U.S. patent application number 15/612944 was filed with the patent office on 2017-11-23 for anti-properdin antibodies and uses thereof.
The applicant listed for this patent is The Trustees of the University of Pennsylvania. Invention is credited to Wenchao SONG.
Application Number | 20170334980 15/612944 |
Document ID | / |
Family ID | 47437643 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170334980 |
Kind Code |
A1 |
SONG; Wenchao |
November 23, 2017 |
ANTI-PROPERDIN ANTIBODIES AND USES THEREOF
Abstract
This invention relates to selective inhibition of the
alternative pathway (AP) of the complement system using an
anti-properdin antibody. Specifically, the invention relates to
methods of treating an AP-mediated pathology or AP-mediated
condition in an individual by contacting the individual with an
anti-properdin antibody.
Inventors: |
SONG; Wenchao; (Bryn Mawr,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of the University of Pennsylvania |
Philadelphia |
PA |
US |
|
|
Family ID: |
47437643 |
Appl. No.: |
15/612944 |
Filed: |
June 2, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14130250 |
Apr 4, 2014 |
9701742 |
|
|
PCT/US12/44974 |
Jun 29, 2012 |
|
|
|
15612944 |
|
|
|
|
61503900 |
Jul 1, 2011 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 13/02 20180101; C07K 2317/76 20130101; A61P 27/02 20180101;
A61P 31/04 20180101; A61P 9/10 20180101; A61P 7/00 20180101; C07K
2317/24 20130101; A61P 29/00 20180101; C07K 2317/34 20130101; A61P
17/02 20180101; C07K 16/18 20130101; A61P 13/12 20180101; A61P
43/00 20180101; C07K 2317/92 20130101; A61P 11/06 20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1.-47. (canceled)
48. A composition comprising an antibody that binds to properdin
and competes with the binding of (1) an antibody that comprises the
CDRs: VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID
NO:5; VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ
ID NO: 10, (2) an antibody that comprises the CDRs: VH-CDR1: SEQ ID
NO:13; VH-CDR2: SEQ ID NO:14; VH-CDR3: SEQ ID NO:15; VL-CDR 1: SEQ
ID NO:18; VL-CDR2: SEQ ID NO:19; and VL-CDR3: SEQ ID NO:20, (3) an
antibody that comprises the CDRs: VH-CDR1: SEQ ID NO:23; VH-CDR2:
SEQ ID NO:24; VH-CDR3: SEQ ID NO:25; VL-CDR 1: SEQ ID NO:28;
VL-CDR2: SEQ ID NO:29; and VL-CDR3: SEQ ID NO:30, or (4) an
antibody comprises the CDRs: VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID
NO:34; VH-CDR3: SEQ ID NO:35; VL-CDR 1: SEQ ID NO:38; VL-CDR2: SEQ
ID NO:39; and VL-CDR3: SEQ ID NO:40, to properdin.
49. A composition comprising an antibody that binds to properdin
and competes with the binding of the antibody designated mAB 19.1
to properdin.
50. A composition comprising an antibody that binds to properdin
and competes with the binding of the antibody designated mAB 25 to
properdin.
51. A composition comprising an antibody that binds to properdin
and competes with the binding of the antibody designated mAB 22.1
to properdin.
52. A composition comprising an antibody that binds to properdin
and competes with the binding of the antibody designated mAB 30 to
properdin.
53.-58. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] The complement system provides a first line of host defense
against invading pathogens. Complement also plays a pathogenic role
in human inflammatory diseases. Activation of the complement system
occurs via three different pathways, the classical pathway (CP),
the lectin pathway (LP) and the alternative pathway (AP). The CP is
initiated by antigen-antibody binding. The LP is triggered when
mannose-binding lectins (MBL) interact with surface sugar molecules
on microorganisms. Activation of both pathways leads to the
assembly of the CP C3 convertase C4b2a, although direct cleavage of
C3 by MBL-associated serine proteases can also occur. The AP is a
self-amplification loop driven by the AP C3 convertase, C3bBb. AP
activation can occur secondary to CP or LP activation, or is
initiated independently. In the latter case, a low level
spontaneous C3 `tick-over` generates the initial C3bBb, which
rapidly propagates the AP in the absence of adequate regulation.
Thus, it is generally assumed that AP activation on non-self
surfaces with no or insufficient negative regulation is considered
a default process, whereas autologous cells typically avoid this
outcome with the help of multiple membrane-bound and fluid phase
complement inhibitory proteins. Under certain conditions, altered,
damaged or stressed autologous cells and tissues can also activate
AP and cause inflammatory injury.
[0002] In contrast to the existence of numerous inhibitory
proteins, the plasma protein properdin is the only known positive
regulator of the complement activation cascade. Properdin is a
plasma glycoprotein of approximately 53 kDa with an estimated blood
concentration of 5-10 .mu.g/ml. It exists mostly as dimers, trimers
and tetramers in a fixed ratio, in a head-to-tail conformation. The
currently held view on properdin function is that it facilitates AP
activation by extending the half-life of the nascent C3bBb
convertase. According to this view, properdin plays a facilitating,
but not essential, role in AP activation. Since activation of the
CP and the LP will invariably trigger the AP amplification loop, it
is expected that properdin will also indirectly promote CP- and
LP-mediated complement activation. Thus, based on the prevailing
knowledge prior to this invention, one may not regard properdin as
an attractive anti-complement therapeutic target because it lacks
specificity and is not indispensible for complement activation.
[0003] While all three pathways of complement activation help the
host in fighting microbial infection, recent studies have shown
that complement-mediated pathology in humans, such as age-related
macular degeneration, atypical hemolytic uremic syndrome,
paroxysmal nocturnal hemoglobinuria (PNH), rheumatoid arthritis,
allergic asthma and ischemia reperfusion injury, is mainly mediated
by the AP. Thus, there remains a need in the art for
anti-complement compositions and methods of treating human
inflammatory diseases by selectively inhibiting the AP while
leaving intact the CP and the LP to fight pathogens and to protect
the host from infection. The current invention fulfills this
need.
SUMMARY
[0004] This invention relates to anti-properdin antibody and
methods of inhibiting the alternative pathway (AP) of complement
using an anti-properdin antibody.
[0005] In one embodiment, the invention is a composition comprising
an antibody that specifically binds to properdin. In preferred
embodiments, the properdin is human properdin. In some embodiments,
the antibody of the invention is a monoclonal antibody. In some
embodiments, the antibody of the invention is a humanized antibody.
In some embodiments, the antibody of the invention is a chimeric
antibody.
[0006] In one embodiment, the antibody of the invention comprises
at least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3; SEQ ID NO:5;
VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID
NO:10. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:2. In one embodiment, the antibody of the invention comprises
a light chain comprising the amino acid sequence of SEQ ID NO:7. In
another embodiment, the antibody of the invention comprises a heavy
chain comprising the amino acid sequence of SEQ ID NO:2 and a light
chain comprising the amino acid sequence of SEQ ID NO:7. In one
embodiment, the antibody of the invention specifically binds to an
epitope comprising at least one amino acid of SEQ ID NO:52.
[0007] In one embodiment, the antibody of the invention comprises
at least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:13; VH-CDR2: SEQ ID NO:14; VH-CDR3: SEQ ID
NO:15; VL-CDR1: SEQ ID NO:18; VL-CDR2: SEQ ID NO:19; and VL-CDR3:
SEQ ID NO:20. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:12. In one embodiment, the antibody of the invention
comprises a light chain comprising the amino acid sequence of SEQ
ID NO:17. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:12 and a light chain comprising the amino acid sequence of
SEQ ID NO:17. In one embodiment, the antibody of the invention
specifically binds to an epitope comprising at least one amino acid
of SEQ NO:53.
[0008] In one embodiment, the antibody of the invention comprises
at least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3: SEQ ID
NO:25; VL-CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID NO:29; and VL-CDR3:
SEQ ID NO:30. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:22. In one embodiment, the antibody of the invention
comprises a light chain comprising the amino acid sequence of SEQ
ID NO:27. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:22 and a light chain comprising the amino acid sequence of
SEQ ID NO:27.
[0009] In one embodiment, the antibody of the invention comprises
at least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID
NO:35; VL-CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39; and VL-CDR3:
SEQ ID NO:40. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:32. In one embodiment, the antibody of the invention
comprises a light chain comprising the amino acid sequence of SEQ
ID NO:37. In another embodiment, the antibody of the invention
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:32 and a light chain comprising the amino acid sequence of
SEQ ID NO:37.
[0010] In one embodiment, the antibody of the invention comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:42, In
another embodiment, the antibody of the invention comprises a heavy
chain comprising the amino acid sequence of SEQ ID NO:44. In one
embodiment, the antibody of the invention comprises a light chain
comprising the amino acid sequence of SEQ ID NO:47. In one
embodiment, the antibody of the invention comprises a heavy chain
comprising the amino acid sequence of SEQ ID NO:42 and a light
chain comprising the amino acid sequence of SEQ ID NO:47. In
another embodiment, the antibody of the invention comprises a heavy
chain comprising the amino acid sequence of SEQ ID NO:44 and a
light chain comprising the amino acid sequence of SEQ ID NO:47.
[0011] In one embodiment, the antibody of the invention comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:49. In
one embodiment, the antibody of the invention comprises a light
chain comprising the amino acid sequence of SEQ ID NO:51. In
another embodiment, the antibody of the invention comprises a heavy
chain comprising the amino acid sequence of SEQ ID NO:49 and a
light chain comprising the amino acid sequence of SEQ ID NO:51.
[0012] In one embodiment, the antibody of the invention comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:2 and
SEQ ID NO:63. In one embodiment, the antibody of the invention
comprises a light chain comprising the amino acid sequence of SEQ
ID NO:7 and SEQ ID NO:64. In another embodiment, the antibody of
the invention comprises a heavy chain comprising the amino acid
sequences of SEQ ID NO:2 and SEQ ID NO:63 and a light chain
comprising the amino acid sequences of SEQ ID NO:7 and SEQ ID
NO:64.
[0013] In one embodiment, the antibody of the invention comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:12 and
SEQ ID NO:63. In one embodiment, the antibody of the invention
comprises a light chain comprising the amino acid sequence of SEQ
ID NO:17 and SEQ ID NO:64. In another embodiment, the antibody of
the invention comprises a heavy chain comprising the amino acid
sequences of SEQ ID NO:12 and SEQ ID NO:63 and a light chain
comprising the amino acid sequences of SEQ ID NO:17 and SEQ ID
NO:64.
[0014] In one embodiment, the antibody of the invention comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:22 and
SEQ ID NO:63. In one embodiment, the antibody of the invention
comprises alight chain comprising the amino acid sequence of SEQ ID
NO:27 and SEQ ID NO:64. In another embodiment, the antibody of the
invention comprises a heavy chain comprising the amino acid
sequences of SEQ ID NO:22 and SEQ ID NO:63 and a light chain
comprising the amino acid sequences of SEQ ID NO:27 and SEQ ID
NO:64.
[0015] In one embodiment, the antibody of the invention comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:32 and
SEQ ID NO:63. In one embodiment, the antibody of the invention
comprises a light chain comprising the amino acid sequence of SEQ
ID NO:37 and SEQ NO:64. In another embodiment, the antibody of the
invention comprises a heavy chain comprising the amino acid
sequences of SEQ ID NO:32 and SEQ ID NO:63 and a light chain
comprising the amino acid sequences of SEQ ID NO:37 and SEQ ID
NO:64.
[0016] In one embodiment, the antibody of the invention is an
antibody that binds to properdin and competes with the binding of
at least one of the anti-properdin antibodies described herein. In
another embodiment, the antibody of the invention is an antibody
that binds to properdin and competes with the binding of the
antibody designated mAb 19.1 to properdin. In another embodiment,
the antibody of the invention is an antibody that binds to
properdin and competes with the binding of the antibody designated
mAb 25 to properdin. In another embodiment, the antibody of the
invention is an antibody that binds to properdin and competes with
the binding of the antibody designated mAb 22.1 to properdin. In
another embodiment, the antibody of the invention is an antibody
that binds to properdin and competes with the binding of the
antibody designated mAb 30 to properdin.
[0017] In another embodiment, the invention is a method of treating
an alternative pathway (AP)-mediated pathology in an individual,
comprising the step of administering to said individual at least
one of the anti-properdin antibodies described herein. In various
embodiments, the alternative pathway (AP)-mediated pathology is at
least selected from the group consisting of: macular degeneration,
ischemia reperfusion injury, arthritis, rheumatoid arthritis,
paroxysmal nocturnal hemoglobinuria (PNH) syndrome, atypical
hemolytic uremic (aHUS) syndrome, asthma, organ transplantation
sepsis, inflammation, glomerulonephritis, lupus, and combinations
thereof. In some embodiments, the anti-properdin antibody
selectively inhibits the alternative pathway, but does not inhibit
the classical pathway and the lectin pathway. In some embodiments,
the anti-properdin antibody does not affect the AP amplification
loop of the classical pathway and the lectin pathway. In some
embodiments, the anti-properdin antibody inhibits the generation of
a C3bBb protein.
[0018] In one embodiment, the invention is a transgenic mouse that
expresses human properdin (e.g., SEQ ID NO:67; SEQ ID NO:54), but
does not express murine properdin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The foregoing summary, as well as the following detailed
description of preferred embodiments of the invention, will be
better understood when read in conjunction with the appended
drawings. For the purpose of illustrating the invention, there are
shown in the drawings embodiments which are presently preferred. It
should be understood, however, that the invention is not limited to
the precise arrangements and instrumentalities of the embodiments
shown in the drawings. In the drawings:
[0020] FIG. 1 is a schematic of the complement pathways. Complement
can be activated through three pathways: classical, lectin, and
alternative. The classical pathway is activated when C1q binds to
antibody attached to antigen, activating C1r and C1s which cleave
C4 and C2. The lectin pathway is activated when mannose-binding
lectin (MBL) encounters conserved pathogenic carbohydrate motifs,
activating the MBL-associated serine proteases (MASPs) and again
cleaving C4 and C2. C4 and C2 cleavage products form the classical
and lectin pathways C3 convertase, C4bC2a which cleaves C3 into C3b
and C3a. A second molecule of C3b can associate with C4bC2a to form
the C5 convertase of the classical and lectin pathways, C4bC2aC3b.
The alternative pathway (AP) is activated when C3 undergoes
spontaneous hydrolysis and forms the initial AP C3 convertase,
C3(H2O)Bb, in the presence of Factors B and D, leading to
additional C3 cleavage and the eventual formation of the AP C3
convertase (C3bBb) and AP C5 convertase (C3bBbC3b). Properdin
facilitates AP activation by stabilizing the AP convertases. All
three pathways culminate in the formation of the convertases, which
in turn generate the major effectors of the complement system: the
anaphylatoxins (C4a/C3a/C5a), the membrane attack complex (MAC),
and the opsonins (e.g. C3b). The anaphylatoxins are potent
proinflammatory molecules derived from cleavage of C4, C3, and C5.
The MAC is a terminal assembly of complement components C5b through
C9 which can directly lyse targeted surfaces. C3b induces
phagocytosis of opsonized targets and also serves to amplify
complement activation through the AP.
[0021] FIG. 2 depicts the results of experiments demonstrating
dose-dependent inhibition of LPS-induced AP complement activation
by mAb 19.1, 22.1, 25 and 30. All 4 clones of mAbs effectively
inhibited AP complement activation when added to 50% normal human
serum (NHS) at a final concentration of 5 .mu.g/ml. A sample with
EDTA added (NHSEDTA) served as a negative control (EDTA blocks
complement activation). A sample with no mAb added (0 Ab) served as
the baseline AP complement activation. The experiment was performed
in GVB-EGTA-Mg++ buffer. ELISA plates were coated with LPS
overnight. NHS was preincubated with mAb before addition to plate.
AP complement activation was detected by measuring the amount of C3
deposition on the plate (OD450).
[0022] FIG. 3 depicts the results of experiments demonstrating that
anti-human properdin mAbs inhibit human red blood cell (RBC) lysis
caused by fH and DAF dysfunction. Human RBCs are not lysed in the
absence of human serum (Eh only). They are also resistant to lysis
by human serum (50%) in the absence of fH 19-20, a recombinant fH
fragment that prevents fH interaction with autologous cells
(0fh1920). However, when human RBCs are incubated with 50% human
serum in the presence of 30 .mu.M fH 19-20 and 7.5 .mu.g/ml of a
function-blocking anti-decay accelerating factor (DAF, a membrane
complement regulator) mAb (fh1920+AntiCD55), about 70% of the RBCs
lysed. This lysis was completely inhibited by each of the 4
anti-properdin mAbs (19.1, 22.1, 25, 30) at 5 .mu.g/ml. An RBC
sample treated with distilled water (Eh+DDW) caused complete lysis
and served as a positive control. An RBC sample in normal human
serum treated with EDTA (NHSEDTA) served as a negative control (no
lysis, because EDTA blocks complement activation). Lysis assays
were performed in Mg++-EGTA GVB++ buffer to allow only AP
complement activation.
[0023] FIG. 4 depicts the results of experiments evaluating
antibody-sensitized sheep RBCs incubated with 50% normal human
serum (NHS) in the absence or presence of 5 .mu.g/ml of
anti-properdin mAbs. A sample of RBCs with no human serum added
(ShEs Only) showed no lysis; RBCs incubated with 50% NHS with no
anti-properdin mAbs added showed complete lysis (50% NHS); RBCs
incubated with 50% NHS and 5 .mu.g/ml of mAbs 19.1, 22.1, 25 or 30
were also completely lysed, demonstrating that the mAbs had no
inhibitory effect on classical pathway-mediated complement lysis of
sensitized sheep RBCs. RBCs incubated with 50% NHS in the presence
of EDTA (NHSEDTA) had no lysis, demonstrating the lysis was
mediated by complement; sheep RBCs treated with distilled water
(Es+DDW) served as a 100% lysis control.
[0024] FIG. 5, comprising FIGS. 5A-5C, depicts the results of
epitope mapping for mAb 19.1 and 25 and generation of deletion
mutants of human properdin (fP). FIG. 5a depicts the deduced amino
acid sequence of human properdin (SEQ ID NO:54). The signal peptide
is underlined. The mature protein starts at residue 28. FIG. 5b
depicts the amino acid sequences of the 7 thrombospondin repeat
(TSR) domains of human properdin. They are as follows: TSR0 (SEQ ID
NO:55), TSR1 (SEQ ID NO:56), TSR2 (SEQ ID NO:57), TSR3 (SEQ ID
NO:58), TSR4 (SEQ ID NO:59), TSR5 (SEQ ID NO:60), TSR6 (SEQ ID
NO:61). FIG. 5c depicts the results of epitope mapping for mAb 19.1
and 25 and generation of deletion mutants of human properdin (fP).
Human properdin (fP) is composed of 7 thrombospondin repeat (TSR)
domains which are numbered 0 to 6. Individual TSR domains (and in
some instances two TSR domains) have been deleted and the mutant
proteins were expressed in Chinese hamster ovary (CHO) cells. All
TSR deletion mutants are expressed at the expected sizes except
TSR5 deletion mutant which is substantially smaller than the
expected size. It is likely that TSR5 deletion mutant was subjected
to proteolysis. The size of TSR5 deletion is similar to that of
TSR5+6 deletion mutant, suggesting that TSR6 may have been
proteolytically removed in the TSR5 deletion mutant. CHO cell
lysates were analyzed by Western blot using a polyclonal goat
anti-human fP antibody. M: molecular weight (MW) marker.
[0025] FIG. 6 depicts the results of epitope mapping for mAb 19.1
and 25 and ELISA assays of mAb 19.1 and 25 binding to human
properdin deletion mutants. CHO cell lysates were coated onto ELISA
plates and detected with mAb 19.1 or 25. A third mAb 29.3 which
binds to a different epitope from 19.1 and 25 was used as a control
to confirm protein expression. The results show that both mAb 19.1
and 25 reacted with the following deletion mutants: dTSR0, dTSR1,
dTSR2, dTSR3, dTSR4. Thus, it can be concluded that the epitopes
for mAb 19.1 and 25 are not located in TSR 0-4. Furthermore, mAb
19.1 lost binding to dTSR5 and dTSR 5+6 but retained binding to
dTSR6, suggesting that its epitope is located in TSR5. mAb 25 lost
binding to dTSR5, dTSR5+6 and dTSR6, suggesting that its epitope is
located in TSR 5-6. However, because dTSR5 has undergone
proteolytic degradation leading to possible TSR6 removal (FIG. 5),
it is likely that the epitope for mAb 25 is located in TSR6. HuP
refers to full length human properdin transfection which is used as
a positive control. Con refers to untransfected CHO cell lysate
which is used as a negative control for lack of binding. All mutant
proteins contained a 6.times.His tag at the C-terminus.
[0026] FIG. 7 depicts the results of epitope mapping showing the
epitope of mAb 19.1 is mapped to the C-terminal half of TSR5 with
the amino acid sequence:
RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO:52). Because TSR0-4
(i.e., dTSR5+6) did not react with mAb 19.1 whereas TSR0-5 (i.e.,
dTSR6) reacted with 19.1 (FIG. 6), further deletion mutants were
generated: TSR0-4+1/4 TSR5, TSR0-4+1/2 TSR5 and TSR0-4+3/4 TSR5.
Mutant, but not intact properdin proteins contained a 6.times.His
tag at their C-terminus. Western blot analysis using both
anti-human fP and anti-His tag antibodies showed that TSR0-4+3/4
TSR5 was not expressed well. The other two mutants, TSR0-4+1/4 TR5
and TSR0-4+1/2 TSR5, were confirmed to be expressed but neither one
was recognized by mAb 19.1, suggesting they have lost the epitope
for mAb 19.1. Thus, it can be concluded that the key epitope
residues for mAb 19.1 are located within the C-terminal half of
TSR5 (SEQ ID NO:52).
[0027] FIG. 8, comprising FIGS. 8A and 8B, depicts the results of
epitope mapping showing the epitope of mAb 25. In FIG. 8A, the data
indicate the epitope mapped to the C-terminal quarter segment of
TSR6 with the amino acid sequence:
LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO:53). Because TSR0-5 (i.e.,
dTSR6) lost binding to mAb 25, it was concluded that TSR6
constitutes at least part of the epitope of mAb 25. Additional
mutants of TSR6 were generated as follows: TSR0-5+1/4 TSR6,
TSR0-5+1/2 TSR6 and TSR0-5+3/4 TSR6. Mutant, but not intact,
properdin proteins contained a 6.times.His tag at their C-terminus.
As shown by western blot with anti-human fP and anti-His tag
antibodies, all three mutants were successfully expressed. ELISA
binding experiments showed that all three mutants lost binding to
mAb 25. As a positive control, all mutant proteins reacted with mAb
19.1. This result suggests that the last quarter segment of TSR6
(with the sequence designated by SEQ TD NO:53) constitutes a key
part of the epitope of mAb 25. HuP refers to full-length (intact)
human IP transfected CHO cells as a positive control; ConLysate
refers to untransfected CHO cells as negative controls for binding.
In FIG. 8B the data indicate that the epitope of mAb 25 is
dependent on two cysteine residues in TSR6 (SEQ ID NO: 61, shown in
FIG. 5B). These are cysteine 62 (C62) and cysteine 78 (C78) of
TSR6. Single mutation to Alanine (A) of either C62 or C78 in
full-length human properdin did not abolish mAb 25 binding, but
double mutations of C62A and C78A abolished mAb 25 binding. As a
positive control for mutant protein expression, mAb 19.1 showed
reactivity to all samples. This result suggests that C78 within the
last quarter segment of TSR6 (with the sequence designated by SEQ
ID NO: 53), as well as C62 which is located outside SEQ ID NO:53
but within TSR6 (SEQ ID: 61), constitute two critical residues of
the epitope of mAb 25. Binding assays of mAbs 19.1 and 25 was
performed on ELISA plates using homogenates of transfected CHO
cells. HuP refers to full-length (intact) human IP transfected CHO
cells as a positive control; Con refers to untransfected CHO cells
as negative controls for binding. The other samples are CHO cells
transfected with mutant human fP cDNA containing single or double
C62A and C78A mutations.
[0028] FIG. 9 depicts the nucleotide and amino acid sequence of the
variable region sequences of heavy (SEQ ID NO:1; SEQ ID NO:2) and
light (SEQ ID NO:6; SEQ ID NO:7) chains of mAb 19.1, including the
CDRs (VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID
NO:5; VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; VL-CDR3: SEQ ID
NO; 10).
[0029] FIG. 10 depicts the nucleotide and amino acid sequence of
the variable region sequences of heavy (SEQ ID NO:11; SEQ ID NO:12)
and light (SEQ ID NO:16; SEQ ID NO:17) chains of mAb 25, including
the CDRs (VH-CDR1: SEQ ID NO:13; VH-CDR2: SEQ ID NO:14; VH-CDR3:
SEQ ID NO:15; VL-CDR1: SEQ ID NO:18; VL-CDR2: SEQ ID NO:19;
VL-CDR3: SEQ ID NO:20).
[0030] FIG. 11 depicts the nucleotide and amino acid sequence of
the variable region sequences of heavy (SEQ ID NO:21; SEQ ID NO:22)
and light (SEQ ID NO:26; SEQ ID NO:27) chains of mAb 22.1,
including the CDRs (VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24;
VH-CDR3: SEQ ID NO:25; VL-CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID
NO:29; VL-CDR3: SEQ ID NO:30).
[0031] FIG. 12 depicts the nucleotide and amino acid sequence of
the variable region sequences of heavy (SEQ ID NO:31; SEQ ID NO:32)
and light (SEQ ID NO:36; SEQ ID NO:37) chains of mAb 30, including
the CDRs (VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3:
SEQ ID NO:35; VL-CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39;
VL-CDR3: SEQ ID NO:40).
[0032] FIG. 13 depicts the humanized amino acid sequence of the
variable region of the heavy chain of mAb 19.1 (humanized 19.1
VH-4-59-01 (SEQ ID NO:42); humanized 19.1 VH-3-66-04 (SEQ ID
NO:44)) after CDR grafting using two human germline VH sequences
(human VH 4-59-01 (SEQ ID NO:4); human VH 3-66-04 (SEQ ID
NO:43)).
[0033] FIG. 14 depicts the humanized amino acid sequence of the
variable region of the light chain of mAb 19.1 (humanized 19.1
VL-4-1-01 (SEQ ID NO:47)) after CDR grafting using a human germline
VL sequence (human VL 4-1-01 (SEQ ID NO:45); human JK2 (SEQ ID
NO:46)).
[0034] FIG. 15 depicts the humanized amino acid sequence of the
variable region of the heavy chain of mAb 25 (humanized
25-VH-1-69-06 (SEQ ID NO:49)) after CDR grafting using a human
germline VH sequence (human VII 1-69-06 (SEQ ID NO:48)).
[0035] FIG. 16 depicts the humanized amino acid sequence of the
variable region of the light chain of mAb 25 (humanized
25-VL-1-69-06 (SEQ ID NO:51)) after CDR grafting using a human
germline VL sequence (human VL 1-69-06 (SEQ ID NO:50)); human Jk3
(SEQ ID NO:62).
[0036] FIG. 17, comprising FIGS. 17A and 17B, depicts the results
of experiments assessing recombinant chimeric and humanized 19.1
mAbs. FIG. 17A depicts the amino acid sequences of human IgG4
constant heavy region, with Serine 229 to Proline mutation (SEQ ID
NO:63), and human kappa constant light region (SEQ ID NO:64). These
sequences were used to construct chimeric (mouse variable
region+human constant region) and humanized (humanized mouse
variable region+human constant region) anti-properdin antibodies.
FIG. 17B depicts the results of experiments assessing the
expression of recombinant chimeric and humanized 19.1 mAbs. SDS
PAGE analysis of recombinant chimeric 19.1 mAb and two humanized
19.1 mAbs. Construction of the chimeric 19.1 heavy chain was
achieved by joining the VH region of 19.1 with the constant region
of human IgG4 heavy chain. Construction of the chimeric 19.1 light
chain was achieved by joining the VL region of 19.1 with the
constant region of the human kappa chain. Humanized 19.1 heavy and
light chains were constructed in the same way, i.e. humanized VH
region was joined with the constant region of human IgG4 heavy
chain and humanized light chain was joined with the constant region
of human kappa chain. CHO cells were co-transfected with heavy and
light chain cDNAs and stable lines established by drug section. For
the two humanized mAbs, each of the two humanized heavy chains was
paired with the same humanized light chain for transfection.
Expressed mAbs were purified from CHO cell culture medium by
protein G affinity column.
[0037] FIG. 18 depicts the results of experiments measuring the
antigen binding affinities of 19.1, chimeric 19.1 and humanized
19.1 mAbs using Biacore. Purified human fP was coupled onto CM4
chip using the amine coupling method. Biacore analysis was
performed on a Biacore-2000 instrument. The chip was regenerated
between each binding using 50 mM NaOH.
[0038] FIG. 19 depicts the results of experiments measuring the
antigen binding affinities of mAb 25, 22.1 and 30 as determined by
Biacore analysis. Purified human fP was coupled onto CM4 chip using
the amine coupling method. Biacore analysis was performed on a
Biacore-2000 instrument. The chip was regenerated between each
binding using 50 mM NaOH.
[0039] FIG. 20 depicts the results of experiments assessing the
relative activities of 19.1, chimeric 19.1 and humanized 19.1 mAbs
in blocking LPS-induced human AP complement activation. ELISA
plates were coated with LPS overnight, and 50% normal human serum
(NHS) diluted in GVB-Mg++-EGTA was added and incubated at
37.degree. C. for 1 hr before detection of C3 deposition using
anti-C3 antibodies. NHS with no antibody added served as a positive
control (NHS) and NHS with EDTA added served as a negative control
(NHSEDTA). For 19.1 mAb, concentrations of 5 .mu.g/ml and 10
.mu.g/ml were sufficient to inhibit complement activation. For the
chimeric and the two humanized 19.1 mAbs, a concentration of 5
.mu.g/ml was not sufficient to inhibit complement activation.
However, concentrations of 10 .mu.g/ml and 20 .mu.g/ml were
effective at blocking AP complement activation.
[0040] FIG. 21 depicts the results of experiments assessing
relative activities of 19.1, chimeric 19.1 and humanized 19.1 mAbs
in blocking human RBC lysis by human AP complement in the context
of fH and DAF dysfunction. Human RBCs were incubated with 50%
normal human serum in the presence of fH-19-20 (30 .mu.M) and
anti-DAF antibody (7.5 .mu.g/ml). Human serum was diluted in
GVB-Mg++-EGTA and the incubation was carried out at 37.degree. C.
for 1 hr. Before being added to the RBCs, the human serum was
pre-incubated with increasing concentrations of 19.1, chimeric 19.1
and humanized 19.1 mAbs (1-15 .mu.g/ml) at 4.degree. C. for 1 hr.
There was a dose-dependent inhibition of RBC lysis by all 4 mAbs.
However, the EC50 for the chimeric and humanized 19.1 mAbs were
higher than that of 19.1 mAb. This result was consistent with the
data shown in FIG. 20.
[0041] FIG. 22 depicts the results of experiments assessing the
relative activities of 19.1, chimeric 19.1 and humanized 19.1 mAbs
in blocking LPS-induced Rhesus monkey AP complement activation.
ELISA plates were coated with LPS overnight, and 50% normal Rhesus
monkey serum (NRS) diluted in GVB-Mg++-EGTA was added and incubated
at 37.degree. C. for 1 hr before detection of C3 deposition using
anti-human C3 antibodies. NRS with no antibody added served as a
positive control (NRS) and NRS with EDTA added served as a negative
control (NRSEDTA). For 19.1 and chimeric 19.1 mAbs, concentrations
of 10-40 .mu.g/ml were sufficient to inhibit Rhesus monkey
complement activation. For the two humanized 19.1 mAbs,
concentrations of 30 or 40 .mu.g/ml were effective to inhibit
complement activation. Concentrations of 10 or 20 .mu.g/ml also
substantially inhibited Rhesus monkey AP complement activation.
[0042] FIG. 23 depicts the results of experiments assessing the
relative activities of 19.1, chimeric 19.1 and humanized 19.1 mAbs
in blocking LPS-induced Cynomolgus monkey AP complement activation.
ELISA plates were coated with LPS overnight, and 50% normal
Cynomolgus monkey serum (NCS) diluted in GVB-Mg++-EGTA was added
and incubated at 37.degree. C. for 1 hr before detection of C3
deposition using anti-human C3 antibodies. NCS with no antibody
added served as a positive control (NCS) and NCS with EDTA added
served as a negative control (NCSEDTA). For the 19.1 mAb,
concentrations of 10-40 .mu.g/ml were sufficient to inhibit
Cynomolgus monkey AP complement activation. For the chimeric 19.1
mAb, concentrations 20-40 .mu.g/ml were sufficient to inhibit
Cynomolgus monkey AP complement activation but a concentration of
10 .mu.g/ml also significantly inhibited complement activation. For
the two humanized 19.1 mAbs, concentrations of 30 or 40 .mu.g/ml
were effective to inhibit Cynomolgus complement activation.
However, a concentration of 20 .mu.g/ml also substantially
inhibited Cynomolgus monkey AP complement activation. A
concentration of 10 .mu.g/ml also partially inhibited Cynomolgus AP
complement activity.
[0043] FIG. 24 depicts the results of experiments assessing the
inhibition of acidified serum lysis of PNH patient red blood cells
(Ham's test) by mAb 19.1, 25 and humanized 19.1. RBCs from
paroxysmal nocturnal hemoglobinuria (PNH) patients were subjected
to Ham's acidified serum test in the presence or absence of mAbs.
RBCs were incubated with autologous serum (final concentration 83%)
at 37 C for 2 hrs and percent lysis was calculated by measuring the
OD405 of the supernatant, normalized to a sample of RBCs completely
lysed by distilled water (Eh DDW). The incubation mixture was
composed of the following: 240 .mu.l of serum, 25 .mu.l of 1/6 N
HCL (or 25 .mu.l saline for negative controls), 12.5 .mu.l of 50%
(v/v) RBC suspension, 10 .mu.l mAb in saline. A sample of RBCs
incubated with nonacidified autologous serum (NHS) was used as a
negative control (background lysis). In the absence of mAbs, about
50% of RBCs were lysed by acidified serum. This lysis was
completely inhibited by mAb 19.1 at 8 .mu.g/ml and above
concentration, by a humanized 19.1 mAb (#459) at the concentration
of 20 .mu.g/ml and by mAb 25 at the concentration of 8 .mu.g/ml and
above.
[0044] FIG. 25, comprising FIGS. 25A-25E, depicts the generation of
a properdin humanized mouse. A human fP expression vector was
constructed as illustrated in the schematic in FIG. 25A, using the
chicken .beta.-actin promoter with CVM-IE enhancer and the rabbit
.beta.-globin polyA tail for stable expression of the cDNA in
eukaryotic cells. This plasmid was linearized and microinjected
into the zygotes of C57BL/6 mice to produce human fP transgenic
founder mice. By PCR screening (using primers specific to human fP
5'-ATCAGAGGCCTGTGACACC-3' (SEQ ID NO:65) and 5'-CTG
CCCTTGTAGCTCCTCA-3' (SEQ ID NO:66)), positive founder mice (showing
a human fP cDNA fragment of about 800 bp) were identified. Of 40
mice analyzed, five (#15, 20, 24, 27 and 32) were positive (FIG.
25B, red arrows). ELISA assays were performed to detect human fP in
the transgenic positive mice (FIG. 25C). Plate was coated with a
non-blocking mAb against human fP (clone 8.1). After incubation
with diluted serum (10%), human fP was detected by ELISA using an
HRP-conjugated goat-antihuman fP antibody. Normal human serum (NHS)
was used as a positive control. As can be seen, human fP was
detected in NHS and in the sera of the 5 transgenic mice but not in
normal (i.e. non-transgenic) mouse serum (NMS), transgenic-negative
(#29) or in fP.sup.-/- mouse serum. Founder mouse #32 was bred with
WT mice and pups were screened by PCR as described above. Three
representative F1 mice, one PCR-negative (F1-429) and two
PCR-positive (F1-430 and F1-431), were tested by ELISA for the
presence of human IP in their sera (FIG. 25D). As shown, human fP
was detected in the two PCR-positive mice but not in the
PCR-negative mouse. Sera from NHS and the founder parent (#32) were
used as positive controls. This result suggested that the transgene
is stable and can be transmitted through germline. Founder mouse
#32 was then bred with fP.sup.-/- mice to generate fP.sup.-/--human
fP transgene+mice. LPS-induced AP complement activation assay
showed that fP.sup.-/--human fP transgene+mice, but not fP.sup.-/-
mice, had serum AP complement activity indistinguishable from that
of WT mice (FIG. 25E), suggesting that transgenically expressed
human IP was able to rescue the phenotype of fP.sup.-/- mice. WT
serum treated with EDTA was used as a negative control. This result
confirmed that a properdin humanized mouse line was generated.
[0045] FIG. 26 depicts experiments examining the in vivo activity
and kinetics of mAb 25 in "properdin humanized" mice. A properdin
humanized mouse (fP.sup.-/--human fP.sup.-/- transgene+) was
injected with 0.5 mg (i.p.) of mAb 25. Serum samples were collected
before injection (0 hr) and then at various time points after
injection and tested for LPS-induced AP complement activation. As
shown, no AP complement activity was present in fP.sup.-/- mouse
serum or in WT serum treated with EDTA. In contrast, AP complement
activity was detected in WT serum and in the serum of fP humanized
mouse at time 0 hr (before mAb treatment). AP complement activity
in the humanized mouse remained undetectable at 8, 24 and 48 hrs
after mAb treatment but became detectible at 72, 96 and 120 hrs.
These results suggest that at a dosage of 0.5 mg/mouse, mAb 25 was
able to inhibit AP complement activity in vivo for 48 hrs.
[0046] FIG. 27 depicts the results of an experiment demonstrating
that anti-human properdin mAb 19.1 prevents extravascular hemolysis
(EVH). In this EVH model, properdin humanized mice (n=4 per
experimental group) were transfused with red blood cells (RBC) from
Crry/DAF/C3 triple knockout (TKO) mice. Recipient mice (properdin
humanized mice) were treated 6 hrs before RBC transfer with mAb
19.1 (2 mg/mouse, i.p.) or a control mouse IgG1 mAb (MOPC, purified
from MoPC 31C hybridoma, from ACTT). RBCs were harvested from donor
TKO mice, washed in PBS and labeled with CFSE before injection
(through tail vein) into recipient mice, according to previously
published procedure (Miwa et al., 2002, Blood 99: 3707-3716). Each
recipient mouse received RBCs equivalent to 100 .mu.l of blood. At
5 minutes and 6, 24, 48, 72, 96, 120 hours after RBC transfusion,
recipient mice were bled and RBCs were analyzed to determine the
number of CFSE-labeled (i.e. transfused) RBCs remaining in the
circulation. Number of CFSE-labeled RBCs in each recipient was
normalized (as %) to that detected at the 5 min time point. In
control IgG (MOPC)-treated recipient mice, TKO RBCs were rapidly
eliminated through EVH, consistent with previous findings (Miwa et
al., 2002, Blood 99: 3707-3716). However, in recipient mice treated
with anti-human properdin 19.1 mAb, no EVH occurred and the
transfused RBCs persisted, demonstrating that anti-properdin mAb
was effective in preventing EVH.
[0047] FIG. 28 depicts the nucleic acid sequence of human properdin
cDNA (SEQ ID NO:67) used for generating human properdin transgenic
mice.
DETAILED DESCRIPTION OF THE INVENTION
[0048] This invention relates to the inhibition of the alternative
pathway (AP) of complement using an anti-properdin antibody. In
various embodiments, the invention is directed to compositions and
methods for treating an AP-mediated pathology or AP-mediated
condition in an individual by contacting the individual with an
anti-properdin antibody. The AP-mediated pathologies and conditions
that can be treated with the compositions and methods of the
invention include, but are not limited to, macular degeneration,
ischemia reperfusion injury, arthritis, rheumatoid arthritis,
asthma, paroxysmal nocturnal hemoglobinuria (PNH) syndrome,
atypical hemolytic uremic (aHUS) syndrome, sepsis, organ
transplantation, inflammation (including, but not limited to,
inflammation associated with cardiopulmonary bypass surgery and
kidney dialysis), glomerulonephritis (including, but not limited
to, anti-neutrophil cytoplasmic antibody (ANCA)-mediated
glomerulonephritis, lupus, and combinations thereof.
Definitions
[0049] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0050] As used herein, each of the following terms has the meaning
associated with it in this section.
[0051] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0052] The terms "inhibit" and "inhibition," as used herein, means
to reduce, suppress, diminish or block an activity or function by
at least about 10% relative to a control value. Preferably, the
activity is suppressed or blocked by 50% compared to a control
value, more preferably by 75%, and even more preferably by 95%.
[0053] The terms "effective amount" and "pharmaceutically effective
amount" refer to a sufficient amount of an agent to provide the
desired biological result. That result can be reduction and/or
alleviation of the signs, symptoms, or causes of a disease or
disorder, or any other desired alteration of a biological system.
An appropriate effective amount in any individual case may be
determined by one of ordinary skill in the art using routine
experimentation.
[0054] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal,
preferably a mammal, and most preferably a human, having a
complement system, including a human in need of therapy for, or
susceptible to, a condition or its sequelae. The individual may
include, for example, dogs, cats, pigs, cows, sheep, goats, horses,
rats, monkeys, and mice and humans.
[0055] The term "abnormal" when used in the context of organisms,
tissues, cells or components thereof, refers to those organisms,
tissues, cells or components thereof that differ in at least one
observable or detectable characteristic (e.g., age, treatment, time
of day, etc.) from those organisms, tissues, cells or components
thereof that display the "normal" (expected/homeostatic) respective
characteristic. Characteristics which are normal or expected for
one cell, tissue type, or subject, might be abnormal for a
different cell or tissue type.
[0056] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to
deteriorate.
[0057] In contrast, a "disorder" in an animal is a state of health
in which the animal is able to maintain homeostasis, but in which
the animal's state of health is less favorable than it would be in
the absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0058] A disease or disorder is "alleviated" if the severity of a
sign or symptom of the disease or disorder, the frequency with
which such a sign or symptom is experienced by a patient, or both,
is reduced.
[0059] An "effective amount" or "therapeutically effective amount"
of a compound is that amount of compound which is sufficient to
provide a beneficial effect to the subject to which the compound is
administered.
[0060] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression which can be used to communicate the usefulness of a
compound, composition, vector, or delivery system of the invention
in the kit for effecting alleviation of the various diseases or
disorders recited herein. Optionally, or alternately, the
instructional material can describe one or more methods of
alleviating the diseases or disorders in a cell or a tissue of a
mammal. The instructional material of the kit of the invention can,
for example, be affixed to a container which contains the
identified compound, composition, vector, or delivery system of the
invention or be shipped together with a container which contains
the identified compound, composition, vector, or delivery system.
Alternatively, the instructional material can be shipped separately
from the container with the intention that the instructional
material and the compound be used cooperatively by the
recipient.
[0061] A "therapeutic" treatment is a treatment administered to a
subject who exhibits signs of pathology, for the purpose of
diminishing or eliminating those signs.
[0062] As used herein, "treating a disease or disorder" means
reducing the frequency and/or severity of a sign and/or symptom of
the disease, disorder or pathology is experienced by a patient.
Disease, disorder and pathology are used interchangeably
herein.
[0063] The phrase "biological sample" as used herein, is intended
to include any sample comprising a cell, a tissue, or a bodily
fluid in which expression of a nucleic acid or polypeptide can be
detected. Examples of such biological samples include but are not
limited to blood, lymph, bone marrow, biopsies and smears. Samples
that are liquid in nature are referred to herein as "bodily
fluids." Biological samples may be obtained from a patient by a
variety of techniques including, for example, by scraping or
swabbing an area or by using a needle, to obtain bodily fluids.
Methods for collecting various body samples are well known in the
art.
[0064] The term "antibody," as used herein, refers to an
immunoglobulin molecule which is able to specifically bind to a
specific epitope of an antigen. Antibodies can be intact
immunoglobulins derived from natural sources, or from recombinant
sources and can be immunoreactive portions of intact
immunoglobulins. The antibodies in the present invention may exist
in a variety of forms including, for example, polyclonal
antibodies, monoclonal antibodies, intracellular antibodies
("intrabodies"), Fv, Fab, Fab', F(ab)2 and F(ab')2, as well as
single chain antibodies (scFv), heavy chain antibodies, such as
camelid antibodies, and humanized antibodies (Harlow et al., 1999,
Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory
Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc.
Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science
242:423-426).
[0065] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0066] As used herein, the term "heavy chain antibody" or "heavy
chain antibodies" comprises immunoglobulin molecules derived from
camelid species, either by immunization with a peptide and
subsequent isolation of sera, or by the cloning and expression of
nucleic acid sequences encoding such antibodies. The term "heavy
chain antibody" or "heavy chain antibodies" further encompasses
immunoglobulin molecules isolated from an animal with heavy chain
disease, or prepared by the cloning and expression of VH (variable
heavy chain immunoglobulin) genes from an animal.
[0067] A "chimeric antibody" refers to a type of engineered
antibody which contains a naturally-occurring variable region
(light chain and heavy chains) derived from a donor antibody in
association with light and heavy chain constant regions derived
from an acceptor antibody.
[0068] A "humanized antibody" refers to a type of engineered
antibody having its CDRs derived from a non-human donor
immunoglobulin, the remaining immunoglobulin-derived parts of the
molecule being derived from one (or more) human immunoglobulin(s).
In addition, framework support residues may be altered to preserve
binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad
Sci USA, 86:10029-10032; 1991, Hodgson et al., Bio/Technology,
9:421). A suitable human acceptor antibody may be one selected from
a conventional database, e.g., the KABAT database, Los Alamos
database, and Swiss Protein database, by homology to the nucleotide
and amino acid sequences of the donor antibody. A human antibody
characterized by a homology to the framework regions of the donor
antibody (on an amino acid basis) may be suitable to provide a
heavy chain constant region and/or a heavy chain variable framework
region for insertion of the donor CDRs. A suitable acceptor
antibody capable of donating light chain constant or variable
framework regions may be selected in a similar manner. It should be
noted that the acceptor antibody heavy and light chains are not
required to originate from the same acceptor antibody. The prior
art describes several ways of producing such humanized antibodies
(see for example EP-A-0239400 and EP-A-054951).
[0069] The term "donor antibody" refers to an antibody (monoclonal,
and/or recombinant) which contributes the amino acid sequences of
its variable regions, CDRs, or other functional fragments or
analogs thereof to a first immunoglobulin partner, so as to provide
the altered immunoglobulin coding region and resulting expressed
altered antibody with the antigenic specificity and neutralizing
activity characteristic of the donor antibody.
[0070] The term "acceptor antibody" refers to an antibody
(monoclonal and/or recombinant) heterologous to the donor antibody,
which contributes all (or any portion, but in some embodiments all)
of the amino acid sequences encoding its heavy and/or light chain
framework regions and/or its heavy and/or light chain constant
regions to the first immunoglobulin partner. In certain embodiments
a human antibody is the acceptor antibody.
[0071] "CDRs" are defined as the complementarity determining region
amino acid sequences of an antibody which are the hypervariable
regions of immunoglobulin heavy and light chains. See, e.g., Kabat
et al., Sequences of Proteins of Immunological interest, 4th Ed.,
U.S. Department of Health and Human Services, National Institutes
of Health (1987). There are three heavy chain and three light chain
CDRs (or CDR regions) in the variable portion of an immunoglobulin.
Thus, "CDRs" as used herein refers to all three heavy chain CDRs,
or all three light chain CDRs (or both all heavy and all light
chain CDRs, if appropriate). The structure and protein folding of
the antibody may mean that other residues are considered part of
the antigen binding region and would be understood to be so by a
skilled person. See for example Chothia et al., (1989)
Conformations of immunoglobulin hypervariable regions; Nature 342 p
877-883.
[0072] As used herein, an "immunoassay" refers to any binding assay
that uses an antibody capable of binding specifically to a target
molecule to detect and quantify the target molecule.
[0073] By the term "specifically binds," as used herein with
respect to an antibody, is meant an antibody which recognizes a
specific antigen, but does not substantially recognize or bind
other molecules in a sample. For example, an antibody that
specifically binds to an antigen from one species may also bind to
that antigen from one or more species. But, such cross-species
reactivity does not itself alter the classification of an antibody
as specific. In another example, an antibody that specifically
binds to an antigen may also bind to different allelic forms of the
antigen. However, such cross reactivity does not itself alter the
classification of an antibody as specific.
[0074] In some instances, the terms "specific binding" or
"specifically binding", can be used in reference to the interaction
of an antibody, a protein, or a peptide with a second chemical
species, to mean that the interaction is dependent upon the
presence of a particular structure (e.g., an antigenic determinant
or epitope) on the chemical species; for example, an antibody
recognizes and binds to a specific protein structure rather than to
proteins generally. If an antibody is specific for epitope "A", the
presence of a molecule containing epitope A (or free, unlabeled A),
in a reaction containing labeled "A" and the antibody, will reduce
the amount of labeled A bound to the antibody.
[0075] A "coding region" of a gene consists of the nucleotide
residues of the coding strand of the gene and the nucleotides of
the non-coding strand of the gene which are homologous with or
complementary to, respectively, the coding region of an mRNA
molecule which is produced by transcription of the gene.
[0076] A "coding region" of a mRNA molecule also consists of the
nucleotide residues of the mRNA molecule which are matched with an
anti-codon region of a transfer RNA molecule during translation of
the mRNA molecule or which encode a stop codon. The coding region
may thus include nucleotide residues comprising codons for amino
acid residues which are not present in the mature protein encoded
by the mRNA molecule (e.g., amino acid residues in a protein export
signal sequence).
[0077] "Complementary" as used herein to refer to a nucleic acid,
refers to the broad concept of sequence complementarity between
regions of two nucleic acid strands or between two regions of the
same nucleic acid strand. It is known that an adenine residue of a
first nucleic acid region is capable of forming specific hydrogen
bonds ("base pairing") with a residue of a second nucleic acid
region which is antiparallel to the first region if the residue is
thymine or uracil. Similarly, it is known that a cytosine residue
of a first nucleic acid strand is capable of base pairing with a
residue of a second nucleic acid strand which is antiparallel to
the first strand if the residue is guanine. A first region of a
nucleic acid is complementary to a second region of the same or a
different nucleic acid if, when the two regions are arranged in an
antiparallel fashion, at least one nucleotide residue of the first
region is capable of base pairing with a residue of the second
region. Preferably, the first region comprises a first portion and
the second region comprises a second portion, whereby, when the
first and second portions are arranged in an antiparallel fashion,
at least about 50%, and preferably at least about 75%, at least
about 90%, or at least about 95% of the nucleotide residues of the
first portion are capable of base pairing with nucleotide residues
in the second portion. More preferably, all nucleotide residues of
the first portion are capable of base pairing with nucleotide
residues in the second portion.
[0078] The term "DNA" as used herein is defined as deoxyribonucleic
acid.
[0079] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting there from. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0080] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0081] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a peptide naturally present in its
normal context in a living animal is not "isolated," but the same
nucleic acid or peptide partially or completely separated from the
coexisting materials of its natural context is "isolated." An
isolated nucleic acid or protein can exist in substantially
purified form, or can exist in a non-native environment such as,
for example, a host cell.
[0082] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, i.e., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, i.e., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, i.e., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (i.e., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0083] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0084] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences which are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR, and the like,
and by synthetic means.
[0085] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0086] The term "progeny" as used herein refers to a descendent or
offspring and includes the offspring of a mammal, and also included
the differentiated or undifferentiated decedent cell derived from a
parent cell. In one usage, the term progeny refers to a descendent
cell which is genetically identical to the parent. In another use,
the term progeny refers to a descendent cell which is genetically
and phenotypically identical to the parent. In yet another usage,
the term progeny refers to a descendent cell that has
differentiated from the parent cell.
[0087] The term "RNA" as used herein is defined as ribonucleic
acid.
[0088] The term "recombinant DNA" as used herein is defined as DNA
produced by joining pieces of DNA from different sources.
[0089] The term "recombinant polypeptide" as used herein is defined
as a polypeptide produced by using recombinant DNA methods.
[0090] As used herein, "conjugated" refers to covalent attachment
of one molecule to a second molecule.
[0091] "Variant" as the term is used herein, is a nucleic acid
sequence or a peptide sequence that differs in sequence from a
reference nucleic acid sequence or peptide sequence respectively,
but retains essential biological properties of the reference
molecule. Changes in the sequence of a nucleic acid variant may not
alter the amino acid sequence of a peptide encoded by the reference
nucleic acid, or may result in amino acid substitutions, additions,
deletions, fusions and truncations. Changes in the sequence of
peptide variants are typically limited or conservative, so that the
sequences of the reference peptide and the variant are closely
similar overall and, in many regions, identical. A variant and
reference peptide can differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A variant
of a nucleic acid or peptide can be a naturally occurring such as
an allelic variant, or can be a variant that is not known to occur
naturally. Non-naturally occurring variants of nucleic acids and
peptides may be made by mutagenesis techniques or by direct
synthesis
[0092] The term "breeding" is used herein to refer to the
propagation of a species with the result being at least one
offspring.
[0093] The term "natural breeding" is used herein to refer to the
propagation of a species by sexual union.
[0094] The term "inbred animal" is used herein to refer to an
animal that has been interbred with other similar animals of the
same species in order to preserve and fix certain characteristics,
or to prevent other characteristics from being introduced into the
breeding population.
[0095] The term "outbred animal" is used herein to refer to an
animal that breeds with any other animal of the same species
without regard to the preservation of certain characteristics.
[0096] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Description
[0097] This invention relates to the inhibition of the alternative
pathway (AP) of complement using an anti-properdin antibody. In one
embodiment, the invention is directed to methods of treating an
AP-mediated pathology or AP-mediated condition in an individual by
contacting the individual with an anti-properdin antibody.
[0098] In one embodiment, the invention is a method of treating an
AP-mediated pathology in an individual, comprising the step of
administering to said individual an anti-properdin antibody,
thereby inhibiting the generation of a C3bBc protein complex.
Examples of complement-mediated pathologies that can be treated
using the methods of the invention include, but are not limited to
macular degeneration, ischemia reperfusion injury, arthritis,
rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria (PNH)
syndrome, atypical hemolytic uremic (aHUS) syndrome, asthma,
inflammation, glomerulonephritis, lupus, organ transplantation
sepsis, or combinations thereof.
[0099] The ability of the immune system to discriminate between
"self" and "non-self" antigens is vital to the functioning of the
immune system as a specific defense against invading
microorganisms. "Non-self" antigens are those antigens on
substances entering or present in the body which are detectably
different or foreign from the animal's own constituents, whereas
"self" antigens are those which, in the healthy animal, are not
detectably different or foreign from its own constituents. In
various embodiments of the methods described herein, the AP
activation that is inhibited is that which was triggered by at
least one of the group consisting of a microbial antigen, a
non-biological foreign surface, altered self-tissue, or
combinations thereof. One example of a non-biological foreign
surface is blood tubing such as that used in cardio-pulmonary
bypass surgery or kidney dialysis. Examples of altered self-tissues
include apoptotic, necrotic and ischemia-stressed tissues and
cells, or combinations thereof.
[0100] In some embodiments, the anti-properdin antibodies of the
invention selectively inhibit the AP, but do not inhibit the
classical pathway (CP) or the lectin pathway (LP). Generally, the
CP is initiated by antigen-antibody complexes, the LP is activated
by binding of lectins to sugar molecules on microbial surfaces,
while the AP is constitutively active at a low level but can be
quickly amplified on bacterial, viral, and parasitic cell surfaces
due to the lack of regulatory proteins. Host cells are usually
protected from AP complement activation by regulatory proteins. But
in some situations, such as when the regulatory proteins are
defective or missing, the AP can also be activated uncontrollably
on host cells, leading to complement-mediated pathology. The CP
consists of components C1, C2, C4 and converges with the AP at the
C3 activation step. The LP consists of mannose-binding lectins
(MBLs) and MBL-associated serine proteases (hasps) and shares with
the CP the components C4 and C2. The AP consists of components C3
and several factors, such as Factor B, Factor D, properdin and the
fluid phase regulator Factor H. Complement activation consists of
three stages: (a) recognition, (b) enzymatic activation, and (c)
membrane attack leading to cell death. The first phase of CP
complement activation begins with C1. C1 is made up of three
distinct proteins: a recognition subunit, C1q, and the serine
protease subcomponents, C1r and C1s, which are bound together in a
calcium-dependent tetrameric complex, C1r2 s2. An intact C1 complex
is necessary for physiological activation of C1 to result.
Activation occurs when the intact C1 complex binds to
immunoglobulin complexed with antigen. This binding activates C1s
which then cleaves both the C4 and C2 proteins to generate C4a and
C4b, as well as C2a and C2b. The C4b and C2a fragments combine to
form the C3 convertase, C4b2a, which in turn cleaves C3 to form C3a
and C3b. Activation of the LP is initiated by MBL binding to
certain sugars on the target surface and this triggers the
activation of Masps which then cleaves C4 and C2 in a manner
analogous to the activity of C1s of the CP, resulting in the
generation of the C3 convertase, C4b2a. Thus, the CP and LP are
activated by different mechanisms but they share the same
components C4 and C2 and both pathways lead to the generation of
the same C3 convertase, C4b2a. The cleavage of C3 by C4b2a into C3b
and C3a is a central event of the complement pathway for two
reasons. It initiates the AP amplification loop because surface
deposited C3b is a central intermediate of the AP, Both C3a and C3b
are biologically important. C3a is proinflammatory and together
with C5a are referred to as anaphylatoxins. C3b and its further
cleavage products also bind to complement receptors present on
neutrophils, eosinophils, monocytes and macrophages, thereby
facilitating phagocytosis and clearance of C3b-opsonized particles.
Finally, C3b can associate with C4b2a to form the C5 convertase of
the CP and LP to activate the terminal complement sequence, leading
to the production of C5a, a potent proinflammatory mediator, and
the assembly of the lytic membrane attack complex (MAC), C5-C9.
[0101] Because the CP and AP play a critical role in host defense
and many complement-dependent human pathologies are mediated by the
AP, it is desirable to selectively inhibit the AP in the treatment
of such human pathologies. Accordingly, in preferred embodiments of
the methods described herein, the immunity provided by the CP and
LP is maintained, while the AP is selectively inhibited. Thus, in
various embodiments, the anti-properdin antibodies used in the
methods described herein, do not inhibit the CP and LP. In certain
embodiments, the anti-properdin antibodies described herein are
distinct from anti-properdin antibodies developed previously which
inhibit both AP and CP.
[0102] The AP is thought to be constitutively active at a low level
due to spontaneous hydrolysis of C3 to form C3(H2O). C3(H2O)
behaves like C3b in that it can associate with fB, which make fB
susceptible to fD cleavage and activation. The resultant C3(H2O)Bb
then cleaves C3 to produce C3b and C3a to initiate the AP cascade
by forming the C3 convertase of the AP, C3bBb. As the initial C3
convertase generates increasing amounts of C3b, an amplification
loop is established. It should be noted that because the CP and LP
also generate C3b, wherein C3b can bind factor B and engages the
AP, the AP amplification loop also participates in the CP and LP
once these pathways are activated. Thus, the AP consists of two
functional entities: an independent complement activation pathway
that is unrelated to CP or LP and an amplification process that
does participate and contribute to the full manifestation of CP and
LP. In one embodiment, the anti-properdin antibodies used in the
methods described herein selectively inhibit the AP activation
process and do not interfere with the AP amplification loop of the
CP and LP.
[0103] Properdin is structurally composed of an N-terminal domain
and six thrombospondin type I repeat (TSR) domains. Under
physiological conditions, it exists in plasma as cyclic polymers
(dimers, trimers, tetramers), formed by head to tail associations
of monomers. Human properdin is encoded on the short arm of the X
chromosome and its deficiency, especially when combined with C2,
MBL or IgG2 deficiency, constitutes a risk factor for lethal
Neisseria infections.
[0104] It appears that the need for properdin in AP initiation is
variable and depends on the nature of the activating surface. By
way of non-limiting examples, properdin appears to be indispensable
for LPS- and LOS-induced AP activation as well as for AP-mediated
autologous tissue injury such as extravascular hemolysis of
Crry-deficient mouse erythrocytes (see U.S. Pat. App. No.
2010/0263061). The invention described herein discloses that
properdin is essential for human AP complement mediated red blood
cell lysis in the context of and DAF dysfunction/blockade (FIG. 3).
The invention described herein also discloses that properdin is
essential for autologous serum lysis of PNH red blood cells (FIG.
24). On the other hand, zymosan-induced AP activation is moderately
impaired by properdin deficiency, and properdin does not appear to
play a substantial role in CVF- and CP-triggered AP amplification
(see U.S. Pat. App. No. 2010/0263061). AP activation on a given
surface may represent the balance between properdin-dependent
promotion via C3bBb stabilization and factor H (fH)-dependent
inhibition of C3 `tick-over.` An AP activator for which properdin
is not essential may have limited interaction with fH and, as a
result of lacking sufficient fH-dependent inhibition, spontaneous
C3 activation and amplification could occur as a default process
without the help of properdin. Accordingly, in various embodiments
of the invention described herein, inhibition of properdin function
by the anti-properdin antibodies of the invention offers several
advantages, including: it does not compromise the AP amplification
loop of the CP and LP, leaving these pathways fully active for host
defense; it does not completely eliminate AP complement activation
since not all AP activators (i.e. pathogens) require properdin to
trigger this pathway, reducing the degree of impairment in host
defense when compared with other methods of AP inhibition such as
anti-fB and anti-fD antibodies.
[0105] In one embodiment, the activity of the AP that is inhibited
using a method of invention is AP activation induced by at least
one of the group selected from a lipopolysacchride (LPS),
lipooligosaccharide (LOS), pathogen-associated molecular patterns
(PAMPs) and danger-associated molecular patterns (DAMPs). In
another embodiment, the activity of the AP that is inhibited using
a method of invention is the generation of C3bBb protein complex.
In another embodiment, the activity of the AP that is inhibited
using a method of invention is properdin dependent.
[0106] In some embodiments, the methods of the present invention
preserve the ability of the individual to combat an infection
through the CP and LP. In one embodiment, the invention is a method
of inhibiting AP activation induced by bacterial
lipooligosaccharide (LOS) in an individual, comprising the step of
administering to said individual an anti-properdin antibody, and
thereby inhibiting an AP activation induced by bacterial LOS in an
individual. In another embodiment, provided herein is a method of
inhibiting AP activation induced by a bacterial LPS. In certain
embodiments, the AP activation is induced by S. typhosa LPS, and
the inhibitors used in the methods provided herein do not inhibit
AP activity induced by S. minnesota LPS or E. coli LPS. In various
embodiments, the anti-properdin antibodies of the invention
selectively inhibit the AP, but do not inhibit CP-triggered
complement activation, LP-triggered complement activation,
zymosan-induced activation, or cobra venom factor-induced
activation.
[0107] In one embodiment, provided herein is a method of inhibiting
a pathogen-associated molecular pattern-mediated AP activation in
an individual, comprising the step of administering to said
individual an anti-properdin antibody, thereby inhibiting a
PAMP-mediated AP activation in an individual. Examples of PAMPs
whose activation of AP can be inhibited by the methods of the
invention, include, but are not limited to, a muramyl dipeptide
(MDP), a CpG motif from bacterial DNA, double-stranded viral RNAs,
a peptidoglycan, a lipoteichoic acid, an outer surface protein A
from Borrelia burgdorferi, a synthetic mycoplasmal
macrophage-activating lipoprotein-2,
tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), a
dipalmitoyl-CSK4 (P2-CSK4), a monopalmitoyl-CSK4 (PCSK4),
amphotericin B, a triacylated or diacylated bacterial polypeptide,
and combinations thereof.
[0108] In one embodiment, the invention is a method of inhibiting
initiation of AP activation in an individual, comprising the step
of administering to said individual an anti-properdin antibody,
thereby inhibiting initiation of AP activation in an individual. In
another embodiment, provided herein is a method of inhibiting
amplification of AP activation in an individual, comprising the
step of administering to said individual an inhibitor of the AP,
thereby inhibiting amplification of AP activation in an individual.
Examples of these embodiments are PNH patients who suffer from AP
complement-mediated hemolysis and individuals suffering from AP
complement-mediated aHUS, asthma, ischemic/reperfusion injury,
rheumatoid arthritis and ANCA-mediated kidney diseases. In various
embodiments of the invention, diseases and disorders that can be
treated using the compositions and methods of the invention
include, but are not limited to, AP complement-mediated hemolysis,
AP complement-mediated aHUS, asthma, ischemic/reperfusion injury,
rheumatoid arthritis and ANCA-mediated kidney diseases.
[0109] In various embodiments, provided herein are methods of
identifying a potential antibody having inhibitory effects on the
AP, comprising the steps of: a) administering the anti-properdin
antibody to an individual; b) measuring the resulting phenotype of
the individual; and c) comparing the resulting phenotype of the
individual to the phenotype of a properdin.sup.-/- knockout animal
(see U.S. Pat. App. No. 2010/0263061). In another embodiment, the
anti-properdin antibody used in the methods provided herein is
identified by the method of selecting a potential therapeutic
compound using the properdin.sup.-/- knockout animal (see U.S. Pat.
App. No. 2010/0263061).
[0110] In various other embodiments, provided herein are methods of
identifying a potential anti-properdin antibody having inhibitory
effects on the AP. One such method includes the steps of: a)
coating a plate with lipopolysaccharide (LPS); b) washing the plate
to remove unbound LPS; c) adding bovine serum albumin (BSA) in
phosphate buffered saline (PBS); d) washing the plate to remove
unbound BSA; e) adding a mixture of a candidate anti-properdin
antibody compound mixed into human serum; f) washing the plate; g)
adding an anti-human C3 antibody; h) washing the plate to remove
unbound antibody; i) adding TMB Substrate Reagent; j) adding
sulphuric acid to stop the reaction; k) measuring the optical
density at 450 nm; l) comparing the optical density of the plate
containing the candidate anti-properdin antibody compound to the
optical density of a positive comparator control and a negative
comparator control; wherein when the optical density is diminished
as compared with the positive comparator control, the
anti-properdin antibody is identified.
Anti-Properdin Antibodies
[0111] In some embodiments, the invention includes compositions
comprising an antibody that specifically binds to properdin. In one
embodiment, the anti-properdin antibody is a polyclonal antibody.
In another embodiment, the anti-properdin antibody is a monoclonal
antibody. In some embodiments, the anti-properdin antibody is a
chimeric antibody. In further embodiments, the anti-properdin
antibody is a humanized antibody. In preferred embodiments, the
properdin is human properdin.
[0112] In one embodiment, the anti-properdin antibody comprises at
least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:3; VH-CDR2: SEQ ID NO:4; VH-CDR3; SEQ ID NO:5;
VL-CDR1: SEQ ID NO:8; VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID
NO:10. In another embodiment, the anti-properdin antibody comprises
alt of the CDRs of the group consisting of: VH-CDR1: SEQ ID NO:3;
VH-CDR2: SEQ ID NO:4; VH-CDR3: SEQ ID NO:5; VL-CDR1: SEQ ID NO:8;
VL-CDR2: SEQ ID NO:9; and VL-CDR3: SEQ ID NO:10.
[0113] In some embodiments, the anti-properdin antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:2. In
other embodiments, the anti-properdin antibody comprises a light
chain comprising the amino acid sequence of SEQ ID NO:7. In another
embodiment, the anti-properdin antibody is a monoclonal antibody
designated mAb 19.1. The monoclonal anti-properdin antibody
designated mAb 19.1 comprises a heavy chain comprising the amino
acid sequence of SEQ ID NO:2 and a light chain comprising the amino
acid sequence of SEQ ID NO:7. In some embodiments, the monoclonal
anti-properdin antibody designated mAb 19.1 is humanized.
[0114] In some embodiments, the anti-properdin antibody of the
invention binds to an epitope comprising at least one amino acid of
TSR5 (SEQ ID NO:60). In some embodiments, the anti-properdin
antibody of the invention specifically binds to an epitope
comprising at least one amino acid of the amino acid sequence
RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO:52). In some
embodiments, the anti-properdin antibody that specifically binds to
an epitope comprising at least one amino acid of SEQ ID NO:52 is
the mAb designated as mAb 19.1. In some embodiments, the
anti-properdin antibody is an antibody that competes for binding
with the antibody designated as mAb 19.1. In various embodiments,
the epitope to which the antibody of the invention can bind is a
linear epitope or a conformational epitope.
[0115] In one embodiment, the anti-properdin antibody comprises at
least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:13; VH-CDR2: SEQ ID NO:14; VH-CDR3: SEQ ID
NO:15; VL-CDR1: SEQ ID NO:18; VL-CDR2: SEQ ID NO:19; and VL-CDR3:
SEQ ID NO:20. In another embodiment, the anti-properdin antibody
comprises all of the CDRs of the group consisting of: VH-CDR1: SEQ
ID NO:13; VH-CDR2: SEQ ID NO:14; VH-CDR3: SEQ ID NO:15; VL-CDR1:
SEQ ID NO:18; VL-CDR2: SEQ ID NO:19; and VL-CDR3: SEQ ID NO:20.
[0116] In some embodiments, the anti-properdin antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:12. In
other embodiments, the anti-properdin antibody comprises a light
chain comprising the amino acid sequence of SEQ ID NO:17. In
another embodiment, the anti-properdin antibody is a monoclonal
antibody designated mAb 25. The monoclonal anti-properdin antibody
designated mAb 25 comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:12 and a light chain comprising the amino
acid sequence of SEQ ID NO:17. In some embodiments, the monoclonal
anti-properdin antibody designated mAb 25 is humanized.
[0117] In some embodiments, the anti-properdin antibody of the
invention binds to an epitope comprising at least one amino acid of
TSR5 (SEQ ID NO:60) and/or TSR6 (SEQ ID NO:61). In some
embodiments, the anti-properdin antibody of the invention
specifically binds to an epitope comprising at least one amino acid
of the amino acid sequence LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO:53).
In some embodiments, the anti-properdin antibody of the invention
specifically binds to an epitope comprising cysteine 62 (C62),
present in SEQ TD NO:61. In some embodiments, the anti-properdin
antibody of the invention specifically binds to an epitope
comprising cysteine 78 (C78), present in SEQ ID NO:53. In some
embodiments, the anti-properdin antibody that specifically binds to
an epitope comprising the amino acid of SEQ ID NO:53 is the mAb
designated as mAb 25. In some embodiments, the anti-properdin
antibody is an antibody that competes for binding with the antibody
designated as mAb 25. In various embodiments, the epitope to which
the antibody of the invention can bind is a linear epitope or a
conformational epitope.
[0118] In one embodiment, the anti-properdin antibody comprises at
least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3: SEQ ID
NO:25; VL-CDR1: SEQ ID NO:28; VL-CDR2: SEQ ID NO:29; and VL-CDR3:
SEQ ID NO:30. In another embodiment, the anti-properdin antibody
comprises all of the CDRs of the group consisting of: VH-CDR1: SEQ
ID NO:23; VH-CDR2: SEQ ID NO:24; VH-CDR3; SEQ ID NO:25; VL-CDR1:
SEQ ID NO:28; VL-CDR2: SEQ ID NO:29; and VL-CDR3: SEQ ID NO:30.
[0119] In some embodiments, the anti-properdin antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:22. In
other embodiments, the anti-properdin antibody comprises a light
chain comprising the amino acid sequence of SEQ ID NO:27. In
another embodiment, the anti-properdin antibody is a monoclonal
antibody designated mAb 22.1. The monoclonal anti-properdin
antibody designated mAb 22.1 comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO:22 and a light chain comprising
the amino acid sequence of SEQ ID NO:27. In other embodiments, the
anti-properdin antibody is an antibody that competes for binding
with the antibody designated as mAb 22.1. In some embodiments, the
monoclonal anti-properdin antibody designated mAb 22.1 is
humanized.
[0120] In one embodiment, the anti-properdin antibody comprises at
least one of the CDRs selected from the group consisting of:
VH-CDR1: SEQ ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID
NO:35; VL-CDR1: SEQ ID NO:38; VL-CDR2: SEQ ID NO:39; and VL-CDR3:
SEQ ID NO:40. In another embodiment, the anti-properdin antibody
comprises all of the CDRs of the group consisting of: VH-CDR1: SEQ
ID NO:33; VH-CDR2: SEQ ID NO:34; VH-CDR3: SEQ ID NO:35; VL-CDR1:
SEQ ID NO:38; VL-CDR2: SEQ ID NO:39; and VL-CDR3: SEQ ID NO:40.
[0121] In some embodiments, the anti-properdin antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO:32. In
other embodiments, the anti-properdin antibody comprises a light
chain comprising the amino acid sequence of SEQ ID NO:37. In
another embodiment, the anti-properdin antibody is a monoclonal
antibody designated mAb 30. The monoclonal anti-properdin antibody
designated mAb 30 comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:32 and a light chain comprising the amino
acid sequence of SEQ ID NO:37. In other embodiments, the
anti-properdin antibody is an antibody that competes for binding
with the antibody designated as mAb 30. In some embodiments, the
monoclonal anti-properdin antibody designated mAb 30 is
humanized.
[0122] In other embodiments, the anti-properdin antibody comprises
a humanized heavy chain comprising the amino acid sequence of SEQ
ID NO:42. In some embodiments, the anti-properdin antibody
comprises a humanized heavy chain comprising the amino acid
sequence of SEQ ID NO:44. In still other embodiments, the
anti-properdin antibody comprises a humanized light chain
comprising the amino acid sequence of SEQ ID NO:47. In some
embodiments, the anti-properdin antibody comprises a humanized
antibody comprising a heavy chain comprising the amino acid
sequence of SEQ ID NO:42 and a light chain comprising the amino
acid sequence of SEQ ID NO:47. In other embodiments, anti-properdin
antibody comprises a humanized antibody comprising a heavy chain
comprising the amino acid sequence of SEQ ID NO:44 and a light
chain comprising the amino acid sequence of SEQ ID NO:47. In
further embodiments, the anti-properdin antibody is an antibody
that competes for binding with a humanized antibody described
herein.
[0123] In some embodiments, the anti-properdin antibody comprises a
humanized heavy chain comprising the amino acid sequence of SEQ ID
NO:49. In other embodiments, the anti-properdin antibody comprises
a humanized light chain comprising the amino acid sequence of SEQ
ID NO:51. In some embodiments, the anti-properdin antibody
comprises a humanized antibody comprising a heavy chain comprising
the amino acid sequence of SEQ ID NO:49 and a light chain
comprising the amino acid sequence of SEQ ID NO:51. In further
embodiments, the anti-properdin antibody is an antibody that
competes for binding with a humanized antibody described
herein.
[0124] In some embodiments, the anti-properdin antibody comprises a
chimeric heavy chain comprising the amino acid sequences of SEQ ID
NO:2 and a human heavy chain constant region, such as, by way of
non-limiting example, a human IgG4 constant region comprising the
amino acid sequence of SEQ ID NO:63. In other embodiments, the
anti-properdin antibody comprises a chimeric light chain comprising
the amino acid sequence of SEQ ID NO:7 and a human light chain
constant region, such as, by way of non-limiting example, a human
kappa light chain constant region comprising the amino acid
sequence of SEQ ID NO:64. In a certain embodiment, the
anti-properdin antibody comprises a chimeric heavy chain comprising
the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:63 and a
chimeric light chain comprising the amino acid sequences of SEQ ID
NO:7 and SEQ ID NO:64.
[0125] In some embodiments, the anti-properdin antibody comprises a
chimeric heavy chain comprising the amino acid sequences of SEQ ID
NO:12 and a human heavy chain constant region, such as, by way of
non-limiting example, a human IgG4 constant region comprising the
amino acid sequence of SEQ ID NO:63. In other embodiments, the
anti-properdin antibody comprises a chimeric light chain comprising
the amino acid sequence of SEQ ID NO:17 and a human light chain
constant region, such as, by way of non-limiting example, a human
kappa light chain constant region comprising the amino acid
sequence of SEQ ID NO:64. In a certain embodiment, the
anti-properdin antibody comprises a chimeric heavy chain comprising
the amino acid sequences of SEQ ID NO:12 and SEQ ID NO:63 and a
chimeric light chain comprising the amino acid sequences of SEQ ID
NO:17 and SEQ ID NO:64.
[0126] In some embodiments, the anti-properdin antibody comprises a
chimeric heavy chain comprising the amino acid sequences of SEQ ID
NO:22 and a human heavy chain constant region, such as, by way of
non-limiting example, a human IgG4 constant region comprising the
amino acid sequence of SEQ ID NO:63. In other embodiments, the
anti-properdin antibody comprises a chimeric light chain comprising
the amino acid sequence of SEQ ID NO:27 and a human light chain
constant region, such as, by way of non-limiting example, a human
kappa light chain constant region comprising the amino acid
sequence of SEQ ID NO:64. In a certain embodiment, the
anti-properdin antibody comprises a chimeric heavy chain comprising
the amino acid sequences of SEQ ID NO:22 and SEQ ID NO:63 and a
chimeric light chain comprising the amino acid sequences of SEQ ID
NO:27 and SEQ ID NO:64.
[0127] In some embodiments, the anti-properdin antibody comprises a
chimeric heavy chain comprising the amino acid sequences of SEQ ID
NO:32 and a human heavy chain constant region, such as, by way of
non-limiting example, a human IgG4 constant region comprising the
amino acid sequence of SEQ ID NO:63. In other embodiments, the
anti-properdin antibody comprises a chimeric light chain comprising
the amino acid sequence of SEQ ID NO:37 and a human light chain
constant region, such as, by way of non-limiting example, a human
kappa light chain constant region comprising the amino acid
sequence of SEQ ID NO:64. In a certain embodiment, the
anti-properdin antibody comprises a chimeric heavy chain comprising
the amino acid sequences of SEQ ID NO:32 and SEQ ID NO:63 and a
chimeric light chain comprising the amino acid sequences of SEQ ID
NO:37 and SEQ ID NO:64.
Screening Assays
[0128] The present invention has application in various screening
assays, including, determining whether a candidate anti-properdin
antibody can inhibit the AP.
[0129] In some embodiments, the level of AP activity in the
presence of the candidate anti-properdin antibody is compared with
AP activity detected in a positive comparator control. The positive
comparator control comprises AP activation in the absence of added
test compound. In some embodiments, the candidate anti-properdin
antibody is identified as an inhibitor of the AP when the AP
activity in the presence of the candidate anti-properdin antibody
is less than about 70% or the AP activity detected in a positive
comparator control; this corresponds to greater than about 30%
inhibition of AP activity in the presence of the test compound. In
other embodiments, the candidate anti-properdin antibody is
identified as an inhibitor of the AP when the AP activity in the
presence of the candidate anti-properdin antibody is less than
about 80% of the AP activity detected in a positive comparator
control; this corresponds to greater than about 20% inhibition of
AP activity in the presence of the test compound. In still other
embodiments, the candidate anti-properdin antibody is identified as
an inhibitor of the AP when the AP activity in the presence of the
candidate anti-properdin antibody is less than about 90% of the AP
activity detected in a positive comparator control; this
corresponds to greater than about 10% inhibition of AP activity in
the presence of the test compound. In some embodiments, the level
of AP inhibition by the candidate anti-properdin antibody is
compared with the level of inhibition detected in a negative
comparator control.
[0130] A variety of immunoassay formats, including competitive and
non-competitive immunoassay formats, antigen capture assays,
two-antibody sandwich assays, and three-antibody sandwich assays
are useful methods of the invention (Self et al., 1996, Curr. Opin.
Biotechnol. 7:60-65). The invention should not be construed to be
limited to any one type of known or heretofor unknown assay,
provided that the assay is able to detect the inhibition of the
AP.
[0131] Hemolytic assays are included in the methods of the
invention. In various embodiments, red blood cells (RBCs) are
obtained from normal (healthy) individuals or from individuals
displaying signs or symptoms of a disease or disorder, such as, for
example, PNH. In various embodiments, RBCs are lysed with 5% to 75%
normal human serum (NHS) in the presence of a recombinant fH
fragment comprising complement control protein (CCP) repeat 19 and
20 of fH (fH19-20, 5-50 .mu.M) and anti-DAF neutralizing antibodies
(3-20 .mu.g/ml). In some embodiments, RBCs from individuals
displaying signs or symptoms of a disease or disorder, such as PNH,
are lysed with acidified serum. In some embodiments, hemolytic
assays are performed in GVB++-Mg++-EGTA buffer to allow only AP
complement activation, but the skilled artisan will understand that
other appropriate buffers can be used, so long as the buffer allows
only AP complement activation. In one embodiment, the degree of
lysis is calculated by measuring the OD410 of the supernatant of
the incubation mixture, as a measure of the release of hemoglobin
into solution. In various embodiments, at least one anti-properdin
antibody is added, at a concentration from about 1 .mu.g/ml to
about 50 .mu.g/ml, and pre-incubated with serum, and the extent of
inhibition of hemolysis of RBCs is measured.
[0132] Enzyme-linked immunosorbent assays (ELISAs) are useful in
the methods of the invention. An enzyme such as, but not limited
to, horseradish peroxidase (HRP), alkaline phosphatase,
beta-galactosidase or urease can be linked, for example, to an
anti-C3 antibody or to a secondary antibody for use in a method of
the invention. A horseradish-peroxidase detection system may be
used, for example, with the chromogenic substrate
tetramethylbenzidine (TMB), which yields a soluble product in the
presence of hydrogen peroxide that is detectable at 450 nm. Other
convenient enzyme-linked systems include, for example, the alkaline
phosphatase detection system, which may be used with the
chromogenic substrate p-nitrophenyl phosphate to yield a soluble
product readily detectable at 405 nm. Similarly, a
beta-galactosidase detection system may be used with the
chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG)
to yield a soluble product detectable at 410 nm. Alternatively, a
urease detection system may be used with a substrate such as
urea-bromocresol purple (Sigma Immunochemicals, St. Louis, Mo.).
Useful enzyme-linked primary and secondary antibodies can be
obtained from any number of commercial sources.
[0133] Chemiluminescent detection is also useful for detecting the
inhibition of the AP. Chemiluminescent secondary antibodies may be
obtained from any number of commercial sources.
[0134] Fluorescent detection is also useful for detecting the
inhibition of the AP. Useful fluorochromes include, but are not
limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin,
B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and
lissamine-Fluorescein- or rhodamine-labeled antibodies.
[0135] Radioimmunoassays (RIAs) are also useful in the methods of
the invention. Such assays are well known in the art, and are
described for example in Brophy et al. (1990, Biochem. Biophys.
Res. Comm. 167:898-903) and Guechot et al. (1996, Clin. Chem.
42:558-563). Radioimmunoassays are performed, for example, using
Iodine-125-labeled primary or secondary antibody (Harlow et al.,
supra, 1999).
[0136] A signal emitted from a detectable antibody is analyzed, for
example, using a spectrophotometer to detect color from a
chromogenic substrate; a radiation counter to detect radiation,
such as a gamma counter for detection of Iodine-125; or a
fluorometer to detect fluorescence in the presence of light of a
certain wavelength. Where an enzyme-linked assay is used,
quantitative analysis is performed using a spectrophotometer. It is
understood that the assays of the invention can be performed
manually or, if desired, can be automated and that the signal
emitted from multiple samples can be detected simultaneously in
many systems available commercially.
[0137] The methods of the invention also encompass the use of
capillary electrophoresis based immunoassays (CEIA), which can be
automated, if desired. Immunoassays also may be used in conjunction
with laser-induced fluorescence as described, for example, in
Schmalzing et al. (1997, Electrophoresis 18:2184-2193) and Bao
(1997, J. Chromatogr. B. Biomed. Sci. 699:463-480). Liposome
immunoassays, such as flow-injection liposome immunoassays and
liposome immunosensors, may also be used according to the methods
of the invention (Rongen et al., 1997, J. Immunol. Methods
204:105-133).
[0138] Quantitative western blotting may also be used to determine
the level of AP inhibition in the methods of the invention. Western
blots are quantified using well known methods such as scanning
densitometry (Parra et al., 1998, J. Vase. Surg. 28:669-675).
Methods of Administration
[0139] The methods of the invention comprise administering a
therapeutically effective amount of at least one anti-properdin
antibody to an individual identified as having an AP-mediated
pathology. In a preferred embodiment the individual is a mammal
having an AP system. In a more preferred embodiment the individual
is a human.
[0140] The methods of the invention can comprise the administration
of at least one of any of the anti-properdin antibodies described
herein, but the present invention should in no way be construed to
be limited to the anti-properdin antibodies described herein, but
rather should be construed to encompass any anti-properdin
antibodies, both known and unknown, that diminish and reduce AP
activation.
[0141] The method of the invention comprises administering a
therapeutically effective amount of at least one anti-properdin
antibody to an individual wherein a composition of the present
invention comprising an anti-properdin antibody, or a combination
thereof, is used either alone or in combination with other
therapeutic agents. The invention can be used in combination with
other treatment modalities, such as, for example antiinflammatory
therapies, and the like. Examples of antiinflammatory therapies
that can be used in combination with the methods of the invention
include, for example, therapies that employ steroidal drugs, as
well as therapies that employ non-steroidal drugs.
Pharmaceutical Compositions and Therapies
[0142] Administration of an anti-properdin antibody in a method of
treatment of the invention can be achieved in a number of different
ways, using methods known in the art. The therapeutic and
prophylactic methods of the invention thus encompass the use of
pharmaceutical compositions comprising an anti-properdin antibody.
The pharmaceutical compositions useful for practicing the invention
may be administered to deliver a dose of between 1 ng/kg/day and
100 mg/kg/day. In one embodiment, the invention envisions
administration of a dose which results in a concentration of the
anti-properdin antibody of the present invention between 1 .mu.M
and 10 .mu.M in an individual. In another embodiment, the invention
envisions administration of a dose which results in a concentration
of the anti-properdin antibody of the present invention between 1
.mu.M and 10 .mu.M in the plasma of an individual.
[0143] Typically, dosages which may be administered in a method of
the invention to an animal, preferably a human, range in amount
from 0.5 .mu.g to about 50 mg per kilogram of body weight of the
animal. While the precise dosage administered will vary depending
upon any number of factors, including but not limited to, the type
of animal and type of disease state being treated, the age of the
animal and the route of administration. Preferably, the dosage of
the compound will vary from about 1 .mu.g to about 10 mg per
kilogram of body weight of the animal. More preferably, the dosage
will vary from about 3 .mu.g to about 1 mg per kilogram of body
weight of the animal.
[0144] The compound may be administered to an animal as frequently
as several times daily, or it may be administered less frequently,
such as once a day, once a week, once every two weeks, once a month
or even less frequently, such as once every several months or even
once a year or less. The frequency of the dose will be readily
apparent to the skilled artisan and will depend upon any number of
factors, such as, but not limited to, the type and severity of the
disease being treated, the type and age of the animal, etc. The
formulations of the pharmaceutical compositions described herein
may be prepared by any method known or hereafter developed in the
art of pharmacology. In general, such preparatory methods include
the step of bringing the active ingredient into association with a
carrier or one or more other accessory ingredients, and then, if
necessary or desirable, shaping or packaging the product into a
desired single- or multi-dose unit.
[0145] Although the description of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation. Individuals to which administration of the
pharmaceutical compositions of the invention is contemplated
include, but are not limited to, humans and other primates, mammals
including commercially relevant mammals such as non-human primates,
cattle, pigs, horses, sheep, cats, and dogs.
[0146] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for ophthalmic, oral, rectal, vaginal, parenteral,
topical, pulmonary, intranasal, buccal, and routes of
administration. Other contemplated formulations include projected
nanoparticles, liposomal preparations, resealed erythrocytes
containing the active ingredient, and immunologically-based
formulations.
[0147] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. A unit dose is discrete amount of
the pharmaceutical composition comprising a predetermined amount of
the active ingredient. The amount of the active ingredient is
generally equal to the dosage of the active ingredient which would
be administered to an individual or a convenient fraction of such a
dosage such as, for example, one-half or one-third of such a
dosage.
[0148] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the individual
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0149] In addition to the active ingredient, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents.
[0150] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0151] Parenteral administration of a pharmaceutical composition
includes any route of administration characterized by physical
breaching of a tissue of an individual and administration of the
pharmaceutical composition through the breach in the tissue.
Parenteral administration thus includes, but is not limited to,
administration of a pharmaceutical composition by injection of the
composition, by application of the composition through a surgical
incision, by application of the composition through a
tissue-penetrating non-surgical wound, and the like. In particular,
parenteral administration is contemplated to include, but is not
limited to, intravenous, intraocular, intravitreal, subcutaneous,
intraperitoneal, intramuscular, intrasternal injection, and
intratumoral.
[0152] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e. powder or granular) form for reconstitution
with a suitable vehicle (e.g. sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0153] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0154] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient and
which have a diameter in the range from about 0.5 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant may be directed to disperse the powder
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a sealed container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by member have a diameter less than 7 nanometers.
More preferably, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[0155] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0156] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous or dilute alcoholic
solutions or suspensions, optionally sterile, comprising the active
ingredient, and may conveniently be administered using any
nebulization or atomization device. Such formulations may further
comprise one or more additional ingredients including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering agent, a surface active agent, or a preservative
such as methylhydroxybenzoate. The droplets provided by this route
of administration preferably have an average diameter in the range
from about 0.1 to about 200 nanometers.
[0157] The formulations described herein as being useful for
pulmonary delivery are also useful for intranasal delivery of a
pharmaceutical composition of the invention.
[0158] Another formulation suitable for intranasal administration
is a coarse powder comprising the active ingredient and having an
average particle from about 0.2 to 500 micrometers. Such a
formulation is administered in the manner in which snuff is taken
i.e. by rapid inhalation through the nasal passage from a container
of the powder held close to the nares.
[0159] Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1% (w/w) and as much as
100% (w/w) of the active ingredient, and may further comprise one
or more of the additional ingredients described herein.
[0160] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for buccal
administration. Such formulations may, for example, be in the form
of tablets or lozenges made using conventional methods, and may,
for example, 0.1 to 20% (w/w) active ingredient, the balance
comprising an orally dissolvable or degradable composition and,
optionally, one or more of the additional ingredients described
herein. Alternately, formulations suitable for buccal
administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such
powdered, aerosolized, or aerosolized formulations, when dispersed,
preferably have an average particle or droplet size in the range
from about 0.1 to about 200 nanometers, and may further comprise
one or more of the additional ingredients described herein.
[0161] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Remington's
Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co.,
Easton, Pa.), which is incorporated herein by reference.
Human Properdin Mouse
[0162] The invention also includes a transgenic mouse that
expresses human properdin and does not express mouse properdin. To
create a transgenic mouse, a nucleic acid encoding the human
properdin protein can be incorporated into a recombinant expression
vector in a form suitable for expression of the human properdin
protein in a host cell. The term "in a form suitable for expression
of the fusion protein in a host cell" is intended to mean that the
recombinant expression vector includes one or more regulatory
sequences operatively linked to the nucleic acid encoding the human
properdin protein in a manner which allows for transcription of the
nucleic acid into mRNA and translation of the mRNA into the human
properdin protein. The term "regulatory sequence" is art-recognized
and intended to include promoters, enhancers and other expression
control elements (e.g., polyadenylation signals). Such regulatory
sequences are known to those skilled in the art and are described
in 1990, Goeddel, Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. It should be understood that
the design of the expression vector may depend on such factors as
the choice of the host cell to be transfected and/or the amount of
human properdin protein to be expressed.
[0163] A transgenic mouse can be created, for example, by
introducing a nucleic acid encoding the human properdin protein
(typically linked to appropriate regulatory elements, such as a
constitutive or tissue-specific enhancer) into oocyte, e.g., by
microinjection, and allowing the oocyte to develop in a female
foster mouse. Intronic sequences and polyadenylation signals can
also be included in the transgene to increase the efficiency of
expression of the transgene. Methods for generating transgenic
animals, particularly animals such as mice, have become
conventional in the art and ate described, for example, in U.S.
Pat. Nos. 4,736,866 and 4,870,009 and 1986, Hogan et al., A
Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring Harbor
Laboratory. A transgenic founder mouse can be used to breed
additional animals carrying the transgene. Transgenic mice carrying
a transgene encoding the properdin protein of the invention can
further be bred to other transgenic mice carrying other transgenes,
or to other knockout mice, e.g., a knockout mouse that does not
express the murine properdin gene, such as those described in U.S.
Pat. App. No. 2010/0263061. It will be understood that in addition
to transgenic mice, the system described herein can be used to
generate other human properdin expressing animals.
[0164] In one embodiment, the transgenic mouse of the invention
expresses human properdin from a chicken .beta.-actin promoter with
CVM-IE enhancer, but the skilled artisan will understand that the
transgenic mouse of the invention encompasses the expression of
human properdin from other promoters and enhancers. Examples of
promoters useful in the invention include, but are not limited to,
DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin
promoter, globin promoter, ovalbumin promoter, SV40 early promoter,
the Rous sarcoma virus (RSV) promoter, retroviral LTR and
lentiviral LTR. Promoter and enhancer expression systems useful in
the invention also include inducible and/or tissue-specific
expression systems.
[0165] In some embodiments, the human properdin that was inserted
into the mouse genome comprises the nucleic acid sequence of SEQ ID
NO:67 and the amino acid sequence of SEQ ID NO:54.
Kits
[0166] The invention also includes a kit comprising an
anti-properdin antibody, or combinations thereof, of the invention
and an instructional material which describes, for instance,
administering the anti-properdin antibody, or a combinations
thereof, to an individual as a therapeutic treatment or a
non-treatment use as described elsewhere herein. In an embodiment,
this kit further comprises a (preferably sterile) pharmaceutically
acceptable carrier suitable for dissolving or suspending the
therapeutic composition, comprising an anti-properdin antibody, or
combinations thereof, of the invention, for instance, prior to
administering the antibody to an individual. Optionally, the kit
comprises an applicator for administering the antibody.
EXPERIMENTAL EXAMPLES
[0167] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these Examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein.
[0168] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
Example 1
[0169] Anti-human properdin monoclonal antibodies were generated
using the hybridoma method first described by Kohler et al. (1975,
Nature, 256:495) with some modifications. Properdin knockout mice
(fP.sup.-/-) (8 weeks old) were intraperitoneally immunized with 50
.mu.g (in 100 .mu.l PBS) purified human properdin (CompTech Inc)
emulsified with 100 .mu.l Titermax adjuvant (From Sigma). At day 14
and day 21, the mice were again immunized with 50 .mu.g purified
human properdin emulsified with Titermax adjuvant. One week later,
the mice were examined for serum anti-properdin titer. Mice with an
antibody titer of 1:10,000 or higher were used for hybridoma fusion
experiments. Two days prior to fusion experiment, mice were
injected (i.p) again with 50 .mu.g purified human properdin (in 100
.mu.l PBS). Mice were sacrificed by cervical dislocation and spleen
was isolated for preparation of single cell suspension by
mechanical disruption. The spleen cell suspension was washed once
with HYB-SFM (Invitrogen)+10% FBS medium and cells were counted,
and mixed with X63-Ag8.653 myeloma cells (ATCC) in a 2:1 ratio.
Cell mixture was again washed with HYB-SFM medium, and the cell
pellet was prepared by centrifugation (1000 rpm.times.5 min). The
cell pellet was gently disturbed and loosened and then cell fusion
was induced by slowly adding poly ethylene glycol (PEG 1500) (1.5
ml PEG for 3.times.10.sup.8 cells). The cells were left for 1 min
at 37.degree. C. and then 20 ml HYB-SFM medium were added to the
cells in 3 min (1 ml for the first min, 3 mls for the second min
and 16 mls for the third min). The mixture was centrifuged at 1000
rpm for 5 min and the cells were plated in 24 well plates in HAT
medium (10 ml HAT [Sigma H0262], 5 ml Pen/Strep, 500 .mu.l
Gentamicin and 10% FBS in 500 ml HYB-SFM medium). After 2 weeks,
supernatants from wells with visible colonies were withdrawn for
screening of reactivity with purified human properdin by ELISA.
Positive clones were picked up and plated in 96 well plates by
limiting dilution method to obtain single clones after second round
screening by ELISA. Positive clones were expanded in HT-medium (10
ml HT, 5 ml Pen/Strep 500 .mu.l Gentamicin and 10% FBS in 500 ml
HYB-SFM medium). Before antibody collection, the hybridoma cells
were switched to serum-free medium (HYB-SFM) for 2-3 days. Cell
culture medium was collected for mAb purification by protein G
affinity chromatography.
[0170] To clone the cDNAs of the anti-properdin mAbs, total RNAs
were isolated from the hybridoma cells by TRizol reagent (Sigma).
First-strand cDNAs were synthesized by reverse transcription using
Oligo(dT) primer. To amplify the heavy chain cDNAs (for IgG1,
IgG2a/b), the following primers were used in PCR reactions:
5'-GAGGTGAAGCTGGTGGAG(T/A)C(T/A)GG-3' (SEQ ID NO:68) and
5'-GGGGCCAGTGGATAGAC-3' (SEQ ID NO:69). To amplify the k light
chain, the following primers were used: mixture of 4 upstream
primers: 5'-CCAGTTCCGAGCTCCAGATGACCCAGACTCCA-3' (SEQ ID NO:70);
5'-CCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA-3' (SEQ ID NO:71);
5'-CCAGTTCCGAGCTCCAGATGACCCAGTCTCCA-3' (SEQ ID NO:72);
5'-CCAGTTCCGAGCTCGTGATGACACAGTCTCCA-3' (SEQ ID NO:73); downstream
primer: 5'-GTTGGTGCAGCATCAGC-3' (SEQ ID NO:74). The PCR amplicons
were cloned into pCR TOPO TA 2.1 vector (Invitrogen) and sequenced.
To obtain the signal peptide (leader) sequence of the mAbs, the
5'-RACE method was used with a kit (GeneRacer) from Invitrogen. The
complete variable region cDNAs were amplified using specific
primers determined from the 5'-RACE and the initial sequencing
data.
Example 2
[0171] The dose-dependent inhibition of LPS-induced AP complement
activation by mAb 19.1, 22.1, 25 and 30 was examined. All 4 clones
of mAbs effectively inhibited AP complement activation when added
to 50% normal human serum (NHS) at a final concentration of 5
.mu.g/ml (see FIG. 2). ELISA plates (96-well, Nunc) were coated
with 50 LPS solution (40 .mu.g/ml in phosphate-buffered saline
[PBS]) overnight at 4.degree. C. The next day, plates were washed
3.times. with PBS containing 0.05% Tween-20 (PBS-T) and 50 .mu.l
50% normal human serum (NHS) that had been incubated with 1-5
.mu.g/ml anti-properdin mAb for 1 hour at 4.degree. C. was added.
The NHS was diluted with GVB-EGTA-Mg++ (containing 10 mM EGTA and
2.5 mM Mg++ final concentration). The plate was left to incubate at
37.degree. C. for 1 hour, washed 3.times. with PBS-T and then 50
.mu.l HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel)
was added and the plate was left For 1 hour room temperature. The
plate was washed 3.times. with PBS-T and then developed using BD
Pharmingen A+B reagent. The reaction was stopped after 5 min with 2
N H2SO4. AP complement activation was detected by measuring the
amount of C3 deposition on the plate (OD450). A sample with EDTA
added (NHSEDTA) served as a negative control (EDTA blocks
complement activation). A sample with no mAb added (0 Ab) served as
the baseline AP complement activation.
Example 3
[0172] Experiments demonstrating that anti-human properdin mAbs
inhibit human red blood cell (RBC) lysis caused by fH and DAF
dysfunction were conducted (see FIG. 3). Normal human RBCs
(5.times.10.sup.6 cells) were incubated at 37 C for 20 min with 100
.mu.l 50% NHS (diluted with GVB-EGTA-Mg++ containing 10 mM EGTA and
2.5 mM Mg++ final concentration) in the presence of 30 .mu.M
recombinant fH 19-20 and 7.5 .mu.g mouse anti-human DAF (from AbD
Serotec) (2006, Ferreira et al., J Immunol. 177:6308-6316). Lysis
reaction was stopped by addition of 200 .mu.l ice-cold 20 mM EDTA
in PBS. The incubation mixtures were centrifuged for 5 min at 1500
g and the supernatant was collected and measured for OD420 nm.
Before addition to RBCs, NHS was pre-incubated with 0 or 5 .mu.g/ml
anti-properdin antibodies for 1 hour at 4 C. Samples without NHS or
fH19-20 added or with EDTA added were used as negative lysis
controls, and a sample of RBCs lysed completely with 100 .mu.l
distilled water was used as a positive control (100% lysis) against
which % lysis in other samples was normalized.
Example 4
[0173] Experiments evaluating antibody-sensitized sheep RBCs
incubated with 50% normal human serum (NHS) in the absence or
presence of 5.mu.g/ml of anti-properdin mAbs (see FIG. 4).
Antibody-sensitized sheep RBCs (5.times.10.sup.6 cells, from
CompTech Inc) were incubated at 37.degree. C. for 20 min with 100
.mu.l 50% NHS (diluted with GVB++ buffer). Before addition to the
sheep RBCs, NHS was pre-incubated with 0 or 5 .mu.g/ml
anti-properdin antibody for 1 hour at 4 C. Lysis reaction was
stopped by addition of 200 .mu.l ice-cold 20 mM EDTA in PBS. The
incubation mixtures were centrifuged for 5 min at 1500 g and the
supernatant was collected and measured for OD420 nm. Samples
without NHS or with EDTA added were used as negative lysis
controls, and a sample of sheep RBCs lysed completely with 100
.mu.l distilled water was used as a positive control (100% lysis)
against which % lysis in other samples was normalized.
Example 5
[0174] Generation of deletion mutants of human properdin and
confirmation by Western blot of their expression in CHO cells (see
FIG. 5). Human properdin (fP) is composed of 7 thrombospondin
repeat (TSR) domains which are numbered 0 to 6. Individual TSR
domains 0 to 5 (see SEQ ID NO:55, 56, 57, 58, 59 and 60) were
deleted by inverse PCR (1989, Hemsley et al., Nucleic Acid. 5 Res.
17:6545) using full-length human properdin cDNA (SEQ ID NO:67) in
pCMV vector (from Origene) as a template. To delete TSR 6 (SEQ ID
NO:61) or TSR 5-6, normal PCR methods was used, followed by cloning
into an expression vector (the pCAGGS vector was used). The
deletion mutants were transfected into CHO cells using
Lipofectamine reagent (Invitrogen) in 6 well plates in Optimem
medium. After 48 hours, the cells were lysed with 50 mM Tris-HCL,
ph 7.4 containing 150 mM NaCl, 10% glycerol, 1 mM EDTA and a
protease inhibitor cocktail (Roche) and 1% Triton X-100 (250 .mu.l
per well). The lysate was centrifuged 10,000 rpm for 10 min and
protein concentration was determined by BCA protein assay method.
About 100 .mu.g total protein from each sample was used for
SDS-PAGE analysis.
Example 6
[0175] Sandwich ELISA assays of mAb 19.1 and 25 binding to human
properdin deletion mutants were performed for epitope mapping of
mAb 19.1 and 25. (see FIG. 6). ELISA plates were coated with 50
.mu.l of 2 .mu.g/ml of the concerned mAb overnight at 4.degree. C.
The plates were washed 3.times. with PBS-T and then 25 .mu.g (in 50
.mu.l PBS containing 1% BSA) CHO cell lysate proteins were added to
the wells and the plates were incubated for 1 hour at room
temperature. The plates were washed 3.times. with PBS-T and then
captured protein was detected by biotinylated goat anti-human
properdin antibody and HRP-avidin system. A third mAb 29.3 which
binds to a different epitope from 19.1 and 25 was used as a control
to confirm mutant protein expression.
Example 7
[0176] Epitope mapping showed the epitope of mAb 19.1 mapped to the
C-terminal half of TSR5 with the following amino acid sequence:
RGRTCRGRKFDGHRCAGQQQDIRHCYSIQHCP (SEQ ID NO:52) (see FIG. 7). Three
human properdin mutants comprising TSR0-4+1/4 TSR5, TSR0-4+1/2 TSR5
or TSR0-4+3/4 TSR5 were generated by conventional PCR. They were
cloned into pCAGGS and expressed in CHO cells as described in
Example 5. Protein expression was confirmed by Western analysis
using goat anti-human propend in antibody. The blot was stripped
and reprobed with a mouse anti-His tag antibody (Qiagen) to confirm
that the C-terminal His Tag is present (no C-terminal proteolysis).
Sandwich ELISA assays to determine reactivity with mAb 19.1 were
performed as described in Example 6. The mAb 29.3 which binds to a
different epitope from 19.1 was used as a control to confirm mutant
protein expression.
Example 8
[0177] Epitope mapping showed the epitope of mAb 25 mapped to the
C-terminal quarter segment of TSR6 with the following amino acid
sequence:
LVVEEKRPCLHVPACKDPEEEEL (SEQ ID NO:53) (see FIG. 8). Three human
properdin mutants comprising TSR0-5+1/4 TSR6, TSR0-5+1/2 TSR6 or
TSR0-5+3/4 TSR6 were generated by conventional PCR. They were
cloned into pCAGGS and expressed in CHO cells as described in
Example 5. Protein expression was confirmed by Western analysis
using goat anti-human properdin antibody. The blot was stripped and
reprobed with a mouse anti-His tag antibody (Qiagen) to confirm
that the C-terminal His Tag is present (no C-terminal proteolysis).
Sandwich ELISA assays to determine reactivity with mAb 25 were
performed as described in Example 6. mAb 19.1 which binds to a
different epitope from 25 was used as a control to confirm mutant
protein expression. Epitope mapping also showed that the epitope of
mAb 25 is dependent on two cysteine residues in TSR6 (SEQ ID NO:
61, shown in FIG. 5B). These are cysteine 62 (C62) and cysteine 78
(C78) of TSR6. Single mutation to Alanine (A) of either C62 or C78
in fill-length human properdin did not abolish mAb 25 binding, but
double mutations of C62A and C78A abolished mAb 25 binding. As a
positive control for mutant protein expression, mAb 19.1 showed
reactivity to all samples. This result suggests that C78 within the
last quarter segment of TSR6 (with the sequence designated by SEQ
ID NO: 53), as well as C62 which is located outside SEQ ID NO:53
but within TSR6 (SEQ ID: 61), constitute two critical residues of
the epitope of mAb 25. Binding assays of mAbs 19.1 and 25 was
performed on ELISA plates using homogenates of transfected CHO
cells. HuP refers to full-length (intact) human fP transfected CHO
cells as a positive control; Con refers to untransfected CHO cells
as negative controls for binding. The other samples are CHO cells
transfected with mutant human fP cDNA containing single or double
C62A and C78A mutations.
Example 9
[0178] Experiments assessing the expression of recombinant chimeric
and humanized 19.1 mAbs in CHO cells were conducted (see FIG. 17).
Chimeric 19.1 heavy chain cDNA was constructed by cloning the
variable region of mAb 19.1 (SEQ ID NO:1) into the pFUSE-CHIg-hG4
vector (from InvivoGen, containing the human IgG4 heavy chain
constant region, with Serine 229 mutated to Proline) using
EcoRI/NheI sites. Chimeric 19.1 light chain cDNA was constructed by
cloning the variable region of mAb 19.1 (SEQ ID NO:6) into the
pFUSE2-CLIg-hk vector (from InvivoGen, containing the human k light
chain constant region) using AgeI/BsiWI sites. Humanized 19.1 heavy
chain cDNAs were constructed by cloning the humanized heavy chain
variable region of 19.1 (cDNAs encoding SEQ ID NO:42 and SEQ ID
NO:44, synthesized by Genescript) into the pFUSE-CHIg-hG4 vector
(from InvivoGen, containing the human IgG4 heavy chain constant
region, with Serine 229 mutated to Proline) using EcoRI/NheI sites.
Humanized 19.1 light chain cDNA was constructed by cloning the
humanized light chain variable region of 19.1 (cDNA encoding SEQ ID
NO:47, synthesized by Genescript) into the pFUSE2-CLIg-hk vector
(from InvivoGen, containing the human k light chain constant
region) using AgeI/BsiWI sites, CHO cells were co-transfected with
chimeric heavy and light chains of 19.1 or humanized heavy and
light chains of 19.1 (the two humanized heavy chains were paired
with the same humanized light chain) using Lipofectamine reagent.
After transfection, CHO cells were selected with Geocine (1 mg/ml)
and Blastcidine (10 .mu.g/ml) for approximately 7 days.
Drug-resistant cell colonies were picked up, trypsinized and
subjected to limited dilution culture in 96-well plates in the
presence of the same selection drugs. After cells became confluent
in the 96-well plates, the medium was tested for reactivity with
human properdin by ELISA and positive clones were expanded. For
antibody production, stable lines of transfected CHO cells were
grown in DMEM: F12 medium with 10% FRS in 150 cm culture flasks and
after reaching confluence, they were switched to serum free CD-CHO
medium (Invitrogen). After 3 days, the medium was collected and
mAbs were purified by protein G chromatography. Aliquots of the
purified mAbs were analyzed by SDS-PAGE.
Example 10
[0179] Experiments measuring the antigen binding affinities of mAb
19.1, chimeric mAb 19.1, humanized mAb 19.1, mAb 25, mAb 22.1 and
mAb 30 were conducted (see FIGS. 18 and 19). Surface Plasmon
resonance analysis was used to measure the association and
dissociation rate constant for binding of anti-human properdin mAb
to immobilized human properdin using BIAcore 2000 instrument
(Biacore AB, Uppsala, Sweden). Biacore experiments were performed
at 25.degree. C. The carboxylated dextran matrix of a CM4 sensor
chip was used to couple the purified human properdin by amine
coupling chemistry to obtain 200RU surface density, mAbs were
diluted to 150, 75, 35.5, 17.75, 8.87 and 0 nM in HBSET (HEPES
buffered saline with EDTA and Tween 20) buffer and the samples were
injected onto the properdin surface at 30 .mu.l/min (60 .mu.l
injection) for 120 s and dissociation of bound analyte was allowed
to proceed for 900 s. The data were analyzed by the BIA evaluation
software 3.2 assuming bivalent binding model. Regeneration of the
surface was achieved with a 50 .mu.l injection (50 .mu.l/min) of 50
mM NaOH.
Example 11
[0180] Experiments were conducted to assess the relative activities
of 19.1, chimeric 19.1 and two humanized 19.1 mAbs in blocking
LPS-induced human AP complement activation (see FIG. 20). ELISA
plates (96-well, Nunc) were coated with 50 .mu.l LPS solution (40
.mu.g/ml in PBS overnight at 4.degree. C.). The next day, plates
were washed 3.times. with PBS containing 0.05% PBS-T and 50 .mu.l
50% NHS that had been incubated with 0, 5, 10 or 20 .mu.g/ml
anti-properdin mAb for 1 hour at 4.degree. C. was added. The NHS
was diluted with GVB-EGTA-Mg++ (containing 10 mM EGTA and 2.5 mM
Mg++, final concentration). The plate was left to incubate at
37.degree. C. for 1 hour, washed 3.times. with PBS-T and then 50
.mu.l HRP-conjugated goat anti-human C3 antibody (1:4000, Cappel)
was added and the plate was left for 1 hour room temperature. The
plate was washed 3.times. with PBS-T and then developed using BD
Pharmingen A+B reagent. The reaction was stopped after 5 min with 2
N H2SO4. AP complement activation was detected by measuring the
amount of C3 deposition on the plate (OD450). A sample with EDTA
added (NHSEDTA) served as a negative control (EDTA blocks
complement activation). A sample with no mAb added (NHS) served as
the baseline AP complement activation.
Example 12
[0181] Experiments were conducted to assess the relative activities
of 19.1, chimeric 19.1 and two humanized 19.1 mAbs in blocking
human RBC lysis by human AP complement in the context of fH and DAF
dysfunction (see FIG. 21). Normal human RBCs (5.times.10.sup.6
cells) were incubated at 37.degree. C. for 20 min with 100 .mu.l
50% NHS (diluted with GVB-EGTA-Mg++ containing 10 mM EGTA and 2.5
mM Mg-HE final concentration) in the presence of 30 .mu.M
recombinant fH 19-20 and 7.5 .mu.g mouse anti-human DAF (from ADB
Serotec) (2006, Ferreira et al., J Immunol. 177:6308-6316). Before
addition to RBCs, NHS was pre-incubated with 1-15 .mu.g/ml various
anti-properdin mAbs for 1 hour at 4.degree. C. Lysis reaction was
stopped by addition of 200 .mu.l ice-cold 20 mM EDTA in PBS. The
incubation mixtures were centrifuged for 5 min at 1500 g and the
supernatant was collected and measured for OD420 nm. Samples
without NHS or fH19-20 added or with EDTA added were used as
negative lysis controls, and a sample of RBCs lysed completely with
100 .mu.l distilled water was used as a positive control (100%
lysis) against which % lysis in other samples was normalized.
Example 13
[0182] Experiments were conducted to assess the relative activities
of 19.1, chimeric 19.1 and two humanized 19.1 mAbs in blocking
LPS-induced Rhesus monkey and Cynomolgus monkey AP complement
activation (see FIGS. 22 and 23). ELISA plates (96-well, Nunc) were
coated with 50 .mu.l LPS solution (40 .mu.g/ml in PBS overnight at
4.degree. C.). The next day, plates were washed 3.times. with PBS
containing 0.05% PBS-T and 50 .mu.l 50% normal Rhesus monkey serum
(NRS) or normal Cynomolgus monkey serum (NCS) that had been
pre-incubated with 0, 10, 20, 30 or 40 .mu.g/ml anti-properdin mAb
for 1 hour at 4.degree. C. was added. The NRS or NCS was diluted
with GVB-EGTA-Mg++ (containing 10 mM EGTA and 2.5 mM Mg++, final
concentration). The plate was left to incubate at 37.degree. C. for
1 hour, washed 3.times. with PBS-T and then 50 .mu.l HRP-conjugated
goat anti-human C3 antibody (1:4000, Cappel, cross react with
monkey C3) was added and the plate was left for 1 hour room
temperature. The plate was washed 3.times. with PBS-T and then
developed using BD Pharmingen A+B reagent. The reaction was stopped
after 5 min with 2 N H2SO4. AP complement activation was detected
by measuring the amount of C3 deposition on the plate (OD450). A
sample with EDTA added (NRSEDTA or NCSEDTA) served as a negative
control (EDTA blocks complement activation). A sample with no mAb
added (NRS or NCS) served as the baseline AP complement
activation.
Example 14
[0183] Experiments were conducted to assess the inhibition of
acidified serum lysis of PNH red blood cells (Ham's test) by mAb
19.1, 25 and humanized 19.1-459 (see FIG. 24). RBCs from paroxysmal
nocturnal hemoglobinuria (PNH) patients were subjected to Ham's
acidified serum test in the presence or absence of mAbs. RBCs were
incubated with autologous serum (final concentration 83%) at
37.degree. C. for 2 hrs and percent lysis was calculated by
measuring the OD405 of the supernatant, normalized to a sample of
RBCs completely lysed by distilled water (Eh DDW). The incubation
mixture was composed of the following: 240 .mu.l of serum, 25 .mu.l
of 1/6 N HCL (or 25 .mu.l saline for negative controls), 12.5 .mu.l
of 50% (v/v) RBC suspension, 10 .mu.l mAb in saline. A sample of
RBCs incubated with nonacidified autologous serum (NHS) was used as
a negative control (background lysis). In the absence of mAbs,
about 50% of RBCs were lysed by acidified serum. This lysis was
completely inhibited by mAb 19.1 at 8 .mu.g/ml and above
concentration, by a humanized 19.1 mAb (#459) at the concentration
of 20 .mu.g/ml and by mAb 25 at the concentration of 8 .mu.g/ml and
above.
Example 15
[0184] A properdin humanized mouse was generated as follows (see
FIG. 25). A human fP expression vector was constructed in pACGGS
plasmid as illustrated in the schematic in FIG. 25A, using the
chicken .beta.-actin promoter with CVM-IE enhancer and the rabbit
.beta.-globin polyA tail for stable expression of the cDNA in
eukaryotic cells. The human properdin cDNA sequence and its encoded
protein sequence used for constructing the expression vector are
shown in SEQ ID NO:67 and SEQ ID NO:68. The plasmid was linearized
by restriction enzyme digestion and microinjected into the zygotes
of C57BL/6 mice to produce human fP transgenic founder mice. By PCR
screening (using primers specific to human fP
5'-ATCAGAGGCCTGTGACACC-3' (SEQ ID NO:65) and 5'-CTG
CCCTTGTAGCTCCTCA-3' (SEQ ID NO:66) and genomic DNAs isolated from
mouse tails), positive founder mice (showing a human fP cDNA
fragment of about 800 bp) can be identified. Of 40 mice analyzed,
five (#15, 20, 24, 27 and 32) were positive (FIG. 25B, red arrows).
Sandwich ELISA assays were performed to detect human fP in the
transgenic positive mice (FIG. 25C). Plate was coated with a
non-blocking mAb against human fP (clone 8.1). After incubation
with diluted mouse serum (10%), human fP was detected by using an
HRP-conjugated goat anti-human IP antibody. Normal human serum
(NHS) was used as a positive control. By this method, human fP
should be detected in NHS and in the sera of transgene positive
mice (e.g. 15, 20, 24, 27, 32) but not in normal (i.e.
non-transgenic, e.g. 29) mouse or fP.sup.-/--mouse serum.
Transgenic positive founder mice were then bred with WT mice to
establish germ line transmission. The screening of F1 mice from
such mating was accomplished by PCR of tail DNA to detect the
transgene and by sandwich ELISA to detect human properdin in their
sera as described above. After confirming germline transmission,
founder mice were bred with fP.sup.-/- mice to generate fP.sup.-/-
human fP transgene+mice. Restoration of AP complement activity in
fP.sup.-/--human fP transgene+mice was assessed by LPS-induced AP
activation assays as described in Example 2. In this assay, AP
complement activity should be detected in WT mouse serum and in the
serum of fP.sup.-/- mice that are human fP transgene positive but
not in the serum of fP.sup.-/- mice. In this assay, WT mouse serum
treated with EDTA will be used as a negative control for AP
complement activation.
Example 16
[0185] Experiments were conducted to examine the in vivo activity
and kinetics of mAb 25 in "properdin humanized" mice (FIG. 26). A
properdin humanized mouse (fP.sup.-/--human human fP transgene+)
was injected with 0.5 mg (i.p.) of mAb 25. Blood samples (50-75
.mu.l) were collected before injection (0 hr) and then at various
time points after injection by retro-orbital bleeding and sera
prepared. Serum samples were tested for LPS-induced AP complement
activation. For this assay, ELISA plates (96-well, Nunc) were
coated with 50 .mu.l LPS solution (40 .mu./ml in PBS overnight at
4.degree. C.). The next day, plates were washed 3.times. with PBS
containing 0.05% Tween-20 (PBS-T) and 50 .mu.l serially diluted
(starting from 1:10) mouse serum was added to each well. The mouse
serum was diluted with GVB-EGTA-Mg++ (containing 10 mM EGTA and 2.5
mM Mg++ final concentration). The plate was left to incubate at
37.degree. C. for 1 hour, washed 3.times. with PBS-T and then 50
.mu.l HRP-conjugated rabbit anti-mouse C3 antibody (1:2000, Cappel)
was added and the plate was left for 1 hour room temperature. The
plate was washed 3.times. with PBS-T and then developed using BD
Pharmingen A+B reagent. The reaction was stopped after 5 min with 2
N H2SO4. AP complement activation was detected by measuring the
amount of C3 deposition on the plate (OD450). In this illustrated
example, no AP complement activity was present in fP.sup.-/- mouse
serum or in WT serum treated with EDTA. In contrast, AP complement
activity was detected in WT serum and in the serum of fP humanized
mouse at time 0 hr (before mAb 25 treatment). AP complement
activity in the humanized mouse remained suppressed at 8, 24 and 48
hrs after mAb 25 treatment but became detectible at 72, 96 and 120
hrs. These results demonstrate that at a dosage of 0.5 mg/mouse,
mAb 25 was able to inhibit AP complement activity in vivo for at
least 48 hrs.
Example 17
[0186] Experiments were conducted to assess the effect of
anti-human properdin mAb 19.1 on extravascular hemolysis (EVH). In
this EVH model, properdin humanized mice (n=4 per experimental
group) were transfused with red blood cells (RBC) from Crry/DAF/C3
triple knockout (TKO) mice. Recipient mice (properdin humanized
mice) were treated 6 hrs before RBC transfer with mAb 19.1 (2
mg/mouse, i.p.) or a control mouse IgG1 mAb (MOPC, purified from
MoPC 31C hybridoma, from ACTT). RBCs were harvested from donor TKO
mice, washed in PBS and labeled with CFSE before injection (through
tail vein) into recipient mice, according to previously published
procedure (Miwa et al., 2002, Blood 99: 3707-3716). Each recipient
mouse received RBCs equivalent to 100 .mu.l of blood. At 5 minutes
and 6, 24, 48, 72, 96, 120 hours after RBC transfusion, recipient
mice were bled and RBCs were analyzed to determine the number of
CFSE-labeled (i.e. transfused) RBCs remaining in the circulation.
Number of CFSE-labeled RBCs in each recipient was normalized (as %)
to that detected at the 5 min time point. In control IgG
(MOPC)-treated recipient mice, TKO RBCs were rapidly eliminated
through EVH, consistent with previous findings (Miwa et al., 2002,
Blood 99: 3707-3716). However, in recipient mice treated with
anti-human properdin 19.1 mAb, no EVH occurred and the transfused
RBCs persisted, demonstrating that anti-properdin mAb was effective
in preventing EVH (FIG. 27).
[0187] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0188] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
671427DNAMus musculus 1gagcatggct gtcttggggc tgctcttctg cctggtgaca
ttcccaagct gtgtcctatc 60ccaggtgcag ctcaagcagt caggacctgg cctagtgcag
ccctcacaga gcctgtccat 120ctcctgcaca gtctctggtt tctcattaac
tacctatggt gttcactggg ttcgccagtc 180tccaggaaag ggtctggaat
ggctgggagt gatttggagt ggtggagaca cagactataa 240tgcatctttc
atatccagac tgcgcatcaa caaggacact tccaagagcc aagttttctt
300taaaatgaac agtctgcaag ctgatgacac agccatttat tactgtgcca
gaaataagga 360ctattatact aactacgact ttactatgga ctactggggt
caaggaacct cagtcaccgt 420ctcctca 4272140PRTMus musculus 2Met Ala
Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys 1 5 10 15
Val Leu Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln 20
25 30 Pro Ser Gln Ser Leu Ser Ile Ser Cys Thr Val Ser Gly Phe Ser
Leu 35 40 45 Thr Thr Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly
Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asp
Thr Asp Tyr Asn Ala 65 70 75 80 Ser Phe Ile Ser Arg Leu Arg Ile Asn
Lys Asp Thr Ser Lys Ser Gln 85 90 95 Val Phe Phe Lys Met Asn Ser
Leu Gln Ala Asp Asp Thr Ala Tyr Tyr 100 105 110 Cys Ala Arg Asn Lys
Asp Tyr Tyr Thr Asn Tyr Asp Phe Thr Met Asp 115 120 125 Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 310PRTMus musculus
3Gly Phe Ser Leu Thr Thr Tyr Gly Val His 1 5 10 416PRTMus musculus
4Val Ile Trp Ser Gly Gly Asp Thr Asp Tyr Asn Ala Ser Phe Ile Ser 1
5 10 15 514PRTMus musculus 5Asn Lys Asp Tyr Tyr Thr Asn Tyr Asp Phe
Thr Met Asp Tyr 1 5 10 6429DNAMus musculus 6ggcaggggga tcaagatgga
atcacagact caggtcttcc tctccctgct gctctgggta 60tctggtacct gtgggaacat
tatgatgaca cagtcgccat catctctggc tgtgtctgca 120ggagaaaagg
tcactatgag ctgtaagtcc agtcaaagtg ttttatacag ttcaaatcag
180aagaacttct tggcctggta ccagcagaaa ccaggacagt ctcctaaact
gctgatctac 240tgggcatcca ctagggaatc tggtgtccct gatcgcttca
caggcagtgg atctgggaca 300gattttattc ttacgatcaa cagtgtacaa
gttgaagacc aggcagttta ttactgtcac 360caatacctct cctcgtacac
gttcgggggg gggaccaagc tggaaataaa acgggctgat 420gctgcacca
4297132PRTMus musculus 7Met Glu Ser Gln Thr Gln Val Phe Leu Ser Leu
Leu Leu Trp Val Ser 1 5 10 15 Gly Thr Cys Gly Asn Ile Met Met Thr
Gln Ser Pro Ser Ser Leu Ala 20 25 30 Val Ser Ala Gly Glu Lys Val
Thr Met Ser Cys Lys Ser Ser Gln Ser 35 40 45 Val Leu Tyr Ser Ser
Asn Gln Lys Asn Phe Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90
95 Phe Ile Leu Thr Ile Asn Ser Val Gln Val Glu Asp Gln Ala Val Tyr
100 105 110 Tyr Cys His Gln Tyr Leu Ser Ser Tyr Thr Phe Gly Gly Gly
Thr Lys 115 120 125 Leu Glu Ile Lys 130 817PRTMus musculus 8Lys Ser
Ser Gln Ser Val Leu Tyr Ser Ser Asn Gln Lys Asn Phe Leu 1 5 10 15
Ala 97PRTMus musculus 9Trp Ala Ser Thr Arg Glu Ser 1 5 108PRTMus
musculus 10His Gln Tyr Leu Ser Ser Tyr Thr 1 5 11487DNAMus musculus
11aacagcatat gatcagtgtc ctctccaaag tccttgaaca tagactctaa ccatggactg
60gacctgggtc tttctcttcc tcctgtcagt aactgcaggt gtccactccc aggttcagct
120gctgcagtct ggagctgagg tgatgaagcc tggggcctca gtgacccttt
cctgcaaggc 180tattggttac acattcattg actactggat agagtggata
aagcagaggc ctggacatgg 240ccttgagtgg attggagaga tttttcctgg
aagtgggact attaatcaca atgagaagtt 300caaggacaag gccagtttta
gtgctcattc atcctccaac acagcctaca tgcaactcag 360cagactgaca
agtgaggact ctgccatcta ttactgtgca agagagggac tggactattg
420gggccaaggc accactctca cagtctcctc agccaaaacg acacccccat
ctgtctatcc 480actggcc 48712133PRTMus musculus 12Met Asp Trp Thr Trp
Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser
Gln Val Gln Leu Leu Gln Ser Gly Ala Glu Val Met Lys 20 25 30 Pro
Gly Ala Ser Val Thr Leu Ser Cys Lys Ala Ile Gly Tyr Thr Phe 35 40
45 Ile Asp Tyr Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu
50 55 60 Glu Trp Ile Gly Glu Ile Phe Pro Gly Ser Gly Thr Ile Asn
His Asn 65 70 75 80 Glu Lys Phe Lys Asp Lys Ala Ser Phe Ser Ala His
Ser Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Arg Leu Thr
Ser Glu Asp Ser Ala Ile 100 105 110 Tyr Tyr Cys Ala Arg Glu Gly Leu
Asp Tyr Trp Gly Gln Gly Thr Thr 115 120 125 Leu Thr Val Ser Ser 130
1310PRTMus musculus 13Gly Tyr Thr Phe Ile Asp Tyr Trp Ile Glu 1 5
10 1417PRTMus musculus 14Glu Ile Phe Pro Gly Ser Gly Thr Ile Asn
His Asn Glu Lys Phe Lys 1 5 10 15 Asp 155PRTMus musculus 15Glu Gly
Leu Asp Tyr 1 5 16420DNAMus musculus 16ccaaaattca aagacaaaat
ggattttcaa gtgcagattt tcagcttcct gctaatcagt 60gcctcagtca taatatccag
aggacaaatt gttctcaccc agtctccagc aatcatgtct 120gcatctccag
gggagagggt caccatgacc tgcagtgcca gctcaagtgt aagttatata
180tactggtacc agcagaagtc aggcacgtcc cccaaaagat ggatttttga
cacatccaca 240ctggcttctg gagtccctac tcgcttcagt ggcagtgggt
ctgggacctc ttactctctc 300acaatcagca gcatggagac tgaagatgct
gccacttatt actgccagca gtggagtaga 360aacccattca cgttcggttc
ggggacaaag ttggaaataa aacgggctga tgctgcacca 42017128PRTMus musculus
17Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1
5 10 15 Val Ile Ile Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Arg Val Thr Met Thr Cys
Ser Ala Ser 35 40 45 Ser Ser Val Ser Tyr Ile Tyr Trp Tyr Gln Gln
Lys Ser Gly Thr Ser 50 55 60 Pro Lys Arg Trp Ile Phe Asp Thr Ser
Thr Leu Ala Ser Gly Val Pro 65 70 75 80 Thr Arg Phe Ser Gly Ser Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile 85 90 95 Ser Ser Met Glu Thr
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 100 105 110 Ser Arg Asn
Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125
1810PRTMus musculus 18Ser Ala Ser Ser Ser Val Ser Tyr Ile Tyr 1 5
10 197PRTMus musculus 19Asp Thr Ser Thr Leu Ala Ser 1 5 209PRTMus
musculus 20Gln Gln Trp Ser Arg Asn Pro Phe Thr 1 5 21464DNAMus
musculus 21gagcatggct gtcttggggc tgctcttctg cctggtgaca ttcccaagct
gtgtcctatc 60ccaggtgcag ctgaagcagt caggacctgg cctagtgcag ccctcacaga
gcctgtccat 120cacctgcaca gtctctggtt tctcattaac tagctatggt
gtacactggg ttcgccagtc 180tccaggaaag ggtctggagt ggctgggagt
gatatggagt ggtggaagca cagactataa 240tgcagctttc atatccagac
tgagcatcag caaagacact tccaagagcc aagttttctt 300taaaatgaac
agtctgcaac ctgatgacac agccatatat tactgtgcca gaaataaaga
360cttctatagt aactacgact atactatgga actactgggg tcaaggaacc
tcagtcaccg 420tctcctcagc caaaacgaca cccccatctg tctatccact ggcc
46422141PRTMus musculus 22Met Ala Val Leu Gly Leu Leu Phe Cys Leu
Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Gln Val Gln Leu Lys
Gln Ser Gly Pro Gly Leu Val Gln 20 25 30 Pro Ser Gln Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45 Thr Ser Tyr Gly
Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu 50 55 60 Glu Trp
Leu Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80
Ala Phe Ile Ser Arg Leu Ser Ile Ser Lys Asp Thr Ser Lys Ser Gln 85
90 95 Val Phe Phe Lys Met Asn Ser Leu Gln Pro Asp Asp Thr Ala Ile
Tyr 100 105 110 Tyr Cys Ala Arg Asn Lys Asp Phe Tyr Ser Asn Tyr Asp
Tyr Thr Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 130 135 140 2310PRTMus musculus 23Gly Phe Ser Leu Thr Ser
Tyr Gly Val His 1 5 10 2416PRTMus musculus 24Val Ile Trp Ser Gly
Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser 1 5 10 15 2514PRTMus
musculus 25Asn Lys Asp Phe Tyr Ser Asn Tyr Asp Tyr Thr Met Asp Tyr
1 5 10 26414DNAMus musculus 26atggaatcac agactcaggt cttcctctcc
ctgctgctct gggtatctgg tacctgtggg 60aacattatga tgacacagtc gccatcattt
ttggctgtgt ctgcaggaga aaaggtcact 120ttgagctgta agtccagtca
aagtgtttta tacagttcaa atcagaagaa cttcttggcc 180tggtaccagc
agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg
240gaatctggtg tccctgatcg cttcacaggc agtggatctg ggacagattt
tactcttacc 300atcagcagtg tacaagctga agacctggca gtttattact
gtcatcaata cctctcctcg 360tacacgttcg gaggggggac caagctggaa
ataaaacggg ctgatgctgc acca 41427132PRTMus musculus 27Met Glu Ser
Gln Thr Gln Val Phe Leu Ser Leu Leu Leu Trp Val Ser 1 5 10 15 Gly
Thr Cys Gly Asn Ile Met Met Thr Gln Ser Pro Ser Phe Leu Ala 20 25
30 Val Ser Ala Gly Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser
35 40 45 Val Leu Tyr Ser Ser Asn Gln Lys Asn Phe Leu Ala Trp Tyr
Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Val
Gln Ala Glu Asp Leu Ala Val Tyr 100 105 110 Tyr Cys His Gln Tyr Leu
Ser Ser Tyr Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys
130 2817PRTMus musculus 28Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser
Asn Gln Lys Asn Phe Leu 1 5 10 15 Ala 297PRTMus musculus 29Trp Ala
Ser Thr Arg Glu Ser 1 5 308PRTMus musculus 30His Gln Tyr Leu Ser
Ser Tyr Thr 1 5 31451DNAMus musculus 31atggaattga tctgggtctt
tctcttcctc ctgtcagtaa ctgcaggtgt ccactctgag 60gtccagcttc agcagtctgg
agttgagctg gtgaggcctg ggtcctcagt gaagatgtcc 120tgcaagactt
ctggatatac attyacagcc tacggtataa actgggtgaa gcagaggcct
180ggacagggcc tggaatggat tggatatatt tatattggaa atggttatac
tgactacaat 240gagaagttca agggcaaggc cacactgact tcagacacat
cctccagcac agcctacatg 300cagctcagca gcctggcatc tgaggactct
gcaatctatt tctgtcaaga tcggggtggg 360acgaggacta tgctatggac
ttctggggtc aaggaacctc agtcaccgtc tcctcagcca 420aaacaacagc
cccatcggtc tatccatggc c 45132139PRTMus
musculusmisc_feature(48)..(48)Xaa can be any naturally occurring
amino acid 32Met Glu Leu Ile Trp Val Phe Leu Phe Leu Leu Ser Val
Thr Ala Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln Ser Gly
Val Glu Leu Val Arg 20 25 30 Pro Gly Ser Ser Val Lys Met Ser Cys
Lys Thr Ser Gly Tyr Thr Xaa 35 40 45 Thr Ala Tyr Gly Ile Asn Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr
Ile Tyr Ile Gly Asn Gly Tyr Thr Asp Tyr Asn 65 70 75 80 Glu Lys Phe
Lys Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Ser Ser 85 90 95 Thr
Ala Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Ile 100 105
110 Tyr Phe Cys Ala Arg Ser Gly Trp Asp Glu Asp Tyr Ala Met Asp Phe
115 120 125 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135
3310PRTMus musculusmisc_feature(4)..(4)Xaa can be any naturally
occurring amino acid 33Gly Tyr Thr Xaa Thr Ala Tyr Gly Ile Asn 1 5
10 3417PRTMus musculus 34Tyr Ile Tyr Ile Gly Asn Gly Tyr Thr Asp
Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 3511PRTMus musculus 35Ser Gly
Trp Asp Glu Asp Tyr Ala Met Asp Phe 1 5 10 36399DNAMus musculus
36atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tggcagatgt
60gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc
120atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca
gcagaaacag 180agaaaatctc ctcacctcct ggtctatcat gcaaaaacct
tagcagaagg tgtgacatca 240aggttcagtg gcagtggatc aggcacacag
ttttctctga agatccacag cctgcagcct 300gaagattttg ggacttatta
ctgtcaacat cattatggtc ctcctcccac gttcggctcg 360gggacaaagt
tggaaataaa acgggctgat gctgcacca 39937127PRTMus musculus 37Met Ser
Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15
Gly Gly Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20
25 30 Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu
Asn 35 40 45 Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Arg
Lys Ser Pro 50 55 60 His Leu Leu Val Tyr His Ala Lys Thr Leu Ala
Glu Gly Val Thr Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Gln Phe Ser Leu Lys Ile His 85 90 95 Ser Leu Gln Pro Glu Asp Phe
Gly Thr Tyr Tyr Cys Gln His His Tyr 100 105 110 Gly Pro Pro Pro Thr
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120 125 3811PRTMus
musculus 38Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10
397PRTMus musculus 39His Ala Lys Thr Leu Ala Glu 1 5 409PRTMus
musculus 40Gln His His Tyr Gly Pro Pro Pro Thr 1 5 41116PRTHomo
sapiens 41Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro
Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr
Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Tyr Tyr Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro 65 70 75 80 Ser Leu Lys Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110
Tyr Cys Ala Arg 115 42141PRTMus musculus 42Met Lys His Leu Trp Phe
Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser
Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45
Thr Thr Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50
55 60 Glu Trp Ile Gly Val Ile Trp Ser Gly Gly Asp Thr Asp Tyr Asn
Ala 65 70 75 80 Ser Phe Ile Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Asn Lys Asp Tyr Tyr
Thr Asn Tyr Asp Phe Thr Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 130 135 140
4397PRTHomo sapiens 43Glu 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 Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg 44141PRTMus musculus 44Met Ala Val Leu Gly Leu Leu Phe Cys
Leu Val Thr Phe Pro Ser Cys 1 5 10 15 Val Leu Ser Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu 35 40 45 Thr Thr Tyr
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu
Trp Val Ser Val Ile Trp Ser Gly Gly Asp Thr Asp Tyr Asn Ala 65 70
75 80 Ser Phe Ile Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr 85 90 95 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr 100 105 110 Tyr Cys Ala Arg Asn Lys Asp Tyr Tyr Thr Asn
Tyr Asp Phe Thr Met 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Thr Val
Thr Val Ser Ser 130 135 140 4599PRTHomo sapiens 45Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35
40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln Tyr Tyr 85 90 95 Ser Thr Pro 4612PRTHomo sapiens 46Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 47132PRTMus
musculus 47Met Glu Ser Gln Thr Gln Val Phe Leu Ser Leu Leu Leu Trp
Val Ser 1 5 10 15 Gly Thr Cys Gly Asp Ile Val Met Thr Gln Ser Pro
Asp Ser Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn
Cys Lys Ser Ser Gln Ser 35 40 45 Val Leu Tyr Ser Ser Asn Gln Lys
Asn Phe Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Glu Ser Gly Val
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 100 105 110
Tyr Cys His Gln Tyr Leu Ser Ser Tyr Thr Phe Gly Gln Gly Thr Lys 115
120 125 Leu Glu Ile Lys 130 4898PRTHomo sapiens 48Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 49133PRTMus musculus 49Met Asp
Trp Thr Trp Val Phe Leu Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20
25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe 35 40 45 Ile Asp Tyr Trp Ile Glu Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu 50 55 60 Glu Trp Met Gly Glu Ile Phe Pro Gly Ser Gly
Thr Ile Asn His Asn 65 70 75 80 Glu Lys Phe Lys Asp Arg Val Thr Ile
Thr Ala Asp Lys Ser Thr Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg
Glu Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu 115 120 125 Val Thr Val
Ser Ser 130 50115PRTHomo sapiens 50Met Glu Ala Pro Ala Gln Leu Leu
Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Ser
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60
Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 65
70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser 100 105 110 Asn Trp Pro 115 51126PRTMus musculus
51Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1
5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala
Ser Ser Ser 35 40 45 Val Ser Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg 50 55 60 Leu Leu Ile Tyr Asp Thr Ser Thr Leu
Ala Ser Gly Ile Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Glu Pro Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Arg 100 105 110 Asn Pro Phe
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 115 120 125 5232PRTMus
musculus 52Arg Gly Arg Thr Cys Arg Gly Arg Lys Phe Asp Gly His Arg
Cys Ala 1 5 10 15 Gly Gln Gln Gln Asp Ile Arg His Cys Tyr Ser Ile
Gln His Cys Pro 20 25 30 5323PRTMus musculus 53Leu Val Val Glu Glu
Lys Arg Pro Cys Leu His Val Pro Ala Cys Lys 1 5 10 15 Asp Pro Glu
Glu Glu Glu Leu 20 54469PRTHomo sapiens 54Met Ile Thr Glu Gly Ala
Gln Ala Pro Arg Leu Leu Leu Pro Pro Leu 1 5 10 15 Leu Leu Leu Leu
Thr Leu Pro Ala Thr Gly Ser Asp Pro Val Leu Cys 20 25 30 Phe Thr
Gln Tyr Glu Glu Ser Ser Gly Lys Cys Lys Gly Leu Leu Gly 35 40 45
Gly Gly Val Ser Val Glu Asp Cys Cys Leu Asn Thr Ala Phe Ala Tyr 50
55 60 Gln Lys Arg Ser Gly Gly Leu Cys Gln Pro Cys Arg Ser Pro Arg
Trp 65 70 75 80 Ser Leu Trp Ser Thr Trp Ala Pro Cys Ser Val Thr Cys
Ser Glu Gly 85 90 95 Ser Gln Leu Arg Tyr Arg Arg Cys Val Gly Trp
Asn Gly Gln Cys Ser 100 105 110 Gly Lys Val Ala Pro Gly Thr Leu Glu
Trp Gln Leu Gln Ala Cys Glu 115 120 125 Asp Gln Gln Cys Cys Pro Glu
Met Gly Gly Trp Ser Gly Trp Gly Pro 130 135 140 Trp Glu Pro Cys Ser
Val Thr Cys Ser Lys Gly Thr Arg Thr Arg Arg 145 150 155 160 Arg Ala
Cys Asn His Pro Ala Pro Lys Cys Gly Gly His Cys Pro Gly 165 170 175
Gln Ala Gln Glu Ser Glu Ala Cys Asp Thr Gln Gln Val Cys Pro Thr 180
185 190 His Gly Ala Trp Ala Thr Trp Gly Pro Trp Thr Pro Cys Ser Ala
Ser 195 200 205 Cys His Gly Gly Pro His Glu Pro Lys Glu Thr Arg Ser
Arg Lys Cys 210 215 220 Ser Ala Pro Glu Pro Ser Gln Lys Pro Pro Gly
Lys Pro Cys Pro Gly 225 230 235 240 Leu Ala Tyr Glu Gln Arg Arg Cys
Thr Gly Leu Pro Pro Cys Pro Val 245 250 255 Ala Gly Gly Trp Gly Pro
Trp Gly Pro Val Ser Pro Cys Pro Val Thr 260 265 270 Cys Gly Leu Gly
Gln Thr Met Glu Gln Arg Thr Cys Asn His Pro Val 275 280 285 Pro Gln
His Gly Gly Pro Phe Cys Ala Gly Asp Ala Thr Arg Thr His 290 295 300
Ile Cys Asn Thr Ala Val Pro Cys Pro Val Asp Gly Glu Trp Asp Ser 305
310 315 320 Trp Gly Glu Trp Ser Pro Cys Ile Arg Arg Asn Met Lys Ser
Ile Ser 325 330 335 Cys Gln Glu Ile Pro Gly Gln Gln Ser Arg Gly Arg
Thr Cys Arg Gly 340 345 350 Arg Lys Phe Asp Gly His Arg Cys Ala Gly
Gln Gln Gln Asp Ile Arg 355 360 365 His Cys Tyr Ser Ile Gln His Cys
Pro Leu Lys Gly Ser Trp Ser Glu 370 375 380 Trp Ser Thr Trp Gly Leu
Cys Met Pro Pro Cys Gly Pro Asn Pro Thr 385 390 395 400 Arg Ala Arg
Gln Arg Leu Cys Thr Pro Leu Leu Pro Lys Tyr Pro Pro 405 410 415 Thr
Val Ser Met Val Glu Gly Gln Gly Glu Lys Asn Val Thr Phe Trp 420 425
430 Gly Arg Pro Leu Pro Arg Cys Glu Glu Leu Gln Gly Gln Lys Leu Val
435 440 445 Val Glu Glu Lys Arg Pro Cys Leu His Val Pro Ala Cys Lys
Asp Pro 450 455 460 Glu Glu Glu Glu Leu 465 5548PRTHomo sapiens
55Asp Pro Val Leu Cys Phe Thr Gln Tyr Glu Glu Ser Ser Gly Lys Cys 1
5 10 15 Lys Gly Leu Leu Gly Gly Gly Val Ser Val Glu Asp Cys Cys Leu
Asn 20 25 30 Thr Ala Phe Ala Tyr Gln Lys Arg Ser Gly Gly Leu Cys
Gln Pro Cys 35 40 45 5658PRTHomo sapiens 56Ser Pro Arg Trp Ser Leu
Trp Ser Thr Trp Ala Pro Cys Ser Val Thr 1 5 10 15 Cys Ser Glu Gly
Ser Gln Leu Arg Tyr Arg Arg Cys Val Gly Trp Asn 20 25 30 Gly Gln
Cys Ser Gly Lys Val Ala Pro Gly Thr Leu Glu Trp Gln Leu 35 40 45
Gln Ala Cys Glu Asp Gln Gln Cys Cys Pro 50 55 5757PRTHomo sapiens
57Glu Met Gly Gly Trp Ser Gly Trp Gly Pro Trp Glu Pro Cys Ser Val 1
5 10 15 Thr Cys Ser Lys Gly Thr Arg Thr Arg Arg Arg Ala Cys Asn His
Pro 20 25 30 Ala Pro Lys Cys Gly Gly His Cys Pro Gly Gln Ala Gln
Glu Ser Glu 35 40 45 Ala Cys Asp Thr Gln Gln Val Cys Pro 50 55
5864PRTHomo sapiens 58Thr His Gly Ala Trp Ala Thr Trp Gly Pro Trp
Thr Pro Cys Ser Ala 1 5 10 15 Ser Cys His Gly Gly Pro His Glu Pro
Lys Glu Thr Arg Ser Arg Lys 20 25 30 Cys Ser Ala Pro Glu Pro Ser
Gln Lys Pro Pro Gly Lys Pro Cys Pro 35 40 45 Gly Leu Ala Tyr Glu
Gln Arg Arg Cys Thr Gly Leu Pro Pro Cys Pro 50 55 60 5958PRTHomo
sapiens 59Val Ala Gly Gly Trp Gly Pro Trp Gly Pro Val Ser Pro Cys
Pro Val 1 5 10 15 Thr Cys Gly Leu Gly Gln Thr Met Glu Gln Arg Thr
Cys Asn His Pro 20 25 30 Val Pro Gln His Gly Gly Pro Phe Cys Ala
Gly Asp Ala Thr Arg Thr 35 40 45 His Ile Cys Asn Thr Ala Val Pro
Cys Pro 50 55 6064PRTHomo sapiens 60Val Asp Gly Glu Trp Asp Ser Trp
Gly Glu Trp Ser Pro Cys Ile Arg 1 5 10 15 Arg Asn Met Lys Ser Ile
Ser Cys Gln Glu Ile Pro Gly Gln Gln Ser 20 25 30 Arg Gly Arg Thr
Cys Arg Gly Arg Lys Phe Asp Gly His Arg Cys Ala 35 40 45 Gly Gln
Gln Gln Asp Ile Arg His Cys Tyr Ser Ile Gln His Cys Pro 50 55 60
6192PRTHomo sapiens 61Leu Lys Gly Ser Trp Ser Glu Trp Ser Thr Trp
Gly Leu Cys Met Pro 1 5 10 15 Pro Cys Gly Pro Asn Pro Thr Arg Ala
Arg Gln Arg Leu Cys Thr Pro 20 25 30 Leu Leu Pro Lys Tyr Pro Pro
Thr Val Ser Met Val Glu Gly Gln Gly 35 40 45 Glu Lys Asn Val Thr
Phe Trp Gly Arg Pro Leu Pro Arg Cys Glu Glu 50 55 60 Leu Gln Gly
Gln Lys Leu Val Val Glu Glu Lys Arg Pro Cys Leu His 65 70 75 80 Val
Pro Ala Cys Lys Asp Pro Glu Glu Glu Glu Leu 85 90 6212PRTHomo
sapiens 62Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 1 5 10
63327PRTHomo sapiens 63Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305
310 315 320 Leu Ser Leu Ser Pro Gly Lys 325 64106PRTHomo sapiens
64Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1
5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 100 105 6519DNAHomo sapiens 65atcagaggcc
tgtgacacc 196619DNAHomo sapiens 66ctgcccttgt agctcctca
19671603DNAHomo sapiens 67tatcaaccca gataaagcgg gacctcctct
ctggtagagg tgcagggggc agtactcaac 60atgatcacag agggagcgca ggcccctcga
ttgttgctgc cgccgctgct cctgctgctc 120accctgccag ccacaggctc
agaccccgtg ctctgcttca cccagtatga agaatcctcc 180ggcaagtgca
agggcctcct ggggggtggt gtcagcgtgg aagactgctg tctcaacact
240gcctttgcct accagaaacg tagtggtggg ctctgtcagc cttgcaggtc
cccacgatgg 300tccctttggt ccacatgggc cccctgttcg gtgacgtgct
ctgagggctc ccagctgcgg 360taccggcgct gtgtgggctg gaatgggcag
tgctctggaa aggtggcacc tgggaccctg 420gagtggcagc tccaggcctg
tgaggaccag cagtgctgtc ctgagatggg cggctggtct 480ggctgggggc
cctgggagcc ttgctctgtc acctgctcca aagggacccg gacccgcagg
540cgagcctgta atcaccctgc tcccaagtgt gggggccact gcccaggaca
ggcacaggaa 600tcagaggcct gtgacaccca gcaggtctgc cccacacacg
gggcctgggc cacctggggc 660ccctggaccc cctgctcagc ctcctgccac
ggtggacccc acgaacctaa ggagacacga 720agccgcaagt gttctgcacc
tgagccctcc cagaaacctc ctgggaagcc ctgcccgggg 780ctagcctacg
agcagcggag gtgcaccggc ctgccaccct gcccagtggc tgggggctgg
840gggccttggg gccctgtgag cccctgccct gtgacctgtg gcctgggcca
gaccatggaa 900caacggacgt gcaatcaccc tgtgccccag catgggggcc
ccttctgtgc tggcgatgcc 960acccggaccc acatctgcaa cacagctgtg
ccctgccctg tggatgggga gtgggactcg 1020tggggggagt ggagcccctg
tatccgacgg aacatgaagt ccatcagctg tcaagaaatc 1080ccgggccagc
agtcacgcgg gaggacctgc aggggccgca agtttgacgg acatcgatgt
1140gccgggcaac agcaggatat ccggcactgc tacagcatcc agcactgccc
cttgaaagga 1200tcatggtcag agtggagtac ctgggggctg tgcatgcccc
cctgtggacc taatcctacc 1260cgtgcccgcc agcgcctctg cacacccttg
ctccccaagt acccgcccac cgtttccatg 1320gtcgaaggtc agggcgagaa
gaacgtgacc ttctggggga gaccgctgcc acggtgtgag 1380gagctacaag
ggcagaagct ggtggtggag gagaaacgac catgtctaca cgtgcctgct
1440tgcaaagacc ctgaggaaga ggaactctaa cacttctctc ctccactctg
agccccctga 1500ccttccaaac ctcaataaac tagcctcttc gaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1560aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaa 1603
* * * * *