U.S. patent application number 14/864750 was filed with the patent office on 2016-04-07 for pc33718e.
This patent application is currently assigned to PFIZER INC.. The applicant listed for this patent is Pfizer Inc., RINAT NEUROSCIENCE CORP.. Invention is credited to Yasmina Noubia ABDICHE, Javier Fernando CHAPARRO RIGGERS, Bruce Charles GOMES, Julie Jia Li HAWKINS, Hong LIANG, Jaume PONS, Xiayang QIU, Pavel STROP, Yuli WANG.
Application Number | 20160096898 14/864750 |
Document ID | / |
Family ID | 42005569 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160096898 |
Kind Code |
A1 |
LIANG; Hong ; et
al. |
April 7, 2016 |
PC33718E
Abstract
The present invention provides antagonizing antibodies,
antigen-binding portions thereof, and aptamers that bind to
proprotein convertase subtilisin kexin type 9 (PCSK9). Also
provided are antibodies directed to peptides, in which the
antibodies bind to PCSK9. The invention further provides a method
of obtaining such antibodies and antibody-encoding nucleic acid.
The invention further relates to therapeutic methods for use of
these antibodies and antigen-binding portions thereof to reduce
LDL-cholesterol levels and/or for the treatment and/or prevention
of cardiovascular disease, including treatment of
hypercholesterolemia.
Inventors: |
LIANG; Hong; (Hillsborough,
CA) ; ABDICHE; Yasmina Noubia; (Mountain View,
CA) ; CHAPARRO RIGGERS; Javier Fernando; (San Mateo,
CA) ; GOMES; Bruce Charles; (Ashburnham, MA) ;
HAWKINS; Julie Jia Li; (Weston, CT) ; PONS;
Jaume; (San Francisco, CA) ; QIU; Xiayang;
(Mystic, CT) ; STROP; Pavel; (San Mateo, CA)
; WANG; Yuli; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RINAT NEUROSCIENCE CORP.
Pfizer Inc. |
South San Francisco
New York |
CA
NY |
US
US |
|
|
Assignee: |
PFIZER INC.
New York
NY
RINAT NEUROSCIENCE CORP.
South San Francisco
CA
|
Family ID: |
42005569 |
Appl. No.: |
14/864750 |
Filed: |
September 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13857063 |
Apr 4, 2013 |
9175093 |
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14864750 |
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13225265 |
Sep 2, 2011 |
8426363 |
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13857063 |
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12558312 |
Sep 11, 2009 |
8080243 |
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13225265 |
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61096716 |
Sep 12, 2008 |
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61232161 |
Aug 7, 2009 |
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61235643 |
Aug 20, 2009 |
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Current U.S.
Class: |
530/387.3 ;
530/387.9 |
Current CPC
Class: |
A61P 3/06 20180101; A61P
43/00 20180101; C07K 2317/92 20130101; A61K 2039/505 20130101; A61P
9/00 20180101; C07K 2317/76 20130101; C07K 2317/24 20130101; A61K
39/3955 20130101; A61K 45/06 20130101; A61P 9/10 20180101; C07K
16/40 20130101; A61P 3/00 20180101; C07K 2299/00 20130101; C07K
2317/33 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. An isolated antibody that comprises a PCSK9 binding region that
competes with, or recognizes a first epitope of PCSK9 that overlaps
with a second epitope that is recognized by, a monoclonal antibody
selected from the group consisting of 5A10, which is produced by a
hybridoma cell line deposited with the American Type Culture
Collection and assigned accession number PTA-8986; 4A5, which is
produced by a hybridoma cell line deposited with the American Type
Culture Collection and assigned accession number PTA-8985; 6F6,
which is produced by a hybridoma cell line deposited with the
American Type Culture Collection and assigned accession number
PTA-8984, and 7D4, which is produced by a hybridoma cell line
deposited with the American Type Culture Collection and assigned
accession number PTA-8983.
2. An isolated antibody comprising a heavy chain variable region
(VH) complementarity determining region one (CDR1) having the amino
acid sequence shown in SEQ ID NO:59, 60 or 8, a VH CDR2 having the
amino acid sequence shown in SEQ ID NO:61 or 9, and a VH CDR3
having the amino acid sequence shown in SEQ ID NO:10, and having
one or more conservative amino acid substitutions in CDR1, CDR2,
and/or CDR3, wherein the VH CDR1 comprises a substitution at amino
acid position 8 of SEQ ID NO:59, the VH CDR2 comprises a
substitution at one or more of amino acid positions 3, 4, 5, 6, and
7 of SEQ ID NO:9, and/or a VH CDR3 comprises a substitution at one
or both of amino acid positions 7 and 9 of SEQ ID NO: 10; and a
light chain variable region (VL) complementarity determining region
one (CDR1) having the amino acid sequence shown in SEQ ID NO:11, a
VL CDR2 having the amino acid sequence shown in SEQ ID NO:12, and a
VL CDR3 having the amino acid sequence shown in SEQ ID NO:13, and
having one or more conservative amino acid substitutions in CDR1,
CDR2, and/or CDR3, wherein the VL CDR1 comprises a substitution at
one or more amino acid positions 1, 2, 3, 4, 5, 6, 8, and 10 of SEQ
ID NO:11, the VL CDR2 comprises a substitution at one or more amino
acid positions 1, 4, 5, 6, and 7 of SEQ ID NO:12, and/or a VL CDR3
comprises a substitution at one or more amino acid positions 4, 5,
6, 7, 8, or 9 of SEQ ID NO:13.
3. The antibody of claim 2, comprising a VH CDR1 having the amino
acid sequence shown in SEQ ID NO:59, a VH CDR2 having the amino
acid sequence shown in SEQ ID NO:65, 174, 175, 9, 176, or 185, and
a VH CDR3 having the amino acid sequence shown in SEQ ID NO:45,
166, 167, 10, 168, 169, 170, 171, 172, or 10; and a VL CDR1 having
the amino acid sequence shown in SEQ ID NO:30, a VL CDR2 having the
amino acid sequence shown in SEQ ID NO:12, and a VL CDR3 having the
amino acid sequence shown in SEQ ID NO:86, 134, 135, 31, 177, 179,
180, 13, 182, 183, 13, or 186.
4. The antibody of claim 3, comprising a VH CDR1 having the amino
acid sequence shown in SEQ ID NO:59, a VH CDR2 having the amino
acid sequence shown in SEQ ID NO:9 or 185, and a VH CDR3 having the
amino acid sequence shown in SEQ ID NO:10; and a VL CDR1 having the
amino acid sequence shown in SEQ ID NO:30, a VL CDR2 having the
amino acid sequence shown in SEQ ID NO:12, and a VL CDR3 having the
amino acid sequence shown in SEQ ID NO:13.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 13/857,063, filed Apr. 4, 2013, which is a divisional of U.S.
application Ser. No. 13/225,265, filed Sep. 2, 2011, which is a
divisional of U.S. application Ser. No. 12/558,312, filed Sep. 11,
2009, now issued as U.S. Pat. No. 8,080,243, which claims priority,
under 35 USC .sctn.119(e), to the following US provisional
applications, U.S. Appl. No. 61/096,716, filed Sep. 12, 2008, U.S.
Appl. No. 61/232,161, filed Aug. 7, 2009, and U.S. Appl. No.
61/235,643, filed Aug. 20, 2009.
REFERENCE TO SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"PC33718E_SequenceListing.txt" created on Sep. 23, 2015 and having
a size of 52 KB. The sequence listing contained in this .txt file
is part of the specification and is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to antibodies, e.g., full
length antibodies or antigen-binding portions thereof, peptides,
and aptamers that antagonize the activity of extracellular
proprotein convertase subtilisin kexin type 9 (PCSK9), including
its interaction with the low density lipoprotein (LDL) receptor
(LDLR). More specifically, the invention relates to compositions
comprising antagonist PCSK9 antibodies, peptides, and/or aptamers
and methods of using these antibodies and/or peptides and/or
aptamers as a medicament. The antagonist PCSK9 antibodies,
peptides, and aptamers can be used therapeutically to lower
LDL-cholesterol levels in blood, and can be used in the prevention
and/or treatment of cholesterol and lipoprotein metabolism
disorders, including familial hypercholesterolemia, atherogenic
dyslipidemia, atherosclerosis, and, more generally, cardiovascular
disease (CVD).
BACKGROUND OF THE INVENTION
[0004] Millions of people in the U.S. are at risk for heart disease
and resulting cardiac events. CVD and underlying atherosclerosis is
the leading cause of death among all demographic groups, despite
the availability of therapies directed at its multiple risk
factors. Atherosclerosis is a disease of the arteries and is
responsible for coronary heart disease associated with many deaths
in industrialized countries. Several risk factors for coronary
heart disease have now been identified: dyslipidemias,
hypertension, diabetes, smoking, poor diet, inactivity and stress.
The most clinically relevant and common dyslipidemias are
characterized by an increase in beta-lipoproteins (very low density
lipoprotein (VLDL) and LDL) with hypercholesterolemia in the
absence or presence of hypertriglyceridemia (Fredrickson et al.,
1967, N Engl J Med. 276:34-42, 94-103, 148-156, 215-225, and
273-281). There is a long-felt significant unmet need with respect
to CVD with 60-70% of cardiovascular events, heart attacks and
strokes occurring despite the treatment with statins (the current
standard of care in atherosclerosis). Moreover, new guidelines
suggest that even lower LDL levels should be achieved in order to
protect high risk patients from premature CVD [National Cholesterol
Education Program (NCEP), 2004].
[0005] PCSK9, also known as NARC-1, was identified as a protein
with a genetic mutation in some forms of familial
hypercholesterolemia. PCSK9 is synthesized as a zymogen that
undergoes autocatalytic processing at the motif LVFAQ in the
endoplasmic reticulum. Population studies have shown that some
PCSK9 mutations are "gain-of-function" and are found in individuals
with autosomal dominant hypercholesterolemia, while other
"loss-of-function" (LOF) mutations are linked with reduced plasma
cholesterol. Morbidity and mortality studies in this group clearly
demonstrated that reducing PCSK9 function significantly diminished
the risk of cardiovascular disease.
[0006] Of significant importance to the treatment of CVD, a LOF
mutation may sensitize humans to statins, allowing for efficacy at
a lower dose (hence, improving risks associated with safety and
tolerance) and potentially achieving lower plasma cholesterol
levels than with current therapies.
[0007] PCSK9 is secreted into the plasma predominantly by
hepatocytes. Genetic modulation of PCSK9 in mice confirmed the
ability of PCSK9 to regulate blood lipids, and suggested that it
acts to down-regulate hepatic LDLR protein levels.
[0008] The mechanism by which, and the site at which, PCSK9
down-regulates LDLR protein has not been clearly established. When
over-expressed, PCSK9 may act both within the hepatocyte and as a
secreted ligand for LDLR. There is strong evidence that
extracellular PCSK9 binds to cell surface LDLR and promotes LDLR
degradation at an intracellular site. However, it is also possible
that PCSK9 could interact with the LDLR when the two proteins are
translated within the endoplasmic reticulum (ER) and traffic
through endosomal compartments towards the cell membrane. Maxwell
et al., 2005, Curr. Opin. Lipidol. 16:167-172, showed that
PCSK9-mediated LDLR endocytosis and degradation was not altered by
proteosome inhibitors nor was it modulated by different classes of
lysosomal and nonlysosomal proteases. Two naturally occurring
familial hypercholesterolemia mutations, S127R and D129G, have been
reported to be defective in autoprocessing and secretion as levels
of these mutant proteins were greatly reduced or undetectable in
the media of transfected cells. Yet these mutants demonstrated an
enhanced ability to down-regulate LDLR, consistent with their
identification in individuals with high plasma LDL (Homer et al.,
2008, Atherosclerosis 196:659-666; Cameron et al., 2006 Human
Molecular Genetics 15:1551-1558; Lambert et al., 2006, TRENDS in
Endocrinology and Metabolism 17:79-81. Since these mutants
apparently do not get secreted extracellularly, and yet do
downregulate LDLR, this strongly suggests that an intracellular
site of action is physiologically important.
[0009] From the information available in the art, and prior to the
present invention, it remained unclear whether the introduction of
an antibody-, peptide-, or aptamer-based PCSK9 antagonist into the
blood circulation to selectively antagonize extracellular PCSK9
would be effective to reduce hypercholesterolemia and the
associated incidence of CVD and, if so, what properties of a PCSK9
antagonist are needed for such in vivo effectiveness.
SUMMARY OF THE INVENTION
[0010] This invention relates to antagonist antibodies, peptides,
and aptamers that selectively interact with and inhibit PCSK9
function. It is demonstrated for the first time that certain PCSK9
antagonists are effective in vivo to lower blood cholesterol.
[0011] In one embodiment, the invention provides an isolated
antagonist of PCSK9 which comprises an antibody, a peptide, or an
aptamer, which interacts with PCSK9 and when administered to a
subject lowers the LDL-cholesterol level in blood of said subject.
The antagonist can be an antibody, for example, a monoclonal
antibody or human, humanized, or chimeric antibody.
[0012] In another embodiment, the invention provides an isolated
anti-PCSK9 antibody which specifically binds to PCSK9 and which is
a full antagonist of the PCSK9-mediated effect on LDLR levels when
measured in vitro using the LDLR down regulation assay in Huh7
cells disclosed herein.
[0013] In yet another embodiment, the invention provides an
isolated antibody which antagonizes the extracellular interaction
of PCSK9 with the LDLR, as measured by PCSK9 binding to the LDLR in
vitro, and, when administered to a subject, lowers the
LDL-cholesterol level in blood of said subject. Preferably, the
antibody recognizes an epitope on human PCSK9 that overlaps with
more than about 75% of the surface on PCSK9 that interacts with the
EGF-like domain of the LDLR as described in Kwon et al., 2008,
PNAS, 105:1820-1825.
[0014] In yet another embodiment, the invention provides an
antibody that recognizes a first epitope of PCSK9 that overlaps
with a second epitope that is recognized by a monoclonal antibody
selected from the group consisting of 5A10, which is produced by a
hybridoma cell line deposited with the American Type Culture
Collection and assigned accession number PTA-8986; 4A5, which is
produced by a hybridoma cell line deposited with the American Type
Culture Collection and assigned accession number PTA-8985; 6F6,
which is produced by a hybridoma cell line deposited with the
American Type Culture Collection and assigned accession number
PTA-8984, and 7D4, which is produced by a hybridoma cell line
deposited with the American Type Culture Collection and assigned
accession number PTA-8983.
[0015] In another embodiment, the invention provides an antibody to
human PCSK9, wherein the antibody recognizes an epitope on human
PCSK9 comprising amino acid residues 153-155, 194, 195, 197,
237-239, 367, 369, 374-379 and 381 of the PCSK9 amino acid sequence
of SEQ ID NO: 53. Preferably, the antibody epitope on human PCSK9
does not comprise one or more of amino acid residues 71, 72,
150-152, 187-192, 198-202, 212, 214-217, 220-226, 243, 255-258,
317, 318, 347-351, 372, 373, 380, 382, and 383.
[0016] In still another embodiment, the invention provides an
antibody which specifically binds PCSK9 comprising a VH
complementary determining region one (CDR1) having the amino acid
sequence shown in SEQ ID NO:8 (SYYMH), a VH CDR2 having the amino
acid sequence shown in SEQ ID NO:9 (EISPFGGRTNYNEKFKS), and/or VH
CDR3 having the amino acid sequence shown in SEQ ID NO:10
(ERPLYASDL), or a variant thereof having one or more conservative
amino acid substitutions in said sequences of CDR1, CDR2, and/or
CDR3, wherein the variant retains essentially the same binding
specificity as the CDR defined by said sequences. Preferably, the
variant comprises up to about ten amino acid substitutions and,
more preferably, up to about four amino acid substitutions.
[0017] The invention is further directed to an antibody comprising
a VL CDR1 having the amino acid sequence shown in SEQ ID NO:11
(RASQGISSALA), a CDR2 having the amino acid sequence shown in SEQ
ID NO:12 (SASYRYT), and/or CDR3 having the amino acid sequence
shown in SEQ ID NO:13 (QQRYSLWRT), or a variant thereof having one
or more conservative amino acid substitutions in said sequences of
CDR1, CDR2, and/or CDR3, wherein the variant retains essentially
the same binding specificity as the CDR1 defined by said sequences.
Preferably, the variant comprises up to about ten amino acid
substitutions and, more preferably, up to about four amino acid
substitutions.
[0018] In another embodiment, the invention provides an antibody
comprising specific VL CDR1, CDR2, and/or CDR3 sequences, or a
variant thereof having one or more conservative amino acid
substitutions in CDR1, CDR2, and/or CDR3 and further comprising a
VH complementary determining region CDR1 having the amino acid
sequence shown in SEQ ID NO:59, 60, or 8, a VH CDR2 having the
amino acid sequence shown in SEQ ID NO:61 or 9, and/or VH CDR3
having the amino acid sequence shown in SEQ ID NO:10, or a variant
thereof having one or more conservative amino acid substitutions in
said sequences of CDR1, CDR2, and/or CDR3, wherein the variant
retains essentially the same binding specificity as the CDR1, CDR2,
and/or CDR3 defined by said sequences. Preferably, the variant
comprises up to about twenty amino acid substitutions and, more
preferably, up to about eight amino acid substitutions. In another
preferred embodiment, the antibody of the invention has a variable
heavy chain sequence comprising or consisting of SEQ ID NO: 54 and
a variable light chain sequence comprising or consisting of SEQ ID
NO: 53.
[0019] The invention also provides a humanized antibody comprising
polypeptides selected from the groups consisting of SEQ ID NO:14,
SEQ ID NO:15, or both SEQ ID NO:14 and SEQ ID NO:15, or a variant
thereof having one or more conservative amino acid substitutions in
said sequences, wherein the variant retains essentially the same
binding specificity as the antibody defined by said sequence(s). It
also includes an antibody lacking a terminal lysine on the heavy
chain, as this is normally lost in a proportion of antibodies
during manufacture.
[0020] Preferably, the variant comprises up to about twenty amino
acid substitutions and more preferably, up to about eight amino
acid substitutions. Preferably, the antibody further comprises an
immunologically inert constant region, and/or the antibody has an
isotype that is selected from the group consisting of IgG.sub.2,
IgG.sub.4, IgG.sub.2.DELTA.a, IgG.sub.4.DELTA.b, IgG.sub.4.DELTA.c,
IgG.sub.4 S228P, IgG.sub.4.DELTA.b S228P and IgG.sub.4.DELTA.c
S228P. In another preferred embodiment, the constant region is
aglycosylated Fc.
[0021] In one embodiment, the invention provides a method for
reducing a level of LDL, LDL-cholesterol, or total cholesterol in
blood, serum, or plasma of a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an antagonist of the invention.
[0022] In one embodiment, the invention provides a therapeutically
effective amount of an antagonist of the invention for use in
reducing a level of LDL, LDL-cholesterol, or total cholesterol in
blood, serum, or plasma of a subject in need thereof. The invention
further provides the use of a therapeutically effective amount of
an antagonist of the invention in the manufacture of a medicament
for reducing a level of LDL, LDL-cholesterol, or total cholesterol
in blood, serum, or plasma of a subject in need thereof.
[0023] In yet another embodiment, the invention provides a method
of preparing an antibody which specifically binds PCSK9, which
comprises: a) providing a PCSK9-negative host animal; b) immunizing
said PCSK9-negative host animal with PCSK9; and c) obtaining an
antibody. An antibody-producing cell, or an antibody-encoding
nucleic acid from said PCSK9-negative host animal, and preparing an
antibody from said antibody-producing cell or said
antibody-encoding nucleic acid.
[0024] The invention also comprises a method for reducing the level
of LDL in blood of a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
the antibody prepared according to the invention. The subject can
be further treated by administering a statin. In a preferred
embodiment, the subject is a human subject.
[0025] In one embodiment, the antibody is administered in a
formulation as a sterile aqueous solution having a pH that ranges
from about 5.0 to about 6.5 and comprising from about 1 mg/ml to
about 200 mg/ml of antibody, from about 1 millimolar to about 100
millimolar of histidine buffer, from about 0.01 mg/ml to about 10
mg/ml of polysorbate 80, from about 100 millimolar to about 400
millimolar of trehalose, and from about 0.01 millimolar to about
1.0 millimolar of disodium EDTA dihydrate.
[0026] In another embodiment, the invention provides a
therapeutically effective amount of the antibody prepared according
to the invention for use in reducing the level of LDL in blood of a
subject in need thereof. The invention further provides the use of
a therapeutically effective amount of the antibody prepared
according to the invention in the manufacture of a medicament for
reducing the level of LDL in blood of a subject in need thereof.
The therapeutically effective amount can optionally be combined
with a therapeutically effective amount of a statin.
[0027] In another embodiment, the invention provides a hybridoma
cell line that produces a PCSK9-specific antibody or an
antigen-binding portion thereof, wherein the hybridoma cell line is
selected from the group consisting of: [0028] 4A5 having an ATCC
Accession No. of PTA-8985; [0029] 5A10 having an ATCC Accession No.
of PTA-8986; [0030] 6F6 having an ATCC Accession No. of PTA-8984;
and [0031] 7D4 having an ATCC Accession No. of PTA-8983.
[0032] In another embodiment, the invention provides cell line that
recombinantly produces an antibody which specifically binds to
PCSK9 and comprises a heavy chain variable region (VH)
complementary determining region one (CDR1) having the amino acid
sequence shown in SEQ ID NO:8, 59, or 60, a VH CDR2 having the
amino acid sequence shown in SEQ ID NO:9 or 61, and/or VH CDR3
having the amino acid sequence shown in SEQ ID NO:10, or a variant
thereof having one or more conservative amino acid substitutions in
CDR1, CDR2, and/or CDR3, and/or comprises a light chain variable
region (VL) CDR1 having the amino acid sequence shown in SEQ ID
NO:11, a VL CDR2 having the amino acid sequence shown in SEQ ID
NO:12, and/or VL CDR3 having the amino acid sequence shown in SEQ
ID NO:13, or a variant thereof having one or more conservative
amino acid substitutions in CDR1, CDR2, and/or CDR3. Preferably,
the cell line recombinantly produces an antibody comprising SEQ ID
NO: 53 and/or 54, and, more preferably, SEQ ID NO: 14 and/or
15.
BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
[0033] FIG. 1 illustrates the effect of anti-PCSK9 antagonistic
monoclonal antibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 on the
ability of mouse PCSK9 (A) and human PCSK9 (B) to down regulate
LDLR in cultured Huh7 cells. 6F6.G10.3 is a subclone of 6F6, 7D4.4
is a subclone of 7D4, 4A5.G3 is a subclone of 4A5, and 5A10.B8 is a
subclone of 5A10.
[0034] FIG. 2 illustrates the dose-response of anti-PCSK9
antagonist monoclonal antibodies 6F6.G10.3, 7D4.4, 4A5.G3, 5A10.B8,
negative control antibody 42H7, and PBS to block the binding of
recombinant biotinylated human PCSK9 (A) and mouse PCSK9 (B) to
immobilized recombinant LDLR extracellular domain in vitro.
[0035] FIG. 3 illustrates the dose-response of anti-PCSK9
monoclonal antagonist antibodies 6F6.G10.3, 7D4.4, 4A5.G3 and
5A10.B8 to block binding of recombinant biotinylated human PCSK9
(30 nM) to Europium labeled recombinant LDLR extracellular domain
(10 nM) in solution at neutral pH in vitro.
[0036] FIGS. 4A and 4B illustrate comparative epitope binding of
anti-PCSK9 antibodies.
[0037] FIG. 5 illustrates Western blots of binding of anti-PCSK9
antibodies to serum PCSK9 from different species.
[0038] FIG. 6 illustrates the effect of anti-PCSK9 monoclonal
antibody 7D4 on blood cholesterol levels in mice.
[0039] FIG. 7 illustrates (A) the effect of a partial antagonist
polyclonal anti-PCSK9 mAb CRN6 on LDLR down regulation and (B) the
lack of effect on cholesterol levels in mice.
[0040] FIGS. 8A and 8B illustrate the time course of the
cholesterol lowering effect obtained using anti-PCSK9 antagonist
antibody 7D4 in mice.
[0041] FIGS. 9A, 9B and 9C illustrate the dose dependence of the
anti-PCSK9 antagonist mAb 7D4 on the reduction of serum total
cholesterol, HDL and LDL in mice.
[0042] FIGS. 10A and 10B illustrate the dose dependence of the
cholesterol lowering effect of anti-PCSK9 antagonist antibody 5A10
in mice.
[0043] FIG. 11 illustrates the dose dependence of the cholesterol
lowering effect of anti-PCSK9 antagonist antibodies (A) 4A5 and (B)
6F6 in mice.
[0044] FIG. 12 depicts Western blots of anti-PCSK9 antagonist
antibodies effect on liver LDLR levels.
[0045] FIG. 13 illustrates the lack of effect of anti-PCSK9
antagonist antibody 4A5 in an LDLR-/- mouse model.
[0046] FIG. 14 illustrates the effect on total serum cholesterol of
multiple administrations of anti-PCSK9 antagonist antibodies in
mice over a longer time course than seen with a single dose.
[0047] FIGS. 15A-H illustrate the time course of the effects of
anti-PCSK9 antagonistic antibody 7D4 on lipid parameters in a
cynomolgus monkey model.
[0048] FIGS. 16A-D illustrate the dose- and time-response of
anti-PCSK9 antagonistic antibody 7D4 on serum cholesterol levels in
the cynomolgus monkey.
[0049] FIGS. 17A-D illustrate illustrates a comparison of
anti-PCSK9 antagonistic antibodies 4A5, 5A10, 6F6 and 7D4 on serum
cholesterol levels in the cynomolgus monkey.
[0050] FIG. 18 illustrates the time course of the effect of
anti-PCSK9 antagonist antibody 7D4 on plasma cholesterol levels of
cynomolgus monkeys fed a 33.4% kcal fat diet supplemented with 0.1%
cholesterol.
[0051] FIG. 19 illustrates the effect of L1 L3 (humanized
anti-PCSK9 monoclonal antibody) on down regulation of LDLR in Huh7
cells.
[0052] FIG. 20 illustrates the dose-response of L1 L3 humanized
antibody, the mouse precursor 5A10, and negative control antibody
42H7 on blocking the binding of recombinant biotinylated human
PCSK9 (A and B) and mouse PCSK9 (C and D) to immobilized
recombinant LDLR extracellular domain in vitro at pH 7.5 (A and C)
and pH 5.3 (B and D).
[0053] FIG. 21 illustrates the effect on serum cholesterol of
treatment of mice with 10 mg/kg L1L3.
[0054] FIG. 22 illustrates the effect of administration of 5A10
antibody or L1 L3 to cynomolgus monkeys and measurement of changes
in serum HDL (A) and serum LDL (B) as a function of time.
[0055] FIG. 23A depicts the crystal structure of the PCSK9 (light
gray surface representation) bound to the L1 L3 antibody (black
cartoon representation). FIG. 23B depicts the crystal structure of
the PCSK9 (light gray surface representation) bound to the EGF-like
domain of the LDLR (black cartoon representation) (Kwon et al.,
PNAS, 105, 1820-1825, 2008). FIG. 23C shows the surface area
representation of PCSK9 with the L1 L3 epitope shown in dark gray.
FIG. 23D shows the surface area representation of PCSK9 with the
LDLR EGF-like domain epitope shown in dark gray.
[0056] FIGS. 24 A-G depict the substitutions made in the CDRs of
antibody 5A10 in the course of affinity maturation and optimization
and to achieve particular properties. PCSK9 binding associated with
antibodies having these CDR substitutions is also represented. The
number following each sequence is the SEQ ID NO designated for each
sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The present invention relates to antibodies, peptides, and
aptamers that antagonize the function of extracellular PCSK9
including its interaction with the LDLR. More specifically, the
invention relates to methods of making antagonist PCSK9 antibodies,
peptides, and aptamers, compositions comprising these antibodies,
peptides, and/or aptamers, and methods of using these antibodies,
peptides, and/or aptamers as a medicament. The antagonist PCSK9
antibodies and peptides can be used to lower blood LDL-cholesterol
levels, and can be used in the prevention and/or treatment of
cholesterol and lipoprotein metabolism disorders, including
familial hypercholesterolemia, atherogenic dyslipidemia,
atherosclerosis, and, more generally, CVD.
General Techniques
[0058] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;
Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998)
Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
DEFINITIONS
[0059] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also fragments
thereof (such as Fab, Fab', F(ab').sub.2, Fv), single chain (ScFv)
and domain antibodies), and fusion proteins comprising an antibody
portion, and any other modified configuration of the immunoglobulin
molecule that comprises an antigen recognition site. An antibody
includes an antibody of any class, such as IgG, IgA, or IgM (or
sub-class thereof), and the antibody need not be of any particular
class. Depending on the antibody amino acid sequence of the
constant domain of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains
that correspond to the different classes of immunoglobulins are
called alpha, delta, epsilon, gamma, and mu, respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0060] As used herein, "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein, 1975,
Nature 256:495, or may be made by recombinant DNA methods such as
described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may
also be isolated from phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature
348:552-554, for example.
[0061] As used herein, "humanized" antibody refers to forms of
non-human (e.g., murine) antibodies that are chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. Preferably, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Preferred are antibodies having Fc regions modified as described in
WO 99/58572. Other forms of humanized antibodies have one or more
CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, and/or CDR H3) which
are altered with respect to the original antibody, which are also
termed one or more CDRs "derived from" one or more CDRs from the
original antibody.
[0062] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody that can
be produced by a human and/or which has been made using any of the
techniques for making human antibodies known to those skilled in
the art or disclosed herein. This definition of a human antibody
includes antibodies comprising at least one human heavy chain
polypeptide or at least one human light chain polypeptide. One such
example is an antibody comprising murine light chain and human
heavy chain polypeptides. Human antibodies can be produced using
various techniques known in the art. In one embodiment, the human
antibody is selected from a phage library, where that phage library
expresses human antibodies (Vaughan et al., 1996, Nature
Biotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad.
Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol.
Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human
antibodies can also be made by immunization of animals into which
human immunoglobulin loci have been transgenically introduced in
place of the endogenous loci, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the
human antibody may be prepared by immortalizing human B lymphocytes
that produce an antibody directed against a target antigen (such B
lymphocytes may be recovered from an individual or may have been
immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al.,
1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.
[0063] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. As known in
the art, the variable regions of the heavy and light chain each
consist of four framework regions (FR) connected by three
complementarity determining regions (CDRs) that contain
hypervariable regions. The CDRs in each chain are held together in
close proximity by the FRs and, with the CDRs from the other chain,
contribute to the formation of the antigen-binding site of
antibodies. There are at least two techniques for determining CDRs:
(1) an approach based on cross-species sequence variability (i.e.,
Kabat et al. Sequences of Proteins of Immunological Interest, (5th
ed., 1991, National Institutes of Health, Bethesda Md.)); and (2)
an approach based on crystallographic studies of antigen-antibody
complexes (Al-lazikani et al, 1997, J. Molec. Biol. 273:927-948).
As used herein, a CDR may refer to CDRs defined by either approach
or by a combination of both approaches.
[0064] As known in the art a "constant region" of an antibody
refers to the constant region of the antibody light chain or the
constant region of the antibody heavy chain, either alone or in
combination.
[0065] As used herein, the term "PCSK9" refers to any form of PCSK9
and variants thereof that retain at least part of the activity of
PCSK9. Unless indicated differently, such as by specific reference
to human PCSK9, PCSK9 includes all mammalian species of native
sequence PCSK9, e.g., human, canine, feline, equine, and bovine.
One exemplary human PCSK9 is found as Uniprot Accession Number
Q8NBP7 (SEQ ID NO:16).
[0066] As used herein, a "PCSK9 antagonist" refers to an antibody,
peptide, or aptamer that is able to inhibit PCSK9 biological
activity and/or downstream pathway(s) mediated by PCSK9 signaling,
including PCSK9-mediated down-regulation of the LDLR, and
PCSK9-mediated decrease in LDL blood clearance. A PCSK9 antagonist
antibody encompasses antibodies that block, antagonize, suppress or
reduce (to any degree including significantly) PCSK9 biological
activity, including downstream pathways mediated by PCSK9
signaling, such as LDLR interaction and/or elicitation of a
cellular response to PCSK9. For purpose of the present invention,
it will be explicitly understood that the term "PCSK9 antagonist
antibody" encompasses all the previously identified terms, titles,
and functional states and characteristics whereby the PCSK9 itself,
a PCSK9 biological activity (including but not limited to its
ability to mediate any aspect of interaction with the LDLR, down
regulation of LDLR, and decreased blood LDL clearance), or the
consequences of the biological activity, are substantially
nullified, decreased, or neutralized in any meaningful degree. In
some embodiments, a PCSK9 antagonist antibody binds PCSK9 and
prevents interaction with the LDLR. Examples of PCSK9 antagonist
antibodies are provided herein.
[0067] As used herein a "full antagonist" is an antagonist which,
at an effective concentration, essentially completely blocks a
measurable effect of PCSK9. By a partial antagonist is meant an
antagonist that is capable of partially blocking a measurable
effect, but that, even at a highest concentration is not a full
antagonist. By essentially completely is meant at least about 80%,
preferably, at least about 90%, more preferably, at least about
95%, and most preferably, at least about 98% or 99% of the
measurable effect is blocked. The relevant "measurable effects" are
described herein and include down regulation of LDLR by a PCSK9
antagonist as assayed in Huh7 cells in vitro, in vivo decrease in
blood (or plasma) levels of total cholesterol, and in vivo decrease
in LDL levels in blood (or plasma).
[0068] As used herein, the term "clinically meaningful" means at
least a 15% reduction in blood LDL-cholesterol levels in humans or
at least a 15% reduction in total blood cholesterol in mice. It is
clear that measurements in plasma or serum can serve as surrogates
for measurement of levels in blood.
[0069] As used herein, the term "PCSK9 antagonist peptide" or
"PCSK9 antagonist aptamer" includes any conventional peptide or
polypeptide or aptamer that blocks, antagonizes, suppresses or
reduces (to any degree including significantly) PCSK9 biological
activity, including downstream pathways mediated by PCSK9
signaling, such as LDLR interaction and/or elicitation of a
cellular response to PCSK9. PCSK9 antagonist peptides or
polypeptides include Fc fusions comprising the LDLR and soluble
portions of the LDLR, or mutants thereof with higher affinity to
PCSK9.
[0070] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to chains of
amino acids of any length, preferably, relatively short (e.g.,
10-100 amino acids). The chain may be linear or branched, it may
comprise modified amino acids, and/or may be interrupted by
non-amino acids. The terms also encompass an amino acid chain that
has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that the polypeptides can occur as single chains or
associated chains.
[0071] As known in the art, "polynucleotide," or "nucleic acid," as
used interchangeably herein, refer to chains of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a chain by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the chain. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by P(O)S
("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"), P(O)R,
P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or R' is
independently H or substituted or unsubstituted alkyl (1-20 C)
optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0072] A "PCSK9 antagonist aptamer," which comprises a nucleic acid
or protein sequence, is, for example, selected from a large pool of
random sequences and specifically binds PCSK9. The nucleic acid of
the aptamer is double-stranded DNA or single-strand RNA. Nucleic
acid aptamers can include modified bases or functional groups,
including but not limited to 2'-fluorine nucleotides and
2'-O-methyl nucleotides. Aptamers can include hydrophilic polymers,
for example, polyethylene glycol. Aptamers may be made by methods
known in the art and selected for PCSK9 antagonist activity by
routine modification of the methods disclosed in the Examples.
[0073] As used herein, an antibody, peptide, or aptamer "interacts
with" PCSK9 when the equilibrium dissociation constant is equal to
or less than 20 nM, preferably less than about 6 nM, more
preferably less than about 1 nM, most preferably less than about
0.2 nM, as measured by the methods disclosed herein in Example
2.
[0074] An epitope that "preferentially binds" or "specifically
binds" (used interchangeably herein) to an antibody or a
polypeptide is a term well understood in the art, and methods to
determine such specific or preferential binding are also well known
in the art. A molecule is said to exhibit "specific binding" or
"preferential binding" if it reacts or associates more frequently,
more rapidly, with greater duration and/or with greater affinity
with a particular cell or substance than it does with alternative
cells or substances. An antibody "specifically binds" or
"preferentially binds" to a target if it binds with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other substances. For example, an antibody that
specifically or preferentially binds to a PCSK9 epitope is an
antibody that binds this epitope with greater affinity, avidity,
more readily, and/or with greater duration than it binds to other
PCSK9 epitopes or non-PCSK9 epitopes. It is also understood by
reading this definition that, for example, an antibody (or moiety
or epitope) that specifically or preferentially binds to a first
target may or may not specifically or preferentially bind to a
second target. As such, "specific binding" or "preferential
binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
binding means preferential binding.
[0075] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from contaminants), more
preferably, at least 90% pure, more preferably, at least 95% pure,
yet more preferably, at least 98% pure, and most preferably, at
least 99% pure.
[0076] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0077] As known in the art, the term "Fc region" is used to define
a C-terminal region of an immunoglobulin heavy chain. The "Fc
region" may be a native sequence Fc region or a variant Fc region.
Although the boundaries of the Fc region of an immunoglobulin heavy
chain might vary, the human IgG heavy chain Fc region is usually
defined to stretch from an amino acid residue at position Cys226,
or from Pro230, to the carboxyl-terminus thereof. The numbering of
the residues in the Fc region is that of the EU index as in Kabat.
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991. The Fc region of an immunoglobulin generally comprises
two constant domains, CH2 and CH3.
[0078] As used in the art, "Fc receptor" and "FcR" describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native sequence human FcR. Moreover, a preferred FcR is
one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof. FcRs are
reviewed in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92;
Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al.,
1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., 1976 J. Immunol.,
117:587; and Kim et al., 1994, J. Immunol., 24:249).
[0079] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
portion thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
portion thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0080] By an antibody with an epitope that "overlaps" with another
(second) epitope or with a surface on PCSK9 that interacts with the
EGF-like domain of the LDLR is meant the sharing of space in terms
of the PCSK9 residues that are interacted with. To calculate the
percent of overlap, for example, the percent overlap of the claimed
antibody's PCSK9 epitope with the surface of PCSK9 which interacts
with the EGF-like domain of the LDLR, the surface area of PCSK9
buried when in complex with the LDLR is calculated on a per-residue
basis. The buried area is also calculated for these residues in the
PCSK9:antibody complex. To prevent more than 100% possible overlap,
surface area for residues that have higher buried surface area in
the PCSK9:antibody complex than in LDLR:PCSK9 complex is set to
values from the LDLR:PCSK9 complex (100%). Percent surface overlap
is calculated by summing over all of the LDLR:PCSK9 interacting
residues and is weighted by the interaction area.
[0081] A "functional Fc region" possesses at least one effector
function of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity;
Fc receptor binding; antibody-dependent cell-mediated cytotoxicity;
phagocytosis; down-regulation of cell surface receptors (e.g., B
cell receptor), etc. Such effector functions generally require the
Fc region to be combined with a binding domain (e.g., an antibody
variable domain) and can be assessed using various assays known in
the art for evaluating such antibody effector functions.
[0082] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
which differs from that of a native sequence Fc region by virtue of
at least one amino acid modification, yet retains at least one
effector function of the native sequence Fc region. Preferably, the
variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g., from about one to about ten amino acid
substitutions, and preferably, from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and most preferably, at least about 90% sequence
identity therewith, more preferably, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, at least about
99% sequence identity therewith.
[0083] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, one or more of the following: enhancement of LDL
clearance and reducing incidence or amelioration of aberrant
cholesterol and/or lipoprotein levels resulting from metabolic
and/or eating disorders, or including familial
hypercholesterolemia, atherogenic dyslipidemia, atherosclerosis,
and, more generally, cardiovascular disease (CVD).
[0084] "Reducing incidence" means any of reducing severity (which
can include reducing need for and/or amount of (e.g., exposure to)
other drugs and/or therapies generally used for this condition. As
is understood by those skilled in the art, individuals may vary in
terms of their response to treatment, and, as such, for example, a
"method of reducing incidence" reflects administering the PCSK9
antagonist antibody, peptide, or aptamer based on a reasonable
expectation that such administration may likely cause such a
reduction in incidence in that particular individual.
[0085] "Ameliorating" means a lessening or improvement of one or
more symptoms as compared to not administering a PCSK9 antagonist
antibody, peptide, or aptamer. "Ameliorating" also includes
shortening or reduction in duration of a symptom.
[0086] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to effect any one or more beneficial or desired results.
For prophylactic use, beneficial or desired results include
eliminating or reducing the risk, lessening the severity, or
delaying the outset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as reducing
hypercholesterolemia or one or more symptoms of dyslipidemia,
atherosclerosis, CVD, or coronary heart disease, decreasing the
dose of other medications required to treat the disease, enhancing
the effect of another medication, and/or delaying the progression
of the disease of patients. An effective dosage can be administered
in one or more administrations. For purposes of this invention, an
effective dosage of drug, compound, or pharmaceutical composition
is an amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0087] An "individual" or a "subject" is a mammal, more preferably,
a human. Mammals also include, but are not limited to, farm
animals, sport animals, pets, primates, horses, dogs, cats, mice
and rats.
[0088] As used herein, "vector" means a construct, which is capable
of delivering, and, preferably, expressing, one or more gene(s) or
sequence(s) of interest in a host cell.
[0089] Examples of vectors include, but are not limited to, viral
vectors, naked DNA or RNA expression vectors, plasmid, cosmid or
phage vectors, DNA or RNA expression vectors associated with
cationic condensing agents, DNA or RNA expression vectors
encapsulated in liposomes, and certain eukaryotic cells, such as
producer cells.
[0090] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0091] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical acceptable excipient" includes any material which,
when combined with an active ingredient, allows the ingredient to
retain biological activity and is non-reactive with the subject's
immune system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline (PBS) or
normal (0.9%) saline. Compositions comprising such carriers are
formulated by well known conventional methods (see, for example,
Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science
and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).
[0092] The term "k.sub.on", as used herein, refers to the rate
constant for association of an antibody to an antigen.
Specifically, the rate constants (k.sub.on and k.sub.off) and
equilibrium dissociation constants are measured using Fab antibody
fragments (i.e., univalent) and PCSK9.
[0093] The term "k.sub.off", as used herein, refers to the rate
constant for dissociation of an antibody from the antibody/antigen
complex.
[0094] The term "K.sub.D", as used herein, refers to the
equilibrium dissociation constant of an antibody-antigen
interaction.
A. Methods for Preventing or Treating Disorders Associated with
Hypercholesterolemia
[0095] In one aspect, the invention provides a method for treating
or preventing hypercholesterolemia, and/or at least one symptom of
dyslipidemia, atherosclerosis, CVD or coronary heart disease, in an
individual comprising administering to the individual an effective
amount of a PCSK9 antagonist antibody or peptide or aptamer that
antagonizes circulating PCSK9.
[0096] In a further aspect, the invention provides an effective
amount of a PCSK9 antagonist antibody, peptide, or aptamer that
antagonizes circulating PCSK9 for use in treating or preventing
hypercholesterolemia, and/or at least one symptom of dyslipidemia,
atherosclerosis, CVD or coronary heart disease, in an individual.
The invention further provides the use of an effective amount of a
PCSK9 antagonist antibody, peptide, or aptamer that antagonizes
extracellular or circulating PCSK9 in the manufacture of a
medicament for treating or preventing hypercholesterolemia, and/or
at least one symptom of dyslipidemia, atherosclerosis, CVD or
coronary heart disease, in an individual.
[0097] Advantageously, therapeutic administration of the antibody,
peptide, or aptamer results in lower blood cholesterol and/or lower
blood LDL. Preferably, blood cholesterol and/or blood LDL is at
least about 10% or 15% lower than before administration. More
preferably, blood cholesterol and/or blood LDL is at least about
20% lower than before administration of the antibody. Yet more
preferably, blood cholesterol and/or blood LDL is at least 30%
lower than before administration of the antibody. Advantageously,
blood cholesterol and/or blood LDL is at least 40% lower than
before administration of the antibody. More advantageously, blood
cholesterol and/or blood LDL is at least 50% lower than before
administration of the antibody. Very preferably, blood cholesterol
and/or blood LDL is at least 60% lower than before administration
of the antibody. Most preferably, blood cholesterol and/or blood
LDL is at least 70% lower than before administration of the
antibody.
[0098] With respect to all methods described herein, reference to
PCSK9 antagonist antibodies, peptides, and aptamers also include
compositions comprising one or more additional agents. These
compositions may further comprise suitable excipients, such as
pharmaceutically acceptable excipients including buffers, which are
well known in the art. The present invention can be used alone or
in combination with other conventional methods of treatment.
[0099] The PCSK9 antagonist antibody, peptide, or aptamer can be
administered to an individual via any suitable route. It should be
apparent to a person skilled in the art that the examples described
herein are not intended to be limiting but to be illustrative of
the techniques available. Accordingly, in some embodiments, the
PCSK9 antagonist antibody, peptide, or aptamer is administered to
an individual in accord with known methods, such as intravenous
administration, e.g., as a bolus or by continuous infusion over a
period of time, by intramuscular, intraperitoneal,
intracerebrospinal, transdermal, subcutaneous, intra-articular,
sublingually, intrasynovial, via insufflation, intrathecal, oral,
inhalation or topical routes. Administration can be systemic, e.g.,
intravenous administration, or localized. Commercially available
nebulizers for liquid formulations, including jet nebulizers and
ultrasonic nebulizers are useful for administration. Liquid
formulations can be directly nebulized and lyophilized powder can
be nebulized after reconstitution. Alternatively, PCSK9 antagonist
antibody, peptide, or aptamer can be aerosolized using a
fluorocarbon formulation and a metered dose inhaler, or inhaled as
a lyophilized and milled powder.
[0100] In one embodiment, a PCSK9 antagonist antibody, peptide, or
aptamer is administered via site-specific or targeted local
delivery techniques. Examples of site-specific or targeted local
delivery techniques include various implantable depot sources of
the PCSK9 antagonist antibody, peptide, or aptamer or local
delivery catheters, such as infusion catheters, indwelling
catheters, or needle catheters, synthetic grafts, adventitial
wraps, shunts and stents or other implantable devices, site
specific carriers, direct injection, or direct application. See,
e.g., PCT Publ. No. WO 00/53211 and U.S. Pat. No. 5,981,568.
[0101] Various formulations of a PCSK9 antagonist antibody,
peptide, or aptamer may be used for administration. In some
embodiments, the PCSK9 antagonist antibody, peptide, or aptamer may
be administered neat. In some embodiments, PCSK9 antagonist
antibody, peptide, or aptamer and a pharmaceutically acceptable
excipient may be in various formulations. Pharmaceutically
acceptable excipients are known in the art, and are relatively
inert substances that facilitate administration of a
pharmacologically effective substance. For example, an excipient
can give form or consistency, or act as a diluent. Suitable
excipients include but are not limited to stabilizing agents,
wetting and emulsifying agents, salts for varying osmolarity,
encapsulating agents, buffers, and skin penetration enhancers.
Excipients as well as formulations for parenteral and nonparenteral
drug delivery are set forth in Remington, The Science and Practice
of Pharmacy, 20th Ed., Mack Publishing (2000).
[0102] These agents can be combined with pharmaceutically
acceptable vehicles such as saline, Ringer's solution, dextrose
solution, and the like. The particular dosage regimen, i.e., dose,
timing and repetition, will depend on the particular individual and
that individual's medical history.
[0103] PCSK9 antibodies can also be administered via inhalation, as
described herein. Generally, for administration of PCSK9
antibodies, an initial candidate dosage can be about 2 mg/kg. For
the purpose of the present invention, a typical daily dosage might
range from about any of about 3 .mu.g/kg to 30 .mu.g/kg to 300
.mu.g/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending
on the factors mentioned above. For example, dosage of about 1
mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25
mg/kg may be used. For repeated administrations over several days
or longer, depending on the condition, the treatment is sustained
until a desired suppression of symptoms occurs or until sufficient
therapeutic levels are achieved, for example, to reduce blood LDL
levels. An exemplary dosing regimen comprises administering an
initial dose of about 2 mg/kg, followed by a weekly maintenance
dose of about 1 mg/kg of the PCSK9 antibody, or followed by a
maintenance dose of about 1 mg/kg every other week. However, other
dosage regimens may be useful, depending on the pattern of
pharmacokinetic decay that the practitioner wishes to achieve. For
example, in some embodiments, dosing from one to four times a week
is contemplated. In other embodiments dosing once a month or once
every other month or every three months is contemplated. The
progress of this therapy is easily monitored by conventional
techniques and assays. The dosing regimen (including the PCSK9
antagonist(s) used) can vary over time.
[0104] For the purpose of the present invention, the appropriate
dosage of a PCSK9 antagonist antibody, peptide, or aptamer will
depend on the PCSK9 antagonist antibody, peptide, or aptamer (or
compositions thereof) employed, the type and severity of symptoms
to be treated, whether the agent is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical
history and response to the agent, the patient's blood PCSK9
levels, the patient's synthesis and clearance rate for PCSK9, the
patient's clearance rate for the administered agent, and the
discretion of the attending physician. Typically the clinician will
administer a PCSK9 antagonist antibody, peptide, or aptamer until a
dosage is reached that achieves the desired result. Dose and/or
frequency can vary over course of treatment. Empirical
considerations, such as the half-life, generally will contribute to
the determination of the dosage. For example, antibodies that are
compatible with the human immune system, such as humanized
antibodies or fully human antibodies, may be used to prolong
half-life of the antibody and to prevent the antibody being
attacked by the host's immune system. Frequency of administration
may be determined and adjusted over the course of therapy, and is
generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or delay of symptoms, e.g.,
hypercholesterolemia. Alternatively, sustained continuous release
formulations of PCSK9 antagonist antibodies may be appropriate.
Various formulations and devices for achieving sustained release
are known in the art.
[0105] In one embodiment, dosages for an antagonist antibody,
peptide, or aptamer may be determined empirically in individuals
who have been given one or more administration(s) of an antagonist
antibody, peptide, or aptamer. Individuals are given incremental
dosages of a PCSK9 antagonist antibody, peptide, or aptamer. To
assess efficacy, an indicator of the disease can be followed.
[0106] Administration of a PCSK9 antagonist antibody, peptide, or
aptamer in accordance with the method in the present invention can
be continuous or intermittent, depending, for example, upon the
recipient's physiological condition, whether the purpose of the
administration is therapeutic or prophylactic, and other factors
known to skilled practitioners. The administration of a PCSK9
antagonist antibody, peptide, or aptamer may be essentially
continuous over a preselected period of time or may be in a series
of spaced doses.
[0107] In some embodiments, more than one antagonist antibody,
peptide, or aptamer may be present. At least one, at least two, at
least three, at least four, at least five different, or more
antagonist antibodies and/or peptides can be present. Generally,
those PCSK9 antagonist antibodies or peptides may have
complementary activities that do not adversely affect each other. A
PCSK9 antagonist antibody, peptide, or aptamer can also be used in
conjunction with other PCSK9 antagonists or PCSK9 receptor
antagonists. For example, one or more of the following PCSK9
antagonists may be used: an antisense molecule directed to a PCSK9
(including an anti-sense molecule directed to a nucleic acid
encoding PCSK9), a PCSK9 inhibitory compound, and a PCSK9
structural analog. A PCSK9 antagonist antibody, peptide, or aptamer
can also be used in conjunction with other agents that serve to
enhance and/or complement the effectiveness of the agents.
[0108] Acceptable carriers, excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and may
comprise buffers such as phosphate, citrate, and other organic
acids; salts such as sodium chloride; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens, such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0109] Liposomes containing the PCSK9 antagonist antibody, peptide,
or aptamer are prepared by methods known in the art, such as
described in Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA
82:3688; Hwang, et al., 1980, Proc. Natl Acad. Sci. USA 77:4030;
and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced
circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0110] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy, 20th Ed., Mack Publishing (2000).
[0111] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or 'poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0112] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes.
[0113] Therapeutic PCSK9 antagonist antibody, peptide, or aptamer
compositions are generally placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0114] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Infralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.,
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g., egg phospholipids, soybean phospholipids or soybean
lecithin) and water. It will be appreciated that other ingredients
may be added, for example glycerol or glucose, to adjust the
tonicity of the emulsion. Suitable emulsions will typically contain
up to 20% oil, for example, between 5 and 20%. The fat emulsion can
comprise fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1
and 0.5 .mu.m, and have a pH in the range of 5.5 to 8.0.
[0115] The emulsion compositions can be those prepared by mixing a
PCSK9 antagonist antibody, peptide, or aptamer with Intralipid.TM.
or the components thereof (soybean oil, egg phospholipids, glycerol
and water).
[0116] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in preferably
sterile pharmaceutically acceptable solvents may be nebulised by
use of gases. Nebulised solutions may be breathed directly from the
nebulising device or the nebulising device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
B. PCSK9 Antagonists
[0117] The methods of the invention use a PCSK9 antagonist
antibody, peptide, or aptamer, which refers to any peptide or
nucleic acid molecule that blocks, suppresses or reduces (including
significantly reduces) PCSK9 biological activity, including
downstream pathways mediated by PCSK9 signaling, such as
elicitation of a cellular response to PCSK9.
[0118] A PCSK9 antagonist antibody, peptide, or aptamer should
exhibit any one or more of the following characteristics: (a) bind
to PCSK9; (b) block PCSK9 interaction with the LDLR; (c) block or
decrease PCSK9-mediated down-regulation of the LDLR; (d) inhibit
the PCSK9-mediated decrease in LDL blood clearance, (e) increase
LDL clearance in media by cultured hepatocytes, (f) increase blood
LDL clearance by the liver in vivo, (g) sensitize to statins, and
(h) block PCSK9 interaction with other yet to be identified
factors.
[0119] For purposes of this invention, the antibody, peptide, or
aptamer preferably reacts with PCSK9 in a manner that inhibits
PCSK9 signaling function and LDLR interaction. In some embodiments,
the PCSK9 antagonist antibody specifically recognizes primate
PCSK9. In some embodiments, the PCSK9 antagonist antibody binds
primate and rodent PCSK9.
[0120] The antibodies useful in the present invention can encompass
monoclonal antibodies, polyclonal antibodies, antibody fragments
(e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies,
bispecific antibodies, heteroconjugate antibodies, single chain
(ScFv), mutants thereof, fusion proteins comprising an antibody
portion (e.g., a domain antibody), human antibodies, humanized
antibodies, and any other modified configuration of the
immunoglobulin molecule that comprises an antigen recognition site
of the required specificity, including glycosylation variants of
antibodies, amino acid sequence variants of antibodies, and
covalently modified antibodies. The antibodies may be murine, rat,
human, or any other origin (including chimeric or humanized
antibodies).
[0121] In some embodiments, the PCSK9 antagonist antibody is a
monoclonal antibody. The PCSK9 antagonist antibody can also be
humanized. In other embodiments, the antibody is human.
[0122] In some embodiments, the antibody comprises a modified
constant region, such as a constant region that is immunologically
inert, that is, having a reduced potential for provoking an immune
response. In some embodiments, the constant region is modified as
described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Publ. No.
WO99/58572; and/or UK Patent Application No. 9809951.8. The Fc can
be human IgG.sub.2 or human IgG.sub.4. The Fc can be human
IgG.sub.2 containing the mutation A330P331 to S330S331
(IgG.sub.2.DELTA.a), in which the amino acid residues are numbered
with reference to the wild type IgG2 sequence. Eur. J. Immunol.,
1999, 29:2613-2624. In some embodiments, the antibody comprises a
constant region of IgG.sub.4 comprising the following mutations
(Armour et al., 2003, Molecular Immunology 40 585-593):
E233F234L235 to P233V234A235 (IgG.sub.4.DELTA.c), in which the
numbering is with reference to wild type IgG4. In yet another
embodiment, the Fc is human IgG.sub.4 E233F234L235 to P233V234A235
with deletion G236 (IgG.sub.4.DELTA.b). In another embodiment the
Fc is any human IgG.sub.4 Fc (IgG.sub.4, IgG.sub.4.DELTA.b or
IgG.sub.4.DELTA.c) containing hinge stabilizing mutation S228 to
P228 (Aalberse et al., 2002, Immunology 105, 9-19). In another
embodiment, the Fc can be aglycosylated Fc.
[0123] In some embodiments, the constant region is aglycosylated by
mutating the oligosaccharide attachment residue (such as Asn297)
and/or flanking residues that are part of the glycosylation
recognition sequence in the constant region. In some embodiments,
the constant region is aglycosylated for N-linked glycosylation
enzymatically. The constant region may be aglycosylated for
N-linked glycosylation enzymatically or by expression in a
glycosylation deficient host cell.
[0124] The binding affinity (K.sub.D) of a PCSK9 antagonist
antibody to PCSK9 (such as human PCSK9)) can be about 0.002 to
about 200 nM. In some embodiments, the binding affinity is any of
about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM,
about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM,
about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In some
embodiments, the binding affinity is less than any of about 250 nM,
about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM,
about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM,
about 5 pM, or about 2 pM.
[0125] One way of determining binding affinity of antibodies to
PCSK9 is by measuring binding affinity of monofunctional Fab
fragments of the antibody. To obtain monofunctional Fab fragments,
an antibody (for example, IgG) can be cleaved with papain or
expressed recombinantly. The affinity of a PCSK9 Fab fragment of an
antibody can be determined by surface plasmon resonance
(Biacore3000.TM. surface plasmon resonance (SPR) system, Biacore,
INC, Piscataway N.J.) equipped with pre-immobilized streptavidin
sensor chips (SA) using HBS-EP running buffer (0.01M HEPES, pH 7.4,
0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Biotinylated
human PCSK9 (or any other PCSK9) can be diluted into HBS-EP buffer
to a concentration of less than 0.5 .mu.g/mL and injected across
the individual chip channels using variable contact times, to
achieve two ranges of antigen density, either 50-200 response units
(RU) for detailed kinetic studies or 800-1,000 RU for screening
assays. Regeneration studies have shown that 25 mM NaOH in 25% v/v
ethanol effectively removes the bound Fab while keeping the
activity of PCSK9 on the chip for over 200 injections. Typically,
serial dilutions (spanning concentrations of 0.1-10.times.
estimated K.sub.D) of purified Fab samples are injected for 1 min
at 100 .mu.L/minute and dissociation times of up to 2 hours are
allowed. The concentrations of the Fab proteins are determined by
ELISA and/or SDS-PAGE electrophoresis using a Fab of known
concentration (as determined by amino acid analysis) as a standard.
Kinetic association rates (k.sub.on) and dissociation rates
(k.sub.off) are obtained simultaneously by fitting the data
globally to a 1:1 Langmuir binding model (Karlsson, R. Roos, H.
Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6. 99-110)
using the BIAevaluation program. Equilibrium dissociation constant
(K.sub.D) values are calculated as k.sub.off/k.sub.on. This
protocol is suitable for use in determining binding affinity of an
antibody to any PCSK9, including human PCSK9, PCSK9 of another
mammalian (such as mouse PCSK9, rat PCSK9, primate PCSK9), as well
as different forms of PCSK9 (such as a and 13 form). Binding
affinity of an antibody is generally measured at 25.degree. C., but
can also be measured at 37.degree. C.
[0126] The PCSK9 antagonist antibodies may be made by any method
known in the art including the method as provided in Example 1. The
route and schedule of immunization of the host animal are generally
in keeping with established and conventional techniques for
antibody stimulation and production, as further described herein.
General techniques for production of human and mouse antibodies are
known in the art and/or are described herein. A currently preferred
method of making the antibodies comprises the immunization of
PCSK9.sup.- knockout (PCSK9 -/-) animals as disclosed herein.
[0127] It is contemplated that any mammalian subject including
humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human,
hybridoma cell lines. Typically, the host animal is inoculated
intraperitoneally, intramuscularly, orally, subcutaneously,
intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
[0128] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C., 1975,
Nature 256:495-497 or as modified by Buck, D. W., et al., 1982, In
Vitro, 18:377-381. Available myeloma lines, including but not
limited to X63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the PCSK9 monoclonal antibodies of the
subject invention. The hybridomas are expanded and subcloned, if
desired, and supernatants are assayed for anti-immunogen activity
by conventional immunoassay procedures (e.g., radioimmunoassay,
enzyme immunoassay, or fluorescence immunoassay).
[0129] Hybridomas that may be used as a source of antibodies
encompass all derivatives, progeny cells of the parent hybridomas
that produce monoclonal antibodies specific for PCSK9, or a portion
thereof.
[0130] Hybridomas that produce such antibodies may be grown in
vitro or in vivo using known procedures. The monoclonal antibodies
may be isolated from the culture media or body fluids, by
conventional immunoglobulin purification procedures such as
ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired. Undesired
activity, if present, can be removed, for example, by running the
preparation over adsorbents made of the immunogen attached to a
solid phase and eluting or releasing the desired antibodies off the
immunogen. Immunization of a host animal with a human PCSK9, or a
fragment containing the target amino acid sequence conjugated to a
protein that is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups, can yield a population of antibodies (e.g.,
monoclonal antibodies).
[0131] If desired, the PCSK9 antagonist antibody (monoclonal or
polyclonal) of interest may be sequenced and the polynucleotide
sequence may then be cloned into a vector for expression or
propagation. The sequence encoding the antibody of interest may be
maintained in vector in a host cell and the host cell can then be
expanded and frozen for future use. Production of recombinant
monoclonal antibodies in cell culture can be carried out through
cloning of antibody genes from B cells by means known in the art.
See, e.g., Tiller et al., 2008, J. Immunol. Methods 329, 112; U.S.
Pat. No. 7,314,622.
[0132] In an alternative, the polynucleotide sequence may be used
for genetic manipulation to "humanize" the antibody or to improve
the affinity, or other characteristics of the antibody. For
example, the constant region may be engineered to more nearly
resemble human constant regions to avoid immune response if the
antibody is used in clinical trials and treatments in humans. It
may be desirable to genetically manipulate the antibody sequence to
obtain greater affinity to PCSK9 and greater efficacy in inhibiting
PCSK9. It will be apparent to one of skill in the art that one or
more polynucleotide changes can be made to the PCSK9 antagonist
antibody and still maintain its binding ability to PCSK9.
[0133] There are four general steps to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains; (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process; (3) the actual humanizing
methodologies/techniques; and (4) the transfection and expression
of the humanized antibody. See, for example, U.S. Pat. Nos.
4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;
5,693,762; 5,585,089; and 6,180,370.
[0134] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent or
modified rodent V regions and their associated CDRs fused to human
constant domains. See, for example, Winter et al., 1991, Nature
349:293-299; Lobuglio et al., 1989, Proc. Nat. Acad. Sci. USA
86:4220-4224; Shaw et al., 1987, J Immunol. 138:4534-4538; and
Brown et al., 1987, Cancer Res. 47:3577-3583. Other references
describe rodent CDRs grafted into a human supporting framework
region (FR) prior to fusion with an appropriate human antibody
constant domain. See, for example, Riechmann et al., 1988, Nature
332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536; and
Jones et al., 1986, Nature 321:522-525. Another reference describes
rodent CDRs supported by recombinantly engineered rodent framework
regions. See, for example, European Patent Publ. No. 0519596. These
"humanized" molecules are designed to minimize unwanted
immunological response toward rodent anti-human antibody molecules
which limits the duration and effectiveness of therapeutic
applications of those moieties in human recipients. For example,
the antibody constant region can be engineered such that it is
immunologically inert (e.g., does not trigger complement lysis).
See, e.g., PCT Publ. No. WO99/58572; UK Patent Application No.
9809951.8. Other methods of humanizing antibodies that may also be
utilized are disclosed by Daugherty et al., 1991, Nucl. Acids Res.
19:2471-2476 and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867;
5,866,692; 6,210,671; and 6,350,861; and in PCT Publ. No. WO
01/27160.
[0135] In yet another alternative, fully human antibodies may be
obtained by using commercially available mice that have been
engineered to express specific human immunoglobulin proteins.
Transgenic animals that are designed to produce a more desirable or
more robust immune response may also be used for generation of
humanized or human antibodies. Examples of such technology are
Xenomouse.TM. from Abgenix, Inc. (Fremont, Calif.),
HuMAb-Mouse.RTM. and TC Mouse.TM. from Medarex, Inc. (Princeton,
N.J.), and the Veloclmmune.RTM. mouse from Regeneron
Pharmaceuticals, Inc. (Tarrytown, N.Y.).
[0136] In an alternative, antibodies may be made recombinantly and
expressed using any method known in the art. In another
alternative, antibodies may be made recombinantly by phage display
technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717;
5,733,743; and 6,265,150; and Winter et al., 1994, Annu. Rev.
Immunol. 12:433-455. Alternatively, the phage display technology
(McCafferty et al., 1990, Nature 348:552-553) can be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. According to this technique, antibody V domain
genes are cloned in-frame into either a major or minor coat protein
gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B cell. Phage
display can be performed in a variety of formats; see, e.g.,
Johnson, Kevin S. and Chiswell, David J., 1993, Current Opinion in
Structural Biology 3:564-571. Several sources of V-gene segments
can be used for phage display. Clackson et al., 1991, Nature
352:624-628 isolated a diverse array of anti-oxazolone antibodies
from a small random combinatorial library of V genes derived from
the spleens of immunized mice. A repertoire of V genes from
unimmunized human donors can be constructed and antibodies to a
diverse array of antigens (including self-antigens) can be isolated
essentially following the techniques described by Mark et al.,
1991, J. Mol. Biol. 222:581-597, or Griffith et al., 1993, EMBO J.
12:725-734. In a natural immune response, antibody genes accumulate
mutations at a high rate (somatic hypermutation). Some of the
changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling." (Marks et al., 1992, Bio/Technol.
10:779-783). In this method, the affinity of "primary" human
antibodies obtained by phage display can be improved by
sequentially replacing the heavy and light chain V region genes
with repertoires of naturally occurring variants (repertoires) of V
domain genes obtained from unimmunized donors. This technique
allows the production of antibodies and antibody fragments with
affinities in the pM-nM range. A strategy for making very large
phage antibody repertoires (also known as "the mother-of-all
libraries") has been described by Waterhouse et al., 1993, Nucl.
Acids Res. 21:2265-2266. Gene shuffling can also be used to derive
human antibodies from rodent antibodies, where the human antibody
has similar affinities and specificities to the starting rodent
antibody. According to this method, which is also referred to as
"epitope imprinting", the heavy or light chain V domain gene of
rodent antibodies obtained by phage display technique is replaced
with a repertoire of human V domain genes, creating rodent-human
chimeras. Selection on antigen results in isolation of human
variable regions capable of restoring a functional antigen-binding
site, i.e., the epitope governs (imprints) the choice of partner.
When the process is repeated in order to replace the remaining
rodent V domain, a human antibody is obtained (see PCT Publ. No. WO
93/06213). Unlike traditional humanization of rodent antibodies by
CDR grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin.
[0137] It is apparent that although the above discussion pertains
to humanized antibodies, the general principles discussed are
applicable to customizing antibodies for use, for example, in dogs,
cats, primate, equines and bovines. It is further apparent that one
or more aspects of humanizing an antibody described herein may be
combined, e.g., CDR grafting, framework mutation and CDR
mutation.
[0138] Antibodies may be made recombinantly by first isolating the
antibodies and antibody producing cells from host animals,
obtaining the gene sequence, and using the gene sequence to express
the antibody recombinantly in host cells (e.g., CHO cells). Another
method which may be employed is to express the antibody sequence in
plants (e.g., tobacco) or transgenic milk. Methods for expressing
antibodies recombinantly in plants or milk have been disclosed.
See, for example, Peeters, 2001, et al. Vaccine 19:2756; Lonberg,
N. and D. Huszar, 1995, Int. Rev. Immunol 13:65; and Pollock, et
al., 1999, J Immunol Methods 231:147. Methods for making
derivatives of antibodies, e.g., humanized, single chain, etc. are
known in the art.
[0139] Immunoassays and flow cytometry sorting techniques such as
fluorescence activated cell sorting (FACS) can also be employed to
isolate antibodies that are specific for PCSK9.
[0140] The antibodies can be bound to many different carriers.
Carriers can be active and/or inert. Examples of well-known
carriers include polypropylene, polystyrene, polyethylene, dextran,
nylon, amylases, glass, natural and modified celluloses,
polyacrylamides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding antibodies, or will be able to ascertain such, using
routine experimentation. In some embodiments, the carrier comprises
a moiety that targets the myocardium.
[0141] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors (such as expression vectors disclosed in PCT Publ. No. WO
87/04462), which are then transfected into host cells such as E.
coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. See, e.g., PCT Publ. No. WO 87/04462. The DNA also may
be modified, for example, by substituting the coding sequence for
human heavy and light chain constant domains in place of the
homologous murine sequences, Morrison et al., 1984, Proc. Nat.
Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. In that manner, "chimeric" or
"hybrid" antibodies are prepared that have the binding specificity
of a PCSK9 monoclonal antibody herein.
[0142] PCSK9 antagonist antibodies and polypeptides derived from
antibodies can be identified or characterized using methods known
in the art, whereby reduction, amelioration, or neutralization of
PCSK9 biological activity is detected and/or measured. In some
embodiments, a PCSK9 antagonist antibody or polypeptide is
identified by incubating a candidate agent with PCSK9 and
monitoring binding and/or attendant reduction or neutralization of
a biological activity of PCSK9. The binding assay may be performed
with purified PCSK9 polypeptide(s), or with cells naturally
expressing, or transfected to express, PCSK9 polypeptide(s). In one
embodiment, the binding assay is a competitive binding assay, where
the ability of a candidate antibody to compete with a known PCSK9
antagonist for PCSK9 binding is evaluated. The assay may be
performed in various formats, including the ELISA format. In other
embodiments, a PCSK9 antagonist antibody is identified by
incubating a candidate agent with PCSK9 and monitoring binding and
attendant inhibition of LDLR expression and/or blood cholesterol
clearance.
[0143] Following initial identification, the activity of a
candidate PCSK9 antagonist antibody can be further confirmed and
refined by bioassays that are known to test the targeted biological
activities. Alternatively, bioassays can be used to screen
candidates directly. Some of the methods for identifying and
characterizing PCSK9 antagonist antibodies, peptides, or aptamers
are described in detail in the Examples.
[0144] PCSK9 antagonist antibodies may be characterized using
methods well known in the art. For example, one method is to
identify the epitope to which it binds, or "epitope mapping." There
are many methods known in the art for mapping and characterizing
the location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N. Y., 1999). In an additional example,
epitope mapping can be used to determine the sequence to which a
PCSK9 antagonist antibody binds. Epitope mapping is commercially
available from various sources, for example, Pepscan Systems
(Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope
can be a linear epitope, i.e., contained in a single stretch of
amino acids, or a conformational epitope formed by a
three-dimensional interaction of amino acids that may not
necessarily be contained in a single stretch. Peptides of varying
lengths (e.g., at least 4-6 amino acids long) can be isolated or
synthesized (e.g., recombinantly) and used for binding assays with
a PCSK9 antagonist antibody. In another example, the epitope to
which the PCSK9 antagonist antibody binds can be determined in a
systematic screening by using overlapping peptides derived from the
PCSK9 sequence and determining binding by the PCSK9 antagonist
antibody. According to the gene fragment expression assays, the
open reading frame encoding PCSK9 is fragmented either randomly or
by specific genetic constructions and the reactivity of the
expressed fragments of PCSK9 with the antibody to be tested is
determined. The gene fragments may, for example, be produced by PCR
and then transcribed and translated into protein in vitro, in the
presence of radioactive amino acids. The binding of the antibody to
the radioactively labeled PCSK9 fragments is then determined by
immunoprecipitation and gel electrophoresis. Certain epitopes can
also be identified by using large libraries of random peptide
sequences displayed on the surface of phage particles (phage
libraries). Alternatively, a defined library of overlapping peptide
fragments can be tested for binding to the test antibody in simple
binding assays. In an additional example, mutagenesis of an antigen
binding domain, domain swapping experiments and alanine scanning
mutagenesis can be performed to identify residues required,
sufficient, and/or necessary for epitope binding. For example,
domain swapping experiments can be performed using a mutant PCSK9
in which various fragments of the PCSK9 polypeptide have been
replaced (swapped) with sequences from PCSK9 from another species,
or a closely related, but antigenically distinct protein (such as
another member of the proprotein convertase family). By assessing
binding of the antibody to the mutant PCSK9, the importance of the
particular PCSK9 fragment to antibody binding can be assessed.
[0145] Yet another method which can be used to characterize a PCSK9
antagonist antibody is to use competition assays with other
antibodies known to bind to the same antigen, i.e., various
fragments on PCSK9, to determine if the PCSK9 antagonist antibody
binds to the same epitope as other antibodies. Competition assays
are well known to those of skill in the art.
[0146] The crystal structure of the antibody and antibody:antigen
complex can also be used to characterize the antibody. The residues
are identified by calculating the difference in accessible surface
area between the L1L3:PCSK9 crystal structure and PCSK9 structure
alone. PCSK9 residues that show buried surface area upon complex
formation with L1 L3 antibody are included as a part of the
epitope. The solvent accessible surface of a protein is defined as
the locus of the centre of a probe sphere (representing a solvent
molecule of 1.4 .ANG. radius) as it rolls over the Van der Waals
surface of the protein. The solvent accessible surface area is
calculated by generating surface points on an extended sphere about
each atom (at a distance from the atom centre equal to the sum of
the atom and probe radii), and eliminating those that lie within
equivalent spheres associated with neighboring atoms as implemented
in program AREAIMOL (Briggs, P. J., 2000, CCP4 Newsletter No. 38,
CCLRC, Daresbury).
[0147] An expression vector can be used to direct expression of a
PCSK9 antagonist antibody. One skilled in the art is familiar with
administration of expression vectors to obtain expression of an
exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;
6,413,942; and 6,376,471. Administration of expression vectors
includes local or systemic administration, including injection,
oral administration, particle gun or catheterized administration,
and topical administration. In another embodiment, the expression
vector is administered directly to the sympathetic trunk or
ganglion, or into a coronary artery, atrium, ventrical, or
pericardium.
[0148] Targeted delivery of therapeutic compositions containing an
expression vector, or subgenomic polynucleotides can also be used.
Receptor-mediated DNA delivery techniques are described in, for
example, Findeis et al., 1993, Trends Biotechnol. 11:202; Chiou et
al., 1994, Gene Therapeutics: Methods And Applications Of Direct
Gene Transfer (J. A. Wolff, ed.); Wu et al., 1988, J. Biol. Chem.
263:621; Wu et al., 1994, J. Biol. Chem. 269:542; Zenke et al.,
1990, Proc. Natl. Acad. Sci. USA 87:3655; Wu et al., 1991, J. Biol.
Chem. 266:338. Therapeutic compositions containing a polynucleotide
are administered in a range of about 100 ng to about 200 mg of DNA
for local administration in a gene therapy protocol. Concentration
ranges of about 500 ng to about 50 mg, about 1 .mu.g to about 2 mg,
about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g to about 100
.mu.g of DNA can also be used during a gene therapy protocol. The
therapeutic polynucleotides and polypeptides can be delivered using
gene delivery vehicles. The gene delivery vehicle can be of viral
or non-viral origin (see generally, Jolly, 1994, Cancer Gene
Therapy 1:51; Kimura, 1994, Human Gene Therapy 5:845; Connelly,
1995, Human Gene Therapy 1:185; and Kaplitt, 1994, Nature Genetics
6:148). Expression of such coding sequences can be induced using
endogenous mammalian or heterologous promoters. Expression of the
coding sequence can be either constitutive or regulated.
[0149] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publ. Nos. WO 90/07936; WO
94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO
91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No.
2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors
(e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67;
ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and
Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250;
ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV)
vectors (see, e.g., PCT Publ. Nos. WO 94/12649, WO 93/03769; WO
93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration
of DNA linked to killed adenovirus as described in Curiel, 1992,
Hum. Gene Ther. 3:147, can also be employed.
[0150] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
1992, Hum. Gene Ther. 3:147); ligand-linked DNA (see, e.g., Wu, J.,
1989, Biol. Chem. 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publ. Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publ. No. WO 90/11092 and U.S. Pat. No. 5,580,859.
Liposomes that can act as gene delivery vehicles are described in
U.S. Pat. No. 5,422,120; PCT Publ. Nos. WO 95/13796; WO 94/23697;
WO 91/14445; and EP 0524968. Additional approaches are described in
Philip, 1994, Mol. Cell Biol., 14:2411, and in Woffendin, 1994
Proc. Natl. Acad. Sci. 91:1581.
[0151] This invention encompasses compositions, including
pharmaceutical compositions, comprising antibodies described herein
or made by the methods and having the characteristics described
herein. As used herein, compositions comprise one or more
antibodies, peptides, or aptamers that antagonize the interaction
of PCSK9 with the LDLR, and/or one or more polynucleotides
comprising sequences encoding one or more these antibodies or
peptides. These compositions may further comprise suitable
excipients, such as pharmaceutically acceptable excipients
including buffers, which are well known in the art.
[0152] The PCSK9 antagonist antibodies and peptides of the
invention are characterized by any (one or more) of the following
characteristics: (a) bind to PCSK9; (b) block PCSK9 interaction
with the LDLR; (c) decrease PCSK9-mediated down-regulation of the
LDLR; and (d) inhibit PCSK9-mediated inhibition of LDL blood
clearance. Preferably, PCSK9 antibodies have two or more of these
features. More preferably, the antibodies have three or more of the
features. Most preferably, the antibodies have all four
characteristics.
[0153] Accordingly, the invention provides any of the following, or
compositions (including pharmaceutical compositions) comprising any
antibody having a partial light chain sequence and a partial heavy
chain sequence as found in Table 1. The underlined sequences are
CDR sequences according to Kabat and in bold according to
Chothia.
TABLE-US-00001 TABLE 1 Light Chain Heavy Chain mAb Variable Region
Variable Region 4A5 DIVMTQSQKFMSTSV EVQLQQSGPELVKPG GDRVSVTCKASQNVG
ASVKISCKASGYTFT TNVAWYQQKPGQSPK DYYMNWVKQSHGKSL ALIYSASYRYSGVPD
EWIGDINPNNGGTTY RFTGSGSGTDFTLTI NQKFKGKATLTVDKS SNVLSEDLAEYFCQQ
YSTAYMELRSLTSED FYSYPYTFGGGTKLE SAVYYCARWLLFAYW IK GQGTLVTVSA (SEQ
ID NO: 16) (SEQ ID NO: 20) 5A10 DIVMTQSHKFMSTSV QVQLQQPGAELVKPG
GDRVSITCKASQDVS ASVKLSCKASGYTFT TAVAWYQQKPGQSPK SYWMHWVKQRPGQGL
LLIYSASYRYTGVPD EWIGEINPSNGRTNY RFTGSGSGTDFTFTI NEKFKSKATLTVDKS
SSVQAEDLAVYYCQQ SSTAYMQLSSLTSED RYSTPRTFGGGTKLE SAVYYCARERPLYAM IK
DYWGQGTSVTVSS (SEQ ID NO: 17) (SEQ ID NO: 21) 6F6 DIQMTQTTSSLSASL
EVQLQQSGPELVKPG GDRVTISCSASQGIS ASVKISCKASGYTFT NYLNWYQQKPDGTVK
DYYMNWVKQSHGKSL LLIYYTSSLHSGVPS EWIGDINPNNGGTSY RFSGSGSGTDYSLTI
NQKFKGKATLTVDKS SNLEPEDIATYYCQQ SSTAYMELRSLTSED YSKLPFTFGSGTKLE
SAVYYCAGGGIYYRY IK DRNYFDYWGQGTTLT (SEQ ID NO: 18) VSS (SEQ ID NO:
22) 7D4 DIVMTQSHKFMSTSF EVKLVESEGGLVQPG GDRVSITCKASQDVS
SSMKLSCTASGFTFS NALAWYQQKPGHSPK DYYMAWVRQVPEKGL LLIFSASYRYTGVPD
EWVANINYDGSNTSY RFTGSGSGTDFTFTI LDSLKSRFIISRDNA SSVQAEDLAVYYCQQ
KNILYLQMSSLKSED HYSTPWTFGGGTKLE TATYYCAREKFAAMD IK YWGQGTSVTVSS
(SEQ ID NO: 19) (SEQ ID NO: 23) L1L3 DIQMTQSPSSLSASV
QVQLVQSGAEVKKPG GDRVTITCRASQGIS ASVKVSCKASGYTFT SALAWYQQKPGKAPK
SYYMHWVRQAPGQGL LLIYSASYRYTGVPS EWMGEISPFGGRTNY RFSGSGSGTDFTFTI
NEKFKSRVTMTRDTS SSLQPEDIATYYCQQ TSTVYMELSSLRSED RYSLWRTFGQGTKLE
TAVYYCARERPLYAS IK DLWGQGTTVTVSS (SEQ ID NO: 53) (SEQ ID NO:
54)
[0154] The invention also provides CDR portions of antibodies to
PCSK9 (including Chothia and Kabat CDRs). Determination of CDR
regions is well within the skill of the art. It is understood that
in some embodiments, CDRs can be a combination of the Kabat and
Chothia CDR (also termed "combined CDRs" or "extended CDRs"). In
some embodiments, the CDRs are the Kabat CDRs. In other
embodiments, the CDRs are the Chothia CDRs. In other words, in
embodiments with more than one CDR, the CDRs may be any of Kabat,
Chothia, combination CDRs, or combinations thereof.
[0155] The invention also provides methods of making any of these
antibodies or polypeptides. The antibodies of this invention can be
made by procedures known in the art. The polypeptides can be
produced by proteolytic or other degradation of the antibodies, by
recombinant methods (i.e., single or fusion polypeptides) as
described above or by chemical synthesis. Polypeptides of the
antibodies, especially shorter polypeptides up to about 50 amino
acids, are conveniently made by chemical synthesis. Methods of
chemical synthesis are known in the art and are commercially
available. For example, an antibody could be produced by an
automated polypeptide synthesizer employing the solid phase method.
See also, U.S. Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.
[0156] In another alternative, the antibodies and peptides can be
made recombinantly using procedures that are well known in the art.
In one embodiment, a polynucleotide comprises a sequence encoding
the heavy chain and/or the light chain variable regions of antibody
4A5, 5A10, 6F6, 7D4 or L1L3. The sequence encoding the antibody of
interest may be maintained in a vector in a host cell and the host
cell can then be expanded and frozen for future use. Vectors
(including expression vectors) and host cells are further described
herein.
[0157] The invention also encompasses scFv of antibodies of this
invention. Single chain variable region fragments are made by
linking light and/or heavy chain variable regions by using a short
linking peptide. Bird et al., 1988, Science 242:423-426. An example
of a linking peptide is (GGGGS).sub.3 (SEQ ID NO:24), which bridges
approximately 3.5 nm between the carboxy terminus of one variable
region and the amino terminus of the other variable region. Linkers
of other sequences have been designed and used. Bird et al., 1988,
supra. Linkers should be short, flexible polypeptides and
preferably comprised of less than about 20 amino acid residues.
Linkers can in turn be modified for additional functions, such as
attachment of drugs or attachment to solid supports. The single
chain variants can be produced either recombinantly or
synthetically. For synthetic production of scFv, an automated
synthesizer can be used. For recombinant production of scFv, a
suitable plasmid containing polynucleotide that encodes the scFv
can be introduced into a suitable host cell, either eukaryotic,
such as yeast, plant, insect or mammalian cells, or prokaryotic,
such as E. coli. Polynucleotides encoding the scFv of interest can
be made by routine manipulations such as ligation of
polynucleotides. The resultant scFv can be isolated using standard
protein purification techniques known in the art.
[0158] Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see e.g., Holliger, P., et al.,
1993, Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et
al., 1994, Structure 2:1121-1123).
[0159] For example, bispecific antibodies, monoclonal antibodies
that have binding specificities for at least two different
antigens, can be prepared using the antibodies disclosed herein.
Methods for making bispecific antibodies are known in the art (see,
e.g., Suresh et al., 1986, Methods in Enzymology 121:210).
Traditionally, the recombinant production of bispecific antibodies
was based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, with the two heavy chains having different
specificities (Millstein and Cuello, 1983, Nature 305,
537-539).
[0160] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0161] In one approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure, with an immunoglobulin light chain in
only one half of the bispecific molecule, facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations. This approach is described in
PCT Publ. No. WO 94/04690.
[0162] Heteroconjugate antibodies, comprising two covalently joined
antibodies, are also within the scope of the invention. Such
antibodies have been used to target immune system cells to unwanted
cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(PCT Publ. Nos. WO 91/00360 and WO 92/200373; EP 03089).
Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents and techniques
are well known in the art, and are described in U.S. Pat. No.
4,676,980.
[0163] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods of synthetic protein chemistry, including those
involving cross-linking agents. For example, immunotoxins may be
constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0164] Humanized antibody comprising one or more CDRs of antibodies
5A10 or 7D4 or one or more CDRs derived from antibodies 5A10 or 7D4
can be made, for example, using any methods know in the art. For
example, four general steps may be used to humanize a monoclonal
antibody. These are: (1) determining the nucleotide and predicted
amino acid sequence of the starting antibody light and heavy
variable domains; (2) designing the humanized antibody, i.e.,
deciding which antibody framework region to use during the
humanizing process; (3) using the actual humanizing
methodologies/techniques; and (4) transfecting and expressing the
humanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;
5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;
5,585,089; and 6,180,370.
[0165] In the recombinant humanized antibodies, the Fc portion can
be modified to avoid interaction with Fc.gamma. receptor and the
complement and immune systems. The techniques for preparation of
such antibodies are described in WO 99/58572. For example, the
constant region may be engineered to more resemble human constant
regions to avoid immune response if the antibody is used in
clinical trials and treatments in humans. See, for example, U.S.
Pat. Nos. 5,997,867 and 5,866,692.
[0166] Humanized antibody comprising the light or heavy chain
variable regions or one or more CDRs of an antibody or its variants
shown in Table 1, or one or more CDRs derived from the antibody or
its variants shown in Table 2 can be made using any methods known
in the art.
[0167] Humanized antibodies may be made by any method known in the
art.
[0168] The invention encompasses modifications to the antibodies
and polypeptides of the invention variants shown in Table 1,
including functionally equivalent antibodies which do not
significantly affect their properties and variants which have
enhanced or decreased activity and/or affinity. For example, the
amino acid sequence may be mutated to obtain an antibody with the
desired binding affinity to PCSK9. Modification of polypeptides is
routine practice in the art and need not be described in detail
herein. Modification of polypeptides is exemplified in the
Examples. Examples of modified polypeptides include polypeptides
with conservative substitutions of amino acid residues, one or more
deletions or additions of amino acids which do not significantly
deleteriously change the functional activity, or which mature
(enhance) the affinity of the polypeptide for its ligand, or use of
chemical analogs.
[0169] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the half-life of the antibody in the
blood circulation.
[0170] Substitution variants have at least one amino acid residue
in the antibody molecule removed and a different residue inserted
in its place. The sites of greatest interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 2 under the heading of "conservative substitutions." If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in Table
2, or as further described below in reference to amino acid
classes, may be introduced and the products screened.
TABLE-US-00002 TABLE 2 Amino Acid Substitutions Conservative
Original Residue Substitutions Exemplary Substitutions Ala (A) Val
Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp,
Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn;
Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys;
Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile
Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met
(M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P)
Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr
(Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0171] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: [0172]
(1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; [0173] (2)
Polar without charge: Cys, Ser, Thr, Asn, Gln; [0174] (3) Acidic
(negatively charged): Asp, Glu; [0175] (4) Basic (positively
charged): Lys, Arg; [0176] (5) Residues that influence chain
orientation: Gly, Pro; and [0177] (6) Aromatic: Trp, Tyr, Phe,
His.
[0178] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class.
[0179] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an antibody fragment such as an Fv fragment.
[0180] Amino acid modifications can range from changing or
modifying one or more amino acids to complete redesign of a region,
such as the variable region. Changes in the variable region can
alter binding affinity and/or specificity. In some embodiments, no
more than one to five conservative amino acid substitutions are
made within a CDR domain. In other embodiments, no more than one to
three conservative amino acid substitutions are made within a CDR
domain. In still other embodiments, the CDR domain is CDR H3 and/or
CDR L3.
[0181] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Jefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect antibody-dependent cellular cytotoxicity (ADCC). In
particular, CHO cells with tetracycline-regulated expression of
13(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GIcNAc, was
reported to have improved ADCC activity (Umana et al., 1999, Nature
Biotech. 17:176-180).
[0182] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0183] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0184] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g., antibodies, as potential therapeutics is
rarely the native cell, variations in the glycosylation pattern of
the antibodies can be expected (see, e.g., Hse et al., 1997, J.
Biol. Chem. 272:9062-9070).
[0185] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example, using endoglycosidase H (Endo H),
N-glycosidase F, endoglycosidase F1, endoglycosidase F2,
endoglycosidase F3. In addition, the recombinant host cell can be
genetically engineered to be defective in processing certain types
of polysaccharides. These and similar techniques are well known in
the art.
[0186] Other methods of modification include using coupling
techniques known in the art, including, but not limited to,
enzymatic means, oxidative substitution and chelation.
Modifications can be used, for example, for attachment of labels
for immunoassay. Modified polypeptides are made using established
procedures in the art and can be screened using standard assays
known in the art, some of which are described below and in the
Examples.
[0187] In some embodiments of the invention, the antibody comprises
a modified constant region, such as a constant region that is
immunologically inert or partially inert, e.g., does not trigger
complement mediated lysis, does not stimulate ADCC, or does not
activate microglia; or have reduced activities (compared to the
unmodified antibody) in any one or more of the following:
triggering complement mediated lysis, stimulating ADCC, or
activating microglia. Different modifications of the constant
region may be used to achieve optimal level and/or combination of
effector functions. See, for example, Morgan et al., 1995,
Immunology 86:319-324; Lund et al., 1996, J. Immunology 157:4963-9
157:4963-4969; Idusogie et al., 2000, J. Immunology 164:4178-4184;
Tao et al., 1989, J. Immunology 143: 2595-2601; and Jefferis et
al., 1998, Immunological Reviews 163:59-76. In some embodiments,
the constant region is modified as described in Eur. J. Immunol.,
1999, 29:2613-2624; PCT Publ. No. WO99/58572; and/or UK Patent
Application No. 9809951.8. In other embodiments, the antibody
comprises a human heavy chain IgG2 constant region comprising the
following mutations: A330P331 to S330S331 (amino acid numbering
with reference to the wild type IgG2 sequence). Eur. J. Immunol.,
1999, 29:2613-2624. In still other embodiments, the constant region
is aglycosylated for N-linked glycosylation. In some embodiments,
the constant region is aglycosylated for N-linked glycosylation by
mutating the glycosylated amino acid residue or flanking residues
that are part of the N-glycosylation recognition sequence in the
constant region. For example, N-glycosylation site N297 may be
mutated to A, Q, K, or H. See, Tao et al., 1989, J. Immunology 143:
2595-2601; and Jefferis et al., 1998, Immunological Reviews
163:59-76. In some embodiments, the constant region is
aglycosylated for N-linked glycosylation. The constant region may
be aglycosylated for N-linked glycosylation enzymatically (such as
removing carbohydrate by enzyme PNGase), or by expression in a
glycosylation deficient host cell.
[0188] Other antibody modifications include antibodies that have
been modified as described in PCT Publ. No. WO 99/58572. These
antibodies comprise, in addition to a binding domain directed at
the target molecule, an effector domain having an amino acid
sequence substantially homologous to all or part of a constant
domain of a human immunoglobulin heavy chain. These antibodies are
capable of binding the target molecule without triggering
significant complement dependent lysis, or cell-mediated
destruction of the target. In some embodiments, the effector domain
is capable of specifically binding FcRn and/or Fc.gamma.RIIb. These
are typically based on chimeric domains derived from two or more
human immunoglobulin heavy chain C.sub.H2 domains. Antibodies
modified in this manner are particularly suitable for use in
chronic antibody therapy, to avoid inflammatory and other adverse
reactions to conventional antibody therapy.
[0189] The invention includes affinity matured embodiments. For
example, affinity matured antibodies can be produced by procedures
known in the art (Marks et al., 1992, Bio/Technology, 10:779-783;
Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier
et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol.,
155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9;
Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and PCT Publ. No.
WO2004/058184).
[0190] The following methods may be used for adjusting the affinity
of an antibody and for characterizing a CDR. One way of
characterizing a CDR of an antibody and/or altering (such as
improving) the binding affinity of a polypeptide, such as an
antibody, termed "library scanning mutagenesis". Generally, library
scanning mutagenesis works as follows. One or more amino acid
positions in the CDR are replaced with two or more (such as 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino
acids using art recognized methods. This generates small libraries
of clones (in some embodiments, one for every amino acid position
that is analyzed), each with a complexity of two or more members
(if two or more amino acids are substituted at every position).
Generally, the library also includes a clone comprising the native
(unsubstituted) amino acid. A small number of clones, e.g., about
20-80 clones (depending on the complexity of the library), from
each library are screened for binding affinity to the target
polypeptide (or other binding target), and candidates with
increased, the same, decreased, or no binding are identified.
Methods for determining binding affinity are well-known in the art.
Binding affinity may be determined using Biacore surface plasmon
resonance analysis, which detects differences in binding affinity
of about 2-fold or greater. Biacore is particularly useful when the
starting antibody already binds with a relatively high affinity,
for example a K.sub.D of about 10 nM or lower. Screening using
Biacore surface plasmon resonance is described in the Examples,
herein.
[0191] Binding affinity may be determined using Kinexa Biocensor,
scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN),
fluorescence quenching, fluorescence transfer, and/or yeast
display. Binding affinity may also be screened using a suitable
bioassay.
[0192] In some embodiments, every amino acid position in a CDR is
replaced (in some embodiments, one at a time) with all 20 natural
amino acids using art recognized mutagenesis methods (some of which
are described herein). This generates small libraries of clones (in
some embodiments, one for every amino acid position that is
analyzed), each with a complexity of 20 members (if all 20 amino
acids are substituted at every position).
[0193] In some embodiments, the library to be screened comprises
substitutions in two or more positions, which may be in the same
CDR or in two or more CDRs. Thus, the library may comprise
substitutions in two or more positions in one CDR. The library may
comprise substitution in two or more positions in two or more CDRs.
The library may comprise substitution in 3, 4, 5, or more
positions, said positions found in two, three, four, five or six
CDRs. The substitution may be prepared using low redundancy codons.
See, e.g., Table 2 of Balint et al., 1993, Gene 137(1):109-18).
[0194] The CDR may be CDRH3 and/or CDRL3. The CDR may be one or
more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR
may be a Kabat CDR, a Chothia CDR, or an extended CDR.
[0195] Candidates with improved binding may be sequenced, thereby
identifying a CDR substitution mutant which results in improved
affinity (also termed an "improved" substitution). Candidates that
bind may also be sequenced, thereby identifying a CDR substitution
which retains binding.
[0196] Multiple rounds of screening may be conducted. For example,
candidates (each comprising an amino acid substitution at one or
more position of one or more CDR) with improved binding are also
useful for the design of a second library containing at least the
original and substituted amino acid at each improved CDR position
(i.e., amino acid position in the CDR at which a substitution
mutant showed improved binding). Preparation, screening, and
selection of this library is discussed further below.
[0197] Library scanning mutagenesis also provides a means for
characterizing a CDR, in so far as the frequency of clones with
improved binding, the same binding, decreased binding or no binding
also provide information relating to the importance of each amino
acid position for the stability of the antibody-antigen complex.
For example, if a position of the CDR retains binding when changed
to all 20 amino acids, that position is identified as a position
that is unlikely to be required for antigen binding. Conversely, if
a position of CDR retains binding in only a small percentage of
substitutions, that position is identified as a position that is
important to CDR function. Thus, the library scanning mutagenesis
methods generate information regarding positions in the CDRs that
can be changed to many different amino acids (including all 20
amino acids), and positions in the CDRs which cannot be changed or
which can only be changed to a few amino acids.
[0198] Candidates with improved affinity may be combined in a
second library, which includes the improved amino acid, the
original amino acid, and may further include additional
substitutions at that position, depending on the complexity of the
library that is desired, or permitted using the desired screening
or selection method. In addition, if desired, and adjacent amino
acid position can be randomized to at least two or more amino
acids. Randomization of adjacent amino acids may permit additional
conformational flexibility in the mutant CDR, which may, in turn,
permit or facilitate the introduction of a larger number of
improving mutations. The library may also comprise substitution at
positions that did not show improved affinity in the first round of
screening.
[0199] The second library is screened or selected for library
members with improved and/or altered binding affinity using any
method known in the art, including screening using Biacore surface
plasmon resonance analysis, and selection using any method known in
the art for selection, including phage display, yeast display, and
ribosome display.
[0200] The invention also encompasses fusion proteins comprising
one or more fragments or regions from the antibodies or
polypeptides of this invention. In one embodiment, a fusion
polypeptide is provided that comprises at least 10 contiguous amino
acids of a variable light chain region shown in SEQ ID NOs: 53, 16,
17, 18, or 19 and/or at least 10 amino acids of a variable heavy
chain region shown in SEQ ID NOs: 54, 20, 21, 22, or 23. In other
embodiments, a fusion polypeptide is provided that comprises at
least about 10, at least about 15, at least about 20, at least
about 25, or at least about 30 contiguous amino acids of the
variable light chain region and/or at least about 10, at least
about 15, at least about 20, at least about 25, or at least about
30 contiguous amino acids of the variable heavy chain region. In
another embodiment, the fusion polypeptide comprises a light chain
variable region and/or a heavy chain variable region, as shown in
any of the sequence pairs selected from among SEQ ID NOs: 53 and
54, 16 and 20, 17 and 21, 18 and 22, and 19 and 23. In another
embodiment, the fusion polypeptide comprises one or more CDR(s). In
still other embodiments, the fusion polypeptide comprises CDR H3
(VH CDR3) and/or CDR L3 (VL CDR3). For purposes of this invention,
a fusion protein contains one or more antibodies and another amino
acid sequence to which it is not attached in the native molecule,
for example, a heterologous sequence or a homologous sequence from
another region. Exemplary heterologous sequences include, but are
not limited to a "tag" such as a FLAG tag or a 6His tag. Tags are
well known in the art.
[0201] A fusion polypeptide can be created by methods known in the
art, for example, synthetically or recombinantly. Typically, the
fusion proteins of this invention are made by preparing an
expressing a polynucleotide encoding them using recombinant methods
described herein, although they may also be prepared by other means
known in the art, including, for example, chemical synthesis.
[0202] This invention also provides compositions comprising
antibodies or polypeptides conjugated (for example, linked) to an
agent that facilitate coupling to a solid support (such as biotin
or avidin). For simplicity, reference will be made generally to
antibodies with the understanding that these methods apply to any
of the PCSK9 binding and/or antagonist embodiments described
herein. Conjugation generally refers to linking these components as
described herein. The linking (which is generally fixing these
components in proximate association at least for administration)
can be achieved in any number of ways. For example, a direct
reaction between an agent and an antibody is possible when each
possesses a substituent capable of reacting with the other. For
example, a nucleophilic group, such as an amino or sulfhydryl
group, on one may be capable of reacting with a carbonyl-containing
group, such as an anhydride or an acid halide, or with an alkyl
group containing a good leaving group (e.g., a halide) on the
other.
[0203] An antibody or polypeptide of this invention may be linked
to a labeling agent such as a fluorescent molecule, a radioactive
molecule or any others labels known in the art. Labels are known in
the art which generally provide (either directly or indirectly) a
signal.
[0204] The invention also provides compositions (including
pharmaceutical compositions) and kits comprising, as this
disclosure makes clear, any or all of the antibodies and/or
polypeptides described herein.
[0205] The invention also provides isolated polynucleotides
encoding the antibodies and peptides of the invention, and vectors
and host cells comprising the polynucleotide.
[0206] Accordingly, the invention provides polynucleotides (or
compositions, including pharmaceutical compositions), comprising
polynucleotides encoding any of the following: the antibodies 4A5,
5A10, 6F6, 7D4, L1L3, or any fragment or part thereof having the
ability to antagonize PCSK9.
[0207] In another aspect, the invention provides polynucleotides
encoding any of the antibodies (including antibody fragments) and
polypeptides described herein, such as antibodies and polypeptides
having impaired effector function. Polynucleotides can be made and
expressed by procedures known in the art.
[0208] In another aspect, the invention provides compositions (such
as pharmaceutical compositions) comprising any of the
polynucleotides of the invention. In some embodiments, the
composition comprises an expression vector comprising a
polynucleotide encoding the antibody as described herein. In other
embodiment, the composition comprises an expression vector
comprising a polynucleotide encoding any of the antibodies or
polypeptides described herein. In still other embodiments, the
composition comprises either or both of the polynucleotides shown
in SEQ ID NO:25 and SEQ ID NO:26. Expression vectors, and
administration of polynucleotide compositions are further described
herein.
[0209] In another aspect, the invention provides a method of making
any of the polynucleotides described herein.
[0210] Polynucleotides complementary to any such sequences are also
encompassed by the present invention. Polynucleotides may be
single-stranded (coding or antisense) or double-stranded, and may
be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules
include HnRNA molecules, which contain introns and correspond to a
DNA molecule in a one-to-one manner, and mRNA molecules, which do
not contain introns. Additional coding or non-coding sequences may,
but need not, be present within a polynucleotide of the present
invention, and a polynucleotide may, but need not, be linked to
other molecules and/or support materials.
[0211] Polynucleotides may comprise a native sequence (i.e., an
endogenous sequence that encodes an antibody or a portion thereof)
or may comprise a variant of such a sequence. Polynucleotide
variants contain one or more substitutions, additions, deletions
and/or insertions such that the immunoreactivity of the encoded
polypeptide is not diminished, relative to a native immunoreactive
molecule. The effect on the immunoreactivity of the encoded
polypeptide may generally be assessed as described herein. Variants
preferably exhibit at least about 70% identity, more preferably, at
least about 80% identity, yet more preferably, at least about 90%
identity, and most preferably, at least about 95% identity to a
polynucleotide sequence that encodes a native antibody or a portion
thereof.
[0212] Two polynucleotide or polypeptide sequences are said to be
"identical" if the sequence of nucleotides or amino acids in the
two sequences is the same when aligned for maximum correspondence
as described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, or 40 to
about 50, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned.
[0213] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O., 1978, A
model of evolutionary change in proteins--Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure (National Biomedical Research Foundation,
Washington D.C.), Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990,
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, (Academic Press, Inc., San Diego, Calif.);
Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E.
W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971,
Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy (Freeman
Press, San Francisco, Calif.); Wilbur, W. J. and Lipman, D. J.,
1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0214] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) of 20
percent or less, usually 5 to 15 percent, or 10 to 12 percent, as
compared to the reference sequences (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid bases or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the reference sequence (i.e. the window size) and
multiplying the results by 100 to yield the percentage of sequence
identity.
[0215] Variants may also, or alternatively, be substantially
homologous to a native gene, or a portion or complement thereof.
Such polynucleotide variants are capable of hybridizing under
moderately stringent conditions to a naturally occurring DNA
sequence encoding a native antibody (or a complementary
sequence).
[0216] Suitable "moderately stringent conditions" include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC,
overnight; followed by washing twice at 65.degree. C. for 20
minutes with each of 2.times., 0.5.times. and 0.2.times.SSC
containing 0.1% SDS.
[0217] As used herein, "highly stringent conditions" or "high
stringency conditions" are those that: (1) employ low ionic
strength and high temperature for washing, for example 0.015 M
sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash consisting of
0.1.times.SSC containing EDTA at 55.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
[0218] It will be appreciated by those of ordinary skill in the art
that, as a result of the degeneracy of the genetic code, there are
many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless,
polynucleotides that vary due to differences in codon usage are
specifically contemplated by the present invention. Further,
alleles of the genes comprising the polynucleotide sequences
provided herein are within the scope of the present invention.
Alleles are endogenous genes that are altered as a result of one or
more mutations, such as deletions, additions and/or substitutions
of nucleotides. The resulting mRNA and protein may, but need not,
have an altered structure or function. Alleles may be identified
using standard techniques (such as hybridization, amplification
and/or database sequence comparison).
[0219] The polynucleotides of this invention can be obtained using
chemical synthesis, recombinant methods, or PCR. Methods of
chemical polynucleotide synthesis are well known in the art and
need not be described in detail herein. One of skill in the art can
use the sequences provided herein and a commercial DNA synthesizer
to produce a desired DNA sequence.
[0220] For preparing polynucleotides using recombinant methods, a
polynucleotide comprising a desired sequence can be inserted into a
suitable vector, and the vector in turn can be introduced into a
suitable host cell for replication and amplification, as further
discussed herein. Polynucleotides may be inserted into host cells
by any means known in the art. Cells are transformed by introducing
an exogenous polynucleotide by direct uptake, endocytosis,
transfection, F-mating or electroporation. Once introduced, the
exogenous polynucleotide can be maintained within the cell as a
non-integrated vector (such as a plasmid) or integrated into the
host cell genome. The polynucleotide so amplified can be isolated
from the host cell by methods well known within the art. See, e.g.,
Sambrook et al., 1989, supra.
[0221] Alternatively, PCR allows reproduction of DNA sequences. PCR
technology is well known in the art and is described in U.S. Pat.
Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR:
The Polymerase Chain Reaction, Mullis et al., 1994, eds.
(Birkauswer Press, Boston, Mass.).
[0222] RNA can be obtained by using the isolated DNA in an
appropriate vector and inserting it into a suitable host cell. When
the cell replicates and the DNA is transcribed into RNA, the RNA
can then be isolated using methods well known to those of skill in
the art, as set forth in Sambrook et al., 1989, supra, for
example.
[0223] Suitable cloning vectors may be constructed according to
standard techniques, or may be selected from a large number of
cloning vectors available in the art. While the cloning vector
selected may vary according to the host cell intended to be used,
useful cloning vectors will generally have the ability to
self-replicate, may possess a single target for a particular
restriction endonuclease, and/or may carry genes for a marker that
can be used in selecting clones containing the vector. Suitable
examples include plasmids and bacterial viruses, e.g., pUC18,
pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,
pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors
such as pSA3 and pAT28. These and many other cloning vectors are
available from commercial vendors such as BioRad, Strategene, and
Invitrogen.
[0224] Expression vectors generally are replicable polynucleotide
constructs that contain a polynucleotide according to the
invention. It is implied that an expression vector must be
replicable in the host cells either as episomes or as an integral
part of the chromosomal DNA. Suitable expression vectors include
but are not limited to plasmids, viral vectors, including
adenoviruses, adeno-associated viruses, retroviruses, cosmids, and
expression vector(s) disclosed in PCT Publ. No. WO 87/04462. Vector
components may generally include, but are not limited to, one or
more of the following: a signal sequence; an origin of replication;
one or more marker genes; suitable transcriptional controlling
elements (such as promoters, enhancers and terminator). For
expression (i.e., translation), one or more translational
controlling elements are also usually required, such as ribosome
binding sites, translation initiation sites, and stop codons.
[0225] The vectors containing the polynucleotides of interest can
be introduced into the host cell by any of a number of appropriate
means, including electroporation, transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (e.g., where the vector is an infectious agent such as
vaccinia virus). The choice of introducing vectors or
polynucleotides will often depend on features of the host cell.
[0226] The invention also provides host cells comprising any of the
polynucleotides described herein. Any host cells capable of
over-expressing heterologous DNAs can be used for the purpose of
isolating the genes encoding the antibody, polypeptide or protein
of interest. Non-limiting examples of mammalian host cells include
but are not limited to COS, HeLa, NSO, and CHO cells. See also PCT
Publ. No. WO 87/04462. Suitable non-mammalian host cells include
prokaryotes (such as E. coli or B. subtiffis) and yeast (such as S.
cerevisae, S. pombe; or K. lactis). Preferably, the host cells
express the cDNAs at a level of about 5 fold higher, more
preferably, 10 fold higher, even more preferably, 20 fold higher
than that of the corresponding endogenous antibody or protein of
interest, if present, in the host cells. Screening the host cells
for a specific binding to PCSK9 or a PCSK9 domain is effected by an
immunoassay or FACS. A cell overexpressing the antibody or protein
of interest can be identified.
C. Compositions
[0227] The compositions used in the methods of the invention
comprise an effective amount of a PCSK9 antagonist antibody, a
PCSK9 antagonist antibody derived polypeptide, or other PCSK9
antagonists described herein. Examples of such compositions, as
well as how to formulate them, are also described in an earlier
section and below. In one embodiment, the composition further
comprises a PCSK9 antagonist. In another embodiment, the
composition comprises one or more PCSK9 antagonist antibodies. In
other embodiments, the PCSK9 antagonist antibody recognizes human
PCSK9. In still other embodiments, the PCSK9 antagonist antibody is
humanized. In yet other embodiments, the PCSK9 antagonist antibody
comprises a constant region that does not trigger an unwanted or
undesirable immune response, such as antibody-mediated lysis or
ADCC. In other embodiments, the PCSK9 antagonist antibody comprises
one or more CDR(s) of the antibody (such as one, two, three, four,
five, or, in some embodiments, all six CDRs). In some embodiments,
the PCSK9 antagonist antibody is human.
[0228] It is understood that the compositions can comprise more
than one PCSK9 antagonist antibody (e.g., a mixture of PCSK9
antagonist antibodies that recognize different epitopes of PCSK9).
Other exemplary compositions comprise more than one PCSK9
antagonist antibodies that recognize the same epitope(s), or
different species of PCSK9 antagonist antibodies that bind to
different epitopes of PCSK9.
[0229] The composition used in the present invention can further
comprise pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and Practice of Pharmacy 20th
Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may comprise
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g., Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutically acceptable excipients are further described
herein.
[0230] In one embodiment, the antibody is administered in a
formulation as a sterile aqueous solution having a pH that ranges
from about 5.0 to about 6.5 and comprising from about 1 mg/ml to
about 200 mg/ml of antibody, from about 1 millimolar to about 100
millimolar of histidine buffer, from about 0.01 mg/ml to about 10
mg/ml of polysorbate 80, from about 100 millimolar to about 400
millimolar of trehalose, and from about 0.01 millimolar to about
1.0 millimolar of disodium EDTA dihydrate.
[0231] The PCSK9 antagonist antibody and compositions thereof can
also be used in conjunction with other agents that serve to enhance
and/or complement the effectiveness of the agents.
D. Kits
[0232] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
comprising a PCSK9 antagonist antibody (such as a humanized
antibody) or peptide described herein and instructions for use in
accordance with any of the methods of the invention described
herein. Generally, these instructions comprise a description of
administration of the PCSK9 antagonist antibody, peptide, or
aptamer for the above described therapeutic treatments.
[0233] In some embodiments, the antibody is a humanized antibody.
In some embodiments, the antibody is human. In other embodiments,
the antibody is a monoclonal antibody. The instructions relating to
the use of a PCSK9 antagonist antibody generally include
information as to dosage, dosing schedule, and route of
administration for the intended treatment. The containers may be
unit doses, bulk packages (e.g., multi-dose packages) or sub-unit
doses. Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0234] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a PCSK9 antagonist antibody. The
container (e.g., pre-filled syringe or autoinjector) may further
comprise a second pharmaceutically active agent.
[0235] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container.
Mutations and Modifications
[0236] To express the PCSK9 antibodies of the present invention,
DNA fragments encoding V.sub.H and V.sub.L regions can first be
obtained using any of the methods described above. Various
modifications, e.g., mutations, deletions, and/or additions can
also be introduced into the DNA sequences using standard methods
known to those of skill in the art. For example, mutagenesis can be
carried out using standard methods, such as PCR-mediated
mutagenesis, in which the mutated nucleotides are incorporated into
the PCR primers such that the PCR product contains the desired
mutations or site-directed mutagenesis.
[0237] One type of substitution, for example, that may be made is
to change one or more cysteines in the antibody, which may be
chemically reactive, to another residue, such as, without
limitation, alanine or serine. For example, there can be a
substitution of a non-canonical cysteine. The substitution can be
made in a CDR or framework region of a variable domain or in the
constant domain of an antibody. In some embodiments, the cysteine
is canonical.
[0238] The antibodies may also be modified, e.g., in the variable
domains of the heavy and/or light chains, e.g., to alter a binding
property of the antibody. For example, a mutation may be made in
one or more of the CDR regions to increase or decrease the K.sub.D
of the antibody for PCSK9, to increase or decrease k.sub.off, or to
alter the binding specificity of the antibody. Techniques in
site-directed mutagenesis are well-known in the art. See, e.g.,
Sambrook et al. and Ausubel et al., supra.
[0239] A modification or mutation may also be made in a framework
region or constant domain to increase the half-life of a PCSK9
antibody. See, e.g., PCT Publ. No. WO 00/09560. A mutation in a
framework region or constant domain can also be made to alter the
immunogenicity of the antibody, to provide a site for covalent or
non-covalent binding to another molecule, or to alter such
properties as complement fixation, FcR binding and
antibody-dependent cell-mediated cytotoxicity. According to the
invention, a single antibody may have mutations in any one or more
of the CDRs or framework regions of the variable domain or in the
constant domain.
[0240] In a process known as "germlining", certain amino acids in
the V.sub.H and V.sub.L sequences can be mutated to match those
found naturally in germline V.sub.H and V.sub.L sequences. In
particular, the amino acid sequences of the framework regions in
the V.sub.H and V.sub.L sequences can be mutated to match the
germline sequences to reduce the risk of immunogenicity when the
antibody is administered. Germline DNA sequences for human V.sub.H
and V.sub.L genes are known in the art (see e.g., the "Vbase" human
germline sequence database; see also Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publ. No.
91-3242; Tomlinson et al., 1992, J. Mol. Biol. 227:776-798; and Cox
et al., 1994, Eur. J. Immunol. 24:827-836.
[0241] Another type of amino acid substitution that may be made is
to remove potential proteolytic sites in the antibody. Such sites
may occur in a CDR or framework region of a variable domain or in
the constant domain of an antibody. Substitution of cysteine
residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its
homogeneity. Another type of amino acid substitution eliminates
asparagine-glycine pairs, which form potential deamidation sites,
by altering one or both of the residues. In another example, the
C-terminal lysine of the heavy chain of a PCSK9 antibody of the
invention can be cleaved. In various embodiments of the invention,
the heavy and light chains of the PCSK9 antibodies may optionally
include a signal sequence.
[0242] Once DNA fragments encoding the V.sub.H and V.sub.L segments
of the present invention are obtained, these DNA fragments can be
further manipulated by standard recombinant DNA techniques, for
example to convert the variable region genes to full-length
antibody chain genes, to Fab fragment genes, or to a scFv gene. In
these manipulations, a V.sub.L- or V.sub.H-encoding DNA fragment is
operatively linked to another DNA fragment encoding another
protein, such as an antibody constant region or a flexible linker.
The term "operatively linked", as used in this context, is intended
to mean that the two DNA fragments are joined such that the amino
acid sequences encoded by the two DNA fragments remain
in-frame.
[0243] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2 and CH3). The sequences of human
heavy chain constant region genes are known in the art (see e.g.,
Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publ. No. 91-3242) and DNA fragments encompassing
these regions can be obtained by standard PCR amplification. The
heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA,
IgE, IgM or IgD constant region, but most preferably is an IgG1 or
IgG2 constant region. The IgG constant region sequence can be any
of the various alleles or allotypes known to occur among different
individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These
allotypes represent naturally occurring amino acid substitution in
the IgG1 constant regions. For a Fab fragment heavy chain gene, the
V.sub.H-encoding DNA can be operatively linked to another DNA
molecule encoding only the heavy chain CH1 constant region. The CH1
heavy chain constant region may be derived from any of the heavy
chain genes.
[0244] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region,
C.sub.L. The sequences of human light chain constant region genes
are known in the art (see e.g., Kabat, E. A., et al., 1991,
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publ. No.
91-3242) and DNA fragments encompassing these regions can be
obtained by standard PCR amplification. The light chain constant
region can be a kappa or lambda constant region. The kappa constant
region may be any of the various alleles known to occur among
different individuals, such as Inv(1), Inv(2), and Inv(3). The
lambda constant region may be derived from any of the three lambda
genes.
[0245] To create a scFv gene, the V.sub.H- and V.sub.L-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences
can be expressed as a contiguous single-chain protein, with the
V.sub.L and V.sub.H regions joined by the flexible linker (See
e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988,
Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990,
Nature 348:552-554. The single chain antibody may be monovalent, if
only a single V.sub.H and V.sub.L are used, bivalent, if two
V.sub.H and V.sub.L are used, or polyvalent, if more than two
V.sub.H and V.sub.L are used. Bispecific or polyvalent antibodies
may be generated that bind specifically to PCSK9 and to another
molecule.
[0246] In another embodiment, a fusion antibody or immunoadhesin
may be made that comprises all or a portion of a PCSK9 antibody of
the invention linked to another polypeptide. In another embodiment,
only the variable domains of the PCSK9 antibody are linked to the
polypeptide. In another embodiment, the V.sub.H domain of a PCSK9
antibody is linked to a first polypeptide, while the V.sub.L domain
of a PCSK9 antibody is linked to a second polypeptide that
associates with the first polypeptide in a manner such that the
V.sub.H and V.sub.L domains can interact with one another to form
an antigen binding site. In another preferred embodiment, the
V.sub.H domain is separated from the V.sub.L domain by a linker
such that the V.sub.H and V.sub.L domains can interact with one
another. The V.sub.H-linker-V.sub.L antibody is then linked to the
polypeptide of interest. In addition, fusion antibodies can be
created in which two (or more) single-chain antibodies are linked
to one another. This is useful if one wants to create a divalent or
polyvalent antibody on a single polypeptide chain, or if one wants
to create a bispecific antibody.
[0247] In other embodiments, other modified antibodies may be
prepared using PCSK9 antibody encoding nucleic acid molecules. For
instance, "Kappa bodies" (Ill et al., 1997, Protein Eng.
10:949-57), "Minibodies" (Martin et al., 1994, EMBO J. 13:5303-9),
"Diabodies" (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-6448), or "Janusins" (Traunecker et al., 1991, EMBO J.
10:3655-3659 and Traunecker et al., 1992, Int. J. Cancer (Suppl.)
7:51-52) may be prepared using standard molecular biological
techniques following the teachings of the specification.
[0248] Bispecific antibodies or antigen-binding fragments can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmann,
1990, Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J.
Immunol. 148:1547-1553. In addition, bispecific antibodies may be
formed as "diabodies" or "Janusins." In some embodiments, the
bispecific antibody binds to two different epitopes of PCSK9. In
some embodiments, the modified antibodies described above are
prepared using one or more of the variable domains or CDR regions
from a human PCSK9 antibody provided herein.
Generation of Antigen-Specific Antibodies
[0249] More than 500 polyclonal and monoclonal antibodies raised
against recombinant full-length human PCSK9, recombinant full
length mouse PCSK9, and various synthetic peptides were evaluated
for their ability to down regulate total LDLR protein in cultured
Huh7 human liver cells. Among these antibodies were a set of
antibodies raised to and reactive with a set of 12-20 amino acid
residue polypeptides that, based on the structure of PCSK9, were
predicted to cover majority of the protein surface. At the highest
concentration, the best antibodies exhibited only about 60%
blocking activity. Thus, an alternative and heretofore unexplored
approach was employed, namely, the generation of monoclonal
antibodies by immunizing PCSK9 null mice with recombinant
full-length PCSK9 protein. This manner of antibody preparation
yielded antagonist antibodies that show complete blocking of PCSK9
binding to LDLR, complete blocking of PCSK9-mediated lowering of
LDLR levels in Huh7 cells, and lowering of LDLc in vivo including
in mice to levels comparable to that seen in PCSK9 -/- mice, as
shown in Example 7.
[0250] Representative antibodies (hybridomas) of the present
invention were deposited in the American Type Culture Collection
(ATCC) on Feb. 28, 2008, and were assigned the accession numbers in
Table 3. Hybridomas were deposited for antibodies 4A5, 5A10, 6F6
and 7D4.
TABLE-US-00003 TABLE 3 Antibody Reference ATCC Accession No. 4A5
PTA-8985 5A10 PTA-8986 6F6 PTA-8984 7D4 PTA-8983
EXAMPLES
Example 1
Generating and Screening PCSK9 Antagonist Antibodies
[0251] General procedures for immunization of animals for
generating monoclonal antibodies:
[0252] Balb/c or 129/bl6 pcsk9 -/- mice were injected 5 times on a
13 day schedule with 100 .mu.g antigen. PCSK9 -/- (that is, null or
knock-out mice) can be obtained from, or as described by, Rashid et
al., 2005, Proc Natl Acad Sci USA 102: 5374. See also U.S. Pat. No.
7,300,754. For the first 4 injections, antigen was prepared by
mixing the recombinant proteins with adjuvant. Immunogen was given
via injection to the scruff of the neck, the foot pads and
intraperitoneally, approximately every 3 days over the course of 11
days, with the last boost administered i.v., without adjuvant. On
Day 13, the mice were euthanized and their spleens were removed.
Lymphocytes were immortalized by fusion with an established cell
line to make hybridoma clones using standard hybridoma technology,
distributed into 96 well plates. Clones were allowed to grow, then
selected by ELISA screening using the immunizing antigen, as
below.
ELISA Screening of Antibodies:
[0253] Supernatant media from growing hybridoma clones were
screened separately for their ability to bind the recombinant human
PCSK9 or recombinant mouse PCSK9. The assays were performed with
96-well plates coated overnight with 100 .mu.l of a 1 .mu.g/ml
solution of one of the antigens. Excess reagents were washed from
the wells between each step with PBS containing 0.05% Tween-20.
Plates were then blocked with PBS containing 0.5% BSA. Supernatant
was added to the plates and incubated at room temperature for 2
hours. Horse radish peroxidase (HRP) conjugated goat-anti mouse Fc
was added to bind to the mouse antibodies bound to the antigen.
Tetramethyl benzidine was then added as substrate for HRP to detect
the amount of mouse antibody present in the supernatant. The
reaction was stopped and the relative amount of antibody was
quantified by reading the absorbance at 450 nm. Hybridoma clones
that secreted antibodies that are capable of binding to either
mouse or human PCSK9 were selected for further analysis.
PCSK9-Mediated LDLR Down-Regulation in Huh7 Cells:
[0254] Hybridoma clones secreting human or mouse PCSK9 binding
antibodies were expanded and supernatants were harvested. Total
IgGs were purified from approximately 10 ml of the supernatant
using protein A beads, dialyzed into PBS buffer, and the final
volume reduced to yield solutions with 0.7-1 mg/ml of antibodies.
Purified antibodies were then used to test their ability to inhibit
the ability of PCSK9 to mediate LDLR down-regulation in Huh7 cells.
Huh7 cells were plated and allowed to grow to 80% confluency in
RPMI media containing 10% FBS, 4 mM glutamine, and penicillin and
streptavidin in 96 well plates. The medium was changed to one
containing 10% de-lipidated FBS for 8-16 hrs to induce LDLR
expression. Cells were then incubated for 8-16 hours with 40
.mu.l/well of 293 expression media supplemented with 6 .mu.g/ml of
human (preferably) or mouse PCSK9, with or without 70-100 .mu.g/ml
of test antibodies. The PCSK9 and antibody containing media were
removed at the end of incubation, and cells were lysed with 17
.mu.l lysis buffer by shaking at 4 C for an hour. The lysis buffer
consisted of 50 mM glycerol phosphate, 10 mM HEPES pH 7.4, 1%
Triton X-100, 20 mM NaCl, and a cocktail of protease inhibitors
(Roche). Cell lysates were collected and analyzed for LDLR protein
levels via staining of Western blots following SDS polyacrylamide
gel electrophoresis. Hybridoma clones producing antibodies that can
partially or fully rescue LDLR level were selected for further
analysis. By "LDLR down regulation assay" is meant the above assay
using Huh7 cells.
[0255] FIG. 1 illustrates the effect of anti-PCSK9 antagonistic
monoclonal antibodies 7D4.4, 4A5.G3, 6F6.G10.3 and 5A10.B8 on the
ability of human and mouse PCSK9 to down regulate LDLR in cultured
Huh7 cells. 100 nM of mouse or human recombinant PCSK9, and a
serial dilution of 25-800 nM of antibodies were used. A) mouse
PCSK9. B) human PCSK9. The figures are Western blots showing that
the antibodies are in general more effective in blocking the
function of human PCSk9 than mouse PCSK9. The several antibodies
have generally similar affinities for human PCSK9 but vary in their
affinity for murine PCSK9.
Example 2
Determining Antibody Binding Affinity
[0256] The affinities of PCSK9 antibodies to PCSK9 were measured on
a surface plasmon resonance Biacore 3000 biosensor equipped with a
research-grade sensor chip using HBS-EP running buffer (Biacore AB,
Uppsala, Sweden--now GE Healthcare). Rabbit polyclonal anti-Ms IgGs
were amine-coupled at saturating levels onto the chip using a
standard N-hydroxysuccinimide/ethyldimethylaminopropyl carbodiimide
(NHS/EDC) chemistry. The buffer was switched to HBS-EP+1 mg/mL
BSA+1 mg/mL CM-dextran. Full-length PCSK9 IgGs were diluted to
about 15 .mu.g/mL and captured for 1 min at 5 .mu.L/min to give
levels of about 500RU per flow cell, leaving one blank to serve as
a reference channel. 3.73-302 nM hPCSK9 or 2.54-206 nM mPCSK9 were
injected as a 5-membered 3-fold series for 1 min at 100 .mu.L/min.
Dissociation was monitored for 5 min. The chip was regenerated
after the last injection of each titration with two 30 sec pulses
of 100 mM phosphoric acid. Buffer cycles provided blanks for
double-referencing the data, which were then fit globally to a
simple binding model using Biaevaluation software v.4.1. Affinities
were deduced from the quotient of the kinetic rate constants
(K.sub.D=k.sub.off/k.sub.on). The results of Example 2 are shown in
Table 4. These data show that the antibodies have excellent
affinity for murine PCSK9 or human PCSK9, as indicated.
TABLE-US-00004 TABLE 4 Inhibition of K.sub.on for K.sub.off for
K.sub.D LDLR-PCSK9 PCSK9 PCSK9 for PCSK9 mAb ligand bindin
(IC.sub.50) (1/Ms) (1/S) (nM) 4A5 human 0.4 nM 6.66 .times.
10.sup.4 1.89 .times. 10.sup.-4 2.8 5A10 human 0.4 nM 8.47 .times.
10.sup.4 8.55 .times. 10.sup.-5 1 6F6 human 1.5 nM 9.15 .times.
10.sup.4 5.84 .times. 10.sup.-4 6.4 7D4 human 1.5 nM 1.25 .times.
10.sup.5 7.94 .times. 10.sup.-4 6.4 4A5 mouse 3 nM 1.41 .times.
10.sup.5 7.2 .times. 10.sup.-4 5.1 5A10 mouse 3 nM 1.27 .times.
10.sup.5 4.89 .times. 10.sup.-4 3.9 6F6 mouse 10 nM 1.11 .times.
10.sup.5 1.97 .times. 10.sup.-3 17.7 7D4 mouse 1 nM 3.92 .times.
10.sup.4 5.23 .times. 10.sup.-4 1.3
Example 3
Analysis of the Effect of PCSK9 Antibodies on PCSK9-LDLR
Interaction
[0257] PCSK9 has been shown to bind LDLR with an affinity of 180 nM
under neutral pH (Cunningham et al., 2007, Nat Struct Mol Biol,
14(5):413-9). Recombinant mouse or human PCSK9 protein was
biotinylated using the Pierce reagents following the manufacture's
instructions. ELISA plates (Corning Mixisorb) were coated with a
solution of 1 .mu.g/ml recombinant LDLR extracellular domain
(R&D Systems) in each well at 4 C overnight, blocked with 2%
BSA+PBS for 2 hrs at room temperature, and then washed 5 times with
washing buffer (1.times.PBS+0.05% Tween-20). Wells were incubated
with 50 .mu.l of indicated concentrations of biotinylated PCSK9
protein for 1 hr RT. LDLR-PCSK9 binding can be stabilized by adding
50 .mu.l of 4% FDH+4% sucrose+PBS solution and incubate for 5 min.
Wells were washed 5 times with washing buffer, incubated with
1:2000 dilution of HRP conjugated Strepavidin (Invitrogen) for 1 hr
at RT, washed 5 times with washing buffer. TMB substrate was added
to the wells, the solution was incubated 20 to 30 min at RT, and
the reaction was terminated using 1 M phosphoric acid. Signals were
read at 450 nm.
[0258] FIG. 2 illustrates the dose-response of anti-PCSK9
antagonist monoclonal antibodies 6F6.G10.3, 7D4.4, 4A5.G3, 5A10.B8,
negative control antibody 42H7, and PBS on blocking the binding of
recombinant biotinylated human PCSK9 and mouse PCSK9 to immobilized
recombinant LDLR extracellular domain in vitro. Part A) shows human
PCSK9 binding to human LDLR extracellular domain and that 7D4, 4A5,
5A10, and 6F6 are effective in blocking binding, whereas 42H7 and
PBS are not. Part B) shows mouse PCSK9 binding to human LDLR
extracellular domain.
[0259] The interaction can also be evaluated in free solution at
neutral pH. FIG. 3 illustrates the dose-response of anti-PCSK9
monoclonal antagonist antibodies 6F6.G10.3, 7D4.4, 4A5.G3 and
5A10.B8 on blocking binding of recombinant biotinylated human PCSK9
(30 nM) to Europium labeled recombinant LDLR extracellular domain
(10 nM) in solution at neutral pH in vitro. This assay measures
binding in free solution at neutral pH.
Example 4
Epitope Mapping/Binding of Antibodies Using the Crystal Structure
of the L1 L3:PCSK9 Complex, Biacore, and Mutagenesis
[0260] a. Crystal structure of the L1 L3:PCSK9 complex. The
residues were identified by calculating the difference in
accessible surface area between the L1L3:PCSK9 crystal structure
and PCSK9 structure alone. PCSK9 residues that show buried surface
area upon complex formation with L1 L3 antibody were included as a
part of the epitope. The solvent accessible surface of a protein
was defined as the locus of the centre of a probe sphere
(representing a solvent molecule of 1.4 .ANG. radius) as it rolls
over the Van der Waals surface of the protein. The solvent
accessible surface area was calculated by generating surface points
on an extended sphere about each atom (at a distance from the atom
centre equal to the sum of the atom and probe radii), and
eliminating those that lie within equivalent spheres associated
with neighboring atoms as implemented in program AREAIMOL (Briggs,
P. J., 2000, CCP4 Newsletter No. 38, CCLRC, Daresbury).
[0261] The result of the crystal structure analysis are shown in
FIG. 23. FIG. 23A shows the crystal structure of the PCSK9 (light
gray surface representation) bound to the L1 L3 antibody (black
cartoon representation). The epitope for L1 L3 binding to PCSK9
involves residues 153-155, 194, 197, 237-239, 367, 369, 374-379 and
381 of the PCSK9 amino acid sequence (SEQ ID NO:53). By comparison,
the epitope for the LDLR EGF domain binding to PCSK9 involves
residues 153-155, 194, 238, 367, 369, 372, 374-375, and 377-381
(Kwon et al., 2008, PNAS 105: 1820-1825).
[0262] b. Group antibodies and epitopes based on competition in
PCSK9 binding. Full-length IgGs were amine-coupled to a CM5 sensor
chip (three per chip at about 7000RU final), using a standard
EDC/NHS-mediated amine-coupling chemistry. One flow cell was left
unmodified to provide a reference channel. Human-PCSK9 (100 nM) was
premixed with an array of IgGs (final 500 nM) and these complexes
were injected over the chip using 1 min injections at 10 .mu.L/min.
Antibodies that bind to competing epitopes will block the binding
of PCSK9 to the antibody immobilized on the chip. Alternatively, a
classical sandwich approach was used by first injecting human-PCSK9
at 50 nM for 1 min at 10 .mu.L/min (to tether it via the IgG on the
chip) and then binding an array of IgGs (final 500 nM each) for 2
mins each. The immobilized IgGs were regenerated with a mild acid
(Pierce gentle elution buffer+1 M NaCl). Antibodies directed to
known different epitopes were used as controls for positive
sandwich formation in this assay.
[0263] c. Structure-guided mutagenesis to map antibody binding
epitopes. Based on the crystal structure of PCSK9 and the likely
involvement of D374 in LDLR binding (Cunningham et al., 2007, Nat
Struct Mol Biol, 14(5): 413-419), nineteen PCSK9 surface-residue
mutants (F379A, 1369A, R194A, D374Y, D238R, T377R, K222A, R199A,
F216A, R218A, R237A, D192R, D367R, R165A, R167A, A443T, A53V,
I474V, H449A) near or far from the position of D374 were chosen for
mutation to map the antibody binding epitopes.
[0264] d. Mutant and antibody production. The 19 single point
mutants were generated from the previously described wild-type DNA
construct (Cunningham et al., 2007, supra) using standard DNA
techniques. The mutant proteins were expressed using transient
transfection in HEK293T cells and secreted into the cell media. The
mutant proteins were purified with the high-throughput AKTA Xpress
system (GE Healthcare) by Ni.sup.2+ and size-exclusion
chromatography steps, using conditions similar to those described
earlier. Protein concentrations were determined using the LabChip
instrument (BioRad). The PCSK9-blocking murine antibodies 4A5, 7D4,
5A10 and 6F6 were expressed with transient transfection in HEK293F
cells and purified with a protein G column eluted with 0.1 M
Glycine buffer at pH 2.8 and neutralized into 1.0 M Tris at pH
9.0.
[0265] e. The regions of PCSK9 that are contacted by monoclonal
antibodies 5A10 and 7D4 (preparation described later herein) were
determined by protein tomography (Sidec AB, Stockholm, Sweden). The
loops at positions 186-200, 371-379, 176-181, 278-283, 449-453,
402-406, and 236-245 of PCSK9 were proximal to amino acid residues
of the antibody. The sequences corresponding to the loops are shown
in Table 5, and in a preferred embodiment, the antagonists of the
invention bind to one or more of these sequences in PSCK9.
TABLE-US-00005 TABLE 5 PCSK9 Loops Sequence SEQ ID NO. 186-200
DTSIQSDHREIEGRV 1 236-245 GRDAGVAKGA 2 371-379 ASSDCSTCF 3 176-181
GGSLVE 4 278-283 QPVGPL 5 449-453 HGAGW 6 402-406 AEPEL 7
[0266] f. Biacore binding of the mutants to immobilized LDLR.
Recombinant LDLR extracelluar domain protein was immobilized onto a
Biacore SA chip. Each mutant protein was injected to the
Biacore-3000 M) in duplicates at 25 mM to 0.012 mM at five
concentrations (from 1.degree. C., with a running buffer of 50 mM
Tris pH 7.5, 2 mM CaCl.sub.2, 200 mM NaCl, 0.02% P20 and 1 mg/ml
BSA. All the results fit nicely to a 1:1 binding kinetics model. As
expected, mutation at residues in direct contact with the EGF-A
domain (F379A, R194A, 1369A, T377R, D238R) significantly weakens
(by 10-100 fold) LDLR binding. Moreover, three mutants not in
contact with EGF-A (R199A, R218A, K222A) showed weaker binding
(5-15 fold). This new finding suggests that they are involved in
binding other domains of LDLR. Overall, these experiments validate
the integrity and activity of the mutants for subsequent epitope
mapping experiments.
[0267] g. Binding of the mutants to immobilized 4A5, 7D4, 5A10 and
6F6 antibodies. Biotinylated anti-PCSK9 antibodies were immobilized
on SA chips using standard methods. Mutant binding experiments were
performed using Biacore 3000 at 25.degree. C. with a running buffer
of 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.02% P20. Mutants were
tested at 333 nM or 111 nM concentrations in duplicates, with the
ones giving weakened binding compared to the wild-type as the
residues involved in mAb binding (listed below).
TABLE-US-00006 mAb Binding Residues in Descending Order of Mutant
Effects 4A5 R237, F379, 369, R194, R199 & D238 5A10 R194, R237,
I369, D238, R199 6F6 R237, R194, F379, D238, I369, T377, R199 7D4
R237, R194, F379, I369, R199
Example 5
Cloning and Sequencing of Antibodies
[0268] One million hybridoma cells were homogenized using the
QIAshredder spin columns and total RNA was extracted according to
RNAeasy Micro kit from QIAGEN. cDNA was synthesized using
SuperScript III RT kit from Invitrogen. Variable regions from the
PCSK9 antibodies were cloned using the mouse IgG-Primer Sets from
Novagen, which consist of degenerate primers for cloning mouse IgG
heavy chain genes and the mouse kappa or lambda light chains. PCR
cycling conditions were the followings: 1 cycle at 92 C for 2 min;
two cycles at 94 C for 30 sec, 44 C for 30 sec and 72 C for 2 min;
two cycles at 94 C for 30 sec, 46 C for 30 sec and 72 C for 2 min;
two cycles at 94 C for 30 sec, 48 C for 30 sec and 72 C for 2 min;
two cycles at 94 C for 30 sec, 50 C for 30 sec and 72 C for 2 min;
two cycles at 94 C for 30 sec, 52 C for 30 sec and 72 C for 2 min;
followed by 35 cycles at 94 C for 30 sec, 54 C for 30 sec and 72 C
for 45 sec. The resulting PCR products were cloned into Topo-TA
cloning vector from Invitrogen and sequenced. The cloned antibody
sequences were confirmed by N-terminal sequencing of the first 10
amino acids of the original antibodies produced from ascites.
Example 6
Generation of Antigens for Immunization
[0269] Recombinant human PCSK9 protein was produced as reported
Cunningham et al., 2007, Nat Struct Mol Biol, 14(5):413-9. To
produce recombinant mouse PCSK9 protein, the cDNA of mouse PCSK9
was cloned into mammalian expression vector PRK5 with the addition
of a 6-His tag at the C-terminus by methods known in the art,
transiently transfected and expressed in HEK293 cells. Recombinant
protein was purified from conditioned media using a Ni column.
[0270] Surface peptides of human and mouse PCSK9 were selected
based on PCSK9 protein structure, and synthesized by Elim
Biopharmaceuticals.
Example 7
PCSK9-Specific Antibodies as PCSK9 Antagonists
1. Identification of PCSK9-Specific Antagonist Antibodies
[0271] a. Identification of PCSK9-Blocking Antibodies
[0272] Murine antibodies to human and/or mouse PCSK9 were generated
by immunizing mice with human-PCSK9 and mouse-PCSK9 synthetic
peptides as prepared in Example 6 or recombinant proteins, and
screening antibodies by ELISA assay using human and/or mouse PCSK9
recombinant protein as the antigens as described in Example 1 and
other standard hybridoma procedures. Over 500 positive clones were
obtained and allowed to grow to confluency in 6 well plates with 10
ml media. Media supernatant were collected and total IgGs in the
conditioned media were purified using mAb Select (Pierce). The
ability of purified and concentrated mouse IgGs to inhibit mouse
and human PCSK9 function was tested in Huh7 cells using the methods
described in Example 1. Hybridoma clones expressing IgGs that
showed some degrees of blocking were expanded and retested. 60
promising clones were subcloned, expanded, and injected into either
Balb/c or nude mice to produce ascites. Antibodies purified from
ascites fluid were retested for their ability to inhibit the down
regulation of LDLR by human or mouse PCSK9 in Huh7 cells. Four
hybridoma clones, 4A5, 5A10, 6F6, and 7D4, were identified as being
able to completely inhibit human PCSK9 function, and at least
partially inhibit mouse PCSK9 function. To determine IC.sub.50 of
each of these blocking antibodies, a serial dilution of IgGs were
used in the assay, starting from 100 .mu.g/ml to 3.125 .mu.g/ml,
with human and mouse PCSK9 concentration being constant at 6
.mu.g/ml.
b. Effect of PCSK9 Antagonists on PCSK9-LDLR Binding
[0273] PCSK9 has been shown to be co-localized with LDLR in
cellular compartments (Lagace et al., 2006, J Clin Inv,
116(11):2995-3005. Recombinant PCSK9 protein also binds to LDLR
extracellular domain in vitro (Fisher et al., 2007, JBC,
282(28):20502-12. To determine the relationship between inhibition
of PCSK9 mediated down-regulation of LDLR and inhibition of
PCSK9-LDLR binding by antibodies, we tested the PCSK9 antibodies
that partially or completely blocked PCSK9 function on LDLR and
representatives of antibodies that do not block. All partial
antagonistic antibodies also partially inhibited LDLR extracellular
domain binding to PCSK9, except one. Antagonistic antibodies that
can completely block PCSK9 function, namely 4A5, 5A10, 6F6 and 7D4
also completely inhibited LDLR extracellular domain binding to
PCSK9 (Table 5). IC.sub.50 values of these four antibodies
correlates with their binding affinity to PCSK9.
c. Epitope Determination of the Blocking Antibodies
[0274] FIG. 4 illustrates the epitope binning of anti-PCSK9
antibodies. Part A) shows epitope information of anti-PCSK9 mAbs,
determined by binding to synthetic 13-18-mer peptides or epitope
binding via Biacore. Part B) shows the ability of immobilized
antibodies 6F6, 5A10 and 4A5 to bind to human PCSK9 premixed with
the mAbs indicated on the y axis by Biacore assay.
[0275] Another monoclonal anti-PCSK9 antibody, termed 6G7, binds to
recombinant mouse PCSK9 but not human PCSK9. See Table 6. 6G7, 4A5,
5A10, 6F6, and 7D4 mutually exclude each other's binding to mouse
PCSK9. Chimera analysis between mouse and human PCSK9 reveals that
6G7 binding to PCSK9 requires the catalytic domain. See Table 6.
Thus the binding sites of 4A5, 5A10, 6F6, and 7D4 overlap the
catalytic site and/or the epitope bound by 6G7.
TABLE-US-00007 TABLE 6 Recombinant protein 6G7 binding Human PCSK9
No Human pro + human catalytic + mouse C-term No Human pro + mouse
catalytic + mouse C-term Yes Mouse pro + human catalytic + human
C-term No Mouse Pro + mouse catalytic + human C-term Yes Mouse
PCSK9 yes
d. Determining Sequences Species Specificity of Anti-PCSK9
Antibodies
[0276] To determine the species specificity of the anti-PCSK9
antibodies, antibodies were incubated with plasma from different
species and the resultant complexes were purified and probed by an
independent anti PCSK9 antibody on Western blots. The antibodies
4A5, 5A10, 6F6, and 7D4 recognized human, cynomolgus monkey, mouse,
and rat PCSK9. See FIG. 5. Antibody 6G7 recognized only murine
PCSK9 and an unrelated control antibody 42H7 did not recognize any
tested PCSK9. Id.
e. Determining Sequences of Antagonist PCSK9 Antibodies
[0277] The amino acid sequences of the variable domains of PCSK9
antibodies 4A5, 5A10, 6F6, and 7D4 were determined using the method
described in Example 5. The sequences indicate that the antibodies
are related but different from each other. Table 1 shows the amino
acid sequences of the variable regions of each antibody. Table 7
shows the CDR sequences of the light chains and heavy chains of
Table 1 as identified by the Kabat and Chotia methods.
TABLE-US-00008 TABLE 7 Blocking PCSK9 Antibodies and
Antigen-binding CDR Sequences according to Kabat (underlined) and
Chotia (bold). VL CDR1 VL CDR2 VL CDR3 4A5 KASQNVGTNVA SASYRYS
QQFYSYPYT (SEQ ID NO: 27) (SEQ ID NO: 28) (SEQ ID NO: 29) 5A10
KASQDVSTAVA SASYRYT QQRYSTPRT (SEQ ID NO: 30) (SEQ ID NO: 12) (SEQ
ID NO: 31) 6F6 SASQGISNYLN YTSSLHS QQYSKLPFT (SEQ ID NO: 32) (SEQ
ID NO: 33) (SEQ ID NO: 55) 7D4 KASQDVSNALA SASYRYT QQHYSTPWT (SEQ
ID NO: 34) (SEQ ID NO: 12) (SEQ ID NO: 35) L1L3 RASQGISSALA SASYRYT
QQRYSLWRT (SEQ ID NO: 11) (SEQ ID NO: 12) (SEQ ID NO: 13) VH CDR1
VH CDR2 VH CDR3 4A5 GYTFTDYYMN DINPNNGGTTYNQKF WLLFAY (SEQ ID NOs:
KG (SEQ ID NOs: (SEQ ID NO: 40) 56(whole), 38 and 39) 36 and 37)
5A10 GYTFTSYWMH EINPSNGRTNYNEKF ERPLYAMDY (SEQ ID NOs: KS (SEQ ID
NO: (SEQ ID NO: 45) 57(whole), 43 and 44) 41 and 42) 6F6 GYTFTDYYMN
DINPNNGGTSYNQKF GGIYYRYDRNYFDY (SEQ ID NOs: KG (SEQ ID NO: (SEQ ID
NO: 47) 56(whole), 38 and 46) 36 and 37) 7D4 GFTFSDYYMA
NINYDGSNTSYLDSL EKFAAMDY (SEQ ID NOs: KS (SEQ ID NOs: (SEQ ID NO:
52) 58(whole), 50 and 51) 48 and 49) L1L3 GYTFTSYYMH
EISPFGGRTNYNEKF ERPLYASDL SEQ ID NOs: KS (SEQ ID NO: (SEQ ID NO:
10) 59(whole), 9 and 61) 60, and 8.
[0278] Anti-PCSK9 IgGs 4A5, 5A10 and 6F6 were amine coupled to the
Biacore chip. hPCSK9 (100 nM) was mixed with 500 nM of 4A5, 5A10,
6F6 or 7D4 in various ratios and injected for 1 min at 10
.mu.l/min. The four antibodies mutually blocked one another
irrespective of the assay orientation tested, suggesting that they
all bind to competing epitopes. In contrast, they are able to form
sandwich complexes with other non-fully-blocking antibodies that
were mapped to specific regions using synthetic peptides.
2. Effect of PCSK9 Specific Antibodies as PCSK9 Antagonist In
Vivo
[0279] a. PCSK9 Antagonist Antibodies Lower Serum Cholesterol in
Mice
[0280] To determine if PCSK9 antagonist monoclonal antibodies can
affect cholesterol levels in vivo by inhibiting the function of
extracellular PCSK9, the effect of 7D4 was tested against mouse
PCSK9 in vitro, on serum cholesterol when injected into mice. 6 to
7 week old male C57/bl6 mice were kept on a 12 hr light/dark cycle,
bled to collect approximately 70 .mu.l serum on day -7. Antagonist
PCSK9 antibody 7D4, and a control isotype matching monoclonal
antibody were injected into male 7 week old C57/bl6 mice via i.p.
injections on days 0, 1, 2, and 3. Mice were sacrificed on day 4
without fasting, and serum samples were collected. All frozen serum
samples were sent to IDEXX laboratories for total cholesterol,
triglyceride, HDL cholesterol and LDL cholesterol measurements.
FIG. 6 shows that 7D4 lowered serum cholesterol by 48%, while the
control antibody did not have any significant affect. Both the
amount and percentage of reduction are similar to what was reported
for PCSK9-/- mice (PCSK9 knock-out mice), suggesting that one can
achieve complete or near complete inhibition of PCSK9 function
through blocking extracellular PCSK9 only, and that intracellular
PCSK9 plays little or no role in down-regulating LDLR under normal
physiological conditions. As expected, liver LDLR levels were
induced in animals treated with 7D4 compared to those treated with
a control antibody (FIG. 6).
b. A Partially Blocking Antibody Had No Effect on Blood Cholesterol
Levels
[0281] FIG. 7 illustrates that a partial antagonist polyclonal
anti-PCSK9 mAb CRN6 does not affect cholesterol levels in mice. Two
groups of 8 week old C57/bl6 mice (n=10 mice/group) were bled and
tested for cholesterol levels on day -7; dosed with 15 mg/kg/day of
CRN6 or a control antibody by i.v. administration on days 0, 1, 2
and 3; and then bled and tested for cholesterol levels 24 hrs after
the final dose. FIG. 7A shows that CRN6 antibody partially blocks
PCSK9 mediated down regulation of LDLR in Huh7 cells in vitro. FIG.
7B shows that administration of CRN6 antibody does not affect serum
cholesterol levels in mice.
c. Prolonged Effect on Serum Cholesterol by Antagonist PCSK9 mAb in
Mice.
[0282] A time course study was performed to determine the time of
onset and duration of the cholesterol lowering effect of PCSK9
antagonist antibodies in mice. MAb 7D4 or saline control were each
injected i.v. at 10 mg/kg or 3 ml/kg in 48 6-week-old C57/bl6 mice.
Eight mice from each treatment group were sacrificed on days 1, 2,
4, 7, 14 and 21 after injection. A single injection of 7D4 produced
a fast and prolonged lowering effect on serum cholesterol. A 25%
reduction in serum cholesterol was seen at 24 hrs after injection.
See FIG. 8. Maximum drop of serum cholesterol was observed at the 7
day time point. At 21 days, the reduction in cholesterol is no
longer statistically significant. Part B) shows HDL cholesterol.
LDL cholesterol levels were very low.
[0283] FIG. 9 illustrates that the anti-PCSK9 antagonist mAb 7D4
dose dependently reduces serum total cholesterol, HDL, and LDL in
mice. Six groups of 8 week old C57/bl6 mice (n=8/group) were bled
and tested for basal cholesterol levels on day -7 and administered
with the indicated doses of antibodies or saline on days 0, 1, 2,
and 3 by i. p. bolus injection. Serum samples were collected and
tested for cholesterol levels 24 hrs after the last dose. FIG. 9A
shows total cholesterol levels, which decreased to less than 60% of
control after administration of 3 to 30 mg/kg/day. The maximal
effect on total cholesterol was seen at 10 mg/kg, and statistically
significant reduction at 1 mg/kg. FIG. 9B shows HDL levels, which
decreased to less than 70% after administration of 3 to 30
mg/kg/day. FIG. 9C shows LDL levels, which decreased to nearly zero
at all tested doses of 0.3 mg/kg/day and above.
d. Dose Response of Antagonist Antibodies Specific to PCSK9 in
Mice
[0284] FIG. 10 illustrates that anti-PCSK9 antagonist antibody 5A10
dose dependently lowers cholesterol levels in mice. FIG. 10A shows
six groups of 8 week old C57/bl6 mice (n=8/group) to which were
administered the indicated doses of antibodies or saline daily on
days 0, 1, 2, and 3 by i.v. bolus injection. Serum samples were
collected and tested for cholesterol levels 24 hrs after the last
dose and showed a graduated decrease with increasing dose of
antibody. FIG. 10B shows five groups of 8 week old C57/bl6 mice
(n=8/group) to which were administered the indicated doses of
antibodies or saline on day 0 by i. p. bolus injection. Serum
samples were collected and tested for cholesterol levels on day 7
and also showed .a graduated decrease with increasing doses of
antibody.
[0285] FIG. 11 illustrates that anti-PCSK9 antagonist antibodies
4A5 and 6F6 lower cholesterol levels in mice in a dose-dependent
fashion. Eight week old C57/bl6 mice (n=8/group) were administered
the indicated doses of antibodies or saline on day 0 by i.p. bolus
injection. Serum samples were collected and tested for cholesterol
levels on day 7. In FIG. 11A, the antibody 4A5 showed a graduated
decrease in total serum cholesterol with increasing dose of
antibody. In FIG. 11B, the antibody 6F6 showed decrease in total
serum cholesterol at 10 mg/kg/day.
[0286] Anti-PCSK9 antagonist antibodies 4A5, 5A10, 6F6 and 7D4
increase liver LDLR levels in mice as found by Western blot
analysis. See FIG. 12. For 4A5, 5A10 and 6F6, 8 week old C57/bl6
mice were administered with 10 mg/kg of antibodies or saline on day
0 by i.v. bolus injection, animals were sacrificed on day 7, and
whole liver lysate of 3 individual animals were analyzed for LDLR
and GAPDH protein levels by Western. For 7D4, 8 week old Bl6/c57
mice were administered with 10 mg/kg of antibodies on days 0, 1, 2,
and 3 via i. p. bolus injection, animals were sacrificed on day 4,
and whole liver lysate of 3 individual animals were analyzed for
LDLR and GAPDH protein levels by Western blot. All antibody-treated
mice showed high levels of LDLR as compared to the PBS control
mice.
[0287] FIG. 13 illustrates that anti-PCSK9 antagonist antibody has
no effect in the LDLR-/- mouse. Eight week old LDLR-/- mice (LDLR
KO mice) were administered 10 mg/kg 4A5 or saline on day 0 by i.p.
bolus injection. Serum samples (from n=9-10 mice) were collected
and tested for cholesterol levels on day 7. Administration of the
antibody did not appreciably alter the levels of total serum
cholesterol, HDL, or LDL.
[0288] FIG. 14 illustrates that multiple treatments of anti-PCSK9
antagonist antibodies in mice can substantially decrease total
serum cholesterol. Eight week old C57/bl6 mice were administered
the indicated doses of antibodies or PBS on days 0, 7, 14 and 21 by
i.v. bolus injection. Serum samples (n=5-11 mice) were collected
and tested for cholesterol levels on day 28.
Example 8
PCSK9 Antagonist Antibodies Lower Serum LDL in Non-Human
Primates
[0289] To test the in vivo effect of antibodies to PCSK9, antibody
7D4 was tested in cynomolgus monkeys. Four 3-4 year old cynomolgus
monkey were injected with vehicle (PBS+0.01% Tween 20) on day 0,
and 10 mg/kg 7D4 on day 7. Plasma lipid profiles were analyzed on
days 0, 2, 7, 9, 11, 14, 21 and 28 following overnight fasting. A
single injection of 10 mg/kg 7D4 produced a dramatic reduction in
plasma LDL (60%) (FIG. 15A) and LDL particle numbers (FIG. 15D) in
all 4 animals, while having minimal effect on their HDL levels
(FIG. 15B) and HDL particle numbers (FIG. 15E). Total cholesterol
(FIG. 15C) was also reduced following 7D4 treatment, while
triglyceride level (FIG. 15F) was not significantly affected. Total
7D4 (G), and total PCSK9 levels (H) were also measured.
[0290] FIGS. 16A-D illustrate the dose-response of anti-PCSK9
antibody 7D4 on serum cholesterol levels in the cynomolgus monkey.
Two male and two female cynomolgus monkeys 3-5 years of age in each
group were given the indicated dose of 7D4 on day 7 and an equal
volume of saline on day 0 by i.v. bolus injection. Plasma samples
were taken at indicated time points and plasma LDL levels were
measured.
[0291] FIG. 17 illustrates a comparison of anti-PCSK9 antibodies
4A5 (FIG. 17A), 5A10 (FIG. 17B), 6F6 (FIG. 17C) and 7D4 (FIG. 17D)
on serum cholesterol levels in the cynomolgus monkey. Two male and
two female cynomolgus monkeys 3-6 years of age in each group were
given 1 mg/kg of the indicated antibody on day 0 by i.v. bolus
injection. Plasma samples were taken at indicated time points,
plasma LDL levels were measured and normalized to that on day
-2.
[0292] FIG. 18 illustrates the effect of anti-PCSK9 antagonist
antibody 7D4 on plasma cholesterol levels of cynomolgus monkeys fed
a 33.4% kcal fat diet supplemented with 0.1% cholesterol. Six 3-5
year old cynomolgus monkeys were put on high-fat diet for 16 weeks.
Three monkeys were treated with 10 mg/kg 7D4 and three with saline
on the indicated date. LDL levels of individual monkeys were
measured and normalized to that of the treatment day.
Example 9
Humanized Anti-PCSK9 Antibody
[0293] The murine monoclonal antibody 5A10 was humanized and
affinity matured to provide the L1 L3 antibody. L1 L3 has an
affinity for murine PCSK9 of 200 pM and an affinity for human PCSK9
of 100 pM when measured by Biacore. L1 L3 completely inhibits the
PCSK9-mediated down regulation of LDLR in cultured Huh7 cells when
incubated with 100 nM human or murine PCSK9 antibody. See FIG.
19.
[0294] FIG. 20 illustrates the dose-response of L1L3, mouse
precursor 5A10, and negative control antibody 42H7 to block the
binding of recombinant biotinylated human PCSK9 and mouse PCSK9 to
immobilized recombinant LDLR extracellular domain in vitro. FIG.
20A shows human PCSK9 binding to human LDLR extracellular domain at
pH 7.5. FIG. 20B shows human PCSK9 binding to human LDLR
extracellular domain at pH 5.3. FIG. 20C shows mouse PCSK9 binding
to human LDLR extracellular domain at pH 7.5. FIG. 20D shows mouse
PCSK9 binding to human LDLR extracellular domain at pH 5.3.
[0295] FIG. 21 shows the effect on serum cholesterol of treatment
with 10 mg/kg L1 L3 in mice. Two groups (n=8/group) of 8 week old
C57/bl6 mice were dosed with 10 mg/kg L1 L3 or an equal volume of
saline by i. p. injection on day 0. Serum samples were collected
and assayed for cholesterol levels on days 2, 4 and 7. L1 L3
decreased total serum cholesterol by about 40% at days 2 and 4. In
another study, when 10 mg/kg of L1 L3 was administered as a single
intraperitoneal (IP) dose to C57BL/6 mice fed a normal diet (n=10),
serum cholesterol levels were reduced by 47% compared to saline
treated controls, 4 days post treatment. When L1 L3 was
administered as a single IP dose at 0, 0.1, 1, 10 and 80 mg/kg
(n=6/group) in a dose-response experiment in male Sprague-Dawley
rats fed a normal diet, serum cholesterol levels were
dose-dependently reduced, with maximum effect of 50% seen at 10 and
80 mg/kg, 48 hours post dosing. The duration of the cholesterol
repression was also dose dependent, ranging from 1 to 21 days.
[0296] The amino acid sequence of L1 L3 fully humanized heavy chain
(SEQ ID NO:15) is shown in Table 8. The sequence of the variable
region is underlined (SEQ ID NO: 54).
TABLE-US-00009 TABLE 8 qvqlvqsqae vkkpgasvkv sckasgytft syymhwvrqa
pqqqlewmqe ispfqgrtny 60 nekfksrvtm trdtststvy melsslrsed
tavyycarer plyasdlwgq qttvtvssas 120 tkgpsvfpla pcsrstsest
aalgclvkdy fpepvtvswn sgaltsgvht fpavlqssgl 180 yslssvvtvp
ssnfgtqtyt cnvdhkpsnt kvdktverkc cvecppcpap pvagpsvflf 240
ppkpkdtlmi srtpevtcvv vdvshedpev qfnwyvdgve vhnaktkpre eqfnstfrvv
300 svltvvhqdw ingkeykckv snkglpssie ktisktkgqp repqvytlpp
sreemtknqv 360 sltclvkgfy psdiavewes ngqpennykt tppmldsdgs
fflyskltvd ksrwqqgnvf 420 scsvmhealh nhytqkslsl spgk 444
[0297] The amino acid sequence of L1 L3 fully humanized light chain
(SEQ ID NO:14) is shown in Table 9. The variable region is
underlined (SEQ ID NO: 53).
TABLE-US-00010 TABLE 9 diqmtqspss lsasvgdrvt itcrasqgis salawyqqkp
gkapklliys asyrytgvps 60 rfsgsgsgtd ftftisslqp ediatyycqq
ryslwrtfgq gtkleikrtv aapsvfifpp 120 sdeqlksgta svvcllnnfy
preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 180 lskadyekhk
vyacevthqg lsspvtksfn rgec 214
[0298] FIG. 22 shows the effect of intravenous administration of an
effective dose (3 mg/kg) of antibody 5A10 (solid circles) or
antibody L1 L3 (solid squares) to each of four cynomolgus monkeys
at day zero. The change in serum HDL (FIG. 22A) and serum LDL (FIG.
22B) was measured from -2 to +28 days. Both antibodies resulted in
greater than about 70% decrease in serum LDL levels by about seven
days, an effect that substantially persisted for about six more
days in the animals administered L1L3. All the animals showed
normal liver and kidney function and near-normal hematocrits.
[0299] L1 L3 dose-dependently reduced LDL-C, with a maximum effect
observed in the 10 mg/kg group, which maintained a 70% reduction in
LDL-C levels until day 21 post-dosing, and fully recovered by day
31. HDL-C levels were not affected by L1 L3 treatment in all dose
groups. The animals in the 3 mg/kg dose group (n=4) were also given
two additional IV doses of 3 mg/kg L1 L3 on study days 42 and 56
(2-weeks apart). These two additional doses again lowered LDL-C and
maintained LDL-C levels below 50% for 4 weeks. LDL-C levels
returned to normal two weeks later. Serum HDL-C levels remained
unchanged throughout the study.
[0300] The efficacy of L1 L3 in non-human primates with
hypercholesterolemia and pharmacodynamic interactions between L1 L3
and HMG-CoA reductase inhibiting statins were investigated. Prior
to the initiation of the study, the LDL-C levels of a cohort of
cynomolgus monkeys (n=12) were elevated to an average of 120 mg/dL,
compared to the normal average levels of 50 mg/dL, by feeding with
a diet containing 35% fat (wt/wt) and 600 ppm cholesterol for over
18 months. Surprisingly, no effect was observed on serum total
cholesterol or LDL-C levels after the daily administration of a
medium-dose (10 mg/animal) of Crestor.RTM. (rosuvastatin calcium)
for 6 weeks, and after a subsequent daily administration of
high-dose (20 mg/kg) for 2 weeks. A single administration of 3
mg/kg L1 L3 with Crestor.RTM. or vehicle treatment for 2 weeks,
effectively lowered serum LDL-C levels by 56% by day 5 post
treatment, and gradually recovered in 2.5 to 3 weeks while not
affecting HDL-C levels. Upon switching the animals to daily
administration of 50 mg/kg Zocor.RTM. (simvastatin), their LDL-C
levels reached a maximal reduction of 43% at day 5, and stabilized
thereafter. After 3 weeks of 50 mg/kg/day Zocor.RTM.
administration, these animals were treated with a single dose of 3
mg/kg L1 L3 while still receiving 50 mg/kg/day Zocor.RTM..
Administration of L1 L3 resulted in another additional 65%
reduction in LDL-C, in addition to the 43% reduction by Zocor.RTM.,
by day 5, and returned to pre-dosing levels within 2 weeks.
[0301] Other CDR amino acid substitutions were made to 5A10 in the
course of humanization and affinity maturation and to achieve
particular properties. The sequences of the modified CDRs and the
PCSK9 binding abilities of the antibodies containing these modified
CDRs are listed in FIGS. 24 A-G. The numbers following each
sequence in FIGS. 24 A-G represent the SEQ ID NO for that
sequence.
[0302] The disclosures of all references cited herein are hereby
incorporated by reference herein.
Sequence CWU 1
1
188115PRTHomo sapiens 1Asp Thr Ser Ile Gln Ser Asp His Arg Glu Ile
Glu Gly Arg Val 1 5 10 15 210PRTHomo sapiens 2Gly Arg Asp Ala Gly
Val Ala Lys Gly Ala 1 5 10 39PRTHomo sapiens 3Ala Ser Ser Asp Cys
Ser Thr Cys Phe 1 5 46PRTHomo sapiens 4Gly Gly Ser Leu Val Glu 1 5
56PRTHomo sapiens 5Gln Pro Val Gly Pro Leu 1 5 65PRTHomo sapiens
6His Gly Ala Gly Trp 1 5 75PRTHomo sapiens 7Ala Glu Pro Glu Leu 1 5
85PRTArtificialVARIABLE HEAVY CHAIN CDR 8Ser Tyr Tyr Met His 1 5
917PRTArtificialVARIABLE HEAVY CHAIN CDR 9Glu Ile Ser Pro Phe Gly
Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser
109PRTArtificialVARIABLE HEAVY CHAIN CDR 10Glu Arg Pro Leu Tyr Ala
Ser Asp Leu 1 5 1111PRTArtificialVARIABLE LIGHT CHAIN CDR 11Arg Ala
Ser Gln Gly Ile Ser Ser Ala Leu Ala 1 5 10 127PRTArtificialVARIABLE
LIGHT CHAIN CDR 12Ser Ala Ser Tyr Arg Tyr Thr 1 5
139PRTArtificialVARIABLE LIGHT CHAIN CDR 13Gln Gln Arg Tyr Ser Leu
Trp Arg Thr 1 5 14214PRTArtificialHUMANIZED L1L3 LIGHT CHAIN 14Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Arg Tyr Ser Leu Trp Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145
150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
15444PRTArtificialHUMANIZED L1L3 HEAVY CHAIN 15Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile Ser Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
Val 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 Glu Arg Pro Leu Tyr Ala Ser Asp
Leu Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190 Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
Cys Val Glu Cys 210 215 220 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 290 295
300 Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Thr 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 405 410 415
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420
425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
16108PRTMus musculus 16Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met
Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala
Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr
Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Leu Ser 65 70 75 80 Glu
Asp Leu Ala Glu Tyr Phe Cys Gln Gln Phe Tyr Ser Tyr Pro Tyr 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
17108PRTMus musculus 17Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr
Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu
Asp Leu Ala Val Tyr Tyr Cys Gln Gln Arg Tyr Ser Thr Pro Arg 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
18107PRTMus musculus 18Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala
Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Phe 85 90
95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 19108PRTMus
musculus 19Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser
Phe Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp
Val Ser Asn Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly His
Ser Pro Lys Leu Leu Ile 35 40 45 Phe Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala
Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 20115PRTMus
musculus 20Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly
Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly
Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Tyr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Trp Leu Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser Ala 115 21118PRTMus musculus 21Gln Val Gln Leu Gln Gln Pro
Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His
Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55
60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 22123PRTMus
musculus 22Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly
Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly
Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Gly
Gly Gly Ile Tyr Tyr Arg Tyr Asp Arg Asn Tyr Phe Asp Tyr 100 105 110
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 23117PRTMus
musculus 23Glu Val Lys Leu Val Glu Ser Glu Gly Gly Leu Val Gln Pro
Gly Ser 1 5 10 15 Ser Met Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Val Pro Glu
Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Tyr Asp Gly Ser
Asn Thr Ser Tyr Leu Asp Ser Leu 50 55 60 Lys Ser Arg Phe Ile Ile
Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Lys Phe Ala Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110
Val Thr Val Ser Ser 115 2415PRTArtificialLINKING PEPTIDE 24Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15
25642DNAArtificialHUMANIZED LIGHT CHAIN NUCLEOTIDE SEQUENCE
25gatatacaaa tgacacaatc tccatcctct ctttccgcat cagtcggcga ccgcgtaacc
60atcacatgta gagcttctca aggcatctcc tccgccctcg catggtacca acaaaaacca
120ggtaaagccc caaaactcct catatactca gcttcataca gatacaccgg
cgtaccctca 180agattctcag gttcaggctc tggaacagac tttactttca
ccatttcatc actccaaccc 240gaagacatag ctacatatta ctgccaacaa
agatacagcc tctggagaac atttggccaa 300ggaacaaaac tcgagatcaa
acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gt 642261332DNAArtificialHUMANIZED HEAVY
CHAIN NUCLEOTIDE SEQUENCE 26caagttcaac tcgttcaatc tggagcagaa
gtaaaaaaac ctggcgcctc tgttaaagta 60agttgtaaag catccggtta cacattcaca
tcatattaca tgcattgggt aagacaagcc 120cctggacaag gactcgaatg
gatgggtgaa atctctcctt ttggcggccg aacaaactat 180aatgaaaaat
ttaaatcccg cgtaactatg acccgagaca catccacatc tactgtttat
240atggaacttt cctcactgcg ttctgaagac actgctgttt attactgtgc
acgcgaaaga 300cctctctacg cttccgatct ctggggccaa ggaacaacgg
tcaccgtctc ctcagcctcc 360accaagggcc catctgtctt cccactggcc
ccatgctccc gcagcacctc cgagagcaca 420gccgccctgg gctgcctggt
caaggactac ttcccagaac ctgtgaccgt gtcctggaac 480tctggcgctc
tgaccagcgg cgtgcacacc ttcccagctg tcctgcagtc ctcaggtctc
540tactccctca gcagcgtggt gaccgtgcca tccagcaact tcggcaccca
gacctacacc 600tgcaacgtag atcacaagcc aagcaacacc aaggtagata
agaccgtgga gagaaagtgt 660tgtgtggagt gtccaccttg tccagcccct
ccagtggccg gaccatccgt gttcctgttc 720cctccaaagc caaaggacac
cctgatgatc tccagaaccc cagaggtgac ctgtgtggtg 780gtggacgtgt
cccacgagga cccagaggtg cagttcaact ggtatgtgga cggagtggag
840gtgcacaacg ccaagaccaa gccaagagag gagcagttca actccacctt
cagagtggtg 900agcgtgctga ccgtggtgca ccaggactgg ctgaacggaa
aggagtataa gtgtaaggtg 960tccaacaagg gactgccatc cagcatcgag
aagaccatct ccaagaccaa gggacagcca 1020agagagccac aggtgtatac
cctgccccca tccagagagg agatgaccaa gaaccaggtg 1080tccctgacct
gtctggtgaa gggattctat ccatccgaca tcgccgtgga gtgggagtcc
1140aacggacagc cagagaacaa ctataagacc acccctccaa tgctggactc
cgacggatcc 1200ttcttcctgt attccaagct gaccgtggac aagtccagat
ggcagcaggg aaacgtgttc 1260tcttgttccg tgatgcacga ggccctgcac
aaccactata cccagaagag cctgtccctg 1320tctccaggaa ag 13322711PRTMus
musculus 27Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10
287PRTMus musculus 28Ser Ala Ser Tyr Arg Tyr Ser 1 5 299PRTMus
musculus 29Gln Gln Phe Tyr Ser Tyr Pro Tyr Thr 1 5 3011PRTMus
musculus 30Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10
319PRTMus musculus 31Gln Gln Arg Tyr Ser Thr Pro Arg Thr 1 5
3211PRTMus musculus 32Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn 1
5 10 337PRTMus musculus 33Tyr Thr Ser Ser Leu His Ser 1 5
3411PRTMus musculus 34Lys Ala Ser Gln Asp Val Ser Asn Ala Leu Ala 1
5 10 359PRTMus musculus 35Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5
367PRTMus musculus 36Gly Tyr Thr Phe Thr Asp Tyr 1 5 375PRTMus
musculus 37Asp Tyr Tyr Met Asn 1 5 386PRTMus musculus 38Asn Pro Asn
Asn Gly Gly 1 5 3917PRTMus musculus 39Asp Ile Asn Pro Asn Asn Gly
Gly Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 406PRTMus
musculus 40Trp Leu Leu Phe Ala Tyr 1 5 417PRTMus musculus 41Gly Tyr
Thr Phe Thr Ser Tyr 1 5 425PRTMus musculus 42Ser Tyr Trp Met His 1
5 436PRTMus musculus 43Asn Pro Ser Asn Gly Arg 1 5
4417PRTMus musculus 44Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 459PRTMus musculus 45Glu Arg Pro
Leu Tyr Ala Met Asp Tyr 1 5 4617PRTMus musculus 46Asp Ile Asn Pro
Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly
4714PRTMus musculus 47Gly Gly Ile Tyr Tyr Arg Tyr Asp Arg Asn Tyr
Phe Asp Tyr 1 5 10 487PRTMus musculus 48Gly Phe Thr Phe Ser Asp Tyr
1 5 495PRTMus musculus 49Asp Tyr Tyr Met Ala 1 5 506PRTMus musculus
50Asn Tyr Asp Gly Ser Asn 1 5 5116PRTMus musculus 51Asn Ile Asn Tyr
Asp Gly Ser Asn Thr Ser Tyr Leu Asp Ser Leu Lys 1 5 10 15 528PRTMus
musculus 52Glu Lys Phe Ala Ala Met Asp Tyr 1 5 53107PRThomo sapiens
53Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser
Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Arg Tyr Ser Leu Trp Arg 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 54118PRThomo sapiens 54Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Glu Ile Ser Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu
Lys Phe 50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val 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 Glu Arg Pro Leu Tyr Ala
Ser Asp Leu Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser
115 559PRTArtificial SequenceSynthetic 55Gln Gln Tyr Ser Lys Leu
Pro Phe Thr 1 5 5610PRTArtificial SequenceSynthetic 56Gly Tyr Thr
Phe Thr Asp Tyr Tyr Met Asn 1 5 10 5710PRTArtificial
SequenceSynthetic 57Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10
5810PRTArtificial SequenceSynthetic 58Gly Phe Thr Phe Ser Asp Tyr
Tyr Met Ala 1 5 10 5910PRTArtificial SequenceSynthetic 59Gly Tyr
Thr Phe Thr Ser Tyr Tyr Met His 1 5 10 607PRTArtificial
SequenceSynthetic 60Gly Tyr Thr Phe Thr Ser Tyr 1 5
616PRTArtificial SequenceSynthetic 61Ser Pro Phe Gly Gly Arg 1 5
628PRTArtificial SequenceSynthetic 62Gln Asp Val Ser Thr Ala Val
Ala 1 5 6311PRTArtificial SequenceSynthetic 63Gly Gly Thr Arg Val
Val Ser Thr Ala Val Ala 1 5 10 6410PRTArtificial SequenceSynthetic
64Arg Gly Asp Phe Val Ser Thr Ala Val Ala 1 5 10 6517PRTArtificial
SequenceSynthetic 65Glu Ile Asn Pro Ser Gly Gly Arg Thr Asn Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Ser 6617PRTArtificial SequenceSynthetic
66Glu Ile Asn Pro Ser Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1
5 10 15 Ser 6717PRTArtificial SequenceSynthetic 67Glu Ile Asn Pro
Ser Thr Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser
6817PRTArtificial SequenceSynthetic 68Glu Ile Asn Pro Ser Ile Gly
Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser 6917PRTArtificial
SequenceSynthetic 69Glu Ile Asn Pro Ser Asp Ser Arg Thr Asn Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Ser 7017PRTArtificial SequenceSynthetic
70Glu Ile Asn Pro Ser Gly Asn Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1
5 10 15 Ser 7117PRTArtificial SequenceSynthetic 71Glu Ile Asn Pro
Ser Ser Ser Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser
729PRTArtificial SequenceSynthetic 72Glu Arg Pro Leu Tyr Ala Met
Asp Tyr 1 5 739PRTArtificial SequenceSynthetic 73Glu Arg Pro Leu
Tyr Ala Ala Asp Tyr 1 5 749PRTArtificial SequenceSynthetic 74Glu
Arg Pro Leu Tyr Ala Ile Asp Tyr 1 5 759PRTArtificial
SequenceSynthetic 75Glu Arg Pro Leu Tyr Ala Arg Asp Tyr 1 5
769PRTArtificial SequenceSynthetic 76Glu Arg Pro Leu Tyr Ala Gly
Asp Tyr 1 5 779PRTArtificial SequenceSynthetic 77Glu Arg Pro Leu
Tyr Ala Lys Asp Tyr 1 5 789PRTArtificial SequenceSynthetic 78Glu
Arg Pro Leu Tyr Ala Pro Asp Tyr 1 5 799PRTArtificial
SequenceSynthetic 79Glu Arg Pro Leu Tyr Ala Ser Asp Tyr 1 5
809PRTArtificial SequenceSynthetic 80Glu Arg Pro Leu Tyr Ala Leu
Asp Tyr 1 5 819PRTArtificial SequenceSynthetic 81Glu Arg Pro Leu
Tyr Ala Val Asp Tyr 1 5 829PRTArtificial SequenceSynthetic 82Glu
Arg Pro Leu Tyr Ala Trp Asp Tyr 1 5 839PRTArtificial
SequenceSynthetic 83Glu Arg Pro Leu Tyr Ala His Asp Tyr 1 5
849PRTArtificial SequenceSynthetic 84Glu Arg Pro Leu Tyr Ala Phe
Asp Tyr 1 5 859PRTArtificial SequenceSynthetic 85Glu Arg Pro Leu
Tyr Ala Thr Asp Tyr 1 5 869PRTArtificial SequenceSynthetic 86Gln
Gln Arg Phe Ser Thr Pro Arg Thr 1 5 879PRTArtificial
SequenceSynthetic 87Gln Gln Arg Tyr Ser Asp Trp Arg Thr 1 5
889PRTArtificial SequenceSynthetic 88Gln Gln Arg Tyr Ser Ser Trp
Arg Thr 1 5 899PRTArtificial SequenceSynthetic 89Gln Gln Arg Tyr
Ser Thr Ala Arg Thr 1 5 909PRTArtificial SequenceSynthetic 90Gln
Gln Arg Tyr Ser Leu Tyr Arg Thr 1 5 919PRTArtificial
SequenceSynthetic 91Gln Gln Arg Tyr Ser Phe Trp Arg Thr 1 5
929PRTArtificial SequenceSynthetic 92Gln Gln Arg Tyr Ser Pro Trp
Arg Thr 1 5 939PRTArtificial SequenceSynthetic 93Gln Gln Arg Tyr
Ser Gly Trp Arg Thr 1 5 949PRTArtificial SequenceSynthetic 94Gln
Gln Arg Tyr Ser Ile Trp Arg Thr 1 5 959PRTArtificial
SequenceSynthetic 95Gln Gln Arg Tyr Ser Ala Trp Arg Thr 1 5
969PRTArtificial SequenceSynthetic 96Gln Gln Arg Tyr Ser Leu Phe
Arg Thr 1 5 979PRTArtificial SequenceSynthetic 97Gln Gln Arg Tyr
Ser Thr Arg Arg Thr 1 5 989PRTArtificial SequenceSynthetic 98Gln
Gln Arg Tyr Ser Thr Leu Tyr Thr 1 5 999PRTArtificial
SequenceSynthetic 99Gln Gln Arg Tyr Ser Thr Trp Arg Thr 1 5
1009PRTArtificial SequenceSynthetic 100Gln Gln Arg Tyr Ser Leu Ala
Arg Thr 1 5 1019PRTArtificial SequenceSynthetic 101Gln Gln Arg Tyr
Ser Ser Glu Arg Thr 1 5 1029PRTArtificial SequenceSynthetic 102Gln
Gln Arg Tyr Gly Thr Ala Arg Thr 1 5 1039PRTArtificial
SequenceSynthetic 103Gln Gln Arg Tyr Ser Gln Ala Arg Thr 1 5
1049PRTArtificial SequenceSynthetic 104Gln Gln Arg Tyr Ser Leu His
Arg Thr 1 5 1059PRTArtificial SequenceSynthetic 105Gln Gln Arg Tyr
Ser Gly Val Arg Thr 1 5 1069PRTArtificial SequenceSynthetic 106Gln
Gln Arg Tyr Ser Gln Ser Arg Thr 1 5 1079PRTArtificial
SequenceSynthetic 107Gln Gln Arg Tyr Ser Ala Glu Arg Thr 1 5
1089PRTArtificial SequenceSynthetic 108Gln Gln Arg Tyr Ser Gln Phe
Arg Thr 1 5 1099PRTArtificial SequenceSynthetic 109Gln Gln Arg Tyr
Ser Ser Arg Arg Thr 1 5 1109PRTArtificial SequenceSynthetic 110Gln
Gln Arg Tyr Ser Cys Ser Arg Thr 1 5 1119PRTArtificial
SequenceSynthetic 111Gln Gln Arg Tyr Ser Thr Asn Arg Arg 1 5
1129PRTArtificial SequenceSynthetic 112Gln Gln Arg Tyr Ser Arg Trp
Arg Thr 1 5 1139PRTArtificial SequenceSynthetic 113Gln Gln Arg Tyr
Ser Pro Tyr Arg Thr 1 5 1149PRTArtificial SequenceSynthetic 114Gln
Gln Arg Tyr Ser Tyr Trp Arg Thr 1 5 1159PRTArtificial
SequenceSynthetic 115Gln Gln Arg Tyr Ser Gly Phe Arg Thr 1 5
1169PRTArtificial SequenceSynthetic 116Gln Gln Arg Tyr Ser Tyr Trp
Arg Thr 1 5 1179PRTArtificial SequenceSynthetic 117Gln Gln Arg Tyr
Ser Phe Lys Arg Thr 1 5 1189PRTArtificial SequenceSynthetic 118Gln
Gln Arg Tyr Ser Ala Arg Arg Thr 1 5 1199PRTArtificial
SequenceSynthetic 119Gln Gln Arg Tyr Ser Arg Tyr Arg Thr 1 5
1209PRTArtificial SequenceSynthetic 120Gln Gln Arg Tyr Ser Leu Gln
Arg Thr 1 5 1219PRTArtificial SequenceSynthetic 121Gln Gln Arg Tyr
Ser Thr Ser Arg Thr 1 5 1229PRTArtificial SequenceSynthetic 122Gln
Gln Arg Tyr Ser His Ala Arg Thr 1 5 1239PRTArtificial
SequenceSynthetic 123Gln Gln Arg Tyr Ser Lys Tyr Arg Thr 1 5
1249PRTArtificial SequenceSynthetic 124Gln Gln Arg Tyr Ser Gln Ser
Arg Thr 1 5 1259PRTArtificial SequenceSynthetic 125Gln Gln Arg Tyr
Ser Thr Ala Phe Thr 1 5 1269PRTArtificial SequenceSynthetic 126Gln
Gln Arg Tyr Ser Thr Cys Cys Thr 1 5 1279PRTArtificial
SequenceSynthetic 127Gln Gln Arg Tyr Ser Thr Asp Arg Thr 1 5
1289PRTArtificial SequenceSynthetic 128Gln Gln Arg Tyr Ser Glu Asp
Arg Thr 1 5 1298PRTArtificial SequenceSynthetic 129Gln Gln Arg Tyr
Val Gly Arg Thr 1 5 1309PRTArtificial SequenceSynthetic 130Gln Gln
Arg Tyr Ser Leu Ser Arg Thr 1 5 1319PRTArtificial SequenceSynthetic
131Gln Gln Arg Tyr Ser Leu Gly Arg Thr 1 5 1329PRTArtificial
SequenceSynthetic 132Gln Gln Arg Tyr Ser Arg Ala Arg Thr 1 5
1339PRTArtificial SequenceSynthetic 133Gln Gln Arg Tyr Ser His Ala
Arg Thr 1 5 1349PRTArtificial SequenceSynthetic 134Gln Gln Arg Tyr
Ser Thr Pro Asp Thr 1 5 1359PRTArtificial SequenceSynthetic 135Gln
Gln Arg Tyr Gln Gln Pro Arg Thr 1 5 13617PRTArtificial
SequenceSynthetic 136Glu Ile Gln Val Ser Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 13717PRTArtificial
SequenceSynthetic 137Glu Ile Asn Pro Trp Gln Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 13817PRTArtificial
SequenceSynthetic 138Glu Ile Asn Pro Val Gln Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 13917PRTArtificial
SequenceSynthetic 139Glu Ile Ser Pro Tyr Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14017PRTArtificial
SequenceSynthetic 140Glu Ile Gln Glu Ser Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14117PRTArtificial
SequenceSynthetic 141Glu Ile Ser Pro Ile Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14217PRTArtificial
SequenceSynthetic 142Glu Ile Asn Pro Glu His Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14317PRTArtificial
SequenceSynthetic 143Glu Ile Asn Pro Ser Glu Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14417PRTArtificial
SequenceSynthetic 144Glu Ile Asn Pro Trp Met Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14517PRTArtificial
SequenceSynthetic 145Glu Ile Asn Pro Gln Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14617PRTArtificial
SequenceSynthetic 146Glu Ile Asn Pro Val Lys Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14717PRTArtificial
SequenceSynthetic 147Glu Ile Gly Pro Trp Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14817PRTArtificial
SequenceSynthetic 148Glu Ile Asn Pro Ile Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 14917PRTArtificial
SequenceSynthetic 149Glu Ile Gln Ile Ser Gly Gly Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 15017PRTArtificial
SequenceSynthetic 150Glu Ile Asn Pro Gln Gly Thr Arg Thr Asn Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Ser 1519PRTArtificial
SequenceSynthetic 151Glu Arg Pro Leu Tyr Ala Ser Asp Ser 1 5
1529PRTArtificial SequenceSynthetic 152Glu Arg Pro Leu Tyr Ala Ser
Asp Arg 1 5 1539PRTArtificial SequenceSynthetic 153Glu Arg Pro Leu
Tyr Ala Met Asp Arg 1 5 1549PRTArtificial SequenceSynthetic 154Glu
Arg Pro Leu Tyr Ala Asn Asp Ala 1 5 1559PRTArtificial
SequenceSynthetic 155Glu Arg Pro Leu Tyr Ala Asn Asp Val 1 5
1569PRTArtificial SequenceSynthetic 156Glu Arg Pro Leu Tyr Ala His
Asp Val 1 5 1579PRTArtificial SequenceSynthetic 157Glu Arg Pro Leu
Tyr Ala Ser Asp Tyr 1 5 1589PRTArtificial SequenceSynthetic 158Glu
Arg Pro Leu Tyr Ala Ser Asp Val 1 5 1599PRTArtificial
SequenceSynthetic 159Glu Arg Pro Leu Tyr Ala Ser Asp Ala 1 5
1609PRTArtificial SequenceSynthetic 160Glu Arg Pro Leu Tyr Ala Asn
Asp Ser 1 5 1619PRTArtificial SequenceSynthetic 161Glu Arg Pro Leu
Tyr Ala Thr Asp Leu 1 5 1629PRTArtificial SequenceSynthetic 162Glu
Arg Pro Leu Tyr Ala Ser Asp Ser 1 5 1639PRTArtificial
SequenceSynthetic 163Glu Arg Pro Leu Tyr Ala Asn Asp Met 1 5
1649PRTArtificial SequenceSynthetic 164Glu Arg Pro Leu Tyr Ala His
Asp Leu 1 5 1659PRTArtificial SequenceSynthetic 165Glu Arg Pro Leu
Tyr Ala His Asp Ile 1 5 1669PRTArtificial SequenceSynthetic 166Glu
Arg Pro Leu Tyr Ala Asn Asp Val 1 5 1679PRTArtificial
SequenceSynthetic 167Glu Arg Pro Leu Tyr Ala Ser Asp Tyr 1 5
1689PRTArtificial SequenceSynthetic 168Glu Arg Pro Leu Tyr Ala Ser
Asp Arg 1 5 1699PRTArtificial SequenceSynthetic 169Glu Arg Pro Leu
Tyr Ala Ser Asp Val 1 5 1709PRTArtificial SequenceSynthetic 170Glu
Arg Pro Leu Tyr Ala His Asp Val 1 5 1719PRTArtificial
SequenceSynthetic 171Glu Arg Pro Leu Tyr Ala Asn Asp Met 1 5
1729PRTArtificial SequenceSynthetic 172Glu Arg Pro Leu Tyr Ala His
Asp Leu 1 5 17317PRTArtificial SequenceSynthetic 173Glu Ile Asn Pro
Trp Gln Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser
17417PRTArtificial SequenceSynthetic 174Glu Ile Asn Pro Val Gln Gly
Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser
17517PRTArtificial SequenceSynthetic 175Glu Ile Ser Pro Tyr Gly Gly
Arg Thr Asn Tyr Asn Glu
Lys Phe Lys 1 5 10 15 Ser 17617PRTArtificial SequenceSynthetic
176Glu Ile Gly Pro Trp Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15 Ser 1779PRTArtificial SequenceSynthetic 177Gln Gln Arg
Tyr Ser Asp Trp Arg Thr 1 5 1789PRTArtificial SequenceSynthetic
178Gln Gln Arg Tyr Ser Ser Trp Arg Thr 1 5 1799PRTArtificial
SequenceSynthetic 179Gln Gln Arg Tyr Ser Ala Glu Arg Thr 1 5
1809PRTArtificial SequenceSynthetic 180Gln Gln Arg Tyr Ser Leu His
Arg Thr 1 5 1819PRTArtificial SequenceSynthetic 181Gln Gln Arg Tyr
Ser Ser Glu Arg Thr 1 5 1829PRTArtificial SequenceSynthetic 182Gln
Gln Arg Tyr Ser Leu Gln Arg Thr 1 5 1839PRTArtificial
SequenceSynthetic 183Gln Gln Arg Tyr Ser Thr Arg Arg Thr 1 5
1849PRTArtificial SequenceSynthetic 184Gln Gln Arg Tyr Ser Asp Trp
Arg Thr 1 5 18517PRTArtificial SequenceSynthetic 185Glu Ile Ser Pro
Tyr Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser
1869PRTArtificial SequenceSynthetic 186Gln Gln Arg Tyr Ser Arg Ser
Arg Thr 1 5 1877PRTArtificial SequenceSynthetic 187Asp Ala Ser Asn
Arg Ala Thr 1 5 188692PRTHomo sapiens 188Met Gly Thr Val Ser Ser
Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Leu
Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25 30 Asp Glu
Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu 35 40 45
Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 50
55 60 His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val
Val 65 70 75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg
Thr Ala Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr
Leu Thr Lys Ile Leu 100 105 110 His Val Phe His Gly Leu Leu Pro Gly
Phe Leu Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu Leu Ala Leu
Lys Leu Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp Ser Ser Val
Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg 145 150 155 160 Ile Thr
Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly 165 170 175
Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp 180
185 190 His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn
Val 195 200 205 Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser
Lys Cys Asp 210 215 220 Ser His Gly Thr His Leu Ala Gly Val Val Ser
Gly Arg Asp Ala Gly 225 230 235 240 Val Ala Lys Gly Ala Ser Met Arg
Ser Leu Arg Val Leu Asn Cys Gln 245 250 255 Gly Lys Gly Thr Val Ser
Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg 260 265 270 Lys Ser Gln Leu
Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro 275 280 285 Leu Ala
Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu 290 295 300
Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp 305
310 315 320 Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile
Thr Val 325 330 335 Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu
Gly Thr Leu Gly 340 345 350 Thr Asn Phe Gly Arg Cys Val Asp Leu Phe
Ala Pro Gly Glu Asp Ile 355 360 365 Ile Gly Ala Ser Ser Asp Cys Ser
Thr Cys Phe Val Ser Gln Ser Gly 370 375 380 Thr Ser Gln Ala Ala Ala
His Val Ala Gly Ile Ala Ala Met Met Leu 385 390 395 400 Ser Ala Glu
Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 405 410 415 His
Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp 420 425
430 Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr
435 440 445 His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser
Ala His 450 455 460 Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg
Cys Ala Pro Asp 465 470 475 480 Glu Glu Leu Leu Ser Cys Ser Ser Phe
Ser Arg Ser Gly Lys Arg Arg 485 490 495 Gly Glu Arg Met Glu Ala Gln
Gly Gly Lys Leu Val Cys Arg Ala His 500 505 510 Asn Ala Phe Gly Gly
Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu 515 520 525 Leu Pro Gln
Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 530 535 540 Ser
Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr 545 550
555 560 Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys
Pro 565 570 575 Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val
Gly His Arg 580 585 590 Glu Ala Ser Ile His Ala Ser Cys Cys His Ala
Pro Gly Leu Glu Cys 595 600 605 Lys Val Lys Glu His Gly Ile Pro Ala
Pro Gln Glu Gln Val Thr Val 610 615 620 Ala Cys Glu Glu Gly Trp Thr
Leu Thr Gly Cys Ser Ala Leu Pro Gly 625 630 635 640 Thr Ser His Val
Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val 645 650 655 Arg Ser
Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val 660 665 670
Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser 675
680 685 Gln Glu Leu Gln 690
* * * * *