U.S. patent application number 14/094560 was filed with the patent office on 2014-06-12 for anti-pcsk9 and methods for treating lipid and cholesterol disorders.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Tatyana Churakova, Joseph A. Hedrick, Diane Hollenbaugh, Frederick James Monsma.
Application Number | 20140161798 14/094560 |
Document ID | / |
Family ID | 40261953 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140161798 |
Kind Code |
A1 |
Hedrick; Joseph A. ; et
al. |
June 12, 2014 |
ANTI-PCSK9 AND METHODS FOR TREATING LIPID AND CHOLESTEROL
DISORDERS
Abstract
The present invention provides compositions and methods for
treating disorders of cholesterol and lipid metabolism by
administration of an anti-PCSK9 antibody or a peptide inhibitor of
PCSK9.
Inventors: |
Hedrick; Joseph A.; (South
River, NJ) ; Monsma; Frederick James; (Summit,
NJ) ; Churakova; Tatyana; (Sunnyvale, CA) ;
Hollenbaugh; Diane; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Family ID: |
40261953 |
Appl. No.: |
14/094560 |
Filed: |
December 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12739761 |
Oct 6, 2010 |
8598320 |
|
|
PCT/US2008/081311 |
Oct 27, 2008 |
|
|
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14094560 |
|
|
|
|
60982922 |
Oct 26, 2007 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/139.1; 435/69.6; 530/387.2; 530/387.3; 530/387.9 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/56 20130101; A61P 9/10 20180101; C07K 2317/34 20130101;
C07K 16/40 20130101; A61K 39/3955 20130101; A61P 3/06 20180101;
A61P 29/00 20180101; C07K 2317/565 20130101; A61K 31/397 20130101;
A61P 43/00 20180101; A61K 2039/505 20130101; A61K 45/06
20130101 |
Class at
Publication: |
424/133.1 ;
530/387.9; 424/139.1; 530/387.3; 530/387.2; 435/69.6 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 45/06 20060101 A61K045/06; A61K 31/397 20060101
A61K031/397; A61K 39/395 20060101 A61K039/395 |
Claims
1. An isolated antibody or antigen-binding fragment thereof
comprising one or more members selected from the group consisting
of: (i) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 11B5
heavy chain immunoglobulin which comprises the amino acid sequence
of SEQ ID NO: 10; (ii) CDR-H1, CDR-H2 and CDR-H3 of the variable
region the 75B9 heavy chain immunoglobulin which comprises the
amino acid sequence of SEQ ID NO: 18; (iii) CDR-H1, CDR-H2 and
CDR-H3 of the variable region the 77D10 heavy chain immunoglobulin
which comprises the amino acid sequence of SEQ ID NO: 26; (iv)
CDR-H1, CDR-H2 and CDR-H3 of the variable region the 29C10 heavy
chain immunoglobulin which comprises the amino acid sequence of SEQ
ID NO: 34; (v) CDR-H1, CDR-H2 and CDR-H3 of the variable region the
22D11 heavy chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 42; (vi) CDR-H1, CDR-H2 and CDR-H3 of the
variable region the 1F11/1G11 heavy chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 50; (vii) CDR-L1,
CDR-L2 and CDR-L3 of the variable region the 11B5 light chain
immunoglobulin which comprises the amino acid sequence of SEQ ID
NO: 14; (viii) CDR-L1, CDR-L2 and CDR-L3 of the variable region the
75B9 light chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 22; (ix) CDR-L1, CDR-L2 and CDR-L3 of the
variable region the 77D10 light chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 30; (x) CDR-L1,
CDR-L2 and CDR-L3 of the variable region the 29C10 light chain
immunoglobulin which comprises the amino acid sequence of SEQ ID
NO: 38; (xi) CDR-L1, CDR-L2 and CDR-L3 of the variable region the
22D11 light chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 46; and (xii) CDR-L1, CDR-L2 and CDR-L3 of
the variable region the 1F11/1G11 light chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 54.
2. A composition comprising the antibody or antigen-binding
fragment thereof of claim 1 in association with a further
chemotherapeutic agent.
3. The composition of claim 2 wherein the further chemotherapeutic
agent is a cardiovascular agent, an adrenergic blocker, an
antihypertensive agent, an angiotensin system inhibitor, an
angiotensin-converting enzyme inhibitor, a coronary vasodilator, a
diuretic, an adrenergic stimulant or an HMG-CoA reductase
inhibitor.
4. The composition of claim 2 wherein the further chemotherapeutic
agent is ezetimibe, lovastatin, atorvastatin, pravastatin,
rosuvastatin, fluvastatin, rivastatin, simvastatin, an azetidinone,
bunolol hydrochloride, acebutolol, alprenolol hydrochloride,
atenolol, carteolol hydrochloride, celiprolol hydrochloride;
cetamolol hydrochloride, labetalol hydrochloride, esmolol
hydrochloride, levobetaxolol hydrochloride, levobunolol
hydrochloride, nadolol, practolol, propranolol hydrochloride,
sotalol hydrochloride, timolol, timolol maleate, bisoprolol;
bisoprolol fumarate, nebivalol, cicloprolol hydrochloride,
dexpropranolol hydrochloride, diacetolol hydrochloride, dilevalol
hydrochloride, exaprolol hydrochloride, flestolol sulfate, metalol
hydrochloride, metoprolol 2-Propanol, metoprolol tartrate,
pamatolol sulfate, penbutolol sulfate, practolol, tiprenolol
hydrochloride or tolamolol.
5. The composition of claim 4 wherein the further chemotherapeutic
agent is ezetimibe optionally in association with simvastatin.
6. The antibody or antigen-binding fragment thereof of claim 1,
which is a humanized antibody.
7. The antibody or antigen-binding fragment thereof of claim 1,
which is an antibody which is a monoclonal antibody, a labeled
antibody, a bivalent antibody, a polyclonal antibody, a bispecific
antibody, a chimeric antibody, a recombinant antibody, an
anti-idiotypic antibody, a humanized antibody or a bispecific
antibody.
8. The antibody or antigen-binding fragment thereof of claim 1,
which is an antigen-binding fragment which is a camelized single
domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a
(dsfv).sub.2, a dsFv-dsfv', a bispecific ds diabody, an Fv, an Fab,
an Fab', an F(ab').sub.2, or a domain antibody.
9. The antibody or antigen-binding fragment thereof of claim 1
which is linked to an immunoglobulin constant region.
10. The antibody or antigen-binding fragment thereof of claim 9
wherein the constant region is a K light chain, .gamma.1 heavy
chain, .gamma.2 heavy chain, .gamma.3 heavy chain or .gamma.4 heavy
chain.
11. A pharmaceutical composition comprising the antibody or
fragment of claim 1 in association with a pharmaceutically
acceptable carrier.
12. An isolated antibody or antigen-binding fragment thereof
comprising one or more members selected from the group consisting
of: TABLE-US-00020 (i) HCDR1 comprising the amino acid sequence
(SEQ ID NO: 11) G F N I K D T Y M H, HCDR2 comprising the amino
acid sequence (SEQ ID NO: 12) R I D P A N G H T E Y D P K F Q D and
HCDR3 comprising the amino acid sequence (SEQ ID NO: 13) S Y F G S
I F A Y; (ii) HCDR1 comprising the amino acid sequence (SEQ ID NO:
19) G F N I K D T Y I H, HCDR2 comprising the amino acid sequence
(SEQ ID NO: 20) R I D P A N G H T E Y D P K F Q G and HCDR3
comprising the amino acid sequence (SEQ ID NO: 21) S Y Y G S I F A
Y; (iii) HCDR1 comprising the amino acid sequence (SEQ ID NO: 27) G
F N I K D Y Y I H, HCDR2 comprising the amino acid sequence (SEQ ID
NO: 28) W I D P E N G D T E Y A P K F Q G and HCDR3 comprising the
amino acid sequence (SEQ ID NO: 29) Y Y R Y D D G T W F P Y; (iv)
HCDR1 comprising the amino acid sequence (SEQ ID NO: 35) G F N I K
D T Y I H, HCDR2 comprising the amino acid sequence (SEQ ID NO: 36)
W I D P A N G Y T K Y A P N F Q G and HCDR3 comprising the amino
acid sequence (SEQ ID NO: 37) G Y Y R Y Y S L D Y; (v) HCDR1
comprising the amino acid sequence (SEQ ID NO: 43) G F T F S N H D
M A, HCDR2 comprising the amino acid sequence (SEQ ID NO: 44) S I T
P S G G T T Y Y R D S V E G and HCDR3 comprising the amino acid
sequence (SEQ ID NO: 45) Q N Y Y D G S Y Y Y G L Y Y F D Y; (vi)
HCDR1 comprising the amino acid sequence (SEQ ID NO: 51) G Y T F T
D Y Y M N, HCDR2 comprising the amino acid sequence (SEQ ID NO: 52)
D I N P N N G G A I Y N Q K F K G and HCDR3 comprising the amino
acid sequence (SEQ ID NO: 53) G I I T E I A E D F; (vii) LCDR1
comprising the amino acid sequence (SEQ ID NO: 15) S A S S S V S Y
L Y, LCDR2 comprising the amino acid sequence (SEQ ID NO: 16) R S S
H R A S and LCDR3 comprising the amino acid sequence (SEQ ID NO:
17) H Q Y Q S Y P P T; (viii) LCDR1 comprising the amino acid
sequence (SEQ ID NO: 23) S A S S S V S Y L F, LCDR2 comprising the
amino acid sequence (SEQ ID NO: 24) R T S Y L A S and LCDR3
comprising the amino acid sequence (SEQ ID NO: 25) H Q Y H T Y P P
T; (ix) LCDR1 comprising the amino acid sequence (SEQ ID NO: 31) R
A S G N I H S Y L A, LCDR2 comprising the amino acid sequence (SEQ
ID NO: 32) N A K T L P D and LCDR3 comprising the amino acid
sequence (SEQ ID NO: 33) Q H F W N T P W T; (x) LCDR1 comprising
the amino acid sequence (SEQ ID NO: 39) R A S Q D I S N Y L N,
LCDR2 comprising the amino acid sequence (SEQ ID NO: 40) Y S S R L
H S and LCDR3 comprising the amino acid sequence (SEQ ID NO: 41) Q
Q G K T L P L T; (xi) LCDR1 comprising the amino acid sequence (SEQ
ID NO: 47) R S S Q S L V Y S D G N T Y L H, LCDR2 comprising the
amino acid sequence (SEQ ID NO: 48) R V S N R F S and LCDR3
comprising the amino acid sequence (SEQ ID NO: 49) L Q S T H F P P
T; and (xii) LCDR1 comprising the amino acid sequence (SEQ ID NO:
55) K A S Q N V G T N V V, LCDR2 comprising the amino acid sequence
(SEQ ID NO: 56) S A S Y R Y S and LCDR3 comprising the amino acid
sequence (SEQ ID NO: 57) Q Q Y K T Y P Y T.
13. A composition comprising the antibody or antigen-binding
fragment thereof of claim 12 in association with a further
chemotherapeutic agent.
14. The composition of claim 13 wherein the further
chemotherapeutic agent is a cardiovascular agent, an adrenergic
blocker, an antihypertensive agent, an angiotensin system
inhibitor, an angiotensin-converting enzyme inhibitor, a coronary
vasodilator, a diuretic, an adrenergic stimulant or an HMG-CoA
reductase inhibitor.
15. The composition of claim 13 wherein the further
chemotherapeutic agent is ezetimibe, lovastatin, atorvastatin,
pravastatin, rosuvastatin, fluvastatin, rivastatin, simvastatin, an
azetidinone, bunolol hydrochloride, acebutolol, alprenolol
hydrochloride, atenolol, carteolol hydrochloride, celiprolol
hydrochloride; cetamolol hydrochloride, labetalol hydrochloride,
esmolol hydrochloride, levobetaxolol hydrochloride, levobunolol
hydrochloride, nadolol, practolol, propranolol hydrochloride,
sotalol hydrochloride, timolol, timolol maleate, bisoprolol;
bisoprolol fumarate, nebivalol, cicloprolol hydrochloride,
dexpropranolol hydrochloride, diacetolol hydrochloride, dilevalol
hydrochloride, exaprolol hydrochloride, flestolol sulfate, metalol
hydrochloride, metoprolol 2-Propanol, metoprolol tartrate,
pamatolol sulfate, penbutolol sulfate, practolol, tiprenolol
hydrochloride, tolamolol fenspiride hydrochloride, proroxan,
alfuzosin hydrochloride, labetalol hydrochloride, bretylium
tosylate, dihydroergtamine mesylate, carvedilol, labetalol,
bretylium tosylate, phentolamine mesylate, solypertine tartrate,
zolertine hydrochloride, candesartan cilexetil, telmisartan,
candesartan; losartan potassium, benazepril hydrochloride,
captopril, fosinopril, moexipril hydrochloride, perindopril
erbumine, quinapril, ramipril, enalapril maleate, lisinopril,
delapril, spirapril, delapril hydrochloride, libenzapril,
pentopril, perindopril 1H-Indole-2-carboxylic acid, quinaprilat,
spirapril hydrochloride, spiraprilat, teprotide, zofenopril,
amlodipine besylate, clentiazem maleate, isradipine, nimodipine,
felodipine, nilvadipine; diltiazem hydrochloride, verapamil
hydrochloride, teludipine hydrochloride, belfosdil, fostedil,
ranolazine, butoprozine hydrochloride, tosifen, molsidomine,
ranolazine hydrochloride, tosifen, diltiazem hydrochloride,
isosorbide dinitrate, sosorbide mononitrate, nitroglycerin,
verapamil hydrochloride, chromonar, clonitate, droprenilamine,
lidoflazine, prenylamine, propatyl nitrate, mioflazine
hydrochloride, mixidine, molsidomine, isosorbide mononitrate,
erythrityl tetranitrate, clonitrate, dipyridamole; nicorandil,
pyridinecarboxamide, nifedipine; perhexyline maleate; oxprenolol,
pentrinitrol, verapamil, althiazide, benzthiazide, buthiazide,
chlorothiazide, spironolactone, triamterene, guanfacine
hydrochloride, methyldopa-hydrochlorothiazide combined with
Hydrochlorothiazide, methyldopa-chlorothiazide, clonidine
hydrochloride, chlorthalidone, clonidine hydrochloride, clonidine,
althiazide, benzthiazide, captopril, carvedilol, chlorothiazide,
clonidine hydrochloride, cyclothiazide, delapril hydrochloride,
dilevalol hydrochloride, delapril hydrochloride, doxazosin
mesylate, fosinopril sodium, moexipril hydrochloride, monatepil
maleate, metoprolol succinate, guanfacine hydrochloride,
methyldopa, quinaprilat, quinapril hydrochloride, primidolol,
prazosin hydrochloride, pelanserin hydrochloride, phenoxybenzamine
hydrochloride, candesartan cilexetil, telmisartan, candesartan,
amlodipine maleate, terazosin hydrochloride, bevantolol
hydrochloride or ramipril.
16. The composition of claim 15 wherein the further
chemotherapeutic agent is ezetimibe optionally in association with
simvastatin.
17. The antibody or antigen-binding fragment thereof of claim 12,
which is a humanized antibody.
18. The antibody or antigen-binding fragment thereof of claim 12,
which is a humanized antibody.
19. The antibody or antigen-binding fragment thereof of claim 12,
which is an antibody which is a monoclonal antibody, a labeled
antibody, a bivalent antibody, a polyclonal antibody, a bispecific
antibody, a chimeric antibody, a recombinant antibody, an
anti-idiotypic antibody, a humanized antibody or a bispecific
antibody.
20. The antibody or antigen-binding fragment thereof of claim 12,
which is an antigen-binding fragment which is a camelized single
domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a
(dsfv).sub.2, a dsFv-dsfv', a bispecific ds diabody, an Fv, an Fab,
an Fab', an F(ab').sub.2, or a domain antibody.
21. The antibody or antigen-binding fragment thereof of claim 12
which is linked to an immunoglobulin constant region.
22. The antibody or antigen-binding fragment thereof of claim 21
wherein the constant region is a .kappa. light chain, .gamma.1
heavy chain, .gamma.2 heavy chain, .gamma.3 heavy chain or .gamma.4
heavy chain.
23. A pharmaceutical composition comprising the antibody or
fragment of claim 12 in association with a pharmaceutically
acceptable carrier.
24. The isolated antibody or antigen-binding fragment thereof of
claim 12 comprising one or more members selected from the group
consisting of: TABLE-US-00021 (a) an immunoglobulin heavy chain
comprising the amino acid sequence: (SEQ ID NO: 10) E V Q L Q Q S G
A E L V K P G A S V T L S C T A S G F N I K D T Y M H W V N Q R P E
Q G L V W I G R I D P A N G H T E Y D P K F Q D K A T I T T D T S S
N T A Y L H L S S L T S G D T A V Y Y C A R S Y E G S I F A Y W G Q
G T L V T V S A; (b) an immunoglobulin light chain comprising the
amino acid sequence: (SEQ ID NO: 14) Q I V L T Q S P A I M S A S P
G E K V T I S C S A S S S V S Y L Y W Y Q Q K P G S S P K P W I F R
S S H R A S G V P A R F S G S G S G T S Y S L T I S S N E A E D A A
T Y Y C H Q Y Q S Y P P T F G G G T K L E I K R A; (c) an
immunoglobulin heavy chain comprising the amino acid sequence: (SEQ
ID NO: 18) E V Q L Q Q S G A D L V K P G A S V K L S C T A S G F N
I K D T Y I H W V K Q R P E Q G L E W I G R I D P A N G H T E Y D P
K F Q G R A T L T T D T S S N T A Y L Q L F S L T S E D S A V Y F C
A R S Y Y G S I F A Y W G Q G T L V T V S A; (d) an immunoglobulin
light chain comprising the amino acid sequence: (SEQ ID NO: 22) Q I
V L T Q S P A I M S A S P G E K V T I S C S A S S S V S Y L F W Y Q
Q K P G S S P K P W I F R T S Y L A S G V P A R F S G S G S G T S F
S L T I S S N E A E D A A T Y Y C H Q Y H T Y P P T F G G G T K L E
I K R A; (e) an immunoglobulin heavy chain comprising the amino
acid sequence: (SEQ ID NO: 26) E V Q L Q Q S G A E L V R S G A S V
K L S C T T S G F N I K D Y Y I H W V K Q R P E Q G L E W I G W I D
P E N G D T E Y A P K F Q G K A T M T A D T S S N T A Y L Q L S S L
T S A D T A V Y Y C N A Y Y R Y D D G T W F P Y W G Q G T L V T V S
A; (f) an immunoglobulin light chain comprising the amino acid
sequence: (SEQ ID NO: 30) D I Q L T Q S P A S L S A S V G E T V T I
T C R A S G N I H S Y L A W Y Q Q K Q G K S P Q F L V D N A K T L P
D G V P S R F S V S G S G T Q Y S L K I N S L Q P E D F G T Y Y C Q
H F W N T P W T F G G G T K L E I K R A; (g) an immunoglobulin
heavy chain comprising the amino acid sequence: (SEQ ID NO: 34) E V
L L Q Q S V A E L V R P G A S V R L S C T A S G F N I K D T Y I H W
V R Q R P E Q G L E W F G W I D P A N G Y T K Y A P N F Q G K A T L
T T D T S S N T A Y L H L S S L T S E D S A I Y Y C A R G Y Y R Y Y
S L D Y W G Q G T S V T V S S; (h) an immunoglobulin light chain
comprising the amino acid sequence: (SEQ ID NO: 38) D I Q M T Q T T
S S L S A S L G D R V T I S C R A S Q D I S N Y L N W Y Q Q K P D G
T V K L L I Y Y S S R L H S G V P S R F S G R G S G T D Y S L T I S
T L E Q E D I A T Y F C Q Q G K T L P L T F G A G T K L E L K R A;
(i) an immunoglobulin heavy chain comprising the amino acid
sequence: (SEQ ID NO: 42) E V Q L V D S G G G L V Q P G R S L K L S
C A A S G F T F S N H D M A W V R Q A P T K G L E W V A S I T P S G
G T T Y Y R D S V E G R F T V S R D N V K S S L H L Q M D S L T S E
D T A T Y Y C A R Q N Y Y D G S Y Y Y G L Y Y F D Y W G Q G V M V T
V S S; (j) an immunoglobulin light chain comprising the amino acid
sequence: (SEQ ID NO: 46) D V L M T Q T P V S L P V S L G G Q V S I
S C R S S Q S L V Y S D G N T Y L H W Y L Q K P G Q S P Q L L I Y R
V S N R F S G V P D R F S G S G S G T D F T L K I S R V E P E D L G
L Y Y C L Q S T H F P P T F G S G T K L E I K R A; (k) an
immunoglobulin heavy chain comprising the amino acid sequence: (SEQ
ID NO: 50) E V Q L Q Q S G P E L V K P G A S V K I S C K V S G Y T
F T D Y Y M N W V K Q S H G K S L E W I G D I N P N N G G A I Y N Q
K F K G K A T L T V D K S S S I A Y M E L R S L T S E D S A V Y Y C
T S G I I T E I A E D F W G Q G T T L T V S S; and (l) an
immunoglobulin light chain comprising the amino acid sequence: (SEQ
ID NO: 54) D I V M T Q S Q K F M S T S V G D R V S V T C K A S Q N
V G T N V V W Y Q Q K P G Q S P K A L I H S A S Y R Y S G V P D R F
K G S G S G T D F T L T I T N V Q S E D L A G F F C Q Q Y K T Y P Y
T F G G G T Q L E I K R A.
25. A composition comprising the antibody or antigen-binding
fragment thereof of claim 24 in association with a further
chemotherapeutic agent.
26. The composition of claim 25 wherein the further
chemotherapeutic agent is a cardiovascular agent, an adrenergic
blocker, an antihypertensive agent, an angiotensin system
inhibitor, an angiotensin-converting enzyme inhibitor, a coronary
vasodilator, a diuretic, an adrenergic stimulant or an HMG-CoA
reductase inhibitor.
27. The composition of claim 25 wherein the further
chemotherapeutic agent is ezetimibe, lovastatin, atorvastatin,
pravastatin, rosuvastatin, fluvastatin, rivastatin, simvastatin, an
azetidinone, bunolol hydrochloride, acebutolol, alprenolol
hydrochloride, atenolol, carteolol hydrochloride, celiprolol
hydrochloride; cetamolol hydrochloride, labetalol hydrochloride,
esmolol hydrochloride, levobetaxolol hydrochloride, levobunolol
hydrochloride, nadolol, practolol, propranolol hydrochloride,
sotalol hydrochloride, timolol, timolol maleate, bisoprolol;
bisoprolol fumarate, nebivalol, cicloprolol hydrochloride,
dexpropranolol hydrochloride, diacetolol hydrochloride, dilevalol
hydrochloride, exaprolol hydrochloride, flestolol sulfate, metalol
hydrochloride, metoprolol 2-Propanol, metoprolol tartrate,
pamatolol sulfate, penbutolol sulfate, practolol, tiprenolol
hydrochloride, tolamolol fenspiride hydrochloride, proroxan,
alfuzosin hydrochloride, labetalol hydrochloride, bretylium
tosylate, dihydroergtamine mesylate, carvedilol, labetalol,
bretylium tosylate, phentolamine mesylate, solypertine tartrate,
zolertine hydrochloride, candesartan cilexetil, telmisartan,
candesartan; losartan potassium, benazepril hydrochloride,
captopril, fosinopril, moexipril hydrochloride, perindopril
erbumine, quinapril, ramipril, enalapril maleate, lisinopril,
delapril, spirapril, delapril hydrochloride, libenzapril,
pentopril, perindopril 1H-Indole-2-carboxylic acid, quinaprilat,
spirapril hydrochloride, spiraprilat, teprotide, zofenopril,
amlodipine besylate, clentiazem maleate, isradipine, nimodipine,
felodipine, nilvadipine; diltiazem hydrochloride, verapamil
hydrochloride, teludipine hydrochloride, belfosdil, fostedil,
ranolazine, butoprozine hydrochloride, tosifen, molsidomine,
ranolazine hydrochloride, tosifen, diltiazem hydrochloride,
isosorbide dinitrate, sosorbide mononitrate, nitroglycerin,
verapamil hydrochloride, chromonar, clonitate, droprenilamine,
lidoflazine, prenylamine, propatyl nitrate, mioflazine
hydrochloride, mixidine, molsidomine, isosorbide mononitrate,
erythrityl tetranitrate, clonitrate, dipyridamole; nicorandil,
pyridinecarboxamide, nifedipine; perhexyline maleate; oxprenolol,
pentrinitrol, verapamil, althiazide, benzthiazide, buthiazide,
chlorothiazide, spironolactone, triamterene, guanfacine
hydrochloride, methyldopa-hydrochlorothiazide combined with
Hydrochlorothiazide, methyldopa-chlorothiazide, clonidine
hydrochloride, chlorthalidone, clonidine hydrochloride, clonidine,
althiazide, benzthiazide, captopril, carvedilol, chlorothiazide,
clonidine hydrochloride, cyclothiazide, delapril hydrochloride,
dilevalol hydrochloride, delapril hydrochloride, doxazosin
mesylate, fosinopril sodium, moexipril hydrochloride, monatepil
maleate, metoprolol succinate, guanfacine hydrochloride,
methyldopa, quinaprilat, quinapril hydrochloride, primidolol,
prazosin hydrochloride, pelanserin hydrochloride, phenoxybenzamine
hydrochloride, candesartan cilexetil, telmisartan, candesartan,
amlodipine maleate, terazosin hydrochloride, bevantolol
hydrochloride or ramipril.
28. The composition of claim 27 wherein the further
chemotherapeutic agent is ezetimibe optionally in association with
simvastatin.
29. The antibody or antigen-binding fragment thereof of claim 24,
which is a humanized antibody.
30. The antibody or antigen-binding fragment thereof of claim 24,
which is a humanized antibody.
31. The antibody or antigen-binding fragment thereof of claim 24,
which is an antibody which is a monoclonal antibody, a labeled
antibody, a bivalent antibody, a polyclonal antibody, a bispecific
antibody, a chimeric antibody, a recombinant antibody, an
anti-idiotypic antibody, a humanized antibody or a bispecific
antibody.
32. The antibody or antigen-binding fragment thereof of claim 24,
which is an antigen-binding fragment which is a camelized single
domain antibody, a diabody, an scfv, an scfv dimer, a dsfv, a
(dsfv).sub.2, a dsFv-dsfv', a bispecific ds diabody, an Fv, an Fab,
an Fab', an F(ab').sub.2, or a domain antibody.
33. The antibody or antigen-binding fragment thereof of claim 24
which is linked to an immunoglobulin constant region.
34. The antibody or antigen-binding fragment thereof of claim 33
wherein the constant region is a .kappa. light chain, .gamma.1
heavy chain, .gamma.2 heavy chain, .gamma.3 heavy chain or .gamma.4
heavy chain.
35. A pharmaceutical composition comprising the antibody or
fragment of claim 24 in association with a pharmaceutically
acceptable carrier.
36. A pharmaceutical composition comprising an isolated antibody or
antigen-binding fragment thereof or isolated EGF-A polypeptide
which binds specifically to PCSK9, which antibody or fragment or
polypeptide inhibits binding between PCSK9 and LDL receptor; and a
pharmaceutically acceptable carrier.
37. The pharmaceutical composition of claim 36 wherein the antibody
binds specifically to a PCSK9 catalytic domain or to a domain of
PCSK9 which interacts with an LDL receptor EGF-A domain.
38. An isolated polypeptide comprising an amino acid sequence
comprising about 90% or more amino acid sequence similarity to a
fragment of the human LDL receptor which fragment consists of amino
acids beginning at about amino acid position 314 and ending at
about amino acid position 355 of said receptor; wherein said
polypeptide optionally comprises one or more properties selected
from the group consisting of: (i) binds to PCSK9; (ii) competes
with LDL receptor or an anti-PCSK9 antibody or antigen-binding
fragment thereof for binding to PCSK9; (iii) reduces total
cholesterol level when administered to an animal; (iv) reduces low
density lipoprotein cholesterol level when administered to an
animal; (v) reduces apolipoprotein B level when administered to an
animal; (vi) reduces total cholesterol/high density lipoprotein
ratio when administered to an animal; and (vii) reduces low density
lipoprotein/high density lipoprotein ratio when administered to an
animal; or a pharmaceutical composition thereof comprising a
pharmaceutically acceptable carrier.
39. The polypeptide of claim 38 which consists of the amino acid
sequence of SEQ ID NO: 3; or a pharmaceutical composition thereof
comprising a pharmaceutically acceptable carrier.
40. A method for reducing: total cholesterol level; low density
lipoprotein cholesterol level; apolipoprotein B level; total
cholesterol/high density lipoprotein ratio; or low density
lipoprotein/high density lipoprotein ratio; in a subject,
comprising administering, to said subject, a therapeutically
effective amount of a PCSK9 antagonist EGF-A polypeptide; or an
antibody or antigen-binding fragment thereof that binds
specifically to PCSK9; which antibody or fragment or EGF-A
polypeptide inhibits binding between PCSK9 and LDL receptor;
optionally in association with a further chemotherapeutic
agent.
41. The method of claim 40 wherein the antibody or fragment binds
specifically to a PCSK9 catalytic domain or to a domain of PCSK9
which interacts with an LDL receptor EGF-A domain.
42. A method for treating or preventing hypercholesterolemia,
hyperlipidemia, hypertriglyceridaemia, sitosterolemia,
atherosclerosis, arteriosclerosis, coronary heart disease, vascular
inflammation or xanthoma, in an subject, comprising administering,
to said subject, a therapeutically effective amount of an EGF-A
polypeptide or an antibody or antigen-binding fragment thereof that
binds specifically to PCSK9, which antibody or fragment or EGF-A
polypeptide inhibits binding between PCSK9 and LDL receptor;
optionally in association with a further chemotherapeutic
agent.
43. The method of claim 42 wherein the antibody binds specifically
to a PCSK9 catalytic domain or to a domain of PCSK9 which interacts
with an LDL receptor EGF-A domain.
44. A method for reducing: total cholesterol level; low density
lipoprotein cholesterol level; apolipoprotein B level; total
cholesterol/high density lipoprotein ratio; or low density
lipoprotein/high density lipoprotein ratio; or for treating:
hypercholesterolemia; hyperlipidemia; hypertriglyceridaemia;
sitosterolemia; atherosclerosis; arteriosclerosis; coronary heart
disease; vascular inflammation; or xanthoma, in a subject,
comprising administering, to said subject, a therapeutically
effective amount of the antibody or antigen-binding fragment
thereof of claim 1.
45. The method of claim 44 wherein the antibody or antigen-binding
fragment thereof of claim 1 in administered in association with a
further chemotherapeutic agent.
46. The method of claim 45 wherein the further chemotherapeutic
agent is a cardiovascular agent, an adrenergic blocker, an
antihypertensive agent, an angiotensin system inhibitor, an
angiotensin-converting enzyme inhibitor, a coronary vasodilator, a
diuretic, an adrenergic stimulant or an HMG-CoA reductase
inhibitor.
47. The method of claim 46 wherein the further chemotherapeutic
agent is ezetimibe, lovastatin, atorvastatin, pravastatin,
rosuvastatin, fluvastatin, rivastatin, simvastatin, an azetidinone,
bunolol hydrochloride, acebutolol, alprenolol hydrochloride,
atenolol, carteolol hydrochloride, celiprolol hydrochloride;
cetamolol hydrochloride, labetalol hydrochloride, esmolol
hydrochloride, levobetaxolol hydrochloride, levobunolol
hydrochloride, nadolol, practolol, propranolol hydrochloride,
sotalol hydrochloride, timolol, timolol maleate, bisoprolol;
bisoprolol fumarate, nebivalol, cicloprolol hydrochloride,
dexpropranolol hydrochloride, diacetolol hydrochloride, dilevalol
hydrochloride, exaprolol hydrochloride, flestolol sulfate, metalol
hydrochloride, metoprolol 2-Propanol, metoprolol tartrate,
pamatolol sulfate, penbutolol sulfate, practolol, tiprenolol
hydrochloride, tolamolol fenspiride hydrochloride, proroxan,
alfuzosin hydrochloride, labetalol hydrochloride, bretylium
tosylate, dihydroergtamine mesylate, carvedilol, labetalol,
bretylium tosylate, phentolamine mesylate, solypertine tartrate,
zolertine hydrochloride, candesartan cilexetil, telmisartan,
candesartan; losartan potassium, benazepril hydrochloride,
captopril, fosinopril, moexipril hydrochloride, perindopril
erbumine, quinapril, ramipril, enalapril maleate, lisinopril,
delapril, spirapril, delapril hydrochloride, libenzapril,
pentopril, perindopril 1H-Indole-2-carboxylic acid, quinaprilat,
spirapril hydrochloride, spiraprilat, teprotide, zofenopril,
amlodipine besylate, clentiazem maleate, isradipine, nimodipine,
felodipine, nilvadipine; diltiazem hydrochloride, verapamil
hydrochloride, teludipine hydrochloride, belfosdil, fostedil,
ranolazine, butoprozine hydrochloride, tosifen, molsidomine,
ranolazine hydrochloride, tosifen, diltiazem hydrochloride,
isosorbide dinitrate, sosorbide mononitrate, nitroglycerin,
verapamil hydrochloride, chromonar, clonitate, droprenilamine,
lidoflazine, prenylamine, propatyl nitrate, mioflazine
hydrochloride, mixidine, molsidomine, isosorbide mononitrate,
erythrityl tetranitrate, clonitrate, dipyridamole; nicorandil,
pyridinecarboxamide, nifedipine; perhexyline maleate; oxprenolol,
pentrinitrol, verapamil, althiazide, benzthiazide, buthiazide,
chlorothiazide, spironolactone, triamterene, guanfacine
hydrochloride, methyldopa-hydrochlorothiazide combined with
Hydrochlorothiazide, methyldopa-chlorothiazide, clonidine
hydrochloride, chlorthalidone, clonidine hydrochloride, clonidine,
althiazide, benzthiazide, captopril, carvedilol, chlorothiazide,
clonidine hydrochloride, cyclothiazide, delapril hydrochloride,
dilevalol hydrochloride, delapril hydrochloride, doxazosin
mesylate, fosinopril sodium, moexipril hydrochloride, monatepil
maleate, metoprolol succinate, guanfacine hydrochloride,
methyldopa, quinaprilat, quinapril hydrochloride, primidolol,
prazosin hydrochloride, pelanserin hydrochloride, phenoxybenzamine
hydrochloride, candesartan cilexetil, telmisartan, candesartan,
amlodipine maleate, terazosin hydrochloride, bevantolol
hydrochloride or ramipril.
48. The method of claim 47 wherein the further chemotherapeutic
agent is ezetimibe and, optionally, simvastatin.
49. The method of claim 44 wherein the antibody or antigen-binding
fragment thereof is a humanized antibody.
50. A method for reducing: total cholesterol level; low density
lipoprotein cholesterol level; apolipoprotein B level; total
cholesterol/high density lipoprotein ratio; or low density
lipoprotein/high density lipoprotein ratio; or for treating:
hypercholesterolemia; hyperlipidemia; hypertriglyceridaemia;
sitosterolemia; atherosclerosis; arteriosclerosis; coronary heart
disease; vascular inflammation; or xanthoma, in a subject,
comprising administering, to said subject, a therapeutically
effective amount of the antibody or antigen-binding fragment
thereof of claim 24.
51. The method of claim 50 wherein the antibody or antigen-binding
fragment thereof of claim 1 in administered in association with a
further chemotherapeutic agent.
52. The method of claim 51 wherein the further chemotherapeutic
agent is a cardiovascular agent, an adrenergic blocker, an
antihypertensive agent, an angiotensin system inhibitor, an
angiotensin-converting enzyme inhibitor, a coronary vasodilator, a
diuretic, an adrenergic stimulant or an HMG-CoA reductase
inhibitor.
53. The method of claim 51 wherein the further chemotherapeutic
agent is ezetimibe, lovastatin, atorvastatin, pravastatin,
rosuvastatin, fluvastatin, rivastatin, simvastatin, an azetidinone,
bunolol hydrochloride, acebutolol, alprenolol hydrochloride,
atenolol, carteolol hydrochloride, celiprolol hydrochloride;
cetamolol hydrochloride, labetalol hydrochloride, esmolol
hydrochloride, levobetaxolol hydrochloride, levobunolol
hydrochloride, nadolol, practolol, propranolol hydrochloride,
sotalol hydrochloride, timolol, timolol maleate, bisoprolol;
bisoprolol fumarate, nebivalol, cicloprolol hydrochloride,
dexpropranolol hydrochloride, diacetolol hydrochloride, dilevalol
hydrochloride, exaprolol hydrochloride, flestolol sulfate, metalol
hydrochloride, metoprolol 2-Propanol, metoprolol tartrate,
pamatolol sulfate, penbutolol sulfate, practolol, tiprenolol
hydrochloride, tolamolol fenspiride hydrochloride, proroxan,
alfuzosin hydrochloride, labetalol hydrochloride, bretylium
tosylate, dihydroergtamine mesylate, carvedilol, labetalol,
bretylium tosylate, phentolamine mesylate, solypertine tartrate,
zolertine hydrochloride, candesartan cilexetil, telmisartan,
candesartan; losartan potassium, benazepril hydrochloride,
captopril, fosinopril, moexipril hydrochloride, perindopril
erbumine, quinapril, ramipril, enalapril maleate, lisinopril,
delapril, spirapril, delapril hydrochloride, libenzapril,
pentopril, perindopril 1H-Indole-2-carboxylic acid, quinaprilat,
spirapril hydrochloride, spiraprilat, teprotide, zofenopril,
amlodipine besylate, clentiazem maleate, isradipine, nimodipine,
felodipine, nilvadipine; diltiazem hydrochloride, verapamil
hydrochloride, teludipine hydrochloride, belfosdil, fostedil,
ranolazine, butoprozine hydrochloride, tosifen, molsidomine,
ranolazine hydrochloride, tosifen, diltiazem hydrochloride,
isosorbide dinitrate, sosorbide mononitrate, nitroglycerin,
verapamil hydrochloride, chromonar, clonitate, droprenilamine,
lidoflazine, prenylamine, propatyl nitrate, mioflazine
hydrochloride, mixidine, molsidomine, isosorbide mononitrate,
erythrityl tetranitrate, clonitrate, dipyridamole; nicorandil,
pyridinecarboxamide, nifedipine; perhexyline maleate; oxprenolol,
pentrinitrol, verapamil, althiazide, benzthiazide, buthiazide,
chlorothiazide, spironolactone, triamterene, guanfacine
hydrochloride, methyldopa-hydrochlorothiazide combined with
Hydrochlorothiazide, methyldopa-chlorothiazide, clonidine
hydrochloride, chlorthalidone, clonidine hydrochloride, clonidine,
althiazide, benzthiazide, captopril, carvedilol, chlorothiazide,
clonidine hydrochloride, cyclothiazide, delapril hydrochloride,
dilevalol hydrochloride, delapril hydrochloride, doxazosin
mesylate, fosinopril sodium, moexipril hydrochloride, monatepil
maleate, metoprolol succinate, guanfacine hydrochloride,
methyldopa, quinaprilat, quinapril hydrochloride, primidolol,
prazosin hydrochloride, pelanserin hydrochloride, phenoxybenzamine
hydrochloride, candesartan cilexetil, telmisartan, candesartan,
amlodipine maleate, terazosin hydrochloride, bevantolol
hydrochloride or ramipril.
54. The method of claim 53 wherein the further chemotherapeutic
agent is ezetimibe and, optionally, simvastatin.
55. The method of claim 50 wherein the antibody or antigen-binding
fragment thereof is a humanized antibody.
56. A method for producing an antibody or antigen-binding fragment
thereof of claim 1 comprising introducing one or more
polynucleotides into one or more host cells; which polynucleotides
encodes and direct expression of said heavy and/or light chain
immunoglobulins; and growing said host cells under conditions
whereby said heavy and light chain immunoglobulins are
expressed.
57. The method of claim 56 wherein said polynucleotide encoding and
directing expression of said light chain and said polynucleotide
encoding and directing expression of said heavy chain are in
separate host cells.
Description
[0001] This application claims the benefit of U.S. patent
application No. 60/982,922, filed Oct. 26, 2007, which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The field of the present invention relates to methods and
compositions for treating disorders of cholesterol homeostasis by
administering an anti-PCSK9 antibody or antigen-binding fragment
thereof.
BACKGROUND OF THE INVENTION
[0003] Atherosclerotic coronary heart disease (CHD) represents the
major cause for death and cardiovascular morbidity in the western
world. Risk factors for atherosclerotic coronary heart disease
include hypertension, diabetes mellitus, family history, male
gender, cigarette smoke, high serum cholesterol, high low density
lipoprotein (LDL) cholesterol levels and low high density
lipoprotein (HDL) cholesterol levels. In general, a total
cholesterol level in excess of about 225-250 mg/dl is associated
with significant elevation of risk of CHD.
[0004] A variety of clinical studies have demonstrated that
elevated levels of total cholesterol or LDL cholesterol promote
human atherosclerosis. Epidemiologic investigations have
established that cardiovascular morbidity and mortality vary
directly with the level of total cholesterol and LDL
cholesterol.
[0005] One method for lowering LDL cholesterol levels is by
administration of HMG-CoA reductase inhibiting drugs. These drugs
antagonize HMG-CoA reductase and cholesterol synthesis in the liver
and increase the number of hepatic LDL receptors on the
cell-surface to enhance uptake and catabolism of LDL. A drawback of
such an approach is that these drugs commonly suffer from a
disadvantageous side-effect profile, including, for example, liver
toxicity. An alternate approach is to modulate the LDL receptor
pathway directly.
[0006] PCSK9 (proprotein convertase subtilisin/kexin type 9) is a
serine protease family member that binds to and regulates LDL
receptor expression on the surface of cells. Inhibition of the LDL
receptor-PCSK9 interaction is an attractive approach to the
treatment of cholesterol disorders. Inhibition of interactions
between large proteins (i.e., protein-protein interactions or PPI)
by the use of antibodies or small molecule inhibitors is, however,
generally regarded as being particularly difficult and challenging.
Large proteins such as PCSK9, with a molecular weight of about 74
KDa, and LDLR, with a molecular weight of about 160 KDa
(glycosylated on cell surface; 115 KDa in immature form), are
likely to exhibit extensive intermolecular contacts over a large
area. The existence of extensive contacts makes it unlikely that a
given antibody or small molecule inhibitor will successfully block
their binding.
SUMMARY OF THE INVENTION
[0007] The present invention surprisingly has overcome the
technical difficulties associated with the blocking of
intermolecular interactions between large proteins and has
demonstrated that blockage of the PCSK9-LDLR interaction with an
antibody or peptide is possible. As discussed in detail herein,
this, in turn, provides a novel method by which to treat
cholesterol disorders.
[0008] The present invention provides, in part, a method for
reducing total cholesterol level, low density lipoprotein
cholesterol level, apolipoprotein B level, total cholesterol/high
density lipoprotein ratio or low density lipoprotein/high density
lipoprotein ratio, in a subject (e.g., a human), comprising
administering, to said subject, a therapeutically effective amount
of an antibody or antigen-binding fragment thereof (e.g.,
monoclonal antibody, polyclonal antibody or recombinant antibody)
or EGF-A polypeptide that binds specifically to PCSK9 which
antibody or fragment or polypeptide inhibits binding between PCSK9
and LDL receptor; optionally in association with a further
chemotherapeutic agent (e.g., ezetimibe and/or simvastatin). In an
embodiment of the invention, the antibody or fragment or EGF-A
polypeptide binds specifically to a PCSK9 catalytic domain or to a
domain of PCSK9 which interacts with an LDL receptor EGF-A
domain.
[0009] The present invention further provides, in part, a method
for treating or preventing hypercholesterolemia, hyperlipidemia,
hypertriglyceridemia, sitosterolemia, atherosclerosis,
arteriosclerosis, coronary heart disease, vascular inflammation or
xanthoma, in an subject, comprising administering, to said subject,
a therapeutically effective amount of an antibody or
antigen-binding fragment thereof (e.g., monoclonal antibody,
polyclonal antibody or recombinant antibody) or EGF-A polypeptide
that binds specifically to PCSK9, which antibody or fragment or
polypeptide inhibits binding between PCSK9 and LDL receptor;
optionally in association with a further therapeutic agent (e.g.,
ezetimibe and/or simvastatin). In an embodiment of the invention,
the antibody or fragment or EGF-A polypeptide binds specifically to
a PCSK9 catalytic domain or to a domain of PCSK9 which interacts
with an LDL receptor EGF-A domain.
[0010] The present invention also provides, in part, a
pharmaceutical composition comprising an antibody or
antigen-binding fragment thereof (e.g., monoclonal antibody,
polyclonal antibody or recombinant antibody) or EGF-A polypeptide
which binds specifically to PCSK9, which antibody or fragment or
polypeptide inhibits binding between PCSK9 and LDL receptor, and a
pharmaceutically acceptable carrier; optionally in association with
a further chemotherapeutic agent (e.g., ezetimibe and/or
simvastatin).
[0011] The present invention also provides an isolated polypeptide
comprising an amino acid sequence comprising about 90% or more
amino acid sequence similarity to a fragment of the human LDL
receptor which fragment consists of amino acids beginning at about
amino acid position 314 and ending at about amino acid position 355
of said receptor wherein said polypeptide; wherein said polypeptide
optionally comprises one or more properties selected from the group
consisting of: (i) binds to PCSK9; (ii) competes with LDL receptor
or an anti-PCSK9 antibody or antigen-binding fragment thereof for
binding to PCSK9; (iii) reduces total cholesterol level when
administered to an animal; (iv) reduces low density lipoprotein
cholesterol level when administered to an animal; (v) reduces
apolipoprotein B level when administered to an animal; (vi) reduces
total cholesterol/high density lipoprotein ratio when administered
to an animal; and (vii) reduces low density lipoprotein/high
density lipoprotein ratio when administered to an animal; or a
pharmaceutical composition thereof comprising a pharmaceutically
acceptable carrier. In an embodiment of the invention, the
polypeptide consists of the amino acid sequence of SEQ ID NO: 3; or
a pharmaceutical composition thereof comprising a pharmaceutically
acceptable carrier.
[0012] The present invention provides an isolated antibody or
antigen-binding fragment thereof comprising one or more members
selected from the group consisting of:
(i) CDR-H1, CDR-H2 and CDR-H3 of the variable region the 11B5 heavy
chain immunoglobulin which comprises the amino acid sequence of SEQ
ID NO: 10; (ii) CDR-H1, CDR-H2 and CDR-H3 of the variable region
the 75B9 heavy chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 18; (iii) CDR-H1, CDR-H2 and CDR-H3 of the
variable region the 77D10 heavy chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 26; (iv) CDR-H1,
CDR-H2 and CDR-H3 of the variable region the 29C10 heavy chain
immunoglobulin which comprises the amino acid sequence of SEQ ID
NO: 34; (v) CDR-H1, CDR-H2 and CDR-H3 of the variable region the
22D11 heavy chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 42; (vi) CDR-H1, CDR-H2 and CDR-H3 of the
variable region the 1F11/1G11 heavy chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 50; (vii) CDR-H1,
CDR-H2 and CDR-H3 of the variable region the 1185 light chain
immunoglobulin which comprises the amino acid sequence of SEQ ID
NO: 14; (viii) CDR-H1, CDR-H2 and CDR-H3 of the variable region the
75B9 light chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 22; (ix) CDR-H1, CDR-H2 and CDR-H3 of the
variable region the 77D10 light chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 30; (x) CDR-H1,
CDR-H2 and CDR-H3 of the variable region the 29C10 light chain
immunoglobulin which comprises the amino acid sequence of SEQ ID
NO: 38; (xi) CDR-H1, CDR-H2 and CDR-H3 of the variable region the
22D11 light chain immunoglobulin which comprises the amino acid
sequence of SEQ ID NO: 46; and (xii) CDR-H1, CDR-H2 and CDR-H3 of
the variable region the 1F11/1G11 light chain immunoglobulin which
comprises the amino acid sequence of SEQ ID NO: 54. The present
invention also provides an isolated antibody or antigen-binding
fragment thereof comprising one or more members selected from the
group consisting of:
TABLE-US-00001 (i) HCDR1 comprising the amino acid sequence (SEQ ID
NO: 11) G F N I K D T Y M H, HCDR2 comprising the amino acid
sequence (SEQ ID NO: 12) R I D P A N G H T E Y D P K F Q D and
HCDR3 comprising the amino acid sequence (SEQ ID NO: 13) S Y F G S
I F A Y; (ii) HCDR1 comprising the amino acid sequence (SEQ ID NO:
19) G F N I K D T Y I H, HCDR2 comprising the amino acid sequence
(SEQ ID NO: 20) R I D P A N G H T E Y D P K F Q G and HCDR3
comprising the amino acid sequence (SEQ ID NO: 21) S Y Y G S I F A
Y; (iii) HCDR1 comprising the amino acid sequence (SEQ ID NO: 27) G
F N I K D Y Y I H, HCDR2 comprising the amino acid sequence (SEQ ID
NO: 28) W I D P E N G D T E Y A P K F Q G and HCDR3 comprising the
amino acid sequence (SEQ ID NO: 29) Y Y R Y D D G T W F P Y; (iv)
HCDR1 comprising the amino acid sequence (SEQ ID NO: 35) G F N I K
D T Y I H, HCDR2 comprising the amino acid sequence (SEQ ID NO: 36)
W I D P A N G Y T K Y A P N F Q G and HCDR3 comprising the amino
acid sequence (SEQ ID NO: 37) G Y Y R Y Y S L D Y; (v) HCDR1
comprising the amino acid sequence (SEQ ID NO: 43) G F T F S N H D
M A, HCDR2 comprising the amino acid sequence (SEQ ID NO: 44) S I T
P S G G T T Y Y R D S V E G and HCDR3 comprising the amino acid
sequence (SEQ ID NO: 45) Q N Y Y D G S Y Y Y G L Y Y F D Y; (vi)
HCDR1 comprising the amino acid sequence (SEQ ID NO: 51) G Y T F T
D Y Y M N, HCDR2 comprising the amino acid sequence (SEQ ID NO: 52)
D I N P N N G G A I Y N Q K F K G and HCDR3 comprising the amino
acid sequence (SEQ ID NO: 53) G I I T E I A E D F; (vii) LCDR1
comprising the amino acid sequence (SEQ ID NO: 15) S A S S S V S Y
L Y, LCDR2 comprising the amino acid sequence (SEQ ID NO: 16) R S S
H R A S and LCDR3 comprising the amino acid sequence (SEQ ID NO:
17) H Q Y Q S Y P P T; (viii) LCDR1 comprising the amino acid
sequence (SEQ ID NO: 23) S A S S S V S Y L F, LCDR2 comprising the
amino acid sequence (SEQ ID NO: 24) R T S Y L A S and LCDR3
comprising the amino acid sequence (SEQ ID NO: 25) H Q Y H T Y P P
T; (ix) LCDR1 comprising the amino acid sequence (SEQ ID NO: 31) R
A S G N I H S Y L A, LCDR2 comprising the amino acid sequence (SEQ
ID NO: 32) N A K T L P D and LCDR3 comprising the amino acid
sequence (SEQ ID NO: 33) Q H F W N T P W T; (x) LCDR1 comprising
the amino acid sequence (SEQ ID NO: 39) R A S Q D I S N Y L N,
LCDR2 comprising the amino acid sequence (SEQ ID NO: 40) Y S S R L
H S and LCDR3 comprising the amino acid sequence (SEQ ID NO: 41) Q
Q G K T L P L T; (xi) LCDR1 comprising the amino acid sequence (SEQ
ID NO: 47) R S S Q S L V Y S D G N T Y L H, LCDR2 comprising the
amino acid sequence (SEQ ID NO: 48) R V S N R F S and LCDR3
comprising the amino acid sequence (SEQ ID NO: 49) L Q S T H F P P
T; and (xii) LCDR1 comprising the amino acid sequence (SEQ ID NO:
55) K A S Q N V G T N V V, LCDR2 comprising the amino acid sequence
(SEQ ID NO: 56) S A S Y R Y S and LCDR3 comprising the amino acid
sequence (SEQ ID NO: 57) Q Q Y K T Y P Y T.
Furthermore, the present invention provides an isolated antibody or
antigen-binding fragment thereof of claim xxx comprising one or
more members selected from the group consisting of:
TABLE-US-00002 (a) an immunoglobulin heavy chain comprising the
amino acid sequence: (SEQ ID NO: 10) E V Q L Q Q S G A E L V K P G
A S V T L S C T A S G F N I K D T Y M H W V N Q R P E Q G L V W I G
R I D P A N G H T E Y D P K F Q D K A T I T T D T S S N T A Y L H L
S S L T S G D T A V Y Y C A R S Y F G S I F A Y W G Q G T L V T V S
A; (b) an immunoglobulin light chain comprising the amino acid
sequence: (SEQ ID NO: 14) Q I V L T Q S P A I M S A S P G E K V T I
S C S A S S S V S Y L Y W Y Q Q K P G S S P K P W I F R S S H R A S
G V P A R F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q
Y Q S Y P P T F G G G T K L E I K R A; (c) an immunoglobulin heavy
chain comprising the amino acid sequence: (SEQ ID NO: 18) E V Q L Q
Q S G A D L V K P G A S V K L S C T A S G F N I K D T Y I H W V K Q
R P E Q G L E W I G R I D P A N G H T E Y D P K F Q G R A T L T T D
T S S N T A Y L Q L F S L T S E D S A V Y F C A R S Y Y G S I F A Y
W G Q G T L V T V S A; (d) an immunoglobulin light chain comprising
the amino acid sequence: (SEQ ID NO: 22) Q I V L T Q S P A I M S A
S P G E K V T I S C S A S S S V S Y L F W Y Q Q K P G S S P K P W I
F R T S Y L A S G V P A R F S G S G S G T S F S L T I S S M E A E D
A A T Y Y C H Q Y H T Y P P T F G G G T K L E I K R A; (e) an
immunoglobulin heavy chain comprising the amino acid sequence: (SEQ
ID NO: 26) E V Q L Q Q S G A E L V R S G A S V K L S C T T S G F N
I K D Y Y I H W V K Q R P E Q G L E W I G W I D P E N G D T E Y A P
K F Q G K A T M T A D T S S N T A Y L Q L S S L T S A D T A V Y Y C
N A Y Y R Y D D G T W F P Y W G Q G T L V T V S A; (f) an
immunoglobulin light chain comprising the amino acid sequence: (SEQ
ID NO: 30) D I Q L T Q S P A S L S A S V G E T V T I T C R A S G N
I H S Y L A W Y Q Q K Q G K S P Q F L V D N A K T L P D G V P S R F
S V S G S G T Q Y S L K I N S L Q P E D E G T Y Y C Q H F W N T P W
T F G G G T K L E I K R A; (g) an immunoglobulin heavy chain
comprising the amino acid sequence: (SEQ ID NO: 34) E V L L Q Q S V
A E L V R P G A S V R L S C T A S G F N I K D T Y T H W V R Q R P E
Q G L E W F G W I D P A N G Y T K Y A P N F Q G K A T L T T D T S S
N T A Y L H L S S L T S E D S A I Y Y C A R G Y Y R Y Y S L D Y W G
Q G T S V T V S S; (h) an immunoglobulin light chain comprising the
amino acid sequence: (SEQ ID NO: 38) D I Q M T Q T T S S L S A S L
G D R V T I S C R A S Q D I S N Y L N W Y Q Q K P D G T V K L L I Y
Y S S R L H S G V P S R F S G R G S G T D Y S L T I S T L E Q E D I
A T Y F C Q Q G K T L P L T F G A G T K L E L K R A; (i) an
immunoglobulin heavy chain comprising the amino acid sequence: (SEQ
ID NO: 42) E V Q L V D S G G G L V Q P G R S L K L S C A A S G F T
F S N H D M A W V R Q A P T K G L E W V A S I T P S G G T T Y Y R D
S V E G R F T V S R D N V K S S L H L Q M D S L T S E D T A T Y Y C
A R Q N Y Y D G S Y Y Y G L Y Y F D Y W G Q G V M V T V S S; (j) an
immunoglobulin light chain comprising the amino acid sequence: (SEQ
ID NO: 46) D V L M T Q T P V S L P V S L G G Q V S I S C R S S Q S
L V Y S D G N T Y L H W Y L Q K P G Q S P Q L L I Y R V S N R F S G
V P D R F S G S G S G T D F T L K I S R V E P E D L G L Y Y C L Q S
T H F P P T F G S G T K L E I K R A; (k) an immunoglobulin heavy
chain comprising the amino acid sequence: (SEQ ID NO: 50) E V Q L Q
Q S G P E L V K P G A S V K I S C K V S G Y T F T D Y Y M N W V K Q
S H G K S L E W I G D I N P N N G G A I Y N Q K F K G K A T L T V D
K S S S I A Y M E L R S L T S E D S A V Y Y C T S G I I T E I A E D
F W G Q G T T L T V S S; and (l) an immunoglobulin light chain
comprising the amino acid sequence: (SEQ ID NO: 54) D I V M T Q S Q
K F M S T S V G D R V S V T C K A S Q N V G T N V V W Y Q Q K P G Q
S P K A L I H S A S Y R Y S G V P D R F K G S G S G T D F T L T I T
N V Q S E D L A G F F C Q Q Y K T Y P Y T F G G G T Q L E I K R
A.
[0013] Embodiments of the invention include, e.g., compositions
comprising any of the antibodies or polypeptides of the present
invention association with a further chemotherapeutic agent, e.g.,
a cardiovascular agent, an adrenergic blocker, an antihypertensive
agent, an angiotensin system inhibitor, an angiotensin-converting
enzyme (ACE) inhibitor, a coronary vasodilator, a diuretic, an
adrenergic stimulant or an HMG-CoA reductase inhibitor. In an
embodiment of the invention, the further chemotherapeutic agent is
ezetimibe, lovastatin, atorvastatin, pravastatin, rosuvastatin,
fluvastatin, rivastatin, simvastatin, an azetidinone, bunolol
hydrochloride, acebutolol, alprenolol hydrochloride, atenolol,
carteolol hydrochloride, celiprolol hydrochloride; cetamolol
hydrochloride, labetalol hydrochloride, esmolol hydrochloride,
levobetaxolol hydrochloride, levobunolol hydrochloride, nadolol,
practolol, propranolol hydrochloride, sotalol hydrochloride,
timolol, timolol maleate, bisoprolol; bisoprolol fumarate,
nebivolol, cicloprolol hydrochloride, dexpropranolol hydrochloride,
diacetolol hydrochloride, dilevalol hydrochloride, exaprolol
hydrochloride, flestolol sulfate, metalol hydrochloride, metoprolol
2-Propanol, metoprolol tartrate, pamatolol sulfate, penbutolol
sulfate, practolol, tiprenolol hydrochloride or tolamolol.
Embodiments of the invention also include those wherein the
antibody or fragment is a humanized antibody, a monoclonal
antibody, a labeled antibody, a bivalent antibody, a polyclonal
antibody, a bispecific antibody, a chimeric antibody, a recombinant
antibody, an anti-idiotypic antibody, a humanized antibody, a
bispecific antibody, a camelized single domain antibody, a diabody,
an scfv, an scfv dimer, a dsfv, a (dsfv).sub.2, a dsFv-dsfv', a
bispecific ds diabody, an Fv, an Fab, an Fab', an F(ab').sub.2, or
a domain antibody. In an embodiment of the invention, the antibody
or antigen-binding fragment thereof is linked to an immunoglobulin
constant region, e.g., a .kappa. light chain, .gamma.1 heavy chain,
.gamma.2 heavy chain, .gamma.3 heavy chain or .gamma.4 heavy chain.
The present invention also provides a pharmaceutical composition
comprising an antibody or antigen-binding fragment thereof of the
present invention in association with a pharmaceutically acceptable
carrier.
[0014] The present invention also provides a method for reducing
total cholesterol level; low density lipoprotein cholesterol level;
apolipoprotein B level; total cholesterol/high density lipoprotein
ratio; or low density lipoprotein/high density lipoprotein ratio;
or for treating hypercholesterolemia; hyperlipidemia;
hypertriglyceridemia; sitosterolemia; atherosclerosis;
arteriosclerosis; coronary heart disease; vascular inflammation; or
xanthoma, in a subject, comprising administering, to said subject,
a therapeutically effective amount of any polypeptide or antibody
or antigen-binding fragment thereof of the present invention as set
forth herein; or pharmaceutical composition thereof; optionally, in
association with a further chemotherapeutic agent, e.g., as set
forth herein.
[0015] The present invention also provides a method for producing
an antibody or antigen-binding fragment thereof of claim 1
comprising introducing one or more polynucleotides into one or more
host cells; which polynucleotides encodes and direct expression of
said heavy and/or light chain immunoglobulins; and growing said
host cells under conditions whereby said heavy and light chain
immunoglobulins are expressed; e.g., wherein said polynucleotide
encoding and directing expression of said light chain and said
polynucleotide encoding and directing expression of said heavy
chain are in separate host cells.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIGS. 1A-1D show anti-human PCSK9 antibody mature variable
region amino acid sequences. CDRs are underscored.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention includes methods and compositions for
an innovative method for treating cholesterol disorders. The
methods and compositions of the present invention are useful for
treating cholesterol disorders by modulating the LDL receptor
pathway. Specifically, the methods and compositions of the present
invention antagonize the interaction between PCSK9 and LDLR and
thereby lead to increased clearance of LDL from the bloodstream. In
spite of formidable technical difficulties associated with blocking
PPIs, the present invention provides a method for targeting and
blocking this interaction, thus, leading to a beneficial effect
with regard to blood cholesterol levels.
[0018] The term low density lipoprotein receptor (LDLR) includes
any such receptor, e.g., human LDLR along with any allelic variant
thereof. In one embodiment of the invention, full-length LDLR
comprises the following amino acid sequence (see e.g., GenBank
NM.sub.--000527.2):
TABLE-US-00003 (SEQ ID NO: 1)
MGPWGWKLRWTVALLLAAAGTAVGDRCERNEFQCQDGKCISYKWVCDGSA
ECQDGSDESQETCLSVTCKSGDFSCGGRVNRCIPQFWRCDGQVDCDNGSD
EQGCPPKTCSQDEFRCHDGKCISRQFVCDSDRDCLDGSDEASCPVLTCGP
ASFQCNSSTCIPQLWACDNDPDCEDGSDEWPQRCRGLYVFQGDSSPCSAF
EFHCLSGECIHSSWRCDGGPDCKDKSDEENCAVATCRPDEFQCSDGNCIH
GSRQCDREYDCKDMSDEVGCVNVTLCEGPNKFKCHSGECITLDKVCNMAR
DCRDWSDEPIKECGTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQR
RCEDIDECQDPDTCSQLCVNLEGGYKCQCEEGFQLDPHTKACKAVGSIAY
LFFTNRHEVRKMTLDRSEYTSLIPNLRNVVALDTEVASNRIYWSDLSQRM
ICSTQLDRAHGVSSYDTVISRDIQAPDGLAVDWIHSNIYWTDSVLGTVSV
ADTKGVKRKTLFRENGSKPRAIVVDPVHGFMYWTDWGTPAKIKKGGLNGV
DIYSLVTENIQWPNGITLDLLSGRLYWVDSKLHSISSIDVNGGNRKTILE
DEKRLAHPFSLAVFEDKVFWTDIINEAIFSANRLTGSDVNLLAENLLSPE
DMVLFHNLTQPRGVNWCERTTLSNGGCQYLCLPAPQINPHSPKFTCACPD
GMLLARDMRSCLTEAEAAVATQETSTVRLKVSSTAVRTQHTTTRPVPDTS
RLPGATPGLTTVEIVTMSHQALGDVAGRGNEKKPSSVRALSIVLPIVLLV
FLCLGVFLLWKNWRLKNINSINFDNPVYQKTTEDEVHICHNQDGYSYPSR QMVSLEDDVA
[0019] In an embodiment of the invention, a soluble LDLR fragment
comprises the following amino acid sequence (Yamamoto et al., Cell
(1984) 39:27-38):
TABLE-US-00004 (SEQ ID NO: 2)
AVGDRCERNEFQCQDGKCISYKWVCDGSAECQDGSDESQETCLSVTCKSG
DFSCGGRVNRCIPQFWRCDGQVDCDNGSDEQGCPPKTCSQDEFRCHDGKC
ISRQFVCDSDRDCLDGSDEASCPVLTCGPASFQCNSSTCIPQLWACDNDP
DCEDGSDEWPQRCRGLYVFQGDSSPCSAFEFHCLSGECIHSSWRCDGGPD
CKDKSDEENCAVATCRPDEFQCSDGNCIHGSRQCDREYDCKDMSDEVGCV
NVTLCEGPNKFKCHSGECITLDKVCNMARDCRDWSDEPIKECGTNECLDN
NGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDIDECQDPDTCSQLCVNL
EGGYKCQCEEGFQLDPHTKACKAVGSIAYLFFTNRHEVRKMTLDRSEYTS
LIPNLRNVVALDTEVASNRIYWSDLSQRMICSTQLDRAHGVSSYDTVISR
DIQAPDGLAVDWIHSNIYWTDSVLGTVSVADTKGVKRKTLFRENGSKPRA
IVVDPVHGFMYWTDWGTPAKIKKGGLNGVDIYSLVTENIQWPNGITLDLL
SGRLYWVDSKLHSISSIDVNGGNRKTILEDEKRLAHPFSLAVFEDKVFWT
DIINEAIFSANRLTGSDVNLLAENLLSPEDMVLFHNLTQPRGVNWCERTT
LSNGGCQYLCLPAPQINPHSPKFTCACPDGMLLARDMRSCLTEAEAAVAT
QETSTVRLKVSSTAVRTQHTTTRPVPDTSRLPGATPGLTTVEIVTMSHQA
LGDVAGRGNEKKPSSVR
[0020] In an embodiment of the invention, the EGF-A domain of LDL
receptor comprises the amino acid sequence:
GTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDI (SEQ ID NO: 3)
[0021] The term "subject" includes any animal, e.g., a mammal such
as a human.
PCSK9
[0022] The present invention includes compositions and methods
comprising antibodies and antigen-binding fragment thereof which
bind specifically to PCSK9, for example, human PCSK9. In some
embodiments of the invention, specific PCSK9 polypeptide sequences
or antigenic fragments thereof from various species set forth below
may be used as an antigen:
TABLE-US-00005 HUMAN gi|31317307|ref|NP_777596.2| proprotein con-
vertase subtilisin/kexin type 9 preproprotein [Homo sapiens] (SEQ
ID NO: 4) MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEED
GLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQA
QAARRGYLTKILHVEHGLLPGELVKMSGDLLELALKLPHVDYIEEDSSVF
AQSIPWNLERITPPRYRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRV
MVTDFENVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSL
RVLNCQGKGTVSGTLIGLEEIRKSQLVQPVGPLVVLLPLAGGYSRVLNAA
CQRLARAGVVLVTAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGT
LGTNEGRCVDLEAPGEDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMML
SAEPELTLAELRQRLIHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHG
AGWQLFCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFSRSGKRRGERM
EAQGGKLVCRAHNAFGGEGVYAIARCCLLPQANCSVHTAPPAEASMGTRV
HCHQQGHVLTGCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREASIHASC
CHAPGLECKVKEHGIPAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAV
DNTCVVRSRDVSTTGSTSEGAVTAVAICCRSRHLAQASQELQ CHIMP
gi|114556790|ref|XP_001154126.1| PREDICTED: proprotein convertase
subtilisin/kexin type 9 [Pan troglodytes] (SEQ ID NO: 5)
MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDEDGLAEAPEHGTTATFH
RCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQAQAARRGYLTKILHVF
HGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSVFAQSIPWNLERITPPR
YRADEYQPPDGGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPEEDGTR
EHRQASKCDSHGTHLAGVVSGRDAGVAKGASMRSLRVLNCQGKGTVSGTL
IGLEEIRKSQLVQPVGPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAA
GNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTLGTNEGRCVDLEAPG
EDIIGASSDCSTCFVSQSGTSQAAAHVAGIAAMMLSAEPELTLAELRQRL
IHFSAKDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQLFCRTVWSAHS
GPTRMATAVARCAPDEELLSCSSFSRSGKRRGERMEAQGGKLVCRAHNAF
GGEGVYAIARCCLLPQANCSIHTAPPAEAGMGTRVHCHQQGHVLTGCSSH
WEVEDLGTHKPPMLRPRGQPNQCVGHREASIHASCCRAPGLECKVKEHGI
PAPQEQVTVACEEGWTLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTAG
STSEEAVAAVAICCRSRHLAQASQELQ MOUSE gi|23956352|ref|NP_705793.1|
proprotein con- vertase subtilisin/kexin type 9 [Mus musculus] (SEQ
ID NO: 6) MGTHCSAWLRWPLLPLLPPLLLLLLLLCPTGAGAQDEDGDYEELMLALPS
QEDGLADEAAHVATATFRRCSKEAWRLPGTYIVVLMEETQRLQIEQTAHR
LQTRAARRGYVIKVLHIFYDLFPGFLVKMSSDLLGLALKLPHVEYIEEDS
FVFAQSIPWNLERIIPAWHQTEEDRSPDGSSQVEVYLLDTSIQGAHREIE
GRVTITDFNSVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGTSL
HSLRVLNCQGKGTVSGTLIGLEFIRKSQLIQPSGPLVVLLPLAGGYSRIL
NAACRHLARTGVVLVAAAGNFRDDACLYSPASAPEVITVGATNAQDQPVT
LGTLGTNEGRCVDLEAPGKDIIGASSDCSTCFMSQSGTSQAAAHVAGIVA
RMLSREPTLTLAELRQRLTHFSTKDVINMAWFPEDQQVLTPNLVATLPPS
THETGGQLLCRTVWSAHSGPTRTATATARCAPEEELLSCSSFSRSGRRRG
DWIEAIGGQQVCKALNAFGGEGVYAVARCCLVPRANCSIHNTPAARAGLE
THVHCHQKDHVLTGCSFHWEVEDLSVRRQPALRSRRQPGQCVGHQAASVY
ASCCHAPGLECKIKEHGISGPSEQVTVACEAGWTLTGCNVLPGASLTLGA
YSVDNLCVARVHDTARADRTSGEATVAAAICCRSRPSAKASWVQ RAT
gi|77020250|ref|NP_954862.2| proprotein con- vertase
subtilisin/kexin type 9 [Rattus norvegicus] (SEQ ID NO: 7)
MGIRCSTWLRWPLSPQLLLLLLLCPTGSRAQDEDGDYEELMLALPSQEDS
LVDEASHVATATFRRCSKEAWRLPGTYVVVLMEETQRLQVEQTAHRLQTW
AARRGYVIKVLHVEYDLFPGELVKMSSDLLGLALKLPHVEYIEEDSLVFA
QSIPWNLERIIPAWQQTEEDSSPDGSSQVEVYLLDTSIQSGHREIEGRVT
ITDFNSVPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGTSLHSLR
VLNCQGKGTVSGTLIGLEFIRKSQLIQPSGPLVVLLPLAGGYSRILNTAC
QRLARTGVVLVAAAGNFRDDACLYSPASAPEVITVGATNAQDQPVTLGTL
GTNEGRCVDLEAPGKDIIGASSDCSTCYMSQSGTSQAAAHVAGIVAMMLN
RDPALTLAELRQRLILESTKDVINMAWFPEDQRVLTPNRVATLPPSTQET
GGQLLCRTVWSAHSGPTRTATATARCAPEEELLSCSSFSRSGRRRGDRIE
AIGGQQVCKALNAFGGEGVYAVARCCLLPRVNCSIHNTPAARAGPQTPVH
CHQKDHVLTGCSFHWEVENLRAQQQPLLRSRHQPGQCVGHQEASVHASCC
HAPGLECKIKEHGIAGPAEQVTVACEAGWTLTGCNVLPGASLPLGAYSVD
NVCVARIRDAGRADRTSEEATVAAAICCRSRPSAKASWVHQ
EGF-A
[0023] The present invention also provides methods for reducing
total cholesterol level, low density lipoprotein cholesterol level,
apolipoprotein B level, total cholesterol/high density lipoprotein
ratio or low density lipoprotein/high density lipoprotein ratio, in
a subject, by administering, to the subject, a polypeptide
comprising an LDL receptor EGF-A domain optionally in association
with a further chemotherapeutic agent (e.g., as set forth herein)
or a pharmaceutical composition thereof which comprises a
pharmaceutically acceptable carrier. The EGF-A domain of LDL
receptor binds to PCSK9 and, without being bound by any particular
theory or mechanism, may reduce the activity of PCSK9 by competing
with the full, endogenous LDL receptor for binding to PCSK9. For
example, in an embodiment of the invention, the EGF-A domain
comprises or consists of the following amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 3)
GTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDI.
[0024] In an embodiment of the invention, human LDL receptor
comprises the following amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 1) 1 mgpwgwklrw tvalllaaag tavgdrcern
efqcgdgkci sykwvcdgsa ecqdgsdesq 61 etclsvtcks gdfscggrvn
rcipqfwrcd gqvdcdngsd eqgcppktcs qdefrchdgk 121 cisrqfvcds
drdcldgsde ascpvltcgp asfqcnsstc ipqlwacdnd pdcedgsdew 181
pqrcrglyvf qgdsspcsaf efhclsgeci hsswrcdggp dckdksdeen cavatcrpde
241 fqcsdgncih gsrqcdreyd ckdmsdevgc vnvtlcegpn kfkchsgeci
tldkvcnmar 301 dcrdwsdepi kecgtnecld nnggcshvcn dlkigyeclc
pdgfqlvaqr rcedidecqd 361 pdtcsqlcvn leggykcqce egfqldphtk
ackavgsiay lfftnrhevr kmtldrseyt 421 slipnlrnvv aldtevasnr
iywsdlsqrm icstqldrah gvssydtvis rdiqapdgla 481 vdwihsniyw
tdsvlgtvsv adtkgvkrkt lfrengskpr aivvdpvhgf mywtdwgtpa 541
kikkgglngv diyslvteni qwpngitldl lsgrlywvds klhsissidv nggnrktile
601 dekrlahpfs lavfedkvfw tdiineaifs anrltgsdvn llaenllspe
dmvlfhnltq 661 prgvnwcert tlsnggcqyl clpapqinph spkftcacpd
gmllardmrs clteaeaava 721 tgetstvrlk vsstavrtqh tttrpvpdts
rlpgatpglt tveivtmshq algdvagrgn 781 ekkpssvral sivlpivllv
flclgvfllw knwrlknins infdnpvyqk ttedevhich 841 nqdgysypsr
qmvsleddva
[0025] The EGF-A domain is underscored and in bold faced text. See
also Genbank accession nos.: NP.sub.--000518, EAW84170, BAD92646.1
or AAF24515.1.
[0026] The present invention also comprises an isolated polypeptide
comprising or consisting of an EGF-A polypeptide, for example, in a
pharmaceutical composition which includes a pharmaceutically
acceptable carrier. The present invention also provides such a
polypeptide optionally fused to any other heterologous polypeptide
which is not naturally contiguous with the other, immediately
adjacent LDL receptor sequences as well as methods of treatments
comprising administration of the fused polypeptide e.g., as
discussed herein. Any such polypeptide may be referred to herein as
an "EGF-A polypeptide" and a polynucleotide encoding an EGF-A
polypeptide may be referred to as an "EGF-A polynucleotide".
[0027] For example, in an embodiment of the invention, the EGF-A
polynucleotide comprises the following nucleotide sequence:
TABLE-US-00008 (SEQ ID NO: 9)
GGTACTAATGAATGTCTTGATAATAATGGTGGTTGTTCTCATGTTTGTAA
TGATCTTAAAATTGGTTATGAATGTCTTTGTCCTGATGGTTTTCAACTTG
TTGCTCAACGTCGTTGTGAAGATATT
[0028] As discussed above, the present invention also includes
fusions which include the EGF-A polypeptides and polynucleotides of
the present invention and a second polypeptide or polynucleotide
moiety, which may be referred to as a "tag". The fused polypeptides
of the invention may be conveniently constructed, for example, by
insertion of a polynucleotide of the invention or fragment thereof
into an expression vector. The fusions of the invention may include
tags which facilitate purification or detection. Such tags include
glutathione-S-transferase (GST), hexahistidine (His6) tags, maltose
binding protein (MBP) tags, hemagglutinin (HA) tags, cellulose
binding protein (CBP) tags and myc tags. Detectable tags such as
.sup.32P, .sup.35S, .sup.3H, .sup.14C, .sup.18F, .sup.125I,
.sup.131I, .sup.113mIn, .sup.76Br, .sup.67Ga, .sup.99mTc,
.sup.123I, .sup.111In and .sup.68Ga may also be used to label the
polypeptides and polynucleotides of the invention. Methods for
constructing and using such fusions are very conventional and well
known in the art.
[0029] Any isolated polynucleotide encoding such an EGF-A
polypeptide of the present invention also forms part of the present
invention along with any vector comprising such a polynucleotide
and a host cell (e.g., bacterial host cell or eukaryotic cell)
comprising such a vector. Such polynucleotides operably associated
with an expression control sequence (e.g., a promoter) also form
part of the present invention.
[0030] In general, a "promoter" or "promoter sequence" is a DNA
regulatory region capable of binding an RNA polymerase in a cell
(e.g., directly or through other promoter-bound proteins or
substances) and initiating transcription of a coding sequence. A
promoter sequence is, in general, bounded at its 3' terminus by the
transcription initiation site and extends upstream (5' direction)
to include the minimum number of bases or elements necessary to
initiate transcription at any level. Within the promoter sequence
may be found a transcription initiation site (conveniently defined,
for example, by mapping with nuclease S1), as well as protein
binding domains (consensus sequences) responsible for the binding
of RNA polymerase. The promoter may be operably associated with
other expression control sequences, including enhancer and
repressor sequences or with a nucleic acid of the invention.
Promoters which may be used to control gene expression include, but
are not limited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos.
5,385,839 and 5,168,062), the SV40 early promoter region (Benoist,
et al., (1981) Nature 290:304-310), the promoter contained in the
3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,
(1980) Cell 22:787-797), the herpes thymidine kinase promoter
(Wagner, et al., (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445),
the regulatory sequences of the metallothionein gene (Brinster, et
al., (1982) Nature 296:39-42); prokaryotic expression vectors such
as the .beta.-lactamase promoter (Villa-Komaroff, et al., (1978)
Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter
(DeBoer, et al., (1983) Proc. Natl. Acad. Sci. USA 80:21-25); see
also "Useful proteins from recombinant bacteria" in Scientific
American (1980) 242:74-94; and promoter elements from yeast or
other fungi such as the Gal 4 promoter, the ADC (alcohol
dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or
the alkaline phosphatase promoter.
[0031] A coding sequence is "under the control of", "functionally
associated with" or "operably associated with" transcriptional and
translational control sequences in a cell or other expression
system when the sequences direct RNA polymerase mediated
transcription of the coding sequence into RNA, preferably mRNA,
which then may be RNA spliced (if it contains introns) and,
optionally, translated into a protein encoded by the coding
sequence.
[0032] The terms "express" and "expression" mean allowing or
causing the information in a gene, RNA or DNA sequence to become
manifest; for example, producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene. A DNA sequence is expressed in or by a cell to
form an "expression product" such as an RNA (e.g., mRNA) or a
protein. The expression product itself may also be said to be
"expressed" by the cell.
[0033] The term "vector" includes a vehicle (e.g., a plasmid) by
which a DNA or RNA sequence can be introduced into a host cell, so
as to transform the host and, optionally, promote expression and/or
replication of the introduced sequence.
[0034] Vectors that can be used in this invention include plasmids,
viruses, bacteriophage, integratable DNA fragments, and other
vehicles that may facilitate introduction of the nucleic acids into
the genome of the host. Plasmids are the most commonly used form of
vector but all other forms of vectors which serve a similar
function and which are, or become, known in the art are suitable
for use herein. See, e.g., Pouwels, et al., Cloning
[0035] Vectors: A Laboratory Manual, 1985 and Supplements,
Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,
Boston, Mass.
[0036] The term "expression system" includes, for example, a host
cell and compatible vector which, under suitable conditions, can
express a protein or nucleic acid which is carried by the vector
and introduced to the host cell. Common expression systems include
E. coli host cells and plasmid vectors, insect host cells and
Baculovirus vectors, and mammalian host cells and vectors.
[0037] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. A representative vector for
amplifying DNA is pBR322 or many of its derivatives (e.g., pUC18 or
19). Vectors that can be used to express the EGF-A polypeptides
include, but are not limited to, those containing the lac promoter
(pUC-series); trp promoter (pBR322-trp); Ipp promoter (the
pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters
such as ptac (pDR540). See Brosius et al., "Expression Vectors
Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in
Rodriguez and Denhardt (eds.) Vectors: A Survey of Molecular
Cloning Vectors and Their Uses, 1988, Buttersworth, Boston, pp.
205-236. Many polypeptides can be expressed, at high levels, in an
E. coli/T7 expression system as disclosed in U.S. Pat. Nos.
4,952,496, 5,693,489 and 5,869,320 and in Davanloo, P., et al.,
(1984) Proc. Natl. Acad. Sci. USA 81: 2035-2039; Studier, F. W., et
al., (1986) J. Mol. Biol. 189: 113-130; Rosenberg, A. H., et al.,
(1987) Gene 56: 125-135; and Dunn, J. J., et al., (1988) Gene
68:259.
[0038] The present invention comprises methods of expression an
EGF-A polypeptide of the present invention comprising introducing a
vector comprising a polynucleotide encoding said polypeptide (e.g.,
operably associated with an expression control sequence such as a
promoter) into a suitable host cell and propagating (e.g., growing
in a suitable liquid growth medium) the host cell under conditions
which are suitable to expression of the polypeptide from the
polynucleotide in the vector.
[0039] Also part of the present invention is any isolated
polypeptide with about 90% or more amino acid sequence similarity
or identity to that of SEQ ID NO: 3. In an embodiment of the
invention, such a polypeptide must bind to PCSK9 or a functional
fragment thereof; or inhibit binding of PCSK9 and LDL receptor or
any functional fragment of either; or must exhibit the ability to
reduce total cholesterol level, low density lipoprotein cholesterol
level, apolipoprotein B level, total cholesterol/high density
lipoprotein ratio or low density lipoprotein/high density
lipoprotein ratio in a subject, such as an acceptable animal model
(e.g., a mammal such as a dog, primate, rabbit, mouse or rat) or a
human.
[0040] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook, et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985));
Transcription And Translation (B. D. Hames & S. J. Higgins,
eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));
Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994).
[0041] The term "host cell" includes any cell of any organism that
is selected, modified, transfected, transformed, grown, or used or
manipulated in any way, for the production of a substance by the
cell, for example the expression or replication, by the cell, of a
gene, a DNA or RNA sequence or a protein.
[0042] The terms "isolated polynucleotide" or "isolated
polypeptide" include a polynucleotide (e.g., RNA or DNA molecule,
or a mixed polymer) or a polypeptide, respectively, which are
partially or fully separated from other components that are
normally found in cells or in recombinant DNA expression systems.
These components include, but are not limited to, cell membranes,
cell walls, ribosomes, polymerases, serum components and extraneous
genomic sequences.
[0043] An isolated polynucleotide or polypeptide will, in an
embodiment of the invention, be an essentially homogeneous
composition of molecules but may contain some heterogeneity.
[0044] The present invention contemplates any superficial or slight
modification to the amino acid or nucleotide sequences which
correspond to the EGF-A polypeptides and polynucleotides of the
invention. In particular, the present invention contemplates
sequence conservative variants of the nucleic acids which encode
the EGF-A polypeptides of the invention. "Sequence-conservative
variants" of an EGF-A polynucleotide sequence are those in which a
change of one or more nucleotides in a given codon results in no
alteration in the amino acid encoded at that position.
Function-conservative variants of the EGF-A polypeptides of the
invention are also contemplated by the present invention.
"Function-conservative variants" are those in which one or more
amino acid residues in an EGF-A polypeptide have been changed
without altering the overall conformation and/or function of the
polypeptide (e.g., ability to bind to PCSK9 or inhibit binding of
PCSK9 and an LDL receptor or fragment thereof), including, but, by
no means limited to, replacement of an amino acid with one having
similar properties. Amino acids with similar properties are well
known in the art. For example, polar/hydrophilic amino acids which
may be interchangeable include asparagine, glutamine, serine,
cysteine, threonine, lysine, arginine, histidine, aspartic acid and
glutamic acid; nonpolar/hydrophobic amino acids which may be
interchangeable include glycine, alanine, valine, leucine,
isoleucine, proline, tyrosine, phenylalanine, tryptophan and
methionine; acidic amino acids which may be interchangeable include
aspartic acid and glutamic acid and basic amino acids which may be
interchangeable include histidine, lysine and arginine.
[0045] The present invention includes polynucleotides encoding
EGF-A (e.g., SEQ ID NO: 9) and functional fragments thereof as well
as nucleic acids which hybridize to the polynucleotides. In an
embodiment of the invention, the nucleic acids hybridize under low
stringency conditions, under moderate stringency conditions or
under high stringency conditions. A nucleic acid molecule is
"hybridizable" to another nucleic acid molecule, such as a cDNA,
genomic DNA, or RNA, when a single stranded form of the nucleic
acid molecule can anneal to the other nucleic acid molecule under
the appropriate conditions of temperature and solution ionic
strength (see Sambrook, et al., supra). The conditions of
temperature and ionic strength determine the "stringency" of the
hybridization. Typical low stringency hybridization conditions are
55.degree. C., 5.times.SSC, 0.1% SDS, 0.25% milk, and no formamide
at 42.degree. C.; or 30% formamide, 5.times.SSC, 0.5% SDS at
42.degree. C. Typical, moderate stringency hybridization conditions
are similar to the low stringency conditions except the
hybridization is carried out in 40% formamide, with 5.times. or
6.times.SSC at 42.degree. C. High stringency hybridization
conditions are similar to low stringency conditions except the
hybridization conditions are carried out in 50% formamide, 5.times.
or 6.times.SSC and, optionally, at a higher temperature (e.g.,
higher than 42.degree. C.: 57.degree. C., 59.degree. C., 60.degree.
C., 62.degree. C., 63.degree. C., 65.degree. C. or 68.degree. C.).
In general, SSC is 0.15M NaCl and 0.015M Na-citrate. Hybridization
requires that the two nucleic acids contain complementary
sequences, although, depending on the stringency of the
hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the higher
the stringency under which the nucleic acids may hybridize. For
hybrids of greater than 100 nucleotides in length, equations for
calculating the melting temperature have been derived (see
Sambrook, et al., supra, 9.50-9.51). For hybridization with shorter
nucleic acids, i.e., oligonucleotides, the position of mismatches
becomes more important, and the length of the oligonucleotide
determines its specificity (see Sambrook, et al., supra). In an
embodiment of the invention, such a polynucleotide encodes a
polypeptide that must bind to PCSK9 or a functional fragment
thereof; or inhibit binding of PCSK9 and LDL receptor or any
functional fragment of either; or must exhibit the ability to
reduce total cholesterol level, low density lipoprotein cholesterol
level, apolipoprotein B level, total cholesterol/high density
lipoprotein ratio or low density lipoprotein/high density
lipoprotein ratio in a subject, such as an acceptable animal model
(e.g., a mammal such as a dog, primate, rabbit, mouse or rat) or a
human.
[0046] Also included in the present invention are polynucleotides
comprising nucleotide sequences and polypeptides comprising amino
acid sequences which are at least about 70% identical, preferably
at least about 80% identical, more preferably at least about 90%
identical and most preferably at least about 95% identical (e.g.,
95%, 96%, 97%, 98%, 99%, 100%) to a reference nucleotide sequence
(SEQ ID NO: 9) or reference amino acid sequence (SEQ ID NO: 3),
when the comparison is performed by a BLAST algorithm wherein the
parameters of the algorithm are selected to give the largest match
between the respective sequences over the entire length of the
respective reference sequences. Polypeptides comprising amino acid
sequences which are at least about 70% similar, preferably at least
about 80% similar, more preferably at least about 90% similar and
most preferably at least about 95% similar (e.g., 95%, 96%, 97%,
98%, 99%, 100%) to the reference EGF-A amino acid sequence of SEQ
ID NO: 3, when the comparison is performed with a BLAST algorithm
wherein the parameters of the algorithm are selected to give the
largest match between the respective sequences over the entire
length of the respective reference sequences, are also included in
the present invention.
[0047] Sequence identity refers to exact matches between the
nucleotides or amino acids of two sequences which are being
compared. Sequence similarity refers to both exact matches between
the amino acids of two polypeptides which are being compared in
addition to matches between nonidentical, biochemically related
amino acids. Biochemically related amino acids which share similar
properties and may be interchangeable are discussed above.
[0048] The following references regarding the BLAST algorithm are
herein incorporated by reference: BLAST ALGORITHMS: Altschul, S.
F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al.,
(1993) Nature Genet. 3:266-272; Madden, T. L., et al., (1996) Meth.
Enzymol. 266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids
Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656;
Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock,
J. M., et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT
SCORING SYSTEMS: Dayhoff, M. O., et al., "A model of evolutionary
change in proteins." in Atlas of Protein Sequence and Structure,
(1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl.
Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al.,
"Matrices for detecting distant relationships." in Atlas of Protein
Sequence and Structure, (1978) vol. 5, suppl. 3." M. O. Dayhoff
(ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;
Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J.,
et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc.
Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al.,
(1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S.,
et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S.,
et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A.,
et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F.
"Evaluating the statistical significance of multiple distinct local
alignments." in Theoretical and Computational Methods in Genome
Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y..
Antibodies
[0049] The present invention includes methods and compositions
comprising anti-PCSK9 antibodies and antigen-binding fragments
thereof. The term anti-PCSK9 antibody or the like includes any
antibody that binds specifically to PCSK9 (e.g., human PCSK9). The
anti-PCSK9 antibodies and antigen-binding fragments thereof used in
the present invention include antibodies and fragments which were
raised against or bind to the whole PCSK9 protein as well as
antibodies raised against or bind to particular short epitopes
within PCSK9, e.g., the catalytic domain of PCSK9 or a portion
thereof (e.g., binding to an VFAQSIPWNLER epitope (SEQ ID NO: 8))
or a C-terminal domain of PCSK9 (i.e., C-terminal relative to the
cat domain of PCSK9)-having amino acids SRSGKRRGERMEA (amino acids
490-502 of SEQ ID NO: 4). In an embodiment of the invention, the
anti-PCSK9 antibody or antigen-binding fragment thereof binds to
the domain of PCSK9 which interacts with the EGF-A domain of the
LDL receptor.
[0050] Specific isolated mouse anti-human PCSK9 antibody
immunoglobulin sequences of the present invention are set forth
below. CDR sequences for each immunoglobulin chain are
underscored.
TABLE-US-00009 11B5 immunoglobulin heavy chain (SEQ ID NO: 10) E V
Q L Q Q S G A E L V K P G A S V T L S C T A S G F N I K D T Y M H W
V N Q R P E Q G L V W I G R I D P A N G H T E Y D P K F Q D K A T I
T T D T S S N T A Y L H L S S L T S G D T A V Y Y C A R S Y F G S I
F A Y W G Q G T L V T V S A HCDR1: (SEQ ID NO: 11) G F N I K D T Y
M H HCDR2: (SEQ ID NO: 12) R I D P A N G H T E Y D P K F Q D HCDR3:
(SEQ ID NO: 13) S Y F G S I F A Y 11B5 immunoglobulin light chain
(SEQ ID NO: 14) Q I V L T Q S P A I M S A S P G E K V T I S C S A S
S S V S Y L Y W Y Q Q K P G S S P K P W I F R S S H R A S G V P A R
F S G S G S G T S Y S L T I S S M E A E D A A T Y Y C H Q Y Q S Y P
P T F G G G T K L E I K R A LCDR1: (SEQ ID NO: 15) S A S S V S Y L
Y LCDR2: (SEQ ID NO: 16) R S S H R A S LCDR3: (SEQ ID NO: 17) H Q Y
Q S Y P P T 75B9 immunoglobulin heavy chain (SEQ ID NO: 18) E V Q L
Q Q S G A D L V K P G A S V K L S C T A S G F N I K D T Y I H W V K
Q R P E Q G L E W I G R I D P A N G H T E Y D P K F Q G R A T L T T
D T S S N T A Y L Q L F S L T S E D S A V Y F C A R S Y Y G S I F A
Y W G Q G T L V T V S A HCDR1: (SEQ ID NO: 19) G F N I K D T Y I H
HCDR2: (SEQ ID NO: 20) R I D P A N G H T E Y D P K F Q G HCDR3:
(SEQ ID NO: 21) S Y Y G S I F AY 75B9 immunoglobulin light chain
(SEQ ID NO: 22) Q I V L T Q S P A I M S A S P G E K V T I S C S A S
S S V S Y L F W Y Q Q K P G S S P K P W I F R T S Y L A S G V P A R
F S G S G S G T S F S L T I S S M E A E D A A T Y Y C H Q Y H T Y P
P T F G G G T K L E I K R A LCDR1: (SEQ ID NO: 23) S A S S S V S Y
L F LCDR2: (SEQ ID NO: 24) R T S Y L A S LCDR3: (SEQ ID NO: 25) H Q
Y H T Y P P T 77D10 immunoglobulin heavy chain (SEQ ID NO: 26) E V
Q L Q Q S G A E L V R S G A S V K L S C T T S G F N I K D Y Y I H W
V K Q R P E Q G L E W I G W I D P E N G D T E Y A P K F Q G K A T M
T A D T S S N T A Y L Q L S S L T S A D T A V Y Y C N A Y Y R Y D D
G T W F P Y W G Q G T L V T V S A HCDR1: (SEQ ID NO: 27) G F N I K
D Y Y I H HCDR2: (SEQ ID NO: 28) W I D P E N G D T E Y A P K F Q G
HCDR3: (SEQ ID NO: 29) Y Y R Y D D G T W F P Y 77D10 immunoglobulin
light chain (SEQ ID NO: 30) D I Q L T Q S P A S L S A S V G E T V T
I T C R A S G N I H S Y L A W Y Q Q K Q G K S P Q F L V D N A K T L
P D G V P S R F S V S G S G T Q Y S L K I N S L Q P E D F G T Y Y C
Q H F W N T P W T F G G G T K L E I K R A LCDR1: (SEQ ID NO: 31) R
A S G N I H S Y L A LCDR2: (SEQ ID NO: 32) N A K T L P D LCDR3:
(SEQ ID NO: 33) Q H F W N T P W T 29C10 immunoglobulin heavy chain
(SEQ ID NO: 34) E V L L Q Q S V A E L V R P G A S V R L S C T A S G
F N I K D T Y I H W V R Q R P E Q G L E W G G W I D P A N G Y T K Y
A P N F Q G K A T L T T D T S S N T A Y L H L S S L T S E D S A I Y
Y C A R G Y Y R Y Y S L D Y W G Q G T S V T V S S HCDR1: (SEQ ID
NO: 35) G F N I K D T Y I H HCDR2: (SEQ ID NO: 36) W I D P A N G Y
T K Y A P N F Q G HCDR3: (SEQ ID NO: 37) G Y Y R Y Y S L D Y 29C10
immunoglobulin light chain (SEQ ID NO: 38) D I Q M T Q T T S S L S
A S L G D R V T I S C R A S Q D I S N Y L N W Y Q Q K P D G T V K L
L I Y Y S S R L H S G V P S R F S G R G S G T D Y S L T I S T L E Q
E D I A T Y F C Q Q G K T L P L T F G A G T K L E L K R A LCDR1:
(SEQ ID NO: 39) R A S Q D I S N Y L N LCDR2: (SEQ ID NO: 40) Y S S
R L H S LCDR3: (SEQ ID NO: 41) Q Q G K T L P L T 22D11
immunoglobulin heavy chain (SEQ ID NO: 42) E V Q L V D S G G G L V
Q P G R S L K L S C A A S G F T F S N H D M A W V R Q A P T K G L E
W V A S I T P S G G T T Y Y R D S V E G R F T V S R D N V K S S L H
L Q M D S L T S E D T A T Y Y C A R Q N Y Y D G S Y Y Y G L Y Y F D
Y W G Q G V M V T V S S HCDR1: (SEQ ID NO: 43) G F T F S N H D M A
HCDR2: (SEQ ID NO: 44) S I T P S G G T T Y Y R D S V E G HCDR3:
(SEQ ID NO: 45) Q N Y Y D G S Y Y Y G L Y Y F D Y 22D11
immunoglobulin light chain (SEQ ID NO: 46) D V L M T Q T P V S L P
V S L G G Q V S I S C R S S Q S L V Y S D G N T Y L H W Y L Q K P G
Q S P Q L L I Y R V S N R F S G V P D R F S G S G S G T D F T L K I
S R V E P E D L G L Y Y C L Q S T H F P P T F G S G T K L E I K R A
LCDR1: (SEQ ID NO: 47) R S S Q S L V Y S D G N T Y L H LCDR2: (SEQ
ID NO: 48) R V S N R F S LCDR3: (SEQ ID NO: 49) L Q S T H F P P T
1F11/1G11 immunoglobulin heavy chain (SEQ ID NO: 50) E V Q L Q Q S
G P E L V K P G A S V K I S C K V S G Y T F T D Y Y M N W V K Q S H
G K S L E W I G D I N P N N G G A I Y N Q K F K G K A T L T V D K S
S S I A Y M E L R S L T S E D S A V Y Y C T S G I I T E I A E D F W
G Q G T T L T V S S HCDR1: (SEQ ID NO: 51) G Y T F T D Y Y M N
HCDR2: (SEQ ID NO: 52) D I N P N N G G A I Y N Q K F K G HCDR3:
(SEQ ID NO: 53) G I I T E I A E D F 1F11/1G11 immunoglobulin light
chain (SEQ ID NO: 54) D I V M T Q S Q K F M S T S V G D R V S V T C
K A S Q N V G T N V V W Y Q Q K P G Q S P K A L I H S A S Y R Y S G
V P D R F K G S G S G T D F T L T I T N V Q S E D L A G F F C Q Q Y
K T Y P Y T F G
G G T Q L E I K R A LCDR1: (SEQ ID NO: 55) K A S Q N V G T N V V
LCDR2: (SEQ ID NO: 56) S A S Y R Y S LCDR3: (SEQ ID NO: 57) Q Q Y K
T Y P Y T
[0051] The present invention includes isolated polypeptides (e.g.,
antibodies and antigen-binding fragments thereof) comprising one or
more (e.g., 3) CDRs taken from the light and/or heavy chain
immunoglobulin set forth above as defined by the convention set
forth in Kabat, "Sequences of Proteins of Immunological Interest"
(National Institutes of Health, Bethesda, Md., 1987 and 1991) or in
Chothia et al., J. Mol. Biol. 196:901 (1987); Nature 342:878
(1989); and J. Mol. Biol. 186:651 (1989) (Kabat or Chothia) or
Al-Lazikani et al., J. Mol. Biol. 273: 927-948 (1997).
[0052] Thus, the invention includes any antibody or antigen-binding
fragment thereof comprising one or more of the light chain and/or
heavy chain immunoglobulin CDRs set forth above. In an embodiment
of the invention, the antibody or fragment comprises all 3 light
chain and/or all 3 heavy chain CDRs, e.g., in the order specified
above. Embodiments of the invention include anti-PCSK9 antibodies
and antigen-binding fragments thereof, e.g., as set forth above
wherein, the antibodies or fragments are monoclonal antibodies,
camelized single domain antibodies, polyclonal antibodies,
bispecific antibodies, chimeric antibodies, recombinant antibodies,
anti-idiotypic antibodies, humanized antibodies, bispecific
antibodies, diabodies, single chain antibodies, disulfide Fvs
(dsfv), Fvs, Fabs, Fab' s, F(ab').sub.2s and domain antibodies.
Thus, the term antibody covers, but is not limited to, monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies). The term antigen-binding fragment of an
antibody encompasses a fragment or a derivative of an antibody,
typically including at least a portion of the antigen-binding or
variable regions (e.g., one or more CDRs) of the parental antibody,
that retains at least some of the binding specificity of the
parental antibody. Examples of antibody antigen-binding fragments
include, but are not limited to, Fab, Fab', F(ab').sub.2, and Fv
fragments; dsFv; (dsFv).sub.2, ds diabodies; dsFv-dsFv';
single-chain antibody molecules, e.g., sc-Fv, sc-Fv dimers
(bivalent diabodies); bispecific diabodies; and multispecific
antibodies formed from antibody fragments.
[0053] The present invention includes anti-PCSK9 antibodies and
antigen-binding fragments thereof which binds specifically to
PCSK9, for example, human PCSK9. In an embodiment of the invention
an antibody or fragment that binds specifically to human PCSK9
binds preferentially to human PCSK9 as compared to that of rat,
mouse or chimp PCSK9. Preferential binding to human PCSK9 means
binding with an affinity which is greater than that of rat, mouse
or chimp PCSK9 binding to any degree (e.g., 1%, 10%, 50%, 100%, or
10.times. higher affinity). In an embodiment of the invention, an
anti-human PCSK9 antibody binds to human PCSK9 without any
detectable binding to any other species of PCSK9 (e.g., no
detectable binding to a mouse or rat PCSK9). Specific anti-PCSK9
binding refers to binding of the antibody to PCSK9 or an antigenic
fragment thereof with a Kd at least about 100-fold higher than that
of any other protein that might be bound and a minimum Kd of about
500 nM.
[0054] Any suitable method for generating antibodies may be used.
For example, a recipient may be immunized with PCSK9 or an
immunogenic fragment thereof. Any suitable method of immunization
can be used. Such methods can include adjuvants, other
immunostimulants, repeated booster immunizations, and the use of
one or more immunization routes. Any suitable source of PCSK9 can
be used as the immunogen for the generation of the antibodies and
fragments of the compositions and methods disclosed herein. Such
forms include, but are not limited whole protein, peptide(s), and
epitopes generated through recombinant, synthetic, chemical or
enzymatic degradation means known in the art.
[0055] Any form of the antigen can be used to generate the antibody
that is sufficient to generate a biologically active antibody.
Thus, the eliciting antigen may be a single epitope, multiple
epitopes, or the entire protein alone or in combination with one or
more immunogenicity enhancing agents known in the art. The
eliciting antigen may be an isolated full-length protein, a cell
surface protein (e.g., immunizing with cells transfected with at
least a portion of the antigen), or a soluble protein fragment.
[0056] Any suitable method can be used to elicit an antibody with
the desired biologic properties to inhibit PCSK9. Monoclonal
antibodies (mAbs) may be prepared from various mammalian hosts,
such as mice, rats, other rodents, humans, other primates, etc.
Description of techniques for preparing such monoclonal antibodies
may be found in, e.g., Stites et al. (eds.) BASIC AND CLINICAL
IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos, Calif.,
and references cited therein; Harlow and Lane (1988) ANTIBODIES: A
LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES:
PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y.
Thus, monoclonal antibodies may be obtained by a variety of
techniques familiar to researchers skilled in the art. Typically,
spleen cells from an animal immunized with a desired antigen are
immortalized, commonly by fusion with a myeloma cell. See Kohler
and Milstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods
of immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods known in the art. See,
e.g., Doyle et al. (eds. 1994 and periodic supplements) CELL AND
TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley and Sons, New
York, N.Y. Colonies arising from single immortalized cells are
screened for production of antibodies of the desired specificity
and affinity for the antigen, and yield of the monoclonal
antibodies produced by such cells may be enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate host. Alternatively, one may isolate DNA sequences that
encode a monoclonal antibody or an antigen binding fragment thereof
by screening a DNA library from human B cells according, e.g., to
the general protocol outlined by Huse et al. (1989) Science
246:1275-1281.
[0057] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse et al.
supra; and Ward et al. (1989) Nature 341:544-546. The polypeptides
and antibodies of the present invention may be used with or without
modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance that
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly U.S. Pat. No.
4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033; or made in transgenic mice, see Mendez et al.
(1997) Nature Genetics 15:146-156.
[0058] Mice which produce human immunoglobulins when immunized with
an given antigen are also available in the art. See e.g., Lonberg,
N., et al., (1994) Nature 368(6474): 856-859; Lonberg, N. (1994)
Handbook of Experimental Pharmacology 113:49-101; Lonberg, N., et
al., (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F., et
al., (1995) Ann. N.Y. Acad. Sci. 764:536-546); Taylor, L., et al.,
(1992) Nucleic Acids Research 20:6287-6295; Chen, J., et al.,
(1993) International Immunology 5: 647-656; Tuaillon, et al.,
(1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi, et al.,
(1993) Nature Genetics 4:117-123; Chen, J., et al., (1993) EMBO J.
12: 821-830; Tuaillon, et al., (1994) J. Immunol. 152:2912-2920;
Lonberg, et al., (1994) Nature 368(6474): 856-859; Lonberg, N.
(1994) Handbook of Experimental Pharmacology 113:49-101; Taylor,
L., et al., (1994) International Immunology 6: 579-591; Lonberg,
N., et al., (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding,
F., et al., (1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D.,
et al., (1996) Nature Biotechnology 14: 845-851 and Harding, et
al., (1995) Annals NY Acad. Sci. 764:536-546. See further, U.S.
Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;
5,877,397; 5,661,016; 5,814,318; 5,874, 299; 5,770,429 and
5,545,807; and International Patent Application Publication Nos. WO
98/24884; WO 94/25585; WO 93/12227; WO 92/22645 and WO
92/03918.
[0059] A "Fab fragment" is comprised of one light chain and the
C.sub.H1 and variable regions of one heavy chain. The heavy chain
of a Fab molecule cannot form a disulfide bond with another heavy
chain molecule.
[0060] An "Fc" region contains two heavy chain fragments comprising
the C.sub.H1 and C.sub.H2 domains of an antibody. The two heavy
chain fragments are held together by two or more disulfide bonds
and by hydrophobic interactions of the C.sub.H3 domains.
[0061] A "Fab' fragment" contains one light chain and a portion of
one heavy chain that contains the V.sub.H domain and the C.sub.H1
domain and also the region between the C.sub.H1 and C.sub.H2
domains, such that an interchain disulfide bond can be formed
between the two heavy chains of two Fab' fragments to form a
F(ab').sub.2 molecule.
[0062] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0063] "Disulfide stabilized Fv fragments" and "dsFv" include
molecules having a variable heavy chain (V.sub.H) and/or a variable
light chain (V.sub.L) which are linked by a disulfide bridge.
[0064] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0065] The term "single-chain Fv" or "scFv" antibody refers to
antibody fragments comprising the V.sub.H and V.sub.L domains of an
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains which
enables the V.sub.H and V.sub.L chains to pair and form a binding
site (e.g., 5-12 residues long). For a review of scFv, see
Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol.
113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.
269-315. See also, International Patent Application Publication No.
WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.
[0066] A "domain antibody" is an immunologically functional
immunoglobulin fragment containing only the variable region of a
heavy chain or the variable region of a light chain. In some
instances, two or more V.sub.H regions are covalently joined with a
peptide linker to create a bivalent domain antibody. The two
V.sub.H regions of a bivalent domain antibody may target the same
or different antigens.
[0067] A "bivalent antibody" comprises two antigen-binding sites.
In some instances, the two binding sites have the same antigen
specificities. However, bivalent antibodies may be bispecific. For
example, the present invention comprises scfv dimers and dsfv
dimers, each of which scfv and dsfv moieties may have a common or
different antigen binding specificity.
[0068] In an embodiment of the invention, a (dsfv).sub.2 comprises
three peptide chains: two V.sub.H moieties linked by a peptide
linker and bound by disulfide bridges to two V.sub.L moieties. In
an embodiment of the invention, a bispecific ds diabody comprises a
VH.sub.1-VL.sub.2 (tethered by a peptide linker) linked, by a
disulfide bridge between the VH.sub.1 and VL.sub.1, to a
VL.sub.1-VH.sub.2 moiety (also tethered by a peptide linker). In an
embodiment of the invention, a bispecific dsfv-dsfv' also comprises
three peptide chains: a VH.sub.1-VH.sub.2 moiety wherein the heavy
chains are linked by a peptide linker (e.g., a long flexible
linker) and are bound to VL.sub.1 and VL.sub.2 moieties,
respectively, by disulfide bridges; wherein each disulfide paired
heavy and light chain has a different antigen specificity. In an
embodiment of the invention, an scfv dimer (a bivalent diabody)
comprises a V.sub.H-V.sub.L moiety wherein the heavy and light
chains are bound to by a peptide linker and dimerized with another
such moiety such that V.sub.Hs of one chain coordinate with the
V.sub.Ls of another chain and form two identical binding sites. In
an embodiment of the invention a bispecific diabody comprises
VH.sub.1-VL.sub.2 moiety (linked by a peptide linker) associated
with a VL.sub.1-VH.sub.2 (linked by a peptide linker), wherein the
VH.sub.1 and VL.sub.1 coordinate and the VH.sub.2 and VL.sub.2
coordinate and each coordinated set has diverse antigen
specificities.
[0069] The term "monoclonal antibody", as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic epitope. In contrast, conventional (polyclonal) antibody
preparations typically include a multitude of antibodies directed
against (or specific for) different epitopes. 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
recombinantly or by the hybridoma method first described by Kohler
et al. (1975) Nature 256: 495, or may be made by recombinant DNA
methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson et al. (1991) Nature
352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597,
for example. See also Presta (2005) J. Allergy Clin. Immunol.
116:731.
[0070] Monoclonal antibodies include "chimeric" antibodies
(immunoglobulins) in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl.
Acad. Sci. USA 81: 6851-6855). For example, variable domains are
obtained from an antibody from an experimental animal (the
"parental antibody"), such as a mouse, and the constant domain
sequences are obtained from human antibodies, so that the resulting
chimeric antibody will be less likely to elicit an adverse immune
response in a human subject than the parental mouse antibody.
[0071] A recombinant antibody or antigen-binding fragment thereof
of the invention is, in an embodiment of the invention, an antibody
which is produced recombinantly, e.g., expressed from a
polynucleotide which has been introduced into an organism (e.g., a
plasmid containing a polynucleotide encoding the antibody or
fragment transformed into a bacterial cell (e.g., E. coli) or a
mammalian cell (e.g., CHO cell)), followed by isolation of the
antibody or fragment from the organism.
[0072] The present invention comprises methods for expressing any
antibody or antigen-binding fragment thereof or polypeptide of the
present invention. For example, an embodiment of the present
invention comprises a process for producing an immunoglobulin
molecule or an immunologically functional immunoglobulin fragment
comprising at least the variable domains of the immunoglobulin
heavy and/or light chains, in a host cell (e.g., a single host
cell), such as a CHO cell (e.g., CHO-K1 or DXB11 cell), comprising
the steps of (i) transforming said host cell with a first
polynucleotide encoding at least the variable domain of the
immunoglobulin heavy chain and a second polynucleotide encoding at
least the variable domain of the immunoglobulin light chain, and
(ii) independently expressing said first polynucleotide and said
second polynucleotide so that said immunoglobulin heavy and light
chains are produced as separate molecules in said transformed host
cell. In an embodiment of the invention, the polynucleotides are
operably linked to a promoter such as a CMV promoter. The present
invention also comprises a process for producing an immunoglobulin
molecule or an immunologically functional immunoglobulin fragment
comprising introducing transforming a first haploid yeast host cell
(e.g., Pichia such as Pichia pastoris) with a first polynucleotide
encoding at least the variable domain of the immunoglobulin heavy
chain or light chain and transforming a second haploid yeast host
cell (e.g., Pichia such as Pichia pastoris) with a second
polynucleotide encoding the other chain, allowing the haploid cells
to form polyploids, such as diploids, (e.g., via mating), selecting
the polyploids from the haploids, and growing the polyploids under
conditions wherein the heavy and light chains are expressed in the
polyploids and, optionally, secreted into the culture medium.
[0073] In an embodiment of the invention, polynucleotides
introduced into a host cell remain ectopic whereas in another
embodiment of the invention, the polynucleotide is integrated into
the chromosomal DNA of the host cell.
[0074] In an embodiment of the invention, a method for producing an
antibody or fragment further comprises isolating the chains that
are expressed from the host cell and/or culture medium.
[0075] The present invention also includes camelized single domain
antibodies. See, e.g., Muyldermans et al. (2001) Trends Biochem.
Sci. 26:230; Reichmann et al. (1999) J. Immunol. Methods 231:25; WO
94/04678; WO 94/25591; U.S. Pat. No. 6,005,079, which are hereby
incorporated by reference in their entireties). Camelidae (camels,
dromedaries and llamas) comprise IgG antibodies in which are devoid
of light chains and therefore called `heavy-chain` IgGs or HCAb
(for heavy-chain antibody). HCAbs typically have a molecular weight
of .about.95 kDa since they consist only of the heavy-chain
variable domains. Although the HCAbs are devoid of light chains,
they have an authentic antigen-binding repertoire (Hamers-Casterman
et al., Nature (1993) 363:446-448; Nguyen et al., Adv. Immunol.
(2001) 79:261-296; Nguyen et al., Immunogenetics. (2002) 54:39-47).
In one embodiment, the present invention provides single domain
antibodies comprising two V.sub.H domains with modifications such
that single domain antibodies are formed.
[0076] As used herein, the term "diabodies" refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy chain variable domain (V.sub.H) connected to a
light chain variable domain (V.sub.L) in the same polypeptide chain
(V.sub.H-V.sub.L or V.sub.L-V.sub.H). By using a linker that is too
short to allow pairing between the two domains on the same chain,
the domains are forced to pair with the complementary domains of
another chain and create two antigen-binding sites. Diabodies are
described more fully in, e.g., EP 404,097; WO 93/11161; and
Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.
For a review of engineered antibody variants generally see Holliger
and Hudson (2005) Nat. Biotechnol. 23:1126-1136.
[0077] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from both human and
non-human (e.g., mouse or rat) antibodies. 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 hypervariable loops (CDRs) correspond to those of a
non-human immunoglobulin, and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
sequence. An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions, also called CDRs. Several public sources for human
framework sequence are available including, e.g., V-base (MRC
Center for Protein Engineering). The humanized antibody may
optionally comprise at least a portion of a human immunoglobulin
constant region (Fc). For example, an embodiment of the invention
includes humanized anti-PCSK9 (e.g., anti-human PCSK9) antibodies
comprising the specific mouse immunoglobulin CDRs and human
immunoglobulin framework regions which are set forth herein, e.g.,
fused to a human immunoglobulin constant region. The following U.S.
patents are herein incorporated by reference: U.S. Pat. Nos.
5,585,089, 5,693,761, 5,693,762 and 6,180,370. For example,
embodiments of the invention include those wherein any of the
following groups of CDRs are included within a humanized antibody
of the invention:
TABLE-US-00010 (i) HCDR1 comprising the amino acid sequence (SEQ ID
NO: 11) G F N I K D T Y M H, HCDR2 comprising the amino acid
sequence (SEQ ID NO: 12) R I D P A N G H T E Y D P K F Q D and
HCDR3 comprising the amino acid sequence (SEQ ID NO: 13) S Y F G S
I F A Y; (ii) HCDR1 comprising the amino acid sequence (SEQ ID NO:
19) G F N I K D T Y I H, HCDR2 comprising the amino acid sequence
(SEQ ID NO: 20) R I D P A N G H T E Y D P K F Q G and HCDR3
comprising the amino acid sequence (SEQ ID NO: 21) S Y Y G S I F A
Y; (iii) HCDR1 comprising the amino acid sequence (SEQ ID NO: 27) G
F N I K D Y Y I H, HCDR2 comprising the amino acid sequence (SEQ ID
NO: 28) W I D P E N G D T E Y A P K F Q G and HCDR3 comprising the
amino acid sequence (SEQ ID NO: 29) Y Y R Y D D G T W F P Y; (iv)
HCDR1 comprising the amino acid sequence (SEQ ID NO: 35) G F N I K
D T Y I H, HCDR2 comprising the amino acid sequence (SEQ ID NO: 36)
W I D P A N G Y T K Y A P N F Q G and HCDR3 comprising the amino
acid sequence (SEQ ID NO: 37) G Y Y R Y Y S L D Y; (v) HCDR1
comprising the amino acid sequence (SEQ ID NO: 43) G F T F S N H D
M A, HCDR2 comprising the amino acid sequence (SEQ ID NO: 44) S I T
P S G G T T Y Y R D S V E G and HCDR3 comprising the amino acid
sequence (SEQ ID NO: 45) Q N Y Y D G S Y Y Y G L Y Y F D Y; (vi)
HCDR1 comprising the amino acid sequence (SEQ ID NO: 51) G Y T F T
D Y Y M N, HCDR2 comprising the amino acid sequence (SEQ ID NO: 52)
D I N P N N G G A I Y N Q K F K G and HCDR3 comprising the amino
acid sequence (SEQ ID NO: 53) G I I T E I A E D F; (vii) LCDR1
comprising the amino acid sequence (SEQ ID NO: 15) S A S S S V S Y
L Y, LCDR2 comprising the amino acid sequence (SEQ ID NO: 16) R S S
H R A S and LCDR3 comprising the amino acid sequence (SEQ ID NO:
17) H Q Y Q S Y P P T; (viii) LCDR1 comprising the amino acid
sequence (SEQ ID NO: 23) S A S S S V S Y L F, LCDR2 comprising the
amino acid sequence (SEQ ID NO: 14) R T S Y L A S and LCDR3
comprising the amino acid sequence (SEQ ID NO: 25) H Q Y H T Y P P
T; (ix) LCDR1 comprising the amino acid sequence (SEQ ID NO: 31) R
A S G N I H S Y L A, LCDR2 comprising the amino acid sequence (SEQ
ID NO: 32) N A K T L P D and LCDR3 comprising the amino acid
sequence (SEQ ID NO: 33) Q H F W N T P W T; (x) LCDR1 comprising
the amino acid sequence (SEQ ID NO: 39) R A S Q D I S N Y L N,
LCDR2 comprising the amino acid sequence (SEQ ID NO: 40) Y S S R L
H S and LCDR3 comprising the amino acid sequence (SEQ ID NO: 41) Q
Q G K T L P L T; (xi) LCDR1 comprising the amino acid sequence (SEQ
ID NO: 47) R S S Q S L V Y S D G N T Y L H, LCDR2 comprising the
amino acid sequence (SEQ ID NO: 48) R V S N R F S and LCDR3
comprising the amino acid sequence (SEQ ID NO: 49) L Q S T H F P P
T; or (xii) LCDR1 comprising the amino acid sequence (SEQ ID NO:
55) K A S Q N V G T N V V, LCDR2 comprising the amino acid sequence
(SEQ ID NO: 56) S A S Y R Y S and LCDR3 comprising the amino acid
sequence (SEQ ID NO: 57) Q Q Y K T Y P Y T.
[0078] The present invention also includes isolated polypeptides
comprising the amino acid sequences of the immunoglobulin chains
set forth herein along with isolated polynucleotides encoding said
polypeptides, vectors comprising the polynucleotides and isolated
host cells comprising the polynucleotides and vectors (e.g., CHO
cells, bacterial cells such as E. coli and fungal cells such as S.
cerevisiae and Pichia such as Pichia pastoris). Methods for making
the polypeptides are also included: comprising introducing a
polynucleotide or vector into a host cell and culturing the host
cell under condition whereby the polypeptide can be expressed and,
optionally, secreted and, optionally, isolating the
polypeptide.
[0079] Therapeutic Methods, Administration and Pharmaceutical
Formulations
[0080] The present invention provides methods for treating or
preventing disorders of cholesterol or lipid homeostasis and
disorders associated therewith, e.g., hypercholesterolemia,
hyperlipidemia, hypertriglyceridemia, sitosterolemia,
atherosclerosis, arteriosclerosis, coronary heart disease, vascular
inflammation and xanthoma by administering a therapeutically
effective amount of an anti-PCSK9 antibody (e.g., as set forth
herein) or antigen-binding fragment thereof or an EGF-A
polypeptide.
[0081] The term hypercholesterolemia includes, e.g., familial and
non-familial hypercholesterolemia. Familial hypercholesterolemia
(FHC) is an autosomal dominant disorder characterized by elevation
of serum cholesterol bound to low density lipoprotein (LDL).
Familial hypercholesterolemia includes both heterozygous FHC and
homozygous FHC.
[0082] Hyperlipidemia is an elevation of lipids in the bloodstream.
These lipids include cholesterol, cholesterol esters, phospholipids
and triglycerides. Hyperlipidemia includes for example, type I,
IIa, IIb, III, IV and V.
[0083] Sitosterolemia is a rare inherited plant sterol storage
disease. In general, the metabolic defect in the affected patient
causes hyperabsorption of sitosterol from the gastrointestinal
tract, decreased hepatic secretion of sitosterol with subsequent
decreased elimination, and altered cholesterol synthesis.
[0084] Atherosclerosis includes hardening of arteries associated
with deposition of fatty substances, cholesterol, cellular waste
products, calcium and fibrin in the inner lining of an artery. The
buildup that results is called plaque.
[0085] Arteriosclerosis includes the diffuse build-up and
deposition of calcium in artery walls which leads to hardening.
[0086] The present invention also provides methods for improving
blood cholesterol markers associated with increased risk of heart
disease. These markers include high total cholesterol, high LDL,
high total cholesterol to HDL ratio and high LDL to HDL ratio.
[0087] In general, a total cholesterol of less than 200 mg/dL is
considered desirable, 200-239 mg/dL is considered borderline high
and 240 mg/dL and above is considered high.
[0088] In general, a blood LDL level of less than 100 mg/dL is
considered optimal; 100-129 mg/dL is considered near optimal/above
optimal, 130-159 mg/dL is considered borderline high, 160-189 mg/dL
is considered high and 190 mg/dL and above is considered very
high.
[0089] In general, HDL levels considered normal are at least 35-40
mg/dL.
[0090] Another indicator of heart disease risk is the ratio of
total cholesterol to HDL. In general, a very low risk of heart
disease correlates with a ratio of <3.4 (men) or <3.3
(women); a low risk is associated with a ratio of 4.0 (men) or 3.8
(women), an average risk is associated with a ratio of 5.0 (men) or
4.5 (women), a moderate risk is associated with a ratio of 9.5
(men) or 7.0 (women) and a high risk is associated with a ratio of
>23 (men) or >11 (women).
[0091] A further indicator of heart disease risk is the ratio of
LDL to HDL. In general, a very low risk is associated with a ratio
of 1 (men) or 1.5 (women), an average risk is associated with a
ratio of 3.6 (men) or 3.2 (women), a moderate risk is associated
with a ratio of 6.3 (men) or 5.0 (women) and a high risk is
associated with a ratio of 8 (men) or 6.1 (women).
[0092] In an embodiment of the invention, anti-PCSK9 antibodies and
antigen-binding fragments thereof or EGF-A polypeptides of the
invention are formulated into a pharmaceutical formulation which
comprises a pharmaceutically acceptable carrier. For general
information concerning formulations, see, e.g., Gilman, et al.,
(eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; A. Gennaro (ed.), Remington's Pharmaceutical
Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa.;
Avis, et al., (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral
Medications Dekker, N.Y.; Lieberman, et al., (eds.) (1990)
Pharmaceutical Dosage Forms: Tablets Dekker, New York; and
Lieberman, et al., (eds.) (1990), Pharmaceutical Dosage Forms:
Disperse Systems Dekker, New York, Kenneth A. Walters (ed.) (2002)
Dermatological and Transdermal Formulations (Drugs and the
Pharmaceutical Sciences), Vol 119, Marcel Dekker.
[0093] The anti-PCSK9 antibodies and antigen-binding fragments
thereof or EGF-A polypeptides of the present invention can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the antibody or fragment is combined
in admixture with a pharmaceutically acceptable carrier. Carriers,
excipients or stabilizers are nontoxic to recipients at the dosages
and concentrations employed, and include buffers such as phosphate,
citrate and other organic acids; antioxidants including ascorbic
acid; low molecular weight polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as Tween.TM., Pluronics.TM. or PEG. The
formulations to be used for in vivo administration must be sterile.
This is readily accomplished by filtration through sterile
filtration membranes, prior to or following lyophilization and
reconstitution. Therapeutic or pharmaceutical compositions or
formulations herein generally are 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.
[0094] The route of administration of the antibodies and
antigen-binding fragments thereof of the invention are, in an
embodiment of the invention, by a parenteral route (e.g.,
intravenous, subcutaneous, intraarterial, intratumoral,
intramuscular, intraperitoneal).
[0095] Dosages and desired anti-PCSK9 or EGF-A polypeptide
concentrations of pharmaceutical compositions of the present
invention may vary depending on the particular use envisioned. The
determination of the appropriate dosage or route of administration
is well within the skill of an ordinary physician. Animal
experiments provide reliable guidance for the determination of
effective doses for human therapy. Interspecies scaling of
effective doses can be performed following the principles laid down
by Mordenti, J. and Chappell, W. "The use of interspecies scaling
in toxicokinetics" In Toxicokinetics and New Drug Development,
Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42 96.
[0096] When used to treat a disorder in a subject (e.g., as
discussed herein), a therapeutically effective dosage or amount of
anti-PCSK9 antibody or antigen-binding fragment thereof or EGF-A
polypeptides is administered to the subject. In an embodiment of
the invention, a therapeutically effective dosage is a dosage
sufficient to decrease total serum cholesterol, decrease blood LDL
levels or increase blood HDL levels to any degree whatsoever. In an
embodiment of the invention, a therapeutically effective dosage of
anti-PCSK9 antibody or antigen-binding fragment thereof (e.g., as
set forth herein) for treatment of hypercholesterolemia,
hyperlipidemia, hypertriglyceridemia, sitosterolemia,
atherosclerosis, arteriosclerosis, coronary heart disease, vascular
inflammation or xanthoma or for treatment of any blood marker of
heart disease risk (e.g., as discussed herein) is about 0.1 mg/kg
(body weight)/week to about 1.0 mg/kg/week. A therapeutically
effective dosage of soluble PCSK9 EGF-A polypeptide is, in an
embodiment of the invention, about 0.25 mg/kg/week to about 25
mg/kg/week.
[0097] When possible, administration and dosage of an agent (e.g.,
further therapeutic agents discussed herein) is done according to
the schedule listed in the product information sheet of the agents,
in the Physicians' Desk Reference 2003 (Physicians' Desk Reference,
57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition
(November 2002), as well as therapeutic protocols well known in the
art.
[0098] A physician or clinician may monitor the blood cholesterol
levels in a subject being treated or about to be treated with an
anti-PCSK9 antibody or antigen-binding fragment thereof or EGF-A
polypeptide and make adjustments to the subject's treatment regimen
as needed to reach a positive medical outcome.
Further Chemotherapeutic Agents
[0099] The present invention provides methods and compositions for
treating disorders of lipid and cholesterol metabolism (e.g., any
set forth herein) by administration of an anti-PCSK9 antibody or
antigen-binding fragment thereof or EGF-A polypeptide. The
antibodies may, in an embodiment of the invention, be provided or
administered in association with any additional or further
chemotherapeutic agent. In an embodiment of the invention, the
further chemotherapeutic agent is a cardiovascular agent, an
adrenergic blocker, an antihypertensive agent, an angiotensin
system inhibitor, an angiotensin-converting enzyme (ACE) inhibitor,
a coronary vasodilator, a diuretic or an adrenergic stimulant. In
an embodiment of the invention, the further therapeutic agent is a
cholesterol lowering medication such as an HMG-CoA reductase
inhibitor.
[0100] Cardiovascular agents which may used in connection with the
present invention include those for treatment or prevention of
lipid and/or cholesterol disorders or hypertension and other
cardiovascular disorders and diseases. Disorders of lipid or
cholesterol metabolism may be caused or aggravated by hypertension.
Hypertension is defined as persistently high blood pressure.
Generally, adults are classified as being hypertensive when
systolic blood pressure is persistently above 140 mmHg or when
diastolic blood pressure is above 90 mmHg. Long-term risks for
cardiovascular mortality increase in a direct relationship with
persistent blood pressure. Examples of antihypertensive agents
which may be used in the present invention include e.g., calcium
channel blockers, angiotensin-converting enzyme (ACE) inhibitors,
angiotensin-II receptor antagonists, diuretics, adrenergic blockers
including beta-adrenergic receptor blockers and alpha-adrenergic
receptor blockers or diuretics.
[0101] Other cardiac drugs that may be provided in association with
an anti-PCSK9 antibody or antigen-binding fragment thereof includes
anti-anginal agents, such as adrenergic stimulants or coronary
vasodilators and HMG-CoA reductase inhibitors.
[0102] HMG-CoA reductase inhibitors inhibit the HMG-CoA reductase
enzyme and, thus, reduce production of cholesterol in the body of a
subject. HMG-CoA reductase inhibitors include, e.g., lovastatin,
atorvastatin, pravastatin, rosuvastatin, fluvastatin, rivastatin
and simvastatin
##STR00001##
[0103] Adrenergic blockers include those compounds which are
.beta.-receptor inhibitors and/or .alpha.-receptor inhibitors.
Adrenergic blockers which are .beta.-receptor inhibitors include a
class of drugs that antagonize the cardiovascular effects of
catecholamines in hypertension, angina pectoris, and cardiac
arrhythmias. .beta.-adrenergic receptor blockers include, but are
not limited to, bunolol hydrochloride (1(2H)-Naphthalenone,
5-[3-(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3,4-dihydro-,hydrochlori-
de, CAS RN 31969-05-8 which can be obtained from Parke-Davis);
acebutolol (.+-.N-[3-Acetyl-4-[2-hydroxy-3-[(1
methylethyl)amino]propoxy]phenyl]-butanamide, or
(.+-.)-3'-Acetyl-4'-[2-hydroxy-3-(isopropylamino)
propoxy]butyranilide); acebutolol hydrochloride (such as
N-3-acetyl-4-[2-hydroxy-3-[1-methyl-ethyle)amino]propoxy]phenyl]-,
monohydrocochloride,
(.+-.-;-3'-Acetyl-4'-[2-hydroxy-3-(isopropylamino)propoxy]butyranilide
monohydrochloride, for example, SECTRAL.RTM. Capsules available
from Wyeth-Ayerst); alprenolol hydrochloride (2-Propanol,
1-[(1-methylethypamino]-3-[2-(2-propenyl)phenoxy]-,hydrochloride,
CAS RN 13707-88-5 see Netherlands Patent Application No.
6,605,692); atenolol (such as benzeneacetamide
4-[2'-hydroxy-3'-[(1-methylethyl)amino]propoxy]-, for example,
TENORMIN.RTM. I.V. Injection available from AstraZeneca); carteolol
hydrochloride (such as
5-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3,4-dihydro-2(1H)-quino-
linone monohydrochloride, for example, Cartrol.RTM. Filmtab.RTM.
Tablets available from Abbott); Celiprolol hydrochloride
(3-[3-Acetyl-4-[3-(tert-butylamino)-2-hydroxypropoxyl]phenyl]-1,1-diethyl-
urea monohydrochloride, CAS RN 57470-78-7, also see in U.S. Pat.
No. 4,034,009); cetamolol hydrochloride (Acetamide,
2-[2-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-phenoxy]-N-methyl-,m-
onohydrochloride, CAS RN 77590-95-5, see also U.S. Pat. No.
4,059,622); labetalol hydrochloride (such as
5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino]ethyl]salicylamide
monohydrochloride, for example, NORMODYNE.RTM. Tablets available
from Schering; esmolol hydrochloride ((.+-.)-Methyl
p-[2-hydroxy-3-(isopropylamino) propoxy]hydrocinnamate
hydrochloride, for example, BREVIBLOC.RTM.Injection available from
Baxter); levobetaxolol hydrochloride (such as
(S)-1-[p-[2-(cyclopropylmethoxy)ethyl]phenoxy]-3-(isopropylamino)-2-propa-
nol hydrochloride, for example, BETAXON.TM. Ophthalmic Suspension
available from Alcon); levobunolol hydrochloride (such as
(-)-5-[3-(tert-Butylamino)-2-hydroxypropoxy]-3,4-dihydro-1(2H)-naphthalen-
one hydrochloride, for example, BETAGAN.RTM. Liquifilm.RTM. with C
CAP.RTM.Compliance Cap available from Allergan); nadolol (such as
1-(tert-butylamino)-3-[(5,6,7,8-tetrahydro-cis-6,7-dihydroxy-1-naphthyl)o-
xy]-2-propanol, for example, Nadolol Tablets available from Mylan);
practolol (Acetamide,
N-[4-[4-[2-hydroxy-3-[1-methylethyl)amino]-propoxy]phenyl]-, CAS RN
6673-35-4, see also U.S. Pat. No. 3,408,387); propranolol
hydrochloride (1-(Isopropylamino)-3-(1-naphthyloxy)-2-propanol
hydrochloride CAS RN 318-98-9); sotalol hydrochloride (such as
d,l-N-[4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-phenyl]methane-sulfona-
mide monohydrochloride, for example, BETAPACE AF.TM. Tablets
available from Berlex);timolol (2-Propanol,
1-[(1,1-dimethylethypamino]-3-[[4-4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]-
oxy]-,hemihydrate, (S)-, CAS RN 91524-16-2); timolol maleate
(S)-1-[(1,1-dimethylethyl)amino]-3-[[4-(4-morpholinyl)-1,2,5-thiadiazol-3-
-yl]oxy]-2-propanol (Z)-2-butenedioate (1:1) salt, CAS RN
26921-17-5); bisoprolol (2-Propanol,
1-[4-[[2-(1-methylethoxy)ethoxy]-methyl]phenoxyl]-3-[(1-methylethyl)amino-
]-, (.+-.), CAS RN 66722-44-9); bisoprolol fumarate (such as
(.+-.)-1-[4-[[2-(1-Methylethoxy)
ethoxy]methyl]phenoxy]-3-[(1-methylethyl)amino]-2-propanol
(E)-2-butenedioate (2:1) (salt), for example, ZEBETA.TM. Tablets
available from Lederle Consumer); nebivalol
(2H-1-Benzopyran-2-methanol,
.alpha..alpha.'-[iminobis(methylene)]bis[6-fluoro-3,4-dihydro-, CAS
RN 99200-09-6 see also U.S. Pat. No. 4,654,362); cicloprolol
hydrochloride, such 2-Propanol,
1-[4-[2-(cyclopropylmethoxy)ethoxy]phenoxy]-3-[1-methylethyl)amino]-,hydr-
ochloride, A.A.S. RN 63686-79-3); and dexpropranolol hydrochloride
(2-Propanol,
1-[1-methylethyl)-amino]-3-(1-naphthalenyloxy)-hydrochloride (CAS
RN 13071-11-9); diacetolol hydrochloride (Acetamide,
N-[3-acetyl-4-[2-hydroxy-3-[(1-methyl-ethypamino]propoxyl]phenyl]-,monohy-
drochloride CAS RN 69796-04-9);dilevalol hydrochloride (Benzamide,
2-hydroxy-5-[1-hydroxy-2-[1-methyl-3-phenylpropyl)amino]ethyl]-,monohydro-
chloride, CAS RN 75659-08-4); exaprolol hydrochloride (2-Propanol,
1-(2-cyclohexylphenoxy)-3-[(1-methylethyl)amino]-,hydrochloride CAS
RN 59333-90-3); flestolol sulfate (Benzoic acid,
2-fluoro-,3-[[2-[aminocarbonyl)amino]-1-dimethylethyl]amino]-2-hydroxypro-
pyl ester, (.+-.)-sulfate (1:1) (salt), CAS RN 88844-73-9; metalol
hydrochloride(Methanesulfonamide,
N-[4-[1-hydroxy-2-(methylamino)propyl]phenyl]-,monohydrochloride
CAS RN 7701-65-7);metoprolol 2-Propanol,
1-[4-(2-methoxyethyl)phenoxy]-3-[1-methylethyl)amino]-; CAS RN
37350-58-6);metoprolol tartrate (such as 2-Propanol,
1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-, for
example, LOPRESSOR.RTM. available from Novartis); pamatolol sulfate
(Carbamic acid,
[2-[4[-2-hydroxy-3-[(1-methylethyl)amino]propoxyl]phenylFethyl]-,me-
thyl ester, (.+-.) sulfate (salt) (2:1), CAS RN 59954-01-7);
penbutolol sulfate (2-Propanol,
1-(2-cyclopentylphenoxy)-3-[1,1-dimethylethypamino]1, (S)--,
sulfate (2:1) (salt), CAS RN 38363-32-5); practolol (Acetamide,
N-[4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phenyl]-, CAS RN
6673-35-4;) tiprenolol hydrochloride (Propanol,
1-[(1-methylethyl)amino]-3-[2-(methylthio)-phenoxy]-,
hydrochloride, (.+-.), CAS RN 39832-43-4); tolamolol (Benzamide,
4-[2-[[2-hydroxy-3-(2-methylphenoxy)-propyl]amino]ethoxyl]-, CAS RN
38103-61-6).
[0104] Adrenergic receptors which are .alpha.-receptor inhibitors
act to block vasoconstriction induced by endogenous catecholamines.
The resulting fall in peripheral resistance leads to a fall in mean
blood pressure. The magnitude of this effect is dependent upon the
degree of sympathetic tone at the time the antagonist is
administered.
[0105] Suitable adrenergic receptors which are .alpha.-receptor
inhibitors include, but are not limited to, fenspiride
hydrochloride (which may be prepared as disclosed in U.S. Pat. No.
3,399,192 herein incorporated by reference); proroxan (CAS RN
33743-96-3); alfuzosin hydrochloride (CAS RN: 81403-68-1); and
labetalol hydrochloride as described above or combinations
thereof.
[0106] Adrenergic blockers with .alpha. and .beta. receptor
inhibitor activity which may be used with the present invention
include, but are not limited to, bretylium tosylate (CAS RN:
61-75-6); dihydroergtamine mesylate (such as ergotaman-3',
6',18-trione,9,-10-dihydro-12'-hydroxy-2'-methyl-5'-(phenylmethyl)-,(5'(a-
lpha))-, monomethanesulfonate, for example, DHE 45.RTM. Injection
available from Novartis); carvedilol (such as
(.+-.)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propan-
ol, for example, COREG.RTM. Tablets available from SmithKline
Beecham); labetalol (such as
5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl)amino]ethyl]salicylamide
monohydrochloride, for example, NORMODYNE.RTM. Tablets available
from Schering); bretylium tosylate (Benzenemethanaminium,
2-bromo-N-ethyl-N,N-dimethyl-, salt with 4-methylbenzenesulfonic
acid (1:1) CAS RN 61-75-6); phentolamine mesylate (Phenol,
3-[[(4,5-dihydro-1H-imidazol-2-yl)methyl](4-methylphenyl)amino]-,
monomethanesulfonate (salt) CAS RN 65-28-1); solypertine tartrate
(5H-1,3-Dioxolo[4,5-f]indole,
7-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-,
(2R,3R)-2,3-dihydroxybutanedioate (1:1) CAS RN 5591-43-5);
zolertine hydrochloride (Piperazine,
1-phenyl-4-[2-(1H-tetrazol-5-yl]ethyl]-, monohydrochloride (8Cl,
9Cl) CAS RN 7241-94-3)
[0107] An angiotensin system inhibitor is an agent that interferes
with the function, synthesis or catabolism of angiotensin II. These
agents which may be used in the present invention include but are
not limited to angiotensin-converting enzyme (ACE) inhibitors,
angiotensin II antagonists, angiotensin II receptor antagonists,
agents that activate the catabolism of angiotensin II and agents
that prevent the synthesis of angiotensin I from which angiotensin
II is ultimately derived. The renin-angiotensin system is involved
in the regulation of hemodynamics and water and electrolyte
balance. Factors that lower blood volume, renal profusion, or the
concentration of Na.sup.+ in plasma tend to activate the system,
while factors that increase these parameters tend to suppress its
function. Angiotensin I and angiotensin II are synthesized by the
enzymatic renin-angiotensin pathway. The synthetic process is
initiated when the enzyme renin acts on angiotensinogen, a
pseudoglobulin in blood plasma, to produce the cecapeptide
angiotensin I. Angiotensin I is converted by angiotensin-converting
enzyme (ACE) to angiotensin II. The latter is an active pressor
substance which has been implicated as a causative agent in several
forms of hypertension in various mammalian species.
[0108] Angiotensin II receptor antagonists are compounds which
interfere with the activity of angiotensin II by binding to
angiotensin II receptors and interfering with its activity.
Angiotensin II receptor antagonists which may be used in the
present invention are well known and include peptide compounds and
non-peptide compounds. Non-limiting examples of angiotensin II
receptor antagonists include: candesartan cilexetil
(1H-Benzimidazole-7-carboxylic acid,
2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-,
1-[[(cyclohexyloxy)carbonyl]oxy]ethyl ester) CAS RN 145040-37-5);
telmisartan([1,1'-Biphenyl]-2-carboxylic acid,
4'-[(1,4'-dimethyl-2'-propyl[2,6'-bi-1H-benzimidazol]-1'-yl)methyl]-
CAS RN 144701-48-4); candesartan (1H-Benzimidazole-7-carboxylic
acid,
2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]- CAS
RN 139481-59-7); losartan potassium (1H-Imidazole-5-methanol,
2-butyl-4-chloro-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-,
monopotassium Irbesartan1,3-Diazaspiro[4.4]non-1-en-4-one,
2-butyl-3-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl[- CAS
RN 138402-11-6).
[0109] Angiotensin-converting enzyme (ACE), is an enzyme which
catalyzes the conversion of angiotensin Ito angiotensin II. ACE
inhibitors which may be used in the present invention include amino
acids and derivatives thereof, peptides, including di and tri
peptides and antibodies to ACE which intervene renin-angiotensin
system by inhibiting the activity of ACE thereby reducing or
eliminating the formation of pressor substance angiotensin II. ACE
inhibitors have been used medically to treat hypertension,
congestive heart failure, myocardial infarction and renal disease.
Suitable ACE inhibitors include, but are not limited to, benazepril
hydrochloride (such as
3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]amino]-2,3,4,5-tetrahydro-2-o-
xo-1H-1-(3S)-benzazepine-1-acetic acid monohydrochloride, for
example, LOTREL.RTM. Capsules available from Novartis); captopril
(such as 1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline, for
example, CAPTOPRIL Tablets available from Mylan); fosinopril (such
as L-proline, 4-cyclohexyl-1-[[[2-methyl-1-(1-oxopropoxy) propoxy]
(4-phenylbutyl) phosphinyl]acetyl]-, sodium salt, trans--, for
example, MONOPRIL.RTM. Tablets available from Bristol-Myers
Squibb); moexipril hydrochloride (such as
[3S-[2[R*(R*)],3R*]]-2-[2-[[1-(Ethoxycarbonyl)-3-phenylpropyl]am-
ino]-1-oxopropyl]-1,2,3,4-tetrahydro-6,7-dimethoxy-3-isoquinolinecarboxyli-
c acid, monohydrochloride, for example, UNIRETIC.RTM. Tablets
available from Schwarz); perindopril erbumine (such as
2S,3aS,7aS)-1-[(S)--N--[(S)-1-Carboxybutyl]alanyl]hexahydro-2-indolinecar-
boxylic acid, 1-ethyl ester, compound with tert-butylamine (1:1),
for example, ACEON.RTM. Tablets available from Solvay); quinapril
(such as
[3S-[2[R*(R*)],3R*]]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-ox-
opropyl]-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid,
monohydrochloride, for example, ACCURETIC.RTM. Tablets available
from Parke-Davis); ramipril (such as
2-aza-bicyclo[3.3.0]-octane-3-carboxylic acid derivative, for
example, ALTACE.RTM. Capsules available from Monarch); enalapril
maleate (such as
(S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline,
(Z)-2-butenedioate salt (1:1), for example, VASOTEC.RTM. Tablets
available from Merck); lisinopril (such as (S)-1-[N,
2-(1-carboxy-3-phenylpropyl)-L-lysyl]-L-proline dihydrate, for
example, PRINZIDE.RTM. Tablets available from Merck); delapril
(which may be prepared as disclosed in U.S. Pat. No. 4,385,051);
and spirapril (which may be prepared as disclosed in U.S. Pat. No.
4,470,972); benazeprilat (1H-1-Benzazepine-1-acetic acid,
3-[[(1S)-1-carboxy-3-phenylpropyl]amino]-2,3,4,5-tetrahydro-2-oxo-,
(3S)- CAS RN 86541-78-8); delapril hydrochloride (Glycine,
N-[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]L-alanyl-N-(2,3-dihydro-1H-inde-
n-2-yl)-, monohydrochloride CAS RN 83435-67-0); fosinopril sodium
(L-Proline,
4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-oxopropoxy)propoxy](4-phenylbuty-
l)phosphinyl]acetyl]-, sodium salt, (4S)- CAS RN 88889-14-9);
libenzapril (1H-1-Benzazepine-1-acetic acid,
3-[[(1S)-5-amino-1-carboxypentyl]amino]-2,3,4,5-tetrahydro-2-oxo-,
(3S)- CAS RN 109214-55-3); pentopril (1H-Indole-1-pentanoic acid,
2-carboxy-2,3-dihydro-.alpha.,.gamma.-dimethyl-.delta.-oxo-,.alpha.-ethyl
ester, (.alpha.R,.gamma.R,2S)- CAS RN 82924-03-6); perindopril
1H-Indole-2-carboxylic acid,
1-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)butyl]amino]-1-oxopropyl]octahydro-,
(2S,3aS,7aS)- CAS RN 82834-16-0); quinapril hydrochloride
(3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,-
2,3,4-tetrahydro-, monohydrochloride, (3S)- CAS RN 82586-55-);
quinaprilat (3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl]amino]-1-oxopropyl]-1,2,3,4-tet-
rahydro-, (3S)- CAS RN 82768-85-2); spirapril hydrochloride
(1,4-Dithia-7-azaspiro[4.4]nonane-8-carboxylic acid,
7-[(25)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-,
monohydrochloride, (8S)- CAS RN 94841-17-5); spiraprilat
1(,4-Dithia-7-azaspiro[4.4]nonane-8-carboxylic acid,
7-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl]amino]-1-oxopropyl]-,
(8S)- CAS RN 83602-05-5); teprotide (Bradykinin potentiator BPP9a
CAS RN 35115-60-7); lisinopril (L-Proline,
N2-[(1S)-1-carboxy-3-phenylpropyl]-L-lysyl- CAS RN 76547-98-3);
zofenopril (L-Proline,
1-[(2S)-3-(benzoylthio)-2-methyl-1-oxopropyl]-4-(phenylthio)-,
calcium salt (2:1), (4S)- CAS RN 81938-43-4).
[0110] "Calcium channel blockers" are a chemically diverse class of
compounds having important therapeutic value in the control of a
variety of diseases including several cardiovascular disorders such
as hypertension, angina, and cardiac arrhythmias (Fleckenstein,
Cir. Res. V. 52 (suppl. 1), p. 13-16 (1983); Fleckenstein,
Experimental Facts and Therapeutic Prospects, John Wiley, New York
(1983); McCall, D., Curr. Pract Cardiol., v. 10, p. 1-11 (1985)).
Calcium channel blockers are a heterogeneous group of drugs that
prevent or slow the entry of calcium into cells by regulating
cellular calcium channels (Remington, The Science and Practice of
Pharmacy, Nineteenth Edition, Mack Publishing Company, Eaton, Pa.,
p. 963 (1995)). Calcium channel blockers useful in the present
invention include but are not limited to, the besylate salt of
amlodipine (such as
3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-
-methyl-3,5-pyridinedicarboxylate benzenesulphonate, for example,
NORVASC.RTM. available from Pfizer); clentiazem maleate
(1,5-Benzothiazepin-4(5H)-one,
3-(acetyloxy)-8-chloro-5-[2-(dimethylamino)ethyl]-2,3-dihydro-2-(4-methox-
yphenyl)-(2S-cis)-, (Z)-2-butenedioate (1:1), see also U.S. Pat.
No. 4,567,195); isradipine (3,5-Pyridinedicarboxylic acid,
4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-,methyl 1-methylethyl
ester,
(.+-.)-4(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedi-
carboxylate, see also U.S. Pat. No. 4,466,972); nimodipine (such as
is isopropyl (2-methoxyethyl)
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate,
for example, NIMOTOP.RTM. available from Bayer); felodipine (such
as ethyl methyl
4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate-
, for example, PLENDIL.RTM. Extended-Release Tablets available from
AstraZeneca LP); nilvadipine (3,5-Pyridinedicarboxylic acid,
2-cyano-1,4-dihydro-6-methyl-4-(3-nitrophenyl)-,3-methyl
5-(1-methylethyl) ester, also see U.S. Pat. No. 3,799,934);
nifedipine (such as 3,5-pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester, for
example, PROCARDIA XL.RTM. Extended Release Tablets available from
Pfizer); diltiazem hydrochloride (such as
1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-
-dihydro-2(4-methoxyphenyl)-, monohydrochloride, (+)-cis., for
example, TIAZAC.RTM. Capsules available from Forest); verapamil
hydrochloride (such as benzeneacetronitrile,
(alpha)-[[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimeth-
oxy-(alpha)-(1-methylethyl) hydrochloride, for example,
ISOPTIN.RTM. SR Tablets available from Knoll Labs); teludipine
hydrochloride (3,5-Pyridinedicarboxylic acid,
2-[(dimethylamino)methyl]-4-[2-[(1E)-3-(1,1-dimethylethoxy)-3-oxo-1-prope-
nyl]phenyl]-1,4-dihydro-6-methyl-, diethyl ester,
monohydrochloride) CAS RN 108700-03-4); belfosdil (Phosphonic acid,
[2-(2-phenoxyethyl)-1,3-propanediyl]bis-, tetrabutyl ester CAS RN
103486-79-9); fostedil (Phosphonic acid,
[[4-(2-benzothiazolyl)phenyl]methyl]-, diethyl ester CAS RN
75889-62-2).
[0111] Cardiovascular agents of the present invention which also
act as "anti-anginal agents" are useful in the present invention.
Angina includes those symptoms that occur when myocardial oxygen
availability is insufficient to meet myocardial oxygen demand.
Non-limiting examples of these agents include: ranolazine
(hydrochloride 1-piperazineacetamide,
N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,
dihydrochloride CAS RN 95635-56-6); betaxolol hydrochloride
(2-Propanol, 1-[4-[2
(cyclopropylmethoxy)ethyl]phenoxy]-3-[(1-methylethyl)amino]-,
hydrochloride CAS RN 63659-19-8); butoprozine hydrochloride
(Methanone,
[4-[3(dibutylamino)propoxy]phenyl](2-ethyl-3-indolizinyl)-,
monohydrochloride CAS RN 62134-34-3); cinepazet
maleatel-piperazineacetic acid,
44'-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-, ethyl ester,
(2Z)-2-butenedioate (1:1) CAS RN 50679-07-7); tosifen
(Benzenesulfonamide,
4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl[- CAS RN
32295-18-4); verapamilhydrochloride (Benzeneacetonitrile,
.alpha.-[3-[[2-(3,4-dimethoxyphenyl)ethyl]nethylamino]propyl]-3,4-dimetho-
xy-.alpha.-(1-methylethyl)-, monohydrochloride CAS RN 152-11-4);
molsidomine (1,2,3-Oxadiazolium,
5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-, inner salt CAS RN
25717-80-0); ranolazine hydrochloride (1-piperazineacetamide,
N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-,
dihydrochloride CAS RN 95635-56-6); tosifen (Benzenesulfonamide,
4-methyl-N-[[[(1S)-1-methyl-2-phenylethyl]amino]carbonyl]- CAS RN
32295-18-4).
[0112] "Coronary vasodilators" may act to reduce angina systems by
increasing the oxygen supply to the heart. Coronary vasodilators
useful in the present invention include, but are not limited to,
diltiazem hydrochloride (such as
1,5-Benzothiazepin-4(5H)-one,3-(acetyloxy)-5[2-(dimethylamino)ethyl]-2,-3-
-dihydro-2(4-methoxyphenyl)-, monohydrochloride, (+)-cis, for
example, TIAZAC.RTM. Capsules available from Forest); isosorbide
dinitrate (such as 1,4:3,6-dianhydro-D-glucitol 2,5-dinitrate, for
example, 1SORDIL.RTM. TITRADOSE.RTM. Tablets available from
Wyeth-Ayerst); sosorbide mononitrate (such as
1,4:3,6-dianhydro-D-glucitol, 5-nitrate, an organic nitrate, for
example, Ismo.RTM. Tablets available from Wyeth-Ayerst);
nitroglycerin (such as 2,3 propanetriol trinitrate, for example,
NITROSTAT.RTM. Tablets available from Parke-Davis); verapamil
hydrochloride (such as benzeneacetonitrile,
(.+-.)-(alpha)[3[[2-(3,4
dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimethoxy-(alpha)-(1-methyl-
ethyl) hydrochloride, for example, COVERA HS.RTM. Extended-Release
Tablets available from Searle); chromonar (which may be prepared as
disclosed in U.S. Pat. No. 3,282,938); clonitate (Annalen 1870
155); droprenilamine (which may be prepared as disclosed in German
Patent No. 2,521,113); lidoflazine (which may be prepared as
disclosed in U.S. Pat. No. 3,267,104); prenylamine (which may be
prepared as disclosed in U.S. Pat. No. 3,152,173); propatyl nitrate
(which may be prepared as disclosed in French Patent No.
1,103,113); mioflazine hydrochloride (1-piperazineacetamide,
3-(aminocarbonyl)-4-[4,4-bis(4-fluorophenyl)butyl]-N-(2,6-dichlorophenyl)-
-, dihydrochloride CAS RN 83898-67-3); mixidine (Benzeneethanamine,
3,4-dimethoxy-N-(1-methyl-2-pyrrolidinylidene)-Pyrrolidine,
2-[(3,4-dimethoxyphenethyl)imino]-1-methyl-1-Methyl-2-[(3,4-dimethoxyphen-
ethyl)imino]pyrrolidine CAS RN 27737-38-8); molsidomine
(1,2,3-Oxadiazolium, 5-[(ethoxycarbonyl)amino]-3-(4-morpholinyl)-,
inner salt CAS RN 25717-80-0); isosorbide mononitrate (D-Glucitol,
1,4:3,6-dianhydro-, 5-nitrate CAS RN 16051-77-7); erythrityl
tetranitrate (1,2,3,4-Butanetetrol, tetranitrate, (2R,3S)-rel- CAS
RN 7297-25-8); clonitrate (1,2-Propanediol, 3-chloro-, dinitrate
(7CI, 8CI, 901) CAS RN 2612-33-1); dipyridamole Ethanol,
2,2',2'',2'''-[(4,8-di-1-piperidinylpyrimido[5,4-d]pyrimidine-2,6-diyl)di-
nitrilo]tetrakis- CAS RN 58-32-2); nicorandil (CAS RN 65141-46-0
3-);
pyridinecarboxamide(N-[2-(nitrooxy)ethyl]-Nisoldipine-3,5-Pyridinedicarbo-
xylic acid, 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, methyl
2-methylpropyl ester CAS RN 63675-72-9);
nifedipine-3,5-Pyridinedicarboxylic acid,
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-, dimethyl ester CAS RN
21829-25-4); perhexyline maleate (Piperidine,
2-(2,2-dicyclohexylethyl)-, (2Z)-2-butenedioate (1:1) CAS RN
6724-53-4); oxprenolol hydrochloride2-Propanol,
1-[(1-methylethyl)amino]-3-[2-(2-propenyloxy)phenoxy]-,
hydrochloride CAS RN 6452-73-9); pentrinitrol (1,3-Propanediol,
2,2-bis[(nitrooxy)methyl]-, mononitrate (ester) CAS RN 1607-17-6);
verapamil (Benzeneacetonitrile,
.alpha.-[3-[[2-(3,4-dimethoxyphenyl)ethyl]methylamino]propyl]-3,4-dimetho-
xy-.alpha.-(1-methylethyl)- CAS RN 52-53-9).
[0113] The term "diuretic" includes compounds that increase the
excretion of solutes (mainly NaCl) and water. In general, the
primary goal of diuretic therapy is to reduce extracellular fluid
volume in order to lower blood pressure or rid the body of excess
interstitial fluid (edema). Non-limiting examples of diuretics
which may be used within the scope of this invention include
althiazide (which may be prepared as disclosed in British Patent
No. 902,658); benzthiazide (which may be prepared as disclosed in
U.S. Pat. No. 3,108,097); buthiazide (which may be prepared as
disclosed in British Patent Nos. 861,367); chlorothiazide (which
may be prepared as disclosed in U.S. Pat. No. 2,809,194);
spironolactone (CAS Number 52-01-7); and triamterene (CAS Number
396-01-0).
[0114] "Adrenergic stimulants" useful as cardiovascular agents in
the present invention include, but are not limited to, guanfacine
hydrochloride (such as N-amidino-2-(2,6-dichlorophenyl) acetamide
hydrochloride, for example, TENEX.RTM. Tablets available from
Robins); methyldopa-hydrochlorothiazide (such as
levo-3-(3,4-dihydroxyphenyl)-2-methylalanine) combined with
Hydrochlorothiazide (such as 6-chloro-3,4-dihydro-2H
-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide, for example, the
combination as, for example, ALDORIL.RTM. Tablets available from
Merck); methyldopa-chlorothiazide (such as 6-chloro-2 H
-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide and methyldopa as
described above, for example, ALDOCLORr.RTM. Tablets available from
Merck); clonidine hydrochloride (such as
2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride and
chlorthalidone (such as
2-chloro-5-(1-hydroxy-3-oxo-1-isoindolinyl)benzenesulfonamide), for
example, COMBIPRES.RTM. Tablets available from Boehringer
Ingelheim); clonidine hydrochloride (such as
2-(2,6-dichlorophenylamino)-2-imidazoline hydrochloride, for
example, CATAPRES.RTM. Tablets available from Boehringer
Ingelheim); clonidine (1H-Imidazol-2-amine,
N-(2,6-dichlorophenyl)-4,5-dihydro- CAS RN 4205-90-7).
[0115] The anti-PCSK9 antibodies and antigen-binding fragments
thereof and EGF-A polypeptides may also be administered in
association with any azetidinone which inhibits intestinal
cholesterol absorption. Such azetidinones include ezetimibe
##STR00002##
[0116] Further chemotherapeutic agents that may be administered in
association with an anti-PCSK9 antibody or antigen-binding fragment
thereof or EGF-A polypeptide include fish oil, eicosaepenanoic
acid, docosahexanoic acid, linoleic acid, niacin, fibrates such as
fenofibrate, gemfibrozil and bile acid sequestrants such as
cholestyramine, colestipol and colesevelam.
[0117] Other chemotherapeutic agents include althiazide
(2H-1,2,4-Benzothiadiazine-7-sulfonamide,
6-chloro-3,4-dihydro-3-[(2-propenylthio)methyl]-, 1,1-dioxide CAS
RN 5588-16-9); benzthiazide
(2H-1,2,4-Benzothiadiazine-7-sulfonamide,
6-chloro-3-[[(phenylmethyl)thio]methyl]-, 1,1-dioxide CAS RN
91-33-8); captopril (L-Proline,
1-[(2S)-3-mercapto-2-methyl-1-oxopropyl]- CAS RN 62571-86-2);
carvedilol (2-Propanol,
1-(9H-carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy)ethyl]amino]- CAS
RN 72956-09-3), chlorothiazide (sodium 2-Propanol,
1-(9H-carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy)ethyl]amino]- CAS
RN 72956-09-3); clonidine hydrochloride (1H-Imidazol-2-amine,
N-(2,6-dichlorophenyl)-4,5-dihydro-, monohydrochloride CAS RN
4205-91-8); cyclothiazide (2H-1,2,4-Benzothiadiazine-7-sulfonamide,
3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide
CAS RN 2259-96-3); delapril hydrochloride
(2H-1,2,4-Benzothiadiazine-7-sulfonamide,
3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide
CAS RN 2259-96-3); dilevalol hydrochloride
(2H-1,2,4-Benzothiadiazine-7-sulfonamide,
3-bicyclo[2.2.1]hept-5-en-2-yl-6-chloro-3,4-dihydro-, 1,1-dioxide
CAS RN 2259-96-3); delapril hydrochloride (Glycine,
N-[1S)-1-(ethoxycarbonyl)-3-phenylpropyl]L-alanyl-N-(2,3-dihydro-1H-inden-
-2-yl)-, monohydrochloride CAS RN 83435-67-0); doxazosin mesylate
(piperazine,
1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-[(2,3-dihydro-1,4-benzodioxin--
2-yl)carbonyl]-, monomethanesulfonate CAS RN 77883-43-3);
fosinopril sodium (L-Proline,
4-cyclohexyl-1-[[(R)-[(1S)-2-methyl-1-(1-oxopropoxy)propox);
moexipril hydrochloride (3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,-
2,3,4-tetrahydro-6,7-dimethoxy-, monohydrochloride, (3S)- CAS RN
82586-52-5); monatepil maleate (1-piperazinebutanamide,
N-(6,11-dihydrodibenzo(b,e)thiepin-11-yl)-4-(4-fluorophenyl)-,
(.+-.)-, (Z)-2-butenedioate (1:1)
(.+-.)-N-(6,11-Dihydrodibenzo(b,e)thiepin-11-yl)-4-(p-fluorophenyl)-1-pip-
erazinebutyramide maleate (1:1) CAS RN 132046-06-1), Metoprolol
succinate (Butanedioic acid, compd. with
1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)amino]-2-propanol
(1:2) CAS RN 98418-47-4); guanfacine hydrochloride
(Benzeneacetamide, N-(aminoiminomethyl)-2,6-dichloro-,
monohydrochloride CAS RN 29110-48-3; methyldopa (L-Tyrosine,
3-hydroxy-.alpha.-methyl- CAS RN 555-30-6); quinaprilat
(3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-carboxy-3-phenylpropyl]amino]-1-oxopropyl]-1,2,3,4-tet-
rahydro-, (3S)- CAS RN 82768-85-2); quinapril hydrochloride
(3-Isoquinolinecarboxylic acid,
2-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]-1,-
2,3,4-tetrahydro-, monohydrochloride, (3S)- CAS RN 82586-55-8);
Primidolol (2,4(1H,3H)-Pyrimidinedione,
1-[2-[[2-hydroxy-3-(2-methylphenoxy)propyl]amino]ethyl]-5-methyl-
CAS RN 67227-55-8); prazosin hydrochloride (piperazine,
1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furanylcarbonyl)-,
monohydrochloride CAS RN 19237-84-4); pelanserin hydrochloride
2,4(1H,3H)-Quinazolinedione, 3-[3-(4-phenyl-1-piperazinyl)propyl]-,
monohydrochloride CAS RN 42877-18-9); phenoxybenzamine
hydrochloride (Benzenemethanamine,
N-(2-chloroethyl)-N-(1-methyl-2-phenoxyethyl)-, hydrochloride CAS
RN 63-92-3); candesartan cilexetil (1 H-Benzimidazole-7-carboxylic
acid,
2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-ylynethyl]-,
1-[[(cyclohexyloxy)carbonyl]oxy]ethyl ester CAS RN 145040-37-5);
telmisartan (1,1'-Biphenyl]-2-carboxylic acid,
4'-[(1,4'-dimethyl-2'-propyl[2,6'-bi-1H-benzimidazol]-1'-yl)methyl]-
CAS RN 144701-48-4); candesartan1H-Benzimidazole-7-carboxylic acid,
2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]- CAS
RN 139481-59-7); amlodipine besylate-3,5-Pyridinedicarboxylic acid,
2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-,
3-ethyl 5-methyl ester, monobenzenesulfonate CAS RN 111470-99-6
Amlodipine maleate 3,5-Pyridinedicarboxylic acid,
2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-,
3-ethyl 5-methyl ester, (2Z)-2-butenedioate (1:1) CAS RN
88150-47-4); terazosin hydrochloride (piperazine,
1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-[(tetrahydro-2-furanyl)carbony-
l]-, monohydrochloride CAS RN 63074-08-8); bevantolol hydrochloride
(2-Propanol,
1-[[2-(3,4-dimethoxyphenyl)ethyl]amino]-3-(3-methylphenoxy)-,
hydrochloride CAS RN 42864-78-8); ramipril
(Cyclopenta[b]pyrrole-2-carboxylic acid,
1-[(2S)-2-[[(1S)-1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]oct-
ahydro-, (2S,3aS,6aS)--CAS RN 87333-19-5).
Screening Assays
[0118] The present invention further provides a method for
identifying a substance which is a PCSK9 inhibitor or an inhibitor
of PCSK9/LDL receptor binding or an inhibitor of PCSK9/EGF-A domain
binding; or a substance which reduces total cholesterol level, low
density lipoprotein cholesterol level, apolipoprotein B level,
total cholesterol/high density lipoprotein ratio or low density
lipoprotein/high density lipoprotein ratio; or a substance which
treats or prevents hypercholesterolemia, hyperlipidemia,
hypertriglyceridaemia, sitosterolemia, atherosclerosis,
arteriosclerosis, coronary heart disease, vascular inflammation or
xanthoma in a subject comprising contacting PCSK9 polypeptide or a
functional fragment thereof and a polypeptide comprising LDL
receptor EGF-A domain (e.g., SEQ ID NO: 3) and a sample to be
tested for the presence of said substance; wherein said substance
is identified if less binding of PCSK9 and the polypeptide
comprising LDL receptor EGF-A domain is observed in the presence of
the sample than in the absence of the sample. Any such substance
identified in such an assay also forms part of the present
invention.
[0119] An embodiment of the invention further comprises a
negative-control assay wherein said PCSK9 and EGF-A polypeptides
are contacted with a substance known not to inhibit binding of
PCSK9 and LDL receptor or EGF-A domain thereof wherein the assay is
determined to be operating properly if more binding of the PCSK9
and LDL receptor or EGF-A domain thereof is observed than in the
present of an inhibitor of said binding.
[0120] An embodiment of the invention further comprises a
positive-control assay wherein said PCSK9 and EGF-A polypeptides
are contacted with a substance known to inhibit binding of PCSK9
and LDL receptor or EGF-A domain thereof wherein the assay is
determined to be operating properly if less binding of the PCSK9
and LDL receptor or EGF-A domain thereof is observed than in the
present of an inhibitor of said binding.
[0121] Binding of the substance can be determined using any of
several methods known in the art.
EXAMPLES
[0122] The following information is provided for more clearly
describing the present invention and should not be construed to
limit the present invention. Any and all of the compositions and
methods described below fall within the scope of the present
invention.
Example 1
Soluble EGF-A Domain of the LDL Receptor or Anti-PCSK9 Antibody
Blocks the LDLR-PCSK9 Interaction
[0123] In this example, a soluble peptide encoding the EGF-A domain
of the human LDL receptor as well as various anti-PCSK9 antibodies
are shown to block interactions between PCSK9 and the receptor.
[0124] The following are the AlphaScreen methods and materials for
the antibody and EGF-A inhibition of PCSK9-LDLR interaction
experiments:
[0125] Purified PCSK9 carrying a C-terminal FLAG tag was purified
from HEK293 cells stably expressing PCSK9. Purity was estimated, by
silver staining, at greater than >90%. Anti-PCSK9-catalytic
domain antibody was purchased from Cayman Chemical (Rabbit
anti-murine-PCSK9 polyclonal antibody, Cayman Chemical; Ann Arbor,
Mich.; Cat.#10008811). This rabbit anti-mouse/human PCSK9-catalytic
domain antibody recognizes an epitope with the following amino acid
sequence: VFAQSIPWNLER (SEQ ID NO: 8). A rabbit anti-human PCSK9
C-terminal domain antibody (which binds specifically to
SRSGKRRGERMEA (amino acids 490-502 of SEQ ID NO: 4)) and a rabbit
anti-human PCSK9 whole protein antibody were also used.
[0126] Soluble EGF-A domain peptide was synthetically generated
using standard methods. 2.5 ng of purified, C-terminally FLAG
tagged PCSK9 (2.5 ng/2.5 ul/well of a ProxiPlate-384 Plus
(Perkin-Elmer; Waltham, Mass.) in 2.5 microliters of buffer (25 mM
HEPES, 0.1M NaCl, 0.1% BSA, pH 7.5) was pre-incubated with 2.5
microliters of either anti-PCSK9 antibody or EGF-A domain peptide
at the indicated concentrations for 30 minutes at room temperature.
Next, 5 ul buffer containing 2.5 ng purified HIS-tagged soluble
LDLR(R&D Systems; Minneapolis, Minn.; Cat.#2148-LD/CF; SEQ ID
NO: 2) and 5 ng biotinylated anti-FLAG antibody (BioM2 monoclonal
antibody, Sigma; St. Louis, Mo.; Cat.# F 9291), and the mixture,
was incubated at room temperature for a further 30 minutes.
Finally, under low light conditions, 5 ul of a donor/acceptor bead
mixture (AlphaScreen Histidine Detection Kit, Perkin-Elmer;
Waltham, Mass.; Cat.#6760619C) was added and alphaScreen signal was
detected using the 2103 EnVision Multilabel Plate Reader
(Perkin-Elmer; Waltham, Mass.). The donor/acceptor bead mixture was
prepared in dark conditions by adding 10 ul Nickel-Acceptor bead
and 10 ul SA-Donor bead to 2.2 ml buffer for one 384-well plate and
incubated in dark at room temperature for at least 2 hours.
[0127] The data generated in these experiments is set forth below
in Tables 1-3.
TABLE-US-00011 TABLE 1 Inhibition of PCSK9-LDLR interaction by
soluble LDLR-EGF-A domain in the presence or absence of added
calcium*. Values are the mean of three replicates. No calcium 2 mM
Calcium log[EGFA]uM (mean counts) (mean counts) 2.30103 8476
4098.333 1.823909 25291 6320.667 1.346787 74404.33 27519 0.869666
132192.7 80542.67 0.392545 182225.3 143204.7 -0.08458 205094.7
202404.3 -0.5617 214258 234140.3 -1.03882 219281 246093.7 -1.51594
221429 240820.7 -1.99306 220878 257794 -2.47018 206909.3 254980.3
*A lower number of counts represents less observed PCSK9/LDL
receptor binding.
[0128] These data indicate that the PCSK9-LDLR interaction is
inhibited by soluble EGF-A domain in the presence or absence of
calcium. A slightly greater level of inhibition was observed in the
presence of calcium than in the absence of calcium. In the presence
of calcium, the IC50 was about 3 micromolar and in the absence of
calcium the IC50 was about 12 micromolar.
TABLE-US-00012 TABLE 2 Inhibition of PCSK9-LDLR interaction by
anti-PCSK9 antibodies (catalytic domain (Cat) and C-terminal domain
(CT)) log[antibody] ug/ml Control IgG Anti-PCSK9 (Cat) Anti-PCSK9
(CT) 1.826075 113607.7 6840 37878 1.348954 135127.7 25815 87575.33
0.871832 139744.7 55616 108605.7 0.394711 135829.3 93545.67
119019.3 -0.08241 140328.3 94768 127197 -0.55953 139224.7 128021.3
127770.3 -1.03665 127405.7 124136.7 129751.7 -1.51377 133565.3
126417.7 131044.3 -1.9909 135194.7 129937.3 131160.7 -2.46802
140051.7 131985.3 128822.3
[0129] These data demonstrate that an anti-PCSK9 catalytic domain
antibody and an anti-PCSK9 C-terminal domain antibody inhibits the
interaction between PCSK9 and LDL receptor. The anti-PCSK9 raised
against a C-terminal domain was less effective at inhibiting the
interaction. A non-specific IgG control was ineffective at
inhibiting the interaction and is shown for comparison. Values in
this table are the mean of three replicates.
TABLE-US-00013 TABLE 3 Inhibition of PCSK9-LDLR interaction by
anti-PCSK9 antibodies (catalytic domain (Cat) and whole protein
(whole)) Anti-PCSK9 log[antibody] ug/ml Control IgG Anti-PCSK9
(Cat) (whole) 1.826075 61920.67 2101.33 1005.33 1.348954 74394.33
12259.00 1435.00 0.871832 79625.67 33347.33 14236.67 0.394711
79552.00 60074.00 52994.00 -0.08241 82751.00 70040.00 75020.33
-0.55953 88110.00 77438.00 85241.00 -1.03665 84421.00 79789.00
82679.00 -1.51377 83144.67 85603.67 87349.33 -1.9909 80747.33
81462.67 80731.67 -2.46802 78151.33 80556.33 83171.00 -2.94514
82101.00 85405.33 80429.00
[0130] These data demonstrate that the PCSK9-LDLR interaction is
inhibited by antibodies that bind the PCSK9 catalytic domain
peptide and antibodies raised against the whole PCSK9 protein. A
non-specific IgG control was ineffective at inhibiting the
interaction and is shown for comparison. These values are mean of
three replicates.
Example 2
Anti-PCSK9 Antibody Treatment of Cells Leads to Enhanced LDL
Uptake
[0131] Incubation of cells with soluble PCSK9 leads to a decrease
in the ability of the cells to absorb and clear low density
lipoprotein (LDL) from the medium. This example demonstrates that
inhibition of PCSK9 with an anti-PCSK9 antibody antagonizes this
effect of PCSK9 and leads to increased levels of LDL uptake and
clearance.
[0132] The uptake assays were performed as follows: HepG2 cells at
10,000 cells/well in 384 Collagen I coated plate were seeded and
grown overnight. On the next day, a serial dilution of
bacu-PCSK9-WT in MEM-1% BSA was made, mixing with a fixed
concentration of anti-mouse-cat domain PCSK9 polyclonal antibody
(Cayman Chemical; Ann Arbor, Mich.; cat#10008811; raised against
mouse PCSK9 antigen VFAQSIPWNLER (SEQ ID NO: 8) and cross reacts
with human PCSK9) or anti-human PCSK9 polyclonal antibody (R&D
Systems, Minneapolis, Minn.; cat# AF3888) at a final concentration
of 100 ug/ml respectively. These mixtures were incubated for 1 hour
at 4.degree. C., then medium was aspirated out from the HepG2 cells
and 25 ul/well of the mixtures was added to HepG2 cells in 384
plate and incubated for 6 hours and 18 hours, respectively. After
the indicated incubation time, the mixture was removed from the
HepG2 cells, washed 1.times. with PBS, then Dil-LDL (Dil label is
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)
was added at 10 ug/ml in MEM-1% BSA to the cells, then incubation
followed at room temperature for 90 minutes. DiI-LDL was removed
afterwards, the cells were fixed with Prefer fixative (glyoxal,
ethanol, buffer; Anatech LTD.; Hayward, Calif.; cat#414) for 20
minutes, then the cells were washed with PBS two times and DiI-LDL
uptake into the cells was read using a fluorescence intensity
reader Analyst. The data generated in these assays are set forth
below in Table 4.
TABLE-US-00014 TABLE 4 LDL uptake in the presence of PCSK9 and
anti-PCSK9 antibodies. [PCSK9] ug/ml control anti-CAT anti-all 6
hour incubation 15 20675218 22419420 18606777 5 21873226 25617426
31744993 1.67 23647886 29483684 30994715 0 29824887 28736616
30124921 18 hour incubation 15 20163100 21843043 17106209 5
24214957 25921384 27741479 1.67 27777510 32019111 29054521 0
31232393 32526668 28127286
[0133] Table 4 shows uptake of dil-labeled LDL by human HepG2 liver
cells treated with the indicated amounts of purified, recombinant
PCSK9 (control) or PCSK9 in the presence of either polyclonal
antibody raised against the catalytic domain peptide (anti-CAT) or
antibody raised against the whole protein (anti-all).
PCSK9-dependent uptake of LDL by the cells increased in a manner
dependent upon the presence of anti-PCSK9 antibody in the mixture.
Higher numbers indicated greater LDL uptake and preservation of
LDLR despite the presence of PCSK9.
Example 3
Characterization of Anti-PCSK9 Antibodies
[0134] The 75B9, 77D10, 11B5, 22D11, 1F11/1G11 and 29C10 anti-PCSK9
antibodies were characterized. The antibodies were found to
effectively inhibit interaction between PCSK9 and the LDL receptor
as well as inhibit PCSK9-mediated LDL receptor degradation. In
vivo, the antibodies were found to inhibit PCSK9 and, thereby
increase the levels of LDL receptor present. Moreover, the
antibodies neutralized the in vivo cholesterol increase observed
when exogenous PCSK9 was added.
Inhibition of PCSK9-Mediated LDLR Degradation with Anti-PCSK9
Antibodies.
[0135] The 75B9, 77D10, 11B5, 22D11, 1F11/1G11 and 29C10 antibodies
were evaluated in an in-cell western assay for their ability to
inhibit the degradation of the LDL receptor by PCSK9 in HepG2
cells.
[0136] In-cell western was performed to quantitate anti-PCSK9
antibody inhibition of PCSK9-mediated LDLR degradation. HepG2 cells
were seeded in 384-well collagen I coated plates and treated with
anti-PCSK9 antibody and/or PCSK9 (100 nM) for 18 hours. Detection
of LDLR and .beta.-actin was performed according to the
manufacturer's protocol (Li-Cor Biosciences; Lincoln, Nebr.) using
the antibodies described above in conjunction with IRDye 800CW goat
anti-rabbit (Li-Cor) and IRDye 680 goat anti-mouse (Li-Cor). The
assay was read on an Odyssey infrared imaging system (Li-Cor) and
the signal for LDLR protein in each well normalized to .beta.-actin
content. The data from these experiments are set forth below in
Table 5.
TABLE-US-00015 TABLE 5 Level of inhibition of each anti-PCSK9
antibody observed in in-cell western assay Relative level of
inhibition of LDLR Treatment degradation observed* 75B9 anti-PCSK9
antibody (IgG2a) ++ 77D10 anti-PCSK9 antibody (IgG2b) ++ 11B5
anti-PCSK9 antibody (IgG2a) ++ 22D11 anti-PCSK9 antibody (IgG1) +
1F11/1G11 anti-PCSK9 antibody (IgG2a) +++ 29C10 anti-PCSK9 antibody
(IgG2a) + No PCSK9 control 100% +PCSK9/no antibody control 0%
*Greater number of + symbols indicates a greater level of
inhibition relative to other treatments. Relative to the "+PCSK9/no
antibody" control, the antibodies tested inhibited PCSK9-mediated
degradation of LDL receptor with various potencies in the HepG2
cells.
AlphaScreen Binding Assays
[0137] An Amplified Luminescent Proximity Homogeneous Assay (ALPHA,
Perkin-Elmer; Waltham, Mass.) capable of directly determining the
interaction between PCSK9-FLAG and a putative binding partner was
used to determine the effect of anti-PCSK9 antibodies on this
interaction. This technique required that "donor" and "acceptor"
beads be brought into proximity via protein--protein interaction,
resulting in increased luminescence (Ullman et al., Proc. Nat.
Acad. Sci. (1994) 91:5426-5430). In the basic assay, LDL receptor
binding to PCSK9 was determined as follows: 5 .mu.l of recombinant
receptor at the appropriate concentrations was incubated with 2.5
.mu.l PCSK9-FLAG (1.4 .mu.g/ml, 30 min). About 2.5 .mu.l of
biotinylated anti-Flag-M2 antibody (1.8 .mu.g/ml) was added and the
mixture incubated for 1 hour. Afterward 5 .mu.l of streptavidin
donor bead and nickel chelate acceptor bead (1:1 mixture) was added
and the assay incubated overnight. AlphaScreen signal (counts per
second) was analyzed using an EnVision microplate reader
(Perkin-Elmer). All data points were determined in triplicate.
Assays were carried out at 23.degree. C. in buffer containing 25 mM
HEPES, 0.1 M NaCl, pH 7.4, 0.1% BSA. The inhibition assays were
determined similarly with slight adjustments to assay volumes and
protein concentrations. Briefly, 5 .mu.l of 1.25 .mu.g/ml of
PCSK9-Flag and 1.25 .mu.g/ml of His-tagged LDL receptor was
incubated with 2.5 .mu.l of antibody at the appropriate
concentrations for 30 minutes followed by the addition of 2.5 .mu.l
of anti-Flag-BioM2 (1.8 .mu.g/ml) and a 1 hour incubation. The data
from these assays are set forth below in Table 6.
TABLE-US-00016 TABLE 6 Level of anti-PCSK9 antibody mediated
inhibition of PCSK9/LDLR interaction % inhibition at 450 nM of
Treatment IC.sub.50 (nM) antibody 75B9 anti-PCSK9 antibody (IgG2a)
0.5 100 77D10 anti-PCSK9 antibody (IgG2b) 38 62 11B5 anti-PCSK9
antibody (IgG2a) 0.4 100 22D11 anti-PCSK9 antibody (IgG1) ND ND
1F11/1G11 anti-PCSK9 antibody (IgG2a) 0.049* 88* 29C10 anti-PCSK9
antibody (IgG2a) 13 71 *Average of two measurements.
[0138] The antibodies tested inhibited PCSK9/LDL receptor
interaction with various potencies (expressed in terms of IC.sub.50
and % inhibition relative to the assay performed with no antibody
added).
Effect of Anti-PCSK9 on In Vivo Inhibition of LDLR Degradation and
Plasma Cholesterol
[0139] The ability of an anti-PCSK9 antibody (1F11/1G11) to inhibit
LDL receptor degration in mice was evaluated in these assays along
with the ability of the antibodies to modulate cholesterol
levels.
In Vivo Procedure:
[0140] Time-0: Male C57BL/6j mice were injected, intraperitoneal,
with 400 .mu.g of anti-human PCSK9 Antibody, 1F11/1G11.
Time-2 hours: Mice were then injected, intravenously, with 10 .mu.g
of human PCSK9 Protein. Time-4 hours: Mice were terminated and
plasma and liver samples were collected.
Analysis:
[0141] Plasma lipoprotein profiles of individual mice (0.1 mL
plasma) were determined by fast protein liquid chromatography
(FPLC) with a Pharmacia Superose 6 column. FPLC fraction
cholesterol levels were determined using Wako Cholesterol Enzymatic
colorimetric method.
[0142] Protein levels of Liver LDL Receptor were determined by
Western Blot. 40 .mu.g of each liver homogenate was separated on an
Invitrogen 4-12% NuPAGE gel, transferred to PVDF membrane and
incubated with goat anti-mouse LDL receptor antibody (rabbit
anti-GAPDH antibody was used as a control for equal loading).
Membranes were then incubated with corresponding HRP-linked
antibodies and visualization was accomplished with high performance
chemiluminescence film. Band intensities were quantified using
ImageQuant 5.2 software.
[0143] LDL receptor levels were expressed in Arbitrary Units (AU)
as a ratio of LDL receptor band intensity to GAPDH band intensity.
The results of the LDL receptor measurements are set forth in
tables 7 and 8 below. Two separate analyses of the data are
presented.
TABLE-US-00017 TABLE 7 Lightest exposure of both LDLR panel vs.
GAPDH panel Mouse LDLR GAPDH LDLR/GAPDH Mean % Change 107 13714
50469 0.272 0.235 -48.0 108 13766 49467 0.278 (Antibody 109 12508
47790 0.262 and PCSK9) 110 5977 46744 0.128 111 1724 46456 0.037
0.081 -82.0 112 912 44130 0.021 (Saline and 113 2631 51367 0.051
PCSK9) 114 10217 47345 0.216 115 23237 48860 0.476 0.452 Control
116 18589 47694 0.390 (Saline only) 117 16577 47554 0.349 118 27116
45755 0.593
TABLE-US-00018 TABLE 8 Darker exposure of LDLR panel vs. lightest
GAPDH panel. Mouse LDLR GAPDH LDLR/GAPDH Mean % Change 107 19883
45242 0.439 0.377 -35.2 108 18057 45634 0.396 (Antibody 109 18642
43767 0.426 and PCSK9) 110 10669 42898 0.249 111 2817 43967 0.064
0.123 -78.8 112 1204 43206 0.028 (Saline and 113 4252 47846 0.089
PCSK9) 114 13848 44288 0.313 115 27675 44510 0.622 0.582 Control
116 23016 45090 0.510 (Saline only) 117 19837 44526 0.446 118 32897
43792 0.751
[0144] The 1F11/1G11 antibody reduced the levels of PCSK9 mediated
LDL receptor degration in the mice tested.
TABLE-US-00019 TABLE 9 Plasma cholesterol levels in mice tested
under indicated conditions. Treatment Plasma total cholesterol
level (mg/dl) Anti-PCSK9 and PCSK9 72.05 Saline and PCSK9 83.28
Saline only 74.88
[0145] The 1F11/1G11 antibody reduced the level of PCSK9-mediated
cholesterol increase in the mice tested.
[0146] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0147] Patents, patent applications, publications, product
descriptions, and protocols are cited throughout this application,
the disclosures of which are incorporated herein by reference in
their entireties for all purposes.
Sequence CWU 1
1
571860PRTHomo sapiens 1Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr
Val Ala Leu Leu Leu 1 5 10 15 Ala Ala Ala Gly Thr Ala Val Gly Asp
Arg Cys Glu Arg Asn Glu Phe 20 25 30 Gln Cys Gln Asp Gly Lys Cys
Ile Ser Tyr Lys Trp Val Cys Asp Gly 35 40 45 Ser Ala Glu Cys Gln
Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu 50 55 60 Ser Val Thr
Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn 65 70 75 80 Arg
Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp 85 90
95 Asn Gly Ser Asp Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp
100 105 110 Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe
Val Cys 115 120 125 Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu
Ala Ser Cys Pro 130 135 140 Val Leu Thr Cys Gly Pro Ala Ser Phe Gln
Cys Asn Ser Ser Thr Cys 145 150 155 160 Ile Pro Gln Leu Trp Ala Cys
Asp Asn Asp Pro Asp Cys Glu Asp Gly 165 170 175 Ser Asp Glu Trp Pro
Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly 180 185 190 Asp Ser Ser
Pro Cys Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu 195 200 205 Cys
Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp 210 215
220 Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu
225 230 235 240 Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg
Gln Cys Asp 245 250 255 Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu
Val Gly Cys Val Asn 260 265 270 Val Thr Leu Cys Glu Gly Pro Asn Lys
Phe Lys Cys His Ser Gly Glu 275 280 285 Cys Ile Thr Leu Asp Lys Val
Cys Asn Met Ala Arg Asp Cys Arg Asp 290 295 300 Trp Ser Asp Glu Pro
Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp 305 310 315 320 Asn Asn
Gly Gly Cys Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr 325 330 335
Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys 340
345 350 Glu Asp Ile Asp Glu Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu
Cys 355 360 365 Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln Cys Glu Glu
Gly Phe Gln 370 375 380 Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val
Gly Ser Ile Ala Tyr 385 390 395 400 Leu Phe Phe Thr Asn Arg His Glu
Val Arg Lys Met Thr Leu Asp Arg 405 410 415 Ser Glu Tyr Thr Ser Leu
Ile Pro Asn Leu Arg Asn Val Val Ala Leu 420 425 430 Asp Thr Glu Val
Ala Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gln 435 440 445 Arg Met
Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser 450 455 460
Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala 465
470 475 480 Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Asp Ser Val
Leu Gly 485 490 495 Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg
Lys Thr Leu Phe 500 505 510 Arg Glu Asn Gly Ser Lys Pro Arg Ala Ile
Val Val Asp Pro Val His 515 520 525 Gly Phe Met Tyr Trp Thr Asp Trp
Gly Thr Pro Ala Lys Ile Lys Lys 530 535 540 Gly Gly Leu Asn Gly Val
Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile 545 550 555 560 Gln Trp Pro
Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr 565 570 575 Trp
Val Asp Ser Lys Leu His Ser Ile Ser Ser Ile Asp Val Asn Gly 580 585
590 Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro
595 600 605 Phe Ser Leu Ala Val Phe Glu Asp Lys Val Phe Trp Thr Asp
Ile Ile 610 615 620 Asn Glu Ala Ile Phe Ser Ala Asn Arg Leu Thr Gly
Ser Asp Val Asn 625 630 635 640 Leu Leu Ala Glu Asn Leu Leu Ser Pro
Glu Asp Met Val Leu Phe His 645 650 655 Asn Leu Thr Gln Pro Arg Gly
Val Asn Trp Cys Glu Arg Thr Thr Leu 660 665 670 Ser Asn Gly Gly Cys
Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn 675 680 685 Pro His Ser
Pro Lys Phe Thr Cys Ala Cys Pro Asp Gly Met Leu Leu 690 695 700 Ala
Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala 705 710
715 720 Thr Gln Glu Thr Ser Thr Val Arg Leu Lys Val Ser Ser Thr Ala
Val 725 730 735 Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr
Ser Arg Leu 740 745 750 Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu
Ile Val Thr Met Ser 755 760 765 His Gln Ala Leu Gly Asp Val Ala Gly
Arg Gly Asn Glu Lys Lys Pro 770 775 780 Ser Ser Val Arg Ala Leu Ser
Ile Val Leu Pro Ile Val Leu Leu Val 785 790 795 800 Phe Leu Cys Leu
Gly Val Phe Leu Leu Trp Lys Asn Trp Arg Leu Lys 805 810 815 Asn Ile
Asn Ser Ile Asn Phe Asp Asn Pro Val Tyr Gln Lys Thr Thr 820 825 830
Glu Asp Glu Val His Ile Cys His Asn Gln Asp Gly Tyr Ser Tyr Pro 835
840 845 Ser Arg Gln Met Val Ser Leu Glu Asp Asp Val Ala 850 855 860
2767PRTHomo sapiens 2Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe
Gln Cys Gln Asp Gly 1 5 10 15 Lys Cys Ile Ser Tyr Lys Trp Val Cys
Asp Gly Ser Ala Glu Cys Gln 20 25 30 Asp Gly Ser Asp Glu Ser Gln
Glu Thr Cys Leu Ser Val Thr Cys Lys 35 40 45 Ser Gly Asp Phe Ser
Cys Gly Gly Arg Val Asn Arg Cys Ile Pro Gln 50 55 60 Phe Trp Arg
Cys Asp Gly Gln Val Asp Cys Asp Asn Gly Ser Asp Glu 65 70 75 80 Gln
Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp Glu Phe Arg Cys His 85 90
95 Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys Asp Ser Asp Arg Asp
100 105 110 Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro Val Leu Thr
Cys Gly 115 120 125 Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys Ile
Pro Gln Leu Trp 130 135 140 Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp
Gly Ser Asp Glu Trp Pro 145 150 155 160 Gln Arg Cys Arg Gly Leu Tyr
Val Phe Gln Gly Asp Ser Ser Pro Cys 165 170 175 Ser Ala Phe Glu Phe
His Cys Leu Ser Gly Glu Cys Ile His Ser Ser 180 185 190 Trp Arg Cys
Asp Gly Gly Pro Asp Cys Lys Asp Lys Ser Asp Glu Glu 195 200 205 Asn
Cys Ala Val Ala Thr Cys Arg Pro Asp Glu Phe Gln Cys Ser Asp 210 215
220 Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp Arg Glu Tyr Asp Cys
225 230 235 240 Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn Val Thr
Leu Cys Glu 245 250 255 Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu
Cys Ile Thr Leu Asp 260 265 270 Lys Val Cys Asn Met Ala Arg Asp Cys
Arg Asp Trp Ser Asp Glu Pro 275 280 285 Ile Lys Glu Cys Gly Thr Asn
Glu Cys Leu Asp Asn Asn Gly Gly Cys 290 295 300 Ser His Val Cys Asn
Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro 305 310 315 320 Asp Gly
Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu 325 330 335
Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val Asn Leu Glu Gly 340
345 350 Gly Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His
Thr 355 360 365 Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr Leu Phe
Phe Thr Asn 370 375 380 Arg His Glu Val Arg Lys Met Thr Leu Asp Arg
Ser Glu Tyr Thr Ser 385 390 395 400 Leu Ile Pro Asn Leu Arg Asn Val
Val Ala Leu Asp Thr Glu Val Ala 405 410 415 Ser Asn Arg Ile Tyr Trp
Ser Asp Leu Ser Gln Arg Met Ile Cys Ser 420 425 430 Thr Gln Leu Asp
Arg Ala His Gly Val Ser Ser Tyr Asp Thr Val Ile 435 440 445 Ser Arg
Asp Ile Gln Ala Pro Asp Gly Leu Ala Val Asp Trp Ile His 450 455 460
Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly Thr Val Ser Val Ala 465
470 475 480 Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe Arg Glu Asn
Gly Ser 485 490 495 Lys Pro Arg Ala Ile Val Val Asp Pro Val His Gly
Phe Met Tyr Trp 500 505 510 Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys
Lys Gly Gly Leu Asn Gly 515 520 525 Val Asp Ile Tyr Ser Leu Val Thr
Glu Asn Ile Gln Trp Pro Asn Gly 530 535 540 Ile Thr Leu Asp Leu Leu
Ser Gly Arg Leu Tyr Trp Val Asp Ser Lys 545 550 555 560 Leu His Ser
Ile Ser Ser Ile Asp Val Asn Gly Gly Asn Arg Lys Thr 565 570 575 Ile
Leu Glu Asp Glu Lys Arg Leu Ala His Pro Phe Ser Leu Ala Val 580 585
590 Phe Glu Asp Lys Val Phe Trp Thr Asp Ile Ile Asn Glu Ala Ile Phe
595 600 605 Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn Leu Leu Ala
Glu Asn 610 615 620 Leu Leu Ser Pro Glu Asp Met Val Leu Phe His Asn
Leu Thr Gln Pro 625 630 635 640 Arg Gly Val Asn Trp Cys Glu Arg Thr
Thr Leu Ser Asn Gly Gly Cys 645 650 655 Gln Tyr Leu Cys Leu Pro Ala
Pro Gln Ile Asn Pro His Ser Pro Lys 660 665 670 Phe Thr Cys Ala Cys
Pro Asp Gly Met Leu Leu Ala Arg Asp Met Arg 675 680 685 Ser Cys Leu
Thr Glu Ala Glu Ala Ala Val Ala Thr Gln Glu Thr Ser 690 695 700 Thr
Val Arg Leu Lys Val Ser Ser Thr Ala Val Arg Thr Gln His Thr 705 710
715 720 Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu Pro Gly Ala Thr
Pro 725 730 735 Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser His Gln
Ala Leu Gly 740 745 750 Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro
Ser Ser Val Arg 755 760 765 342PRTHomo sapiens 3Gly Thr Asn Glu Cys
Leu Asp Asn Asn Gly Gly Cys Ser His Val Cys 1 5 10 15 Asn Asp Leu
Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln 20 25 30 Leu
Val Ala Gln Arg Arg Cys Glu Asp Ile 35 40 4692PRTHomo sapiens 4Met
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 5677PRTPan troglodytes 5Met 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 Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr 35 40
45 Phe His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val
50 55 60 Val Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg
Thr Ala 65 70 75 80 Arg Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr
Leu Thr Lys Ile 85 90 95 Leu His Val Phe His Gly Leu Leu Pro Gly
Phe Leu Val Lys Met Ser 100 105 110 Gly Asp Leu Leu Glu Leu Ala Leu
Lys Leu Pro His Val Asp Tyr Ile 115 120 125 Glu Glu Asp Ser Ser Val
Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu 130 135 140 Arg Ile Thr Pro
Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp 145 150 155 160 Gly
Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser 165 170
175 Asp His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn
180 185 190 Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser
Lys Cys 195 200 205 Asp Ser His Gly Thr His Leu Ala Gly Val Val Ser
Gly Arg Asp Ala 210 215 220 Gly Val Ala Lys Gly Ala Ser Met Arg Ser
Leu Arg Val Leu Asn Cys 225 230 235 240 Gln Gly Lys Gly Thr Val Ser
Gly Thr Leu Ile Gly Leu Glu Phe Ile 245 250 255 Arg Lys Ser Gln Leu
Val Gln Pro Val Gly Pro Leu Val Val Leu Leu 260 265 270 Pro Leu Ala
Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg 275 280 285 Leu
Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg 290 295
300 Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr
305 310 315 320 Val Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu
Gly Thr Leu 325 330 335 Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe
Ala Pro Gly Glu Asp 340 345 350 Ile Ile Gly Ala Ser Ser Asp Cys Ser
Thr Cys Phe Val Ser Gln Ser 355 360 365 Gly Thr Ser Gln Ala Ala Ala
His Val Ala Gly Ile Ala Ala Met Met 370 375 380 Leu Ser Ala Glu Pro
Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu 385 390 395 400 Ile His
Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu 405 410 415
Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser 420
425 430 Thr His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser
Ala 435 440 445 His Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg
Cys Ala Pro 450 455 460 Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser
Arg Ser Gly Lys Arg 465 470 475 480 Arg Gly Glu Arg Met Glu Ala Gln
Gly Gly Lys Leu Val Cys Arg Ala 485 490 495 His Asn Ala Phe Gly Gly
Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys 500 505 510 Leu Leu Pro Gln
Ala Asn Cys Ser Ile His Thr Ala Pro Pro Ala Glu 515 520 525 Ala Gly
Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu 530 535 540
Thr Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys 545
550 555 560 Pro Pro Met Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val
Gly His 565 570 575 Arg Glu Ala Ser Ile His Ala Ser Cys Cys Arg Ala
Pro Gly Leu Glu 580 585 590 Cys Lys Val Lys Glu His Gly Ile Pro Ala
Pro Gln Glu Gln Val Thr 595 600 605 Val Ala Cys Glu Glu Gly Trp Thr
Leu Thr Gly Cys Ser Ala Leu Pro 610 615 620 Gly Thr Ser His Val Leu
Gly Ala Tyr Ala Val Asp Asn Thr Cys Val 625 630 635 640 Val Arg Ser
Arg Asp Val Ser Thr Ala Gly Ser Thr Ser Glu Glu Ala 645 650 655 Val
Ala Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala 660 665
670 Ser Gln Glu Leu Gln 675 6694PRTMus musculus 6Met Gly Thr His
Cys Ser Ala Trp Leu Arg Trp Pro Leu Leu Pro Leu 1 5 10 15 Leu Pro
Pro Leu Leu Leu Leu Leu Leu Leu Leu Cys Pro Thr Gly Ala 20 25 30
Gly Ala Gln Asp Glu Asp Gly Asp Tyr Glu Glu Leu Met Leu Ala Leu 35
40 45 Pro Ser Gln Glu Asp Gly Leu Ala Asp Glu Ala Ala His Val Ala
Thr 50 55 60 Ala Thr Phe Arg Arg Cys Ser Lys Glu Ala Trp Arg Leu
Pro Gly Thr 65 70 75 80 Tyr Ile Val Val Leu Met Glu Glu Thr Gln Arg
Leu Gln Ile Glu Gln 85 90 95 Thr Ala His Arg Leu Gln Thr Arg Ala
Ala Arg Arg Gly Tyr Val Ile 100 105 110 Lys Val Leu His Ile Phe Tyr
Asp Leu Phe Pro Gly Phe Leu Val Lys 115 120 125 Met Ser Ser Asp Leu
Leu Gly Leu Ala Leu Lys Leu Pro His Val Glu 130 135 140 Tyr Ile Glu
Glu Asp Ser Phe Val Phe Ala Gln Ser Ile Pro Trp Asn 145 150 155 160
Leu Glu Arg Ile Ile Pro Ala Trp His Gln Thr Glu Glu Asp Arg Ser 165
170 175 Pro Asp Gly Ser Ser Gln Val Glu Val Tyr Leu Leu Asp Thr Ser
Ile 180 185 190 Gln Gly Ala His Arg Glu Ile Glu Gly Arg Val Thr Ile
Thr Asp Phe 195 200 205 Asn Ser Val Pro Glu Glu Asp Gly Thr Arg Phe
His Arg Gln Ala Ser 210 215 220 Lys Cys Asp Ser His Gly Thr His Leu
Ala Gly Val Val Ser Gly Arg 225 230 235 240 Asp Ala Gly Val Ala Lys
Gly Thr Ser Leu His Ser Leu Arg Val Leu 245 250 255 Asn Cys Gln Gly
Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu 260 265 270 Phe Ile
Arg Lys Ser Gln Leu Ile Gln Pro Ser Gly Pro Leu Val Val 275 280 285
Leu Leu Pro Leu Ala Gly Gly Tyr Ser Arg Ile Leu Asn Ala Ala Cys 290
295 300 Arg His Leu Ala Arg Thr Gly Val Val Leu Val Ala Ala Ala Gly
Asn 305 310 315 320 Phe Arg Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser
Ala Pro Glu Val 325 330 335 Ile Thr Val Gly Ala Thr Asn Ala Gln Asp
Gln Pro Val Thr Leu Gly 340 345 350 Thr Leu Gly Thr Asn Phe Gly Arg
Cys Val Asp Leu Phe Ala Pro Gly 355 360 365 Lys Asp Ile Ile Gly Ala
Ser Ser Asp Cys Ser Thr Cys Phe Met Ser 370 375 380 Gln Ser Gly Thr
Ser Gln Ala Ala Ala His Val Ala Gly Ile Val Ala 385 390 395 400 Arg
Met Leu Ser Arg Glu Pro Thr Leu Thr Leu Ala Glu Leu Arg Gln 405 410
415 Arg Leu Ile His Phe Ser Thr Lys Asp Val Ile Asn Met Ala Trp Phe
420 425 430 Pro Glu Asp Gln Gln Val Leu Thr Pro Asn Leu Val Ala Thr
Leu Pro 435 440 445 Pro Ser Thr His Glu Thr Gly Gly Gln Leu Leu Cys
Arg Thr Val Trp 450 455 460 Ser Ala His Ser Gly Pro Thr Arg Thr Ala
Thr Ala Thr Ala Arg Cys 465 470 475 480 Ala Pro Glu Glu Glu Leu Leu
Ser Cys Ser Ser Phe Ser Arg Ser Gly 485 490 495 Arg Arg Arg Gly Asp
Trp Ile Glu Ala Ile Gly Gly Gln Gln Val Cys 500 505 510 Lys Ala Leu
Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Val Ala Arg 515 520 525 Cys
Cys Leu Val Pro Arg Ala Asn Cys Ser Ile His Asn Thr Pro Ala 530 535
540 Ala Arg Ala Gly Leu Glu Thr His Val His Cys His Gln Lys Asp His
545 550 555 560 Val Leu Thr Gly Cys Ser Phe His Trp Glu Val Glu Asp
Leu Ser Val 565 570 575 Arg Arg Gln Pro Ala Leu Arg Ser Arg Arg Gln
Pro Gly Gln Cys Val 580 585 590 Gly His Gln Ala Ala Ser Val Tyr Ala
Ser Cys Cys His Ala Pro Gly 595 600 605 Leu Glu Cys Lys Ile Lys Glu
His Gly Ile Ser Gly Pro Ser Glu Gln 610 615 620 Val Thr Val Ala Cys
Glu Ala Gly Trp Thr Leu Thr Gly Cys Asn Val 625 630 635 640 Leu Pro
Gly Ala Ser Leu Thr Leu Gly Ala Tyr Ser Val Asp Asn Leu 645 650 655
Cys Val Ala Arg Val His Asp Thr Ala Arg Ala Asp Arg Thr Ser Gly 660
665 670 Glu Ala Thr Val Ala Ala Ala Ile Cys Cys Arg Ser Arg Pro Ser
Ala 675 680 685 Lys Ala Ser Trp Val Gln 690 7691PRTRattus
norvegicus 7Met Gly Ile Arg Cys Ser Thr Trp Leu Arg Trp Pro Leu Ser
Pro Gln 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Cys Pro Thr Gly Ser
Arg Ala Gln Asp 20 25 30 Glu Asp Gly Asp Tyr Glu Glu Leu Met Leu
Ala Leu Pro Ser Gln Glu 35 40 45 Asp Ser Leu Val Asp Glu Ala Ser
His Val Ala Thr Ala Thr Phe Arg 50 55 60 Arg Cys Ser Lys Glu Ala
Trp Arg Leu Pro Gly Thr Tyr Val Val Val 65 70 75 80 Leu Met Glu Glu
Thr Gln Arg Leu Gln Val Glu Gln Thr Ala His Arg 85 90 95 Leu Gln
Thr Trp Ala Ala Arg Arg Gly Tyr Val Ile Lys Val Leu His 100 105 110
Val Phe Tyr Asp Leu Phe Pro Gly Phe Leu Val Lys Met Ser Ser Asp 115
120 125 Leu Leu Gly Leu Ala Leu Lys Leu Pro His Val Glu Tyr Ile Glu
Glu 130 135 140 Asp Ser Leu Val Phe Ala Gln Ser Ile Pro Trp Asn Leu
Glu Arg Ile 145 150 155 160 Ile Pro Ala Trp Gln Gln Thr Glu Glu Asp
Ser Ser Pro Asp Gly Ser 165 170 175 Ser Gln Val Glu Val Tyr Leu Leu
Asp Thr Ser Ile Gln Ser Gly His 180 185 190 Arg Glu Ile Glu Gly Arg
Val Thr Ile Thr Asp Phe Asn Ser Val Pro 195 200 205 Glu Glu Asp Gly
Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp Ser 210 215 220 His Gly
Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly Val 225 230 235
240 Ala Lys Gly Thr Ser Leu His Ser Leu Arg Val Leu Asn Cys Gln Gly
245 250 255 Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile
Arg Lys 260 265 270 Ser Gln Leu Ile Gln Pro Ser Gly Pro Leu Val Val
Leu Leu Pro Leu 275 280 285 Ala Gly Gly Tyr Ser Arg Ile Leu Asn Thr
Ala Cys Gln Arg Leu Ala 290 295 300 Arg Thr Gly Val Val Leu Val Ala
Ala Ala Gly Asn Phe Arg Asp Asp 305 310 315 320 Ala Cys Leu Tyr Ser
Pro Ala Ser Ala Pro Glu Val Ile Thr Val Gly 325 330 335 Ala Thr Asn
Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly Thr 340 345 350 Asn
Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Lys Asp Ile Ile 355 360
365 Gly Ala Ser Ser Asp Cys Ser Thr Cys Tyr Met Ser Gln Ser Gly Thr
370 375 380 Ser Gln Ala Ala Ala His Val Ala Gly Ile Val Ala Met Met
Leu Asn 385 390 395 400 Arg Asp Pro Ala Leu Thr Leu Ala Glu Leu Arg
Gln Arg Leu Ile Leu 405 410 415 Phe Ser Thr Lys Asp Val Ile Asn Met
Ala Trp Phe Pro Glu Asp Gln 420 425 430 Arg Val Leu Thr Pro Asn Arg
Val Ala Thr Leu Pro Pro Ser Thr Gln 435 440 445 Glu Thr Gly Gly Gln
Leu Leu Cys Arg Thr Val Trp Ser Ala His Ser 450 455 460 Gly Pro Thr
Arg Thr Ala Thr Ala Thr Ala Arg Cys Ala Pro Glu Glu 465 470 475 480
Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Arg Arg Arg Gly 485
490 495 Asp Arg Ile Glu Ala Ile Gly Gly Gln Gln Val Cys Lys Ala Leu
Asn 500 505 510 Ala Phe Gly Gly Glu Gly Val Tyr Ala Val Ala Arg Cys
Cys Leu Leu 515 520 525 Pro Arg Val Asn Cys Ser Ile His Asn Thr Pro
Ala Ala Arg Ala Gly 530 535 540 Pro Gln Thr Pro Val His Cys His Gln
Lys Asp His Val Leu Thr Gly 545 550 555 560 Cys Ser Phe His Trp Glu
Val Glu Asn Leu Arg Ala Gln Gln Gln Pro 565 570 575 Leu Leu Arg Ser
Arg His Gln Pro Gly Gln Cys Val Gly His Gln Glu 580 585 590 Ala Ser
Val His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys Lys 595 600 605
Ile Lys Glu His Gly Ile Ala Gly Pro Ala Glu Gln Val Thr Val Ala 610
615 620 Cys Glu Ala Gly Trp Thr Leu Thr Gly Cys Asn Val Leu Pro Gly
Ala 625 630 635 640 Ser Leu Pro Leu Gly Ala Tyr Ser Val Asp Asn Val
Cys Val Ala Arg 645 650 655 Ile Arg Asp Ala Gly Arg Ala Asp Arg Thr
Ser Glu Glu Ala Thr Val 660 665 670 Ala Ala Ala Ile Cys Cys Arg Ser
Arg Pro Ser Ala Lys Ala Ser Trp 675 680 685 Val His Gln 690
812PRTHomo sapiens 8Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg
1 5 10 913PRTHomo sapiens 9Ser Arg Ser Gly Lys Arg Arg Gly Glu Arg
Met Glu Ala 1 5 10 10118PRTMus musculus 10Glu Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Thr Leu
Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met
His Trp Val Asn Gln Arg Pro Glu Gln Gly Leu Val Trp Ile 35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly His Thr Glu Tyr Asp Pro Lys Phe 50
55 60 Gln Asp Lys Ala Thr Ile Thr Thr Asp Thr Ser Ser Asn Thr Ala
Tyr 65 70 75 80 Leu His Leu Ser Ser Leu Thr Ser Gly Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Tyr Phe Gly Ser Ile Phe Ala Tyr
Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115
1110PRTMus musculus 11Gly Phe Asn Ile Lys Asp Thr Tyr Met His 1 5
10 1217PRTMus musculus 12Arg Ile Asp Pro Ala Asn Gly His Thr Glu
Tyr Asp Pro
Lys Phe Gln 1 5 10 15 Asp 139PRTMus musculus 13Ser Tyr Phe Gly Ser
Ile Phe Ala Tyr 1 5 14108PRTMus musculus 14Gln Ile Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Leu 20 25 30 Tyr Trp
Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Phe 35 40 45
Arg Ser Ser His Arg Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50
55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala
Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr Gln Ser Tyr
Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Ala 100 105 1510PRTMus musculus 15Ser Ala Ser Ser Ser Val Ser Tyr
Leu Tyr 1 5 10 167PRTMus musculus 16Arg Ser Ser His Arg Ala Ser 1 5
179PRTMus musculus 17His Gln Tyr Gln Ser Tyr Pro Pro Thr 1 5
18118PRTMus musculus 18Glu Val Gln Leu Gln Gln Ser Gly Ala Asp Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro
Ala Asn Gly His Thr Glu Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Arg
Ala Thr Leu Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Leu Phe Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90
95 Ala Arg Ser Tyr Tyr Gly Ser Ile Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ala 115 1910PRTMus musculus 19Gly
Phe Asn Ile Lys Asp Thr Tyr Ile His 1 5 10 2017PRTMus musculus
20Arg Ile Asp Pro Ala Asn Gly His Thr Glu Tyr Asp Pro Lys Phe Gln 1
5 10 15 Gly 219PRTMus musculus 21Ser Tyr Tyr Gly Ser Ile Phe Ala
Tyr 1 5 22108PRTMus musculus 22Gln Ile Val Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Ser Cys
Ser Ala Ser Ser Ser Val Ser Tyr Leu 20 25 30 Phe Trp Tyr Gln Gln
Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Phe 35 40 45 Arg Thr Ser
Tyr Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Phe Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Thr Tyr Pro Pro
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100
105 2310PRTMus musculus 23Ser Ala Ser Ser Ser Val Ser Tyr Leu Phe 1
5 10 247PRTMus musculus 24Arg Thr Ser Tyr Leu Ala Ser 1 5 259PRTMus
musculus 25His Gln Tyr His Thr Tyr Pro Pro Thr 1 5 26121PRTMus
musculus 26Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser
Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn
Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly
Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met
Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser
Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala
Tyr Tyr Arg Tyr Asp Asp Gly Thr Trp Phe Pro Tyr Trp Gly 100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 2710PRTMus musculus
27Gly Phe Asn Ile Lys Asp Tyr Tyr Ile His 1 5 10 2817PRTMus
musculus 28Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys
Phe Gln 1 5 10 15 Gly 2912PRTMus musculus 29Tyr Tyr Arg Tyr Asp Asp
Gly Thr Trp Phe Pro Tyr 1 5 10 30109PRTMus musculus 30Asp Ile Gln
Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu
Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Phe Leu Val
35 40 45 Asp Asn Ala Lys Thr Leu Pro Asp Gly Val Pro Ser Arg Phe
Ser Val 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His
Phe Trp Asn Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Arg Ala 100 105 3111PRTMus musculus 31Arg Ala Ser Gly
Asn Ile His Ser Tyr Leu Ala 1 5 10 327PRTMus musculus 32Asn Ala Lys
Thr Leu Pro Asp 1 5 339PRTMus musculus 33Gln His Phe Trp Asn Thr
Pro Trp Thr 1 5 34119PRTMus musculus 34Glu Val Leu Leu Gln Gln Ser
Val Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Arg Leu Ser
Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His
Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp Phe 35 40 45 Gly
Trp Ile Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Ala Pro Asn Phe 50 55
60 Gln Gly Lys Ala Thr Leu Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80 Leu His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr
Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Arg Tyr Tyr Ser Leu Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115
3510PRTMus musculus 35Gly Phe Asn Ile Lys Asp Thr Tyr Ile His 1 5
10 3617PRTMus musculus 36Trp Ile Asp Pro Ala Asn Gly Tyr Thr Lys
Tyr Ala Pro Asn Phe Gln 1 5 10 15 Gly 3710PRTMus musculus 37Gly Tyr
Tyr Arg Tyr Tyr Ser Leu Asp Tyr 1 5 10 38109PRTMus musculus 38Asp
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 Arg Ala Ser Gln Asp 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 Ser Ser Arg Leu His Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp Tyr Ser Leu Thr
Ile Ser Thr Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys
Gln Gln Gly Lys Thr Leu Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys Arg Ala 100 105 3911PRTMus musculus 39Arg Ala
Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 407PRTMus musculus 40Tyr
Ser Ser Arg Leu His Ser 1 5 419PRTMus musculus 41Gln Gln Gly Lys
Thr Leu Pro Leu Thr 1 5 42126PRTMus musculus 42Glu Val Gln Leu Val
Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn His 20 25 30 Asp
Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val 35 40
45 Ala Ser Ile Thr Pro Ser Gly Gly Thr Thr Tyr Tyr Arg Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Val Lys Ser Ser
Leu His 65 70 75 80 Leu Gln Met Asp Ser Leu Thr Ser Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Gln Asn Tyr Tyr Asp Gly Ser Tyr
Tyr Tyr Gly Leu Tyr Tyr 100 105 110 Phe Asp Tyr Trp Gly Gln Gly Val
Met Val Thr Val Ser Ser 115 120 125 4310PRTMus musculus 43Gly Phe
Thr Phe Ser Asn His Asp Met Ala 1 5 10 4417PRTMus musculus 44Ser
Ile Thr Pro Ser Gly Gly Thr Thr Tyr Tyr Arg Asp Ser Val Glu 1 5 10
15 Gly 4517PRTMus musculus 45Gln Asn Tyr Tyr Asp Gly Ser Tyr Tyr
Tyr Gly Leu Tyr Tyr Phe Asp 1 5 10 15 Tyr 46114PRTMus musculus
46Asp Val Leu Met Thr Gln Thr Pro Val Ser Leu Pro Val Ser Leu Gly 1
5 10 15 Gly Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr
Ser 20 25 30 Asp Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Pro Glu Asp
Leu Gly Leu Tyr Tyr Cys Leu Gln Ser 85 90 95 Thr His Phe Pro Pro
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala
4716PRTMus musculus 47Arg Ser Ser Gln Ser Leu Val Tyr Ser Asp Gly
Asn Thr Tyr Leu His 1 5 10 15 487PRTMus musculus 48Arg Val Ser Asn
Arg Phe Ser 1 5 499PRTMus musculus 49Leu Gln Ser Thr His Phe Pro
Pro Thr 1 5 50119PRTMus musculus 50Glu 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 Val 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 Ala Ile Tyr Asn Gln Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ile 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 Thr Ser Gly Ile Ile Thr Glu Ile Ala Glu Asp Phe
Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115
5110PRTMus musculus 51Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Asn 1 5
10 5217PRTMus musculus 52Asp Ile Asn Pro Asn Asn Gly Gly Ala Ile
Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 5310PRTMus musculus 53Gly Ile
Ile Thr Glu Ile Ala Glu Asp Phe 1 5 10 54109PRTMus musculus 54Asp
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 Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala
Leu Ile 35 40 45 His Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp
Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Thr Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Gly Phe Phe Cys
Gln Gln Tyr Lys Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Gln Leu Glu Ile Lys Arg Ala 100 105 5511PRTMus musculus 55Lys Ala
Ser Gln Asn Val Gly Thr Asn Val Val 1 5 10 567PRTMus musculus 56Ser
Ala Ser Tyr Arg Tyr Ser 1 5 579PRTMus musculus 57Gln Gln Tyr Lys
Thr Tyr Pro Tyr Thr 1 5
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