.degree. C. for 45 seconds. The cell/DNA mixture immediately are
transferred back to ice and incubated on ice for 2 minutes. 450
.mu.L pre-warmed (37.degree. C.) SOC media is added to the cell/DNA
mixture, and the resulting SOC/cell/DNA mixture is incubated at
37.degree. C. with shaking. The cells in SOC (2-200 .mu.L) are
plated and spread on LB-carbenicillin plates, which are incubated
overnight at 37.degree. C. The plates harboring bacterial colonies
are removed from the incubator, sealed with parafilm and stored at
4.degree. C. Glycerol stocks of individual transformed colonies are
prepared by standard methods and stored at -70.degree. C.
[0880] C. Assessing Protein Expression of Fusion Proteins in
Mammalian Cell Culture
[0881] Protein concentration from the expi293 cell culture
supernatant (see Example 14) was assessed by ELISA and
qualitatively by western blot. Protein expression after western
blotting was scored on a 1 to 5 scale where 1 represents the
highest expression and 5 represents no expression.
[0882] Six samples were tested for each construct. The results are
set forth in the tables below. The results show that by ELISA and
Western Blotting of the fusion proteins set forth in SEQ ID NOs:
1004, 1007-1009 and 1013 were poorly expressed. The proteins set
forth in SEQ ID NOs: 1005, 1010 and 1011 had the highest expression
as assessed by qualitative western blot and ELISA.
TABLE-US-00044 TABLE Protein Expression Assessed by ELISA Sample
ELISA Titer u-PA ELISA SEQ ID (mg/L) (mg/L) 1004 0.4 0.4 1005 75.39
149.7 1006 2.2 2.6 1007 0.3 0.3 1008 0.06 0.1 1009 0.26 0.3 1010
15.89 17.3 1011 21.66 19.3 1012 7.5 16.2 1013 5.46 4.7 R squared =
0.9993; limit of detection = 0.109 ng/mL; limit of quantitation =
l.375 ng/mL
TABLE-US-00045 TABLE Protein Expression Assessed by Western
Blotting SEQ ID NO: SDS-PAGE/Western Ranking 1004 4 1005 1 1006 3
1007 3 1008 5 1009 5 1010 1 1011 1 1012 3 1013 4
[0883] D. Fusion Protein Activation Strategies
[0884] Various strategies were employed for u-PA activation. The
proteins set forth in SEQ ID NOs: 1004-1005, 1011 and 1015 were
activated by plasmin, as detailed above in Example 14. The proteins
set forth in SEQ ID NOs: 1010, 1014 and 1016 were expected to be
activated by intracellular furin during expression. The protein set
forth in SEQ ID NO: 1006-1008 were anticipated to be auto-activated
during expression. The proteins set forth in SEQ ID NOs: 1017 and
1018 were activated by SUMO protease treatment. To activate the
SUMO-u-PA constructs, typically 10 Units of SUMO protease per 1 mg
of protein was added and allowed to incubate overnight at 4.degree.
C. Further purification on a HisTrap nickel chelation column would
effectively remove the His-tagged SUMO moiety.
TABLE-US-00046 SEQ ID NO: Activation Sequence Activation Strategy
1004 uPA wt w/Cys Plasmin treatment 1005 Full-length uPA w/Cys
Plasmin treatment 1006 No activation sequence Secretion signal
cleavage during expression generates activated protease 1007 No
activation sequence Secretion signal cleavage during expression
generates activated protease 1008 No activation sequence Secretion
signal cleavage during expression generates activated protease 1009
C3 activation sequence C3 sequence autoactivated post expression
1010 uPA Furin w/o Cys Furin activation during expression;
activiation not necessary during downstream processing 1011
Full-length WT uPA w/Cys Plasmin treatment 1012 No activation
sequence Secretion signal cleavage during expression generates
activated protease 1013 No activation sequence Secretion signal
cleavage during expression generates activated protease 1014 Furin
with Cys Intracellular activation by Furin during expression;
activation not necessary during downstream processing 1015 uPA with
Cys Plasmin treatment 1016 Furin without Cys Intracellular
activation by Furin during expression; activation not necessary
during downstream processing 1017 SUMO SUMO protease treatment 1018
SUMO SUMO protease treatment
[0885] E. Measuring Enzyme Activity
[0886] Volume specific activity of expi293 HEK supernatants
containing the u-PA fusion proteins on 40 .mu.M human C3 FRET
peptide was assessed as described in Example 15. The interpolated,
dilution-adjusted initial rate (nM EDANS/min/.mu.L sample) was
calculated. Fusion proteins set forth in SEQ ID NOs: 1004, 1005,
1008 and 1011-1013 showed no activity. Fusion proteins set forth in
SEQ ID NOs: 1006, 1007, 1009 and 1010 demonstrated u-PA protease
activity. Modified u-PA with a Furin activation sequence N-terminal
to u-PA with an Ig FC fusion at the C-terminus (set forth in SEQ ID
NO: 1010) showed the highest activity. The results are set forth in
the table below.
TABLE-US-00047 SEQ ID Activity (nM Can auto- No Activity on NO.
EDANS/min/.mu.L sample) activate Mouse C3 1004 -0.1 1005 -0.1 1006
3.7 X X 1007 3.2 X X 1008 0.3 X 1009 2.7 X 1010 56.0 X 1011 -0.1
1012 0.0 X 1013 0.1 X
[0887] F. Affinity Purification
[0888] The fusion proteins set forth in SEQ ID NOs: 1010 and 1011
had the greatest expression as assessed by western blotting and
ELISA. The Furin-variant u-PA-Fc and full-length uPA-Fc fusion
proteins set forth in SEQ ID NOs: 1010 and 1011, respectively, were
Protein A affinity purified using the manufacturer's recommended
conditions (GE Healthcare).
[0889] G. Purification and Activity Assessment of High-Expressing
Fusion Proteins
[0890] After affinity purification, protein concentrations were
assessed by absorbance at 280 nm and by the u-PA ELISA (see Example
15), while purity was evaluated by SDS-PAGE gel electrophoresis and
analytical size exclusion chromatography (SEC), as described above
in Example 14. Both fusion proteins were expressed. Purity, when
assessed by gel electrophoresis and staining, and by analytical
size exclusion chromatography (SEC), was deemed to be poor for the
fusion protein whose sequence is set forth in SEQ ID NO: 1010 (u-PA
Furin w/o Cys). Purity by gel electrophoresis was deemed good for
the fusion protein set forth in SEQ ID NO: 1011 (full-length WT uPA
w/Cys). The results are set forth in the table below:
TABLE-US-00048 Total Concentration Total Protein SEQ ID Volume
(mg/mL) Yield (mg) Purity Purity by NO. Column (mL) A280 ELISA A280
ELISA by Gel SEC 1010 ProA 8.6 4.23 0.60 36.38 5.19 Poor Poor 1011
ProA 13.6 1.69 1.04 22.98 14.09 Good Poor
[0891] u-PA enzyme activity after affinity purification was
assessed on the Human C3 FRET Peptide assay as described above in
Example 15. The results are set forth in the table below:
TABLE-US-00049 ELISA: Activity Volumetric A280: Relative Relative
to Specific A280: Enzyme Activity to the ELISA: Enzyme variant u-PA
SEQ Activity Specific Activity u-PA SEQ ID Specific Activity
protease domain ID NO. (nmol/min/mL) (nmol/min/mg) NO: 21
(nmol/min/mg) (SEQ ID NO 21) 1010 466.81 110.36 2% 773.05 45% 1011
0.10 0.06 0% 0.10 0%
[0892] The u-PA enzyme activity after affinity purification was
assessed on the Human C3 FRET Peptide assay as described above in
Example 15. Enzyme activity was assessed after plasmin activation
as described above in Example 14. The results are set forth in the
table below:
TABLE-US-00050 Relative activity Relative (nmol/min/ Enzyme
activity Enzyme nmol) specific (nmol/min/ specific compared to SEQ
activity mg) activity to u-PA ID (nmol/min/ to u-PA SEQ (nmol/min/
SEQ ID NO. mg) ID NO: 21 MW nmol) NO: 21 21 692 100% 28429 19.7
100% 1011 198 29% 144385 14.3 73% 1015 178 26% 95301 20.2 103%
[0893] H. Description of Fusion Proteins with Modified u-PA
Polypeptides that Cleave C3
[0894] Fusion proteins with u-PA polypeptides are described below.
Exemplary sequences are provided in the following discussion.
[0895] 1. Catalytic Domains
[0896] The catalytic domain (protease domain) can be any of the
protease domains of the modified u-PA polypeptides provided herein
(see Example 2, Table 14, which provides the ED.sub.50 for protease
domains containing various modifications as described herein and in
the Examples), particularly any with an ED.sub.50 less than 100 nM,
as described in Example 2, or less than 50 nM, 30 nM, or 10 nM.
Exemplary of the modified uPA protease domain is that set forth in
SEQ ID NO:21, except, when using it to activate to produce a two
chain polypeptide, residue 122 (by chymotrypsin numbering) is C,
not S as in SEQ ID NO:21. Tab The modified uPA polypeptide protease
domains:
TABLE-US-00051 SEQ ID NO: 987: IIGGEFTTIE NQPWFAAIYQ RYEGGSEYYR
CGGSLISPCWVISATHCFIP QPKKEDYIVY LGRSRLNSNT QGEMKFEVEN LILHKDYSAD
IAAQHNDIALLKIRSKEGRC AQPSRTIQTI CLPSMYNDPQ FGTSCEITGF GKENSTDRLY
PEQLKMTVVKLISHRECQQP HYYGSEVTTK MLCAADPQWK TDSCQGDSGG PLVCSLQGRM
LTGIVSWGRGCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL;
[0897] SEQ ID NO: 21 modified uPA polypeptide protease domain
containing a C122S mutation: IIGGEFTTIENQPWFAAIYQRY EGGSEYYRCG
GSLISPCWVI SATHCFIPQP KKEDYIVYLG RSRLNSNTQGEMKFEVENLI LHKDYSADIA
AQHNDIALLK IRSKEGRCAQ PSRTIQTISL PSMYNDPQFGTSCEITGFGK ENSTDRLYPE
QLKMTVVKLI SHRECQQPHY YGSEVTTKML CAADPQWKTDSCQGDSGGPL VCSLQGRMTL
TGIVSWGRGC ALKDKPGVYT RVSHFLPWIR SHTKEENGLAL contain amino acid
replacements compared to the native uPA polypeptide protease domain
SEQ ID NO: 2:
TABLE-US-00052 IIGGEFTTIE NQPWFAAIYR RHRGGSVTYV
CGGSL[I/M]SPCWVISATHCFID YPKKEDYIVY LGRSRLNSNT QGEMKFEVEN
LILHKDYSAD TLAHHNDIALLKIRSKEGRC AQPSRTIQTI CLPSMYNDPQ FGTSCEITGF
GKENSTDYLY PEQLKMTVVKLISHRECQQP HYYGSEVTTK MLCAADPQWK TDSCQGDSGG
PLVCSLQGRM TLTGIVSWGRGCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL;
or compared to the protease domain in which the C at residue 122
(by chymotrypsin numbering) is S as set forth in SEQ ID NO:5:
TABLE-US-00053 IIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISAT
HCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSAD
TLAHHNDIALLKIRSKEGRCAQPSRTIQTISLPSMYNDPQFGTSC
EITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTK
MLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALK
DKPGVYTRVSHFLPWIRSHTKEENGLAL.
[0898] As shown herein, the modified u-PA polypeptides, exhibit
altered proteolytic activity and biochemical properties compared to
the native u-PA. The u-PA polypeptides are modified to have
increased activity for cleaving C3, as described herein, and to
have reduced activity for cleaving their native substrate.
[0899] 2. Secretion Signals
[0900] To ensure the extracellular secretion of modified u-PA
polypeptide during protein expression, a secretion signal can be
included in the construct to direct secretion of the encoded
polypeptide upon expression. Many such signals are known to those
of skill in the art. These include the native u-PA signal, and
others, such as the mKLC secretion signal (SEQ ID NO: 999
(METDTLLLWVLLLWVPGSTG)) sequence or the human IL2 secretion signal
(SEQ ID NO: 1000 (MYRMQLLSCI ALSLALVTNS), and others exemplified
herein. Signal sequences generally occur on the N-terminus of a
polypeptide to direct secretion; they are removed by the host
cell.
[0901] 3. Fusion Partners
[0902] In addition to amino acid replacement, insertion or
deletion, additional modifications can be added to the uPA
polypeptide to create a fusion or chimeric protein. Numerous fusion
partners and their properties are known to those of skill in the
art. These include, for example, fusion partners that can
multimerized, those that can increase serum half-life, and those
that alter binding properties or target the polypeptide. For
example, modified and native uPA polypeptides can be linked to an
antibody fragment or multimerization domain such as the Fc region
of an immunoglobulin polypeptide IgG1 (SEQ ID NO: 992 (DKT
HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE
VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP
REPQVYTLPP SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS
FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG)) or Collagen II
scFv (SEQ ID NO: 993 (QVQLQQPGADL VRPGVSVKLSCKASGYTFTS YWMNWVKQRP
GQGLEWIGMI HPSDSETRLS QKFKDKATLT VDKSSSTAYMQLSSPTSEDS AVYYCARLKP
GGTWFAYWGQ GTLVTVSAGG GGSGGGGSGG GGSGGSDIVLTQSPASLTVS LGQRATISCR
ASKSVDSYGN SFMEWYQQKP GQPPKLLIYR ASNLESGIPARFSGSGSRTD FTLTINPVEA
DDVATYYCQQ SNEDPYTFGG GTKLEIK)) to alter pharmacological properties
of the u-PA polypeptides.
[0903] To alter pharmacological properties, the uPA polypeptides,
for example, can be fused to a ligand or polypeptide, such as an
antibody, or other protein, such as human serum albumin (HSA; SEQ
ID NO: 991): DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKT
CVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL
QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELL
FFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKAS SAKQRLKCASLQKF
GERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDR
ADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
ESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCC
AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK
KVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH
EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSE
KERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA
EEGKKLVAASQAALGL), or a hyaluronic acid binding domain (HABD), such
as TSG-6 (SEQ ID NO: 994) ERAAGVYHREA RSGKYKLTYAEAKAVCEFEG
GHLATYKQLE AARKIGFHVC AAGWMAKGRV GYPIVKPGPN CGFGKTGIIDYGIRLNRSER
WDAYCYNPHA KE.
[0904] Typically, the u-PA protease activity remains functionally
active within the resulting fusion protein, but the fusion peptide
may change the pharmacokinetic and pharmacodynamic parameters of
the u-PA polypeptide. Other fusion proteins containing a u-PA
modified polypeptide can be created with a growth factor or a
receptor to alter pharmacokinetic and pharmacodynamic
properties.
[0905] 4. Activation Sequences
[0906] The u-PA polypeptide can be produced in an inactive form
(zymogen) and modified posttranslationally via proteolytic cleavage
to generate a mature and activated form. To do so, an activation
sequence is included on the the native or modified u-PA polypeptide
to suppress its enzymatic activity. After protein expression,
cleavage of the activation sequence produces a mature u-PA protein.
Examples of activations sequences, include, but are not limited, to
the wild-type u-PA activation sequence (SEQ ID NO: 997
(QCGQKTLRPRFK) or SEQ ID NO: 998 (QSGQKTLRPRFK)) or a furin
cleavage sequence (SEQ ID NOS: 995, 996, 1041, or 1044 (such as
QCGQKTLRRRKR, or QSGQKTLRRRKR, or QSGKTLRRKR, or QSGQKTLRRKR). A
disulfide linkage can be maintained between the cysteine within the
activation sequence and the cysteine (C122 by chymotrypsin
numbering) within the u-PA catalytic domain. Upon cleavage of the
activation sequence, the activated molecule retains a covalent
linkage between the N-terminal fragment activation or full
N-terminal domain.
[0907] 5. Linkers
[0908] To join the u-PA polypeptide with other polypeptide
sequences, a short, flexible sequence of amino acids (linker) is
used. Examples of linkers include but are not limited to GGSSGG or
GGGGS or AGS (such as those set forth in SEQ ID NOS: 1001-1003 and
1024-1030), as well as those discussed in the detailed description.
Longer linkages with concatenations of these sequences repeated
also are included such that a linker has the sequence
(GGSSGG).sub.n+1, where n is 0 or an integer between 1 and 20.
Other linkers are set forth in the Sequence Listing and in the
Detailed Description.
[0909] 6. Other Modification of u-PA
[0910] Other peptide sequences such as 6.times.His SUMO (such as
those set forth in SEQ ID NOS: 990, and 1031-1033 (DGHHHHHHGS
LQDSEVNQEA KPEVKPEVKP ETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEM
DSL(T/R) FLYDGI (E/R) IQADQ (T/A)PED LDMEDNDIIE AHREQIGG)) can be
added to facilitate the expression, secretion or purification of
u-PA polypeptides. Additional chemical and posttranslational
modification to the altered or native u-PA polypeptide can include
but are not limited to a conjugation to a polymer such as
PEGylation, PASylation, and sialylation to alter pharmacodynamic
properties of the u-PA polypeptide.
[0911] 7. Exemplary Modified u-PA Polypeptides with N-Terminal
Fusions
TABLE-US-00054 Other N Sequence Fusion Activation Catalytic
terminal Seq signal partner Linker Sequence domain domain ID
(residue (residue (residue (residue (residue (residue No. Name
nos.) nos.) nos.) nos.) nos.) nos.) 1004 Fc-u-PA METDTLLL
DKTHTCPPCP AGS QCGQKTLRP IIGGEFTTIE (SEQ ID WVLLLWVP APELLGGPSV
(247-249) RFK NQPWFAAIYQ NO: 987) GSTG FLFPPKPKDT (250-261)
RYEGGSEYYR (1-20) LMISRTPEVT CGGSLISPCW CVVVDVSHED VISATHCFIP
PEVKFNWYVD QPKKEDYIVY GVEVHNAKTK LGRSRLNSNT PREEQYNSTY QGEMKFEVEN
RVVSVLTVLH LILHKDYSAD QDWLNGKEYK IAAQHNDIAL CKVSNKALPA LKIRSKEGRC
PIEKTISKAK AQPSRTIQTI GQPREPQVYT CLPSMYNDPQ LPPSRDELTK FGTSCEITGF
NQVSLTCLVK GKENSTDRLY GFYPSDIAVE PEQLKMTVVK WESNGQPENN LISHRECQQP
YKTTPPVLDS HYYGSEVTTK DGSFFLYSKL MLCAADPQWK TVDKSRWQQG TDSCQGDSGG
NVFSCSVMHE PLVCSLQGRM ALHNHYTQKS TLTGIVSWGR LSLSPG GCALKDKPGV
(21-240) YTRVSHFLPW IRSHTKEENG LAL (262-514) 1005 N-term METDTLLL
-- -- QCGQKTLRP IIGGEFTTIE SNELHQVPSN u-PA-u- WVLLLWVP RFK
NQPWFAAIYQ CDCLNGGTCV PA GSTG (167-178) RYEGGSEYYR SNKYFSNIHW (SEQ
ID (1-20) CGGSLISPCW CNCPKKFGGQ NO: 987) VISATHCFIP HCEIDKSKTC
QPKKEDYIVY YEGNGHFYRG LGRSRLNSNT KASTDTMGRP QGEMKFEVEN CLPWNSATVL
LILHKDYSAD QQTYHAHRSD IAAQHNDIAL ALQLGLGKHN LKIRSKEGRC YCRNPDNRRR
AQPSRTIQTI PWCYVQVGLK CLPSMYNDPQ PLVQECMVHD FGTSCEITGF CADGKKPSSP
GKENSTDRLY PEELKF PEQLKMTVVK (21-166) LISHRECQQP HYYGSEVTTK
MLCAADPQWK TDSCQGDSGG PLVCSLQGRM TLTGIVSWGR GCALKDKPGV YTRVSHFLPW
IRSHTKEENG LAL (179-431) * u-PA protease domain of SEQ ID NO: 21
(with and without the C122S replacement)
[0912] FIG. 2 sets forth schematics of the u-PA polypeptides with
N-terminal fusions, such as the N-terminal fusion polypeptides set
forth in SEQ ID NOS: 1004 and 1005.
[0913] 8. Exemplary Modified u-PA Polypeptides with C-Terminal
Fusions
TABLE-US-00055 Sequence SEQ signal Linker Other ID (residue
Catalytic (residue Activation Fusion N terminal No. Name nos.)
domain nos.) Sequence partner domain 1006 uPA MYRMQLLSCI
IIGGEFTTIENQP GGSSGG None DKTHTCPPCPAPELL (SEQ ID ALSLALVTNS
WFAAIYQRYEGGS (274-279) GGPSVFLFPPKPKDT NO: 21)- (1-20)
EYYRCGGSLISPC LMISRTPEVTCVVVD Fc (No WVISATHCFIPQP VSHEDPEVKFNWYVD
PP) KKEDYIVYLGRSR GVEVHNAKTKPREEQ LNSNTQGEMKFEV YNSTYRVVSVLTVLH
ENLILHKDYSADI QDWLNGKEYKCKVSN AAQHNDIALLKIR KALPAPIEKTISKAK
SKEGRCAQPSRTI GQPREPQVYTLPPSR QTISLPSMYNDPQ DELTKNQVSLTCLVK
FGTSCEITGFGKE GFYPSDIAVEWESNG NSTDRLYPEQLKM QPENNYKTTPPVLDS
TVVKLISHRECQQ DGSFFLYSKLTVDKS PHYYGSEVTTKML RWQQGNVFSCSVMHE
CAADPQWKTDSCQ ALHNHYTQKSLSLSP GDSGGPLVCSLQG G RMTLTGIVSWGRG
(280-505) CALKDKPGVYTRV SHFLPWIRSHTKE ENGLAL (21-273) 1007 uPA
MYRMQLLSCI IIGGEFTTIE GGSSGG None DAHKSEVAHRFKDLG (SEQ ID
ALSLALVTNS NQPWFAAIYQ (274-279) EENFKALVLIAFAQY NO: 21)- (1-20)
RYEGGSEYYR LQQCPFEDHVKLVNE HSA CGGSLISPCW VTEFAKTCVADESAE (No PP)
VISATHCFIP NCDKSLHTLFGDKLC QPKKEDYIVY TVATLRETYGEMADC LGRSRLNSNT
CAKQEPERNECFLQH QGEMKFEVEN KDDNPNLPRLVRPEV LILHKDYSAD
DVMCTAFHDNEETFL IAAQHNDIAL KKYLYEIARRHPYFY LKIRSKEGRC
APELLFFAKRYKAAF AQPSRTIQTI TECCQAADKAACLLP SLPSMYNDPQ
KLDELRDEGKASSAK FGTSCEITGF QRLKCASLQKFGERA GKENSTDRLY
FKAWAVARLSQRFPK PEQLKMTVVK AEFAEVSKLVTDLTK LISHRECQQP
VHTECCHGDLLECAD HYYGSEVTTK DRADLAKYICENQDS MLCAADPQWK
ISSKLKECCEKPLLE TDSCQGDSGG KSHCIAEVENDEMPA PLVCSLQGRM
DLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMF GCALKDKPGV
LYEYARRHPDYSVVL YTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENG
CAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ (21-273) NCELFEQLGEYKFQN
ALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAE DYLSVVLNQLCVLHE
KTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFH ADICTLSEKERQIKK
QTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAE EGKKLVAASQAALGL
(280-864) 1008 uPA MYRMQLLSCI IIGGEFTTIE GGSSGG None
QVQLQQPGADLVRPG (SEQ ID ALSLALVTNS NQPWFAAIYQ (274-279)
VSVKLSCKASGYTFT NO: 21)- (1-20) RYEGGSEYYR SYWMNWVKQRPGQGL C2scFv
CGGSLISPCW EWIGMIHPSDSETRL (No PP) VISATHCFIP SQKFKDKATLTVDKS
QPKKEDYIVY SSTAYMQLSSPTSED LGRSRLNSNT SAVYYCARLKPGGTW QGEMKFEVEN
FAYWGQGTLVTVSAG LILHKDYSAD GGGSGGGGSGGGGSG IAAQHNDIAL
GSDIVLTQSPASLTV LKIRSKEGRC SLGQRATISCRASKS AQPSRTIQTI
VDSYGNSFMEWYQQK SLPSMYNDPQ PGQPPKLLIYRASNL FGTSCEITGF
ESGIPARFSGSGSRT GKENSTDRLY DFTLTINPVEADDVA PEQLKMTVVK
TYYCQQSNEDPYTFG LISHRECQQP GGTKLEIK HYYGSEVTTK (280-527) MLCAADPQWK
TDSCQGDSGG PLVCSLQGRM TLTGIVSWGR GCALKDKPGV YTRVSHFLPW IRSHTKEENG
LAL (21-273) 1009 uPA MYRMQLLSCI IIGGEFTTIENQP GGSSGG None
ERAAGVYHRE (SEQ ID ALSLALVTNS WFAAIYQRYEGGS (274-279) ARSGKYKLTY
NO: 21)- (1-20) EYYRCGGSLISPC AEAKAVCEFE HABD WVISATHCFIPQP
GGHLATYKQL (No PP) KKEDYIVYLGRSR EAARKIGFHV LNSNTQGEMKFEV
CAAGWMAKGR ENLILHKDYSAD VGYPIVKPGP IAAQHNDIAL NCGFGKTGII LKIRSKEGRC
DYGIRLNRSE AQPSRTIQTI RWDAYCYNPH SLPSMYNDPQ AKE FGTSCEITGF
(280-382) GKENSTDRLY PEQLKMTVVK LISHRECQQP HYYGSEVTTK MLCAADPQWK
TDSCQGDSGG PLVCSLQGRM TLTGIVSWGR GCALKDKPGV YTRVSHFLPW IRSHTKEENG
LAL (21-273) 1010 uPA METDTLLLWV IIGGEFTTIENQP GGSSGG QSGQKTL
DKTHTCPPCP (SEQ ID LLLWVPGSTG WFAAIYQRYEGGS (285-290) RRKR
APELLGGPSV NO: 21)- (1-20) EYYRCGGSLISPC (21-31) FLFPPKPKDT Fc
WVISATHCFIPQP LMISRTPEVT (Furin) KKEDYIVYLGRSR CVVVDVSHED
LNSNTQGEMKFEV PEVKFNWYVD ENLILHKDYSADI GVEVHNAKTK AAQHNDIALLKIR
PREEQYNSTY SKEGRCAQPSRTI RVVSVLTVLH QTISLPSMYNDPQ QDWLNGKEYK
FGTSCEITGFGKE CKVSNKALPA NSTDRLYPEQLKM PIEKTISKAK TVVKLISHRECQQ
GQPREPQVYT PHYYGSEVTTKML LPPSRDELTK CAADPQWKTDSCQ NQVSLTCLVK
GDSGGPLVCSLQG GFYPSDIAVE RMTLTGIVSWGRG WESNGQPENN CALKDKPGVYTRV
YKTTPPVLDS SHFLPWIRSHTKE DGSFFLYSKL ENGLAL TVDKSRWQQG (32-284)
NVFSCSVMHE ALHNHYTQKS LSLSPG (291-516) 1011 uPA N- METDTLLLWV
IIGGEFTTIENQ GGSSGG QCGQ DKTHTCPPCP SNELHQVPSNCDCL Term- LLLWVPGSTG
PWFAAIYQRYEG (432-437) KTLRPRFK APELLGGPSV NGGTCVSNKYFSNI uPA
(1-20) GSEYYRCGGSLI (167-178) FLFPPKPKDT HWCNCPKKFGGQHC (SEQ ID
SPCWVISATHCF LMISRTPEVT EIDKSKTCYEGNGH NO: 987)- IPQPKKEDYIVY
CVVVDVSHED FYRGKASTDTMGRP Fc LGRSRLNSNTQG PEVKFNWYVD CLPWNSATVLQQTY
EMKFEVENLILH GVEVHNAKTK HAHRSDALQLGLGK KDYSADIAAQHN PREEQYNSTY
HNYCRNPDNRRRPW DIALLKIRSKEG RVVSVLTVLH CYVQVGLKPLVQEC RCAQPSRTIQTI
QDWLNGKEYK MVHDCADGKKPSSP CLPSMYNDPQFG CKVSNKALPA PEELKF
TSCEITGFGKEN PIEKTISKAK (21-166) STDRLYPEQLKM GQPREPQVYT
TVVKLISHRECQ LPPSRDELTK QPHYYGSEVTTK NQVSLTCLVK MLCAADPQWKTD
GFYPSDIAVE SCQGDSGGPLVC WESNGQPENN SLQGRMTLTGIV YKTTPPVLDS
SWGRGCALKDKP DGSFFLYSKL GVYTRVSHFLPW TVDKSRWQQG IRSHTKEENGLA
NVFSCSVMHE L ALHNHYTQKS (179-431) LSLSPG (438-663) 1012 WT
MYRMQLLSCI IIGGEFTTIENQP GGSSGG None DKTHTCPPCPAPELL uPA ALSLALVTNS
WFAAIYRRHRGGS (274-279) GGPSVFLFPPKPKDT with (1-20) VTYVCGGSLMSPC
LMISRTPEVTCVVVD C122S- WVISATHCFIDYP VSHEDPEVKFNWYVD Fc (No
KKEDYIVYLGRSR GVEVHNAKTKPREEQ PP) LNSNTQGEMKFEV YNSTYRVVSVLTVLH
ENLILHKDYSADT QDWLNGKEYKCKVSN LAHHNDIALLKIR KALPAPIEKTISKAK
SKEGRCAQPSRTI GQPREPQVYTLPPSR QTISLPSMYNDPQ DELTKNQVSLTCLVK
FGTSCEITGFGKE GFYPSDIAVEWESNG NSTDYLYPEQLKM QPENNYKTTPPVLDS
TVVKLISHRECQQ DGSFFLYSKLTVDKS PHYYGSEVTTKML RWQQGNVFSCSVMHE
CAADPQWKTDSCQ ALHNHYTQKSLSLSP GDSGGPLVCSLQG G RMTLTGIVSWGRG
(280-505) CALKDKPGVYTRV SHFLPWIRSHTKE ENGLAL (21-273) 1013 WT
MYRMQLLSCI IIGGEFTTIEN GGSSGG None DAHKSEVAHRFKDLG uPA ALSLALVTNS
QPWFAAIYRRH (274-279) EENFKALVLIAFAQY with (1-20) RGGSVTYV
LQQCPFEDHVKLVNE C122S - CGGSLMSPCW VTEFAKTCVADESAE HSA VISATHCFID
NCDKSLHTLFGDKLC (No PP) YPKKEDYIVY TVATLRETYGEMADC LGRSRLNSNT
CAKQEPERNECFLQH QGEMKFEVEN KDDNPNLPRLVRPEV LILHKDYSAD
DVMCTAFHDNEETFL TLAHHNDIAL KKYLYEIARRHPYFY LKIRSKEGRC
APELLFFAKRYKAAF AQPSRTIQTI TECCQAADKAACLLP SLPSMYNDPQ
KLDELRDEGKASSAK FGTSCEITGF QRLKCASLQKFGERA GKENSTDYLY
FKAWAVARLSQRFPK PEQLKMTVVK AEFAEVSKLVTDLTK LISHRECQQP
VHTECCHGDLLECAD HYYGSEVTTK DRADLAKYICENQDS MLCAADPQWK
ISSKLKECCEKPLLE TDSCQGDSGG KSHCIAEVENDEMPA PLVCSLQGRM
DLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMF GCALKDKPGV
LYEYARRHPDYSVVL YTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENG
CAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ (21-273) NCELFEQLGEYKFQN
ALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAE DYLSVVLNQLCVLHE
KTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFH ADICTLSEKERQIKK
QTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAE EGKKLVAASQAALGL
(280-864) 1036 uPA N- METDTLLLWV IIGGEFTTIENQ GGSSGG QCGQ
DKTHTCPPCPAPELL SNELHQVPSNCDC term- LLLWVPGSTG PWFAAIYQRYEG
(432-437) KTLRRRKR GGPSVFLFPPKPKDT LNGGTCVSNKYFS uPA (1-20)
GSEYYRCGGSLI (167-178) LMISRTPEVTCVVVD NIHWCNCPKKFGG (SEQ ID
SPCWVISATHCF VSHEDPEVKFNWYVD QHCEIDKSKTCYE NO: 987)- IPQPKKEDYIVY
GVEVHNAKTKPREEQ GNGHFYRGKASTD Fc (Furin) LGRSRLNSNTQG
YNSTYRVVSVLTVLH TMGRPCLPWNSAT EMKFEVENLILH QDWLNGKEYKCKVSN
VLQQTYHAHRSDA KDYSADIAAQHN KALPAPIEKTISKAK LQLGLGKHNYCRN
DIALLKIRSKEG GQPREPQVYTLPPSR PDNRRRPWCYVQV RCAQPSRTIQTI
DELTKNQVSLTCLVK GLKPLVQECMVHD CLPSMYNDPQFG GFYPSDIAVEWESNG
CADGKKPSSPPEE
TSCEITGFGKEN QPENNYKTTPPVLDS LKF STDRLYPEQLKM DGSFFLYSKLTVDKS
(21-166) TVVKLISHRECQ RWQQGNVFSCSVMHE QPHYYGSEVTTK ALHNHYTQKSLSLSP
MLCAADPQWKTD G SCQGDSGGPLVC (438-663) SLQGRMTLTGIV SWGRGCALKDKP
GVYTRVSHFLPW IRSHTKEENGLA L (179-431) 1014 uPA N- METDTLLLWV
IIGGEFTTIENQ GGSSGG QCGQ DAHKSEVAHRFKDLG SNELHQVPSNCDCL term-
LLLWVPGSTG PWFAAIYQRYEG (432-437) KTLRRRKR EENFKALVLIAFAQY
NGGTCVSNKYFSNI uPA (1-20) GSEYYRCGGSLI (167-178) LQQCPFEDHVKLVNE
HWCNCPKKFGGQHC (SEQ ID SPCWVISATHCF VTEFAKTCVADESAE EIDKSKTCYEGNGH
NO: 987)- IPQPKKEDYIVY NCDKSLHTLFGDKLC FYRGKASTDTMGRP HSA (Furin)
LGRSRLNSNTQG TVATLRETYGEMADC CLPWNSATVLQQTY EMKFEVENLILH
CAKQEPERNECFLQH HAHRSDALQLGLGK KDYSADIAAQHN KDDNPNLPRLVRPEV
HNYCRNPDNRRRPW DIALLKIRSKEG DVMCTAFHDNEETFL CYVQVGLKPLVQEC
RCAQPSRTIQTI KKYLYEIARRHPYFY MVHDCADGKKPSSP CLPSMYNDPQFG
APELLFFAKRYKAAF PEELKF TSCEITGFGKEN TECCQAADKAACLLP (21-166)
STDRLYPEQLKM KLDELRDEGKASSAK TVVKLISHRECQ QRLKCASLQKFGERA
QPHYYGSEVTTK FKAWAVARLSQRFPK MLCAADPQWKTD AEFAEVSKLVTDLTK
SCQGDSGGPLVC VHTECCHGDLLECAD SLQGRMTLTGIV DRADLAKYICENQDS
SWGRGCALKDKP ISSKLKECCEKPLLE GVYTRVSHFLPW KSHCIAEVENDEMPA
IRSHTKEENGLA DLPSLAADFVESKDV L CKNYAEAKDVFLGMF (179-431)
LYEYARRHPDYSVVL LLRLAKTYETTLEKC CAAADPHECYAKVFD EFKPLVEEPQNLIKQ
NCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAE
DYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFH
ADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAE
EGKKLVAASQAALGL (438-1022) 1015 uPA N- METDTLLLWV IIGGEFTTIE GGSSGG
QCGQ DAHKSEVAHRFKDLG SNELHQVPSN term- LLLWVPGSTG NQPWFAAIYQ
(432-437) KTLRPRFK EENFKALVLIAFAQY CDCLNGGTCV uPA (1-20) RYEGGSEYYR
(167-178) LQQCPFEDHVKLVNE SNKYFSNIHW (SEQ ID CGGSLISPCW
VTEFAKTCVADESAE CNCPKKFGGQ NO: 987)- VISATHCFIP NCDKSLHTLFGDKLC
HCEIDKSKTC HSA QPKKEDYIVY TVATLRETYGEMADC YEGNGHFYRG LGRSRLNSNT
CAKQEPERNECFLQH KASTDTMGRP QGEMKFEVEN KDDNPNLPRLVRPEV CLPWNSATVL
LILHKDYSAD DVMCTAFHDNEETFL QQTYHAHRSD IAAQHNDIAL KKYLYEIARRHPYFY
ALQLGLGKHN LKIRSKEGRC APELLFFAKRYKAAF YCRNPDNRRR AQPSRTIQTI
TECCQAADKAACLLP PWCYVQVGLK CLPSMYNDPQ KLDELRDEGKASSAK PLVQECMVHD
FGTSCEITGF QRLKCASLQKFGERA CADGKKPSSP GKENSTDRLY FKAWAVARLSQRFPK
PEELKF PEQLKMTVVK AEFAEVSKLVTDLTK (21-166) LISHRECQQP
VHTECCHGDLLECAD HYYGSEVTTK DRADLAKYICENQDS MLCAADPQWK
ISSKLKECCEKPLLE TDSCQGDSGG KSHCIAEVENDEMPA PLVCSLQGRM
DLPSLAADFVESKDV TLTGIVSWGR CKNYAEAKDVFLGMF GCALKDKPGV
LYEYARRHPDYSVVL YTRVSHFLPW LLRLAKTYETTLEKC IRSHTKEENG
CAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ 179-431 NCELFEQLGEYKFQN
ALLVRYTKKVPQVST PTLVEVSRNLGKVGS KCCKHPEAKRMPCAE DYLSVVLNQLCVLHE
KTPVSDRVTKCCTES LVNRRPCFSALEVDE TYVPKEFNAETFTFH ADICTLSEKERQIKK
QTALVELVKHKPKAT KEQLKAVMDDFAAFV EKCCKADDKETCFAE EGKKLVAASQAALGL
(438-1022) 1016 uPA METDTLLLWV IIGGEFTTIE GGSSGG QSGQKTL
DAHKSEVAHRFKDLG (SEQ ID LLLWVPGSTG NQPWFAAIYQ (286-291) RRRKR
EENFKALVLIAFAQY NO: 21)- (1-20) RYEGGSEYYR (21-32) LQQCPFEDHVKLVNE
HSA CGGSLISPCW VTEFAKTCVADESAE (Furin) VISATHCFIP NCDKSLHTLFGDKLC
QPKKEDYIVY TVATLRETYGEMADC LGRSRLNSNT CAKQEPERNECFLQH QGEMKFEVEN
KDDNPNLPRLVRPEV LILHKDYSAD DVMCTAFHDNEETFL IAAQHNDIAL
KKYLYEIARRHPYFY LKIRSKEGRC APELLFFAKRYKAAF AQPSRTIQTI
TECCQAADKAACLLP SLPSMYNDPQ KLDELRDEGKASSAK FGTSCEITGF
QRLKCASLQKFGERA GKENSTDRLY FKAWAVARLSQRFPK PEQLKMTVVK
AEFAEVSKLVTDLTK LISHRECQQP VHTECCHGDLLECAD HYYGSEVTTK
DRADLAKYICENQDS MLCAADPQWK ISSKLKECCEKPLLE TDSCQGDSGG
KSHCIAEVENDEMPA PLVCSLQGRM DLPSLAADFVESKDV TLTGIVSWGR
CKNYAEAKDVFLGMF GCALKDKPGV LYEYARRHPDYSVVL YTRVSHFLPW
LLRLAKTYETTLEKC IRSHTKEENG CAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ
(33-285) NCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGS
KCCKHPEAKRMPCAE DYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDE
TYVPKEFNAETFTFH ADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFV
EKCCKADDKETCFAE EGKKLVAASQAALGL (292-876) 1017 SUMO- METDTLLLWV
IIGGEFTTIE GGSSGG -- DAHKSEVAHRFKDLG DGHHHHHHGSLQD uPA LLLWVPGSTG
NQPWFAAIYQ (382-387) EENFKALVLIAFAQY SEVNQEAKPEVKP (SEQ ID (1-20)
RYEGGSEYYR LQQCPFEDHVKLVNE EVKPETHINLKVS NO: 21)- CGGSLISPCW
VTEFAKTCVADESAE DGSSEIFFKIKKT HSA VISATHCFIP NCDKSLHTLFGDKLC
TPLRRLMEAFAKR QPKKEDYIVY TVATLRETYGEMADC QGKEMDSLTFLYD LGRSRLNSNT
CAKQEPERNECFLQH GIEIQADQTPEDL QGEMKFEVEN KDDNPNLPRLVRPEV
DMEDNDIIAHREQ LILHKDYSAD DVMCTAFHDNEETFL IGG IAAQHNDIAL
KKYLYEIARRHPYFY (21-128) LKIRSKEGRC APELLFFAKRYKAAF AQPSRTIQTI
TECCQAADKAACLLP SLPSMYNDPQ KLDELRDEGKASSAK FGTSCEITGF
QRLKCASLQKFGERA GKENSTDRLY FKAWAVARLSQRFPK PEQLKMTVVK
AEFAEVSKLVTDLTK LISHRECQQP VHTECCHGDLLECAD HYYGSEVTTK
DRADLAKYICENQDS MLCAADPQWK ISSKLKECCEKPLLE TDSCQGDSGG
KSHCIAEVENDEMPA PLVCSLQGRM DLPSLAADFVESKDV TLTGIVSWGR
CKNYAEAKDVFLGMF GCALKDKPGV LYEYARRHPDYSVVL YTRVSHFLPW
LLRLAKTYETTLEKC IRSHTKEENG CAAADPHECYAKVFD LAL EFKPLVEEPQNLIKQ
(129-381) NCELFEQLGEYKFQN ALLVRYTKKVPQVST PTLVEVSRNLGKVGS
KCCKHPEAKRMPCAE DYLSVVLNQLCVLHE KTPVSDRVTKCCTES LVNRRPCFSALEVDE
TYVPKEFNAETFTFH ADICTLSEKERQIKK QTALVELVKHKPKAT KEQLKAVMDDFAAFV
EKCCKADDKETCFAE EGKKLVAASQAALGL (388-972) 1018 SUMO- METDTLLLWV
IIGGEFTTIE GGSSGG -- DKTHTCPPCPAPELL DGHHHHHHGSLQD uPA LLLWVPGSTG
NQPWFAAIYQ (382-387) GGPSVFLFPPKPKDT SEVNQEAKPEVKP (SEQ ID (1-20)
RYEGGSEYYR LMISRTPEVTCVVVD EVKPETHINLKVS NO: 21)- CGGSLISPCW
VSHEDPEVKFNWYVD DGSSEIFFKIKKT Fc VISATHCFIP GVEVHNAKTKPREEQ
TPLRRLMEAFAKR QPKKEDYIVY YNSTYRVVSVLTVLH QGKEMDSLTFLYD LGRSRLNSNT
QDWLNGKEYKCKVSN GIEIQADQTPEDL QGEMKFEVEN KALPAPIEKTISKAK
DMEDNDIIEAHRE LILHKDYSAD GQPREPQVYTLPPSR QIGG IAAQHNDIAL
DELTKNQVSLTCLVK (21-128) LKIRSKEGRC GFYPSDIAVEWESNG AQPSRTIQTI
QPENNYKTTPPVLDS SLPSMYNDPQ DGSFFLYSKLTVDKS FGTSCEITGF
RWQQGNVFSCSVMHE GKENSTDRLY ALHNHYTQKSLSLSP PEQLKMTVVK G LISHRECQQP
(388-613) HYYGSEVTTK MLCAADPQWK TDSCQGDSGG PLVCSLQGRM TLTGIVSWGR
GCALKDKPGV YTRVSHFLPW IRSHTKEENG LAL (129-381) 1037 6xHis- --
IIGGEFTTIENQ -- MGHHHHHHGSLQD sumo- PWFAAIYQRYEG SEVNQEAKPEVKP uPA
GSEYYRCGGSLI EVKPETHINLKVS (SEQ ID SPCWVISATHCF DGSSEIFFKIKKT NO:
987) IPQPKKEDYIVY TPLRRLMEAFAKR LGRSRLNSNTQG QGKEMDSLRFLYD
EMKFEVENLILH GIRIQADQAPEDL KDYSADIAAQHN DMEDNDIIEAHRE DIALLKIRSKEG
QIGG RCAQPSRTIQTI (1-108) CLPSMYNDPQFG TSCEITGFGKEN STDRLYPEQLKM
TVVKLISHRECQ QPHYYGSEVTTK MLCAADPQWKTD SCQGDSGGPLVC SLQGRMTLTGIV
SWGRGCALKDKP GVYTRVSHFLPW IRSHTKEENGLA L (109-361) 1038 6xHis --
IIGGEFTTIENQ -- MGHHHHHHGSLQD sumo- PWFAAIYQRYEG SEVNQEAKPEVKP uPA
GSEYYRCGGSLI EVKPETHINLKVS (SEQ ID SPCWVISATHCF DGSSEIFFKIKKT NO:
21) IPQPKKEDYIVY TPLRRLMEAFAKR LGRSRLNSNTQG QGKEMDSLRFLYD
EMKFEVENLILH GIRIQADQAPEDL KDYSADIAAQHN DMEDNDIIEAHRE DIALLKIRSKEG
QIGG RCAQPSRTIQTI (1-108) SLPSMYNDPQFG TSCEITGFGKEN STDRLYPEQLKM
TVVKLISHRECQ QPHYYGSEVTTK MLCAADPQWKTD SCQGDSGGPLVC SLQGRMTLTGIV
SWGRGCALKDKP
GVYTRVSHFLPW IRSHTKEENGLA L (109-361) 1039 6xHis -- IIGGEFTTIENQ --
GGSCK MGHHHHHHGSLQD sum- PWFAAIYQRYEG (362-366) SEVNQEAKPEVKP uPA
GSEYYRCGGSLI EVKPETHINLKVS (SEQ ID SPCWVISATHCF DGSSEIFFKIKKT NO:
21)- IPQPKKEDYIVY TPLRRLMEAFAKR GGSCK LGRSRLNSNTQG QGKEMDSLRFLYD
EMKFEVENLILH GIRIQADQAPEDL KDYSADIAAQHN DMEDNDIIEAHRE DIALLKIRSKEG
QIGG RCAQPSRTIQTI (1-108) SLPSMYNDPQFG TSCEITGFGKEN STDRLYPEQLKM
TVVKLISHRECQ QPHYYGSEVTTK MLCAADPQWKTD SCQGDSGGPLVC SLQGRMTLTGIV
SWGRGCALKDKP GVYTRVSHFLPW IRSHTKEENGLA L (109-361)
[0914] FIG. 3 sets forth schematics of the u-PA polypeptides with
C-terminal fusions, such as the C-terminal fusion polypeptides set
forth in SEQ ID NOS: 1006-1018 and 1036.
[0915] I. Assays for assessing u-PA amounts and complement pathway
activity
[0916] The u-PA polypeptide fusions were produced after
transfection of the pcDNA3_4 vector encoding altered and native
u-PA polypeptide fusion in a mammalian expression system in
Expi293.TM. cells. The u-PA polypeptides that correctly expressed
in Expi293.TM. cells were purified on a HiTrap Protein A HP or
CaptureSelect.TM. Human Albumin Affinity Matrix and processed for
bioanalytical assays such as a u-PA ELISA to examine the uPA titers
or a C3 FRET proteolytic cleavage assay to examine u-PA polypeptide
catalytic activity. The results are set forth in the table
below:
TABLE-US-00056 Total Proteolytic Proteolytic amount of uPA uPA
activity activity on SEQ protein ELISA ELISA on C3 C3 relative ID
from 1L titer titer relative to to uPA NO: A280 culture' (mg/L)
(mg/L) A280 ELISA titer 1004 0.4 0.4 1005 149.7 75.39 1006 2.6 2.2
1007 0.3 0.3 1008 0.1 0.06 1009 0.3 0.26 1010 4.23 36.4 mg 17.3
15.89 110.36 773.05 mg/ml 1011 1.69 23.0 mg 19.3 21.66 0.06 0.1
mg/ml 1012 16.2 7.5 1013 4.7 5.46 1036 3.32 42.5 mg mg/ml 1014
1015u 3.43 70.3 mg mg/ml 1015a 1.16 44.7 mg mg/ml 1015i 1016 1017
3.32 89.3 mg mg/ml 1018 1.10 11 mg mg/ml
[0917] 1. u-PA ELISA levels
[0918] An enzyme linked immunosorbent assay (ELISA) is used to
measure the presence of u-PA polypeptides (see, e.g., Example 15).
Typically, the measurement of u-PA is an indirect measure of the
binding of u-PA to a capture antibody (PA1-36166 at 1.0 ug/mL,
Invitrogen). The captured u-PA polypeptide is then detected with a
detection antibody (PA1-36015 at 0.25 ug/mL) which is recognized by
the HRP conjugated anti Goat antibody (Rockland, 605-403-B69). The
HRP enzyme triggers a colorimetric reaction upon addition of the
TMB substrate. Using the u-PA ELISA method, four u-PA polypeptides
were identified to express at high uPA titer levels (SEQ ID NOS:
1005, 1010, 1011, 1012, 1015), two u-PA polypeptides at medium
titers (SEQ ID NOS: 1006 and 1013), and u-PA polypeptides set forth
in SEQ ID NOS: 1004, 1007-1009 did not express.
[0919] 2. Enzyme Activity (Human C3 FRET Peptide).
[0920] The proteolytic activity of uPA polypeptides on human C3 was
measured in vitro using a human C3 FRET peptide RHQARASHL
EDANS/DABCYL produced by Genscript (lot #94045990005/PE6379) (see,
e.g., Example 15). The N-terminal side of the peptide is labeled
with a DABCYL fluorophore, and the C-terminal side is labeled with
an EDANS fluorophore. Cleavage of the peptide separates the
EDANS/DABCYL FRET pair to generate a fluorescent signal, which is
measured in a multi-well plate reader. The rate of generation of
fluorescence intensity is interpolated against an EDANS standard
curve to yield the EDANS product generation rate. The product
generation rate is multiplied by the dilution factor to yield a
volumetric specific activity in units of nmol product per minute of
reaction per mL of sample (nmol/min/mL). The volumetric specific
activity indicates the total amount of active enzyme in the sample.
The second specific activity is calculated by dividing the
volumetric specific activity by the sample enzyme concentration to
yield an enzyme specific activity in units of nmol product per
minute of reaction per mg of enzyme (nmol/min/mg). The enzyme
specific activity indicates the intrinsic activity of uPA
polypeptides in the sample regardless of the concentration. Using
the human C3 FRET activity assay, uPA polypeptide set forth in SEQ
ID NO: 1010 was shown to be active.
[0921] Since modifications will be apparent to those of skill in
this art, it is intended that this invention be limited only by the
scope of the appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210222143A9).
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210222143A9).
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