U.S. patent application number 14/981853 was filed with the patent office on 2016-12-08 for anti-pcsk9 antibodies, formulations, dosing, and methods of use.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Monica Kong Beltran, Nageshwar R. Budha, Cecilia Pui Chi Chiu, John Douglas Davis, Ada Hui, Daniel K. Kirchhofer, Ganesh A. Kolumam, Xanthe Lam, Wei Li, Lin Luis, Paul Moran, Andrew Peterson, Whittemore Tingley, Yan Wu.
Application Number | 20160355606 14/981853 |
Document ID | / |
Family ID | 48746111 |
Filed Date | 2016-12-08 |
United States Patent
Application |
20160355606 |
Kind Code |
A1 |
Wu; Yan ; et al. |
December 8, 2016 |
ANTI-PCSK9 ANTIBODIES, FORMULATIONS, DOSING, AND METHODS OF USE
Abstract
The invention provides anti-PCSK9 antibodies, formulations,
dosing regimens, and methods of using the same.
Inventors: |
Wu; Yan; (South San
Francisco, CA) ; Chiu; Cecilia Pui Chi; (South San
Francisco, CA) ; Kirchhofer; Daniel K.; (South San
Francisco, CA) ; Peterson; Andrew; (South San
Francisco, CA) ; Kolumam; Ganesh A.; (South San
Francisco, CA) ; Beltran; Monica Kong; (South San
Francisco, CA) ; Moran; Paul; (South San Francisco,
CA) ; Li; Wei; (South San Francisco, CA) ;
Lam; Xanthe; (South San Francisco, CA) ; Luis;
Lin; (South San Francisco, CA) ; Hui; Ada;
(South San Francisco, CA) ; Tingley; Whittemore;
(South San Francisco, CA) ; Davis; John Douglas;
(South San Francisco, CA) ; Budha; Nageshwar R.;
(South San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
48746111 |
Appl. No.: |
14/981853 |
Filed: |
December 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13918755 |
Jun 14, 2013 |
9266961 |
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14981853 |
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61786280 |
Mar 14, 2013 |
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61660605 |
Jun 15, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/34 20130101; C07K 2317/94 20130101; A61K 39/39591
20130101; C07K 16/40 20130101; A61P 43/00 20180101; A61K 2039/505
20130101; C07K 2317/55 20130101; C07K 2317/92 20130101; A61P 3/06
20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. An anti-PCSK9 antibody comprising a heavy chain and light chain
variable domain comprising six hypervariable region (HVR)
sequences: (i) HVR-H1 comprising GFTFX.sub.1X.sub.2X.sub.3X.sub.4IH
(SEQ ID NO:28), wherein X.sub.1 is S or T; X.sub.2 is G, R or S;
X.sub.3 is H, T or Y; X.sub.4 is A or T; (ii) HVR-H2 comprising
RISPANGNTNYADSVKG (SEQ ID NO:4); (iii) HVR-H3 comprising
WIGSRELYIMDY (SEQ ID NO:5); (iv) HVR-L1 comprising
RASQDVSX.sub.1AVA (SEQ ID NO:29), wherein X.sub.1 is S or T; (v)
HVR-L2 comprising SASX.sub.1LYS (SEQ ID NO:30), wherein X.sub.1 is
F or S; and (vi) HVR-L3 comprising
QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37), wherein X.sub.1
is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is L, P or Q;
X.sub.4 is A, H, P or S.
2-100. (canceled)
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 13/918,755, filed Jun. 14, 2013, which claims the priority
benefit of U.S. Provisional Application Ser. No. 61/660,605, filed
Jun. 15, 2012, and 61/786,280, filed Mar. 14, 2013, the entire
disclosures of which are expressly incorporated by reference herein
in their entireties.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392014510SEQLIST.txt, date recorded: Jun. 13, 2013, size: 39
KB).
FIELD OF THE INVENTION
[0003] The present invention relates to anti-PCSK9 antibodies,
antibody formulations, dosing regimens, and methods of using the
same.
BACKGROUND OF THE INVENTION
[0004] Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a
member of the mammalian subtilisin family of proprotein
convertases. PCSK9 plays a critical role in cholesterol metabolism
by controlling the levels of low density lipoprotein (LDL)
particles that circulate in the bloodstream. Elevated levels of
PCSK9 have been shown to reduce LDL-receptor levels in the liver,
resulting in high levels of LDL-cholesterol in the plasma and
increased susceptibility to coronary artery disease. (Peterson et
al., J Lipid Res. 49(7):1595-9 (2008)). Therefore, it would be
highly advantageous to produce a therapeutic-based antagonist of
PCSK9 that inhibits or antagonizes the activity of PCSK9 and the
corresponding role PCSK9 plays in various therapeutic
conditions.
SUMMARY OF THE INVENTION
[0005] The invention is in part based on a variety of antibodies to
PCSK9. PCSK9 presents as an important and advantageous therapeutic
target, and the invention provides antibodies as therapeutic and
diagnostic agents for use in targeting pathological conditions
associated with expression and/or activity of PCSK9. Accordingly,
the invention provides methods, compositions, kits and articles of
manufacture related to PCSK9.
[0006] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a variable domain comprising at least one, two,
three, four, five or six hypervariable region (HVR) sequences
selected from the group consisting of:
[0007] (i) HVR-H1 comprising GFTFX.sub.1X.sub.2X.sub.3X.sub.4IH
(SEQ ID NO: 28), wherein X.sub.1 is S or T; X.sub.2 is G, R or S;
X.sub.3 is H, T or Y; X.sub.4 is A or T;
[0008] (ii) HVR-H2 comprising RISPANGNTNYADSVKG (SEQ ID NO:4);
[0009] (iii) HVR-H3 comprising WIGSRELYIMDY (SEQ ID NO:5);
[0010] (iv) HVR-L1 comprising RASQDVSX.sub.1AVA (SEQ ID NO:29),
wherein X.sub.1 is S or T;
[0011] (v) HVR-L2 comprising SASX.sub.1LYS (SEQ ID NO:30), wherein
X.sub.1 is F or S; and
[0012] (vi) HVR-L3 comprising QQSYX.sub.1X.sub.2X.sub.3X.sub.4T
(SEQ ID NO:31) or QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37),
wherein X.sub.1 is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is
L, P or Q; X.sub.4 is A, H, P or S.
[0013] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a variable domain comprising the following six
HVR sequences:
[0014] (i) HVR-H1 comprising GFTFX.sub.1X.sub.2X.sub.3X.sub.4IH
(SEQ ID NO:28), wherein X.sub.1 is S or T; X.sub.2 is G, R or S;
X.sub.3 is H, T or Y; X.sub.4 is A or T;
[0015] (ii) HVR-H2 comprising RISPANGNTNYADSVKG (SEQ ID NO:4);
[0016] (iii) HVR-H3 comprising WIGSRELYIMDY (SEQ ID NO:5);
[0017] (iv) HVR-L1 comprising RASQDVSX.sub.1AVA (SEQ ID NO:29),
wherein X.sub.1 is S or T;
[0018] (v) HVR-L2 comprising SASX.sub.1LYS (SEQ ID NO:30), wherein
X.sub.1 is F or S; and
[0019] (vi) HVR-L3 comprising QQSYX.sub.1X.sub.2X.sub.3X.sub.4T
(SEQ ID NO:31) or QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37),
wherein X.sub.1 is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is
L, P or Q; X.sub.4 is A, H, P or S.
[0020] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a variable domain comprising at least one, two,
three, four, five or six hypervariable region (HVR) sequences
selected from the group consisting of:
[0021] (i) HVR-H1 comprising
GFTFX.sub.1X.sub.2X.sub.3X.sub.4IX.sub.5 (SEQ ID NO: 45), wherein
X.sub.1 is S or T; X.sub.2 is G, R or S; X.sub.3 is H, T or Y;
X.sub.4 is A or T; X.sub.5 is H or N;
[0022] (ii) HVR-H2 comprising RISPANGNTNYADSVKG (SEQ ID NO:4);
[0023] (iii) HVR-H3 comprising WIGSRELYIMDY (SEQ ID NO:5);
[0024] (iv) HVR-L1 comprising RASQDVSX.sub.1AVA (SEQ ID NO:29),
wherein X.sub.1 is S or T;
[0025] (v) HVR-L2 comprising SASX.sub.1LYS (SEQ ID NO:30), wherein
X.sub.1 is F or S; and
[0026] (vi) HVR-L3 comprising QQSYX.sub.1X.sub.2X.sub.3X.sub.4T
(SEQ ID NO:31) or QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37),
wherein X.sub.1 is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is
L, P or Q; X.sub.4 is A, H, P or S.
[0027] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a variable domain comprising the following six
HVR sequences:
[0028] (i) HVR-H1 comprising
GFTFX.sub.1X.sub.2X.sub.3X.sub.4IX.sub.5 (SEQ ID NO: 45), wherein
X.sub.1 is S or T; X.sub.2 is G, R or S; X.sub.3 is H, T or Y;
X.sub.4 is A or T; X.sub.5 is H or N;
[0029] (ii) HVR-H2 comprising RISPANGNTNYADSVKG (SEQ ID NO:4);
[0030] (iii) HVR-H3 comprising WIGSRELYIMDY (SEQ ID NO:5);
[0031] (iv) HVR-L1 comprising RASQDVSX.sub.1AVA (SEQ ID NO:29),
wherein X.sub.1 is S or T;
[0032] (v) HVR-L2 comprising SASX.sub.1LYS (SEQ ID NO:30), wherein
X.sub.1 is F or S; and
[0033] (vi) HVR-L3 comprising QQSYX.sub.1X.sub.2X.sub.3X.sub.4T
(SEQ ID NO:31) or QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37),
wherein X.sub.1 is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is
L, P or Q; X.sub.4 is A, H, P or S.
[0034] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a variable domain comprising at least one, two,
three, four, five or six hypervariable region (HVR) sequences
selected from the group consisting of:
[0035] (i) HVR-H1 comprising
GFTFX.sub.1X.sub.2X.sub.3X.sub.4IX.sub.5 (SEQ ID NO: 45), wherein
X.sub.1 is S or T; X.sub.2 is G, R or S; X.sub.3 is H, T or Y;
X.sub.4 is A or T; X.sub.5 is H or N;
[0036] (ii) HVR-H2 comprising RISPANGNTNYADSVKG (SEQ ID NO:4);
[0037] (iii) HVR-H3 comprising WIGSRELYIMDY (SEQ ID NO:5);
[0038] (iv) HVR-L1 comprising RASQDVSTAVA (SEQ ID NO:7);
[0039] (v) HVR-L2 comprising SASFLYS (SEQ ID NO:8); and
[0040] (vi) HVR-L3 comprising QQSYX.sub.1X.sub.2X.sub.3X.sub.4T
(SEQ ID NO:31) or QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37),
wherein X.sub.1 is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is
L, P or Q; X.sub.4 is A, H, P or S.
[0041] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a variable domain comprising the following six
HVR sequences:
[0042] (i) HVR-H1 comprising
GFTFX.sub.1X.sub.2X.sub.3X.sub.4IX.sub.5 (SEQ ID NO: 45), wherein
X.sub.1 is S or T; X.sub.2 is G, R or S; X.sub.3 is H, T or Y;
X.sub.4 is A or T; X.sub.5 is H or N;
[0043] (ii) HVR-H2 comprising RISPANGNTNYADSVKG (SEQ ID NO:4);
[0044] (iii) HVR-H3 comprising WIGSRELYIMDY (SEQ ID NO:5);
[0045] (iv) HVR-L1 comprising RASQDVSTAVA (SEQ ID NO:7);
[0046] (v) HVR-L2 comprising SASFLYS (SEQ ID NO:8); and
[0047] (vi) HVR-L3 comprising QQSYX.sub.1X.sub.2X.sub.3X.sub.4T
(SEQ ID NO:31) or QQAYX.sub.1X.sub.2X.sub.3X.sub.4T (SEQ ID NO:37),
wherein X.sub.1 is P, R or T; X.sub.2 is A, I, S or T; X.sub.3 is
L, P or Q; X.sub.4 is A, H, P or S.
[0048] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:42, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5. In certain
embodiments, the antibody further comprises (a) HVR-L1 comprising
the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7; (b) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:26;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, or SEQ ID NO:33.
[0049] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises (a) HVR-L1 comprising the amino acid sequence of
SEQ ID NO:6 or SEQ ID NO:7; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:8 or SEQ ID NO:26; and (c) HVR-L3 comprising
the amino acid sequence of SEQ ID NO: 9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:33.
In certain embodiments, the antibody further comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, or SEQ ID NO:42, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:4, and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:5.
[0050] In one embodiment, an antibody or an antibody fragment that
binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0051] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:1;
[0052] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0053] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0054] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:6;
[0055] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:26; and
[0056] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:9.
[0057] In one embodiment, an antibody or an antibody fragment that
binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0058] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:1;
[0059] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0060] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0061] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0062] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0063] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:9.
[0064] In one embodiment, an antibody or an antibody fragment that
binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0065] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:1;
[0066] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0067] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0068] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0069] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0070] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:10.
[0071] In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises: [0072] (1) an HVR-H1 comprising the amino acid
sequence of SEQ ID NO:1; [0073] (2) an HVR-H2 comprising the amino
acid sequence of SEQ ID NO:4; [0074] (3) an HVR-H3 comprising the
amino acid sequence of SEQ ID NO:5; [0075] (4) an HVR-L1 comprising
the amino acid sequence of SEQ ID NO:7; [0076] (5) an HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and [0077] (6)
an HVR-L3 comprising the amino acid sequence of SEQ ID NO:11.
[0078] In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0079] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2;
[0080] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0081] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0082] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0083] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0084] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:12.
[0085] In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0086] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:42;
[0087] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0088] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0089] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0090] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0091] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:12.
[0092] In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0093] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:3;
[0094] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0095] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0096] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0097] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0098] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:13.
[0099] In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0100] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:3;
[0101] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0102] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0103] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0104] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0105] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:33.
[0106] In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises:
[0107] (1) an HVR-H1 comprising the amino acid sequence of SEQ ID
NO:1;
[0108] (2) an HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4;
[0109] (3) an HVR-H3 comprising the amino acid sequence of SEQ ID
NO:5;
[0110] (4) an HVR-L1 comprising the amino acid sequence of SEQ ID
NO:7;
[0111] (5) an HVR-L2 comprising the amino acid sequence of SEQ ID
NO:8; and
[0112] (6) an HVR-L3 comprising the amino acid sequence of SEQ ID
NO:14.
[0113] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises (a) a VH sequence having at least 95% sequence
identity to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:27, or SEQ ID NO:43; or (b) a VL sequence
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:34, or SEQ ID NO:44.
[0114] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a VH sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:27, or SEQ ID NO:43. In certain embodiments,
the antibody further comprises a VL sequence of SEQ ID NO: 18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:34, or SEQ ID NO:44.
[0115] In one embodiment, an antibody or an antibody fragment that
binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a VH sequence of SEQ ID NO:15 and a VL sequence
of SEQ ID NO:18. In another embodiment, an antibody or an antibody
fragment that binds to PCSK9 or a fragment thereof is provided,
wherein the antibody comprises a VH sequence of SEQ ID NO:27 and a
VL sequence of SEQ ID NO:44. In another embodiment, an antibody or
an antibody fragment that binds to PCSK9 or a fragment thereof is
provided, wherein the antibody comprises a VH sequence of SEQ ID
NO:15 and a VL sequence of SEQ ID NO:19. In another embodiment, an
antibody or an antibody fragment that binds to PCSK9 or a fragment
thereof is provided, wherein the antibody comprises a VH sequence
of SEQ ID NO:27 and a VL sequence of SEQ ID NO:19. In another
embodiment, an antibody or an antibody fragment that binds to PCSK9
or a fragment thereof is provided, wherein the antibody comprises a
VH sequence of SEQ ID NO:27 and a VL sequence of SEQ ID NO:20. In
another embodiment, an antibody or an antibody fragment that binds
to PCSK9 or a fragment thereof is provided, wherein the antibody
comprises a VH sequence of SEQ ID NO:16 and a VL sequence of SEQ ID
NO:21. In another embodiment, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody comprises a VH sequence of SEQ ID NO:43 and a VL sequence
of SEQ ID NO:21. In another embodiment, an antibody or an antibody
fragment that binds to PCSK9 or a fragment thereof is provided,
wherein the antibody comprises a VH sequence of SEQ ID NO:17 and a
VL sequence of SEQ ID NO:22. In another embodiment, an antibody or
an antibody fragment that binds to PCSK9 or a fragment thereof is
provided, wherein the antibody comprises a VH sequence of SEQ ID
NO:27 and a VL sequence of SEQ ID NO:23. In another embodiment, an
antibody or an antibody fragment that binds to PCSK9 or a fragment
thereof is provided, wherein the antibody comprises a VH sequence
of SEQ ID NO:17 and a VL sequence of SEQ ID NO:34.
[0116] In certain embodiments, an antibody or an antibody fragment
that binds to PCSK9 or a fragment thereof is provided, wherein the
antibody binds to an epitope within a fragment of PCSK9. In certain
embodiments, an antibody or an antibody fragment that binds to
PCSK9 or a fragment thereof is provided, wherein the antibody binds
to an epitope within a fragment of PCSK9 comprising amino acids 376
to 379 of human PCSK9 amino acid sequence of SEQ ID NO:24. In
certain embodiments, the functional and/or structural epitope of an
antibody according to this invention includes residue D238 of human
PCSK9. In certain embodiments, the functional and/or structural
epitope of an antibody according to this invention includes residue
A239 of human PCSK9. In certain embodiments, the functional and/or
structural epitope of an antibody according to this invention
includes residues D238 and A239 of human PCSK9. In certain
embodiments, the functional and/or structural epitope of an
antibody according to this invention includes residue E366 of human
PCSK9. In certain embodiments, the functional and/or structural
epitope of an antibody according to this invention includes residue
D367 of human PCSK9. In certain embodiments, the functional and/or
structural epitope of an antibody according to this invention
includes residues E366 and D367 of human PCSK9. In certain
embodiments, the functional and/or structural epitope of an
antibody according to this invention includes residue H391 of human
PCSK9. In certain embodiments, the functional and/or structural
epitope of an antibody according to this invention includes
residues E366, D367 and H391 of human PCSK9. According to another
embodiment, the functional and/or structural epitope of an antibody
according to this invention includes residues A239 and H391 of
human PCSK9. In certain embodiments, the functional and/or
structural epitope of includes one or more of residues A239, A341,
E366, D367 and H391 of human PCSK9. In certain embodiments, the
functional and/or structural epitope of includes one or more of
residues near A239, A341, E366, D367 and H391 of human PCSK9. In
certain embodiments, the functional and/or structural epitope of an
antibody according to this invention comprises (i) at least one
residue selected from the group consisting of R194 and E195, (ii)
at least one residue selected from the group consisting of D238 and
A239, (iii) at least one residue selected from the group consisting
of A341 and Q342, and (iv) at least one residue selected from the
group consisting of E366, D367, 1369, S376, T377, C378, F379, S381
and H391, of human PCSK9. In certain embodiments, the functional
and/or structural epitope comprises one, two, three, four, five,
six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or
all of the following residues: R194, E195, D238, A239, A341, Q342,
E366, D367, 1369, S376, T377, C378, F379, S381 and H391 of human
PCSK9.
[0117] In certain embodiments, the anti-PCSK9 antibody is a
monoclonal antibody. In certain embodiments, the anti-PCSK9
antibody is humanized. In certain embodiments, the anti-PCSK9
antibody is a human antibody. In certain embodiments, at least a
portion of the framework sequence of the anti-PCSK9 antibody is a
human consensus framework sequence. In one embodiment, the antibody
is an antibody fragment selected from a Fab, Fab'-SH, Fv, scFv, or
(Fab').sub.2 fragment.
[0118] In one aspect, a nucleic acid encoding any of the above
anti-PCSK9 antibodies is provided. In one embodiment, a vector
comprising the nucleic acid is provided. In one embodiment, the
vector is an expression vector. In one embodiment, a host cell
comprising the vector is provided. In one embodiment, the host cell
is eukaryotic. In another embodiment, the host cell is mammalian.
In yet another embodiment, the host cell is prokaryotic. In one
embodiment, a method of making an anti-PCSK9 antibody is provided,
wherein the method comprises culturing the host cell under
conditions suitable for expression of the nucleic acid encoding the
antibody, and isolating the antibody. In certain embodiment, the
method further comprises recovering the anti-PCSK9 antibody from
the host cell. In certain embodiments, a composition comprising any
of the anti-PCSK9 antibodies described herein is provided. In one
embodiment, the composition further comprises a pharmaceutically
acceptable carrier.
[0119] In one aspect, provided herein is a pharmaceutical
composition comprising an anti-PCSK9 antibody at about 100 to about
225 mg/mL, arginine succinate at about 180 to about 220 mM,
polysorbate at about 0.01% to about 0.03%, and pH at about 5.2 to
about 6.2. In certain embodiments, the anti-PCSK9 antibody or
antibody fragment in the composition is at about 150 mg/mL,
arginine succinate in the composition is at about 200 mM, and
polysorbate 20 in the composition is about 0.02%, and pH at about
5.5. In certain embodiments, the composition is suitable for
subcutaneous administration. In certain embodiments, the viscosity
of the composition is less than about 10 cP at 25.degree. C. Any
anti-PCSK9 antibodies known in the art or described herein may be
formulated into the composition.
[0120] In one aspect, provided herein is a pharmaceutical
composition comprising an anti-PCSK9 antibody at about 150 to about
225 mg/mL, histidine acetate at about 10 to about 30 mM, arginine
acetate at about 150 to about 170 mM, polysorbate at about 0.01% to
about 0.03%, and pH at about 5.8 to about 6.2. In certain
embodiments, the anti-PCSK9 antibody or antibody fragment in the
composition is at about 200 mg/mL, histidine acetate in the
composition is at about 20 mM, arginine acetate in the composition
is at about 160 mM, and polysorbate 20 in the composition is about
0.02%, and pH at about 6.0. In certain embodiments, the composition
is suitable for subcutaneous administration. In certain
embodiments, the viscosity of the composition is less than about 10
cP at 25.degree. C. Any anti-PCSK9 antibodies known in the art or
described herein may be formulated into the composition.
[0121] In one aspect, provided herein is a subcutaneous
administration device containing an anti-PCSK9 antibody or a
composition comprising an anti-PCSK9 antibody described herein. In
certain embodiments, the device is for delivering to an individual
a flat dose in the range of about 200 to about 1200 mg of the
antibody. In certain embodiments, the device is a pre-filled
syringe (e.g., 0.5-mL, 1-mL, 1.25-mL, 1.5-mL, 1.75-mL, 2-mL,
2.25-mL, or 2.5-mL syringe). In certain embodiments, the device is
a 1-mL pre-filled syringe and the antibody concentration in the
pre-filled syringe is about 200 mg/mL. In certain embodiments, the
device is a 1.5-mL pre-filled syringe and the antibody
concentration in the pre-filled syringe is about 200 mg/mL. In
certain embodiments, the device is a 2-mL pre-filled syringe and
the antibody concentration in the pre-filled syringe is about 200
mg/mL. In certain embodiments, the device is a 2.25-mL pre-filled
syringe and the antibody concentration in the pre-filled syringe is
about 200 mg/mL. In certain embodiments, the device is a 2.5-mL
pre-filled syringe and the antibody concentration in the pre-filled
syringe is about 200 mg/mL.
[0122] In one aspect, the invention concerns methods of inhibiting
binding of PCSK9 to LDL-receptor (LDLR) in a subject, said method
comprising administering to the subject an effective amount of any
of the anti-PCSK9 antibodies described herein. In another aspect,
the invention concerns methods of reducing a level of cholesterol
in a subject, said method comprising administering to the subject
an effective amount of any of the anti-PCSK9 antibodies described
herein. In one embodiment, the cholesterol is LDL-cholesterol. In
another aspect, the invention concerns methods of reducing a level
of LDL-cholesterol in a subject, said method comprising
administering to the subject an effective amount of any of the
anti-PCSK9 antibodies described herein. In certain embodiments, the
invention concerns methods of lowering serum LDL-cholesterol level
in a subject, said method comprising administering to the subject
an effective amount of any one of the anti-PCSK9 antibodies
described herein. In another aspect, the invention concerns methods
of treating a condition associated with elevated level of
LDL-cholesterol in a subject, said method comprising administering
to the subject an effective amount of any one of the anti-PCSK9
antibodies described herein.
[0123] In one aspect, the invention concerns methods of treating a
cholesterol related disorder. An exemplary and non-limiting list of
cholesterol related disorders contemplated is provided herein under
"Compositions and Methods." In certain embodiments, the cholesterol
related disorder is hypercholesterolemia. In certain embodiments,
the invention concerns methods of treating hypercholesterolemia
comprising administering to the subject an effective amount of any
one of the anti-PCSK9 antibodies described herein. In certain
embodiments, the invention concerns methods of preventing and/or
treating atherosclerosis and/or cardiovascular diseases. In certain
embodiments, the invention concerns methods of reducing the risk of
recurrent cardiovascular events in an individual comprising
administering to the individual an amount effective of any one of
the anti-PCSK9 antibodies described herein.
[0124] In one aspect, the invention concerns methods for treating
any disease or condition which can be improved, ameliorated,
inhibited or prevented by removal, inhibition or reduction of PCSK9
activity. In certain embodiments, diseases or disorders that are
either treatable or preventable through the use of statins can also
be treated using any one of the anti-PCSK9 antibodies described
herein. In certain embodiments, disorders or disease that can
benefit from the prevention of cholesterol synthesis or increased
LDLR expression can also be treated using any one of the anti-PCSK9
antibodies described herein.
[0125] In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200 mg, 220
mg, 380 mg, 400 mg, 600 mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or
1200 mg per dose every two weeks, every month, every two months, or
every three months. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
200 mg, 220 mg, 380 mg, 400 mg, 600 mg, 760 mg, 800 mg, 1000 mg,
1140 mg, or 1200 mg per dose every two weeks. In certain
embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 200 mg, 220 mg, 380 mg,
400 mg, 600 mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or 1200 mg per
dose every month. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
200 mg, 220 mg, 380 mg, 400 mg, 600 mg, 760 mg, 800 mg, 1000 mg,
1140 mg, or 1200 mg per dose every two months. In certain
embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 200 mg, 220 mg, 380 mg,
400 mg, 600 mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or 1200 mg per
dose every three months.
[0126] In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200-1200 mg,
200-1000 mg, 200-800 mg, 200-600 mg, 200-400 mg, 400-1200 mg,
400-1000 mg, 400-800 mg, 400-600 mg, 600-1200 mg, 600-1000 mg,
600-800 mg, 800-1200 mg, 800-1000 mg, 800-900 mg, 750-850 mg,
750-800 mg, 775-825 mg, 350-450 mg, 375-425 mg, or 375-400 mg per
dose every two weeks, every month, every two months, or every three
months. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200-1200 mg,
200-1000 mg, 200-800 mg, 200-600 mg, 200-400 mg, 400-1200 mg,
400-1000 mg, 400-800 mg, 400-600 mg, 600-1200 mg, 600-1000 mg,
600-800 mg, 800-1200 mg, 800-1000 mg, 800-900 mg, 750-850 mg,
750-800 mg, 775-825 mg, 350-450 mg, 375-425 mg, or 375-400 mg per
dose every two weeks. In certain embodiments of the methods
described herein, the anti-PCSK9 antibody is administered
subcutaneously at 200-1200 mg, 200-1000 mg, 200-800 mg, 200-600 mg,
200-400 mg, 400-1200 mg, 400-1000 mg, 400-800 mg, 400-600 mg,
600-1200 mg, 600-1000 mg, 600-800 mg, 800-1200 mg, 800-1000 mg,
800-900 mg, 750-850 mg, 750-800 mg, 775-825 mg, 350-450 mg, 375-425
mg, or 375-400 mg per dose every month. In certain embodiments of
the methods described herein, the anti-PCSK9 antibody is
administered subcutaneously at 200-1200 mg, 200-1000 mg, 200-800
mg, 200-600 mg, 200-400 mg, 400-1200 mg, 400-1000 mg, 400-800 mg,
400-600 mg, 600-1200 mg, 600-1000 mg, 600-800 mg, 800-1200 mg,
800-1000 mg, 800-900 mg, 750-850 mg, 750-800 mg, 775-825 mg,
350-450 mg, 375-425 mg, or 375-400 mg per dose every two months. In
certain embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 200-1200 mg, 200-1000
mg, 200-800 mg, 200-600 mg, 200-400 mg, 400-1200 mg, 400-1000 mg,
400-800 mg, 400-600 mg, 600-1200 mg, 600-1000 mg, 600-800 mg,
800-1200 mg, 800-1000 mg, 800-900 mg, 750-850 mg, 750-800 mg,
775-825 mg, 350-450 mg, 375-425 mg, or 375-400 mg per dose every
three months.
[0127] In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200 mg, 220
mg, 380 mg, 400 mg, 600 mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or
1200 mg per dose every 2 weeks, every 4 weeks, every 6 weeks, every
8 weeks, every 10 weeks, or every 12 weeks. In certain embodiments
of the methods described herein, the anti-PCSK9 antibody is
administered subcutaneously at 200 mg, 220 mg, 380 mg, 400 mg, 600
mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or 1200 mg per dose every 2
weeks. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200 mg, 220
mg, 380 mg, 400 mg, 600 mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or
1200 mg per dose every 4 weeks. In certain embodiments of the
methods described herein, the anti-PCSK9 antibody is administered
subcutaneously at 200 mg, 220 mg, 380 mg, 400 mg, 600 mg, 760 mg,
800 mg, 1000 mg, 1140 mg, or 1200 mg per dose every 6 weeks. In
certain embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 200 mg, 220 mg, 380 mg,
400 mg, 600 mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or 1200 mg per
dose every 8 weeks. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
200 mg, 220 mg, 380 mg, 400 mg, 600 mg, 760 mg, 800 mg, 1000 mg,
1140 mg, or 1200 mg per dose every 10 weeks. In certain embodiments
of the methods described herein, the anti-PCSK9 antibody is
administered subcutaneously at 200 mg, 220 mg, 380 mg, 400 mg, 600
mg, 760 mg, 800 mg, 1000 mg, 1140 mg, or 1200 mg per dose every 12
weeks.
[0128] In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200-1200 mg,
200-1000 mg, 200-800 mg, 200-600 mg, 200-400 mg, 400-1200 mg,
400-1000 mg, 400-800 mg, 400-600 mg, 600-1200 mg, 600-1000 mg,
600-800 mg, 800-1200 mg, 800-1000 mg, 800-900 mg, 750-850 mg,
750-800 mg, 775-825 mg, 350-450 mg, 375-425 mg, or 375-400 mg per
dose every 2 weeks, every 4 weeks, every 6 weeks, every 8 weeks,
every 10 weeks, or every 12 weeks. In certain embodiments of the
methods described herein, the anti-PCSK9 antibody is administered
subcutaneously at 200-1200 mg, 200-1000 mg, 200-800 mg, 200-600 mg,
200-400 mg, 400-1200 mg, 400-1000 mg, 400-800 mg, 400-600 mg,
600-1200 mg, 600-1000 mg, 600-800 mg, 800-1200 mg, 800-1000 mg,
800-900 mg, 750-850 mg, 750-800 mg, 775-825 mg, 350-450 mg, 375-425
mg, or 375-400 mg per dose every 2 weeks. In certain embodiments of
the methods described herein, the anti-PCSK9 antibody is
administered subcutaneously at 200-1200 mg, 200-1000 mg, 200-800
mg, 200-600 mg, 200-400 mg, 400-1200 mg, 400-1000 mg, 400-800 mg,
400-600 mg, 600-1200 mg, 600-1000 mg, 600-800 mg, 800-1200 mg,
800-1000 mg, 800-900 mg, 750-850 mg, 750-800 mg, 775-825 mg,
350-450 mg, 375-425 mg, or 375-400 mg per dose every 4 weeks. In
certain embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 200-1200 mg, 200-1000
mg, 200-800 mg, 200-600 mg, 200-400 mg, 400-1200 mg, 400-1000 mg,
400-800 mg, 400-600 mg, 600-1200 mg, 600-1000 mg, 600-800 mg,
800-1200 mg, 800-1000 mg, 800-900 mg, 750-850 mg, 750-800 mg,
775-825 mg, 350-450 mg, 375-425 mg, or 375-400 mg per dose every 6
weeks. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 200-1200 mg,
200-1000 mg, 200-800 mg, 200-600 mg, 200-400 mg, 400-1200 mg,
400-1000 mg, 400-800 mg, 400-600 mg, 600-1200 mg, 600-1000 mg,
600-800 mg, 800-1200 mg, 800-1000 mg, 800-900 mg, 750-850 mg,
750-800 mg, 775-825 mg, 350-450 mg, 375-425 mg, or 375-400 mg per
dose every 8 weeks. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
200-1200 mg, 200-1000 mg, 200-800 mg, 200-600 mg, 200-400 mg,
400-1200 mg, 400-1000 mg, 400-800 mg, 400-600 mg, 600-1200 mg,
600-1000 mg, 600-800 mg, 800-1200 mg, 800-1000 mg, 800-900 mg,
750-850 mg, 750-800 mg, 775-825 mg, 350-450 mg, 375-425 mg, or
375-400 mg per dose every 10 weeks. In certain embodiments of the
methods described herein, the anti-PCSK9 antibody is administered
subcutaneously at 200-1200 mg, 200-1000 mg, 200-800 mg, 200-600 mg,
200-400 mg, 400-1200 mg, 400-1000 mg, 400-800 mg, 400-600 mg,
600-1200 mg, 600-1000 mg, 600-800 mg, 800-1200 mg, 800-1000 mg,
800-900 mg, 750-850 mg, 750-800 mg, 775-825 mg, 350-450 mg, 375-425
mg, or 375-400 mg per dose every 12 weeks.
[0129] In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 600 mg per
dose every 8 weeks. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
800 mg per dose every 8 weeks. In certain embodiments of the
methods described herein, the anti-PCSK9 antibody is administered
subcutaneously at 800 mg per dose every 10 weeks. In certain
embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 800 mg per dose every 12
weeks. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 760 mg per
dose every 8 weeks. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
760 mg per dose every 10 weeks. In certain embodiments of the
methods described herein, the anti-PCSK9 antibody is administered
subcutaneously at 760 mg per dose every 12 weeks. In certain
embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 400 mg per dose every 4
weeks. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 400 mg per
dose every 8 weeks. In certain embodiments of the methods described
herein, the anti-PCSK9 antibody is administered subcutaneously at
400 mg per dose every 12 weeks. In certain embodiments of the
methods described herein, the anti-PCSK9 antibody is administered
subcutaneously at 380 mg per dose every 4 weeks. In certain
embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 380 mg per dose every 8
weeks. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 380 mg per
dose every 12 weeks. In certain embodiments of the methods
described herein, the anti-PCSK9 antibody is administered
subcutaneously at 220 mg per dose every 2 weeks. In certain
embodiments of the methods described herein, the anti-PCSK9
antibody is administered subcutaneously at 220 mg per dose every 4
weeks. In certain embodiments of the methods described herein, the
anti-PCSK9 antibody is administered subcutaneously at 220 mg per
dose every 8 weeks.
[0130] In certain embodiments of the methods described herein,
subjects receiving the anti-PCSK9 antibody are monitored for LDL-c
levels and if their levels drop below 25 or 15 mg/dL, then their
dose is adjusted down to around 50% or 25% of the initial dose by
adjusting the dose and/or frequency of administration. In certain
embodiments of the methods described herein, a subject is
administered an initial dose of 800 mg of anti-PCSK9 antibody every
8 weeks, the LDL-c levels of the subject are monitored and if the
subject's LDL-c levels drop below 25 mg/dL, the dose is adjusted to
200 mg of anti-PCSK9 antibody every 8 weeks. In certain embodiments
of the methods described herein, a subject is administered an
initial dose of 800 mg of anti-PCSK9 antibody every 8 weeks, the
LDL-c levels of the subject are monitored and if the subject's
LDL-c levels drop below 15 mg/dL, the dose is adjusted to 200 mg of
anti-PCSK9 antibody every 8 weeks. In certain embodiments of the
methods described herein, a subject is administered an initial dose
of 760 mg of anti-PCSK9 antibody every 8 weeks, the LDL-c levels of
the subject are monitored and if the subject's LDL-c levels drop
below 25 mg/dL, the dose is adjusted to 200 mg of anti-PCSK9
antibody every 8 weeks. In certain embodiments of the methods
described herein, a subject is administered an initial dose of 760
mg of anti-PCSK9 antibody every 8 weeks, the LDL-c levels of the
subject are monitored and if the subject's LDL-c levels drop below
15 mg/dL, the dose is adjusted to 200 mg of anti-PCSK9 antibody
every 8 weeks. In certain embodiments of the methods described
herein, a subject is administered an initial dose of 760 mg of
anti-PCSK9 antibody every 8 weeks, the LDL-c levels of the subject
are monitored and if the subject's LDL-c levels drop below 25
mg/dL, the dose is adjusted to 190 mg of anti-PCSK9 antibody every
8 weeks. In certain embodiments of the methods described herein, a
subject is administered an initial dose of 760 mg of anti-PCSK9
antibody every 8 weeks, the LDL-c levels of the subject are
monitored and if the subject's LDL-c levels drop below 15 mg/dL,
the dose is adjusted to 190 mg of anti-PCSK9 antibody every 8
weeks. In certain embodiments of the methods described herein, a
subject is administered an initial dose of 400 mg of anti-PCSK9
antibody every 4 weeks, the LDL-c levels of the subject are
monitored and if the subject's LDL-c levels drop below 25 mg/dL,
the dose is adjusted to 100 mg of anti-PCSK9 antibody every 4
weeks. In certain embodiments of the methods described herein, a
subject is administered an initial dose of 400 mg of anti-PCSK9
antibody every 4 weeks, the LDL-c levels of the subject are
monitored and if the subject's LDL-c levels drop below 15 mg/dL,
the dose is adjusted to 100 mg of anti-PCSK9 antibody every 4
weeks. In certain embodiments of the methods described herein, a
subject is administered an initial dose of 380 mg of anti-PCSK9
antibody every 4 weeks, the LDL-c levels of the subject are
monitored and if the subject's LDL-c levels drop below 25 mg/dL,
the dose is adjusted to 100 mg of anti-PCSK9 antibody every 4
weeks. In certain embodiments of the methods described herein, a
subject is administered an initial dose of 380 mg of anti-PCSK9
antibody every 4 weeks, the LDL-c levels of the subject are
monitored and if the subject's LDL-c levels drop below 15 mg/dL,
the dose is adjusted to 100 mg of anti-PCSK9 antibody every 4
weeks.
[0131] In certain embodiments, any of the foregoing subcutaneous
doses are administered using a subcutaneous administration device.
In certain embodiments, the device is a pre-filled syringe (e.g.,
0.5-mL, 1-mL, 1.25-mL, 1.5-mL, 1.75-mL, 2-mL, 2.25-mL, or 2.5-mL
syringe). In certain embodiments, the device is a 1-mL pre-filled
syringe and the antibody concentration in the pre-filled syringe is
about 200 mg/mL. In certain embodiments, the device is a 1.5-mL
pre-filled syringe and the antibody concentration in the pre-filled
syringe is about 200 mg/mL. In certain embodiments, the device is a
2-mL pre-filled syringe and the antibody concentration in the
pre-filled syringe is about 200 mg/mL. In certain embodiments, the
device is a 2.25-mL pre-filled syringe and the antibody
concentration in the pre-filled syringe is about 200 mg/mL. In
certain embodiments, the device is a 2.5-mL pre-filled syringe and
the antibody concentration in the pre-filled syringe is about 200
mg/mL. In certain embodiments, more than one syringe may be used to
obtain the full flat dose, e.g., one syringe, two syringes, three
syringes, or four syringes. In alternative embodiments, a high
volume, single use, subcutaneous infusion device may be used to
obtain the full flat dose, e.g., a dose that can administer 3 mL, 4
mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL or 10 mL.
[0132] In certain embodiments, the dose is 800 mg and it is
administered every 8 weeks using two 2.25 mL syringes containing an
anti-PCSK9 antibody at 200 mg/mL concentration. In certain
embodiments, the dose is 800 mg and it is administered every 8
weeks using three 2.25 mL syringes containing an anti-PCSK9
antibody at 200 mg/mL concentration. In certain embodiments, the
dose is 800 mg and it is administered every 10 weeks using two 2.25
mL syringes containing an anti-PCSK9 antibody at 200 mg/mL
concentration. In certain embodiments, the dose is 800 mg and it is
administered every 10 weeks using three 2.25 mL syringes containing
an anti-PCSK9 antibody at 200 mg/mL concentration. In certain
embodiments, the dose is 800 mg and it is administered every 12
weeks using two 2.25 mL syringes containing an anti-PCSK9 antibody
at 200 mg/mL concentration. In certain embodiments, the dose is 800
mg and it is administered every 12 weeks using three 2.25 mL
syringes containing an anti-PCSK9 antibody at 200 mg/mL
concentration. In certain embodiments, the dose is 800 mg and it is
administered every 8 weeks using a high volume, single use,
subcutaneous infusion device containing 4 mL of an anti-PCSK9
antibody at 200 mg/mL.
[0133] In certain embodiments, the dose is 760 mg and it is
administered every 8 weeks using two 2.25 mL syringes containing an
anti-PCSK9 antibody at 200 mg/mL concentration. In certain
embodiments, the dose is 760 mg and it is administered every 10
weeks using two 2.25 mL syringes containing an anti-PCSK9 antibody
at 200 mg/mL concentration. In certain embodiments, the dose is 760
mg and it is administered every 12 weeks using two 2.25 mL syringes
containing an anti-PCSK9 antibody at 200 mg/mL concentration.
[0134] In certain embodiments, the dose is 600 mg and it is
administered every 8 weeks using two 2.25 mL syringes containing an
anti-PCSK9 antibody at 200 mg/mL concentration. In certain
embodiments, the dose is 600 mg and it is administered every 12
weeks using two 2.25 mL syringes containing an anti-PCSK9 antibody
at 200 mg/mL concentration.
[0135] In certain embodiments, the dose is 400 mg and it is
administered every 4 weeks using one 2.5 mL syringe containing an
anti-PCSK9 antibody at 200 mg/mL concentration. In certain
embodiments, the dose is 400 mg and it is administered every 4
weeks using two 2.25 mL syringes containing an anti-PCSK9 antibody
at 200 mg/mL concentration. In certain embodiments, the dose is 380
mg and it is administered every 4 weeks using one 2.25 mL syringe
containing an anti-PCSK9 antibody at 200 mg/mL concentration.
[0136] In certain embodiments, the methods described herein further
comprise administering to the subject an effective amount of a
second medicament, wherein the anti-PCSK9 antibody is the first
medicament. In one embodiment, the second medicament elevates the
level of LDLR protein. In another embodiment, the second medicament
reduces the level of LDL-cholesterol. In another embodiment, the
second medicament comprises a statin. In another embodiment, the
statin is selected from the group consisting of atorvastatin,
fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,
rosuvastatin, simvastatin, and any combination thereof. In another
embodiment, the second medicament elevates the level of
HDL-cholesterol. In certain embodiments, the subject or the
individual is human.
[0137] In one aspect, the invention concerns a method of detecting
PCSK9 protein in a sample suspected of containing the PCSK9
protein, the method comprising (a) contacting the sample with the
anti-PCSK9 antibody described herein; and (b) detecting formation
of a complex between the anti-PCSK9 antibody and the PCSK9 protein.
In one embodiment, the anti-PCSK9 antibody is detectably
labeled.
[0138] Any embodiment described herein or any combination thereof
applies to any and all anti-PCSK9 antibodies, methods and uses of
the invention described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0139] FIG. 1 shows heavy chain HVR sequences, H1 (SEQ ID NOS 1, 1,
1, 1, 1, 2, 42, 3, 3, 1, respectively, in order of appearance), H2
(all disclosed as SEQ ID NO: 4), and H3 (all disclosed as SEQ ID
NO: 5), and light chain HVR sequences, L1 (SEQ ID NOS: 6, 7, 7, 7,
7, 7, 7, 7, 7 and 7, respectively, in order of appearance), L2 (SEQ
ID NOS: 26, 8, 8, 8, 8, 8, 8, 8, 8 and 8, respectively, in order of
appearance) and L3 (SEQ ID NOS: 9, 9, 10, 10, 11, 12, 12, 13, 33
and 14, respectively, in order of appearance), of anti-PCSK9
antibodies.
[0140] FIG. 2A-B show the amino acid sequences of (A) the heavy
chain variable domains (SEQ ID NOS: 15, 27, 15, 27, 27, 16, 43, 17,
17 and 27, respectively, in order of appearance) and (B) light
chain variable domains (SEQ ID NOS: 18, 44, 19, 19, 20, 21, 21, 22,
34 and 23, respectively, in order of appearance) of anti-PCSK9
antibodies. Positions are numbered according to Kabat and
hypervariable regions are boxed.
[0141] FIG. 3A-D show dissociation constants of the anti-PCSK9
antibodies (IgG) against (A) human PCSK9, (B) murine PCSK9, (C)
cyno PCSK9 and rat PCSK9, and (D) rhesus PCSK9.
[0142] FIG. 4. Anti-PCSK9 antibodies inhibit binding of PCSK9 to
LDLR in a competition binding ELISA. Blank (no antibody; open
square) and control antibody (open circle) are shown in dashed
lines. Anti-PCSK9 antibodies are shown in solid lines. IC.sub.50
values of anti-PCSK9 antibodies are shown in the table.
[0143] FIG. 5. Different concentrations of anti-PCSK9 antibodies
were incubated with 15 .mu.g/ml PCSK9 and added to HepG2 cells for
4 hours. Cells were processed for FACS analysis of surface LDLR.
The data indicate that the anti-PCSK9 antibodies effectively
prevented LDLR downregulation. The positive control is cells not
treated with PCSK9.
[0144] FIG. 6. Western blot with anti-LDLR antibody showing that 30
.mu.g of PCSK9 for 1 hr significantly downregulated LDLR levels in
mouse liver.
[0145] FIG. 7. Western blot with anti-LDLR antibody showing that
all five anti-PCSK9 antibodies prevented LDLR downregulation in
mouse liver. The bottom immunoblot is a pool of 4 livers (10 .mu.g
of protein from each liver) per treatment group.
[0146] FIG. 8 shows anti-PCSK9 antibody concentrations in sera of
C57JBL/6 mice after single I.V. injection. Shown are the average
concentrations of the dosing groups 0.5 mg/kg; 5 mg/kg; and 20
mg/kg (n=3).
[0147] FIG. 9 shows comparison of anti-PCSK9 antibody
concentrations in sera of C57JBL/6 WT and PCSK9.sup.-/- mice after
single I.V. injection of 5 mg/kg anti-PCSK9 antibody. The average
concentrations of each dosing group are shown (n=3).
[0148] FIG. 10 shows anti-PCSK9 antibody concentrations in sera of
individual cynomolgus monkey after single I.V. injection. Three
dosing groups are included: 5 mg/kg; 20 mg/kg; and 60 mg/kg.
[0149] FIG. 11 shows anti-PCSK9 antibody concentrations in sera of
cynomolgus monkeys after single I.V. injection. Shown are the
average concentrations of the dosing groups 5 mg/kg, 20 mg/kg, and
60 mg/kg (n=3).
[0150] FIG. 12 shows total cholesterol level in the sera of mice
treated with a single dose (10 mg/kg body weight) of either control
(Crtl) or anti-PCSK9 antibody. Cholesterol levels were measured at
different days as indicated in the figure.
[0151] FIG. 13 shows total cholesterol level in the sera from the
mice treated with single dose (10 mg/kg body weight) of either
control or anti-PCSK9 antibody.
[0152] FIG. 14 shows a schematic of the Phase I trial design
including cohorts A-J. Each cohort included six patients treated
with the active agent and two patients treated with placebo, for a
total of 8 patients per cohort and 80 total patients.
[0153] FIG. 15 shows pharmacokinetic data for study cohorts A-J.
Results from the single dose cohorts A-E and J are shown in the
left panel and results from the multiple dose cohorts F-I are shown
in the right panel. Red arrows indicate timing of drug
administration.
[0154] FIG. 16 shows mean absolute change from baseline in LDL-c
(mg/dL) levels for the single dose cohorts.
[0155] FIG. 17 shows mean percent change in baseline in LDL-c
levels for the single dose cohorts.
[0156] FIG. 18 shows mean absolute change from baseline in LDL-c
(mg/dL) levels for the multiple dose cohorts.
[0157] FIG. 19 shows mean percent change in baseline in LDL-c
levels for the multiple dose cohorts.
[0158] FIG. 20 shows the viscosity of anti-PCSK9 as a function of
protein concentration in a formulation of 200 mM arginine
succinate, 0.02% PS20, pH 5.5.
[0159] FIG. 21 shows size exclusion chromatography (SEC) (left
panel) and turbidity (right panel) analyses for control and
agitated anti-PCSK9 samples containing various concentrations of
Polysorbate 20 (PS20) in 2cc glass vials.
[0160] FIG. 22 shows oxidation of methionine and tryptophan
residues in anti-PCSK9 under various conditions by peptide
mapping.
[0161] FIG. 23 shows oxidation of methionine and tryptophan
residues in and adjacent to CDRs of anti-PCSK9 under various
conditions by peptide mapping.
[0162] FIG. 24 shows ion exchange chromatography (IEC) (left panel)
and SEC (right panel) pH rate profiles for 200 mg/mL anti-PCSK9
from pH 5.0 to 6.5 (200 mM arginine succinate, 0.02% PS20 at pH
5.0-6.0 or 20 mM histidine HCL, 160 mM arginine HCl, 0.02% PS20 at
pH 6.5).
[0163] FIG. 25 shows percent main peak (left panel) and percent
high molecular weight species (HMWS) (right panel) data by SEC for
anti-PCSK9 during frozen storage in HCl (200 mg/mL anti-PCSK9 in 20
mM histidine HCl, 160 mM arginine HCl, 0.02% PS20, pH 6.0) and
Acetate (200 mg/mL anti-PCSK9 in 20 mM histidine acetate, 160 mM
arginine acetate, 0.02% PS20, pH 6.0) formulations.
[0164] FIG. 26 shows counter-ion effects on 200 mg/mL anti-PCSK9 at
pH 6.0 by CE-SDS (top), SEC (middle), and IEC (bottom) after 1
month at 40.degree. C. storage.
[0165] FIG. 27 shows the study design of a phase II clinical trial,
including an overview of study dose cohorts, anti-PCSK9 antibody
dose regimen, and number of patients in each arm of the trial.
[0166] FIG. 28 shows mean pharmacokinetics (+/- standard deviation)
(left panel) and mean total PCSK9 (+/- standard error) (right
panel) in patients receiving anti-PCSK9 antibody or placebo.
[0167] FIG. 29 shows the absolute change from baseline in direct
LDL cholesterol observed in patients receiving anti-PCSK9 antibody
or placebo.
[0168] FIG. 30 shows the relative change from baseline in direct
LDL cholesterol observed in patients receiving anti-PCSK9 antibody
or placebo.
[0169] FIG. 31 shows the absolute change from baseline in total
cholesterol observed in patients receiving anti-PCSK9 antibody or
placebo.
[0170] FIG. 32 shows the relative change from baseline in total
cholesterol observed in patients receiving anti-PCSK9 antibody or
placebo.
[0171] FIG. 33 shows the absolute change from baseline in non-HDL
cholesterol in patients receiving anti-PCSK9 antibody or
placebo.
[0172] FIG. 34 shows the relative change from baseline in non-HDL
cholesterol in patients receiving anti-PCSK9 antibody or
placebo.
[0173] FIG. 35 shows the absolute change from baseline in
apolipoprotein B in patients receiving anti-PCSK9 antibody or
placebo.
[0174] FIG. 36 shows the relative change from baseline in
apolipoprotein B in patients receiving anti-PCSK9 antibody or
placebo.
[0175] FIG. 37A shows the proportion of patients with direct LDL-c
values less than or equal to 15 mg/dL for at least one visit after
receiving anti-PCSK9 antibody or placebo, and FIG. 37B shows the
results of experiments performed to determine the proportion of
patients with direct LDL-c values less than or equal to 25 mg/dL
for at least one visit after receiving anti-PCSK9 antibody or
placebo.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0176] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds.,
(2003)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A
LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed.
(1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods
in Molecular Biology, Humana Press; Cell Biology: A Laboratory
Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell
Culture (R. I. Freshney), ed., 1987); Introduction to Cell and
Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;
Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons;
Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain
Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: A Practical Approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal Antibodies: A Practical Approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:
Principles and Practice of Oncology (V. T. DeVita et al., eds.,
J.B. Lippincott Company, 1993).
I. DEFINITIONS
[0177] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March,
Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th
ed., John Wiley & Sons (New York, N.Y. 1992), provide one
skilled in the art with a general guide to many of the terms used
in the present application. All references cited herein, including
patent applications and publications, are incorporated by reference
in their entirety.
[0178] For purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
It is to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not
intended to be limiting. In the event that any definition set forth
below conflicts with any document incorporated herein by reference,
the definition set forth below shall control.
[0179] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin heavy chain is that
of the EU index as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), expressly incorporated
herein by reference. The "EU index as in Kabat" refers to the
residue numbering of the human IgG.sub.1 EU antibody.
[0180] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0181] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0182] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0183] The terms "anti-PCSK9 antibody", "anti-PCSK9", "PCSK9
antibody" or "an antibody that binds to PCSK9" refers to an
antibody that is capable of binding PCSK9 with sufficient affinity
such that the antibody is useful as a diagnostic and/or therapeutic
agent in targeting PCSK9. In one embodiment, the extent of binding
of an anti-PCSK9 antibody to an unrelated, non-PCSK9 protein is
less than about 10% of the binding of the antibody to PCSK9 as
measured, e.g., by a radioimmunoassay (RIA). In certain
embodiments, an antibody that binds to PCSK9 has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, .ltoreq.0.1 nM, <0.01 nM, or .ltoreq.0.001 nM
(e.g. 10.sup.-8M or less, e.g. from 10.sup.-8 M to 10.sup.-13M,
e.g., from 10.sup.-9 M to 10.sup.-13 M). In certain embodiments, an
anti-PCSK9 antibody binds to an epitope of PCSK9 that is conserved
among PCSK9 from different species.
[0184] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0185] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments. Papain digestion of antibodies produces
two identical antigen-binding fragments, called "Fab" fragments,
each with a single antigen-binding site, and a residual "Fc"
fragment, whose name reflects its ability to crystallize readily.
Pepsin treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0186] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein. In certain embodiments, the
epitope is determined based on the crystal structure of the
anti-PCSK9 antibody Fab fragment bound to PCSK9.
[0187] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0188] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0189] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0190] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). 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 may be bivalent or bispecific. Diabodies are described
more fully in, for example, EP 404,097; WO 1993/01161; Hudson et
al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are
also described in Hudson et al., Nat. Med. 9:129-134 (2003).
[0191] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0192] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0193] The "Fab" fragment contains the heavy- and light-chain
variable domains and also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0194] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In certain embodiments, a human
IgG heavy chain Fc region extends from Cys226, or from Pro230, to
the carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0195] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0196] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0197] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three HVRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0198] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0199] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0200] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0201] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0202] The term "hypercholesterolemia," as used herein, refers to a
condition in which cholesterol levels are elevated above a desired
level. In certain embodiments, the LDL-cholesterol level is
elevated above the desired level. In certain embodiments, the serum
LDL-cholesterol levels are elevated above the desired level.
[0203] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0204] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0205] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0206] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0207] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0208] "Isolated nucleic acid encoding an anti-PCSK9 antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0209] The term "monoclonal antibody," as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0210] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0211] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0212] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0213] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0214] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0215] The term "pharmaceutical formulation" or "pharmaceutical
composition" refers to a preparation which is in such form as to
permit the biological activity of an active ingredient contained
therein to be effective, and which contains no additional
components which are unacceptably toxic to a subject to which the
formulation would be administered.
[0216] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0217] The term "proprotein convertase subtilisin kexin type 9,"
"PCSK9," or "NARC-1," as used herein, refers to any native PCSK9
from any vertebrate source, including mammals such as primates
(e.g. humans) and rodents (e.g., mice and rats), unless otherwise
indicated. The term encompasses "full-length," unprocessed PCSK9 as
well as any form of PCSK9 that results from processing in the cell
or any fragment thereof. The term also encompasses naturally
occurring variants of PCSK9, e.g., splice variants or allelic
variants.
[0218] The term "PCSK9 activity" or "biological activity" of PCSK9,
as used herein, includes any biological effect of PCSK9. In certain
embodiments, PCSK9 activity includes the ability of PCSK9 to
interact or bind to a substrate or receptor. In certain
embodiments, the biological activity of PCSK9 is the ability of
PCSK9 to bind to a LDL-receptor (LDLR). In certain embodiments,
PCSK9 binds to and catalyzes a reaction involving LDLR. In certain
embodiments, PCSK9 activity includes the ability of PCSK9 to
decrease or reduce the availability of LDLR. In certain
embodiments, the biological activity of PCSK9 includes the ability
of PCSK9 to increase the amount of LDL in a subject. In certain
embodiments, the biological activity of PCSK9 includes the ability
of PCSK9 to decrease the amount of LDLR that is available to bind
to LDL in a subject. In certain embodiments, the biological
activity of PCSK9 includes the ability of PCSK9 to decrease the
amount of LDLR that is available to bind to LDL. In certain
embodiments, biological activity of PCSK9 includes any biological
activity resulting from PCSK9 signaling.
[0219] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York, 1994), pp. 269-315.
[0220] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, decreasing the rate of disease progression,
amelioration or palliation of the disease state, and remission or
improved prognosis. In some embodiments, antibodies of the
invention are used to delay development of a disease or to slow the
progression of a disease.
[0221] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007)). A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0222] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0223] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0224] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se.
[0225] It is understood that aspect and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
II. COMPOSITIONS AND METHODS
[0226] In one aspect, the invention is based, in part, on
experimental and clinical results obtained with anti-PCSK9
antibodies. Results obtained indicate that blocking biological
activity of PCSK9 with anti-PCSK9 antibodies leads to a prevention
of reduction in LDLR. In addition, the results demonstrate that
administration of anti-PCSK9 antibody reduces total LDL-cholesterol
level in a subject. Accordingly, PCSK9 antibodies of the invention,
as described herein, provide important therapeutic and diagnostic
agents for use in targeting pathological conditions associated with
PCSK9, e.g., cholesterol related disorders.
[0227] In certain embodiments, a "cholesterol related disorder"
includes any one or more of the following: hypercholesterolemia,
heart disease, metabolic syndrome, diabetes, coronary heart
disease, stroke, cardiovascular diseases, Alzheimers disease and
generally dyslipidemias, which can be manifested, for example, by
an elevated total serum cholesterol, elevated LDL, elevated
triglycerides, elevated VLDL, and/or low HDL. Some non-limiting
examples of primary and secondary dyslipidemias that can be treated
using an anti-PCSK9 antibody, either alone, or in combination with
one or more other agents include the metabolic syndrome, diabetes
mellitus, familial combined hyperlipidemia, familial
hypertriglyceridemia, familial hypercholesterolemias, including
heterozygous hypercholesterolemia, homozygous hypercholesterolemia,
familial defective apoplipoprotein B-100; polygenic
hypercholesterolemia; remnant removal disease, hepatic lipase
deficiency; dyslipidemia secondary to any of the following: dietary
indiscretion, hypothyroidism, drugs including estrogen and
progestin therapy, beta-blockers, and thiazide diuretics; nephrotic
syndrome, chronic renal failure, Cushing's syndrome, primary
biliary cirrhosis, glycogen storage diseases, hepatoma,
cholestasis, acromegaly, insulinoma, isolated growth hormone
deficiency, and alcohol-induced hypertriglyceridemia. Anti-PCSK9
antibodies described herein can also be useful in preventing or
treating atherosclerotic diseases, such as, for example, coronary
heart disease, coronary artery disease, peripheral arterial
disease, stroke (ischaemic and hemorrhagic), angina pectoris, or
cerebrovascular disease and acute coronary syndrome, myocardial
infarction. In certain embodiments, the anti-PCSK9 antibodies
described herein are useful in reducing the risk of: nonfatal heart
attacks, fatal and non-fatal strokes, certain types of heart
surgery, hospitalization for heart failure, chest pain in patients
with heart disease, and/or cardiovascular events because of
established heart disease such as prior heart attack, prior heart
surgery, and/or chest pain with evidence of clogged arteries. In
certain embodiments, the anti-PCSK9 antibodies and methods
described herein can be used to reduce the risk of recurrent
cardiovascular events.
[0228] A. Exemplary Anti-PCSK9 Antibodies
[0229] In one aspect, the invention provides isolated antibodies
that bind to PCSK9. In certain embodiments, an anti-PCSK9 antibody
modulates PCSK9 activity.
[0230] In one aspect, the invention provides an anti-PCSK9 antibody
comprising at least one, two, three, four, five, or six HVRs
selected from (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:42; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8 or SEQ
ID NO:26; and (f) HVR-L3 comprising the amino acid sequence of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO:14, or SEQ ID NO:33.
[0231] In one aspect, the invention provides an anti-PCSK9 antibody
comprising six HVRs comprising (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:42;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:4; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO:5; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID
NO:7; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8
or SEQ ID NO:26; and (f) HVR-L3 comprising the amino acid sequence
of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14 or SEQ ID NO:33.
[0232] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:42; (b) HVR-H2 comprising
the amino acid sequence of SEQ ID NO:4; and (c) HVR-H3 comprising
the amino acid sequence of SEQ ID NO:5. In one embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO:5. In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:5 and HVR-L3
comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID
NO:33. In a further embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:5, HVR-L3
comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID
NO:33, and HVR-H2 comprising the amino acid sequence of SEQ ID
NO:4. In a further embodiment, the antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, or SEQ ID NO:42; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:4; and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:5.
[0233] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:6 or SEQ ID NO:7; (b) HVR-L2 comprising the
amino acid sequence of SEQ ID NO:8 or SEQ ID NO:26; and (c) HVR-L3
comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID
NO:33. In one embodiment, the antibody comprises (a) HVR-L1
comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7;
(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8 or SEQ
ID NO:26; and (c) HVR-L3 comprising the amino acid sequence of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ NO:14, or SEQ NO:33.
[0234] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:42,
(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:4, and
(iii) HVR-H3 comprising an amino acid sequence selected from SEQ ID
NO:5; and (b) a VL domain comprising at least one, at least two, or
all three VL HVR sequences selected from (i) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7, (ii) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:26,
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, or SEQ ID NO:33.
[0235] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:4;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:5; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:6; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:26; and (f)
HVR-L3 comprising an amino acid sequence of SEQ ID NO:9. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:9. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:10. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:11. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:12. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:42; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:12. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:3; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:13. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:1; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:14. In another
aspect, the invention provides an antibody comprising (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:3; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5; (d) HVR-L1
comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2
comprising the amino acid sequence of SEQ ID NO:8; and (f) HVR-L3
comprising an amino acid sequence of SEQ ID NO:33.
[0236] In certain embodiments, the anti-PCSK9 antibody is
humanized. In one embodiment, an anti-PCSK9 antibody comprises HVRs
as in any of the above embodiments, and further comprises an
acceptor human framework, e.g., a human immunoglobulin framework or
a human consensus framework.
[0237] In another aspect, an anti-PCSK9 antibody comprises a heavy
chain variable domain (VH) sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the
amino acid sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:27, or SEQ ID NO:43. In certain embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-PCSK9 antibody comprising that sequence
retains the ability to bind to PCSK9. In certain embodiments, a
total of 1 to 10 amino acids have been substituted, inserted and/or
deleted in SEQ ID NO:15, SEQ ID NO:16, SEQ NO:17, SEQ ID NO:27, or
SEQ ID NO:43. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-PCSK9 antibody comprises the VH sequence in
SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:27, or SEQ ID
NO:43, including post-translational modifications of that sequence.
In a particular embodiment, the VH comprises one, two or three HVRs
selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:42, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:4, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:5.
[0238] In another aspect, an anti-PCSK9 antibody is provided,
wherein the antibody comprises a light chain variable domain (VL)
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% sequence identity to the amino acid sequence of SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:34, or SEQ ID NO:44. In certain embodiments, a
VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-PCSK9 antibody comprising that sequence
retains the ability to bind to PCSK9. In certain embodiments, a
total of 1 to 10 amino acids have been substituted, inserted and/or
deleted in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:34, or SEQ ID NO:44. In
certain embodiments, the substitutions, insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally,
the anti-PCSK9 antibody comprises the VL sequence in SEQ ID NO:18,
SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:34, or SEQ ID NO:44, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1
comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7;
(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:8 or SEQ
ID NO:26; and (c) HVR-L3 comprising the amino acid sequence of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
SEQ ID NO:14, or SEQ ID NO:33.
[0239] In another aspect, an anti-PCSK9 antibody is provided,
wherein the antibody comprises a VH as in any of the embodiments
provided above, and a VL as in any of the embodiments provided
above. In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:15 and SEQ ID NO:18, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:27 and SEQ ID NO:44, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:15 and SEQ ID NO:19, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:27 and SEQ ID NO:19, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:27 and SEQ ID NO:20, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:16 and SEQ ID NO:21, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:43 and SEQ ID NO:21, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:17 and SEQ ID NO:22, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:27 and SEQ ID NO:23, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:17 and SEQ ID NO:34, respectively, including
post-translational modifications of those sequences.
[0240] In another aspect, an anti-PCSK9 antibody comprises a heavy
chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to the amino acid sequence
of SEQ ID NO:35. In certain embodiments, a heavy chain sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-PCSK9 antibody comprising that sequence retains the ability to
bind to PCSK9. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:35. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-PCSK9 antibody heavy chain comprises the VH
sequence in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:27,
or SEQ ID NO:43, including post-translational modifications of that
sequence. In a particular embodiment, the heavy chain comprises
one, two or three HVRs selected from: (a) HVR-H1 comprising the
amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or
SEQ ID NO:42, (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:4, and (c) HVR-H3 comprising the amino acid sequence of SEQ
ID NO:5.
[0241] In another aspect, an anti-PCSK9 antibody is provided,
wherein the antibody comprises a light chain having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the amino acid sequence of SEQ ID NO:36. In certain
embodiments, a light chain sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-PCSK9
antibody comprising that sequence retains the ability to bind to
PCSK9. In certain embodiments, a total of 1 to 10 amino acids have
been substituted, inserted and/or deleted in SEQ ID NO:36. In
certain embodiments, the substitutions, insertions, or deletions
occur in regions outside the HVRs (i.e., in the FRs). Optionally,
the anti-PCSK9 antibody light chain comprises the VL sequence in
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:34, or SEQ ID NO:44, including
post-translational modifications of that sequence. In a particular
embodiment, the light chain comprises one, two or three HVRs
selected from (a) HVR-L1 comprising the amino acid sequence of SEQ
ID NO:6 or SEQ ID NO:7; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:8 or SEQ ID NO:26; and (c) HVR-L3 comprising
the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:33.
[0242] In another aspect, an anti-PCSK9 antibody is provided,
wherein the antibody comprises a heavy chain as in any of the
embodiments provided above, and a light chain as in any of the
embodiments provided above. In one embodiment, the antibody
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:35, and a light chain comprising the amino acid sequence of
SEQ ID NO:36. In certain embodiments, SEQ ID NO:35 is truncated by
one or two amino acids at the C-terminus, e.g., it does not contain
K451, or G450 and K451. In certain embodiments, P449 in SEQ ID
NO:35 is amidated.
TABLE-US-00001 Antibody 508.20.33b heavy chain amino acid sequence
(SEQ ID NO: 35): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSTAIHWVRQAPGKGLEW
VARISPANGNTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY
YCARWIGSRELYIMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody 508.20.33b light chain amino
acid sequence (SEQ ID NO: 36):
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLL
IYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYPA
LHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC
[0243] In certain embodiments, SEQ ID NO:35 is truncated by one or
two amino acids at the C-terminus, e.g., it does not contain K451,
or G450 and K451 (e.g., the heavy chain comprises amino acids 1-449
of SEQ ID NO:35 or amino acids 1-450 of SEQ ID NO:35). In certain
embodiments, P449 in SEQ ID NO:35 is amidated.
[0244] In certain embodiments, functional epitopes can be mapped by
combinatorial alanine scanning. In this process, a combinatorial
alanine-scanning strategy can be used to identify amino acids in
the PCSK9 protein that are necessary for interaction with
anti-PCSK9 antibodies. In certain embodiments, the epitope is
conformational and crystal structure of anti-PCSK9 antibody Fab
fragment bound to PCSK9 may be employed to identify the epitopes.
In one aspect, the invention provides an antibody that binds to the
same epitope as any of the anti-PCSK9 antibody provided herein. For
example, in certain embodiments, an antibody is provided that binds
to the same epitope as an anti-PCSK9 antibody comprising a VH
sequence of SEQ ID NO:15 and a VL sequence of SEQ ID NO:19. In
certain embodiments, an antibody is provided that binds to the same
epitope as an anti-PCSK9 antibody comprising a VH sequence of SEQ
ID NO:27 and a VL sequence of SEQ ID NO:19. In certain embodiments,
an antibody is provided that binds to the same epitope as an
anti-PCSK9 antibody comprising a VH sequence of SEQ ID NO:27 and a
VL sequence of SEQ ID NO:20. In certain embodiments, an antibody is
provided that binds to the same epitope as an anti-PCSK9 antibody
comprising a VH sequence of SEQ ID NO:16 and a VL sequence of SEQ
ID NO:21. In certain embodiments, an antibody is provided that
binds to the same epitope as an anti-PCSK9 antibody comprising a VH
sequence of SEQ ID NO:43 and a VL sequence of SEQ ID NO:21. In
certain embodiments, an antibody is provided that binds to the same
epitope as an anti-PCSK9 antibody comprising a VH sequence of SEQ
ID NO:17 and a VL sequence of SEQ ID NO:22. In certain embodiments,
an antibody is provided that binds to the same epitope as an
anti-PCSK9 antibody comprising a VH sequence of SEQ ID NO:27 and a
VL sequence of SEQ ID NO:23. In certain embodiments, an antibody is
provided that binds to the same epitope as an anti-PCSK9 antibody
comprising a VH sequence of SEQ ID NO:17 and a VL sequence of SEQ
ID NO:34.
[0245] In one aspect, the invention provides an anti-PCSK9
antibody, or antigen binding fragment thereof, that binds to human
PCSK9 competitively with any one of the antibodies described
herein. In certain embodiments, competitive binding may be
determined using an ELISA assay. For example, in certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:15 and a VL sequence of SEQ ID NO:19. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:27 and a VL sequence of SEQ ID NO:19. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:27 and a VL sequence of SEQ ID NO:20. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:16 and a VL sequence of SEQ ID NO:21. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:43 and a VL sequence of SEQ ID NO:21. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:17 and a VL sequence of SEQ ID NO:22. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:27 and a VL sequence of SEQ ID NO:23. In certain
embodiments, an antibody is provided that binds to PCSK9
competitively with an anti-PCSK9 antibody comprising a VH sequence
of SEQ ID NO:17 and a VL sequence of SEQ ID NO:34.
[0246] In certain embodiments, an antibody is provided that binds
to an epitope within a fragment of PCSK9 as described herein. In
certain embodiments, an antibody is provided that binds to an
epitope within a fragment of PCSK9 comprising amino acids 376 to
379 of human PCSK9 amino acid sequence of SEQ ID NO:24. In certain
embodiments, the functional and/or structural epitope of an
antibody according to this invention includes residue D238 of human
PCSK9. In certain embodiments, the functional and/or structural
epitope of an antibody according to this invention includes residue
A239 of human PCSK9. In certain embodiments, the functional and/or
structural epitope of an antibody according to this invention
includes residues D238 and A239 of human PCSK9. In certain
embodiments, the functional and/or structural epitope of an
antibody according to this invention includes residue E366 of human
PCSK9. In certain embodiments, the functional and/or structural
epitope of an antibody according to this invention includes residue
D367 of human PCSK9. In certain embodiments, the functional and/or
structural epitope of an antibody according to this invention
includes residues E366 and D367 of human PCSK9. In certain
embodiments, the functional and/or structural epitope of an
antibody according to this invention includes residue H391 of human
PCSK9. In certain embodiments, the functional and/or structural
epitope of an antibody according to this invention includes
residues E366, D367 and H391 of human PCSK9. According to another
embodiment, the functional and/or structural epitope of an antibody
according to this invention includes residues A239 and H391 of
human PCSK9. In certain embodiments, the functional and/or
structural epitope of includes one or more of residues A239, A341,
E366, D367 and H391 of human PCSK9. In certain embodiments, the
functional and/or structural epitope of includes one or more of
residues near A239, A341, E366, D367 and H391 of human PCSK9. In
certain embodiments, the functional and/or structural epitope of an
antibody according to this invention comprises (i) at least one
residue selected from the group consisting of R194 and E195, (ii)
at least one residue selected from the group consisting of D238 and
A239, (iii) at least one residue selected from the group consisting
of A341 and Q342, and (iv) at least one residue selected from the
group consisting of E366, D367, 1369, S376, T377, C378, F379, S381
and H391, of human PCSK9. In certain embodiments, the functional
and/or structural epitope comprises one, two, three, four, five,
six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or
all of the following residues: R194, E195, D238, A239, A341, Q342,
E366, D367, 1369, S376, T377, C378, F379, S381 and H391 of human
PCSK9.
[0247] In a further aspect of the invention, an anti-PCSK9 antibody
according to any of the above embodiment is a monoclonal antibody,
including a chimeric, humanized or human antibody. In one
embodiment, an anti-PCSK9 antibody is an antibody fragment, e.g., a
Fv, Fab, Fab', scFv, diabody, or F(ab').sub.2 fragment. In another
embodiment, the antibody is a full length antibody, e.g., an intact
IgG.sub.1 antibody or other antibody class or isotype as defined
herein.
[0248] In a further aspect, an anti-PCSK9 antibody according to any
of the above embodiments may incorporate any of the features,
singly or in combination, as described in Sections 1-7 below:
[0249] 1. Antibody Affinity
[0250] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8M to
10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13 M).
[0251] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA) performed with the Fab version of an antibody
of interest and its antigen as described by the following assay.
Solution binding affinity of Fabs for antigen is measured by
equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.
293:865-881(1999)). To establish conditions for the assay,
MICROTITER.RTM. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/ml of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta et al.,
Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0252] According to another embodiment, Kd is measured using
surface plasmon resonance assays using a BIACORE.RTM.-2000 or a
BIACORE.RTM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized antigen CM5 chips at .about.10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10.sup.6
M.sup.-1 s.sup.-1 by the surface plasmon resonance assay above,
then the on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic) with a stirred
cuvette.
[0253] 2. Antibody Fragments
[0254] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0255] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0256] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0257] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0258] 3. Chimeric and Humanized Antibodies
[0259] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0260] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0261] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0262] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0263] 4. Human Antibodies
[0264] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0265] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HuMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0266] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
1E3-cell hybridoma technology are also described in Li et al.,
Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional
methods include those described, for example, in U.S. Pat. No.
7,189,826 (describing production of monoclonal human IgM antibodies
from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0267] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0268] 5. Library-Derived Antibodies
[0269] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0270] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0271] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0272] 6. Multispecific Antibodies
[0273] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for PCSK9 and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of PCSK9. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express PCSK9. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0274] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0275] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g., US 2006/0025576A1).
[0276] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to
PCSK9 as well as another, different antigen (see, e.g., US
2008/0069820).
[0277] 7. Antibody Variants
[0278] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0279] a) Substitution, Insertion, and Deletion Variants
[0280] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table A under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00002 TABLE A Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0281] Amino acids may be grouped according to common side-chain
properties:
[0282] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0283] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0284] (3) acidic: Asp, Glu;
[0285] (4) basic: His, Lys, Arg;
[0286] (5) residues that influence chain orientation: Gly, Pro;
[0287] (6) aromatic: Trp, Tyr, Phe.
[0288] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0289] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g., a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0290] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0291] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0292] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0293] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g., for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0294] b) Glycosylation Variants
[0295] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0296] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0297] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e.g., complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (EU numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application
No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et
al., especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0298] Antibody variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0299] c) Fc Region Variants
[0300] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0301] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc(RIII only, whereas monocytes express Fc(RI,
Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337
(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0302] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0303] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0304] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0305] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0306] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0307] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0308] d) Cysteine Engineered Antibody Variants
[0309] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0310] e) Antibody Derivatives
[0311] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0312] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0313] B. Recombinant Methods and Compositions
[0314] Anti-PCSK9 antibodies described herein may be produced using
recombinant methods and compositions, e.g., as described in U.S.
Pat. No. 4,816,567. In one embodiment, isolated nucleic acid
encoding an anti-PCSK9 antibody described herein is provided. Such
nucleic acid may encode an amino acid sequence comprising the VL
and/or an amino acid sequence comprising the VH of the antibody
(e.g., the light and/or heavy chains of the antibody). In certain
embodiments, an isolated nucleic acid encoding an anti-PCSK9 heavy
chain variable region is provided wherein the nucleic acid
comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid
sequence of SEQ ID NO:38 or SEQ ID NO:39. In certain embodiments,
an isolated nucleic acid encoding an anti-PCSK9 light chain
variable region is provided wherein the nucleic acid comprises a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the nucleic acid sequence of
SEQ ID NO:40 or SEQ ID NO:41. In certain embodiments, an isolated
nucleic acid encoding an anti-PCSK9 heavy chain variable region and
an anti-PCSK9 light chain variable region is provided, wherein the
nucleic acid encoding the heavy chain variable region comprises a
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the nucleic acid sequence of
SEQ ID NO:38 or SEQ ID NO:39 and the nucleic acid encoding the
light chain variable region comprises a sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
identity to the nucleic acid sequence of SEQ ID NO:40 or SEQ ID
NO:41. In certain embodiments, an isolated nucleic acid encoding an
anti-PCSK9 heavy chain variable region is provided wherein the
nucleic acid comprises SEQ ID NO: 38 or 39. In certain embodiments,
an isolated nucleic acid encoding an anti-PCSK9 light chain
variable region is provided wherein the nucleic acid comprises SEQ
ID NO: 40 or 41. In certain embodiments, an isolated nucleic acid
encoding an anti-PCSK9 heavy chain variable region and light chain
variable region is provided, wherein the nucleic acid encoding the
heavy chain comprises SEQ ID NO:38 and the nucleic acid encoding
the light chain comprises SEQ ID NO:40. In certain embodiments, an
isolated nucleic acid encoding an anti-PCSK9 heavy chain variable
region and light chain variable region is provided, wherein the
nucleic acid encoding the heavy chain comprises SEQ ID NO:39 and
the nucleic acid encoding the light chain comprises SEQ ID
NO:41.
TABLE-US-00003 Antibody 508.20.33b Full Length Heavy Chain Nucleic
Acid Sequence (SEQ ID NO: 38) GAA GTTCAGCTGG TGGAGTCTGG CGGTGGCCTG
GTGCAGCCAG GGGGCTCACT CCGTTTGTCC TGTGCAGCTT CTGGCTTCAC CTTCTCTAGT
ACTGCTATTC ACTGGGTGCG TCAGGCCCCG GGTAAGGGCC TGGAATGGGT TGCTAGGATT
TCTCCTGCTA ACGGTAATAC TAACTATGCC GATAGCGTCA AGGGCCGTTT CACTATAAGC
GCAGACACAT CCAAAAACAC AGCCTACCTA CAAATGAACA GCTTAAGAGC TGAGGACACT
GCCGTCTATT ATTGTGCTCG TTGGATCGGG TCCCGGGAGC TGTACATTAT GGACTACTGG
GGTCAAGGAA CCCTGGTCAC CGTCTCCTCG GCCTCCACCA AGGGCCCATC GGTCTTCCCC
CTGGCACCCT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG
GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG
CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACT
GTGCCCTCTA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC
AACACCAAGG TGGACAAGAA AGTTGAGCCC AAATCTTGTG ACAAAACTCA CACATGCCCA
CCGTGCCCAG CACCTGAACT CCTGGGGGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC
AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC
CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC
AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTACC GTGTGGTCAG CGTCCTCACC
GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC
CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG
GTGTACACCC TGCCCCCATC CCGGGAAGAG ATGACCAAGA ACCAGGTCAG CCTGACCTGC
CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG
GAGAACAACT ACAAGACCAC GCCTCCCGTG CTGGACTCCG ACGGCTCCTT CTTCCTCTAC
AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG
ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA
Antibody 508.20.33b Heavy Chain Variable Region Nucleic Acid
Sequence (SEQ ID NO: 39) GAA GTTCAGCTGG TGGAGTCTGG CGGTGGCCTG
GTGCAGCCAG GGGGCTCACT CCGTTTGTCC TGTGCAGCTT CTGGCTTCAC CTTCTCTAGT
ACTGCTATTC ACTGGGTGCG TCAGGCCCCG GGTAAGGGCC TGGAATGGGT TGCTAGGATT
TCTCCTGCTA ACGGTAATAC TAACTATGCC GATAGCGTCA AGGGCCGTTT CACTATAAGC
GCAGACACAT CCAAAAACAC AGCCTACCTA CAAATGAACA GCTTAAGAGC TGAGGACACT
GCCGTCTATT ATTGTGCTCG TTGGATCGGG TCCCGGGAGC TGTACATTAT GGACTACTGG
GGTCAAGGAA CCCTGGTCAC CGTCTCCTCG Antibody 508.20.33b Full Length
Light Chain Nucleic Acid Sequence (SEQ ID NO: 40) GA TATCCAGATG
ACCCAGTCCC CGAGCTCCCT GTCCGCCTCT GTGGGCGATA GGGTCACCAT CACCTGCCGT
GCCAGTCAGG ATGTGTCCAC TGCTGTAGCC TGGTATCAAC AGAAACCAGG AAAAGCTCCG
AAGCTTCTGA TTTACTCGGC ATCCTTCCTC TACTCTGGAG TCCCTTCTCG CTTCTCTGGT
AGCGGTTCCG GGACGGATTT CACTCTGACC ATCAGCAGTC TGCAGCCGGA AGACTTCGCA
ACTTATTACT GTCAGCAAGC CTATCCGGCC CTACACACGT TCGGACAGGG TACCAAGGTG
GAGATCAAAC GAACTGTGGC TGCACCATCT GTCTTCATCT TCCCGCCATC TGATGAGCAG
TTGAAATCTG GAACTGCTTC TGTTGTGTGC CTGCTGAATA ACTTCTATCC CAGAGAGGCC
AAAGTACAGT GGAAGGTGGA TAACGCCCTC CAATCGGGTA ACTCCCAGGA GAGTGTCACA
GAGCAGGACA GCAAGGACAG CACCTACAGC CTCAGCAGCA CCCTGACGCT GAGCAAAGCA
GACTACGAGA AACACAAAGT CTACGCCTGC GAAGTCACCC ATCAGGGCCT GAGCTCGCCC
GTCACAAAGA GCTTCAACAG GGGAGAGTGT Antibody 508.20.33b Light Chain
Variable Region Nucleic Acid Sequence (SEQ ID NO: 41) GA TATCCAGATG
ACCCAGTCCC CGAGCTCCCT GTCCGCCTCT GTGGGCGATA GGGTCACCAT CACCTGCCGT
GCCAGTCAGG ATGTGTCCAC TGCTGTAGCC TGGTATCAAC AGAAACCAGG AAAAGCTCCG
AAGCTTCTGA TTTACTCGGC ATCCTTCCTC TACTCTGGAG TCCCTTCTCG CTTCTCTGGT
AGCGGTTCCG GGACGGATTT CACTCTGACC ATCAGCAGTC TGCAGCCGGA AGACTTCGCA
ACTTATTACT GTCAGCAAGC CTATCCGGCC CTACACACGT TCGGACAGGG TACCAAGGTG
GAGATCAAAC GA
[0315] In a further embodiment, one or more vectors (e.g.,
expression vectors) comprising such nucleic acid are provided. In a
further embodiment, a host cell comprising such nucleic acid is
provided. In one such embodiment, a host cell comprises (e.g., has
been transformed with): (1) a vector comprising a nucleic acid that
encodes an amino acid sequence comprising the VL of the antibody
and an amino acid sequence comprising the VH of the antibody, or
(2) a first vector comprising a nucleic acid that encodes an amino
acid sequence comprising the VL of the antibody and a second vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VH of the antibody. In one embodiment, the host cell
is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid
cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of
making an anti-PCSK9 antibody is provided, wherein the method
comprises culturing a host cell comprising a nucleic acid encoding
the antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0316] For recombinant production of an anti-PCSK9 antibody,
nucleic acid encoding an antibody, e.g., as described above, is
isolated and inserted into one or more vectors for further cloning
and/or expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0317] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.). After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0318] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0319] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0320] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0321] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
[0322] C. Assays
[0323] Anti-PCSK9 antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0324] 1. Binding Assays and Other Assays
[0325] In one aspect, an anti-PCSK9 antibody of the invention is
tested for its PCSK9 binding activity, e.g., by known methods such
as ELISA, Western blot, etc. Numerous types of competitive binding
assays can be used to determine if an anti-PCSK9 antibody competes
with another, for example: solid phase direct or indirect
radioimmunoassay (RIA), solid phase direct or indirect enzyme
immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et
al., 1983, Methods in Enzymology 9:242-253); solid phase direct
biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.
137:3614-3619) solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see, e.g., Harlow and Lane, 1988,
Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid
phase direct label RIA using 1-125 label (see, e.g., Morel et al.,
1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin
EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and
direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol.
32:77-82). Typically, such an assay involves the use of purified
antigen bound to a solid surface or cells bearing either of these,
an unlabelled test antigen binding protein and a labeled reference
antigen binding protein. Competitive inhibition is measured by
determining the amount of label bound to the solid surface or cells
in the presence of the test antigen binding protein. Usually the
test antigen binding protein is present in excess. Antigen binding
proteins identified by competition assay (competing antigen binding
proteins) include antigen binding proteins binding to the same
epitope as the reference antigen binding proteins and antigen
binding proteins binding to an adjacent epitope sufficiently
proximal to the epitope bound by the reference antigen binding
protein for steric hindrance to occur. Additional details regarding
methods for determining competitive binding are provided in the
examples herein. Usually, when a competing antigen binding protein
is present in excess, it will inhibit (e.g., reduce) specific
binding of a reference antigen binding protein to a common antigen
by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%
or 75% or more. In certain embodiments, binding is inhibited by at
least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
[0326] In one aspect of the invention, competition assays may be
used to identify an antibody that competes with anti-PCSK9 antibody
508.20.04a, 508.20.04b, 508.20.06, 508.20.28a, 508.20.28b,
508.20.33a, 508.20.33b or 508.20.84 for binding to PCSK9. In
certain embodiments, such a competing antibody binds to the same
epitope (e.g., a linear or a conformational epitope) that is bound
by anti-PCSK9 antibody 508.20.04a, 508.20.04b, 508.20.06,
508.20.28a, 508.20.28b, 508.20.33a, 508.20.33b and/or 508.20.84.
Detailed exemplary methods for mapping an epitope to which an
antibody binds are provided in Morris (1996) "Epitope Mapping
Protocols," in Methods in Molecular Biology vol. 66 (Humana Press,
Totowa, N.J.).
[0327] In an exemplary competition assay, immobilized PCSK9 is
incubated in a solution comprising a first labeled antibody that
binds to PCSK9 (e.g., anti-PCSK9 antibody 508.20.04a, 508.20.04b,
508.20.06, 508.20.28a, 508.20.28b, 508.20.33a, 508.20.33b or
508.20.84) and a second unlabeled antibody that is being tested for
its ability to compete with the first antibody for binding to
PCSK9. The second antibody may be present in a hybridoma
supernatant. As a control, immobilized PCSK9 is incubated in a
solution comprising the first labeled antibody but not the second
unlabeled antibody. After incubation under conditions permissive
for binding of the first antibody to PCSK9, excess unbound antibody
is removed, and the amount of label associated with immobilized
PCSK9 is measured. If the amount of label associated with
immobilized PCSK9 is substantially reduced in the test sample
relative to the control sample, then that indicates that the second
antibody is competing with the first antibody for binding to PCSK9.
See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14
(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0328] 2. Activity Assays
[0329] In one aspect, assays are provided for identifying
anti-PCSK9 antibodies thereof having biological activity.
Biological activity of the anti-PCSK9 antibodies may include, e.g.,
blocking, antagonizing, suppressing, interfering, modulating and/or
reducing one or more biological activities of PCSK9. Antibodies
having such biological activity in vivo and/or in vitro are
provided.
[0330] In certain embodiments, anti-PCSK9 antibody binds human
PCSK9 and prevents interaction with the LDLR. In certain
embodiments, anti-PCSK9 antibody binds specifically to human PCSK9
and/or substantially inhibits binding of human PCSK9 to LDLR by at
least about 20%-40%, 40-60%, 60-80%, 80-85%, or more (for example,
by measuring binding in an in vitro competitive binding assay). In
certain embodiments, the invention provides isolated anti-PCSK9
antibodies which specifically bind to PCSK9 and which antagonize
the PCSK9-mediated effect on LDLR levels when measured in vitro
using the LDLR down regulation assay in HepG2 cells disclosed
herein.
[0331] D. Immunoconjugates
[0332] The invention also provides immunoconjugates comprising an
anti-PCSK9 antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0333] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0334] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PANII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0335] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125 Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or I123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0336] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0337] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0338] E. Methods and Compositions for Diagnostics and
Detection
[0339] In certain embodiments, any of the anti-PCSK9 antibodies
provided herein is useful for detecting the presence of PCSK9 in a
biological sample. The term "detecting" as used herein encompasses
quantitative or qualitative detection. In certain embodiments, a
biological sample is blood, serum or other liquid samples of
biological origin. In certain embodiments, a biological sample
comprises a cell or tissue.
[0340] In one embodiment, an anti-PCSK9 antibody for use in a
method of diagnosis or detection is provided. In a further aspect,
a method of detecting the presence of PCSK9 in a biological sample
is provided. In certain embodiments, the method comprises detecting
the presence of PCSK9 protein in a biological sample. In certain
embodiments, PCSK9 is human PCSK9. In certain embodiments, the
method comprises contacting the biological sample with an
anti-PCSK9 antibody as described herein under conditions permissive
for binding of the anti-PCSK9 antibody to PCSK9, and detecting
whether a complex is formed between the anti-PCSK9 antibody and
PCSK9. Such method may be an in vitro or in vivo method. In one
embodiment, an anti-PCSK9 antibody is used to select subjects
eligible for therapy with an anti-PCSK9 antibody, e.g. where PCSK9
or LDL-cholesterol is a biomarker for selection of patients.
[0341] Exemplary disorders that may be diagnosed using an antibody
of the invention include cholesterol related disorders (which
includes "serum cholesterol related disorders"), including any one
or more of the following: hypercholesterolemia, heart disease,
metabolic syndrome, diabetes, coronary heart disease, stroke,
cardiovascular diseases, Alzheimers disease and generally
dyslipidemias, which can be manifested, for example, by an elevated
total serum cholesterol, elevated LDL, elevated triglycerides,
elevated very low density lipoprotein (VLDL), and/or low HDL. In
one aspect, the invention provides a method for treating or
preventing hypercholesterolemia, and/or at least one symptom of
dyslipidemia, atherosclerosis, cardiovascular disease (CVD) or
coronary heart disease, in an individual comprising administering
to the individual an effective amount of anti-PCSK9 antibody. In
certain embodiments, the invention provides an effective amount of
an anti-PCSK9 antibody for use in treating or preventing
hypercholesterolemia, and/or at least one symptom of dyslipidemia,
atherosclerosis, CVD or coronary heart disease, in a subject. The
invention further provides the use of an effective amount of an
anti-PCSK9 antibody that antagonizes extracellular or circulating
PCSK9 in the manufacture of a medicament for treating or preventing
hypercholesterolemia, and/or at least one symptom of dyslipidemia,
atherosclerosis, CVD or coronary heart disease, in an
individual.
[0342] In certain embodiments, labeled anti-PCSK9 antibodies are
provided. Labels include, but are not limited to, labels or
moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0343] F. Pharmaceutical Formulations
[0344] This invention also encompasses compositions, including
pharmaceutical formulations, comprising an anti-PCSK9 antibody, and
polynucleotides comprising sequences encoding an anti-PCSK9
antibody. In certain embodiments, compositions comprise one or more
antibodies that bind to PCSK9, or one or more polynucleotides
comprising sequences encoding one or more antibodies that bind to
PCSK9. These compositions may further comprise suitable carriers,
such as pharmaceutically acceptable excipients including buffers,
which are well known in the art.
[0345] Pharmaceutical formulations of an anti-PCSK9 antibody as
described herein are prepared by mixing such antibody having the
desired degree of purity with one or more optional pharmaceutically
acceptable carriers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Pharmaceutically acceptable
carriers are generally nontoxic to recipients at the dosages and
concentrations employed, and include, but are not limited to:
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Exemplary pharmaceutically acceptable
carriers herein further include insterstitial drug dispersion
agents such as soluble neutral-active hyaluronidase glycoproteins
(sHASEGP), for example, human soluble PH-20 hyaluronidase
glycoproteins, such as rHuPH20 (HYLENEX.RTM., Baxter International,
Inc.). Certain exemplary sHASEGPs and methods of use, including
rHuPH20, are described in US Patent Publication Nos. 2005/0260186
and 2006/0104968. In one aspect, a sHASEGP is combined with one or
more additional glycosaminoglycanases such as chondroitinases.
[0346] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0347] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide statin. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0348] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0349] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0350] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0351] In one aspect, the invention provides a composition
comprising an anti-PCSK9 antibody at about 100 to about 225 mg/mL,
arginine succinate at about 180 to about 220 mM, polysorbate at
about 0.01% to about 0.03%, and pH at about 5.2 to about 5.8. In
certain embodiments, the composition is suitable for subcutaneous
administration. In certain embodiments, the viscosity of the
composition is less than about 25 cP at 25.degree. C., less than
about 20 cP at 25.degree. C., less than about 15 cP at 25.degree.
C., less than about 12 cP at 25.degree. C., or less than about 10
cP at 25.degree. C. In certain embodiments, the composition is
stable for at least one month, at least two months, at least three
months, at least four months, at least five months, or at least six
months at 2-8.degree. C. In some embodiments, the composition is in
a 0.5-mL, 1-mL, 1.25-mL, 1.5-mL, 1.75-mL, 2-mL, 2.25-mL, or 2.5-mL
pre-filled syringe. In certain embodiments, the antibody in the
composition is about any of 110, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,
220, and 225 mg/mL, including concentrations between any of these
concentrations. In certain embodiments, arginine succinate in the
composition is about any of 180, 185, 190, 200, 210 and 220 mM,
including concentrations between any of these concentrations. In
certain embodiments, polysorbate (e.g., polysorbate 20, polysorbate
80) in the composition is about any of 0.01%, 0.015%, 0.02%,
0.025%, and 0.03%, including concentrations between any of these
concentrations. In certain embodiments, the composition has a pH at
any of 5.0, 5.2, 5.4, 5.5, 5.6, 5.8, 5.9, 6.0, 6.1 and 6.2,
including pH between any of these values. In certain embodiments,
the anti-PCSK9 antibody in the composition is at about 150 mg/mL,
arginine succinate in the composition is at about 200 mM, and
polysorbate 20 in the composition is about 0.02%, and pH at about
5.5.
[0352] In one aspect, the invention provides a composition
comprising an anti-PCSK9 antibody at about 150 to about 225 mg/mL,
histidine acetate at about 10 to about 30 mM, arginine acetate at
about 150 to about 170 mM, polysorbate at about 0.01% to about
0.03%, and pH at about 5.8 to about 6.2. In certain embodiments,
the composition is suitable for subcutaneous administration. In
certain embodiments, the viscosity of the composition is less than
about 25 cP at 25.degree. C., less than about 20 cP at 25.degree.
C., less than about 15 cP at 25.degree. C., less than about 12 cP
at 25.degree. C., or less than about 10 cP at 25.degree. C. In
certain embodiments, the composition is stable for at least one
month, at least two months, at least three months, at least four
months, at least five months, or at least six months at 2-8.degree.
C. In some embodiments, the composition is in a 0.5-mL, 1-mL,
1.25-mL, 1.5-mL, 1.75-mL, 2-mL, 2.25-mL, or 2.5-mL pre-filled
syringe. In certain embodiments, the antibody in the composition is
about any of 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,
205, 210, 215, 220, and 225 mg/mL, including concentrations between
any of these concentrations. In certain embodiments, histidine
acetate in the composition is about any of 10, 15, 20, 25, and 30
mM, including concentrations between these concentrations. In
certain embodiments, arginine acetate in the composition is about
any of 150, 155, 160, 165, and 170 mM, including concentrations
between any of these concentrations. In certain embodiments,
polysorbate (e.g., polysorbate 20, polysorbate 80) in the
composition is about any of 0.01%, 0.015%, 0.02%, 0.025%, and
0.03%, including concentrations between any of these
concentrations. In certain embodiments, the composition has a pH at
any of 5.8, 5.9, 6.0, 6.1 and 6.2, including pH between any of
these values. In certain embodiments, the anti-PCSK9 antibody in
the composition is at about 200 mg/mL, histidine acetate in the
composition is at about 20 mM, arginine acetate in the composition
is at about 160 mM, and polysorbate 20 in the composition is about
0.02%, and pH at about 6.0.
[0353] Also provided herein is a subcutaneous administration device
containing the anti-PCSK9 antibody in a composition described
herein, for delivering to an individual a flat dose in the range of
200 mg to 1200 mg of the antibody. A complete dose for one
administration may be in one or more of the devices. In certain
embodiments, the concentration of the antibody in the device is
about 200 mg/mL. In certain embodiments, the device is a pre-filled
syringe (e.g., 0.5-mL syringe, 1-mL syringe, 1.25-mL syringe,
1.5-mL syringe, 1.75-mL syringe, 2-mL syringe, 2.25-mL syringe, or
2.5-mL syringe) or a high volume, single use, subcutaneous infusion
device (e.g., for delivery of from 1-10 mL, 2-8 mL, 3-6 mL, 4-5 mL,
or 4, 5, 6, 7, 8, 9, or 10 mL).
[0354] G. Therapeutic Methods and Compositions
[0355] Any of the anti-PCSK9 antibodies provided herein may be used
in therapeutic methods.
[0356] In one aspect, an anti-PCSK9 antibody for use as a
medicament is provided. In another aspect, an anti-PCSK9 antibody
for use in treating conditions associated with cholesterol related
disorder is provided. In certain embodiments, an anti-PCSK9
antibody for use in treating conditions associated with elevated
level of LDL-cholesterol is provided. In certain embodiments, an
anti-PCSK9 antibody for use in a method of treatment is provided.
In certain embodiments, the invention provides an anti-PCSK9
antibody for use in a method of treating an individual having
conditions associated with elevated level of LDL-cholesterol
comprising administering to the individual an effective amount of
the anti-PCSK9 antibody. In certain embodiments, the methods and
uses described herein further comprise administering to the
individual an effective amount of at least one additional
therapeutic agent, e.g., statin. In certain embodiments, the
invention provides an anti-PCSK9 antibody for use in reducing
LDL-cholesterol level in a subject. In further embodiments, the
invention provides an anti-PCSK9 antibody for use in lowering serum
LDL-cholesterol level in a subject. In certain embodiments, the
invention provides an anti-PCSK9 antibody for use in increasing
availability of LDLR in a subject. In certain embodiments, the
invention provides an anti-PCSK9 antibody for use in inhibiting
binding of PCSK9 to LDLR in a subject. In certain embodiments, the
invention provides an anti-PCSK9 antibody for use in a method of
reducing LDL-cholesterol level in an individual comprising
administering to the individual an effective of the anti-PCSK9
antibody to reduce the LDL-cholesterol level. In certain
embodiments, the invention provides an anti-PCSK9 antibody for use
in a method of lowering serum LDL-cholesterol level in an
individual comprising administering to the individual an effective
of the anti-PCSK9 antibody to lower the serum LDL-cholesterol
level. In certain embodiments, the invention provides an anti-PCSK9
antibody for use in a method of increasing availability of LDLR in
an individual comprising administering to the individual an
effective of the anti-PCSK9 antibody to increase availability of
LDLR. In certain embodiments, the invention provides an anti-PCSK9
antibody for use in a method of inhibiting binding of PCSK9 to LDLR
in an individual comprising administering to the individual an
effective of the anti-PCSK9 antibody to inhibit the binding of
PCSK9 to LDLR. An "individual" or "subject" according to any of the
embodiments described herein is preferably a human.
[0357] In a further aspect, the invention provides for the use of
an anti-PCSK9 antibody in the manufacture or preparation of a
medicament. In one embodiment, the medicament is for treatment of
cholesterol related disorder. In certain embodiments, the
cholesterol related disorder is hypercholesterolemia. In another
embodiment, the medicament is for use in a method of treating
hypercholesterolemia comprising administering to an individual
having hypercholesterolemia an effective amount of the
medicament.
[0358] In certain embodiments, the disorder treated is any disease
or condition which is improved, ameliorated, inhibited or prevented
by removal, inhibition or reduction of PCSK9 activity. In certain
embodiments, diseases or disorders that are generally addressable
(either treatable or preventable) through the use of statins can
also be treated. In certain embodiments, disorders or disease that
can benefit from the prevention of cholesterol synthesis or
increased LDLR expression can also be treated by anti-PCSK9
antibodies of the present invention. In certain embodiments,
individuals treatable by the anti-PCSK9 antibodies and therapeutic
methods of the invention include individuals indicated for LDL
apheresis, individuals with PCSK9-activating mutations (gain of
function mutations, "GOF"), individuals with heterozygous Familial
Hypercholesterolemia (heFH), individuals with primary
hypercholesterolemia who are statin intolerant or statin
uncontrolled, and individuals at risk for developing
hypercholesterolemia who may be preventably treated. Other
indications include dyslipidemia associated with secondary causes
such as Type 2 diabetes mellitus, cholestatic liver diseases
(primary biliary cirrhosis), nephrotic syndrome, hypothyroidism,
obesity, and the prevention and treatment of atherosclerosis and
cardiovascular diseases. In certain embodiments, the individuals
treatable by the anti-PCSK9 antibodies and therapeutic methods
described herein include individuals with LDL-c levels of 90-250
mg/dL and with coronary heart disease (CHD) or a CHD risk
equivalent as described in detail in Example 12.
[0359] In certain embodiments, the methods described herein
comprise administering an anti-PCSK9 antibody to an individual
suffering from coronary heart disease. In certain embodiments, an
individual with coronary heart disease has a history of documented
myocardial infarction. In certain embodiments, an individual with
coronary heart disease refers to an individual who has had a prior
coronary revascularization procedure (e.g., percutaneous coronary
intervention or coronary artery bypass graft). In certain
embodiments, an individual with coronary heart disease refers to an
individual having at least one coronary atherosclerotic lesion with
50% diameter stenosis (e.g., as determined by coronary angiography
including invasive coronary angiography or cardiac computed
tomography coronary angiography).
[0360] In certain embodiments, the methods described herein
comprise administering an anti-PCSK9 antibody to an individual
having at least one CHD risk equivalent. In certain embodiments, an
individual with a CHD risk equivalent is an individual having one
or more forms of clinical atherosclerotic disease, such as, for
example, peripheral arterial disease (e.g., ankle/brachial blood
pressure index of <0.85, prior percutaneous or surgical
peripheral arterial revascularization procedure, prior
non-traumatic amputation of a lower extremity due to peripheral
artery disease, or 50% diameter stenosis on prior vascular
imaging), carotid artery disease (e.g., carotid atherosclerotic
lesion with 50% diameter stenosis or prior cutaneous or surgical
carotid revascularization procedure), prior ischemic stroke, or
abdominal aortic aneurysm. In certain embodiments, an individual
with a CHD risk equivalent is an individual having type II
diabetes. In certain embodiments, an individual with a CHD risk
equivalent is an individual having type I diabetes coupled with
organ damage (e.g., retinopathy, neuropathy, or nephropathy
including microalbuminuria). In certain embodiments, an individual
with a CHD risk equivalent is an individual having moderate to
severe chronic kidney disease.
[0361] In certain embodiments, the methods described herein
comprise administering an anti-PCSK9 antibody to an individual
having one or more of the following risk factors: age (.gtoreq.45
years for men or .gtoreq.55 years for women), smoking (within 1
month), hypertension (systolic blood pressure .gtoreq.140 mmHg,
diastolic blood pressure .gtoreq.90 mmHg, or taking an
antihypertensive medication), low HDL cholesterol (<40 mg/dL),
or a family history of premature CHD.
[0362] In certain embodiments, the methods and uses described
herein further comprises administering to the individual an
effective amount of at least one additional therapeutic agent,
e.g., statin. In certain embodiments, the additional therapeutic
agent is for preventing and/or treating atherosclerosis and/or
cardiovascular diseases. In certain embodiment, the additional
therapeutic agent is for use in a method of reducing the risk of
recurrent cardiovascular events. In certain embodiments, the
additional therapeutic agent is for elevating the level of
HDL-cholesterol in a subject.
[0363] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the anti-PCSK9 antibodies provided
herein, e.g., for use in any of the above therapeutic methods. In
one embodiment, a pharmaceutical formulation comprises any of the
anti-PCSK9 antibodies provided herein and a pharmaceutically
acceptable carrier. In another embodiment, a pharmaceutical
formulation comprises any of the anti-PCSK9 antibodies provided
herein and at least one additional therapeutic agent, e.g.,
statin.
[0364] Antibodies of the invention can be used either alone or in
combination with other agents in a therapy. For instance, an
antibody of the invention may be co-administered with at least one
additional therapeutic agent. In certain embodiments, such
additional therapeutic agent elevates the level of LDLR. In certain
embodiments, an additional therapeutic agent is a LDL-cholesterol
lowering drugs such as statin, e.g., atorvastatin, fluvastatin,
lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,
simvastatin, or any combination thereof, e.g., VYTORIN.RTM.,
ADVICOR.RTM. or SIMCOR.RTM.. In certain embodiments, an additional
therapeutic agent is a HDL-cholesterol raising drugs.
[0365] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the anti-PCSK9 antibody of the
invention can occur prior to, simultaneously, and/or following,
administration of the additional therapeutic agent and/or
adjuvant.
[0366] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. Dosing can be by any suitable
route, e.g., by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
[0367] Anti-PCSK9 antibodies of the invention would be formulated,
dosed, and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The antibody need not
be, but is optionally formulated with one or more agents currently
used to prevent or treat the disorder in question. The effective
amount of such other agents depends on the amount of antibody
present in the formulation, the type of disorder or treatment, and
other factors discussed above. These are generally used in the same
dosages and with administration routes as described herein, or
about from 1 to 99% of the dosages described herein, or in any
dosage and by any route that is empirically/clinically determined
to be appropriate.
[0368] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 mg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of
antibody can be an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 mg/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered.
[0369] In certain embodiments, a flat-fixed dosing regimen is used
to administer anti-PCSK9 antibody to an individual. Depending on
the type and severity of the disease an exemplary flat-fixed dosage
might range from 10 to 1200 mg of anti-PCSK9 antibody. One
exemplary dosage of the antibody would be in the range from about
10 mg to about 1000 mg. Another exemplary dosage of the antibody
would be in the range from about 100 mg to about 600 mg. Another
exemplary dosage of the antibody would be in the range from about
200 mg to about 800 mg. Another exemplary dosage of the antibody
would be in the range from about 350 mg to about 400 mg. Another
exemplary dosage of the antibody would be in the range from about
750 mg to about 800 mg. In certain embodiments, 150 mg, 200 mg, 220
mg, 300 mg, 380 mg, 400 mg, 500 mg, 600 mg, 700 mg, 760 mg, 800 mg,
1000 mg, 1140 mg, or 1200 mg of anti-PCSK9 antibody is administered
to an individual. In certain embodiments, the flat dose of the
anti-PCSK9 antibody is administered every 2 weeks, every 4 weeks,
every 6 weeks, every 8 weeks, every 10 weeks, or every 12 weeks. In
certain embodiments, the flat dose of the anti-PCSK9 antibody is
administered no more frequently than once every 2 weeks, every 4
weeks, every 6 weeks, every 8 weeks, every 10 weeks, or every 12
weeks. In certain embodiments, the flat dose of the anti-PCSK9
antibody is administered every month, every 1.5 months, every 2
months, every 2.5 months, or every 3 months. In certain
embodiments, the flat dose of the anti-PCSK9 antibody is
administered no more frequently than once every month, every 1.5
months, every 2 months, every 2.5 months, or every 3 months. In
certain embodiments, the anti-PCSK9 antibody is administered
subcutaneously. However, other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional
techniques and assays.
[0370] In certain embodiments, the flat, fixed, subcutaneous dose
to be administered is provided in a volume that is less than or
equal to 5 mL, 4.5 mL, 4 mL, 3.8 mL, 3.5 mL, 3 mL, 2.5 mL, 2 mL,
1.9 mL, 1.5 mL, or 1 mL. In certain embodiments, the flat, fixed,
subcutaneous dose is 800 mg in a total volume of less than or equal
to 4 mL. In certain embodiments, the flat, fixed, subcutaneous dose
is 760 mg in a total volume of less than or equal to 3.8 mL. In
certain embodiments, the flat, fixed, subcutaneous dose is 600 mg
in a total volume of less than or equal to 3 mL. In certain
embodiments, the flat, fixed, subcutaneous dose is 400 mg in a
total volume of less than or equal to 2 mL. In certain embodiments,
the flat, fixed, subcutaneous dose is 380 mg in a total volume of
less than or equal to 1.9 mL.
[0371] In certain embodiments, the LDL-cholesterol level in the
individual treated by the methods described herein is reduced by at
least about 45%, at least about 50%, at least about 55%, or at
least about 60% from baseline. In some embodiments, the
LDL-cholesterol level in the individual treated by the methods
described is reduced at least about 45%, at least about 50%, at
least about 55%, or at least about 60% from baseline, and maintains
at the reduced level for at least two weeks, at least one month, at
least two months, or three months after last dosing. In some
embodiments, the LDL-cholesterol level in the individual treated by
the methods described is reduced at least about 45%, at least about
50%, at least about 55%, or at least about 60% from baseline within
about 1 week, about 10 days, or about 2 weeks of the initial dose.
In some embodiments, the LDL-cholesterol level in the individual
treated by the methods described is reduced at least about 45%, at
least about 50%, at least about 55%, or at least about 60% from
baseline within about 1 week, about 10 days, or about 2 weeks of
the initial dose, and maintains at the reduced level for at least
two weeks, at least one month, at least two months, or three months
after last dosing. In certain embodiments, the LDL-cholesterol
level in the individual treated by the methods described is reduced
at least about 45% and maintains at the reduced level for at least
about six weeks, at least about 7 weeks or at least about 1.5
months. In certain embodiments, the LDL-cholesterol level in the
individual treated by the methods described is reduced at least
about 45% within about 1 week from the initial dose and maintains
at the reduced level for at least about six weeks, at least about 7
weeks or at least about 1.5 months. In certain embodiments, the
LDL-cholesterol level in the individual treated by the methods
described is reduced at least about 50% and maintains at the
reduced level for at least about four weeks or at least about 1
month. In certain embodiments, the LDL-cholesterol level in the
individual treated by the methods described is reduced at least
about 50% within about 10 days from the initial dose and maintains
at the reduced level for at least about four weeks or at least
about 1 month. In certain embodiments, the LDL-cholesterol level in
the individual treated by the methods described is reduced at least
about 50% and maintains at the reduced level for at least about
eight weeks or at least about 2 months. In certain embodiments, the
LDL-cholesterol level in the individual treated by the methods
described is reduced at least about 50% within about 10 days from
the initial dose and maintains at the reduced level for at least
about eight weeks or at least about 2 months. In certain
embodiments, the LDL-cholesterol level in the individual treated by
the methods described is reduced at least about 55% and maintains
at the reduced level for at least about two weeks. In certain
embodiments, the LDL-cholesterol level in the individual treated by
the methods described is reduced at least about 55% within about 2
weeks of the initial dose and maintains at the reduced level for at
least about two weeks. As used herein, a "baseline" level (such as
baseline level for LDL-cholesterol level) in an individual refers
to the level before an administration of an anti-PCSK9 antibody
described herein to the individual. In certain embodiments, the
baseline may be a mean or average of two or more measurements
obtained before administration of an anti-PCSK9 antibody.
[0372] In certain embodiments, the LDL-cholesterol level in the
individual treated by the methods described herein is reduced by at
least about 60 mg/dL, at least about 70 mg/dL, at least about 75
mg/dL, at least about 80 mg/dL, or at least about 90 mg/dL from
baseline. In some embodiments, the LDL-cholesterol level in the
individual treated by the methods described is reduced by at least
about 60 mg/dL, at least about 70 mg/dL, at least about 75 mg/dL,
at least about 80 mg/dL, or at least about 90 mg/dL from baseline,
and maintains at the reduced level for at least two weeks, at least
one month, at least two months, or three months after last dosing.
In some embodiments, the LDL-cholesterol level in the individual
treated by the methods described is reduced by at least about 60
mg/dL, at least about 70 mg/dL, at least about 75 mg/dL, at least
about 80 mg/dL, or at least about 90 mg/dL from baseline within
about 1 week, about 10 days, or about 2 weeks of the initial dose.
In some embodiments, the LDL-cholesterol level in the individual
treated by the methods described is reduced by at least about 60
mg/dL, at least about 70 mg/dL, at least about 75 mg/dL, at least
about 80 mg/dL, or at least about 90 mg/dL from baseline within
about 1 week, about 10 days, or about 2 weeks of the initial dose,
and maintains at the reduced level for at least two weeks, at least
one month, at least two months, or three months after last dosing.
In certain embodiments, the LDL-cholesterol level in the individual
treated by the methods described is reduced by at least about 60
mg/dL or 70 mg/dL and maintains at the reduced level for at least
about six weeks, at least about 7 weeks or at least about 1.5
months. In certain embodiments, the LDL-cholesterol level in the
individual treated by the methods described is reduced by at least
about 60 mg/dL or 70 mg/dL within about 1 week from the initial
dose and maintains at the reduced level for at least about six
weeks, at least about 7 weeks or at least about 1.5 months. In
certain embodiments, the LDL-cholesterol level in the individual
treated by the methods described is reduced by at least about 80
mg/dL and maintains at the reduced level for at least about four
weeks or at least about 1 month. In certain embodiments, the
LDL-cholesterol level in the individual treated by the methods
described is reduced by at least about 80 mg/dL within about 10
days from the initial dose and maintains at the reduced level for
at least about four weeks or at least about 1 month. In certain
embodiments, the LDL-cholesterol level in the individual treated by
the methods described is reduced by at least about 90 mg/dL and
maintains at the reduced level for at least about two weeks. In
certain embodiments, the LDL-cholesterol level in the individual
treated by the methods described is reduced by at least about 90
mg/dL within about 2 weeks of the initial dose and maintains at the
reduced level for at least about two weeks.
[0373] In certain embodiments, the reduction in LDL-cholesterol
levels is maintained within a certain range between dosings. In
certain embodiments, upon administration of a dose of an anti-PCSK9
antibody, LDL-cholesterol levels are reduced to a nadir of at least
about 45%, at least about 50%, at least about 55%, or at least
about 60% from baseline and do not increase beyond about 40%, 45%,
50%, 55%, or 60% below baseline before the next dosing of the
anti-PCSK9 antibody. In certain embodiments, upon administration of
a dose of an anti-PCSK9 antibody, LDL-cholesterol levels are
reduced to a nadir of at least about 45% from baseline and do not
increase beyond about 40% or 45% below baseline before the next
dosing of the anti-PCSK9 antibody. In certain embodiments, upon
administration of a dose of an anti-PCSK9 antibody, LDL-cholesterol
levels are reduced to a nadir of at least about 50% from baseline
and do not increase beyond about 40%, 45%, or 50% below baseline
before the next dosing of the anti-PCSK9 antibody. In certain
embodiments, upon administration of a dose of an anti-PCSK9
antibody, LDL-cholesterol levels are reduced to a nadir of at least
about 55% from baseline and do not increase beyond about 40%, 45%,
50%, or 55% below baseline before the next dosing of the anti-PCSK9
antibody. In certain embodiments, upon administration of a dose of
an anti-PCSK9 antibody, LDL-cholesterol levels are reduced to a
nadir of at least about 60% from baseline and do not increase
beyond about 40%, 45%, 50%, 55%, or 60% below baseline before the
next dosing of the anti-PCSK9 antibody.
[0374] In certain embodiments, the reduction in LDL-cholesterol
levels is maintained within a certain range between dosings. In
certain embodiments, upon administration of a dose of an anti-PCSK9
antibody, LDL-cholesterol levels are reduced to a nadir of at least
about 60 mg/dL, at least about 70 mg/dL, at least about 75 mg/dL,
at least about 80 mg/dL, or at least about 90 mg/dL below baseline
and do not increase beyond about 55 mg/dL, 60 mg/dL, 65 mg/dL, 70
mg/dL, 75 mg/dL, 80 mg/dL or 90 mg/dL below baseline before the
next dosing of the anti-PCSK9 antibody. In certain embodiments,
upon administration of a dose of an anti-PCSK9 antibody,
LDL-cholesterol levels are reduced to a nadir of at least about 60
mg/dL below baseline and do not increase beyond about 55 mg/dL or
60 mg/dL below baseline before the next dosing of the anti-PCSK9
antibody. In certain embodiments, upon administration of a dose of
an anti-PCSK9 antibody, LDL-cholesterol levels are reduced to a
nadir of at least about 70 mg/dL below baseline and do not increase
beyond about 55 mg/dL, 60 mg/dL, 65 mg/dL, or 70 mg/dL below
baseline before the next dosing of the anti-PCSK9 antibody. In
certain embodiments, upon administration of a dose of an anti-PCSK9
antibody, LDL-cholesterol levels are reduced to a nadir of at least
about 75 mg/dL below baseline and do not increase beyond about 55
mg/dL, 60 mg/dL, 65 mg/dL, 70 mg/dL, or 75 mg/dL below baseline
before the next dosing of the anti-PCSK9 antibody. In certain
embodiments, upon administration of a dose of an anti-PCSK9
antibody, LDL-cholesterol levels are reduced to a nadir of at least
about 80 mg/dL below baseline and do not increase beyond about 55
mg/dL, 60 mg/dL, 65 mg/dL, 70 mg/dL, 75 mg/dL, or 80 mg/dL below
baseline before the next dosing of the anti-PCSK9 antibody. In
certain embodiments, upon administration of a dose of an anti-PCSK9
antibody, LDL-cholesterol levels are reduced to a nadir of at least
about 90 mg/dL below baseline and do not increase beyond about 55
mg/dL, 60 mg/dL, 65 mg/dL, 70 mg/dL, 75 mg/dL, 80 mg/dL or 90 mg/dL
below baseline before the next dosing of the anti-PCSK9
antibody.
[0375] In one embodiment, an anti-PCSK9 antibody is administered to
a subject at a dose of 800 mg every 8 weeks, wherein the level of
LDL-cholesterol in the subject is reduced by at least 50% below
baseline within 10 days and does not increase to more than 40% or
45% below baseline before the next dose. In one embodiment, an
anti-PCSK9 antibody is administered to a subject at a dose of 760
mg every 8 weeks, wherein the level of LDL-cholesterol in the
subject is reduced by at least 45% below baseline within 14 days
and does not increase to more than 35% or 40% below baseline before
the next dose. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at a dose of 400 mg every 4 weeks,
wherein the level of LDL-cholesterol in the subject is reduced by
at least 50% below baseline within 10 days and does not increase to
more than 45% or 50% below baseline before the next dose. In one
embodiment, an anti-PCSK9 antibody is administered to a subject at
a dose of 380 mg every 4 weeks, wherein the level of
LDL-cholesterol in the subject is reduced by at least 50% below
baseline within 10 days and does not increase to more than 45% or
50% below baseline before the next dose.
[0376] In certain embodiments, subjects receiving the anti-PCSK9
antibody are monitored for LDL-c levels and if their levels drop
below 25 or 15 mg/dL, then their dose is adjusted down to around
50% or 25% of the initial dose, by reducing the total amount of
antibody administered to around 50% or 25% of the initial dose
administered and keeping the frequency of injections the same, by
keeping the total amount of antibody administered the same but
decrease the frequency by 2-fold or 4-fold (e.g., from once every 4
weeks to once every 8 weeks or 16 weeks), or a combination thereof
(e.g., by reducing the dose and changing the frequency of
administration). In certain embodiments, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 800 mg every 8
weeks. The subject is monitored and if the LDL-c levels of the
subject drop below 25 or 15 mg/dL, then the subject is switched to
a dose of 400 mg every 8 weeks, 400 mg every 16 weeks, 380 mg every
8 weeks, 380 mg every 16 weeks, 200 mg every 8 weeks, 200 mg every
4 weeks, 190 mg every 8 weeks, 190 mg every 4 weeks, 760 mg every
16 weeks or 4 months, or 760 mgs every 24 weeks or 6 month 800 mg
every 16 weeks or 4 months, or 800 mgs every 24 weeks or 6 months.
In one embodiment, an anti-PCSK9 antibody is administered to a
subject at an initial dose of 800 mg every 8 weeks, the subject is
monitored and if the subject's LDL-c levels drop below 25 mg/dL,
the subject is switched to a dose of 200 mg every 8 weeks. In one
embodiment, an anti-PCSK9 antibody is administered to a subject at
an initial dose of 800 mg every 8 weeks, the subject is monitored
and if the subject's LDL-c levels drop below 15 mg/dL, the subject
is switched to a dose of 200 mg every 8 weeks. In certain
embodiments, an anti-PCSK9 antibody is administered to a subject at
an initial dose of 760 mg every 8 weeks. The subject is monitored
and if the LDL-c levels of the subject drop below 25 or 15 mg/dL,
then the subject is switched to a dose of 380 mg every 8 weeks, 380
mg every 16 weeks, 200 mg every 4 weeks, 200 mg every 8 weeks, 190
mg every 8 weeks, 190 mg every 4 weeks, 760 mg every 16 weeks or 4
months, or 760 mgs every 24 weeks or 6 months. In one embodiment,
an anti-PCSK9 antibody is administered to a subject at an initial
dose of 760 mg every 8 weeks, the subject is monitored and if the
subject's LDL-c levels drop below 25 mg/dL, the subject is switched
to a dose of 190 mg or 200 mg every 8 weeks. In one embodiment, an
anti-PCSK9 antibody is administered to a subject at an initial dose
of 760 mg every 8 weeks, the subject is monitored and if the
subject's LDL-c levels drop below 15 mg/dL, the subject is switched
to a dose of 190 mg or 200 mg every 8 weeks. In certain
embodiments, an anti-PCSK9 antibody is administered to a subject at
an initial dose of 400 mg every 4 weeks. The subject is monitored
and if the LDL-c levels of the subject drop below 25 or 15 mg/dL,
then the subject is switched to a dose of 200 mg every 4 weeks, 200
mg every 8 weeks, 100 mg every 4 weeks, 400 mg every 8 weeks, 400
mgs every 16 weeks or 3 months, 50 mgs every 2 weeks, or 25 mgs
every 2 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 100 mg
every 4 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 100 mg
every 4 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 200 mg
every 8 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 200 mg
every 8 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 50 mg
every 2 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 50 mg
every 2 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 25 mg
every 2 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 400 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 25 mg
every 2 weeks. In certain embodiments, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks. The subject is monitored and if the LDL-c levels of the
subject drop below 25 mg/dL or 15 mg/dL, then the subject is
switched to a dose of 200 mg every 4 weeks, 200 mg every 8 weeks,
190 mg every 4 weeks, 100 mg every 4 weeks, 90 mg every 4 weeks,
380 mg every 8 weeks, 380 mgs every 16 weeks or 3 months, 50 mg
every 2 weeks, or 25 mg every 2 weeks. In one embodiment, an
anti-PCSK9 antibody is administered to a subject at an initial dose
of 380 mg every 4 weeks, the subject is monitored and if the
subject's LDL-c levels drop below 25 mg/dL, the subject is switched
to a dose of 100 mg every 4 weeks. In one embodiment, an anti-PCSK9
antibody is administered to a subject at an initial dose of 380 mg
every 4 weeks, the subject is monitored and if the subject's LDL-c
levels drop below 15 mg/dL, the subject is switched to a dose of
100 mg every 4 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 200 mg
every 8 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 200 mg
every 8 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 190 mg
every 8 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 190 mg
every 8 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 50 mg
every 2 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 50 mg
every 4 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 25 mg/dL, the subject is switched to a dose of 25 mg
every 2 weeks. In one embodiment, an anti-PCSK9 antibody is
administered to a subject at an initial dose of 380 mg every 4
weeks, the subject is monitored and if the subject's LDL-c levels
drop below 15 mg/dL, the subject is switched to a dose of 25 mg
every 2 weeks.
[0377] It is understood that any of the above formulations or
therapeutic methods may be carried out using an immunoconjugate of
the invention in place of or in addition to an anti-PCSK9
antibody.
[0378] H. Articles of Manufacture and Kits
[0379] In another aspect of the invention, an article of
manufacture or kit containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. In certain embodiments, the article of manufacture or kit
comprises a container containing one or more of the anti-PCSK9
antibodies or the compositions described herein. In certain
embodiments, the article of manufacture or kit comprises a
container and a label or package insert on or associated with the
container. Suitable containers include, for example, bottles,
vials, syringes, IV solution bags, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is by itself or combined with
another composition effective for treating, preventing and/or
diagnosing the condition and may have a sterile access port (for
example the container may be an intravenous solution bag or a vial
having a stopper pierceable by a hypodermic injection needle). At
least one active agent in the composition is an anti-PCSK9 antibody
of the invention. The label or package insert indicates that the
composition is used for treating the condition of choice. Moreover,
the article of manufacture or kit may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises an anti-PCSK9 antibody of the invention; and
(b) a second container with a composition contained therein,
wherein the composition comprises a further cytotoxic or otherwise
therapeutic agent. In certain embodiments, the second container
comprises a second therapeutic agent, wherein the second
therapeutic agent is a cholesterol-lowering drug of the "statin"
class. In certain embodiments, the statin is and/or comprises
atorvastatin (e.g., LIPITOR.RTM. or Torvast), fluvastatin (e.g.,
LESCOL.RTM.), lovastatin (e.g., MEVACOR.RTM., ALTOCOR.TM., or
ALTOPREV.RTM.), mevastatin (pitavastatin (e.g., LIVALO.RTM. or
PITAVA.RTM.), pravastatin (e.g., PRAVACHOL.RTM., SELEKTINE.RTM.,
LIPOSTAT.RTM.), rosuvastatin (e.g., CRESTOR.RTM.), simvastatin
(e.g., ZOCOR.RTM., LIPEX.RTM.), or any combination thereof, e.g.,
VYTORIN.RTM., ADVICOR.RTM. or SIMCOR.RTM..
[0380] The article of manufacture or kit in this embodiment of the
invention may further comprise a package insert indicating that the
compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0381] It is understood that any of the above articles of
manufacture or kit may include an immunoconjugate of the invention
in place of or in addition to an anti-PCSK9 antibody.
III. EXAMPLES
[0382] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1
Generation of Anti-PCSK9 Antibodies
[0383] Residue numbers are according to Kabat (Kabat et al.,
Sequences of proteins of immunological interest, 5th Ed., Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)).
[0384] Library Sorting and Screening to Identify Anti-PCSK9
Antibodies
[0385] Biotinylated human PCSK9 generated in-house was used as
antigen for library sorting. The phage libraries were sorted five
rounds against biotinylated PCSK9 in solution phase. For the first
round of sorting, 20 .mu.g/mL biotinylated PCSK9 was added to
antibody phage libraries VH (see, e.g., Lee et al., J. Immunol.
Meth. 284:119-132, 2004) and VH/VL (see Liang et al., JMB. 366:
815-829, 2007) pre-blocked with phage blocking buffer PBST
(phosphate-buffered saline (PBS) and 1% (w/v) bovine serum albumin
(BSA) and 0.05% (v/v) TWEEN.RTM. 20) and incubated overnight at
room temperature. The following day 120 .mu.l of PBST/BSA
pre-absorbed DYNABEADS.RTM. MyOne.TM. Streptavidin T1 (Invitrogen,
Carlsbad, Calif.) was added to each library and incubated for 1
hour at room temperature. The beads were then washed three times
with PBT (PBS with 0.05% TWEEN.RTM. 20), and bound phage were
eluted with 1 mL 50 mM HCl and 500 mM NaCl for 30 minutes and
neutralized with 400 .mu.L of 1 M Tris base (pH7.5). Recovered
phages were amplified in E. coli XL-1 Blue cells. During the
subsequent selection rounds, incubation of antibody phage with the
biotinylated PCSK9 was reduced to 2-3 hours, and the phage bound
antigen was captured for 30 minutes on neutravidin-coated (Catalog
#89890, 10 .mu.g/ml, Fisher Scientific, Waltham, Mass.) or
streptavidin-coated (Catalog #21125, 10 .mu.g/ml, Fisher
Scientific, Waltham, Mass.) Nunc 96 well Maxisorp.TM. immunoplates.
The stringency of plate washing was gradually increased.
[0386] After 5 rounds of panning, significant enrichment was
observed. 96 clones were picked each from VH and VH/VL library
sorting to determine whether they specifically bound to human
PCSK9. The variable regions of these clones were PCR sequenced to
identify unique sequence clones. Unique phage antibodies that bind
human PCSK9 at least 5.times. above background were chosen and
reformatted to full length IgGs for evaluation in in vitro cell
assay.
[0387] Clones of interest were reformatted into IgGs by cloning VL
and VH regions of individual clones into the LPG3 and LPG4 vector
respectively, transiently expressing in mammalian CHO cells, and
purifying with a protein A column.
[0388] Construct Libraries for Affinity Improvement of Clones
Derived from the VH Library
[0389] Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et
al., J. Mol. Biol 340, 1073-1093 (2004)), containing stop codon
(TAA) in all CDR-L3 positions and displaying monovalent Fab on the
surface of M13 bacteriophage served as the library template for
grafting heavy chain variable domains (VH) of clones of interest
from the VH library for affinity maturation. Both hard and soft
randomization strategies were used for affinity maturation. For
hard randomization, one light chain library with selected positions
of the three light chain CDRs was randomized using amino acids
designed to mimic natural human antibodies and the designed DNA
degeneracy was as described in Lee et al. (J. Mol. Biol 340,
1073-1093 (2004)). For soft randomization, residues at positions
91-96 of CDR-L3, 30-33, 35 of CDR-H1, 50, 52, 53-54, 56, and 58 of
CDR-H2, 95-100, 100A, and 100C of CDR-H3, were targeted; and three
different combinations of CDR loops, H1/L3, H2/L3, and H3/L3, were
selected for randomization. To achieve the soft randomization
conditions, which introduced the mutation rate of approximately 50%
at the selected positions, the mutagenic DNA was synthesized with
70-10-10-10 mixtures of bases favoring the wild type nucleotides
(Gallop et al., Journal of Medicinal Chemistry 37:1233-1251
(1994)).
[0390] Phage Sorting Strategy to Generate Affinity Improvement
[0391] For affinity improvement selection, phage libraries were
subjected to five rounds of solution sorting with increasing
stringency. For the first round of solution sorting, 3 O.D./ml in
1% BSA and 0.05% TWEEN.RTM. 20 of phage input were incubated with
100 nM biotinylated PCSK9 (the concentration is based on parental
clone phage 1050 value) in 100 .mu.l buffer containing 1%
SUPERBLOCK.RTM. (Pierce Biotechnology) and 0.05% TWEEN.RTM. 20 for
2 hours at room temperature. The mixture was further diluted
10.times. with 1% SUPERBLOCK.RTM., and 100 .mu.l/well was applied
to neutravidin-coated wells (10 m/ml) for 30 minutes at room
temperature with gentle shaking. The wells were washed with
PBS-0.05% TWEEN.RTM. 20 ten times. To determine background binding,
control wells containing phage were captured on neutravidin-coated
plates. Bound phage was eluted with 150 .mu.l/well 50 mM HCl, 500
mM KCl for 30 minutes, and subsequently neutralized by 50
.mu.l/well of 1M Tris pH8, titered, and propagated for the next
round. Four more rounds of solution sorting were carried out
together with increasing selection stringency. The first couple of
rounds were for on-rate selection by decreasing biotinylated target
protein concentration from 100 nM to 1 nM, and the last two rounds
were for off-rate selection by adding excess amounts of
non-biotinylated target protein (300 to 1000 fold more) to compete
off weaker binders at room temperature.
[0392] High Throughput Affinity Screening ELISA (Single Spot
Competition)
[0393] Colonies were picked from the fifth round of screening.
Colonies were grown overnight at 37.degree. C. in 150 .mu.l/well of
2YT media with 50 .mu.g/ml carbenicillin and 1E10/ml KO7 in 96-well
plate (Falcon). From the same plate, a colony of XL-1 infected
parental phage was picked as control. 96-well Nunc Maxisorp.TM.
plates were coated with 100 .mu.l/well of neutravidin (10 .mu.g/ml)
in PBS at 4.degree. C. overnight. The plates were blocked with 150
.mu.l of 1% BSA and 0.05% TWEEN.RTM. 20 in PBS for 1 hour.
[0394] 35 .mu.l of the phage supernatant was diluted with 350 of
ELISA (enzyme linked immunosorbent assay) buffer (PBS with 0.5%
BSA, 0.05% TWEEN.RTM. 20) with or without 15 nM PCSK9 and let
incubate for 1 hour at room temperature in an F plate (NUNC). 35
.mu.l of 3 .mu.g/ml biotinylated-PCSK9 was then added to each well
and incubated for 15 minutes at room temperature. 95 .mu.l of
mixture was transferred side by side to the neutravidin coated
plates. The plate was gently shaken for 15 min to allow the capture
of biotinylated-PCSK9 bound phage to the plate. The plate was
washed ten times with PBS-0.05% TWEEN.RTM. 20. The binding was
quantified by adding horseradish peroxidase (HRP)-conjugated
anti-M13 antibody in ELISA buffer (1:2500) and incubated for 30
minutes at room temperature. The plates were washed with PBS-0.05%
TWEEN.RTM. 20 ten times. Next, 100 .mu.l/well of a 1:1 ratio of
3,3',5,5'-tetramethylbenzidine (TMB) Peroxidase substrate and
Peroxidase Solution B (H.sub.2O.sub.2) (Kirkegaard-Perry
Laboratories (Gaithersburg, Md.)) was added to the well and
incubated for 5 minutes at room temperature. The reaction was
stopped by adding 100 .mu.l 10.1M Phosphoric Acid (H.sub.3PO.sub.4)
to each well and allowed to incubate for 5 minutes at room
temperature. The O.D. (optical density) of the yellow color in each
well was determined using a standard ELISA plate reader at 450 nm.
The O.D. reduction (%) was calculated by the following
equation:
OD.sub.450nm reduction (%)=[(OD.sub.450nm of wells with
competitor)/(OD.sub.450nm of well with no competitor)]*100
[0395] In comparison to the OD.sub.450nm reduction (%) of the well
of parental phage (100%), clones that had the OD.sub.450nm
reduction (%) lower than 50% were picked for sequence analysis.
Unique clones were selected for phage preparation to determine
binding affinity (phage IC.sub.50) against PCSK9 by comparison to
parental clone (clone 508.20b). Then the most affinity-improved
clones (508.20.04b, 508.20.06, 508.20.28b, 508.20.33b and
508.20.84) were reformatted into human IgG.sub.1 for antibody
production and further BIAcore binding kinetic analysis and other
in vitro or in vivo assay. See FIGS. 1 and 2.
Example 2
Characterization of Anti-PCSK9 Antibodies by BIAcore
[0396] Binding affinities of anti-PCSK9 antibodies were measured by
Surface Plasmon Resonance (SRP) using a BIAcore.TM.-3000
instrument. Anti-PCSK9 human antibodies were captured by mouse
anti-human Fc antibody (Catalog # BR-1008-39, GE Healthcare,
Piscataway, N.J.) coated on CM5 biosensor chips to achieve
approximately 200 response units (RU). For kinetics measurements,
two-fold serial dilutions (500 nM to 0.245 nM) of human, murine,
rhesus, and cyno PCSK9 (Genentech, South San Francisco, Calif.)
were injected in PBT buffer (PBS with 0.05% TWEEN.RTM. 20) at
25.degree. C. with a flow rate of 300 min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) were calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2). The equilibrium dissociation constant
(K.sub.D) was calculated as the ratio k.sub.off/k.sub.on. See FIG.
3.
Example 3
LDLR-PCSK9 Binding Assay
[0397] A competition binding ELISA was performed to investigate the
activity of anti-PCSK9 antibodies in blocking human PCSK9 binding
to LDLR. Briefly, 1 .mu.g/mL of soluble human LDLR extracellular
domain (R&D Systems, Minneapolis, Minn.) was coated on 384-well
MaxiSorp.TM. plate (NALGENE.RTM. NUNC.TM. International, Rochester,
N.Y.) at 4.degree. C. overnight. Then 0.25 .mu.g/mL of biotinylated
human PCSK9 pre-mixed with different concentrations of anti-PCSK9
antibodies and control antibodies were added to the plate and
incubated for 2 hour at room temperature. The binding of PCSK9 to
coated LDLR was detected by adding streptavidin-HRP (GE Healthcare)
and substrate 3, 3', 5, 5'-tetramethyl benzidine (TMBE-1000, Moss,
Inc., Pasadena, Md.). The binding results (OD) were plotted against
antibody concentration and IC.sub.50 values were generated using
KaleidaGraph software. See FIG. 4.
Example 4
Antibodies Prevent LDLR Downregulation on HepG2 Cells
[0398] HepG2 cells were seeded at 1.times.10.sup.5 into a 48-well
plate. The next day, the media was changed to 10% lipoprotein
deficient serum (LPDS, Frederick, Md.). The following day, 15
.mu.g/ml PCSK9 plus/minus anti-PCSK9 antibody were added to cells
for 4 hours at 37.degree. C. Cells were rinsed with PBS and
detached using 2.5 mM EDTA. Cells were incubated with 1:20
anti-LDLR (Progen Biotechnik, Heidelberg, Germany) for 15 minutes,
washed with PBS and incubated with 1:200 goat anti-mouse
ALEXAFLUOR.RTM. 488 from Invitrogen (Carlsbad, Calif.) for 15
minutes. Cells were washed and resuspended in PBS plus 10 .mu.g/ml
propidium iodide. The samples were then analyzed with a dual laser
flow cytometer (FACScan.TM., Becton Dickinson, Franklin Lakes,
N.J.). The data suggest all five of the anti-PCSK9 antibodies
(508.20.04b, 508.20.06, 508.20.28b, 508.20.33b and 508.20.84)
prevent downregulation of LDLR. See FIG. 5.
Example 5
LDLR Downregulation in Mouse Liver
[0399] Normal C57/BL6 mice (Charles River, Wilmington, Mass.) were
treated with 3, 30 or 60 .mu.g of PCSK9 by I.V. administration.
Using the PROTEOEXTRACT.RTM. Native Membrane Protein Extraction Kit
from Calbiochem (Gibbstown, N.J.) according to the manufacturer's
instructions, liver from each mouse was harvested 15 min, 1 hr or 4
hrs after PCSK9 I.V. administration and proteins extracted. As a
control, 5 mice were treated with vehicle only and 8 .mu.g of each
liver lysate were pooled for analysis. Lysates were analyzed by
SDS-PAGE on 8% tris-gly gel (Invitrogen, Carlsbad, Calif.).
Proteins were transferred to nitrocellulose membrane using
IBLOT.RTM. (Invitrogen). The membrane was blocked with 5% nonfat
milk for 1 hour and then incubated with 1:500 anti-LDLR (Abcam,
Cambridge, Mass.) in 5% nonfat milk overnight at 4.degree. C. The
next day, the membrane was washed three times with TBS-T, incubated
with 1:5000 anti-rabbit HRP (GE Healthcare, Piscataway, N.J.) for 1
hour and washed with TBS-T three times. Proteins were visualized
using ECL-Plus (GE Healthcare) and exposed to XAR film (KODAK.RTM.,
Rochester, N.Y.). After an overnight exposure, the membrane was
washed with TBS-T, incubated with 1:500 anti-transferrin receptor
antibody (Invitrogen) for 1 hour, washed with TBS-T, incubated with
1:5000 anti-mouse HRP (GE Healthcare) for 1 hour, washed with TBS-T
and visualized with ECL-Plus. Western blot with anti-LDLR antibody
shows that 30 .mu.g of PCSK9 for 1 hour significantly downregulated
LDLR levels in mouse liver. See FIG. 6.
Example 6
Antibodies Prevent Liver LDLR Downregulation
[0400] Normal C57/BL6 (Charles River) mice were injected with
vehicle or 5 mg/kg anti-PCSK9 antibodies 24 hours prior to
treatment with 30 .mu.g PCSK9 for 1 hour. Liver from each mouse was
harvested using the PROTEOEXTRACT.RTM. Native Membrane Protein
Extraction Kit (Calbiochem) according to the manufacturer's
instructions. Lysates were analyzed by SDS-PAGE on 8% bis-tris gel.
Proteins were transferred to nitrocellulose membrane using
IBLOT.RTM. (Invitrogen). The membrane was blocked with 5% nonfat
milk for 1 hour and then incubated with 1:500 anti-LDLR (Abcam) in
5% nonfat milk overnight at 4.degree. C. The next day, the membrane
was washed three times with TBS-T, incubated with 1:5000
anti-rabbit HRP (GE Healthcare) for 1 hour and washed three times
with TBS-T. Proteins were visualized using ECL-Plus (GE Healthcare)
and exposed to XAR film (KODAK.RTM.). Western blot with anti-LDLR
antibody show that all five anti-PCSK9 antibodies (508.20.84,
508.20.33b, 508.20.04b, 508.20.28b, 508.20.06) prevented LDLR
downregulation in mouse liver. See FIG. 7.
Example 7
Pharmacokinetics of Anti-PCSK9 Antibody
[0401] Anti-PCSK9 antibody concentrations in mouse PK study samples
were determined using anti-human IgG Fc ELISA. Briefly, donkey
anti-human IgG Fc (Jackson ImmunoResearch, West Grove, Pa.) was
used to coat assay plates and goat anti-human IgG Fc HRP conjugate
(Jackson ImmunoResearch, West Grove, Pa.) was used as detection
antibody. The assay was able to measure anti-PCSK9 antibody in up
to 10% mouse serum matrix with assay range of 0.31-20 ng/mL. See
FIGS. 8 and 9.
[0402] Serum anti-PCSK9 antibody concentrations in cynomolgus
monkey PK study samples were determined by anti-PCSK9 antibody
ELISA using recombinant human PCSK9 (Genentech, Inc. South San
Francisco, Calif.) as capture and goat anti-human IgG (H+L) HRP as
detection antibody. The assay was able to measure anti-PCSK9
antibody in up to 2% cynomolgus monkey serum matrix with assay
range of 0.313-50 ng/mL. See FIGS. 10 and 11.
Example 8
Antibodies Reduce Serum Cholesterol Level in Mice
[0403] Eight weeks old male C57BL/6J mice were purchased
commercially from Jackson Laboratory. The mice were on housing for
one week at the holding room before the start of the experiment.
All mice were pre-bled under anesthesia and total cholesterol
levels from the mice were determined using INFINITY.TM. Cholesterol
Reagent (Fisher Diagnostics, Middletown, Va.). The mice were
randomized into 6 different groups with the same level of average
cholesterol level. All mice received a single dose of 10 mg/kg body
weight of either control antibody or anti-PCSK9 antibodies. The
mice were bled on day 3, day 7, day 10 and day 15 and serum total
cholesterol levels were determined using INFINITY.TM. Cholesterol
Reagent (Fisher Diagnostics, Middletown, Va.).
[0404] All five anti-PCSK9 antibodies (508.20.04b, 508.20.06,
508.20.28b, 508.20.33b, 508.20.84) showed a reduction in total
cholesterol levels when a single dose of 10 mg/kg was administered.
The administration of anti-PCSK9 antibody resulted in a significant
reduction in total cholesterol level on day 3 and up to day 10 when
compared to the mice receiving control antibody. See FIG. 12.
Example 9
Enhancement of Statin Effectiveness
[0405] This experiment demonstrates that a combination of
anti-PCSK9 antibody and statin results in a greater reduction in
total cholesterol level compared to anti-PCSK9 antibody alone or
statin alone treatments. See e.g., FIG. 13. Eight weeks old male
C57BL/6J mice was purchased from Jackson Laboratory. The mice were
grouped into 2 different groups. The non-statin mice received
control diet, while statin groups received 0.2% of lovastatin in
the diet for 2 weeks prior to antibody administration (Bioserve,
Frenchtown, N.J.). All the mice were pre-bled and mice were
randomized based on equal average cholesterol level. Mice were bled
on day 3 and the total cholesterol levels were assayed using
INFINITY.TM. Cholesterol Reagent (Fisher Diagnostics, Middletown,
Va.).
[0406] The anti-PCSK9 antibodies showed significant cholesterol
lowering effect. Statin alone treatment resulted in modest
reduction in total cholesterol level, compared to non-statin
groups. The combination of statin plus anti-PCSK9 antibody resulted
in an additional reduction compared to anti-PCSK9 alone in total
cholesterol level. See FIG. 13.
Example 10
X-Ray Crystal Structure of PCSK9 Bound to Fab Fragment of
Anti-PCSK9 Antibody
[0407] Protein Purification and Crystallization
[0408] 210 g of frozen cell paste from 10 L E. coli expression were
thawed in 1 L of lysis buffer (PBS/25 mM EDTA/1 mM PMSF). Cells
were disrupted by Tissuemizer (30 seconds) and the resulting slurry
was passed through a microfluidizer twice. Insoluble matter was
pelleted by centrifugation. Clarified lysate (250 mL at a time) was
loaded onto a Protein G column (cat#17-0618-05, GE Healthcare) at 5
mL/min. The column was then washed with 100 mL of lysis buffer
before eluting the bound Fab fragment of anti-PCSK9 antibody with
150 mL of elution buffer (0.58% acetic acid). 25 mL fractions were
collected during elution. Fractions containing Fab fragment of
anti-PCSK9 antibody were pooled after SDS PAGE analysis.
[0409] 5 mL prepacked SPHP column (GE Healthcare, cat#17-1152-01)
were equilibrated with 50 ml of Buffer A (20 mM MES pH5.5). Pooled
fractions from the prior step were loaded onto the column at 3
mL/min. The column was washed with Buffer A to baseline. Bound Fab
fragment was eluted with buffer B (20 mM MES pH 5.5, 1M NaCl) using
a gradient from 0% to 100% buffer B in 20 column volumes. 2 mL
fractions were collected during elution. The fractions containing
the protein (determined using SDS-PAGE) were pooled and
concentrated to 5 mL before loaded onto a 320 mL S75 gel filtration
column that had been pre-equilibrated with sizing buffer (20 mM
Hepes 7.2, 150 mM NaCl). The sizing buffer was run continuously at
1.5 mL/min for 220 mins while collecting 2 mL fractions. The peak
fractions (A280) were analyzed using SDS-PAGE.
[0410] Human PCSK9 (Genbank EF692496) complementary
deoxyribonucleic acids (cDNAs) containing a histidine (His).sub.8
C-terminal tag (SEQ ID NO:32) were inserted into a mammalian
expression vector (pRK5) with a cytomegalovirus (CMV) promoter
using standard molecular biology techniques. Protein was expressed
by transient transfection of Chinese hamster ovary (CHO) cells and
purified from conditioned media using affinity chromatography on a
nickel-nitrilotriacetic-agarose column (Qiagen) followed by gel
filtration on a SEPHACRYL.RTM. S-200 column (GE Healthcare). The
correct masses of purified proteins were verified by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the
accuracy of amino acid sequences were confirmed by N-terminal
sequencing.
[0411] The purified Fab fragment of anti-PCSK9 antibody and 6.9 mg
of PCSK9 protein were mixed in 2-fold molar excess of the Fab
fragment and incubated at 4.degree. C. for 1 hour before
concentration to 5 mL. The concentrated mixture was then loaded
onto a Superdex 200 size exclusion column (cat#17-1071-01, GE
Healthcare) pre-equilibrated with sizing buffer. The sizing buffer
was continuously run at 1.5 mL/min for 220 mins while collecting 2
mL fractions. The peak fractions (A280) containing both PCSK9 and
Fab fragment of anti-PCSK9 antibody (SDS-PAGE) were pooled and
concentrated to 20 mg/mL. The concentrated complex was then used to
set up crystallization trials. Initial crystals were formed from a
1:1 mixture between protein and reservoir containing 1.3 M
potassium/sodium phosphate at pH 7 using sitting drops. Crysals
were optimized by varying the protein:reservoir ratio in hanging
drops. A selected crystal was treated with mother liquor
supplumented with 25% glycerol and preserved in liquid
nitrogen.
[0412] Structure Determination of the PCSK9:Fab Fragment of
Anti-PCSK9 Antibody Complex
[0413] Diffraction data extending to about 3.5 .ANG. resolution
were collected at synchrotron beamline SSRL 7-1 and integrated and
scaled in space group 1222. Approximate phases were obtained by the
method of molecular replacement, using the previously reported
structure of PCSK9 (Hampton et al., PNAS 104:14609-9 (2007), pdb
accession code 2QTW) and the previously reported structure of an
antibody Fv fragment (Eigenbrot et al., J Mol Biol 229:969-95
(1993), pdb accession code 1FVC). The constant region of Fab
fragment of anti-PCSK9 antibody was placed as a rigid body using a
part of a previously reported homologous structure (Eigenbrot et
al. supra, pdb accession code 1FVD) after partial refinement had
improved phases. The final refined structure has crystallographic
R-values of 25 & 30%. Data collection and refinement statistics
appear in Table 1 below.
TABLE-US-00004 TABLE 1 Data collection space group I222 unit cell
(.ANG., .degree.) a = 92.283, b = 142.523, c = 253.983 V.sub.M
(.ANG..sup.3/Dalton) 2.8 Resolution (.ANG.) 40-3.5 (3.63-3.50)
Rsym.sup.a,b 0.184 (0.807) Number of observations 157526 Unique
reflections 21579 Completeness (%).sup.b 100 (100) I/.sigma.I.sup.b
11 (2.6) Wilson B (.ANG..sup.2) 58 Refinement Resolution (.ANG.)
40-3.5 Number of reflections 20644 (F > 0.sigma.(F)) Final
R.sup.c, R.sub.FREE 0.247, 0.295 complexes/asymmetric unit 1
protein residues 994 solvent molecules 0 atoms 7463 Mean B-factor
(.ANG..sup.2) 86 Rmsd bonds (.ANG.) 0.007 Rmsd angles (.degree.)
1.1 Rmsd bonded Bs (.ANG..sup.2) 2.4/1.9 Number of TLS groups 4
Ramachandran (%) 81.5/16.8/0.6/1.1 .sup.aRsym = .SIGMA.||I| -
|<I>||/.SIGMA.|<I>|, where I is the intensity of a
single observation and <I> the average intensity for symmetry
equivalent observations. .sup.bIn parenthesis, for the highest
resolution shell. .sup.cR = .SIGMA.|Fo - Fc|/.SIGMA.|Fo|, where Fo
and Fc are observed and calculated structure factor amplitudes,
respectively. R.sub.FREE is calculated as R for reflections
sequestered from refinement.
[0414] Determination of Epitope on PCSK9 from the X-Ray
Structure
[0415] A 4 .ANG. criterion was applied using the molecular analysis
program PyMOL. PCSK9 residues within 4 .ANG. of any part of the Fab
fragment of anti-PCSK9 antibody were determined as an epitope.
Based on the analysis, the epitope comprises one or more of the
following residues: R194, E195, D238, A239, A341, Q342, E366, D367,
1369, 5376, T377, C378, F379, 5381 and H391 of human PCSK9.
Example 11
Human Clinical Trial, Single and Multiple Ascending Doses
[0416] A randomized, double-blind, placebo-controlled, single and
multiple dose study was conducted to evaluate, primarily, the
safety and tolerability of single and multiple (four weekly) doses
of study drug (YW508.20.33b reformatted into human IgG.sub.1 having
a heavy chain with SEQ ID NO: 35 and a light chain with SEQ ID NO:
36) administered by subcutaneous (SC) injection to healthy
volunteers with elevated serum low-density lipoprotein cholesterol
(LDL-c) concentration. 80 healthy adult volunteers (men and women)
with elevated serum LDL-c concentrations (130-220 mg/dL) were
randomized into 10 cohorts each containing 8 subjects. Subjects in
each cohort were randomized to receive either study drug or placebo
(6 active and 2 placebo subject per cohort).
[0417] The cohorts were dosed as shown in FIG. 14 and the Table 2.
All doses were administered subcutaneously using syringes,
typically in the abdomen or thigh. The drug product was formulated
as 150 mg/mL antibody in 200 mM arginine succinate, 0.02%
polysorbate 20, pH 5.5. For the multiple dose cohorts, study drug
was administered once per week for four consecutive weeks. The
statin cohorts (H and I), were initially administered atorvastatin
at 20 mg once a day orally for at least 7 days, followed by a
safety and tolerability assessment. If the 20 mg dose was well
tolerated, the dose was increased to 40 mg daily and continued for
a minimum of 21 days prior to initiation of study drug on Day 1.
Subjects in cohorts H and I continued atorvastatin (40 mg PO daily)
until and including Day 35. Treatment was discontinued for any
subject whose direct LDL-c level fell below 25 mg/dL at any point
during the study.
TABLE-US-00005 TABLE 2 Overview of Study Dose Cohorts. Total doses
Follow-up Cohort Dose (mg) administered duration.sup.a Atorvastatin
A 10 1 8 weeks No B 40 1 8 weeks No C 150 1 12 weeks No D 300 1 12
weeks No E 600 1 16 weeks No F 40 4 16 weeks No G 150 4 16 weeks No
H 40 4 16 weeks Yes I 150 4 16 weeks Yes J 800 1 16 weeks No
.sup.a= Time between first dose of study drug and final study
visit.
[0418] Subjects were followed for 8 to 16 weeks following
initiation of study drug with frequent safety, PK and PD
assessments. The following data were evaluated: safety outcomes
(adverse events, abnormalities in hematology, clinical chemistry,
and urinalysis, and incidence of anti-therapeutic antibodies),
pharmacokinetic (PK) profile (including C.sub.max, total serum
apparent clearance (CL/F), apparent volume of distribution (V/F),
total exposure (AUC), t.sub.max, t.sub.1/2, and dose
proportionality (based on AUC)), pharmacodynamics outcomes (percent
and absolute reduction from baseline in LDL-c at day 15 in single
dose cohorts and day 36 in multiple dose cohorts), and percent and
absolute change from baseline over time in total cholesterol,
LDL-c, HDL-c, non-HDL-c, triglycerides, and lipid particle
sub-fractions.
[0419] Early results from the study have not identified a
drug-related, clinically significant pattern of adverse events.
There were no serious or severe adverse events, no discontinuations
for adverse events, and no dose-limiting toxicities. The tested
doses have not defined a maximum tolerated dose. Two moderate
adverse events have been reported: one headache (study drug-treated
subject in the 10-mg single dose cohort) and one radius fracture
(study drug-treated subject in the 600-mg single dose cohort). Five
study drug-treated subjects, all in multiple dose cohorts and
treated with concomitant atorvastatin, were discontinued from study
drug therapy because of LDL-c levels below the protocol-specified
threshold of 25 mg/dL. There were no associated adverse events in
these subjects.
[0420] As shown in FIG. 15 (left panel), there was a dose related
increase in exposure from 10-600 mg for study drug. No differences
in PK were observed between statin treated and untreated groups
(FIG. 15, right panel). There was a saturable clearance of study
drug with a Km of 5.94 ug/mL.
[0421] As shown in FIGS. 16-19 and Tables 3 and 4, study drug
produced clinically meaningful LDL-c reductions in healthy
volunteers, alone and in combination with statin. Pharmacodynamic
(PD) data showed a dose-dependent reduction in LDL-c that was
statistically significant in all cohorts except the 10 mg single
dose cohort. LDL-c decreased by 80-90 mg/dL in the highest dose
groups (300-800 mg in the single dose cohorts), from an average
baseline LDL-c of 160-170 mg/dL. Similar reductions in LDL-c levels
were seen between atorvastatin (cohorts H and I) and non-statin
cohorts (cohorts F and G) (see FIGS. 18 and 19 and Tables 3 and 4).
The differences between cohorts I and G (at day 10) and F and H (at
day 36) are not statistically significant. As shown in FIGS. 16 and
17, at doses .gtoreq.300 mg, the maximal LDL-c effect appears to
saturate but the duration of the effect lengthens. The data support
monthly or less frequent dosing.
TABLE-US-00006 TABLE 3 Absolute Change in LDL-c Levels from
Baseline in Single and Multiple Dose Cohorts. Mean (SD) Change in
LDL (mg/dL) Arm Active Placebo P-value.sup.a Single Dose (at day
15) A (10 mg) -18 (21) -5.6 (15) 0.22 B (40 mg) -45 (32) 0.03 C
(150 mg) -61 (17) <0.001 D (300 mg) -88 (28) <0.001 E (600
mg) -82 (22) <0.001 J (800 mg) -91 (14) <0.001 Multiple Dose
(at day 36) F (40 mg .times. 4) -50 (28) -9.7 (13) 0.016 G (150 mg
.times. 4) -71 (26) 0.001 H (A.sup.b + 40 mg .times. 4) -38 (10) -5
(14) 0.009 I (A.sup.b + 150 mg .times. 4) N/A.sup.c -15 (21)
N/A.sup.c .sup.a= The differences between cohorts I and G (at day
10) and F and H (at day 36) are not statistically significant.
.sup.b= A is Atorvastatin. .sup.c= Multiple subjects in cohort I
(150 mg .times. 4 + Atorvastatin) were discontinued after day 10
due to LDL levels falling below the protocol threshold of <25
mg/dL.
TABLE-US-00007 TABLE 4 Percent Change in LDL-c Levels from Baseline
in Single and Multiple Dose Cohorts. Mean % (SD) Change in LDL Arm
Active Placebo P-value Single Dose (at day 15) A (10 mg) -9.4 (11)
-3.7 (10) 0.3 B (40 mg) -23 (12) 0.008 C (150 mg) -37 (11)
<0.001 D (300 mg) -53 (10) <0.001 E (600 mg) -51 (18)
<0.001 J (800 mg) -58 (4) <0.001 Multiple Dose (at day 36) F
(40 mg .times. 4) -34 (19) -6.2 (8) 0.016 G (150 mg .times. 4) -49
(10) 0.001 H (A.sup.a + 40 mg .times. 4) -48 (17) -5.7 (16) 0.005 I
(A.sup.a + 150 mg .times. 4) -65 (13) -12 (24) 0.014 (at day 10)
.sup.a= A is Atorvastatin.
Example 12
Human Clinical Trial in Patients with Coronary Heart Disease (CHD)
or at High Risk of CHD
[0422] A randomized, double-blind, placebo-controlled, study of
study drug (YW508.20.33b reformatted into human IgG.sub.1 having a
heavy chain with SEQ ID NO: 35 and a light chain with SEQ ID NO:
36) will be conducted to evaluate the safety and efficacy of study
drug on top of standard-of-care (SOC) statin in patients with LDL-c
levels of 90-250 mg/dL and either coronary heart disease (CHD) or a
CHD risk equivalent. Approximately 224 patients (adult men and
women) with serum LDL-c concentrations of 90-250 mg/dL and either
CHD or a CHD risk equivalent will be randomized to one of five
study arms to be administered study drug or a placebo arm, as set
forth below in Table 5. All doses will be administered
subcutaneously using syringes. The drug product is formulated as
150 mg/mL antibody in 200 mM arginine succinate, 0.02% polysorbate
20, pH 5.5.
TABLE-US-00008 TABLE 5 Overview of Study Dose Cohorts. Study Drug
Planned Number Dose Regimen of Patients Arm Dose (mg) Frequency
(weeks) Active Drug Placebo A 400 4 56 -- B 200 8 14 -- C 400 8 28
-- D 800 8 56 -- E 800 12 14 -- F Placebo -- -- 56 (A-F) total --
-- 168 56
[0423] The study will include consecutive periods for screening
(0-4 weeks), run-in (0-6 weeks, if necessary), treatment (24 weeks;
Days 1-169), and follow-up (12 weeks). The study completion visit
at the end of the follow-up period (Day 253) will occur 16 weeks
after the final dose of study drug (Day 141). All patients,
regardless of treatment assignment, will receive SOC treatment,
including statins unless statins are not tolerated. All patients
will continue SOC statin therapy throughout the treatment and
follow-up periods, at the same dose they were receiving during the
run-in period and at enrollment. Other prescription and
over-the-counter (OTC) lipid-modifying therapies are not permitted.
Patients who have been taking a stable dose of SOC statin therapy
(or no statin and have documented intolerance to two or more
statins) and no other lipid-modifying therapy for at least 4 weeks
(or 6 weeks in the case of fibrates) at the time of screening will
not require a run-in period.
[0424] Patients will be monitored to determine efficacy based on
absolute change from baseline in LDL-c concentration at day 169. In
addition, patients will be monitored to determine secondary
efficacy outcomes including absolute change from baseline in LDL-c
concentration for each arm at the nadir for that arm; average value
over time of the change in LDL-c (absolute and percent change) for
each arm, up to Day 169, weighted by the number of weeks between
consecutive LDL-c measurements; percent change from baseline in
LDL-c concentration at Day 169 and at the nadir for each arm;
percent and absolute change from baseline in LDL-c concentration at
all other designated timepoints; and percent and absolute change
from baseline in total cholesterol, non-HDL-c, and apolipoprotein B
at Day 169 and at the nadir for each arm. Finally, patients will
also be monitored for safety including incidence, nature, and
severity of adverse events; incidence and nature of changes in
vital signs, physical findings, and clinical laboratory results
during and following study drug administration; and incidence of
anti-therapeutic antibodies directed against study drug.
[0425] The safety of low LDL-c values will be assessed regularly in
a blinded, exploratory manner. Study drug will be withheld from
patients with two consecutive LDL-c values of <15 mg/dL. This
will not be considered an adverse event. Such patients will be
treated with placebo instead, in blinded fashion, until LDL-c
increases to .gtoreq.50 mg/dL, after which these patients will be
switched to the lowest dosage (200 mg every 8 weeks). All doses of
active drug or placebo will be given according to the study drug
administration schedule, that is, on Days 1, 29 (.+-.2 days), 57
(.+-.2 days), 85 (.+-.2 days), 113 (.+-.4 days), and 141 (.+-.4
days) only.
[0426] The primary efficacy outcome measure is the change from
baseline in LDL-c at Day 169. Baseline LDL-c is defined as the
average of the last two measurements collected before the first
dose of study drug. The treatment comparisons between the study
drug doses and between each of the study drug doses and placebo
will be based on an analysis of covariance (ANCOVA), which will be
performed through a linear regression model adjusting for two
covariates: baseline LDL-c concentration (<120 mg/dL,
.gtoreq.120 mg/dL) and diabetes status (yes, no). The confidence
intervals, as well as the least-square estimates from the ANCOVA
models, will be used to aid in the interpretation of the study
results.
[0427] The eligibility criteria define a population of patients
with high cardiovascular and CHD risk based on risk categories in
the European Society of Cardiology (ESC)/European Atherosclerosis
Society (EAS) and National Cholesterol Education Program Adult
Treatment Panel III (NCEP ATP III) lipid-lowering guidelines. The
study aims to enroll patients who qualify for a therapeutic target
LDL-c level of 70 mg/dL according to these guidelines, but who have
not come close to this goal despite SOC statin therapy, either
because SOC is insufficient or because statins were not tolerated.
These patients are in need of additional safe and effective
LDL-c-lowering therapies.
[0428] CHD refers to a history of documented myocardial infarction,
prior coronary revascularization procedure (percutaneous coronary
intervention or coronary artery bypass graft), or prior coronary
angiography (invasive coronary angiography or cardiac computed
tomography coronary angiography) demonstrating at least one
coronary atherosclerotic lesion with .gtoreq.50% diameter
stenosis.
[0429] A CHD risk-equivalent condition is at least one of the
following:
[0430] 1. One or more forms of clinical atherosclerotic disease:
[0431] a. Peripheral arterial disease (previously documented
ankle/brachial blood pressure index <0.85, prior percutaneous or
surgical peripheral arterial revascularization procedure, prior
non-traumatic amputation of a lower extremity due to peripheral
artery disease, or .gtoreq.50% diameter stenosis on prior vascular
imaging), [0432] b. Carotid artery disease (previously documented
carotid atherosclerotic lesion with .gtoreq.50% diameter stenosis
on imaging or prior cutaneous or surgical carotid revascularization
procedure), [0433] c. Prior ischemic stroke, documented by CT or
MRI brain imaging, not due to embolism of cardiac origin (e.g.,
atrial fibrillation, valvular disease, or left ventricular mural
thrombus) in the opinion of the investigator, or [0434] d.
Abdominal aortic aneurysm with prior surgical or endovascular
repair.
[0435] 2. Diabetes mellitus type 2,
[0436] 3. Diabetes mellitus type 1 with target organ damage
(retinopathy, neuropathy, or nephropathy including
microalbuminuria, as determined by the investigator),
[0437] 4. Moderate to severe chronic kidney disease (manifested by
an estimated glomerular filtration rate of 15-60 mL/min/1.73
m.sup.2 using the Modification of Diet in Renal Disease equation
consistently over at least three measurements spanning at least 3
months, including screening laboratories), or
[0438] 5. Two or more of the CHD risk factors listed below AND
either an absolute 10-year risk of a CHD event .gtoreq.20% (as
determined by the National Cholesterol Education Program Adult
Treatment Panel III guideline modification of the Framingham risk
score) or a 10-year risk of a first fatal atherosclerotic event
.gtoreq.10% (determined by the Systemic Coronary Risk Estimation
system): [0439] a. Age 45 years for men or .gtoreq.55 years for
women, [0440] b. Current cigarette smoking (within 1 month), [0441]
c. Hypertension (screening systolic blood pressure .gtoreq.140
mmHg, diastolic blood pressure .gtoreq.90 mmHg, or taking an
antihypertensive medication to treat hypertension) [0442] d. Low
HDL cholesterol (<40 mg/dL), or [0443] e. Family history of
premature CHD (myocardial infarction or coronary revascularization
in a male first-degree relative <55 years of age or in a female
first-degree relative <65 years of age).
[0444] Standard-of-care statin therapy refers to a therapy meeting
one of the following conditions: (1) high-dose simvastatin (40 mg
daily), atorvastatin (40-80 mg daily), or rosuvastatin (20-40 mg
daily), (2) low-dose simvastatin, atorvastatin, or rosuvastatin and
documented intolerance of a high dose of that statin or of any dose
of another statin, (3) other statin (any dose) and documented
intolerance of simvastatin, atorvastatin, or rosuvastatin (any
dose), or (4) no statin and documented intolerance of at least two
statins (any statin, any dose).
[0445] Diabetes status will be determined based on the presence of
any one of the following, according to patient medical record or
history, or to screening laboratory test results: (1)
HbA.sub.1c>6.5%, (2) fasting plasma glucose .gtoreq.126 mg/dL
(7.0 mmol/L), (3) prior 2-hour plasma glucose .gtoreq.200 mg/dL
(11.1 mmol/L) during an oral glucose tolerance test (the test
should be performed as described by the World Health Organization,
with use of a glucose load containing the equivalent of 75 g of
anhydrous glucose dissolved in water), or (4) currently on an oral
or injectable therapy for a diagnosis of diabetes mellitus.
Example 13
Development of Stable, High Concentration Antibody Formulation
[0446] Initial clinical studies (see Examples 11 and 12) were
carried out using a formulation of anti-PCSK9 antibody
(YW508.20.33b reformatted into human IgG.sub.1 having a heavy chain
with SEQ ID NO: 35 and a light chain with SEQ ID NO: 36) formulated
at 150 mg/mL antibody in 200 mM arginine succinate, 0.02% (w/v)
polysorbate 20 at pH 5.5. However, a formulation with a higher
protein concentration (.gtoreq.200 mg/mL) and increased stability
was desired to facilitate administration of higher subcutaneous
doses that could be delivered monthly or less frequently.
Viscosity of Anti-PCSK9 Formulations
[0447] The viscosity of a 200 mM arginine succinate, 0.02% (w/v)
PS20, pH 5.5 anti-PCSK9 formulation was evaluated at various
protein concentrations. At each protein concentration, the
viscosity was measured at 5, 15, 25 and 40.degree. C. using a
rheometer (Anton Paar Physica MCR 501) with a shear rate of 1000
1/s.
[0448] Viscosity is an important parameter for subcutaneous dosing
of drug solution. A desirable viscosity limit for subcutaneous
delivery using a syringe is <10 cP at ambient temperature. The
viscosity of anti-PCSK9 at 100 to 300 mg/mL in 200 mM arginine
succinate, 0.02% (w/v) PS20, pH 5.5 is presented in Table 6. For
anti-PCSK9, viscosity is protein concentration and temperature
dependent. As protein concentration increases, viscosity also
increases. However, at each concentration, the viscosity can be
lowered by increasing temperature. By increasing the protein
concentration over 200 mg/mL, viscosity of anti-PCSK9 increased
exponentially (FIG. 20). Therefore, anti-PCSK9 at 200 mg/mL was
selected as the target concentration.
TABLE-US-00009 TABLE 6 Viscosity of anti-PCSK9 from 100 to 300
mg/mL antibody concentration. Temp Viscosity (cP).sup.1 (.degree.
C.) 100 mg/mL 150 mg/mL 200 mg/mL 225 mg/mL 250 mg/mL 275 mg/mL 300
mg/mL 5 4.6 .+-. 0.27 8.0 .+-. 0.16 18.6 .+-. 0.17 50.2 .+-. 0.99
76.9 .+-. 0.83 306 .+-. 5.8 603 .+-. 9.2 15 3.1 .+-. 0.10 5.2 .+-.
0.08 11.7 .+-. 0.15 31.9 .+-. 0.33 46.7 .+-. 0.91 179 .+-. 2.8 357
.+-. 4.8 25 2.5 .+-. 0.06 3.8 .+-. 0.09 8.3 .+-. 0.13 22.6 .+-.
0.14 31.0 .+-. 0.84 115 .+-. 0.6 225 .+-. 4.1 40 1.8 .+-. 0.01 2.7
.+-. 0.10 5.7 .+-. 0.26 15.5 .+-. 0.2 18.8 .+-. 0.63 74.8 .+-. 4.3
135 .+-. 3.2 .sup.1200 mM arginine succinate, 0.02% PS20, pH
5.5.
Agitation Study
[0449] An agitation study was performed to assess the minimum
amount of surfactant required to prevent or minimize aggregation of
anti-PCSK9 at 150 mg/mL in 200 mM arginine succinate, pH 5.5.
Polysorbate 20 (PS20) was added to the formulation to achieve
concentrations of 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1% (w/v). All
samples were sterile filtered, and 0.5 mL of each sample was filled
into 2-cc glass vial. Samples were agitated using Glas-Col benchtop
shaker set at 50 cycles/min with a sample displacement of 11 cm for
24 hours at room temperature (RT). The appropriate sample controls
(no shaking) in the corresponding configuration were placed in the
same vicinity of the shaker. All samples were analyzed by
size-exclusion chromatography (SEC) and turbidity by UV measurement
at 340-360 nm absorbance (abs).
[0450] The results are presented in FIG. 21. Without PS20 in the
formulation, the 24-hour agitated sample (at room temperature) had
obvious visible changes when compared to the unshaken control vial.
The agitated sample had a milky appearance with an increase in
turbidity and a 6% decrease in SEC main peak. With the addition of
.gtoreq.0.01% PS20 to the formulation, no differences were observed
by SEC and turbidity measurement between the control (without
agitation) and agitated samples in the vials. These results suggest
that the use of 0.01% PS20 was sufficient to prevent
agitation-induced aggregate formation of anti-PCSK9 at 200 mg/mL.
However, a concentration of 0.02% PS20 was selected as the target
concentration to account for potential degradation of the
surfactant during product storage.
Oxidation Potential of Anti-PCSK9 Formulations
[0451] Oxidation of anti-PCSK9 was determined by trypsin-peptide
map and the site(s) of oxidation was characterized by LC-MS.
Oxidation of anti-PCSK9 was induced by elevated temperature, light
and oxidizing agents such as hydrogen peroxide and
2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH). The
degradation conditions for preparing the oxidative samples are
summarized in Table 7. These oxidized anti-PCSK9 samples were also
evaluated for possible potency loss due to oxidation by measuring
its ability to inhibit PCSK9 binding to low density lipoprotein
receptor domain Fc (LDLR.sub.D-Fc) fusion protein as described in
Example 3.
[0452] For peptide mapping, samples were reduced with 1M
dithiothreitol, alkylated with 2.9 M iodoacetamide, and buffer
exchanged before digestion. Trypsin was used for a 1.5 hour
digestion at 37.degree. C. using an enzyme to protein ratio of
1:25. The digestion was quenched with 10% trifluoroacetic acid
(TFA) to a final pH 2-3. The resulting peptide digestion mixture
was analyzed by reverse-phase liquid chromatography with detection
by mass spectrometry (LC-MS) with a LTQ Orbitrap XL. The peptide
map utilized a linear gradient from 0-40% over 160 minutes at 0.25
mL/min in conjunction with a Phenomenex Jupiter C18 column (5
.mu.m, 2.times.250 mm, 300 .ANG.) maintained at 55.degree. C.
Mobile phases A and B consisted of 0.1% TFA in water and 0.09% TFA
in acetonitrile respectively. Peptides were also detected at 214
and 280 nm abs before MS analysis. LC-MS data was processed by
Mascot software to identify peptides and respective oxidation sites
of anti-PCSK9. Amount of oxidation in a sample was expressed as
"total oxidation per site" or accumulative oxidation since Trp and
Met produce multiple oxidation products and/or oxidation
states.
[0453] Methionine (Met/M) and Tryptophan (Trp/W) are the two common
amino acid residues that are easily oxidized in protein drug
products. W.sub.99 and M.sub.108 located in the
complementarity-determining region (CDR) Ill of the heavy chain and
the three Trp residues (W.sub.36, W.sub.111 and W.sub.486) adjacent
to the CDRs are the potential oxidation sites of anti-PCSK9.
Oxidation of these amino acid residues may result in loss of drug
potency due to their proximity to the CDRs. Peptide mapping
analysis of the degraded samples revealed that oxidation of
anti-PCSK9 mainly occurred at M.sub.256, M.sub.362, M.sub.432 and
M.sub.455 residues of the Fc portion (FIG. 22). When anti-PCSK9 was
degraded by exposing to light (room or UV) and oxidizing agents
such as H.sub.2O.sub.2 and AAPH, the relative amount of oxidation
per site for Met or Trp residues in/adjacent to the CDR was less
than 3% with no significant impact on potency (FIG. 23). Therefore,
anti-PSCK9 is considered not susceptible to oxidation and the use
of antioxidants in the protein formulation is not necessary.
TABLE-US-00010 TABLE 7 Anti-PCSK9 Degradation Conditions for
Oxidation Analysis. Degradation Mode Exposure Condition Expected
Degradation Thermal 2 weeks @ 40.degree. C. Oxidation Light 24
hours of Room Light Photo-oxidation 1.2 million lux hours Oxidizing
1000 ppM H.sub.2O.sub.2 Methionine Oxidation (24 hours @ 5.degree.
C.) Agents 5 mM AAPH Methionine + Tryptophan (24 hours @ 40.degree.
C.) Oxidation
pH Profile and Excipient Studies
[0454] The effect of formulation pH and excipients on anti-PCSK9
was evaluated at a protein concentration of 200 mg/mL. A pH range
of 5.0 to 6.5 in formulations containing arginine succinate,
histidine HCl or histidine acetate as buffer species and arginine
HCl or arginine acetate as solubilizers were assessed for
accelerated stability at 40.degree. C. (see Table 9) and viscosity
at 5.degree. C. and 25.degree. C. (see Table 8). The following
assays were used for the assessment: SEC, ion-exchange
chromatography (IEC), capillary electrophoresis-sodium dodecyl
sulfate (CE-SDS) and potency. A total of seven formulations were
evaluated.
[0455] IEC was performed on an Agilent 1100 HPLC and utilized a
Dionex ProPac.TM. WCX-10 column (4.times.250 mm) with mobile phase
A (20 mM HEPES, pH 7.9) and gradient from 1%-34% mobile phase B (20
mM HEPES, 100 mM NaCl, pH 7.9) in 50 minutes at a flow-rate of 0.9
mL/min. The column was maintained at 35.degree. C. The sample load
was 40 .mu.g, and the separation was monitored at 280 nm abs.
[0456] Effect of pH
[0457] The effect of pH on stability of anti-PCSK9 at 200 mg/mL in
200 mM arginine succinate, 0.02% PS20 was evaluated from pH 5.0,
5.5 and 6.0. As analyzed by SEC, IEC and CE-SDS, increasing the
formulation pH from 5.0 to 6.0 increased the stability of
anti-PCSK9 after 1 month at 40.degree. C. Compared to pH 5.0 and
5.5, the formulation at pH 6.0 had less acidic and basic peak
formation as determined by IEC. The formulation at pH 6.0 also had
a decrease in high molecular weight species (HMWS) as determined by
SEC and low molecular weight species by determined by both SEC and
CE-SDS. For the formulation at pH 6.5, anti-PCSK9 was formulated at
200 mg/mL in 20 mM histidine HCl, 160 mM arginine HCl, and 0.02%
PS20. The degradation rates of anti-PCSK9 at 40.degree. C. for all
formulations at pH 5.0 to 6.5 are shown in Table 9 and the pH rate
profiles for IEC and SEC are presented in FIG. 24. Based on the pH
rate profiles and degradation rates, a target pH 6.0 was
selected.
TABLE-US-00011 TABLE 8 Viscosity of anti-PCSK9 at 200 mg/mL in
Various Formulations. Viscosity Stabilizer/ (cP) Formulation Buffer
Excipients pH 5.degree. C. 25.degree. C. 1 200 mM Arginine 0.02%
PS20 5.0 18.2 7.7 2 Succinate 5.5 18.6 8.3 3 6.0 16.4 7.9 4 20 mM
Histidine 160 mM 6.0 18.0 7.6 5 HCl Arginine HCl, 6.5 17.5 7.3
0.02% PS20 6 20 mM Histidine 160 mM 5.5 16.4 7.7 7 Acetate Arginine
Acetate, 6.0 15.9 7.6 0.02% PS20
[0458] Effect of Buffer Species
[0459] The effect of buffer species on accelerated stability of 200
mg/mL anti-PCSK9 at pH 6.0 was evaluated in formulations containing
the following three buffer systems: (1) 160 mM arginine succinate,
(2) 20 mM histidine HCl and 160 mM arginine HCl, and (3) 20 mM
histidine acetate and 160 mM arginine acetate. All three
formulations contained 0.02% PS20. After 1 month at 40.degree. C.,
anti-PCSK9 had comparable CE-SDS profiles among the three buffer
systems (FIG. 26, top panel). No differences were observed by SEC
between histidine HCl/arginine HCl and histidine acetate/arginine
acetate buffer systems, while the use of the arginine succinate
buffer had a slight increase in a HMWS Peak (FIG. 26, middle
panel). By IEC analysis, the use of histidine HCl/arginine HCl
buffer system in the formulation had less acidic peak formation
when compared to histidine acetate/arginine acetate buffer system
and arginine succinate buffer (FIG. 26, bottom panel). However, the
overall degradation rates of anti-PCSK9 at 40.degree. C. determined
by SEC, IEC and CE-SDS are comparable in all three buffer systems
at pH 6.0 (Table 9).
TABLE-US-00012 TABLE 9 Degradation Rate for 200 mg/mL anti-PCK9 at
40.degree. C. in Various Formulations. % Loss/Month at 40.degree.
C. 200 mM Arginine 20 mM HisHCl, 20 mM HisAce, Succinate 160 mM
ArgHCl 160 mM ArgAce pH pH pH pH pH pH pH 5.0 5.5 6.0 6.0 6.5 5.5
6.0 SEC Main 3.8 2.7 2.2 2.1 2.9 2.1 2.1 Peak IEC Main 33 22 19
15.9 18 20.9 19 Peak CE-SDS 5.1 4.5 3.6 3.8 3.5 4.0 4.3 Main
Peak
[0460] The stability of anti-PCSK9 in two formulations (histidine
HCl, pH 6.0 and histidine acetate, pH 6.0) in a 1 mL syringe was
also evaluated.
[0461] At 5.degree. C., both formulations were stable for up to 6
months (Tables 10 and 11). At accelerated and stress conditions,
formation of acidic variants and aggregation are the major
degradation routes for anti-PCSK9 in liquid formulation. At
30.degree. C./65% relative humidity (RH) and 40.degree. C./75% RH,
the protein degraded faster in histidine acetate than histidine HCl
at pH 6.0 as determined by IEC (Table 11). No differences in
aggregation rate were observed by SEC and CE-SDS for either
formulation under the same storage conditions (Table 12). No
increase in oxidation was observed for both lead formulations when
stored at 5.degree. C. for up to 6 months. Although there was a
slight increase in oxidation of Met256 (.about.2%) in the Fc
portion in both formulations after 6 months at 30.degree. C./65%
RH, increase in oxidation of other Met and Trp residues was not
observed. Loss of potency was not observed in either formulation
for up to 6 months at 5.degree. C. and 30.degree. C./65% RH.
Similar results were obtained using a 2.25 mL syringe.
TABLE-US-00013 TABLE 10 Stability Data for 200 mg/mL anti-PCSK9 in
20 mM Histidine HCl, 160 mM Arginine HCl, 0.02% PS20, pH 6.0 in a
1-mL Syringe. IEC SEC Potency % Temp Timepoint Strength % Main %
Main CE-SDS % Relative .degree. C./% RH Days/Months mg/mL % Acidic
Peak % Basic % HMWS Peak % LMWS Main Peak Potency NA T = 0/0 209
11.6 73.1 15.1 0.7 99.2 0 96.1 114 5 28/1 210 11.9 73.5 14.5 0.6
99.3 0 96.2 101 5 61/2 206 11.7 72.9 15.3 0.6 99.3 0 96.0 100 5
91/3 208 11.6 73.3 15.0 0.6 99.3 0 96.0 101 5 183/6 208 12.4 71.9
15.6 0.7 99.2 0 95.3 103 30/65 28/1 210 15.0 69.5 15.3 0.7 99.1 0.1
95.6 NT 30/65 61/2 206 19.0 64.0 16.9 0.9 98.8 0.2 94.7 91 30/65
91/3 204 21.4 61.9 16.5 1.0 98.5 0.4 94.0 84 30/65 183/6 209 33.9
48.7 17.3 1.4 97.7 0.8 91.2 92 40/75 7/0.25 206 15.0 68.6 16.3 0.8
99.0 0.1 95.5 NT 40/75 14/0.5 206 18.9 64.3 16.7 0.9 98.8 0.2 95.0
NT 40/75 28/1 209 25.9 57.4 16.6 1.1 98.4 0.4 93.6 105 NT = not
tested.
TABLE-US-00014 TABLE 11 Stability Data for 200 mg/mL anti-PCSK9 in
20 mM Histidine Acetate, 160 mM Arginine Acetate, 0.02% PS20, pH
6.0 in a 1-mL syringe. IEC SEC Temp Timepoint Strength % Main %
Main % Main % Relative (.degree. C.) Days/Months mg/mL % Acidic
Peak % Basic % HMWS Peak % LMWS Peak Potency NA T = 0/0 211 11.8
72.7 15.4 0.6 99.3 0 96.2 100 5 28/1 203 12.1 72.8 14.9 0.6 99.4 0
96.2 106 5 61/2 208 11.7 72.8 15.4 0.6 99.3 0 96.0 97 5 91/3 208
11.6 72.9 15.3 0.6 99.3 0 96.0 92 5 183/6 207 12.5 71.6 15.8 0.7
99.2 0 95.6 98 30/65 28/1 209 16.8 67.5 15.6 0.7 99.1 0.1 95.6 NT
30/65 61/2 210 21.6 61.8 16.4 0.9 98.8 0.2 94.7 98 30/65 91/3 205
26.1 57.4 16.3 1.0 98.6 0.3 94.3 87 30/65 183/6 205 41.4 42.5 16.0
1.5 97.6 0.8 91.1 91 40/75 7/0.25 206 17.0 66.6 16.2 0.8 99.0 0.1
95.4 NT 40/75 14/0.5 204 22.5 60.0 16.2 0.9 98.8 0.2 94.5 NT 40/75
28/1 196 31.8 51.9 16.1 1.1 98.4 0.4 93.5 106 NT = not tested.
TABLE-US-00015 TABLE 12 Degradation Rates for anti-PCSK9 in a 1-mL
Syringe at Accelerated Stability Conditions. Histidine Histidine %
Change Per Month HCl.sup.1 Acetate.sup.2 IEC 30.degree. C./65% RH
4.0 5.0 40.degree. C./75% RH 16.7 22.0 SEC 30.degree. C./65% RH 0.2
0.2 40.degree. C./75% RH 0.8 0.9 CE-SDS 30.degree. C./65% RH 0.8
0.8 40.degree. C./75% RH 2.6 2.8 .sup.1Histidine HCl = 200 mg/mL
anti-PCSK9 in 20 mM histidine HCl, 160 mM arginine HCl, 0.02% PS20,
pH 6.0 .sup.2Histidine Acetate = 200 mg/mL anti-PCSK9 in 20 mM
histidine acetate, 160 mM arginine acetate, 0.02% PS20, pH 6.0
Frozen Stability
[0462] Anti-PCSK9 was formulated at 200 mg/mL in the following two
formulations: (1) 20 mM histidine HCl, 160 mM arginine HCl, 0.02%
PS20, pH 6.0; and (2) 20 mM histidine acetate, 160 mM arginine
acetate, 0.02% PS20, pH 6.0. For each formulation, 20 mL of the
drug solution was filled into 25-cc 316L stainless steel minicans.
All minicans were then placed at -20.degree. C. for up to 6 months
for stability analysis.
[0463] No difference was observed by IEC, CE-SDS and potency for
both formulations for up to 6 months at -20.degree. C. However,
aggregates increased by 1.4% in the histidine HCl after 6 months of
frozen storage when compared to only a 0.5% increase in aggregates
in the histidine acetate formulations under the same storage
condition (see FIG. 25). Due to the faster rate of aggregation with
the histidine HCl formulation under frozen storage conditions, the
histidine acetate formulation was selected as the preferred
buffer.
Effect of Sucrose on Frozen Stability
[0464] Sucrose was evaluated for its effect on stabilizing
anti-PCSK9 during frozen storage. Using a lab-scale Millipore
Tangential Flow Filtration (TFF) system equipped with LCGC10
cartridges, anti-PCSK9 was tested in the following two
sucrose-containing formulations: (1) 200 mg/mL anti-PCSK9 in 20 mM
histidine HCl, 130 mM arginine HCl, 60 mM sucrose, 0.02% PS20
(w/v), pH 6.0; and (2) 200 mg/mL anti-PCSK9 in 20 mM histidine
acetate, 100 mM arginine acetate, 60 mM sucrose, 0.02% PS20 (w/v),
pH 6.0. Samples of anti-PCSK9 in the two formulations were placed
at -20.degree. C. for up to 3 months and analyzed by SEC for
aggregation.
[0465] The addition of sucrose (60 mM) had no effect on reducing
aggregation of aPCSK9 in the histidine acetate formulation, but it
did help to slow down aggregation in the histidne HCl formulation
by 0.7% over 3 months at -20.degree. C. However, the addition of
sucrose also increased the viscosity from 7-8 cP to 11-13 cP at
25.degree. C. for both formulations, which was undesirable for a
subcutaneous formulation. Therefore, sucrose was not selected as a
stabilizer for the formulation.
[0466] Based on the results described above, a liquid formulation
consisting of 200 mg/mL anti-PCSK9 in 20 mM histidine acetate, 160
mM arginine acetate, 0.02% PS20 (w/v), pH 6.0 was selected. This
formulation has optimal stability at 2-8.degree. C. and at
-20.degree. C. for storage and improved stability when compared to
the initial formulation at pH 5.5.
Example 14
Human Clinical Trial in Patients with Coronary Heart Disease (CHD)
or at High Risk of CHD
[0467] This Example describes a phase II clinical study and FIGS.
27-37 show interim results for at least 50% of patients at 12
weeks. The study enrolled 248 patients, including 183 patients
treated with study drug and 64 patients treated with placebo. One
patient dropped out prior to the first treatment and 13 patients
discontinued treatment prior to day 85 of the study. 234 patients
completed at least 12 weeks of the study.
[0468] A .about.3:1 randomized, double-blind, placebo-controlled,
study of study drug (YW508.20.33b reformatted into human IgG.sub.1
having a heavy chain with SEQ ID NO: 35 and a light chain with SEQ
ID NO: 36) was conducted to evaluate the safety and efficacy of
study drug on top of standard-of-care (SOC) statin in patients with
fasting serum LDL-c (direct) levels of 90-250 mg/dL and either
coronary heart disease (CHD) or a CHD risk equivalent. Additional
eligibility criteria included weight .gtoreq.45 kg (100 lb); body
mass index of 18-37 kg/m.sup.2; and age between 18 and 80. The
randomization was stratified by LDL-c>120 mg/dL and diabetes
status.
[0469] The eligibility criteria for this phase II clinical study
defined a population of patients with high cardiovascular and CHD
risk based on risk categories in the European Society of Cardiology
(ESC)/European Atherosclerosis Society (EAS) and National
Cholesterol Education Program Adult Treatment Panel III (NCEP ATP
III) lipid-lowering guidelines. This study enrolled patients who
qualified for a therapeutic target LDL-c level of 70 mg/dL
according to these guidelines, but who had not come close to this
goal despite stable SOC statin therapy, either because SOC is
insufficient or because statins were not tolerated.
[0470] Briefly, CHD refers to a history of documented myocardial
infarction, prior coronary revascularization procedure
(percutaneous coronary intervention or coronary artery bypass
graft), or prior coronary angiography (invasive coronary
angiography or cardiac computed tomography coronary angiography)
demonstrating at least one coronary atherosclerotic lesion with
.gtoreq.50% diameter stenosis.
[0471] A patient with a CHD risk-equivalent condition had at least
one of the following:
[0472] 1. One or more forms of clinical atherosclerotic disease:
[0473] a. Peripheral arterial disease (previously documented
ankle/brachial blood pressure index <0.85, prior percutaneous or
surgical peripheral arterial revascularization procedure, prior
non-traumatic amputation of a lower extremity due to peripheral
artery disease, or .gtoreq.50% diameter stenosis on prior vascular
imaging), [0474] b. Carotid artery disease (previously documented
carotid atherosclerotic lesion with .gtoreq.50% diameter stenosis
on imaging or prior cutaneous or surgical carotid revascularization
procedure), [0475] c. Prior ischemic stroke, documented by CT or
MRI brain imaging, not due to embolism of cardiac origin (e.g.,
atrial fibrillation, valvular disease, or left ventricular mural
thrombus) in the opinion of the investigator, or [0476] d.
Abdominal aortic aneurysm with prior surgical or endovascular
repair.
[0477] 2. Diabetes mellitus type 2,
[0478] 3. Diabetes mellitus type 1 with target organ damage
(retinopathy, neuropathy, or nephropathy including
microalbuminuria, as determined by the investigator),
[0479] 4. Moderate to severe chronic kidney disease (manifested by
an estimated glomerular filtration rate of 15-60 mL/min/1.73
m.sup.2 using the Modification of Diet in Renal Disease equation
consistently over at least three measurements spanning at least 3
months, including screening laboratories), or
[0480] 5. Two or more of the CHD risk factors listed below AND
either an absolute 10-year risk of a CHD event .gtoreq.20% (as
determined by the National Cholesterol Education Program Adult
Treatment Panel III guideline modification of the Framingham risk
score) or a 10-year risk of a first fatal atherosclerotic event
.gtoreq.10% (determined by the Systemic Coronary Risk Estimation
system): [0481] a. Age .gtoreq.45 years for men or .gtoreq.55 years
for women, [0482] b. Current cigarette smoking (within 1 month),
[0483] c. Hypertension (screening systolic blood pressure
.gtoreq.140 mmHg, diastolic blood pressure .gtoreq.90 mmHg, or
taking an antihypertensive medication to treat hypertension) [0484]
d. Low HDL cholesterol (<40 mg/dL), or [0485] e. Family history
of premature CHD (myocardial infarction or coronary
revascularization in a male first-degree relative <55 years of
age or in a female first-degree relative <65 years of age).
[0486] Diabetes status was determined based on the presence of any
one of the following, according to patient medical record or
history, or to screening laboratory test results: (1)
HbA.sub.1c.gtoreq.6.5%, (2) fasting plasma glucose .gtoreq.126
mg/dL (7.0 mmol/L), (3) prior 2-hour plasma glucose .gtoreq.200
mg/dL (11.1 mmol/L) during an oral glucose tolerance test (the test
should be performed as described by the World Health Organization,
with use of a glucose load containing the equivalent of 75 g of
anhydrous glucose dissolved in water), or (4) currently on an oral
or injectable therapy for a diagnosis of diabetes mellitus.
[0487] Exclusion criteria included: planned coronary, carotid or
peripheral arterial revascularization procedure or surgery during
study; uncontrolled clinically significant medical disease as
listed in the protocol within 3 months or screening; any acquired
or congenital immunosuppression; any organ transplant other than
the corneal transplant; life expectancy <2 years, in the
investigator's judgment; fasting serum triglyceride levels >=400
mg/dL; history of alcoholism or drug addiction with a year of
screening; use of illicit drugs with 3 months of screening;
pregnancy or not willing to use highly effective contraception;
history of anaphylaxis or anaphylactic reactions.
[0488] 248 patients (adult men and women) with serum LDL-c
concentrations of 90-250 mg/dL and either CHD or a CHD risk
equivalent were randomized to one of five study arms and were
administered study drug or a placebo arm (Arm F). Patients in the
first study arm (Arm A) were administered 400 mg of anti-PCSK9
antibody every 4 weeks; patients in the second study arm (Arm B)
were administered 200 mg of anti-PCSK9 antibody every 8 weeks;
patients in the third study arm (Arm C) were administered 400 mg of
anti-PCSK9 antibody every 8 weeks; patients in the fourth study arm
(Arm D) were administered 800 mg of anti-PCSK9 antibody every 8
weeks; and patients in the fifth study arm (Arm E) were
administered 800 mg of anti-PCSK9 antibody every 12 weeks. An
overview of study dose cohorts, study drug dose regimen, and number
of patients per arm are provided in FIG. 27. All doses were
administered subcutaneously using syringes. The drug product is
formulated as 150 mg/mL antibody in 200 mM arginine succinate,
0.02% polysorbate 20, pH 5.5.
[0489] The demographics of the patients in the study are set forth
below in Table 13, indicating no difference by arm. The patients'
baseline characteristics are set forth below in Table 14,
indicating no difference by arm.
TABLE-US-00016 TABLE 13 Patient Demographics (Mean (SD), unless
noted) 400 mg/4 W 200 mg/8 W 400 mg/8 W 800 mg/8 W 800 mg/12 W
Placebo mITT (n = 57) (n = 23) (n = 30) (n = 50) (n = 23) (n = 64)
(n = 247) Age (years) 66 (8.5) 63 (10.0) 63 (8.1) 64 (8.9) 64 (7.2)
63 (7.8) 64 (8.4) Weight (kg) 89 (15.4) 89 (15.3) 85 (11.7) 83
(17.7) 83 (17.1) 87 (15.1) 86 (15.6) BMI (kg/m) 31 (4.3) 30 (4.5)
30 (4.2) 29 (5.2) 29 (3.7) 30 (5.0) 30 (4.7) Female (%) 24 (42%) 8
(35%) 16 (53%) 24 (48%) 10 (44%) 24 (38%) 106 (43%) Hispanic (%) 1
(2%) 1 (4%) 1 (3%) 1 (2%) 1 (4%) 5 (8%) 10 (4%) Race: White (%) 55
(97%) 19 (83%) 27 (90%) 44 (88%) 23 (100%) 59 (92%) 227 (92%) Race:
Black (%) 1 (2%) 2 (9%) 2 (7%) 5 (10%) 0 3 (5%) 13 (5%) Race: Asian
(%) 0 0 1 (3%) 1 (2%) 0 1 (2%) 3 (1%) Race: Other (%) 1 (2%) 1 (4%)
0 0 0 1 (2%) 3 (1%) Race: Native (%) 0 1 (4%) 0 0 0 0 1 (0.4%)
TABLE-US-00017 TABLE 14 Patient Baseline Characteristics (Mean
(SD), unless noted) 400 mg/4 W 200 mg/8 W 400 mg/8 W 800 mg/8 W 800
mg/12 W Placebo mITT (n = 57) (n = 23) (n = 30) (n = 50) (n = 23)
(n = 64) (n = 247) Pre-diabetic (% 68% 65% 60% 54% 65% 59% 62% FBG
.gtoreq. 100 mg/dl) Statin use (%) 88% 78% 73% 76% 74% 89% 82%
LDL-c .gtoreq. 120 (%) 46% 48% 60% 54% 52% 45% 50% LDL-c (mg/dL)
123 (31.3) 123 (25.3) 133 (35.2) 127 (31.5) 134 (43.8) 122 (31.4)
126 (32.7) Median LDL-c (mg/dL) 117 117 123 118 123 111 117
Triglyceride (mg/dL) 156 (66.3) 146 (60.0) 152 (54.3) 173 (90.8)
144 (37.0) 141 (63.1) 153 (67.8) Median Trig. (mg/dL) 142 132 142
149 145 132 142 Family history 26 (46%) 5 (22%) 13 (43%) 20 (40%) 9
(39%) 18 (28%) 91 (37%) of CHD (% yes) Smoker: never (%) 23 (40%) 6
(26%) 13 (43%) 17 (34%) 7 (30%) 25 (39%) 91 (37%)
[0490] As shown in FIG. 27, the study includes consecutive periods
for screening (0-4 weeks), run-in (0-6 weeks, if necessary),
treatment (24 weeks; Days 1-169), and follow-up (12 weeks). The
study completion visit at the end of the follow-up period (Day 253)
occurs 16 weeks after the final dose of study drug (Day 141). All
patients, regardless of treatment assignment, received
standard-of-care (SOC) treatment, including statins unless statins
were not tolerated. SOC statin therapy refers to a therapy meeting
one of the following conditions: (1) high-dose simvastatin (40 mg
daily), atorvastatin (40-80 mg daily), or rosuvastatin (20-40 mg
daily), (2) low-dose simvastatin, atorvastatin, or rosuvastatin and
documented intolerance of a high dose of that statin or of any dose
of another statin, (3) other statin (any dose) and documented
intolerance of simvastatin, atorvastatin, or rosuvastatin (any
dose), or (4) no statin and documented intolerance of at least two
statins (any statin, any dose). All patients continue SOC statin
therapy throughout the treatment and follow-up periods, at the same
dose they were receiving during the run-in period and at
enrollment. Other prescription and over-the-counter (OTC)
lipid-modifying therapies (e.g., red yeast rice, omega-3 fatty acid
supplements, etc.) are not permitted. Patients who had been taking
a stable dose of SOC statin therapy (or no statin and had
documented intolerance to two or more statins) and no other
lipid-modifying therapy for at least 4 weeks (or 6 weeks in the
case of fibrates) at the time of screening did not require a run-in
period.
[0491] All doses of active drug or placebo are given according to
the study drug administration schedule, that is, on Days 1, 29
(.+-.2 days), 57 (.+-.2 days), 85 (.+-.2 days), 113 (.+-.4 days),
and 141 (.+-.4 days) only. See FIG. 28. Patients are monitored to
determine efficacy based on absolute change from baseline in LDL-c
concentration at day 169. In addition, patients are monitored to
determine secondary efficacy outcomes including absolute change
from baseline in LDL-c concentration for each arm at the nadir for
that arm; average value over time of the change in LDL-c (absolute
and percent change) for each arm, up to Day 169, weighted by the
number of weeks between consecutive LDL-c measurements; percent
change from baseline in LDL-c concentration at Day 169 and at the
nadir for each arm; percent and absolute change from baseline in
LDL-c concentration at all other designated timepoints; and percent
and absolute change from baseline in total cholesterol, non-HDL-c,
and apolipoprotein B at Day 169 and at the nadir for each arm.
[0492] The primary efficacy outcome measure includes the change
from baseline in LDL-c at Day 169. Baseline LDL-c is defined as the
average of the last two measurements collected before the first
dose of study drug. The treatment comparisons between the study
drug doses and between each of the study drug doses and placebo
were based on an analysis of covariance (ANCOVA), which was
performed through a linear regression model adjusting for two
covariates: baseline LDL-c concentration (<120 mg/dL,
.gtoreq.120 mg/dL) and diabetes status (yes, no). The confidence
intervals, as well as the least-square estimates from the ANCOVA
models, were used to aid in the interpretation of the study
results. The secondary efficacy outcome measures include absolute
change in LDL-c at nadir and all time points; weighted average of
change in LDLc per week; percent change from baseline in LDL-c at
Day 169, nadir and all visits; absolute and percent change in total
cholesterol, non-HDL-c, and apolipoprotein B at Day 169 and at the
nadir.
[0493] Table 15 below shows patient disposition after 12 weeks of
treatment.
TABLE-US-00018 TABLE 15 Disposition after 12 weeks of treatment.
400 mg/4 W 200 mg/8 W 400 mg/8 W 800 mg/8 W 800 mg/12 W Placebo
ITT* (n = 57) (n = 23) (n = 30) (n = 51) (n = 23) (n = 64) (n =
248) Completed study 0 0 0 0 0 0 0 Discontinued study 2 (4%) 0 1
(3%) 3 (6%) 0 1 (2%) 7 (3%).sup. Discontinued drug 3 (5%) 0 1 (3%)
4 (8%) 0 2 (3%) 10 (4.0%) Adverse event 1 (2%) 0 0 0 0 0 1 (0.4%)
Protocol violation 2 (4%) 0 0 2 (4%) 0 0 4 (1.6%) Subject choice 0
0 1 (3%) 1 (2%) 0 1 (2%) 3 (1.2%) Sponsor choice 0 0 0 1 (2%) 0 0 1
(0.4%) Other 0 0 0 0 0 1 (2%) 1 (0.4%)
[0494] Interim data of this study are summarized in Table 16 below
and in FIGS. 28-36.
TABLE-US-00019 TABLE 16 Patients' Total Cholesterol, non-HDL-c, and
Apolipoprotein B, Measured from Baseline to Nadir 400 mg/4 W 200
mg/8 W 400 mg/8 W 800 mg/8 W 800 mg/12 W Placebo (n = 57) (n = 23)
(n = 30) (n = 50) (n = 23) (n = 63) TC, mean absolute -99.9 -73.9
-92.3 -102.0 -92.3 -24.4 change (mg/dL) Reduction from placebo 74.9
48.7 64.7 75.5 66.2 95% confidence interval 65.2, 84.6 36.0, 61.5
52.9, 76.4 65.5, 85.5 53.4, 79.0 TC, mean relative -49.4 -37.7
-43.6 -48.6 -44.8 -12.4 change (%) Reduction from placebo 36.7 25.2
30.8 35.8 32.1 95% confidence interval 32.6, 40.8 19.8, 30.7 25.7,
35.8 31.6, 40.1 26.6, 37.6 Non-HDLc, mean abs. -101.6 -76.2 -96.3
-103.3 -95.0 -24.1 ch. (mg/dL) Reduction from placebo 76.7 51.3
68.7 76.9 69.0 95% confidence interval 66.9, 86.5 38.4, 64.1 56.9,
80.5 66.9, 87.0 56.1, 81.9 Non-HDLc, mean rel. -67.3 -52.1 -60.5
-65.9 -59.8 -16.6 change (%) Reduction from placebo 50.3 35.5 43.7
49.1 43.0 95% confidence interval 45.2, 55.4 28.8, 42.2 37.5, 49.8
43.9, 54.3 36.3, 49.7 Apo-B, mean abs. -64.3 -48.5 -59.1 -65.6
-62.8 -15.8 change (mg/dL) Reduction from placebo 48.1 32.3 41.4
48.5 46.0 95% confidence interval 42.0, 54.3 24.2, 40.4 33.9, 48.8
42.2, 54.9 37.8, 54.1 Apo-B, mean relative -63.1 -49.2 -55.8 -62.7
-58.3 -15.7 change (%) Reduction from placebo 47.0 33.4 39.9 46.8
42.3 95% confidence interval 42.2, 51.7 27.1, 39.6 34.2, 45.7 41.9,
51.7 36.1, 48.6
[0495] FIG. 28 provides mean pharmacokinetics (+/- standard
deviation) (left panel) and mean total PCSK9, e.g. both drug-bound
and free PCSK9 (+/- standard error) (right panel).
[0496] FIG. 29 shows the absolute change from baseline in direct
LDL cholesterol observed in patients receiving anti-PCSK9 antibody
or placebo. FIG. 30 shows the relative change from baseline in
direct LDL cholesterol observed in patients receiving anti-PCSK9
antibody or placebo. Patients receiving 400 mg of anti-PCSK9
antibody every 4 weeks and patients receiving 800 mg of anti-PCSK9
antibody every 8 weeks exhibited the highest reduction in direct
LDL-c. This effect was observed within a week of treatment.
Patients receiving 800 mg of anti-PCSK9 antibody every 12 weeks
exhibited the lowest reduction in direct LDL-c.
[0497] FIG. 31 shows the absolute change from baseline in total
cholesterol observed in patients participating in this study. FIG.
32 shows the relative change from baseline in total cholesterol
observed in patients receiving anti-PCSK9 antibody or placebo.
Patients receiving 400 mg of anti-PCSK9 antibody every 4 weeks and
patients receiving 800 mg of anti-PCSK9 antibody every 8 weeks
exhibited the highest reduction in total cholesterol. This effect
was observed within a week of treatment. Patients receiving 800 mg
of anti-PCSK9 antibody every 12 weeks exhibited the lowest
reduction in total cholesterol.
[0498] FIG. 33 shows the absolute change from baseline in non-HDL
cholesterol in patients participating in this study. FIG. 34 shows
the relative change from baseline in non-HDL cholesterol in
patients participating in this study. Patients receiving 400 mg of
anti-PCSK9 antibody every 4 weeks and patients receiving 800 mg of
anti-PCSK9 antibody every 8 weeks exhibited the highest reduction
in non-HDL cholesterol. This effect was observed within a week of
treatment. Patients receiving 800 mg of anti-PCSK9 antibody every
12 weeks exhibited the lowest reduction in non-HDL cholesterol.
[0499] FIG. 35 shows the absolute change from baseline in
apolipoprotein B in patients participating in this study. FIG. 36
shows the relative change from baseline in apolipoprotein B in
patients participating in this study. Patients receiving 400 mg of
anti-PCSK9 antibody every 4 weeks and patients receiving 800 mg of
anti-PCSK9 antibody every 8 weeks exhibited the highest reduction
in apolipoprotein B. This effect was observed within a week of
treatment. Patients receiving 800 mg of anti-PCSK9 antibody every
12 weeks exhibited the lowest reduction in apolipoprotein B.
[0500] Conclusions regarding the efficacy of study drug are
summarized here. The highest dose-dependent reduction in LDL-c on
Day 85, at nadir, and AUC was observed in patients receiving 400 mg
of anti-PCSK9 antibody every 4 weeks and in patients receiving 800
mg of anti-PCSK9 antibody every 8 weeks. The smallest
dose-dependent reduction in LDL-c based on Day 85 analyses was
observed in patients receiving 800 mg of anti-PCSK9 antibody every
12 weeks. The smallest dose-dependent reduction in LDL-c based on
nadir and AUC analyses was observed in patients receiving 200 mg of
anti-PCSK9 antibody every 8 weeks. The reduction was evident within
a week of treatment. Dose-dependent reduction in total cholesterol,
non-HDL-c, and apolipoprotein-B was observed on Day 85 and at
nadir, and the reduction was also evident within a week of
treatment.
[0501] Finally, patients were also monitored for safety including
incidence, nature, and severity of adverse events; incidence and
nature of changes in vital signs, physical findings, and clinical
laboratory results during and following study drug administration;
and incidence of anti-therapeutic antibodies directed against study
drug.
[0502] The safety of low LDL-c values was assessed regularly in a
blinded, exploratory manner. FIG. 37A shows the proportion of
patients with direct LDL-c values less than or equal to 15 mg/dL
for at least one visit after receiving anti-PCSK9 antibody or
placebo, and FIG. 37B shows the proportion of patients with direct
LDL-c values less than or equal to 25 mg/dL for at least one visit
after receiving anti-PCSK9 antibody or placebo. The highest
percentage of patients with LDL-c.ltoreq.15 mg/dL or
LDL-c.ltoreq.25 mg/dL were receiving either 400 mg of drug every
four weeks or 800 mg of drug every 8 weeks. The lowest percent of
patients with LDL-c.ltoreq.25 mg/dL were receiving 200 mg of drug
every 8 weeks. Study drug was withheld from patients with two
consecutive LDL-c values of <15 mg/dL. This was not considered
an adverse event. Such patients were treated with placebo instead,
in blinded fashion, until LDL-c increased to .gtoreq.50 mg/dL,
after which these patients were switched to the lowest dosage (200
mg every 8 weeks).
[0503] Conclusions regarding the safety of study drug are
summarized here. Briefly, anti-PCSK9 antibody was well tolerated in
patients aged 37-80 with elevated baseline LDL-c (90-250 mg/dL),
diagnosed with CHD or a CHD risk equivalent, and who were taking
stable doses of statins or were statin-intolerant. Injection-site
reactions were more common among patients receiving study drug
(25%) vs. placebo (9%). Only 2 injection-site reactions were
moderate (1 placebo, 1 study drug), and the rest were mild in
severity. No other clinically significant imbalances of
treatment-emergent events were observed between study drug-treated
and placebo-treated patients. No clinically relevant imbalances in
laboratory abnormalities were observed. No safety signals were
determined. No deaths were reported, and no new safety concerns
were observed. No patterns were detected in safety laboratory
results.
[0504] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
45110PRTArtificial SequenceSynthetic construct 1Gly Phe Thr Phe Thr
Gly Tyr Ala Ile His1 5 10 210PRTArtificial SequenceSynthetic
construct 2Gly Phe Thr Phe Thr Arg His Thr Ile His1 5 10
310PRTArtificial SequenceSynthetic construct 3Gly Phe Thr Phe Ser
Ser Thr Ala Ile His1 5 10 417PRTArtificial SequenceSynthetic
construct 4Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser
Val Lys1 5 10 15 Gly512PRTArtificial SequenceSynthetic construct
5Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr1 5 10
611PRTArtificial SequenceSynthetic construct 6Arg Ala Ser Gln Asp
Val Ser Ser Ala Val Ala1 5 10 711PRTArtificial SequenceSynthetic
construct 7Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala1 5 10
87PRTArtificial SequenceSynthetic construct 8Ser Ala Ser Phe Leu
Tyr Ser1 5 99PRTArtificial SequenceSynthetic construct 9Gln Gln Ser
Tyr Thr Thr Pro Pro Thr1 5 109PRTArtificial SequenceSynthetic
construct 10Gln Gln Ser Tyr Pro Ala Pro Ala Thr1 5 119PRTArtificial
SequenceSynthetic construct 11Gln Gln Ser Tyr Pro Ser Pro Ala Thr1
5 129PRTArtificial SequenceSynthetic construct 12Gln Gln Ser Tyr
Arg Ile Gln Pro Thr1 5 139PRTArtificial SequenceSynthetic construct
13Gln Gln Ser Tyr Pro Ala Leu His Thr1 5 149PRTArtificial
SequenceSynthetic construct 14Gln Gln Ser Tyr Pro Ala Pro Ser Thr1
5 15121PRTArtificial SequenceSynthetic construct 15Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Tyr 20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu
Tyr Ile Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 16121PRTArtificial SequenceSynthetic construct
16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg
His 20 25 30 Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Ile Gly
Ser Arg Glu Leu Tyr Ile Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 17121PRTArtificial
SequenceSynthetic construct 17Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Thr 20 25 30 Ala Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile
Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr
Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
18107PRTArtificial SequenceSynthetic construct 18Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 19107PRTArtificial SequenceSynthetic construct
19Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Pro Ala Pro Ala 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 20107PRTArtificial
SequenceSynthetic construct 20Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Ser Pro Ala
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
21107PRTArtificial SequenceSynthetic construct 21Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Arg Ile Gln Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 22107PRTArtificial SequenceSynthetic construct
22Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Pro Ala Leu His 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 23107PRTArtificial
SequenceSynthetic construct 23Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Ala Pro Ser
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
24692PRTHomo sapiens 24Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp
Pro Leu Pro Leu Leu1 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 Val65 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 Arg145 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
Gly225 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 Asp305 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 Leu385 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
Asp465 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 Thr545 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 Gly625 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 253636DNAHomo sapiens 25cagcgacgtc gaggcgctca
tggttgcagg cgggcgccgc cgttcagttc agggtctgag 60cctggaggag tgagccaggc
agtgagactg gctcgggcgg gccgggacgc gtcgttgcag 120cagcggctcc
cagctcccag ccaggattcc gcgcgcccct tcacgcgccc tgctcctgaa
180cttcagctcc tgcacagtcc tccccaccgc aaggctcaag gcgccgccgg
cgtggaccgc 240gcacggcctc taggtctcct cgccaggaca gcaacctctc
ccctggccct catgggcacc 300gtcagctcca ggcggtcctg gtggccgctg
ccactgctgc tgctgctgct gctgctcctg 360ggtcccgcgg gcgcccgtgc
gcaggaggac gaggacggcg actacgagga gctggtgcta 420gccttgcgtt
ccgaggagga cggcctggcc gaagcacccg agcacggaac cacagccacc
480ttccaccgct gcgccaagga tccgtggagg ttgcctggca cctacgtggt
ggtgctgaag 540gaggagaccc acctctcgca gtcagagcgc actgcccgcc
gcctgcaggc ccaggctgcc 600cgccggggat acctcaccaa gatcctgcat
gtcttccatg gccttcttcc tggcttcctg 660gtgaagatga gtggcgacct
gctggagctg gccttgaagt tgccccatgt cgactacatc 720gaggaggact
cctctgtctt tgcccagagc atcccgtgga acctggagcg gattacccct
780ccacggtacc gggcggatga ataccagccc cccgacggag gcagcctggt
ggaggtgtat 840ctcctagaca ccagcataca gagtgaccac cgggaaatcg
agggcagggt catggtcacc 900gacttcgaga atgtgcccga ggaggacggg
acccgcttcc acagacaggc cagcaagtgt 960gacagtcatg gcacccacct
ggcaggggtg gtcagcggcc gggatgccgg cgtggccaag 1020ggtgccagca
tgcgcagcct gcgcgtgctc aactgccaag ggaagggcac ggttagcggc
1080accctcatag gcctggagtt tattcggaaa agccagctgg tccagcctgt
ggggccactg 1140gtggtgctgc tgcccctggc gggtgggtac agccgcgtcc
tcaacgccgc ctgccagcgc 1200ctggcgaggg ctggggtcgt gctggtcacc
gctgccggca acttccggga cgatgcctgc 1260ctctactccc cagcctcagc
tcccgaggtc atcacagttg gggccaccaa tgcccaagac 1320cagccggtga
ccctggggac tttggggacc aactttggcc gctgtgtgga cctctttgcc
1380ccaggggagg acatcattgg tgcctccagc gactgcagca cctgctttgt
gtcacagagt 1440gggacatcac aggctgctgc ccacgtggct ggcattgcag
ccatgatgct gtctgccgag 1500ccggagctca ccctggccga gttgaggcag
agactgatcc acttctctgc caaagatgtc 1560atcaatgagg cctggttccc
tgaggaccag cgggtactga cccccaacct ggtggccgcc 1620ctgcccccca
gcacccatgg ggcaggttgg cagctgtttt gcaggactgt atggtcagca
1680cactcggggc ctacacggat ggccacagcc gtcgcccgct gcgccccaga
tgaggagctg 1740ctgagctgct ccagtttctc caggagtggg aagcggcggg
gcgagcgcat ggaggcccaa 1800gggggcaagc tggtctgccg ggcccacaac
gcttttgggg gtgagggtgt ctacgccatt 1860gccaggtgct gcctgctacc
ccaggccaac tgcagcgtcc acacagctcc accagctgag 1920gccagcatgg
ggacccgtgt ccactgccac caacagggcc acgtcctcac aggctgcagc
1980tcccactggg aggtggagga ccttggcacc cacaagccgc ctgtgctgag
gccacgaggt 2040cagcccaacc agtgcgtggg ccacagggag gccagcatcc
acgcttcctg ctgccatgcc
2100ccaggtctgg aatgcaaagt caaggagcat ggaatcccgg cccctcagga
gcaggtgacc 2160gtggcctgcg aggagggctg gaccctgact ggctgcagtg
ccctccctgg gacctcccac 2220gtcctggggg cctacgccgt agacaacacg
tgtgtagtca ggagccggga cgtcagcact 2280acaggcagca ccagcgaagg
ggccgtgaca gccgttgcca tctgctgccg gagccggcac 2340ctggcgcagg
cctcccagga gctccagtga cagccccatc ccaggatggg tgtctgggga
2400gggtcaaggg ctggggctga gctttaaaat ggttccgact tgtccctctc
tcagccctcc 2460atggcctggc acgaggggat ggggatgctt ccgcctttcc
ggggctgctg gcctggccct 2520tgagtggggc agcctccttg cctggaactc
actcactctg ggtgcctcct ccccaggtgg 2580aggtgccagg aagctccctc
cctcactgtg gggcatttca ccattcaaac aggtcgagct 2640gtgctcgggt
gctgccagct gctcccaatg tgccgatgtc cgtgggcaga atgactttta
2700ttgagctctt gttccgtgcc aggcattcaa tcctcaggtc tccaccaagg
aggcaggatt 2760cttcccatgg ataggggagg gggcggtagg ggctgcaggg
acaaacatcg ttggggggtg 2820agtgtgaaag gtgctgatgg ccctcatctc
cagctaactg tggagaagcc cctgggggct 2880ccctgattaa tggaggctta
gctttctgga tggcatctag ccagaggctg gagacaggtg 2940cgcccctggt
ggtcacaggc tgtgccttgg tttcctgagc cacctttact ctgctctatg
3000ccaggctgtg ctagcaacac ccaaaggtgg cctgcgggga gccatcacct
aggactgact 3060cggcagtgtg cagtggtgca tgcactgtct cagccaaccc
gctccactac ccggcagggt 3120acacattcgc acccctactt cacagaggaa
gaaacctgga accagagggg gcgtgcctgc 3180caagctcaca cagcaggaac
tgagccagaa acgcagattg ggctggctct gaagccaagc 3240ctcttcttac
ttcacccggc tgggctcctc atttttacgg gtaacagtga ggctgggaag
3300gggaacacag accaggaagc tcggtgagtg atggcagaac gatgcctgca
ggcatggaac 3360tttttccgtt atcacccagg cctgattcac tggcctggcg
gagatgcttc taaggcatgg 3420tcgggggaga gggccaacaa ctgtccctcc
ttgagcacca gccccaccca agcaagcaga 3480catttatctt ttgggtctgt
cctctctgtt gcctttttac agccaacttt tctagacctg 3540ttttgctttt
gtaacttgaa gatatttatt ctgggttttg tagcattttt attaatatgg
3600tgacttttta aaataaaaac aaacaaacgt tgtcct 3636267PRTArtificial
SequenceSynthetic construct 26Ser Ala Ser Ser Leu Tyr Ser1 5
27121PRTArtificial SequenceSynthetic construct 27Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Tyr 20 25 30
Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu
Tyr Ile Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 2810PRTArtificial SequenceSynthetic construct 28Gly
Phe Thr Phe Xaa Xaa Xaa Xaa Ile His1 5 10 2911PRTArtificial
SequenceSynthetic construct 29Arg Ala Ser Gln Asp Val Ser Xaa Ala
Val Ala1 5 10 307PRTArtificial SequenceSynthetic construct 30Ser
Ala Ser Xaa Leu Tyr Ser1 5 319PRTArtificial SequenceSynthetic
construct 31Gln Gln Ser Tyr Xaa Xaa Xaa Xaa Thr1 5 328PRTArtificial
SequenceSynthetic construct 32His His His His His His His His 1 5
339PRTArtificial SequenceSynthetic construct 33Gln Gln Ala Tyr Pro
Ala Leu His Thr1 5 34107PRTArtificial SequenceSynthetic construct
34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Tyr Pro Ala Leu His 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 35451PRTArtificial
SequenceSynthetic construct 35Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Thr 20 25 30 Ala Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile
Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr
Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160 Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325
330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro385 390 395 400 Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445
Pro Gly Lys 450 36214PRTArtificial SequenceSynthetic construct
36Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Tyr Pro Ala Leu His 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 379PRTArtificial SequenceSynthetic construct 37Gln Gln Ala Tyr
Xaa Xaa Xaa Xaa Thr1 5 381353DNAArtificial SequenceSynthetic
construct 38gaagttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc
actccgtttg 60tcctgtgcag cttctggctt caccttctct agtactgcta ttcactgggt
gcgtcaggcc 120ccgggtaagg gcctggaatg ggttgctagg atttctcctg
ctaacggtaa tactaactat 180gccgatagcg tcaagggccg tttcactata
agcgcagaca catccaaaaa cacagcctac 240ctacaaatga acagcttaag
agctgaggac actgccgtct attattgtgc tcgttggatc 300gggtcccggg
agctgtacat tatggactac tggggtcaag gaaccctggt caccgtctcc
360tcggcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa
gagcacctct 420gggggcacag cggccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 540tcaggactct actccctcag
cagcgtggtg actgtgccct ctagcagctt gggcacccag 600acctacatct
gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag
660cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga
actcctgggg 720ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg
agccacgaag accctgaggt caagttcaac 840tggtacgtgg acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac 900aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
960aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga
gaaaaccatc 1020tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggaa 1080gagatgacca agaaccaggt cagcctgacc
tgcctggtca aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc 1200gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
1260tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac 1320acgcagaaga gcctctccct gtctccgggt aaa
135339363DNAArtificial SequenceSynthetic construct 39gaagttcagc
tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60tcctgtgcag
cttctggctt caccttctct agtactgcta ttcactgggt gcgtcaggcc
120ccgggtaagg gcctggaatg ggttgctagg atttctcctg ctaacggtaa
tactaactat 180gccgatagcg tcaagggccg tttcactata agcgcagaca
catccaaaaa cacagcctac 240ctacaaatga acagcttaag agctgaggac
actgccgtct attattgtgc tcgttggatc 300gggtcccggg agctgtacat
tatggactac tggggtcaag gaaccctggt caccgtctcc 360tcg
36340642DNAArtificial SequenceSynthetic construct 40gatatccaga
tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60atcacctgcc
gtgccagtca ggatgtgtcc actgctgtag cctggtatca acagaaacca
120ggaaaagctc cgaagcttct gatttactcg gcatccttcc tctactctgg
agtcccttct 180cgcttctctg gtagcggttc cgggacggat ttcactctga
ccatcagcag tctgcagccg 240gaagacttcg caacttatta ctgtcagcaa
gcctatccgg ccctacacac gttcggacag 300ggtaccaagg tggagatcaa
acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360tctgatgagc
agttgaaatc tggaactgct tctgttgtgt gcctgctgaa taacttctat
420cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg
taactcccag 480gagagtgtca cagagcagga cagcaaggac agcacctaca
gcctcagcag caccctgacg 540ctgagcaaag cagactacga gaaacacaaa
gtctacgcct gcgaagtcac ccatcagggc 600ctgagctcgc ccgtcacaaa
gagcttcaac aggggagagt gt 64241324DNAArtificial SequenceSynthetic
construct 41gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga
tagggtcacc 60atcacctgcc gtgccagtca ggatgtgtcc actgctgtag cctggtatca
acagaaacca 120ggaaaagctc cgaagcttct gatttactcg gcatccttcc
tctactctgg agtcccttct 180cgcttctctg gtagcggttc cgggacggat
ttcactctga ccatcagcag tctgcagccg 240gaagacttcg caacttatta
ctgtcagcaa gcctatccgg ccctacacac gttcggacag 300ggtaccaagg
tggagatcaa acga 3244210PRTArtificial SequenceSynthetic construct
42Gly Phe Thr Phe Thr Arg His Thr Ile Asn1 5 10 43121PRTArtificial
SequenceSynthetic construct 43Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Thr Arg His 20 25 30 Thr Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile
Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr
Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
44107PRTArtificial SequenceSynthetic construct 44Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 4510PRTArtificial SequenceSynthetic construct 45Gly
Phe Thr Phe Xaa Xaa Xaa Xaa Ile Xaa1 5 10
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