U.S. patent application number 15/653201 was filed with the patent office on 2017-11-30 for anti-htra1 antibodies and methods of use.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Kenneth J. KATSCHKE, Jr., Daniel KIRCHHOFER, Wei-Ching LIANG, Michael Terry LIPARI, Paul M. MORAN, Scott STAWICKI, Menno van LOOKEREN CAMPAGNE, Yan WU.
Application Number | 20170342163 15/653201 |
Document ID | / |
Family ID | 48082468 |
Filed Date | 2017-11-30 |
United States Patent
Application |
20170342163 |
Kind Code |
A1 |
WU; Yan ; et al. |
November 30, 2017 |
ANTI-HtrA1 ANTIBODIES AND METHODS OF USE
Abstract
The invention provides anti-HtrA1 antibodies and methods of
using the same.
Inventors: |
WU; Yan; (Foster City,
CA) ; van LOOKEREN CAMPAGNE; Menno; (San Francisco,
CA) ; KIRCHHOFER; Daniel; (Los Altos, CA) ;
LIPARI; Michael Terry; (Santa Clara, CA) ; KATSCHKE,
Jr.; Kenneth J.; (Fairfield, CA) ; MORAN; Paul
M.; (El Cerrito, CA) ; STAWICKI; Scott; (San
Francisco, CA) ; LIANG; Wei-Ching; (Foster City,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
48082468 |
Appl. No.: |
15/653201 |
Filed: |
July 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13651289 |
Oct 12, 2012 |
9738727 |
|
|
15653201 |
|
|
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61547649 |
Oct 14, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/76 20130101;
A61P 9/10 20180101; C07K 16/40 20130101; A61P 27/02 20180101; C07K
2317/34 20130101; A61P 43/00 20180101; C07K 2317/92 20130101; A61P
27/00 20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. An isolated antibody that binds to HtrA1 competitively with an
antibody comprising a VH sequence of SEQ ID NO:8 and a VL sequence
of SEQ ID NO:7.
2. The antibody of claim 1, wherein competitive binding is
determined using an ELISA assay.
3. An isolated antibody that binds to HtrA1, wherein the antibody
(i) binds to an epitope that includes N224, K248, or both of HtrA1,
and (ii) inhibits HtrA1 with an IC.sub.50 of .ltoreq.30 nM.
4. The antibody of claim 3, wherein the antibody further comprises
one or more of the following properties: (i) binds to HtrA1 with a
ratio of 1 variable domain to one subunit of an HtrA1 trimer, or
(ii) does not prevent trimer formation of HtrA1.
5. The antibody of claim 3, wherein the IC.sub.50 is determined
using a serine protease assay with a substrate having SEQ ID
NO:12.
6-90. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/547,649, filed Oct. 14, 2011, which
application is hereby incorporated by reference in its
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing
submitted in ASCII format via EFS-Web and hereby incorporated by
reference in its entirety. Said ASCII copy, created on Oct. 4,
2012, is named P4761RUS.txt and is 47,451 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to anti-HtrA1 antibodies and
methods of using the same.
BACKGROUND
[0004] The serine protease HtrA1 (PRSS11; Clan PA, family S1)
belongs to an evolutionarily conserved family of HtrA proteins
(Clausen, T., et al., Nat Rev Mol Cell Biol 12:152-62 (2011);
Clausen, T., et al., Mol Cell 10:443-55 (2002)). In humans, HtrA1,
3, and 4 share the same domain architecture: an N-terminal
IGFBP-like module and a Kazal-like module, a protease domain with
trypsin-like fold, and a C-terminal PDZ domain. The physiological
relevance of HtrA1 has been firmly established by the
identification of human loss-of-function mutations causing familial
ischemic cerebral small-vessel disease (Hara, K., et al., N Engl J
Med 360:1729-39 (2009)). The molecular mechanism involves deficient
TGF-.beta. inhibition by HtrA1 resulting in increased TGF-.beta.
signaling (Hara et al., 2009). Dysregulated TGF-.beta. signaling by
aberrant HtrA1 expression may also contribute to arthritic disease
(Oka, C., et al., Development 131:1041-53 (2004); Tsuchiya, A., et
al., Bone 37:323-36 (2005)), perhaps in conjunction with
HtrA1-mediated degradation of various extracellular matrix
components (Chamberland et al., J Biol Chem 284:27352-9 (2009);
Grau, S., et al., J Biol Chem 281:6124-9 (2006); Hadfield, K. D.,
et al., J Biol Chem 283:5928-38 (2008); Tocharus, J., et al., Dev
Growth Differ 46:257-74 (2004); Tsuchiya et al., 2005)), or
indirectly via up-regulation of matrix metalloproteases (Grau et
al., 2006). In addition, human genetic studies identified a strong
correlation between progression of age-related macular degeneration
and a SNP in the HtrA1 promoter region, which results in increased
HtrA1 transcript levels (Dewan, A., et al., Science 314:989-92
(2006); Yang, Z., et al., Science 314:992-3 (2006)). Therefore,
inhibition of HtrA1 enzymatic function is an attractive therapeutic
approach, e.g. in age-related macular degeneration and in arthritic
disease.
SUMMARY
[0005] The invention provides anti-HtrA1 antibodies and methods of
using the same for diagnostic and therapeutic purposes.
[0006] In one aspect, the invention provides an isolated antibody
that binds to HtrA1 competitively with antibody comprising a VH
sequence of SEQ ID NO:8 and a VL sequence of SEQ ID NO:7.
Competitive binding may be determined, for example, using an ELISA
assay.
[0007] In one aspect, the invention provides an isolated antibody
that bind to HtrA1 having one or more of the following properties:
(i) an IC.sub.50 of less than 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10
nM, 5 nM, 3 nM, 2.5 nM, 2 nM, 1 nM, or less for one or more HtrA1
substrates; (ii) binds to HtrA1 with a ratio of 1 variable domain
to one subunit of an HtrA1 trimer (e.g., a Fab binds to an HtrA1
trimer with a ratio of 3 Fab to 1 HtrA1 trimer, and an IgG binds to
an HtrA1 trimer with a ratio of 3 IgG to 2 HtrA1 trimers), (iii)
for antibodies comprising two variable domains, binds to HtrA1 in a
manner that results in the forming a "cage" similar to that shown
in FIG. 9, (iv) does not prevent trimer formation of HtrA1, (v)
binds to one or more residues in Loop C of the HtrA1 protein, (vi)
binds to the protease domain of HtrA1, (vii) binds to an epitope
comprising one or both of amino acids N224 or K248 of SEQ ID NO:13,
or amino acids equivalent thereto in a different HtrA1 sequence
(e.g., amino acids N224 and K248 of SEQ ID NO: 14, see FIGS. 10A
and B); (viii) binds to an epitope comprising one or more of the
following residues of N224, K248, V201, T223, K243, K225, E247 and
H220 of SEQ ID NO: 13, or amino acids equivalent thereto in a
different HtrA1 sequence; (ix) cross-reacts with murine HtrA1; (x)
does not cross-react with HtrA2, HtrA3 and/or HtrA4; (xi) binds to
HtrA1 competitively with an antibody comprising a VH sequence of
SEQ ID NO:8 and a VL sequence of SEQ ID NO:7, (xii) binds to HtrA1
with a dissociation constant of .ltoreq.500 nM, or (xiii) inhibits
complex formation between HtrA1 and .alpha.1-antitrypsin
(A1AT).
[0008] In another aspect, the invention provides an isolated
antibody that binds to HtrA1, wherein the antibody (i) binds to an
epitope that includes N224, K248, or both of HtrA1, and (ii)
inhibits HtrA1 with an IC.sub.50 of .ltoreq.30 nM. In certain
embodiments, the epitope further includes one or more of the
following residues of HtrA1: V201, T223, K243, K225, E247 and H220.
In certain embodiments, the antibody may further comprise one or
more of the following properties: (i) binds to HtrA1 with a ratio
of 1 variable domain to one subunit of an HtrA1 trimer, or (ii)
does not prevent trimer formation of HtrA1. In certain embodiments,
the IC.sub.50 is determined using a serine protease assay with a
substrate having SEQ ID NO: 12, e.g., such as the FRET assay
described herein.
[0009] In certain embodiments, an antibody described herein does
not cross-react with one or more of HtrA2, HtrA3 and HtrA4.
[0010] In certain embodiments, an antibody described herein has a
dissociation constant of 500 nM. The dissociation constant may be
determined, for example, by BIAcore using a Fab.
[0011] In certain embodiments, an antibody described herein is a
monoclonal antibody.
[0012] In certain embodiments, an antibody described herein is a
human, humanized, or chimeric antibody.
[0013] In certain embodiments, an antibody described herein is an
antibody fragment that binds HtrA1.
[0014] In certain embodiments, an antibody described herein
comprises (a) HVR-H3 comprising the amino acid sequence
GTFLTX.sub.pWGHYFDY, wherein X.sub.p is S or T (SEQ ID NO: 27); (b)
HVR-L3 comprising the amino acid sequence
QQX.sub.gX.sub.hX.sub.iX.sub.jPX.sub.kT, wherein X.sub.g is S, V or
D; X.sub.h is Y, D or S; X is T, S, A, D or N; X.sub.j is T, H, N,
S, A, L or R; and X.sub.k is P, T, A or S (SEQ ID NO: 24); and (c)
HVR-H2 comprising the amino acid sequence WIDPYGGDTX.sub.oYADSVKG,
wherein X.sub.o is N or D (SEQ ID NO: 26).
[0015] In certain embodiments, an antibody described herein
comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:6; (b) HVR-L3 comprising the amino acid sequence of SEQ ID NO:3;
and (c) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5.
[0016] In certain embodiments, an antibody described herein
comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:6; (b) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
19; and (c) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5.
[0017] In certain embodiments, an antibody described herein
comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:6; (b) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:22; and (c) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5.
[0018] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence
GFX.sub.lIX.sub.mX.sub.nYYIH, wherein X.sub.l is N, S or T; X.sub.m
is S, D, Y or A; and X.sub.n is G or D (SEQ ID NO: 25); (b) HVR-H2
comprising the amino acid sequence WIDPYGGDTX.sub.oYADSVKG, wherein
X.sub.o is N or D (SEQ ID NO:26); and (c) HVR-H3 comprising the
amino acid sequence GTFLTX.sub.pWGHYFDY, wherein X.sub.p is S or T
(SEQ ID NO: 27).
[0019] In certain embodiments, an antibody described herein further
comprises (a) HVR-L1 comprising the amino acid sequence
RASQX.sub.aX.sub.bX.sub.cX.sub.dX.sub.eX.sub.fA, wherein X.sub.a is
D, S or V; X.sub.b is V or I; X.sub.c is S, N or G; X.sub.d is T or
N; X.sub.e is A or Y; and X.sub.f is V or L (SEQ ID NO: 23); (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (c)
HVR-L3 comprising the amino acid sequence
QQX.sub.gX.sub.hX.sub.iX.sub.jPX.sub.kT, wherein X.sub.g is S, V or
D; X.sub.h is Y, D or S; X.sub.i is T, S, A, D or N; X.sub.j is T,
H, N, S, A, L or R; and X.sub.k is P, T, A or S (SEQ ID NO:
24).
[0020] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5;
and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:6.
[0021] In certain embodiments, an antibody described herein further
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:3.
[0022] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:6.
[0023] In certain embodiments, an antibody described herein further
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:19.
[0024] In certain embodiments, an antibody described herein further
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:22.
[0025] In certain embodiments, an antibody described herein
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 1; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:3.
[0026] In certain embodiments, an antibody described herein
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO: 19.
[0027] In certain embodiments, an antibody described herein
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:21; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:22.
[0028] In certain embodiments, an antibody described herein further
comprises a heavy chain variable domain framework (FR2) sequence of
SEQ ID NO: 17.
[0029] In certain embodiments, an antibody described herein
comprises (a) a VH sequence having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO:8; (b) a VL sequence having
at least 95% sequence identity to the amino acid sequence of SEQ ID
NO:7; or (c) a VH sequence as in (a) and a VL sequence as in
(b).
[0030] In certain embodiments, an antibody described herein
comprises (a) a VH sequence having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO:29; (b) a VL sequence
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO:7; or (c) a VH sequence as in (a) and a VL sequence as in
(b).
[0031] In certain embodiments, an antibody described herein
comprises (a) a VH sequence having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO:29; (b) a VL sequence
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO:28; or (c) a VH sequence as in (a) and a VL sequence as
in (b).
[0032] In certain embodiments, an antibody described herein
comprises (a) a VH sequence having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO:29; (b) a VL sequence
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO:30; or (c) a VH sequence as in (a) and a VL sequence as
in (b).
[0033] In certain embodiments, an antibody described herein
comprises (a) a VH sequence comprising SEQ ID NO: 32; (b) a VL
sequence comprising SEQ ID NO: 31; or (c) a VH sequence comprising
SEQ ID NO: 32 and a VL sequence comprising SEQ ID NO: 31.
[0034] In certain embodiments, an antibody described herein
comprises a VH sequence of SEQ ID NO:8.
[0035] In certain embodiments, an antibody described herein
comprises a VL sequence of SEQ ID NO:7.
[0036] In certain embodiments, an antibody described herein
comprises a VH sequence of SEQ ID NO:30.
[0037] In certain embodiments, an antibody described herein
comprises a VL sequence of SEQ ID NO:28.
[0038] In certain embodiments, an antibody described herein
comprises a VL sequence of SEQ ID NO:29.
[0039] In certain embodiments, an antibody described herein
comprises a VH sequence of SEQ ID NO:8 and a VL sequence of SEQ ID
NO:7.
[0040] In certain embodiments, an antibody described herein
comprises a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID
NO:28.
[0041] In certain embodiments, an antibody described herein
comprises a VH sequence of SEQ ID NO:30 and a VL sequence of SEQ ID
NO:29.
[0042] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence
GFX.sub.lIX.sub.mX.sub.nYYIH, wherein X.sub.1 is N, S or T; X.sub.m
is S, D, Y or A; and X.sub.n is G or D (SEQ ID NO: 25); (b) HVR-H2
comprising the amino acid sequence WIDPYGGDTX.sub.oYADSVKG, wherein
X.sub.o is N or D (SEQ ID NO:26); (c) HVR-H3 comprising the amino
acid sequence GTFLTX.sub.pWGHYFDY, wherein X.sub.p is S or T (SEQ
ID NO: 27); (d) HVR-L1 comprising the amino acid sequence
RASQX.sub.aX.sub.bX.sub.cX.sub.dX.sub.eX.sub.fA, wherein X.sub.a is
D, S or V; X.sub.b is V or I; X.sub.c is S, N or G; X.sub.d is T or
N; X.sub.r is A or Y; and X.sub.f is V or L (SEQ ID NO: 23); (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence
QQX.sub.gX.sub.hX.sub.iX.sub.jPX.sub.kT, wherein X.sub.g is S, V or
D: X.sub.h is Y, D or S; X.sub.i is T, S, A, D or N; X.sub.j is T,
H, N, S, A, L or R; and X.sub.k is P, T, A or S (SEQ ID NO:
24).
[0043] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising an amino acid sequence selected
from: SEQ ID NO:4, 20, and 47-51; (b) HVR-H2 comprising an amino
acid sequence selected from: SEQ ID NO:5 and 52; (c) HVR-H3
comprising an amino acid sequence selected from: SEQ ID NO:6 and
53; (d) HVR-L1 comprising an amino acid sequence selected from: SEQ
ID NO: 1, 18, 21 and 33; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2; and (f) HVR-L3 comprising an amino acid
sequence selected from: SEQ ID NO:3, 19, 22, and 34-46.
[0044] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:3.
[0045] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 18; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:19.
[0046] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.
[0047] In certain embodiments, an antibody described herein
comprises (a) HVR-H3 comprising the amino acid sequence
GTFLTX.sub.pWGHY, wherein X.sub.p is S or T (SEQ ID NO: 89); (b)
HVR-L3 comprising the amino acid sequence
X.sub.gX.sub.hX.sub.iX.sub.jPX.sub.k, wherein X.sub.g is S, V or D;
X.sub.h is Y, D or S; X.sub.i is T, S, A, D or N; X.sub.j is T, H,
N, S, A, L or R; and X.sub.k is P, T. A or S (SEQ ID NO: 86); and
(c) HVR-H2 comprising the amino acid sequence WIDPYGGDTX.sub.o,
wherein X.sub.o is N or D (SEQ ID NO:88).
[0048] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence
GFX.sub.lIX.sub.mX.sub.nYY, wherein X.sub.l is N, S or T; X.sub.m
is S, D, Y or A; and X.sub.n is G or D (SEQ ID NO:87); (b) HVR-H2
comprising the amino acid sequence WIDPYGGDTX.sub.o, wherein
X.sub.o is N or D (SEQ ID NO:88); and (c) HVR-H3 comprising the
amino acid sequence GTFLTX.sub.pWGHY, wherein X.sub.p is S or T
(SEQ ID NO: 89).
[0049] In certain embodiments, an antibody described herein further
comprises (a) HVR-L1 comprising the amino acid sequence
X.sub.aX.sub.bX.sub.cX.sub.dX.sub.eX.sub.f, wherein X.sub.a is D, S
or V; X.sub.b is V or I; X.sub.c is S, N or G; X.sub.d is T or N;
X.sub.e is A or Y; and X.sub.f is V or L (SEQ ID NO: 85); (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:58; and (c)
HVR-L3 comprising the amino acid sequence
X.sub.gX.sub.hX.sub.iX.sub.jPX.sub.k, wherein X.sub.g is S, V or D;
X.sub.h is Y, D or S; X.sub.i is T, S, A, D or N; X.sub.j is T, H,
N, S, A, L or R; and X.sub.k is P, T, A or S (SEQ ID NO: 86).
[0050] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising the amino acid sequence
GFX.sub.lIX.sub.mX.sub.nYY, wherein X.sub.l is N, S or T; X.sub.m
is S, D, Y or A; and X.sub.n is G or D (SEQ ID NO:87); (b) HVR-H2
comprising the amino acid sequence WIDPYGGDTX.sub.o, wherein
X.sub.o is N or D (SEQ ID NO:88); (c) HVR-H3 comprising the amino
acid sequence GTFLTX.sub.pWGHY, wherein X.sub.p is S or T (SEQ ID
NO: 89); (d) HVR-L1 comprising the amino acid sequence
X.sub.aX.sub.bX.sub.cX.sub.dX.sub.eX.sub.f, wherein X.sub.a is D, S
or V; X.sub.b is V or I; X.sub.c is S, N or G; X.sub.d is T or N;
X.sub.e is A or Y; and X.sub.f is V or L (SEQ ID NO: 85); (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:58; and (f)
HVR-L3 comprising the amino acid sequence
X.sub.gX.sub.hX.sub.iX.sub.jPX.sub.k, wherein X, is S, V or D;
X.sub.h is Y, D or S; X.sub.i is T, S, A, D or N; X.sub.j is T, H,
N, S, A, L or R; and X.sub.k is P, T, A or S (SEQ ID NO:86).
[0051] In certain embodiments, an antibody described herein
comprises (a) HVR-H1 comprising an amino acid sequence selected
from: SEQ ID NO:4, 20, 47-51, and 75-80; (b) HVR-H2 comprising an
amino acid sequence selected from: SEQ ID NO:5, 52, and 81-82; (c)
HVR-H3 comprising an amino acid sequence selected from: SEQ ID
NO:6, 53 and 83-84; (d) HVR-L1 comprising an amino acid sequence
selected from: SEQ ID NO: 1, 18, 21, 33, and 54-57; (c) HVR-L2
comprising an amino acid sequence selected from: SEQ ID NO:2 and
58; and (f) HVR-L3 comprising an amino acid sequence selected from:
SEQ ID NO:3, 19, 22, 34-46, and 59-74.
[0052] In certain embodiments, an antibody described herein is a
full length IgG1 or IgG4 antibody.
[0053] In another aspect, an isolated nucleic acid encoding an
anti-HtrA1 antibody described herein is provided.
[0054] In another aspect, a host cell comprising an isolated
nucleic acid encoding an anti-HtrA1 antibody described herein is
provided.
[0055] In another aspect, a method of producing an antibody is
provided. The method may comprise culturing a host cell comprising
an isolated nucleic acid encoding an anti-HtrA1 antibody under
conditions suitable for expression of the nucleic acid encoding the
anti-HtrA1 antibody. The method may further comprise recovering the
anti-HtrA1 antibody from the host cell culture, purifying the
anti-HtrA1 antibody, or formulating the anti-HtrA1 antibody with a
pharmaceutically acceptable excipient.
[0056] In another aspect, an immunoconjugate comprising an
anti-HtrA1 antibody and a cytotoxic agent is provided.
[0057] In another aspect, a pharmaceutical formulation comprising
an anti-HtrA1 antibody and a pharmaceutically acceptable carrier is
provided.
[0058] In another aspect, the application provides an anti-HtrA1
antibody for use as a medicament, e.g., for use in treating
age-related macular degeneration (wet or dry), geographic atrophy,
diabetic retinopathy, retinopathy of prematurity, or polypoidal
choroidal vasculopathy, for inhibiting degeneration of retinal or
photoreceptor cells, or for inhibiting HtrA1 protease activity in
an eye.
[0059] In another aspect, the application provides use of an
anti-HtrA1 antibody for the manufacture of a medicament, e.g., a
medicament for the treatment of age-related macular degeneration
(AMD, wet or dry), geographic atrophy (GA), diabetic retinopathy
(DR), retinopathy of prematurity (ROP), or polypoidal choroidal
vasculopathy (PCV), for inhibiting degeneration of retinal or
photoreceptor cells, or for inhibiting HtrA1 protease activity in
an eye.
[0060] In another aspect, the application provides a method of
treating an individual having age-related macular degeneration (wet
or dry), geographic atrophy, diabetic retinopathy, retinopathy of
prematurity, or polypoidal choroidal vasculopathy, comprising
administering to the individual an effective amount of an
anti-HtrA1 antibody as described herein.
[0061] In another aspect, the application provides a method for
inhibiting retinal or photoreceptor cell degeneration in an
individual comprising administering to the individual an effective
amount of an anti-HtrA1 antibody as described herein to inhibit
retinal or photoreceptor cell degeneration.
[0062] In another aspect, the application provides a method of
inhibiting HtrA1 serine protease activity in an eye of an
individual comprising administering to the individual an effective
amount of an anti-HtrA1 antibody as described herein to inhibit
HtrA1 serine protease activity in the eye.
BRIEF DESCRIPTION OF THE FIGURES
[0063] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0064] FIGS. 1A-B. FIG. 1A shows the light chain variable domain
sequence of anti-HtrA antibody94 (YW505.94) (SEQ ID NO:7). FIG. 1B
shows the heavy chain variable domain sequence of anti-HtrA1
antibody94 (YW505.94) (SEQ ID NO:8). The residues are numbered
according to the Kabat numbering system (Kabat, E. A., et al.,
1991, In: Sequences of proteins of immunological interest, fifth
edition. National Institutes of Health, Bethesda, Md.). The
YW505.94 light chain variable domain sequence is aligned with human
KappaI light chain consensus sequence (SEQ ID NO: 9) and the
YW505.94 heavy chain variable domain sequence is aligned with the
human subgroup III heavy chain sequence (SEQ ID NO: 10). The boxed
sequences are CDRs according to Kabat definitions. The sequence
differences between anti-HtrA1 (YW505.94) and consensus sequences
are shaded.
[0065] FIG. 2. Screening of panel of 13 phage derived antibodies
(IgG). Single concentrations (0.08-0.28 mg/ml final) of IgG were
incubated with HuHtrA1 or HuHtrA1_PD and enzyme activity measured
in the FRET assay. Of the 13 antibodies tested, antibodies
YW503.57, YW504.57, YW504.61 and YW505.94 (also referred to as Ab94
or IgG94) strongly inhibited both HuHtrA1 and HuHtrA1_PD
activities.
[0066] FIG. 3. Inhibition of HuHtrA1 by IgG94 and Fab94. HuHtrA1
was incubated with IgG94 and Fab94 for 20 min at 37.degree. C.
Enzyme activity towards the peptide substrate H2-Opt was measured
and fractional activities (v.sub.i/v.sub.o) calculated from the
determined linear velocities. IgG94 at 300 nM did not inhibit
H2-Opt hydrolysis by trypsin (1 nM) or elastase (1 nM).
[0067] FIG. 4. IgG94 inhibits hydrolysis of fluorescent dye-labeled
casein (BODIPY FL) by HuHtrA1 and MuHtrA1. HuHtrA1 and MuHtrA1 were
incubated with IgG94 for 15 min at 37.degree. C. Hydrolysis of
casein BODIPY FL reagent was measured on a microplate reader at
37.degree. C. and the linear rates of fluorescence increase
determined and expressed as percent of uninhibited rates (% of
control).
[0068] FIG. 5. Specificity of IgG94. MuHtrA1_PD, MuHtrA3_PD and
MuHtrA4_PD were incubated with increasing concentrations of IgG94
and enzyme activities towards the peptide substrate H2-Opt (top
panel) or casein BODIPY FL reagent (bottom panel) determined and
expressed as percentage of uninhibited enzyme activities (% of
control)
[0069] FIG. 6. Inhibition of HuHtrA1-mediated macromolecular
substrate cleavage by IgG94. Increasing concentrations of IgG94
(2.3-150 nM for .beta.-casein; 2-125 nM for decorin; 2.3-150 nM for
biglycan) were incubated with HuHtrA1 (10 nM for .beta.-casein, 125
nM for decorin, 75 nM for biglycan) for 15 min at 37.degree. C. The
substrates .beta.-casein, decorin and biglycan (50 .mu.g/ml) were
added and incubated for 2-14 h. After addition of SDS-sample buffer
the digests were analyzed by SDS-PAGE (non-reducing conditions) and
stained by SimplyBlue Safestain.
[0070] FIGS. 7A-C. Mapping the functional epitope of IgG94 on
HuHtrA1_PD. FIG. 7A shows the results of an ELISA measuring the
binding of IgG94 to HuHtrA1_PD mutants with alanine substitutions
of residues surrounding the active site. The shaded rows indicate
alanine substitutions that resulted in a greater than 5-fold
decrease in binding. FIG. 7B shows the structure of HuHtrA1_PD (PDB
3NWU) (Clausen, T., et al., Nat Rev Mol Cell Biol. 12:152-62
(2011)) with Mutated residues indicated. Medium gray shading shows
mutations without loss of IgG94 binding in initial experiments;
dark gray shading shows a subset of residues with >5-fold loss
in binding affinity. The three monomers forming the protease domain
trimer, shown in surface representation, are shaded light gray.
Catalytic triad residues D250 and S328 are underlined. FIG. 7C
shows a close-up view of the loops harboring the epitope residues
N224 and K248 on monomer (light gray, as cartoon) of HuHtrA1_PD
(PDB 3NWU). Also included is the catalytic residue H220.
[0071] FIGS. 8A-B. SEC-MALLS (size exclusion
chromatography--multi-angle laser light scattering) of the
Fab94:HuHtrA1_PD(S328A) complex (FIG. 8A) and the
IgG94:HuHtrA1_PD(S328A) complex (FIG. 8B). Shown are the elution
peaks by SEC (x-axis) and the masses of individual proteins and
complexes (y-axis; dotted lines across the elution peaks).
[0072] FIG. 9. Hypothetical `cage model` of the IgG:HuHtrA1-PD
complex.
[0073] FIGS. 10A-B. Alignment of human HtrA1 (SEQ ID NO: 13),
murine HtrA1 (SEQ ID NO:14), murine HtrA3 (SEQ ID NO:15), and
murine HtrA4 (SEQ ID NO:16).
[0074] FIG. 11. HtrA1 mRNA expression increases in the retina in a
mouse model of constant light exposure (top left panel). HtrA2
levels do not vary significantly in the same model (top right
panel). HtrA1 expression is significantly higher than HtrA2, HtrA3
and HtrA4 expression levels in the mouse retina at baseline (bottom
panel).
[0075] FIG. 12. Increased bi-polar cell/Muller cell responses in
the absence of HtrA1 expression in a mouse model of constant light
exposure.
[0076] FIG. 13. Sparing of retina in the absence of HtrA1
expression in a mouse model of constant light exposure.
[0077] FIG. 14. Sparing of outer nuclear layer (ONL) photoreceptor
cells in the absence of HtrA1 expression in a mouse model of
constant light exposure.
[0078] FIG. 15. Light chain HVR sequences for affinity-improved
variants of anti-HtrA1 antibody YW505.94a.
[0079] FIG. 16. Heavy chain HVR sequences for affinity-improved
variants of anti-HtrA1 antibody YW505.94a.
[0080] FIG. 17. Results of a phage competition assay demonstrating
the binding of YW505.94a affinity-improved variants against
HuHtrA1.
[0081] FIG. 18. Results of a phage competition assay demonstrating
the binding of YW505.94a affinity-improved variants against
MuHtrA1.
[0082] FIGS. 19A-C. Results of ELISA assay showing levels of HtrA1
protein in rat tissues (FIG. 19A), mouse eye fluid (FIG. 19B) and
mouse retina tissue (FIG. 19C).
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions
[0083] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0084] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0085] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0086] The terms "anti-HtrA1 antibody" and "an antibody that binds
to HtrA1" refer to an antibody that is capable of binding HtrA1
with sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting HtrA1. In one
embodiment, the extent of binding of an anti-HtrA1 antibody to an
unrelated, non-HtrA1 protein is less than about 10% of the binding
of the antibody to HtrA1 as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that binds to HtrA1 has
a dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M). In
certain embodiments, an anti-HtrA1 antibody binds to an epitope of
HtrA1 that is conserved among HtrA1 from different species.
[0087] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0088] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0089] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0090] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0091] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0092] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0093] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0094] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0095] The term "Fe region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fe region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0096] "Framework region" or "FR" refers to variable domain
residues other than hypervariable region (HVR) residues. The FR of
a variable domain generally consists of four FR domains: FR1, FR2,
FR3, and FR4. Accordingly, the HVR and FR sequences generally
appear in the following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0097] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0098] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0099] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0100] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0101] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0102] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. An HVR region as used herein comprise any number of
residues located within positions 24-36 (for L1), 46-56 (for L2),
89-97 (for L3), 26-35B (for H1), 47-65 (for H2), and 93-102 (for
H3). Therefore, an HVR includes residues in positions described
previously:
[0103] A) 24-34 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987);
[0104] B) 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1,
50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991).
[0105] C) 30-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58
(H2), 93-100a-j (H3) (MacCallum et al. J. Mol. Biol. 262:732-745
(1996).
[0106] With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0107] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0108] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0109] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0110] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally, at a chromosomal location that is different
from its natural chromosomal location, or contains only coding
sequences.
[0111] "Isolated nucleic acid encoding an anti-HtrA1 antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0112] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0113] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0114] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0115] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0116] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0117] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0118] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0119] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0120] The term "High-temperature requirement associated A" or
"HtrA1," as used herein, refers to any native HtrA1 from any
vertebrate source, including mammals such as primates (e.g. humans)
and rodents (e.g., mice and rats), unless otherwise indicated. The
term encompasses "full-length," unprocessed HtrA1 as well as any
form of HtrA1 that results from processing in the cell. The term
also encompasses naturally occurring variants of HtrA1, e.g.,
splice variants or allelic variants. The amino acid sequence of an
exemplary HtrA1 is shown in SEQ ID NO: 13. Exemplary fragments of
human HtrA1 include fragments comprising, consisting essentially
of, or consisting of amino acids Q23-P480 or P161-K379.
[0121] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0122] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0123] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
II. Compositions and Methods
[0124] In one aspect, the invention is based, in part, on the
discovery that a reduction of HtrA1 activity has protective effects
on photoreceptor cells in the eye, the outer nuclear layer, and on
elecroretinogram functionality. In certain embodiments, antibodies
that bind to HtrA1 are provided. Antibodies of the invention are
useful, e.g., for the diagnosis or treatment of various diseases
associated with HtrA1 activity, including ocular disorders such as
age-related macular degeneration or geographic atrophy.
[0125] A. Exemplary Anti-HtrA1 Antibodies
[0126] In one aspect, the invention provides isolated antibodies
that bind to HtrA1. In certain embodiments, an anti-HtrA1 antibody
has one or more of the following properties: (i) has an IC.sub.50
of less than 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 3 nM,
2.5 nM, 2 nM, 1 nM, or less for one or more HtrA1 substrates; (ii)
binds to HtrA1 with a ratio of 1 variable domain to one subunit of
an HtrA1 trimer (e.g., a Fab binds to an HtrA1 trimer with a ratio
of 3 Fab to 1 HtrA1 trimer, and an IgG binds to an HtrA1 trimer
with a ratio of 3 IgG to 2 HtrA1 trimers), (iii) for antibodies
comprising two variable domains, binds to HtrA1 in a manner that
results in the forming a "cage" similar to that shown in FIG. 9,
(iv) does not prevent trimer formation of HtrA1, (v) binds to one
or more residues in Loop C of the HtrA1 protein, (vi) binds to the
protease domain of HtrA1, (vii) binds to an epitope comprising one
or both of amino acids N224 or K248 of SEQ ID NO: 13, or amino
acids equivalent thereto in a different HtrA1 sequence (e.g., amino
acids N224 and K248 of SEQ ID NO: 14, see FIGS. 10A-B); (viii)
binds to an epitope comprising one or more of residues N224, K248,
V201, T223, K243, K225, E247 and H220 of SEQ ID NO:13, or amino
acids equivalent thereto in a different HtrA1 sequence; (ix)
cross-reacts with murine HtrA1; (x) does not cross-react with
HtrA2, HtrA3 and/or HtrA4; (xi) binds to HtrA1 competitively with
an antibody comprising a VH sequence of SEQ ID NO:8 and a VL
sequence of SEQ ID NO:7, or (xii) binds to HtrA1 with a
dissociation constant of 5500 nM, or (xiii) inhibits complex
formation between HtrA1 and .alpha.1-antitrypsin (A1AT).
[0127] In one aspect, the invention provides an anti-HtrA1 antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:87; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:88; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:89; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:58; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:86. In one embodiment, the invention provides an anti-HtrA1
antibody comprising at least one, two, three, four, five, or six
HVRs selected from (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:25; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:24. In one embodiment, the invention provides an anti-HtrA1
antibody comprising at least one, two, three, four, five, or six
HVRs selected from (a) HVR-H1 comprising an amino acid sequence
selected from: SEQ ID NO:4, 20, 47-51 and 75-80; (b) HVR-H2
comprising an amino acid sequence selected from: SEQ ID NO:5, 52
and 81-82; (c) HVR-H3 comprising an amino acid sequence selected
from: SEQ ID NO:6, 53 and 83-84; (d) HVR-L1 comprising an amino
acid sequence selected from: SEQ ID NO:1, 18, 21, 33 and 54-57; (e)
HVR-L2 comprising an amino acid sequence selected from: SEQ ID NO:2
and 58; and (f) HVR-L3 comprising an amino acid sequence selected
from: SEQ ID NO:3, 19, 22, 34-46 and 59-74. In one embodiment, the
invention provides an anti-HtrA1 antibody comprising at least one,
two, three, four, five, or six HVRs selected from (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:5; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:6; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO: 1; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:2; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO:3. In one
embodiment, the invention provides an anti-HtrA1 antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 18; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 19. In one
embodiment, the invention provides an anti-HtrA1 antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.
[0128] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:87;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:88; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:89. In
one embodiment, the antibody comprises HVR-H3 comprising the amino
acid sequence of SEQ ID NO:89. In another embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:89
and HVR-L3 comprising the amino acid sequence of SEQ ID NO:86. In a
further embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:89, HVR-L3 comprising the amino
acid sequence of SEQ ID NO:86, and HVR-H2 comprising the amino acid
sequence of SEQ ID NO:88. In a further embodiment, the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:87; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:88; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:89.
[0129] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:25;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:26; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:27. In
one embodiment, the antibody comprises HVR-H3 comprising the amino
acid sequence of SEQ ID NO:27. In another embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:27
and HVR-L3 comprising the amino acid sequence of SEQ ID NO:24. In a
further embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:27, HVR-L3 comprising the amino
acid sequence of SEQ ID NO:24, and HVR-H2 comprising the amino acid
sequence of SEQ ID NO:26. In a further embodiment, the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:26; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:27.
[0130] In one embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VH HVR
sequences selected from (a) HVR-H1 comprising an amino acid
sequence selected from: SEQ ID NO:4, 20, 47-51 and 75-80; (b)
HVR-H2 comprising an amino acid sequence selected from SEQ ID NO:5,
52 and 81-82; and (c) HVR-H3 comprising an amino acid sequence
selected from: SEQ ID NO:6, 53 and 83-84. In one embodiment, the
antibody comprises HVR-H3 comprising an amino acid sequence
selected from SEQ ID NO:6 and 53. In another embodiment, the
antibody comprises HVR-H3 comprising an amino acid sequence
selected from SEQ ID NO:6, 53 and 83-84 and HVR-L3 comprising an
amino acid sequence selected from SEQ ID NO:3, 19, 22, 34-46 and
59-74. In a further embodiment, the antibody comprises HVR-H3
comprising an amino acid sequence selected from SEQ ID NO:6, 53 and
83-84, HVR-L3 comprising an amino acid sequence selected from SEQ
ID NO: 3, 19, 22, 34-46 and 59-74, and HVR-H2 comprising an amino
acid sequence selected from SEQ ID NO:5, 52 and 81-82. In a further
embodiment, the antibody comprises (a) HVR-H1 comprising an amino
acid sequence selected from SEQ ID NO:4, 20, 47-51 and 75-80; (b)
HVR-H2 comprising an amino acid sequence selected from SEQ ID NO:5,
52 and 81-82; and (c) HVR-H3 comprising an amino acid sequence
selected from SEQ ID NO:6, 53 and 83-84.
[0131] In one embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VH HVR
sequences selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:4; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:5; and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:6. In one embodiment, the antibody comprises
HVR-H3 comprising the amino acid sequence of SEQ ID NO:6. In
another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:6 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO:3. In a further embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:6,
HVR-L3 comprising the amino acid sequence of SEQ ID NO:3, and
HVR-H2 comprising the amino acid sequence of SEQ ID NO:5. In a
further embodiment, the antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:4; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:5; and (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:6.
[0132] In one embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VH HVR
sequences selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:20: (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:5; and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:6. In one embodiment, the antibody comprises
HVR-H3 comprising the amino acid sequence of SEQ ID NO:20. In
another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:20 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 19. In a further embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO:20, HVR-L3 comprising the amino acid sequence of SEQ ID NO:
19, and HVR-H2 comprising the amino acid sequence of SEQ ID NO:5.
In another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:20 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO:22. In a further embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO:20, HVR-L3 comprising the amino acid sequence of SEQ ID
NO:22, and HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5. In a further embodiment, the antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:6.
[0133] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:85; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:58; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:86. In one embodiment, the invention provides
an antibody comprising at least one, at least two, or all three VL
HVR sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:23; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:24. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:85; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:58; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:86. In one
embodiment, the antibody comprises (a) HVR-L1 comprising the amino
acid sequence of SEQ ID NO:23; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:24.
[0134] In another embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising an amino acid
sequence selected from SEQ ID NO: 1, 18, 21, 33 and 54-57; (b)
HVR-L2 comprising an amino acid sequence selected from SEQ ID NO:2
and 58; and (c) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:3, 19, 22, 34-46 and 59-74. In one embodiment, the
antibody comprises (a) HVR-L1 comprising an amino acid sequence
selected from SEQ ID NO: 1, 18, 21, 33 and 54-57; (b) HVR-L2
comprising an amino acid sequence selected from SEQ ID NO:2 and 58;
and (c) HVR-L3 comprising an amino acid sequence selected from SEQ
ID NO:3, 19, 22, 34-46 and 59-74.
[0135] In another embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:1; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:3. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:3.
[0136] In another embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:18: (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2: and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 19. In one embodiment, the antibody
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 18; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO: 19.
[0137] In another embodiment, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:21; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:22. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.
[0138] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:87, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:88, and (iii) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:89; and (b) a VL domain comprising at
least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:85,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:58, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:86. In
one embodiment, an antibody of the invention comprises (a) a VH
domain comprising at least one, at least two, or all three VH HVR
sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:25, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:26, and (iii) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:27; and (b) a VL domain comprising at
least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L comprising the amino acid sequence of SEQ ID NO:23,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:24.
[0139] In another embodiment, an antibody of the invention
comprises (a) a VH domain comprising at least one, at least two, or
all three VH HVR sequences selected from (i) HVR-H1 comprising an
amino acid sequence selected from SEQ ID NO:4, 20, 47-51 and 75-80,
(ii) HVR-H2 comprising an amino acid sequence selected from SEQ ID
NO:5, 52 and 81-82, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:6, 53 and 83-84; and (b) a VL
domain comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising an amino acid
sequence selected from SEQ ID NO: 1, 18, 21, 33 and 54-57, (ii)
HVR-L2 comprising an amino acid sequence selected from SEQ ID NO:2
and 58, and (c) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:3, 19, 22, 34-46 and 59-74.
[0140] In another embodiment, an antibody of the invention
comprises (a) a VH domain comprising at least one, at least two, or
all three VH HVR sequences selected from (i) HVR-H1 comprising the
amino acid sequence of SEQ ID NO:4, (ii) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:5, and (iii) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:6; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:3.
[0141] In another embodiment, an antibody of the invention
comprises (a) a VH domain comprising at least one, at least two, or
all three VH HVR sequences selected from (i) HVR-H1 comprising the
amino acid sequence of SEQ ID NO:20, (ii) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:5, and (iii) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:6; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:
18, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:2,
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:19.
[0142] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:20, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:5, and (iii) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:6; and (b) a VL domain comprising at least
one, at least two, or all three VL HVR sequences selected from (i)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:21, (ii)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2, and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.
[0143] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:87; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:88; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:89; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:85; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:58; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:86. In one embodiment, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:25; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:26; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:27; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:23; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:24.
[0144] In another embodiment, the invention provides an antibody
comprising (a) HVR-H1 comprising an amino acid sequence selected
from SEQ ID NO:4, 20, 47-51 and 75-80; (b) HVR-H2 comprising an
amino acid sequence selected from SEQ ID NO:5, 52 and 81-82; (c)
HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:6,
53 and 83-84; (d) HVR-L1 comprising an amino acid sequence selected
from SEQ ID NO: 1, 18, 21, 33 and 54-57; (e) HVR-L2 comprising an
amino acid sequence selected from SEQ ID NO:2 and 58; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:3,
19, 22, 34-46 and 59-74.
[0145] In another embodiment the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:4; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:5;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO: 1; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:3.
[0146] In another embodiment the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 18; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:19.
[0147] In another embodiment the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:5; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:6;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:21; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:2; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.
[0148] In any of the above embodiments, an anti-HtrA1 antibody is
humanized. In one embodiment, an anti-HtrA1 antibody comprises HVRs
as in any of the above embodiments, and further comprises an
acceptor human framework, e.g. a human immunoglobulin framework or
a human consensus framework. In another embodiment, an anti-HtrA1
antibody comprises HVRs as in any of the above embodiments, and
further comprises a VH comprising an FR2 sequence of SEQ ID
NO:17.
[0149] In another aspect, an anti-HtrA1 antibody comprises a heavy
chain variable domain (VH) sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of any one of SEQ ID NO:8 or 29. In certain
embodiments, a VH sequence having at least 90%, 91%. 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but an anti-HtrA1 antibody comprising that
sequence retains the ability to bind to HtrA1. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:8 or 29. In certain
embodiments, substitutions, insertions, or deletions occur in
regions outside the HVRs (i.e., in the FRs). Optionally, the
anti-HtrA1 antibody comprises the VH sequence in SEQ ID NO:8, 29 or
32, including post-translational modifications of that sequence. In
a particular embodiment, the VH comprises one, two or three HVRs
selected from: (a) HVR-H1 comprising an amino acid sequence
selected from: SEQ ID NO:4, 20, 25 and 47-51, (b) HVR-H2 comprising
an amino acid sequence selected from SEQ ID NO:5, 26 and 52, and
(c) HVR-H3 comprising an amino acid sequence selected from SEQ ID
NO:6, 27 and 53.
[0150] In another aspect, an anti-HtrA1 antibody is provided,
wherein the antibody comprises a light chain variable domain (VL)
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of any one of
SEQ ID NO:7, 28 or 30. In certain embodiments, a VL sequence having
at least 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-HtrA1 antibody comprising that sequence retains the ability to
bind to HtrA1. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:7, 28 or 30. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-HtrA antibody comprises the VL
sequence in SEQ ID NO:7, 28, 30 or 31, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1
comprising an amino acid sequence selected from SEQ ID NO: 1, 18,
21, 23 and 33; (b) HVR-L2 comprising the amino acid sequence of SEQ
ID NO:2; and (c) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:3, 19, 22, 24 and 34-36.
[0151] In another aspect, an anti-HtrA1 antibody is provided,
wherein the antibody comprises a VH as in any of the embodiments
provided above, and a VL as in any of the embodiments provided
above. In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:32 and SEQ ID NO:31, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:8 and SEQ ID NO:7, respectively, including post-translational
modifications of those sequences. In one embodiment, the antibody
comprises the VH and VL sequences in SEQ ID NO:29 and SEQ ID NO:28,
respectively, including post-translational modifications of those
sequences. In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:29 and SEQ ID NO:30, respectively, including
post-translational modifications of those sequences.
[0152] In a further aspect, the invention provides an antibody that
binds to the same epitope as an anti-HtrA1 antibody provided
herein. For example, in certain embodiments, an antibody is
provided that binds to the same epitope as an anti-HtrA1 antibody
comprising a VH sequence of SEQ ID NO:8 and a VL sequence of SEQ ID
NO:7. In certain embodiments, an antibody is provided that binds to
an epitope of HtrA1 containing residue N224, K248 or both of SEQ ID
NO: 13, or residues equivalent thereto in a different HtrA1
sequence. In certain embodiments, the epitope of HtrA1 further
comprising one or more of the following residues V201, T223, K243,
K225, E247 and H220 of SEQ ID NO: 13, or amino acids equivalent
thereto in a different HtrA1 sequence. In certain embodiments, an
antibody is provided that binds to an epitope of HtrA1 containing
one or more of residues N224, K248 and V201 or all of the foregoing
of SEQ ID NO: 13, or residues equivalent thereto in a different
HtrA sequence. In certain embodiments, an antibody is provided that
binds to an epitope of HtrA1 containing one or more of residues
N224, K248, V201, T223 and K243 or all of the foregoing of SEQ ID
NO: 13, or residues equivalent thereto in a different HtrA1
sequence. In certain embodiments, an antibody is provided that
binds to an epitope of HtrA1 containing one or more of residues
N224, K248, V201, T223, K243, K225, E247 and H22A or all of the
foregoing of SEQ ID NO: 13, or residues equivalent thereto in a
different HtrA1 sequence. In certain embodiments, the epitope is a
linear epitope. In other embodiments, the epitope is a
conformational epitope.
[0153] In a further aspect of the invention, an anti-HtrA1 antibody
according to any of the above embodiments is a monoclonal antibody,
including a chimeric, humanized or human antibody. In one
embodiment, an anti-HtrA1 antibody is an antibody fragment, e.g., a
Fv, Fab, Fab', scFv, diabody, or F(ab').sub.2 fragment. In another
embodiment, the antibody is a full length antibody, e.g., an intact
IgG1 antibody or other antibody class or isotype as defined
herein.
[0154] In certain embodiments, an anti-HtrA1 antibody according to
any of the above embodiments is not an antibody having a VH
sequence of SEQ ID NO:8 and a VL sequence of SEQ ID NO:7.
[0155] In a further aspect, an anti-HtrA1l antibody according to
any of the above embodiments may incorporate any of the features,
singly or in combination, as described in Sections 1-7 below:
[0156] 1. Antibody Affinity
[0157] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 108 M or less, e.g. from 10.sup.-8 M to
10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M).
[0158] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA) performed with the Fab version of an antibody
of interest and its antigen as described by the following assay.
Solution binding affinity of Fabs for antigen is measured by
equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta et al.,
Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0159] According to another embodiment, Kd is measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 pM) before injection at a flow rate of 5
l/minute to achieve approximately 10 response units (RU) of coupled
protein. Following the injection of antigen, 1 M ethanolamine is
injected to block unreacted groups. For kinetics measurements,
two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected
in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.) surfactant (PBST)
at 25.degree. C. at a flow rate of approximately 25 l/min.
Association rates (k.sub.on) and dissociation rates (k.sub.off) are
calculated using a simple one-to-one Langmuir binding model
(BIACORE.RTM. Evaluation Software version 3.2) by simultaneously
fitting the association and dissociation sensorgrams. The
equilibrium dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999). If the on-rate exceeds 106 M.sup.-1 s.sup.-1 by
the surface plasmon resonance assay above, then the on-rate can be
determined by using a fluorescent quenching technique that measures
the increase or decrease in fluorescence emission intensity
(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25.degree.
C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in
the presence of increasing concentrations of antigen as measured in
a spectrometer, such as a stop-flow equipped spectrophometer (Aviv
Instruments) or a 8000-series SLM-AMINCO.TM. spectrophotometer
(ThermoSpectronic) with a stirred cuvette.
[0160] 2. Antibody Fragments
[0161] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0162] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0163] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0164] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0165] 3. Chimeric and Humanized Antibodies
[0166] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0167] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0168] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0169] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0170] 4. Human Antibodies
[0171] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0172] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0173] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0174] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0175] 5. Library-Derived Antibodies
[0176] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0177] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0178] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0179] 6. Multispecific Antibodies
[0180] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for HtrA1 and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of HtrA1. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express HtrA1. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0181] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):
1547-1553 (1992)); using "diabody" technology for making bispecific
antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)
dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and
preparing trispecific antibodies as described, e.g., in Tutt et al.
J. Immunol. 147: 60 (1991).
[0182] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0183] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to
HtrA1 as well as another, different antigen (see, US 2008/0069820,
for example).
[0184] 7. Antibody Variants
[0185] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0186] a) Substitution, Insertion, and Deletion Variants
[0187] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00001 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
Amino acids may be grouped according to common side-chain
properties:
[0188] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0189] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;
[0190] (3) acidic: Asp, Glu;
[0191] (4) basic: His, Lys, Arg;
[0192] (5) residues that influence chain orientation: Gly, Pro:
[0193] (6) aromatic: Trp, Tyr. Phe.
[0194] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0195] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0196] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0197] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0198] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0199] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0200] b) Glycosylation Variants
[0201] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0202] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0203] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e.g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochenm. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0204] Antibody variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0205] c) Fc Region Variants
[0206] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0207] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et al. Proc. Nat'l Acad. Sci. LISA 83:7059-7063
(1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA
82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et
al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively,
non-radioactive assay methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q
binding assays may also be carried out to confirm that the antibody
is unable to bind C1 q and hence lacks CDC activity. See, e.g., C1q
and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg,
M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding
and in vivo clearance/half life determinations can also be
performed using methods known in the art (see, e.g., Petkova, S. B.
et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).
[0208] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0209] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001)).
[0210] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0211] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0212] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0213] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0214] d) Cysteine Engineered Antibody Variants
[0215] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0216] e) Antibody Derivatives
[0217] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0218] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0219] B. Recombinant Methods and Compositions
[0220] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-HtrA1 antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-HtrA1
antibody is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
[0221] For recombinant production of an anti-HtrA1 antibody,
nucleic acid encoding an antibody, e.g., as described above, is
isolated and inserted into one or more vectors for further cloning
and/or expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0222] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0223] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0224] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0225] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0226] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV 1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0227] C. Assays
[0228] Anti-HtrA1 antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0229] 1. Binding Assays and Other Assays
[0230] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc.
[0231] In another aspect, competition assays may be used to
identify an antibody that competes with Fab94 or IgG94 for binding
to HtrA1. In certain embodiments, such a competing antibody binds
to the same epitope (e.g., a linear or a conformational epitope)
that is bound by Fab94 or IgG94. Detailed exemplary methods for
mapping an epitope to which an antibody binds are provided in
Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular
Biology vol. 66 (Humana Press, Totowa, N.J.).
[0232] In an exemplary competition assay, immobilized HtrA1 is
incubated in a solution comprising a first labeled antibody that
binds to HtrA1 (e.g., Fab94 or IgG94) and a second unlabeled
antibody that is being tested for its ability to compete with the
first antibody for binding to HtrA1. The second antibody may be
present in a hybridoma supernatant. As a control, immobilized HtrA1
is incubated in a solution comprising the first labeled antibody
but not the second unlabeled antibody. After incubation under
conditions permissive for binding of the first antibody to HtrA1,
excess unbound antibody is removed, and the amount of label
associated with immobilized HtrA1 is measured. If the amount of
label associated with immobilized HtrA1 is substantially reduced in
the test sample relative to the control sample, then that indicates
that the second antibody is competing with the first antibody for
binding to HtrA1. See Harlow and Lane (1988) Antibodies: A
Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.).
[0233] 2. Activity Assays
[0234] In one aspect, assays are provided for identifying
anti-HtrA1 antibodies thereof having biological activity.
Biological activity may include, e.g., blocking, antagonizing,
suppressing, interfering, modulating and/or reducing one or more
biological activities of HtrA1. Antibodies having such biological
activity in vivo and/or in vitro are also provided.
[0235] In certain embodiments, an antibody of the invention is
tested for such biological activity. In certain embodiments, an
anti-HtrA1 antibody binds to HtrA1 and reduces or inhibits its
serine protease activity for one or more HtrA1 substrates,
including, for example, the H2-Opt peptide, .beta.-casein or BODIPY
FL casein substrates as described in the Examples below, or any
other suitable HtrA1 substrate. In certain embodiments, an
anti-HtrA1 antibody inhibits HtrA1 serine protease activity with an
IC.sub.50 of less than 50 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5
nM, 3 nM, 2.5 nM, 2 nM, 1 nM, or less for one or more HtrA1
substrates. In certain embodiments, an anti-HtrA1 antibody protects
photoreceptor cells from degradation, protects the thickness of the
outer nuclear layer, or protects electroretinogram functional
activity in an ocular disease model, such as the constant light
exposure mouse model described in the Examples below.
[0236] D. Immunoconjugates
[0237] The invention also provides immunoconjugates comprising an
anti-HtrA1 antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0238] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0239] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0240] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example Tc.sup.99m or I.sup.123, or a spin label for nuclear
magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0241] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0242] The immunoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0243] E. Methods and Compositions for Diagnostics and
Detection
[0244] In certain embodiments, any of the anti-HtrA1 antibodies
provided herein is useful for detecting the presence of HtrA1 in a
biological sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a
biological sample comprises a cell or tissue, such as a sample
comprising photoreceptor cells, retinal pigment epithelium cells,
cells of the outer nuclear layer, the inner nuclear layer, Muller
cells, ciliary epithelium, or retinal tissue.
[0245] In one embodiment, an anti-HtrA1 antibody for use in a
method of diagnosis or detection is provided. In a further aspect,
a method of detecting the presence of HtrA1 in a biological sample
is provided. In certain embodiments, the method comprises
contacting the biological sample with an anti-HtrA1 antibody as
described herein under conditions permissive for binding of the
anti-HtrA1 antibody to HtrA1, and detecting whether a complex is
formed between the anti-HtrA1 antibody and HtrA1. Such method may
be an in vitro or in vivo method. In one embodiment, an anti-HtrA1
antibody is used to select subjects eligible for therapy with an
anti-HtrA1 antibody, e.g. where HtrA1 is a biomarker for selection
of patients.
[0246] In certain embodiments, a patient suitable for treatment
with an anti-HtrA1 antibody may be identified by detecting one or
more polymorphisms in the HtrA1 gene or HtrA1 control sequence,
such as the HtrA1 promoter polymorphism rs11200638(G/A) (see e.g.,
A. DeWan, et al., Science 314: 989-992 (2006)).
[0247] Exemplary disorders that may be diagnosed using an antibody
of the invention include ocular disorders, such as, for example,
wet age-related macular degeneration (AMD), dry age-related macular
degeneration, geographic atrophy (GA), diabetic retinopathy (DA),
retinopathy of prematurity (ROP), or polypoidal choroidal
vasculopathy (PCV).
[0248] In certain embodiments, labeled anti-HtrA antibodies are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction.
[0249] Exemplary labels include, but are not limited to, the
radioisotopes .sup.32P, .sup.14C, .sup.125I, .sup.3H, and
.sup.131I, fluorophores such as rare earth chelates or fluorescein
and its derivatives, rhodamine and its derivatives, dansyl,
umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0250] F. Pharmaceutical Formulations
[0251] Pharmaceutical formulations of an anti-HtrA1 antibody as
described herein are prepared by mixing such antibody having the
desired degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include interstitial drug dispersion agents
such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases such as chondroitinases.
[0252] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0253] The formulation herein may also contain more than one active
ingredient as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, for treating an ocular
disorder associated undesired neovascularization, such as wet AMD,
it may be desirable to further provide an anti-angiogenic therapy,
such as an anti-VEGF therapy like LUCENTIS.TM. (ranibizumab). Such
active ingredients are suitably present in combination in amounts
that are effective for the purpose intended. In other embodiments,
treatment of a disease or disorder associated undesirable ocular
neovascularization may involve a combination of an anti-HtrA1
antibody and photodynamic therapy (e.g., with MACUGEN.TM. or
VISUDYNE.TM.).
[0254] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0255] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0256] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0257] G. Therapeutic Methods and Compositions
[0258] Any of the anti-HtrA1 antibodies provided herein may be used
in therapeutic methods.
[0259] In certain embodiments, an anti-HtrA1 antibody is useful for
inhibiting the degeneration of retinal cells (such as, for example,
retinal pigment epithelium (RPE) cells) or photoreceptor cells in
an eye of a patient suffering from or at risk for developing an
ocular disorder (e.g., a patient having an HtrA1 polymorphism,
increased HtrA1 expression in the eye, or drusen in at least one
eye). In certain embodiments, an anti-HtrA1 antibody may be used
for slowing the progression to late stage geographic atrophy or dry
age-related macular degeneration in an eye of a patient having
drusen in the eye to be treated. In certain embodiments, a patient
suitable for treatment with an anti-HtrA1 antibody may be
identified by detecting the onset of disease in at least one eye of
the patient. For example, certain patients may be selected by
detecting drusen, geographic atrophy, or choroidal
neovascularization in one eye, while the other eye is symptom free.
Such patients may be good candidates for preventative treatment in
the symptom free eye, e.g., to delay the onset or reduce the
severity of such symptoms (e.g., drusen, geographic atrophy, and/or
choroidal neovascularization) in the symptom free eye.
Alternatively, the symptomatic eye may be treated, or both the
symptomatic and symptom free eye may be treated in accordance with
the methods described herein. Accordingly, an anti-HtrA1 antibody
may be used for preventing or inhibiting the progression of an
ocular disorder in an eye of patient, wherein the patient has
developed drusen, wet AMD, or geographic atrophy in the other eye,
but the eye being treated is not yet symptomatic. Alternatively,
the symptomatic eye is treated, or both the symptomatic and the
symptom free eye are treated.
[0260] In another embodiment, an anti-HtrA1 antibody is useful for
treating arthritis.
[0261] In one aspect, an anti-HtrA1 antibody for use as a
medicament is provided. In further aspects, an anti-HtrA1 antibody
for use in treating an ocular disease or disorder, such as, for
example, AMD (wet or dry), GA, DR, PCV or ROP, is provided. In
certain embodiments, an anti-HtrA1 antibody for use in a method of
treatment is provided. In certain embodiments, the invention
provides an anti-HtrA1 antibody for use in a method of treating an
individual having an ocular disease or disorder comprising
administering to the individual an effective amount of the
anti-HtrA1 antibody. In one such embodiment, the method further
comprises administering to the individual an effective amount of at
least one additional therapeutic agent, e.g., as described below.
In further embodiments, the invention provides an anti-HtrA1
antibody for use in inhibiting degeneration of retinal cells (such
as, for example, RPE cells) or photoreceptor cells in an eye of a
patient. In certain embodiments, the invention provides an
anti-HtrA1 antibody for use in a method of inhibiting degeneration
of retinal or photoreceptor cells in an individual comprising
administering to the individual an effective amount of the
anti-HtrA1 antibody to inhibit degeneration of retinal or
photoreceptor cells. In further embodiments, the invention provides
an anti-HtrA1 antibody for use in inhibiting HtrA1 serine protease
activity in an eye of a patient. In certain embodiments, the
invention provides an anti-HtrA1 antibody for use in a method of
inhibiting HtrA1 serine protease activity in an eye of an
individual comprising administering to the individual an effective
amount of the anti-HtrA1 antibody to inhibit HtrA1 serine protease
activity in the eye. An "individual" according to any of the above
embodiments is preferably a human. In further embodiments, the
invention provides an anti-HtrA1 antibody for use in inhibiting PCV
in a patient in need thereof, e.g., a patient having choroidal
vascular networks with polyp-like aneurysmal dilations.
[0262] In a further aspect, the invention provides for the use of
an anti-HtrA1 antibody in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of
an ocular disorder, such as, for example, AMD (wet or dry), GA, DR,
PCV or ROP. In a further embodiment, the medicament is for use in a
method of treating an ocular disorder comprising administering to
an individual having the ocular disorder an effective amount of the
medicament. In one such embodiment, the method further comprises
administering to the individual an effective amount of at least one
additional therapeutic agent, e.g., as described below. In a
further embodiment, the medicament is for inhibiting the
degradation of retinal cells or photoreceptor cells in an eye of an
individual. In a further embodiment, the medicament is for use in a
method of inhibiting degeneration of retinal cells or photoreceptor
cells in an individual comprising administering to the individual
an amount effective of the medicament to inhibit degeneration of
retinal or photoreceptor cells in the individual. In a further
embodiment, the medicament is for inhibiting HtrA1 serine protease
activity in the eye of an individual. In a further embodiment, the
medicament is for use in a method of inhibiting HtrA1 serine
protease activity in an eye of an individual comprising
administering to the individual an amount effective of the
medicament to inhibit HtrA1 serine protease activity in the eye. An
"individual" according to any of the above embodiments may be a
human.
[0263] In a further aspect, the invention provides a method for
treating an ocular disorder, such as, for example, AMD (wet or
dry), GA, DR, PCV or ROP. In one embodiment, the method comprises
administering to an individual having such an ocular disorder an
effective amount of an anti-HtrA1 antibody. In one such embodiment,
the method further comprises administering to the individual an
effective amount of at least one additional therapeutic agent, as
described below. An "individual" according to any of the above
embodiments may be a human.
[0264] In a further aspect, the invention provides a method for
inhibiting degeneration of retinal or photoreceptor cells in an
individual. In one embodiment, the method comprises administering
to the individual an effective amount of an anti-HtrA1 antibody to
inhibit degeneration of retinal or photoreceptor cells in the
individual. In one embodiment, an "individual" is a human.
[0265] In a further aspect, the invention provides a method for
inhibiting HtrA1 serine protease activity in an eye of an
individual. In one embodiment, the method comprises administering
to the individual an effective amount of an anti-HtrA1 antibody to
inhibit HtrA1 serine protease activity in an eye of the individual.
In one embodiment, an "individual" is a human.
[0266] The efficacy of the treatment of an ocular disorder using an
anti-HtrA1 antibody, can be measured by various endpoints commonly
used in evaluating intraocular diseases, such as, for example,
performing an eye exam, measuring intraocular pressure, assessing
visual acuity, measuring slitlamp pressure, assessing intraocular
inflammation, measuring the size of CNV, measuring the leakage of
CNV (e.g., by Fluorescein angiography), measuring the amount of
drusen, measuring the location of drusen, etc. In certain
embodiments, vision loss can be assessed, for example, by measuring
the mean change in best correction visual acuity (BCVA) from
baseline to a desired time point (e.g., where the BCVA is based on
Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity
chart and assessment at a test distance of 4 meters), measuring the
proportion of subjects who lose fewer than 15 letters in visual
acuity at a desired time point compared to baseline, measuring the
proportion of subjects who gain greater than or equal to 15 letters
in visual acuity at a desired tine point compared to baseline,
measuring the proportion of subjects with a visual-acuity Snellen
equivalent of 20/2000 or worse at a desired time point, or
measuring the NEI Visual Functioning Questionnaire.
[0267] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the anti-HtrA1 antibodies provided
herein, e.g., for use in any of the above therapeutic methods. In
one embodiment, a pharmaceutical formulation comprises any of the
anti-HtrA1 antibodies provided herein and a pharmaceutically
acceptable carrier. In another embodiment, a pharmaceutical
formulation comprises any of the anti-HtrA1 antibodies provided
herein and at least one additional therapeutic agent, e.g., as
described below.
[0268] Antibodies of the invention can be used either alone or in
combination with other agents in a therapy. For instance, an
antibody of the invention may be co-administered with at least one
additional therapeutic agent. In certain embodiments, an additional
therapeutic agent is a therapeutic agent suitable for treatment of
an ocular disorder associated with undesirable neovascularization
in the eye, such as, for example, wet AMD. Suitable therapeutic
agents include, for example, anti-angiogenic therapies such as an
anti-VEGF therapy like LUCENTIS.TM. (ranibizumab).
[0269] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the antibody of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent and/or adjuvant. Antibodies of the
invention can also be used in combination with photodynamic therapy
(e.g., with MACUGEN.TM. or VISUDYNE.TM.).
[0270] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. In an exemplary embodiment, an
antibody of the invention (and any additional therapeutic agent)
can be administered by intravitreal injection. Dosing can be by any
suitable route, e.g. by injections, such as intravenous,
intravitreal or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0271] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0272] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered.
[0273] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-HtrA1
antibody.
[0274] H. Articles of Manufacture
[0275] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0276] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an anti-HtrA1 antibody.
TABLE-US-00002 SEQUENCE KEY Light chain HVRs for Anti-HtrA1
Antibody YW505.94 (SEQ ID NO: 1) HVR L1: RASQDVSTAVA (SEQ ID NO: 2)
HVR L2: SASFLYS (SEQ ID NO: 3) HVR L3: QQSYTTPPT Heavy chain HVRs
for Anti-HtrA1 Antibody YW505.94 (SEQ ID NO: 4) HVR H1: GFNISGYYIH
(SEQ ID NO: 5) HVR H2: WIDPYGGDTNYADSVKG (SEQ ID NO: 6) HVR H3:
GTFLTSWGHYFDY Light chain VR for Anti-HtrA1 Antibody YW505.94 (SEQ
ID NO: 7)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKR Heavy chain VR for
Anti-HtrA1 Antibody YW505.94 (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFNISGYYIHWVRQAPGKGLEWVGWIDPYGGDTN
TADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTSWGHYFDYWGQGT Human
HtrA1 (full length sequence in caps, protease domain is underlined,
residues N224 and K248 are shaded) (SEQ ID NO: 13)
mqipraallpllllllaapasaQLSRAGRSAPLAAGCPDRCEPARCPPQPEHCEGGRARDACGCCEVC
GAPEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDANTYA
NLCQLRAASRRSERLHRPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIEL
##STR00001##
ADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRN
SDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKG
KAITKKKYIGIRMMSLTSSKAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISIN
GQSVVSANDVSDVIKRESTLNMVVRRGNEDIMITVIPEEIDP Murine HtrA1 (full
length sequence in caps, protease domain is underlined) (SEQ ID NO:
14)
mqslrttllsllllllaapslalPSGTGRSAPAATVCPEHCDPTRCAPPPTDCEGGRVRDACGCCEVCGA
LEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDAKTYTNL
CQLRAASRRSEKLRQPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELYR
KLPFSKREVPVASGSGFIVSEDGLIVTNAHVVTNKNRVKVELKNGATYEAKIKDVDEKAD
IALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSD
MDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGK
AVTKKKYIGIRMMSLTSSKAKELKDRHRDFPDVLSGAYIIEVIPDTPAEAGGLKENDVIISIN
GQSVVTANDVSDVIKKENTLNMVVRRGNEDIVITVIPEEIDP Murine HtrA3 (protease
domain is underlined) (SEQ ID NO: 15)
MQARALLPATLAILATLAVLALAREPPAAPCPARCDVSRCPSPRCPGGYVPDLCNCCLVC
AASEGEPCGRPLDSPCGDSLECVRGVCRCRWTHTVCGTDGHTYADVCALQAASRRALQ
VSGTPVRQLQKGACPSGLHQLTSPRYKFNFIADVVEKIAPAVVHIELFLRHPLFGRNVPLSS
GSGFIMSEAGLIVTNAHVVSSSSTASGRQQLKVQLQNGDAYEATIQDIDKKSDIATIVIHPK
KKLPVLLLGHSADLRPGEFVVAIGSPFALQNTVTTGIVSTAQRDGKELGLRDSDMDYIQTD
AIINTGNSGGPLVNLDGEVIGINTLKVAAGISFAIPSDRITRFLSEFQNKHVKDWKKRFIGIR
MRTITPSLVEELKAANPDFPAVSSGIYVQEVVPNSPSQRGGIQDGDIIVKVNGRPLADSSEL
QEAVLNESSLLLEVRRGNDDLLFSIIPEVVM Murine HtrA4 (protease domain is
underlined) (SEQ ID NO: 16)
MSFQRLWAVRTQFLLLWLLLPAVPVPWAEARRSRVSLPCPDACDPTRCPTLPTCSAQLAP
VPDRCGCCRVCAAAEGQECGGARGRPCAPRLRCGAPFSRDPSGGAWLGTCGCAEGAED
AVVCGSDGRTYPSLCALRKENRAARQRGALPAVPVQKGACEEAGTTRAGRLRRKYNFIA
AVVEKVAPSVVHLQLFRRSPLTNQEIPSSSGSGFIVSEDGLIVTNAHVLTNQQKIQVELQSG
ARYEATVKDIDHKLDLALIKIEPDTELPVLLLGRSSDLRAGEFVVALGSPFSLQNTVTAGIV
STTQRGGRELGLKNSDIDYIQTDAIINHGNSGGPLVNLDGDVIGINTLKVTAGISFAIPSDRIR
QFLEDYHERQLKGKAPLQKKYLGLRMLPLTLNLLQEMKRQDPEFPDVSSGVFVYEVIQGS
AAASSGLRDHDVIVSINGQPVTTTTDVIEAVKDNDFLSILVLRGSQTLFLTVTPEIIN YW505.95
HC FR2 (SEQ ID NO: 17) WVRQAPGKGLEWVG Light chain HVRs for
Anti-HtrA1 Antibody YW505.94a.28 (SEQ ID NO: 18) HVR L1:
RASQSINTYLA (SEQ ID NO: 2) HVR L2: SASFLYS (SEQ ID NO: 19) HVR L3:
QQSDDTPPT Heavy chain HVRs for Anti-HtrA1 Antibody YW505.94a.28
(SEQ ID NO: 20) HVR H1: GFSISGYYIH (SEQ ID NO: 5) HVR H2:
WIDPYGGDTNYADSVKG (SEQ ID NO: 6) HVR H3: GTFLTSWGHYFDY Light chain
VR for Anti-HtrA1 Antibody YW505.94a.28 (SEQ ID NO: 28)
DIQMTQSPSSLSASVGDRVTITCRASQSINTYLAWYOOKPGKAPKLLIYSASFLYSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYQQSDDTPPTFGQGTKVEIKR Heavy chain VR for
Anti-HtrA1 Antibody YW505.94a.28 (SEQ ID NO: 29)
EVQLVESGGGLVQPGGSLRLSCAASGFSISGYYIHWVRQAPGKGLEWVGWIDPYGGDTN
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTSWGHYFDYWGQGT Light
chain HVRs for Anti-HtrA1 Antibody YW505.94a.54 (SEQ ID NO: 21) HVR
L1: RASQVVGNYLA (SEQ ID NO: 2) HVR L2: SASFLYS (SEQ ID NO: 22) HVR
L3: QQSDDHPPT Heavy chain HVRs for Anti-HtrA1 Antibody YW505.94a.54
(SEQ ID NO: 20) HVR H1: GFSISGYYIH (SEQ ID NO: 5) HVR H2:
WIDPYGGDTNYADSVKG (SEQ ID NO: 6) HVR H3: GTFLTSWGHYFDY Light chain
VR for Anti-HtrA1 Antibody YW505.94a.54 (SEQ ID NO: 30)
DIQMTQSPSSLSASVGDRVTITCRASQVVGNYLAVVYQQKPGKAPKLLIYSASFLYSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSDDHPPTFGQGTKVEIKR Heavy chain VR for
Anti-HtrA1 Antibody YW505.94a.54 (SEQ ID NO: 29)
EVOLVESGGGLVOPGGSLRLSCAASGFSISGYYIHWVRQAPGKGLEWVGWIDPYGGDTN
YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTSWGHYFDYWGQGT Light
chain HVR Consensus Sequences (SEQ ID NO: 23) HVR L1:
RASQX.sub.aX.sub.bX.sub.cX.sub.dX.sub.eX.sub.fA, wherein X.sub.a is
D, S or V; X.sub.b is V or I; X.sub.c is S, N or G; X.sub.d is T or
N; X.sub.e is A or Y; and X.sub.f is V or L; (SEQ ID NO: 2) HVR L2:
SASFLYS (SEQ ID NO: 24) HVR L3:
QQX.sub.gX.sub.hX.sub.iX.sub.jPX.sub.kT, wherein X.sub.g is S, V or
D; X.sub.h is Y, D or S; X.sub.i is T, S, A, D or N; X.sub.j is T,
H, N, S, A, L or R; and X.sub.k is P, T, A or S; Heavy chain HVR
COnsensus Sequences (SEQ ID NO: 25) HVR H1:
GFX.sub.lIX.sub.mX.sub.nYYIH, wherein X.sub.l is N, S or T; X.sub.m
is S, D, Y or A; and X.sub.n is G or D; (SEQ ID NO: 26) HVR H2:
WIDPYGGDTX.sub.oTADSVKG, wherein X.sub.o is N or D; (SEQ ID NO: 27)
HVR H3: GTFLTX.sub.pWGHYFDY, wherein X.sub.p is S or T. Consensus
Light chain VR (SEQ ID NO: 31)
DIQMTQSPSSLSASVGDRVTITCRASQX.sub.aX.sub.bX.sub.cX.sub.dX.sub.eX.sub.fAWYQQ-
KPGKAPKLLIYSASFLYSG
VPSRFSGDSGSGTDFTLTISSLQPEDFATYYCQQX.sub.gX.sub.hX.sub.iX.sub.jPX.sub.kTFGQ-
GTKVEIKR, wherein X.sub.a is D, S or V; X.sub.b is V or I; X.sub.c
is S, N or G; X.sub.d is T or N; X.sub.e is A or Y; X.sub.f is V or
L; X.sub.g is S, V or D; X.sub.h is Y, D or S; X.sub.i is T, S, A,
D or N; X.sub.j is T, H, N, S, A, L or R; and X.sub.k is P, T, A or
S; Consensus Heavy chain VR (SEQ ID NO: 32)
EVQLVESGGGLVQPGGSLRLSCAASGFX.sub.lIX.sub.mX.sub.nYYIHWVRQAPGKGLEWVGWIDPYGG-
D
TX.sub.oYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGTFLTX.sub.pWGHYFDYWGQ
GT, wherein X.sub.l is N, S or T; X.sub.m is S, D, Y or A; X.sub.n
is G or D; X.sub.o is N or D; and X.sub.p is S or T (SEQ ID NO: 33)
YW505.94a51 HVR L1: RASQDVGTYLA (SEQ ID NO: 34) YW505.94a.1 HVR L3:
QQVYSHPPT (SEQ ID NO: 35) YW505.94a.7 HVR L3: QQSYTNPPT (SEQ ID NO:
36) YW505.94a.22 HVR L3: QQSYATPTT (SEQ ID NO: 37) YW505.94a.37 HVR
L3: QQSYSSPAT (SEQ ID NO: 38) YW505.94a.39 HVR L3: QQVYTTPFT (SEQ
ID NO: 39) YW505.94a.40 HVR L3: QQVYATPST (SEQ ID NO: 40)
YW505.94a.42 HVR L3: QQSYNSPAT (SEQ ID NO: 41) YW505.94a.46 HVR L3:
QQSYSTPAT (SEQ ID NO: 42) YW505.94a.50 HVR L3: QQSYTAPTT (SEQ ID
NO: 43) YW505.94a.51 HVR L3: QQDSTLPPT (SEQ ID NO: 44) YW505.94a.52
HVR L3: QQSDAAPPT (SEQ ID NO: 45) YW505.94a.78 HVR L3: QQSYSTPPT
(SEQ ID NO: 46) YW505.94a.89 HVR L3: QQSYTRPPT (SEQ ID NO: 47)
YW505.94a.7 HVR H1: GFSISDYYIH (SEQ ID NO: 48) YW505.94a.26 HVR H1:
GFSIDGYYIH (SEQ ID NO: 49) YW505.94a.38 HVR H1: GFTIYDYYIH (SEQ ID
NO: 50) YW505.94a.78 HVR H1: GFSIAGYYIH (SEQ ID NO: 51)
YW505.94a.82 HVR H1: GFTISDYYIH (SEQ ID NO: 52) YW505.94A.42 HVR
H2: WIDPYGGDTDYADSVKG
(SEQ ID NO: 53) YW505.94A.46 HVR H3: GTFLTTWGHYFDY
TABLE-US-00003 TABLE A HVR and variable domain sequences for
anti-HtrA1 YW505.94 antibody and affinity-improved variants
thereof. SEQ ID NOs H1 H2 H3 L1 L2 L3 HC VR LC VR Consensus 25 26
27 23 2 24 32 31 YW505.94 4 5 6 1 2 3 8 7 YW505.94a 20 5 6 1 2 3 29
7 YW505.94a.28 20 5 6 18 2 19 29 28 YW505.94a.54 20 5 6 21 2 22 29
30 YW505.94a.1 20 5 6 1 2 34 YW505.94a.7 47 5 6 1 2 35 YW505.94a.22
20 5 6 1 2 36 YW505.94a.26 48 5 6 1 2 3 YW505.94a.37 47 5 6 1 2 37
YW505.94a.38 49 5 6 1 2 3 YW505.94a.39 20 5 6 1 2 38 YW505.94a.40
20 5 6 1 2 39 YW505.94a.42 20 52 6 1 2 40 YW505.94a.46 20 5 53 1 2
41 YW505.94a.47 20 52 6 1 2 3 YW505.94a.50 47 5 6 1 2 42
YW505.94a.51 20 5 6 33 2 43 YW505.94a.52 20 5 6 1 2 44 YW505.94a.77
20 5 53 1 2 3 YW505.94a.78 50 5 6 1 2 45 YW505.94a.82 51 5 6 1 2 3
YW505.94a.89 20 52 6 1 2 46
III. Examples
[0277] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1: Generation of Anti-HtrA1 Antibodies
[0278] Phage Display.
[0279] Synthetic antibody libraries displayed bivalent Fab
fragments on the M13 (phage and the diversity was generated by use
of oligo-directed mutagenesis in three complementarity determining
regions (CDRs) of the heavy chain. The details of the Fab libraries
were described previously (Lee, C. V., et al., J Mol Biol.
340:1073-93 (2004); Lee, C. V., et al., J Immunol Methods.
284:119-32 (2004)). Nunc 96-well Maxisorp immunoplates were coated
overnight at 4.degree. C. with MuHtrA1_PD (10 .mu.g/ml) and then
blocked for 1 h at room temperature with phage blocking buffer
(PBS, 1% (w/v) BSA, 0.05% (v/v) Tween 20). The antibody phage
libraries were added to the MuHtrA1_PD-coated plates and incubated
overnight at room temperature. The plates were washed with PBS,
0.05% (v/v) Tween-20 buffer and bound phage were eluted with 50 mM
HCl-500 mM NaCl for 30 min and neutralized with an equal volume of
1 M Tris-HCl, pH 7.5. Recovered phage was amplified in E. coli XL-1
blue cells. During subsequent selection rounds, incubation of
antibody phage with the antigen-coated plates was reduced to 2-3
hours and the stringency of plate washing was gradually increased.
13 identified clones were reformatted to IgG (as described below
for Fab94), expressed in 293 cells and purified by Protein A
affinity chromatography. The 13 purified IgG bound to MuHtrA1_PD in
a binding ELISA.
[0280] Anti-HtrA1 Fab94 and IgG94 Expression and Purification.
[0281] The variable regions of both the heavy and light chains of
Fab94 (YW505.94) were subcloned into a E. coli Fab expression
vector (AEP1). The resulting plasmid was transformed into E. coli.
strain 34B8. The single colony was grown overnight at 37.degree. C.
in 30 ml LB medium supplemented with 50 .mu.g/ml of Carbenicillin.
Five ml of the culture was inoculated into 500 ml of complete
C.R.A.P. medium (Simmons, L. C., et al., J Immunol Methods.
263:133-47 (2002)) supplemented with Carbenicillin (50 pig/ml) and
grown at 30.degree. C. for 24 h. The Fab94 protein was purified
using protein A agarose resin.
[0282] The variable domains of both the light chain and heavy chain
of Fab94 were cloned into a pRK5-based plasmid with human light
chain or heavy chain (human IgG1) constant domain for transient
expression in Chinese hamster ovary (CHO) cells. The IgG94 protein
was purified by use of protein A agarose chromatography.
[0283] The amino acid sequences for the light chain variable region
and heavy chain variable region for Fab94 (YW505.94) are shown in
FIGS. 1A and 1B, respectively.
Example 2: Production of HtrA1 Proteins
[0284] Protein Constructs.
[0285] Full length human HtrA1 (HuHtrA1) (Q23-P480) and full length
murine HtrA1 (MuHtrA1) (P24-P480) were cloned by PCR amplification
from full length clones using Taq polymerase. The primers contained
the nucleotides required for adding portions of the restriction
sites BamHI and EcoRI to the resulting PCR products. The PCR
fragments were ligated into a modified pAcGP67 baculovirus transfer
vector (BD Pharmingen) containing a His.sub.6 sequence (SEQ ID NO:
90) upstream of the BamHI restriction site resulting in an
N-terminal His.sub.6 tag (SEQ ID NO: 90) on the HtrA1 proteins.
[0286] The protease domain of human HtrA1 (HtrA1_PD) lacking the
N-domain and the PDZ domain (e.g., containing residues D161-K379)
was cloned by PCR amplification. The amplified DNA was inserted
into a modified pET vector resulting in an N-terminal His.sub.6 tag
(SEQ ID NO: 90) and a thrombin cleavage site fused upstream of the
D161 codon of HtrA1. HtrA1_PD Ala mutants were generated using the
QuikChange XL site-directed mutagenesis kit according to the
manufacturer's protocol (Agilent Technologies, Santa Clara Calif.).
Constructs were verified by DNA sequencing. The protease domains of
murine HtrA1 (MuHtrA1_PD) (G 156-S367), murine HtrA3 (MuHtrA3_PD)
(P133-F350) and murine HtrA4 (MuHtrA4_PD) (E159-Y371) were cloned
from full length clones using Pfu (Agilent) polymerase. The primers
contained the nucleotides required for adding the restriction sites
Not I and Stu I to the resulting PCR products. The PCR fragments
were ligated into pST23 of an expression vector with a phoA
promoter. This expression vector contains the amino acid sequences
MK(HQ).sub.6MHQSTAA (SEQ ID NO: 1) upstream of the Not I
restriction site resulting in N-terminal fusion of unizyme tag to
the protease domains.
[0287] As described herein, amino acid residues of huHtrA1,
muHtrA1, muHtrA3, and muHtrA4 are made with reference to SEQ ID
NOs: 13-16, respectively. Amino acids positions are specified by
the one letter amino acid code followed by its position within one
of SEQ ID NOs: 13-16. For example, full length huHtrA1 comprises a
sequence starting at glutamine at position 23 of SEQ ID NO:13 and
ending at proline at position 480 of SEQ ID NO:13, e.g., Q23-P480.
Similarly, mutations at a particular position within an HtrA
protein are designated by the starting amino acid, followed by the
position within one of SEQ ID NOs:13-16, followed by the
substituted amino acid. For Example, a mutation of Lysine at
position 248 of HuHtrA1 (SEQ ID NO: 13) to alanine is referred to
as K248A.
[0288] Protein Expression and Purification.
[0289] HuHtrA1 and MuHtrA1 were expressed in Trichoplusia ni insect
cells (Expression Systems LLC, Woodland, Calif.). Harvested insect
cell media (after pH was adjusted to 6.8) had NiCl.sub.2,
CaCl.sub.2, and NaCl added to final concentrations of 1.0, 2.5 and
150 mM respectively.
[0290] HuHtrA1_PD constructs (wildtype and S328A mutant) were
expressed in E. coli BL21 (Stratagene). E. coli cultures were grown
at 37.degree. C. in LB medium containing 50 .mu.g/ml carbenicillin
until A.sub.600 reached 0.8 to 1.0 and then induced with 0.4 mM
IPTG and grown at 16.degree. C. for 24 h. The bacterial cell
pellets were resuspended in 1/10.sup.th culture volume of 50 mM
Tris pH 8.0, 500 mM NaCl and disrupted using a Microfluidizer.
Lysates were centrifuged at 30,000.times.g for 30 min.
[0291] HuHtrA1, MuHtrA1, HuHtrA1_PD and HuHtrA1_PD(S328A) were
purified using nickel-nitrilo-triacetic acid resin (Qiagen). After
loading insect cell media or E. coli lysates, columns were washed
with 10 column volumes (CV) of 50 mM Tris pH 8.0, 500 mM NaCl
followed by 10 CV of 50 mM Tris pH 8.0, 1 M NaCl, 20 mM imidazole.
Proteins were eluted with 50 mM Tris pH 8.0, 200 mM NaCl, 10%
glycerol, 0.25% CHAPS, 300 mM imidazole. Pooled fractions were
further purified by size exclusion chromatography on a S-200 column
(GE Healthcare) and peak fractions corresponding to the trimeric
protein forms were collected. Protein purity was greater than 90%
as assessed by SDS-PAGE and protein concentrations were determined
using the BCA.TM. protein assay (Pierce, Rockford, Ill.).
[0292] HuHtrA1_PD Ala mutants (panel of 15 mutants except S328A)
were expressed as described for the wildtype form of HuHtrA1_PD
(see above). Lysis buffer (PopCulture, EMD Chemical, San Diego
Calif.) was added to bacterial cell pellets at 50 ml lysis
buffer/500 ml pellet. The pellet was suspended completely in lysis
buffer with a polytron and stirred at room temperature for 30 min.
The cell lysate was centrifuged (Beckman, LA-16.250 rotor) at
33,700.times.g for 30 min at 4.degree. C. and the resulting
supernatant was filtered through a 0.22 .mu.m filter unit
(Nalgene). The filtered supernatant was loaded onto a Ni-NTA column
(3 ml, Qiagen) pre-equilibrated with 50 mM Tris, 300 mM NaCl, 10 mM
imidazole, 10% glycerol pH 8.0 (buffer A). Once loaded the column
was washed with 12 CV of buffer A followed by 20 CV of buffer
A+0.1% Triton X-114. The column was washed again with 15 CV of
buffer A to remove detergent. Proteins were eluted with buffer
A+200 mM imidazole, 0.25% CHAPS. Pooled Fractions were further
purified with Superdex 200 (Hi load 16/60, 120 ml, GE Healthcare)
equilibrated with 50 mM Tris pH 8.0, 200 mM NaCl, 10% glycerol,
0.25% CHAPS. The fractions corresponding to the HtrA1_PD trimer
peak were pooled. Protein purity was greater than 90% as assessed
by SDS-PAGE and protein concentrations were determined by use of
the BCA.TM. protein assay (Pierce, Rockford, Ill.).
[0293] MuHtrA1_PD, MuHtrA3_PD and MuHtrA4_PD were expressed in E.
coli 58F3 (Szeto, W., et al., Cancer Res. 61:4197-205 (2001)).
Overnight E. coli cultures, grown at 30.degree. C. in LB medium
containing 50 .mu.g/ml carbenicillin, were diluted ( 1/100 vol)
into a larger culture containing C.R.A.P. medium (Simmons, L. C.,
et al., J Immunol Methods. 263:133-47 (2002)) supplemented with 50
g/ml carbenicillin. After inoculation E. coli were grown for
approximately 20 h at 30.degree. C. MuHtrA1_PD was purified as
described for HuHtrA1_PD (see above). For purification of
MuHtrA3_PD and MuHtrA4_PD, lysis buffer (50 mM Tris pH 8.5, 500 mM
NaCl, 10 mM imidazole, 10% glycerol) was added to the bacterial
cell pellet. The pellet was homogenized in lysis buffer with a
polytron and cells disrupted with a mircofludizer. The cell lysate
was centrifuged and the resulting supernatant filtered through a
0.22 .mu.m filter unit (Nalgene). The filtered supernatant was
loaded onto a 3 ml Ni-NTA column (Qiagen) pre-equilibrated with 25
mM Tris pH 8.5, 500 mM NaCl, 20 mM imidazole, 10% glycerol (buffer
B). After loading, the column was washed with 12 CV of buffer B
followed by 20 CV of buffer B+0.1% Triton X-114. The column was
washed again with 15 CV of buffer B to remove detergent. Proteins
were eluted with 25 mM Tris pH 8.5, 500 mM NaCl, 250 mM imidazole,
10% glycerol, 0.25% CHAPS. Pooled fractions were further purified
on a 120 ml Superdex 75 (GE Healthcare) equilibrated with 25 mM
Tris pH 8.5, 10% glycerol, 0.5 mM TCEP, 0.25% CHAPS. The fractions
corresponding to the protease trimer peak were pooled. Protein
purity was greater than 90% as assessed by SDS-PAGE.
Example 3: Determination of Inhibitory Activity of Anti-HtrA1
Antibodies Using a FRET Assay
[0294] Synthesis of Peptide Substrate.
[0295] The peptide H2-Opt (Mca-IRRVSYSF(Dnp)KK) (SEQ ID NO: 12),
originally described as a substrate for HtrA2 (Martins, L. M., et
al., J Biol Chem. 278:49417-27 (2003)), was synthesized on
Fmoc-Lys(Boc)-wang resin using standard coupling procedures with
HBTU. Fmoc-Lys(DNP)-OH (Anaspec) was incorporated in the P5'
position. The peptide was synthesized up to P5 (Mca,
7-Methoxy-coumarin, Aldrich) and then cleaved from the solid
support using trifluoroacetic acid, triisoproplysilane and water
for 2 hours at room temperature. Peptide was precipitated from
ethyl ether, extracted with acetic acid, acetonitrile, water and
lyophilized. Crude labeled peptide was dissolved and purified on
preparative reverse phase C18 column using acetonitrile/water.
Purified fractions were pooled, lyophilized and analyzed by liquid
chromatography/mass spectrometry (PE/Sciex) and found to be
consistent with their calculated masses.
[0296] Enzymatic Assays with Peptide Substrate.
[0297] HtrA1 was incubated in 96-well black optical bottom plates
(Nalge Nunc Int., Rochester, N.Y.) with IgG94 or Fab94 serially
diluted in 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.25% CHAPS (assay
buffer) for 20 min at 37.degree. C. For initial testing a panel of
13 phage derived anti-HtrA1 antibodies, single concentrations of
IgGs (final concentrations 0.16 mg/ml-0.28 mg/ml) were incubated
with HuHtrA1 or HuHtrA1_PD. A 10 mM stock solution of the peptide
substrate Mca-IRRVSYSF(Dnp)KK (SEQ ID NO: 12) (H2-Opt) in DMSO was
diluted in water to 12.5 .mu.M, pre-warmed at 37.degree. C. and
then added to the reaction mixture. The final concentrations of the
reactants were: 5 nM HuHtrA1 or MuHtrA1, 0.005-300 nM IgG94,
0.02-900 nM Fab94, 2.5 pM H2-Opt. The increase of fluorescence
signal (excitation 328 nm, emission 393 nm) was measured on a
SPECTRAmax M5 microplate reader (Molecular Devices, Sunnyvale,
Calif.) and the linear rates of H2-Opt cleavage (mRFU/min)
determined. Experiments with IgG94 inhibition of trypsin (Roche)
and elastase (MP Biomedicals) were carried out identically, except
that the final enzyme concentrations were 1 nM, the IgG94
concentration was 300 nM and the incubation time was 15 min.
[0298] As shown in FIG. 2, a panel of 13 phage derived antibodies
(IgG) was incubated with HuHtrA1 or HuHtrA1_PD and enzyme activity
measured. Of the 13 antibodies tested, antibodies YW503.57,
YW504.57, YW504.61 and YW505.94 (also referred to as Fab94,
antibody 94, Ab94 or IgG94) strongly inhibited both HuHtrA1 and
HuHtrA1_PD activities.
[0299] As shown in FIG. 3, IgG94 inhibited the enzymatic activity
of HuHtrA1 towards the H2-Opt substrate in a
concentration-dependent fashion with an IC.sub.50 value of 1.8 nM.
Complete inhibition was achieved above 30 nM IgG94. In contrast,
two other members of the trypsin-like serine protease family,
trypsin and elastase, were not inhibited by IgG94 at 300 nM,
indicating that IgG94 has specificity towards HuHtrA1. Fab94 also
inhibited HuHtrA1 in a concentration-dependent manner, but less
potently than IgG94 as indicated by the IC.sub.50 value of 29.1 nM.
These results are in excellent agreement with the K.sub.D value of
31.1 nM determined by surface plasmon resonance experiments (see
Table 1 below). The results show that IgG94 and Fab94 are able to
completely neutralize the enzymatic activity of HuHtrA1 towards a
small synthetic peptide substrate and that this activity is
specific. Moreover, the about 16-fold increase in potency of IgG94
vs. Fab94 is consistent with the avidity effects predicted from the
`cage-like` IgG94:HtrA1 complex based on mass analysis (see e.g.,
Table 2 and FIG. 9).
[0300] The inhibitory potencies of affinity-improved variant
antibody YW505.94a.28 was also determined using the assay described
above with the fluorescence quenched peptide substrate H2-Opt and
1.0 nM of HuHtrA1 or 1.5 nM of HuHtrA1_PD. The results are shown
below.
TABLE-US-00004 YW505.94a.28 HuHtrA1_PD HuHtrA1 format IC.sub.50 nM
IC.sub.50 nM IgG 0.229 0.335 Fab 2.03 1.95
Example 4: Determination of Inhibitory Activity of Anti-HtrA1
Antibodies Using Macromolecular Substrates
[0301] Bovine .beta.-casein (Sigma-Aldrich) was repurified on a
MonoQ ion exchange column to yield highly purified material.
HuHtrA1 was incubated in Eppendorf tubes together with increasing
concentrations of IgG94 in assay buffer for 15 min at 37.degree. C.
after which the macromolecular substrates were added. For
.beta.-casein digestion, the final concentration of reactants were:
10 nM HuHtrA1, 50 .mu.g/ml .beta.-casein, 2.3-150 nM IgG94. For
decorin the final concentrations were: 125 nM HuHtrA1, 50 .mu.g/ml
decorin (R & D Systems), 2-125 nM IgG94. For biglycan the final
concentrations were: 75 nM HuHtrA1, 50 .mu.g/ml biglycan (R & D
Systems), 2.3-150 nM IgG94. After incubation at 37.degree. C. (2 h
for .beta.-casein, 14 h for decorin, 6 h for biglycan), SDS sample
buffer was added and samples boiled and analyzed by SDS-PAGE
(non-reducing).
[0302] Hydrolysis of fluorescent dye-labeled casein, BODIPY FL
casein (Invitrogen), was carried out in 96-well black optical
bottom plates (Nalge Nunc Int., Rochester, N.Y.). HuHtrA1 or
MuHtrA1 were incubated with IgG94 serially diluted in assay buffer
for 15 min at 37.degree. C. After addition of BODIPY FL in assay
buffer, the reactant concentrations were as follows: 30 nM HuHtrA1,
30 nM MuHtrA1, 5 g/ml BODIPY FL, 0.24-250 nM IgG94. The increase of
fluorescence signal (excitation 484 nm, emission 535 nm) was
measured on a SPECTRAmax M5 microplate reader (Molecular Devices,
Sunnyvale, Calif.) and the linear rates of BODIPY FL cleavage
(mRFU/min) determined and expressed as percentage of uninhibited
rates (control).
[0303] As shown in FIG. 6, HuHtrA1 degraded the macromolecular
substrates .beta.-casein, decorin and biglycan (lane 2). In the
absence of HuHtrA1, the substrates remained intact during the
experimental period (FIG. 6, lane 1). Preincubation of HuHtrA1 with
increasing concentrations of IgG94 (FIG. 6, lane 9-lane 3) resulted
in a concentration-dependent inhibition of substrate degradation.
At the highest IgG94 concentrations tested, degradation of
0-casein, decorin and biglycan was completely prevented (FIG. 6,
lane 3). The results show that IgG94 potently and effectively
inhibited HuHtrA1 activity towards three macromolecular
substrates.
[0304] As shown in FIG. 4, IgG94 concentration-dependently
inhibited BODIPY FL hydrolysis by both HuHtrA1 and MuHtrA1 with
IC.sub.50 values of 8.3 nM and 5.4 nM, respectively. The results
show that IgG94 recognizes and completely neutralizes the enzymatic
activities of both HuHtrA1 and MuHtrA1 towards the macromolecular
substrate BODIPY FL.
Example 5: Determination of the Specificity of Anti-HtrA1
Antibodies to HtrA1
[0305] The specificity of IgG94 was assessed by measuring its
activity for inhibiting muHtrA1_PD as compared to its ability to
inhibit the structurally related proteases MuHtrA3_PD and
MuHtrA4_PD (see FIG. 5). The specificity was determined using the
FRET assay as described above in Example 3, except that the
concentrations of MuHtrA1_PD, MuHtrA3_PD and MuHtrA4_PD 6 nM, 20 nM
and 20 nM, respectively, and the concentration range of IgG94 was
0.2-400 nM. The specificity was also analyzed using the BODIPY FL
casein substrate as described above in Example 4, except that the
concentration of MuHtrA1_PD, MuHtrA3_PD and MuHtrA4_PD was 50 nM
and the concentration range of IgG94 was 1.4-1000 nM. IgG94
inhibited MuHtrA1_PD cleavage of H2-Opt (FIG. 5, top panel) and
BODIPY FL (FIG. 5, bottom panel), but did not inhibit cleavage of
the same substrates by MuHtrA3_PD and MuHtrA4_PD up to
concentrations of 400 nM and 1000 nM for H2-Opt and BODIPY FL
substrates, respectively. The results suggest that IgG94 has
excellent specificity and does not impair activities of related
proteases.
Example 6: Determination of Antibody Affinity by BIAcore
[0306] To determine the binding affinity of Fab94 by single-cycle
kinetics, Surface Plasmon Resonance (SRP) measurement with a
BIAcore.TM. T100 instrument was used. Briefly, series S sensor chip
CM5 was activated with EDC and NHS reagents according to the
supplier's instructions, and streptavidin (Pierce) was coupled to
achieve approximately 2000 response units (RU), followed by
blocking unreacted groups with 1M ethanolamine.
[0307] For kinetics measurements, biotinylated HuHtrA1_PD or
MuHtrA1_PD were first injected at 10 l/min flow rate to capture
approximately 100 RU at 3 different flow cells (FC), except for FC1
(reference cell), and then 5-fold serial dilutions of Fab94 (0.48
nM-300 nM) in 0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% surfactant P20
were injected (flow rate: 30 .mu.l/min) with no regeneration
between injections. The sensorgrams were recorded and evaluated by
BIAcore.TM. T100 Evaluation Software (version 2.0) after
subtraction of reference cell signal. Association rates (k.sub.on)
and dissociation rates (k.sub.off) were calculated using a simple
one-to-one Langmuir binding model. The equilibrium dissociation
constant (K.sub.D) was calculated as the ratio k.sub.off/k.sub.on
(see Table 1 below). The binding kinetics of Fab94 to HuHtrA1_PD
and to MuHtrA1_PD were very similar. The K.sub.D values were 31.1
nM and 28.9 nM, respectively.
TABLE-US-00005 TABLE 1 Binding Affinities of Fab94 to HuHtrA1_PD
and MuHtrA1_PD by Surface Plasmon Resonance. k.sub.on k.sub.off
K.sub.D (10.sup.5 M.sup.-1s.sup.-1) (10.sup.4 s.sup.-1) (nM)
HuHtrA1_PD 3.5 109.0 31.1 MuHtrA1_PD 4.5 129.0 28.9
Example 7: Epitope Mapping of IgG94 on HuHtrA1_PD
[0308] To identify the functional binding epitope of IgG94, a panel
of HuHtrA1_PD mutants with individual alanine mutations was
generated for binding experiments. Most of the mutated residues are
located on surface loops surrounding the active site and also
included the catalytic serine (S328) and aspartate (D250). The
mutants were expressed in E. coli and the trimers purified by size
exclusion chromatography as described in Example 2. Binding was
determined using an ELISA assay, which was conducted using a
MAXISORP.TM. microtiter plate coated with HuHtrA1_PD mutants at 2
.mu.g/ml in PBS for 1 h, followed by blocking with PBST buffer
(0.5% BSA and 0.05%/Tween 20 in PBS) for 1 h at room temperature.
Five-fold serially diluted Ig94 (50 nM to 0.0006 nM) in PBST buffer
was added and incubated for 30 min. The plates were washed with PBT
buffer (0.05% Tween 20 in PBS), and HRP-conjugated goat anti-human
IgG (H+L) (Invitrogen) was added (1:5000 in PBST buffer) and
incubated for 1 h. The plates were washed with PBT buffer and
developed by adding tetramethylbenzidine substrate (Kirkegaard and
Perry Laboratories, Gaithersburg, Md.). The absorbance at 450 nm
was plotted as a function of antibody concentration in solution to
determine EC.sub.50 values. The location of mutants effecting
binding were then mapped onto the structure of HtrA1.
[0309] In the binding ELISA, IgG94 showed the greatest reduction in
binding to the mutants HuHtrA1_PD(N224A) and HuHtrA1_PD(K248A) (see
FIG. 7A). Upon repetition of the experiment using a lower
concentration of HtrA1 to coat the plates (1 .mu.g/ml) and a
shortened incubation time (reduced from 1 hour to 20 minutes
incubation), IgG94 also showed at least a 5-fold reduction in
binding to HuHtrA1 mutants V201A, H220A, T223A, K225A, K243A and
E247A (see FIG. 7A). The results indicate that IgG94 binds to an
epitope that includes residues N224 and K248, which are located on
Loop B and Loop C (see FIGS. 7B and 7C). In trypsin-like serine
proteases these loops are known to be part of the specificity
determining region that interacts with substrates. K248 (Loop C) is
very close to the catalytic D250 and N224 to the catalytic H220.
Therefore, binding of IgG94 to these residues may impair substrate
access to the catalytic cleft either by direct steric hindrance or
by an indirect allosteric mechanism as found for neutralizing
anti-HGFA antibodies that also recognize residues in Loop C
(Ganesan, R., et al., Structure 17:1614-1624 (2009); Wu, Y., et
al., Proc Natl Acad Sci USA 104:19784-9 (2007)). Alternatively,
antibody binding may inhibit catalysis by directly influencing the
catalytic triad residues D250 and/or H220.
Example 8: Stoichiometry of Complexes of HtrA1_PD with Fab94 and
IgG94 by Size Exclusion Chromatography--Multi-Angle Light
Scattering (SEC-MALLS)
[0310] A catalytically inactive form HuHtrA1_PD(S328A) was used for
complex formation. The Fab94:HuHtrA1_PD(S328A) and
IgG94:HuHtrA1_PD(S328A) complexes were formed in Tris buffer (50 mM
Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol, 0.25% CHAPS). The
complexes and the individual components alone were incubated at
4.degree. C. overnight and then injected and resolved on a S200
Superdex 10/300 GL column (GE Healthcare) in Tris buffer with a
flow rate of 0.5 mL/min. Data was collected with an 18-angle light
scatter detector (Dawn Helios II with QELS) and a refractive index
detector (Optilab reX) from Wyatt Technologies. Analysis was done
with Astra 5 software to yield molar mass independently of elution
time. Normalization was performed with a control IgG.
[0311] SEC-MALLS experiments show that Fab94 and IgG94 form
complexes with HuHtrA1_PD(S328A) that have distinct masses. The
determined masses and deduced stoichiometry of the complexes are
shown in Table 2.
TABLE-US-00006 TABLE 2 Stoichiometry of HuHtrA1_PD(S328A) in
complex with Fab94 and IgG94 determined by SEC-MALLS. Mass by
Expected Mass MALLS Best-fit mass difference Components* (Da)**
stoichiometry.sup.& (Da) (%) HtrA1_PD 76,755 -- -- -- Fab94
47,400 -- -- -- IgG94 151,200 -- -- -- Fab94:HtrA1_PD 210,100 3:1
218,955 4.2 IgG94:HtrA1_PD 618,600 3:2 607,110 1.9 *HuHtrA1_PD was
HuHtrA1_PD with the catalytic serine mutated to alanine
(HuHtrA1_PD(S328A)); the mass represents the trimer **Average of
two independent experiments .sup.&Ratio of antibody:protease
trimer
[0312] The determined mass of the Fab94:HtrA1_PD(S328A) complex of
210,100 Da is consistent with a complex of 3 Fabs binding to one
HuHtrA1_PD(S328A) trimer (3:1 complex). The difference between the
experimental and the theoretical masses of such a 3:1 complex is
only 4.2%. Therefore, one Fab is able to bind to each
HuHtrA1_PD(S328A) monomer within one HtrA1 homo-trimer.
[0313] The determined mass of the IgG94:HtrA1_PD(S328A) complex of
618,600 Da fits well to a 3:2 stoichiometry, in that 3 IgG
molecules bind to two HuHtrA1_PD(S328A) trimers. The difference
between the experimental and the theoretical mass of such a 3:2
complex is only 1.9%.
[0314] The elucidated stoichiometries are in agreement with the
findings that Fab94 and IgG94 are able to completely inhibit HtrA1
activity, in that each monomer within an HtrA1 trimer is binding to
one Fab or to one Fab arm of an IgG. Thus, in these complexes there
is no `free` HtrA1 monomer available, in agreement with the
complete inhibition of HtrA1 enzyme activity by Fab94 and IgG94. We
propose a `cage` model for the IgG94:HtrA1_PD(S328A) complex in
which the Fab arms of the 3 IgGs are bridging two trimers, each Fab
arm binding to one monomer (see FIG. 9). The model also accounts
for the about 16-fold increased potency of IgG94 over Fab94 in
enzymatic assays (see e.g., FIG. 3) in that IgG94, strongly
benefits from avidity effects in its binding to HtrA1_PD
trimers.
Example 9: Affinity Maturation of YW505.94
[0315] Construct Libraries for Anti-HtrA1 Affinity Maturation.
[0316] Clone YW505.94a was derived from YW505.94 [Ab94] by changing
Kabat residue N28 to serine within CDR H1 in order to remove a
potential N-linked glycosylation site. Phagemid pW0703, derived
from phagemid pV0350-2b (Lee et al., J. Mol. Biol 340, 1073-1093
(2004)), which contains a stop codon (TAA) in all CDR-L3 positions
and displays monovalent Fab on the surface of M13 bacteriophage,
served as a library template for grafting the heavy chain variable
domain (V.sub.H) of clone YW505.94a for affinity maturation. Both
hard and soft randomization strategies were used for affinity
maturation. For hard randomization, one light chain library
(L1/L2/L3hard) with selected positions at three light chain CDRs
was randomized using amino acids designed to mimic a natural human
antibody and the designed DNA degeneracy was as described (Lee et
al., 2004). For soft randomization, selected residues at Kabat
positions 91, 92, 93, 94 and 96 of CDR-L3, 28-35 of CDR-H1, 50-58
of CDR-H2, and 95-100 of CDR-H3 with two different combinations of
CDR loops, L3/H1soft, L3/H2soft and L3/H3soft, were targeted for
randomization. To achieve the soft randomization conditions, which
introduce a mutation rate of approximately 50% at the selected
positions, the mutagenic DNA was synthesized with 70-10-10-10
mixtures of bases favoring the wild type nucleotides (Gallop et
al., J. of Med. Chem. 37, 1233-1251 (1994)).
[0317] Phage Sorting Strategy to Isolate Affinity-Improved
Variants.
[0318] For affinity improvement selection, phage libraries were
subjected to plate sorting for the first round, followed by four
rounds of solution sorting. For the first round of plate sorting,
four libraries (L1/L2/L3hard, L3/H1soft, L3/H2soft and L3/H3soft)
were sorted against a human HtrA1 (HuHtrA1) coated plate (NUNC
Maxisorp plate) separately with phage input at about 3 O.D./ml in
1% BSA and 0.05% Tween 20 for 1 hour at room temperature (RT).
After the first round of plate sorting, four rounds of solution
sorting were performed to increase the stringency of selection. For
solution sorting, 1 O.D./ml of phage propagated from first round of
plate sorting were incubated with 500 nM of biotinylated HuHtrA1 in
100 ul buffer containing 1% Superblock (Pierce biotechnology) and
0.05% Tween20 for at least 1 hour at room temperature (RT). The
mixture was further diluted 10.times. with 1% Superblock and
applied 100 ul/well to neutravidin-coated wells (10 ug/ml) for 15
min at RT with gentle shaking so that biotinylated HuHtrA1 could
bind phage. The wells were washed with PBS-0.05% Tween20 ten times.
To determine background binding, control wells containing phage
without biotinylated HuHtrA1 selection were captured on
neutravidin-coated plates. Bound phage was eluted with 0.1N HCl for
20 min, neutralized by 1/10 volume of 1M Tris pH11, titered, and
propagated for the next round. Next, three more rounds of solution
sorting were carried out using two methods of increasing selection
stringency simultaneously. The first method, which is for on-rate
selection, decreases biotinylated target protein concentration from
10 nM to 0.1 nM. The second method, which is for off-rate
selection, adds excess amounts of non-biotinylated HuHtrA1 protein
(100.about.1000 fold more) to compete off weaker binders. Also, the
phage input was decreased (0.1.about.0.5 O.D/ml) to lower the
background phage binding.
[0319] High Throughput Affinity Screening ELISA (Single Spot
Competition).
[0320] Colonies were picked from the fifth round screening and
grown overnight at 37.degree. C. in 350 ul/well of 2YT media with
50 ug/ml carbenicillin and 1e 10/ml KO7 in 96-well block (QIAgene).
From the same plate, a colony of XL-1 infected parental phage was
picked as a control. 96-well Nunc Maxisorp plates were coated with
100 ul/well of HuHtrA1 protein (2 ug/ml) in PBS at RT for 2 hours.
The plates were blocked with 100 ul of 0.5% BSA and 0.05% Tween in
PBS (PBST buffer) for one hour.
[0321] The phage supernatant was diluted 1:5 in PBST buffer with or
without 5 nM HuHtrA1 in 100 ul total volume and incubated at least
1 hour at RT. Then, 75 ul of mixture were transferred to the
HuHtrA1 coated plates. The plate was gently shaken for 15 min to
allow the capture of unbound phage to the HuHtrA1 coated plate. The
plate was washed five times with 0.05% Tween20 in PBS (PBT buffer).
The binding was quantified by adding horse radish peroxidase
(HRP)-conjugated anti-M13 antibody in ELISA buffer (1:5000) and
incubated for 30 min at RT. The plates were washed with PBT buffer
five times. Next, 100 ul/well of a 1:1 ratio of
3,3',5,5'-tetramethylbenzidine (TMB) Peroxidase substrate and
Peroxidase Solution B (H.sub.2O.sub.2) (Kirkegaard-Perry
Laboratories (Gaithersburg, Md.)) was added to the well and
incubated for 5 minutes at RT. The reaction was stopped by adding
100 ul 1M Phosphoric Acid (H.sub.3PO.sub.4) to each well and
allowed to incubate for 5 minutes at RT. The OD of each well was
determined using a standard ELISA plate reader at 450 nm. The OD
reduction (%) was calculated by the following equation:
OD.sub.450 nmreduction (%)=[(OD.sub.450 nm of wells with
competitor)/(OD.sub.450 nm of well with no competitor)]*100
[0322] In comparison to the OD.sub.450 nm reduction (%) of the well
of parental phage (100%), clones that had the OD.sub.450 nm
reduction (%) lower than 50% were picked for sequence analysis (see
FIGS. 15 and 16). Unique clones were selected for phage preparation
to determine binding affinity (phage IC.sub.50) by phage
competition ELISA (see FIGS. 17 and 18). Then, the most
affinity-improved clones (YW505.94a.28 & YW505.94a.54) were
reformatted into human IgG1 for antibody production and further
BIAcore binding kinetic analysis.
[0323] Phage Competition ELISA to Determine IC.sub.50.
[0324] MAXISORP.TM. microtiter plates were coated with HuHtrA1 at 2
.mu.g/ml in PBS for 2 hr at RT and then blocked with PBST buffer
for another hour at RT. Purified phage from culture supernatants
were incubated with serially diluted HuHtrA1 or MuHtrA1 in PBST
buffer in a tissue-culture microtiter plate for an hour, after
which 80 .mu.l of the mixture was transferred to the HuHtrA1-coated
wells for 15 minutes to capture unbound phage. The plate was washed
with PBT buffer, and HRP-conjugated anti-M13 (Amersham Pharmacia
Biotech) was added (1:5000 in PBST buffer) for one hour. The plate
was washed with PBT buffer and developed by adding
tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories,
Gaithersburg, Md.). The absorbance at 450 nm was plotted as a
function of HuHtrA1 or MuHtrA1 concentration in solution to
determine phage IC.sub.50. This was used as an affinity estimate
for the Fab clone displayed on the surface of the phage. FIG. 17
shows results from a phage competition assay demonstrating the
binding of YW505.94a affinity-improved variants against HuHtrA1.
FIG. 18 shows results from a phage competition assay demonstrating
the binding of YW505.94a affinity-improved variants against
MuHtrA1.
[0325] Antibody Affinity Determinations by BIAcore.
[0326] To determine the binding affinity of HtrA1 antibodies by
single cycle kinetics, Surface Plasmon Resonance (SRP) measurement
with a BIAcore.TM. T100 instrument was used. Briefly, a series S
sensor chip CM5 was activated with EDC and NHS reagents according
to the supplier's instructions, and streptavidin (Pierce) was
coupled to achieve approximately 2000 response units (RU), followed
by blocking un-reacted groups with 1M ethanolamine.
[0327] For kinetics measurements, biotinylated human or murine
HtrA1 was first injected at 10 ul/min flow rate to capture
approximately 100 RU at 3 different flow cells (FC), except for FC
1 (reference), and then 5-fold serial dilutions of anti-HtrA1 Fab
in HBS-P buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 0.005% surfactant
P20) from low (0.48 nM) to high (300 nM) [5 points] were injected
(flow rate: 30 .mu.l/min) one after the other in the same cycle
with no regeneration between injections. The sensorgram was
recorded and subject to reference and buffer subtraction before
evaluation by BIAcore.TM. T100 Evaluation Software (version 2.0).
Association rates (k.sub.on) and dissociation rates (k.sub.off)
were calculated using a simple one-to-one Langmuir binding model.
The equilibrium dissociation constant (Kd) was calculated as the
ratio k.sub.off/k.sub.on. The results are shown in Table 3
below.
TABLE-US-00007 TABLE 3 Binding Affinities of Affinity Matured Fabs
as Compared to the Parent Fab. HuHtrA1 MuHtrA1 k.sub.on k.sub.off
K.sub.D k.sub.on k.sub.off K.sub.D Fab (M.sup.-1s.sup.-1)
(s.sup.-1) (nM) (M.sup.-1s.sup.-1) (s.sup.-1) (nM) YW505.94 3.51e5
109e-4 31.1 4.47e5 129e-4 28.9 YW505.94a 8.13e5 4.48e-2 55 1.48e6
8.56e-2 58 YW505.94a.28 1.69e6 5.74e-3 3.4 2.02e6 9.3e-3 4.6
YW505.94a.54 2.97e6 7.84e-3 2.6 2.82e6 4.55e-3 1.6
Example 10: Sparing of Photoreceptors, Outer Nuclear Layer (ONL),
and Functional Responses in the Absence of HtrA1 Following Constant
Light Exposure
[0328] Construction of HtrA1 Knockout Mouse.
[0329] To generate HtrA1(+/-) embryonic stem cells, linearized
targeting vector DNA was electroporated into ES cells of Balb/c
background to introduce Flox sites in the region 5' of exon 1 and
3' of exon 1 and an introduced neomycin-resistance gene (Neo). ES
clones resistant to neomycin were selected, and exon 1 plus the
neomycin cassette was excised by electroporating the ES cells with
a cre-recombinase-expressing expression plasmid. Homologous
recombination was confirmed by sequencing of the entire HtrA1 locus
and upstream promoter region in the targeted ES cell. Targeted
clones were injected into C57BL/6 blastocysts to generate male
chimeric mice of germline transmission. Interbreeding of +/- female
and +/- male mice was performed to generate -/- female or -/- male
HtrA1 mice on a Balb/c background. Successful deletion of HtrA1 was
confirmed by RT-PCR of ovaries from HtrA1 wt and ko mice.
[0330] Constant Light Exposure Model.
[0331] Male Balb/c Htra1 wt/wt or ko/ko mice, 8-13 weeks old, are
in normal housing (<100 lux in cage) until start of constant
light exposure (CLE). To start CLE, mice are housed singly in
normal cages covered with a wire rack only, without a filter top.
Food pellets and Hydrogels are placed on the bottom of the cage,
and not on the wire top, for nourishment so as to not impede light
entering the cage. Ten cages are placed on a Metro rack outfitted
with fluorescent lights and enclosed in white panels to deliver
.about.1200 lux to mice for up to 14 days. Cages are rotated
counterclockwise around the rack daily during CLE to ensure equal
light exposure. If multiple shelves are used, cages are also
rotated between shelves to ensure equal light exposure.
[0332] Optical Coherence Tomography.
[0333] Optical coherence tomography (OCT) is performed 4-7 days
before light exposure to provide a retinal thickness baseline
measurement. OCT is performed using a Heidelberg Spectralis HRA+OCT
camera. Animals are anesthetized with 70-80 mg/kg ketamine/15 mg/kg
xylazine in 150-300 ul sterile saline, eyes are dilated with 1%
tropicamide drops and retina thickness is measured by OCT.
Artificial tears are used to keep eyes moist to prevent cataracts.
After OCT, animals are allowed to recover from anesthesia and
returned to their cages and light rack.
[0334] Electroretinogram.
[0335] Electroretinograms (ERG) ERGs are performed 7 days before
light exposure and at 15 days post CLE. ERGs are performed and
recorded using a Diagnosys LLC Espion 2 visual electrophysiology
system and a Colordome desktop Ganzfeld as a light source. Mice are
dark adapted overnight in a dark room to equilibrate
photoreceptors. Once dark adapted, all subsequent procedures are
performed in the dark with only a red light for illumination.
Animals are anesthetized with Ketamine, 75-80 mg/kg, &
Xylazine, 7.5-15 mg/kg, IP in 200-300 ul PBS. Mouse body
temperature is maintained at 37.degree. C. using a homeothermic
plate connected to its control unit. Pupils are dilated with 1%
tropicamide. ERGs from both eyes will be recorded simultaneously
with Burian-Allen silver or platinum wire loop electrodes. Mice are
placed on a platform, a reference electrode is inserted
subcutaneously through the forehead, and a ground electrode is
inserted subcutaneously at the base of the tail. Gonak hypermellose
solution is placed on the cornea to establish an electrical contact
between the cornea and the electrode, and protect eyes from drying
during the experiment. Electrodes are placed on the left and right
eyes and mouse inserted into a ColorDome light stimulator. Eyes
will be stimulated with white light (7 flash intensities 4e-5,
2e-5, 0.5, 2, 5, 10, 20 cds/m.sup.2) and signals bandpass-filtered
at 0.15-1000 Hz and sampled at 2 kHz.
[0336] Determination of HtrA mRNA Levels in the Mouse Retina and
Ovary.
[0337] Whole eyes were enucleated, the retina removed and placed in
RLT Buffer (Qiagen) and frozen at -80.degree. C. until
homoginization. Retina's were homogenized and shredded by
gentleMACS M tubes (Miltinyi Biotec). Similar to the retina, ovarys
were isolated from female HtrA1 wt, het and ko mice and homogenized
as described for the retina. RNA was isolated using Qiagen Plus
RNeasy kit; cDNA was generated with High Capacity cDNA kit (Applied
Biosystem); Taqman qPCR was performed using 20 ug cDNA, Taqman Gene
Expression Master Mix (Applied Biosystem) and Taqman Gene
Expression Assay primers (Applied Biosystem) to exon 1-2 (retina
and ovary), exon 3-4 (ovary) and exon 5-6 (ovary) and ran on an ABI
7500 Real-time PCR System (Applied Biosystem). HtrA expression was
normalized to 18s expression (primer from Applied Biosystem).
[0338] As shown in FIG. 11, HtrA1 mRNA expression increases in a
mouse model of constant light exposure. However, in the same model,
HtrA2 levels do not increase in the retina. In addition, HtrA1
levels are are significantly higher than HtrA2, HtrA3 and HtrA4
levels in the retina of a non-exposed mouse.
[0339] In addition, we found that mice lacking HtrA1 expression
show a sparing of photoreceptors (FIG. 13), the outer nuclear layer
(ONL) (FIG. 14), and functional responses (ERG) (FIG. 12) in a
mouse model of light-induced degeneration.
Example 11: Effects of an Anti-HtrA1 Antibody in a Rat Model of
Constant Light Exposure
[0340] Rats will be subjected to constant light exposure as
described above for mice. The efficacy of an anti-HtrA1 antibody to
protect the rat eye from degeneration caused by the light exposure
will be evaluated. Rat eyes will be subject to intravitreal
injections of an anti-HtrA1 antibody at a dose and frequency
suitable to maintain an effective concentration of the anti-HtrA1
antibody in the eye during the course of the experiment. The rat
eyes will be monitored following light exposure to determine
retinal thickness by OCT and functionality by ERG, as described
above for the mouse model.
Example 12: HtrA1 Expression is Abundant in Rat Vitreous and
Accumulates in Mouse Eye Fluid and Eye Tissue Upon Light Stress
[0341] ELISA Assay.
[0342] For analysis of rat tissues, an anti-muHtrA1 rabbit
polyclonal antibody (Genentech) was diluted to 125 ng/mL, while for
analysis of mouse eye fluid and retina tissue, an anti-huHtrA1
mouse monoclonal antibody (Genentech) was diluted to 250 ng/mL,
both in PBS, and coated onto 384-well ELISA plates (Nunc; Neptune,
N.J.) during an overnight incubation at 4.degree. C. Plates were
washed with PBS plus 0.05% Tween-20 and blocked during a two hour
incubation with PBS plus 0.5% bovine serum albumin (BSA). This and
all subsequent incubations were performed at room temperature with
gentle agitation. Recombinant murine HtrA1 standard (Genentech) and
samples from rat or murine tissues were diluted in sample/standard
dilution buffer (PBS, 0.5% BSA, 15 ppm Proclin, 0.05% Tween 20,
0.25% CHAPS, 0.2% BgG, 5 mM EDTA, 0.35M NaCl, (pH 7.4)), added to
washed plates, and incubated for 1.5-2 hours. Plate-bound HtrA1 was
detected during a 1-hour incubation with a biotinylated
anti-muHtrA1 rabbit polyclonal antibody (Genentech) diluted to 100
ng/mL for rat tissues, and for mouse eye fluid and retina tissue,
biotinylated anti-huHtrA1 mouse monoclonal antibody (Genentech) was
diluted to 200 ng/mL, both in assay buffer (PBS, 0.5% BSA, 15 ppm
Proclin, 0.05% Tween 20), followed by a wash step and a 30-minute
incubation with streptavidin-HRP (GE Healthcare; Piscataway, N.J.),
also diluted in assay buffer (1:20,000). After a final wash,
tetramethyl benzidine (KPL, Gaithersburg, Md.) was added, color was
developed for 10-15 minutes, and the reaction was stopped with 1 M
phosphoric acid. The plates were read at 450 nm with a 620 nm
reference using a microplate reader. The concentrations of rat or
murine HtrA1 were calculated from a four-parameter fit of the
muHtrA1 standard curve.
[0343] As shown in FIG. 19A, expression of HtrA1 was low in rat
ovary, brain, spleen, and total eye tissue as compared to the
levels of expression of HtrA1 in rat vitreous. As shown in FIG.
19B, the level of HtrA1 in mouse eye fluid increased in a model of
constant light exposure as compared to a control. As shown in FIG.
19C, the level of HtrA1 in mouse retinal tissue increased in a
model of constant light exposure as compared to a control.
[0344] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
90111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5
10 27PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Ser Ala Ser Phe Leu Tyr Ser 1 5 39PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Gln
Gln Ser Tyr Thr Thr Pro Pro Thr 1 5 410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Gly
Phe Asn Ile Ser Gly Tyr Tyr Ile His 1 5 10 517PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Trp
Ile Asp Pro Tyr Gly Gly Asp Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 613PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Gly Thr Phe Leu Thr Ser Trp Gly His Tyr Phe Asp
Tyr 1 5 10 7108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro
Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105 8116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asn Ile Ser Gly Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asp Pro
Tyr Gly Gly Asp Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Thr Phe Leu Thr Ser Trp Gly His Tyr Phe Asp Tyr Trp
100 105 110 Gly Gln Gly Thr 115 9108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 10107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 1121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Met
Lys His Gln His Gln His Gln His Gln His Gln His Gln Met His 1 5 10
15 Gln Ser Thr Ala Ala 20 1210PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideN-term
McaMOD_RES(9)..(9)Lys(DNP) 12Ile Arg Arg Val Ser Tyr Ser Phe Lys
Lys 1 5 10 13480PRTHomo sapiens 13Met Gln Ile Pro Arg Ala Ala Leu
Leu Pro Leu Leu Leu Leu Leu Leu 1 5 10 15 Ala Ala Pro Ala Ser Ala
Gln Leu Ser Arg Ala Gly Arg Ser Ala Pro 20 25 30 Leu Ala Ala Gly
Cys Pro Asp Arg Cys Glu Pro Ala Arg Cys Pro Pro 35 40 45 Gln Pro
Glu His Cys Glu Gly Gly Arg Ala Arg Asp Ala Cys Gly Cys 50 55 60
Cys Glu Val Cys Gly Ala Pro Glu Gly Ala Ala Cys Gly Leu Gln Glu 65
70 75 80 Gly Pro Cys Gly Glu Gly Leu Gln Cys Val Val Pro Phe Gly
Val Pro 85 90 95 Ala Ser Ala Thr Val Arg Arg Arg Ala Gln Ala Gly
Leu Cys Val Cys 100 105 110 Ala Ser Ser Glu Pro Val Cys Gly Ser Asp
Ala Asn Thr Tyr Ala Asn 115 120 125 Leu Cys Gln Leu Arg Ala Ala Ser
Arg Arg Ser Glu Arg Leu His Arg 130 135 140 Pro Pro Val Ile Val Leu
Gln Arg Gly Ala Cys Gly Gln Gly Gln Glu 145 150 155 160 Asp Pro Asn
Ser Leu Arg His Lys Tyr Asn Phe Ile Ala Asp Val Val 165 170 175 Glu
Lys Ile Ala Pro Ala Val Val His Ile Glu Leu Phe Arg Lys Leu 180 185
190 Pro Phe Ser Lys Arg Glu Val Pro Val Ala Ser Gly Ser Gly Phe Ile
195 200 205 Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala His Val Val
Thr Asn 210 215 220 Lys His Arg Val Lys Val Glu Leu Lys Asn Gly Ala
Thr Tyr Glu Ala 225 230 235 240 Lys Ile Lys Asp Val Asp Glu Lys Ala
Asp Ile Ala Leu Ile Lys Ile 245 250 255 Asp His Gln Gly Lys Leu Pro
Val Leu Leu Leu Gly Arg Ser Ser Glu 260 265 270 Leu Arg Pro Gly Glu
Phe Val Val Ala Ile Gly Ser Pro Phe Ser Leu 275 280 285 Gln Asn Thr
Val Thr Thr Gly Ile Val Ser Thr Thr Gln Arg Gly Gly 290 295 300 Lys
Glu Leu Gly Leu Arg Asn Ser Asp Met Asp Tyr Ile Gln Thr Asp 305 310
315 320 Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu Val Asn Leu
Asp 325 330 335 Gly Glu Val Ile Gly Ile Asn Thr Leu Lys Val Thr Ala
Gly Ile Ser 340 345 350 Phe Ala Ile Pro Ser Asp Lys Ile Lys Lys Phe
Leu Thr Glu Ser His 355 360 365 Asp Arg Gln Ala Lys Gly Lys Ala Ile
Thr Lys Lys Lys Tyr Ile Gly 370 375 380 Ile Arg Met Met Ser Leu Thr
Ser Ser Lys Ala Lys Glu Leu Lys Asp 385 390 395 400 Arg His Arg Asp
Phe Pro Asp Val Ile Ser Gly Ala Tyr Ile Ile Glu 405 410 415 Val Ile
Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp 420 425 430
Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Ser Ala Asn Asp Val 435
440 445 Ser Asp Val Ile Lys Arg Glu Ser Thr Leu Asn Met Val Val Arg
Arg 450 455 460 Gly Asn Glu Asp Ile Met Ile Thr Val Ile Pro Glu Glu
Ile Asp Pro 465 470 475 480 14480PRTMus musculus 14Met Gln Ser Leu
Arg Thr Thr Leu Leu Ser Leu Leu Leu Leu Leu Leu 1 5 10 15 Ala Ala
Pro Ser Leu Ala Leu Pro Ser Gly Thr Gly Arg Ser Ala Pro 20 25 30
Ala Ala Thr Val Cys Pro Glu His Cys Asp Pro Thr Arg Cys Ala Pro 35
40 45 Pro Pro Thr Asp Cys Glu Gly Gly Arg Val Arg Asp Ala Cys Gly
Cys 50 55 60 Cys Glu Val Cys Gly Ala Leu Glu Gly Ala Ala Cys Gly
Leu Gln Glu 65 70 75 80 Gly Pro Cys Gly Glu Gly Leu Gln Cys Val Val
Pro Phe Gly Val Pro 85 90 95 Ala Ser Ala Thr Val Arg Arg Arg Ala
Gln Ala Gly Leu Cys Val Cys 100 105 110 Ala Ser Ser Glu Pro Val Cys
Gly Ser Asp Ala Lys Thr Tyr Thr Asn 115 120 125 Leu Cys Gln Leu Arg
Ala Ala Ser Arg Arg Ser Glu Lys Leu Arg Gln 130 135 140 Pro Pro Val
Ile Val Leu Gln Arg Gly Ala Cys Gly Gln Gly Gln Glu 145 150 155 160
Asp Pro Asn Ser Leu Arg His Lys Tyr Asn Phe Ile Ala Asp Val Val 165
170 175 Glu Lys Ile Ala Pro Ala Val Val His Ile Glu Leu Tyr Arg Lys
Leu 180 185 190 Pro Phe Ser Lys Arg Glu Val Pro Val Ala Ser Gly Ser
Gly Phe Ile 195 200 205 Val Ser Glu Asp Gly Leu Ile Val Thr Asn Ala
His Val Val Thr Asn 210 215 220 Lys Asn Arg Val Lys Val Glu Leu Lys
Asn Gly Ala Thr Tyr Glu Ala 225 230 235 240 Lys Ile Lys Asp Val Asp
Glu Lys Ala Asp Ile Ala Leu Ile Lys Ile 245 250 255 Asp His Gln Gly
Lys Leu Pro Val Leu Leu Leu Gly Arg Ser Ser Glu 260 265 270 Leu Arg
Pro Gly Glu Phe Val Val Ala Ile Gly Ser Pro Phe Ser Leu 275 280 285
Gln Asn Thr Val Thr Thr Gly Ile Val Ser Thr Thr Gln Arg Gly Gly 290
295 300 Lys Glu Leu Gly Leu Arg Asn Ser Asp Met Asp Tyr Ile Gln Thr
Asp 305 310 315 320 Ala Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu
Val Asn Leu Asp 325 330 335 Gly Glu Val Ile Gly Ile Asn Thr Leu Lys
Val Thr Ala Gly Ile Ser 340 345 350 Phe Ala Ile Pro Ser Asp Lys Ile
Lys Lys Phe Leu Thr Glu Ser His 355 360 365 Asp Arg Gln Ala Lys Gly
Lys Ala Val Thr Lys Lys Lys Tyr Ile Gly 370 375 380 Ile Arg Met Met
Ser Leu Thr Ser Ser Lys Ala Lys Glu Leu Lys Asp 385 390 395 400 Arg
His Arg Asp Phe Pro Asp Val Leu Ser Gly Ala Tyr Ile Ile Glu 405 410
415 Val Ile Pro Asp Thr Pro Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp
420 425 430 Val Ile Ile Ser Ile Asn Gly Gln Ser Val Val Thr Ala Asn
Asp Val 435 440 445 Ser Asp Val Ile Lys Lys Glu Asn Thr Leu Asn Met
Val Val Arg Arg 450 455 460 Gly Asn Glu Asp Ile Val Ile Thr Val Ile
Pro Glu Glu Ile Asp Pro 465 470 475 480 15459PRTMus musculus 15Met
Gln Ala Arg Ala Leu Leu Pro Ala Thr Leu Ala Ile Leu Ala Thr 1 5 10
15 Leu Ala Val Leu Ala Leu Ala Arg Glu Pro Pro Ala Ala Pro Cys Pro
20 25 30 Ala Arg Cys Asp Val Ser Arg Cys Pro Ser Pro Arg Cys Pro
Gly Gly 35 40 45 Tyr Val Pro Asp Leu Cys Asn Cys Cys Leu Val Cys
Ala Ala Ser Glu 50 55 60 Gly Glu Pro Cys Gly Arg Pro Leu Asp Ser
Pro Cys Gly Asp Ser Leu 65 70 75 80 Glu Cys Val Arg Gly Val Cys Arg
Cys Arg Trp Thr His Thr Val Cys 85 90 95 Gly Thr Asp Gly His Thr
Tyr Ala Asp Val Cys Ala Leu Gln Ala Ala 100 105 110 Ser Arg Arg Ala
Leu Gln Val Ser Gly Thr Pro Val Arg Gln Leu Gln 115 120 125 Lys Gly
Ala Cys Pro Ser Gly Leu His Gln Leu Thr Ser Pro Arg Tyr 130 135 140
Lys Phe Asn Phe Ile Ala Asp Val Val Glu Lys Ile Ala Pro Ala Val 145
150 155 160 Val His Ile Glu Leu Phe Leu Arg His Pro Leu Phe Gly Arg
Asn Val 165 170 175 Pro Leu Ser Ser Gly Ser Gly Phe Ile Met Ser Glu
Ala Gly Leu Ile 180 185 190 Val Thr Asn Ala His Val Val Ser Ser Ser
Ser Thr Ala Ser Gly Arg 195 200 205 Gln Gln Leu Lys Val Gln Leu Gln
Asn Gly Asp Ala Tyr Glu Ala Thr 210 215 220 Ile Gln Asp Ile Asp Lys
Lys Ser Asp Ile Ala Thr Ile Val Ile His 225 230 235 240 Pro Lys Lys
Lys Leu Pro Val Leu Leu Leu Gly His Ser Ala Asp Leu 245 250 255 Arg
Pro Gly Glu Phe Val Val Ala Ile Gly Ser Pro Phe Ala Leu Gln 260 265
270 Asn Thr Val Thr Thr Gly Ile Val Ser Thr Ala Gln Arg Asp Gly Lys
275 280 285 Glu Leu Gly Leu Arg Asp Ser Asp Met Asp Tyr Ile Gln Thr
Asp Ala 290 295 300 Ile Ile Asn Tyr Gly Asn Ser Gly Gly Pro Leu Val
Asn Leu Asp Gly 305 310 315 320 Glu Val Ile Gly Ile Asn Thr Leu Lys
Val Ala Ala Gly Ile Ser Phe 325 330 335 Ala Ile Pro Ser Asp Arg Ile
Thr Arg Phe Leu Ser Glu Phe Gln Asn 340 345 350 Lys His Val Lys Asp
Trp Lys Lys Arg Phe Ile Gly Ile Arg Met Arg 355 360 365 Thr Ile Thr
Pro Ser Leu Val Glu Glu Leu Lys Ala Ala Asn Pro Asp 370 375 380 Phe
Pro Ala Val Ser Ser Gly Ile Tyr Val Gln Glu Val Val Pro Asn 385 390
395 400 Ser Pro Ser Gln Arg Gly Gly Ile Gln Asp Gly Asp Ile Ile Val
Lys 405 410 415 Val Asn Gly Arg Pro Leu Ala Asp Ser Ser Glu Leu Gln
Glu Ala Val 420 425 430 Leu Asn Glu Ser Ser Leu Leu Leu Glu Val Arg
Arg Gly Asn Asp Asp 435 440 445 Leu Leu Phe Ser Ile Ile Pro Glu Val
Val Met 450 455 16483PRTMus musculus 16Met Ser Phe Gln Arg Leu Trp
Ala Val Arg Thr Gln Phe Leu Leu Leu 1 5 10 15 Trp Leu Leu Leu Pro
Ala Val Pro Val Pro Trp Ala Glu Ala Arg Arg 20 25 30 Ser Arg Val
Ser Leu Pro Cys Pro Asp Ala Cys Asp Pro Thr Arg Cys 35 40 45 Pro
Thr Leu Pro Thr Cys Ser Ala Gly Leu Ala Pro Val Pro Asp Arg 50 55
60 Cys Gly Cys Cys Arg Val Cys Ala Ala Ala Glu Gly Gln Glu Cys Gly
65 70 75 80 Gly Ala Arg Gly Arg Pro Cys Ala Pro Arg Leu Arg Cys Gly
Ala Pro 85 90 95 Phe Ser Arg Asp Pro Ser Gly Gly Ala Trp Leu Gly
Thr Cys Gly Cys 100 105 110 Ala Glu Gly Ala Glu Asp Ala Val Val Cys
Gly Ser Asp Gly Arg Thr 115 120 125 Tyr Pro Ser Leu Cys Ala Leu Arg
Lys Glu Asn Arg Ala Ala Arg Gln 130 135 140 Arg Gly Ala Leu Pro Ala
Val Pro Val Gln Lys Gly Ala Cys Glu Glu 145 150 155 160 Ala Gly Thr
Thr Arg Ala Gly Arg Leu Arg Arg Lys Tyr Asn Phe Ile 165 170 175 Ala
Ala Val Val Glu Lys Val Ala Pro Ser Val Val His Leu Gln Leu 180
185
190 Phe Arg Arg Ser Pro Leu Thr Asn Gln Glu Ile Pro Ser Ser Ser Gly
195 200 205 Ser Gly Phe Ile Val Ser Glu Asp Gly Leu Ile Val Thr Asn
Ala His 210 215 220 Val Leu Thr Asn Gln Gln Lys Ile Gln Val Glu Leu
Gln Ser Gly Ala 225 230 235 240 Arg Tyr Glu Ala Thr Val Lys Asp Ile
Asp His Lys Leu Asp Leu Ala 245 250 255 Leu Ile Lys Ile Glu Pro Asp
Thr Glu Leu Pro Val Leu Leu Leu Gly 260 265 270 Arg Ser Ser Asp Leu
Arg Ala Gly Glu Phe Val Val Ala Leu Gly Ser 275 280 285 Pro Phe Ser
Leu Gln Asn Thr Val Thr Ala Gly Ile Val Ser Thr Thr 290 295 300 Gln
Arg Gly Gly Arg Glu Leu Gly Leu Lys Asn Ser Asp Ile Asp Tyr 305 310
315 320 Ile Gln Thr Asp Ala Ile Ile Asn His Gly Asn Ser Gly Gly Pro
Leu 325 330 335 Val Asn Leu Asp Gly Asp Val Ile Gly Ile Asn Thr Leu
Lys Val Thr 340 345 350 Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp Arg
Ile Arg Gln Phe Leu 355 360 365 Glu Asp Tyr His Glu Arg Gln Leu Lys
Gly Lys Ala Pro Leu Gln Lys 370 375 380 Lys Tyr Leu Gly Leu Arg Met
Leu Pro Leu Thr Leu Asn Leu Leu Gln 385 390 395 400 Glu Met Lys Arg
Gln Asp Pro Glu Phe Pro Asp Val Ser Ser Gly Val 405 410 415 Phe Val
Tyr Glu Val Ile Gln Gly Ser Ala Ala Ala Ser Ser Gly Leu 420 425 430
Arg Asp His Asp Val Ile Val Ser Ile Asn Gly Gln Pro Val Thr Thr 435
440 445 Thr Thr Asp Val Ile Glu Ala Val Lys Asp Asn Asp Phe Leu Ser
Ile 450 455 460 Ile Val Leu Arg Gly Ser Gln Thr Leu Phe Leu Thr Val
Thr Pro Glu 465 470 475 480 Ile Ile Asn 1714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10
1811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Arg Ala Ser Gln Ser Ile Asn Thr Tyr Leu Ala 1 5
10 199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Gln Gln Ser Asp Asp Thr Pro Pro Thr 1 5
2010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gly Phe Ser Ile Ser Gly Tyr Tyr Ile His 1 5 10
2111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Arg Ala Ser Gln Val Val Gly Asn Tyr Leu Ala 1 5
10 229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Gln Gln Ser Asp Asp His Pro Pro Thr 1 5
2311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Consensus peptideMOD_RES(5)..(5)Asp, Ser or
ValMOD_RES(6)..(6)Val or IleMOD_RES(7)..(7)Ser, Asn or
GlyMOD_RES(8)..(8)Thr or AsnMOD_RES(9)..(9)Ala or
TyrMOD_RES(10)..(10)Val or Leu 23Arg Ala Ser Gln Xaa Xaa Xaa Xaa
Xaa Xaa Ala 1 5 10 249PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Consensus peptideMOD_RES(3)..(3)Ser,
Val or AspMOD_RES(4)..(4)Tyr, Asp or SerMOD_RES(5)..(5)Thr, Ser,
Ala, Asp or AsnMOD_RES(6)..(6)Thr, His, Asn, Ser, Ala, Leu or
ArgMOD_RES(8)..(8)Pro, Thr, Ala or Ser 24Gln Gln Xaa Xaa Xaa Xaa
Pro Xaa Thr 1 5 2510PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Consensus peptideMOD_RES(3)..(3)Asn, Ser or
ThrMOD_RES(5)..(5)Ser, Asp, Tyr or AlaMOD_RES(6)..(6)Gly or Asp
25Gly Phe Xaa Ile Xaa Xaa Tyr Tyr Ile His 1 5 10 2617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Consensus
peptideMOD_RES(10)..(10)Asn or Asp 26Trp Ile Asp Pro Tyr Gly Gly
Asp Thr Xaa Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Consensus
peptideMOD_RES(6)..(6)Ser or Thr 27Gly Thr Phe Leu Thr Xaa Trp Gly
His Tyr Phe Asp Tyr 1 5 10 28108PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 28Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Thr Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp
Asp Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 29116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 29Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Ile Ser Gly Tyr 20 25 30 Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Trp Ile Asp Pro Tyr Gly Gly Asp Thr Asn Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Phe Leu Thr Ser Trp Gly His
Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr 115
30108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Val Val Gly Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Asp His Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
31108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Consensus polypeptideMOD_RES(28)..(28)Asp, Ser or
ValMOD_RES(29)..(29)Val or IleMOD_RES(30)..(30)Ser, Asn or
GlyMOD_RES(31)..(31)Thr or AsnMOD_RES(32)..(32)Ala or
TyrMOD_RES(33)..(33)Val or LeuMOD_RES(91)..(91)Ser, Val or
AspMOD_RES(92)..(92)Tyr, Asp or SerMOD_RES(93)..(93)Thr, Ser, Ala,
Asp or AsnMOD_RES(94)..(94)Thr, His, Asn, Ser, Ala, Leu or
ArgMOD_RES(96)..(96)Pro, Thr, Ala or Ser 31Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Xaa Xaa Xaa
Xaa Pro Xaa 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 32116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Consensus polypeptideMOD_RES(28)..(28)Asn, Ser
or ThrMOD_RES(30)..(30)Ser, Asp, Tyr or AlaMOD_RES(31)..(31)Gly or
AspMOD_RES(59)..(59)Asn or AspMOD_RES(104)..(104)Ser or Thr 32 Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Xaa Ile Xaa Xaa Tyr
20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Gly Trp Ile Asp Pro Tyr Gly Gly Asp Thr Xaa Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Phe Leu
Thr Xaa Trp Gly His Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr
115 3311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Arg Ala Ser Gln Asp Val Gly Thr Tyr Leu Ala 1 5
10 349PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Gln Gln Val Tyr Ser His Pro Pro Thr 1 5
359PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Gln Gln Ser Tyr Thr Asn Pro Pro Thr 1 5
369PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Gln Gln Ser Tyr Ala Thr Pro Thr Thr 1 5
379PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 37Gln Gln Ser Tyr Ser Ser Pro Ala Thr 1 5
389PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Gln Gln Val Tyr Thr Thr Pro Pro Thr 1 5
399PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Gln Gln Val Tyr Ala Thr Pro Ser Thr 1 5
409PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Gln Gln Ser Tyr Asn Ser Pro Ala Thr 1 5
419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Gln Gln Ser Tyr Ser Thr Pro Ala Thr 1 5
429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Gln Gln Ser Tyr Thr Ala Pro Thr Thr 1 5
439PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Gln Gln Asp Ser Thr Leu Pro Pro Thr 1 5
449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Gln Gln Ser Asp Ala Ala Pro Pro Thr 1 5
459PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Gln Gln Ser Tyr Ser Thr Pro Pro Thr 1 5
469PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Gln Gln Ser Tyr Thr Arg Pro Pro Thr 1 5
4710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Gly Phe Ser Ile Ser Asp Tyr Tyr Ile His 1 5 10
4810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Gly Phe Ser Ile Asp Gly Tyr Tyr Ile His 1 5 10
4910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Gly Phe Thr Ile Tyr Asp Tyr Tyr Ile His 1 5 10
5010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Gly Phe Ser Ile Ala Gly Tyr Tyr Ile His 1 5 10
5110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Gly Phe Thr Ile Ser Asp Tyr Tyr Ile His 1 5 10
5217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Trp Ile Asp Pro Tyr Gly Gly Asp Thr Asp Tyr Ala
Asp Ser Val Lys 1 5 10 15 Gly 5313PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 53Gly Thr Phe Leu Thr Thr
Trp Gly His Tyr Phe Asp Tyr 1 5 10 546PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Asp
Val Ser Thr Ala Val 1 5 556PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 55Ser Ile Asn Thr Tyr Leu 1 5
566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Asp Val Gly Thr Tyr Leu 1 5 576PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Val
Val Gly Asn Tyr Leu 1 5 586PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 58Ser Ala Ser Phe Leu Tyr 1 5
596PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Ser Tyr Thr Thr Pro Pro 1 5 606PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60Val
Tyr Ser His Pro Pro 1 5 616PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 61Ser Tyr Thr Asn Pro Pro 1 5
626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Ser Tyr Ala Thr Pro Thr 1 5 636PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 63Ser
Asp Asp Thr Pro Pro 1 5 646PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 64Ser Tyr Ser Ser Pro Ala 1 5
656PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Val Tyr Thr Thr Pro Pro 1 5 666PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Val
Tyr Ala Thr Pro Ser 1 5 676PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 67Ser Tyr Asn Ser Pro Ala 1 5
686PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Ser Tyr Ser Thr Pro Ala 1 5 696PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 69Ser
Tyr Thr Ala Pro Thr 1 5 706PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Asp Ser Thr Leu Pro Pro 1 5
716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 71Ser Asp Ala Ala Pro Pro 1 5 726PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Ser
Asp Asp His Pro Pro 1 5 736PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 73Ser Tyr Ser Thr Pro Pro 1 5
746PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 74Ser Tyr Thr Arg Pro Pro 1 5 758PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Gly
Phe Ser Ile Ser Gly Tyr Tyr 1 5 768PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Gly
Phe Ser Ile Ser Asp Tyr Tyr 1 5 778PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Gly
Phe Ser Ile Asp Gly Tyr Tyr 1 5 788PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Gly
Phe Thr Ile Tyr Asp Tyr Tyr 1 5 798PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Gly
Phe Ser Ile Ala Gly Tyr Tyr 1 5 808PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Gly
Phe Thr Ile Ser Asp Tyr Tyr 1 5 8110PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Trp
Ile Asp Pro Tyr Gly Gly Asp Thr Asn 1 5 10 8210PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Trp
Ile Asp Pro Tyr Gly Gly Asp Thr Asp 1 5 10 8310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Gly
Thr Phe Leu Thr Ser Trp Gly His Tyr 1 5 10 8410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Gly
Thr Phe Leu Thr Thr Trp Gly His Tyr 1 5 10 856PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)Asp, Ser or ValMOD_RES(2)..(2)Val or
IleMOD_RES(3)..(3)Ser, Asn or GlyMOD_RES(4)..(4)Thr or
AsnMOD_RES(5)..(5)Ala or TyrMOD_RES(6)..(6)Val or Leu 85Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 866PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(1)..(1)Ser, Val or
AspMOD_RES(2)..(2)Tyr, Asp or SerMOD_RES(3)..(3)Thr, Ser, Ala, Asp
or AsnMOD_RES(4)..(4)Thr, His, Asn, Ser, Ala, Leu or
ArgMOD_RES(6)..(6)Pro, Thr, Ala or Ser 86Xaa Xaa Xaa Xaa Pro Xaa 1
5 878PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(3)..(3)Asn, Ser or ThrMOD_RES(5)..(5)Ser,
Asp, Tyr or AlaMOD_RES(6)..(6)Gly or Asp 87Gly Phe Xaa Ile Xaa Xaa
Tyr Tyr 1 5 8810PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(10)..(10)Asn or Asp 88Trp Ile Asp
Pro Tyr Gly Gly Asp Thr Xaa 1 5 10 8910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(6)..(6)Ser or Thr 89Gly Thr Phe Leu Thr Xaa Trp Gly
His Tyr 1 5 10 906PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 90His His His His His His 1 5
* * * * *