U.S. patent application number 14/459844 was filed with the patent office on 2014-12-04 for nucleic acid encoding antigen binding proteins to proprotein convertase subtilisin kexin type 9 (pcsk9).
The applicant listed for this patent is Amgen Inc.. Invention is credited to Qiong Cao, Teresa Arazas Carabeo, Joyce Chi Yee Chan, Simon Mark Jackson, Randal Robert Ketchem, Chadwick Terence King, Christopher Mehlin, Derek Evan Piper, Bei Shan, Wenyan Shen, Nigel Pelham Clinton Walker.
Application Number | 20140357854 14/459844 |
Document ID | / |
Family ID | 39951467 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140357854 |
Kind Code |
A1 |
Jackson; Simon Mark ; et
al. |
December 4, 2014 |
NUCLEIC ACID ENCODING ANTIGEN BINDING PROTEINS TO PROPROTEIN
CONVERTASE SUBTILISIN KEXIN TYPE 9 (PCSK9)
Abstract
Antigen binding proteins that interact with Proprotein
Convertase Subtilisin Kexin Type 9 (PCSK9) are described. Methods
of treating hypercholesterolemia and other disorders by
administering a pharmaceutically effective amount of an antigen
binding protein to PCSK9 are described. Methods of detecting the
amount of PCSK9 in a sample using an antigen binding protein to
PCSK9 are described.
Inventors: |
Jackson; Simon Mark; (San
Carlos, CA) ; Walker; Nigel Pelham Clinton;
(Burlingame, CA) ; Piper; Derek Evan; (Santa
Clara, CA) ; Shan; Bei; (Redwood City, CA) ;
Shen; Wenyan; (Wayne, PA) ; Chan; Joyce Chi Yee;
(San Francisco, CA) ; King; Chadwick Terence;
(North Vancouver, CA) ; Ketchem; Randal Robert;
(Snohomish, WA) ; Mehlin; Christopher; (Seattle,
WA) ; Carabeo; Teresa Arazas; (Seattle, WA) ;
Cao; Qiong; (Daly City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amgen Inc. |
Thousand Oaks |
CA |
US |
|
|
Family ID: |
39951467 |
Appl. No.: |
14/459844 |
Filed: |
August 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13251909 |
Oct 3, 2011 |
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14459844 |
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12197093 |
Aug 22, 2008 |
8030457 |
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13251909 |
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60957668 |
Aug 23, 2007 |
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61008965 |
Dec 21, 2007 |
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61010630 |
Jan 9, 2008 |
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61086133 |
Aug 4, 2008 |
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Current U.S.
Class: |
536/23.53 |
Current CPC
Class: |
A61K 31/366 20130101;
C07K 2317/76 20130101; A61P 3/06 20180101; A61K 31/40 20130101;
C07K 2317/92 20130101; C07K 2317/14 20130101; A61K 39/395 20130101;
A61P 43/00 20180101; A61K 31/22 20130101; C07K 2299/00 20130101;
A61P 25/28 20180101; C07K 16/40 20130101; A61K 31/505 20130101;
A61K 2039/505 20130101; C12N 15/1137 20130101; A61P 3/00 20180101;
A61K 31/47 20130101; A61K 31/44 20130101; C07K 2317/34 20130101;
A61K 45/06 20130101; A61K 31/66 20130101; A61P 9/00 20180101; A61P
7/00 20180101; A61K 31/405 20130101; A61K 39/3955 20130101; A61P
9/10 20180101; A61K 31/22 20130101; A61K 2300/00 20130101; A61K
31/366 20130101; A61K 2300/00 20130101; A61K 31/40 20130101; A61K
2300/00 20130101; A61K 31/405 20130101; A61K 2300/00 20130101; A61K
31/44 20130101; A61K 2300/00 20130101; A61K 31/47 20130101; A61K
2300/00 20130101; A61K 31/505 20130101; A61K 2300/00 20130101; A61K
31/66 20130101; A61K 2300/00 20130101; A61K 39/395 20130101; A61K
2300/00 20130101; A61K 39/3955 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
536/23.53 |
International
Class: |
C07K 16/40 20060101
C07K016/40 |
Claims
1. (canceled)
2. A monoclonal antibody that comprises a heavy chain variable
region and a light chain variable region and binds to PCSK9,
wherein the antibody comprises a paratope, wherein the paratope
comprises: at least one amino acid in a specified position of a
heavy chain variable region selected from the group consisting of
T30, S31, Y32, G33, W50, S52, F53, Y54, N55, N57, N59, R98, G99,
Y100, and G101.
3. The monoclonal antibody of claim 2, wherein the heavy chain
variable region comprises at least two amino acids in the specified
position selected from the group consisting of T30, S31, Y32, G33,
W50, S52, F53, Y54, N55, N57, N59, R98, G99, Y100, and G101.
4. The monoclonal antibody of claim 2, wherein the heavy chain
variable region comprises at least three amino acids in the
specified position selected from the group consisting of T30, S31,
Y32, G33, W50, S52, F53, Y54, N55, N57, N59, R98, G99, Y100, and
G101.
5. The monoclonal antibody of claim 2, wherein said monoclonal
antibody is a human or a humanized monoclonal antibody.
6. The monoclonal antibody of claim 2, wherein said monoclonal
antibody binds to a D374Y variant of a PCSK9 protein comprising SEQ
ID NO: 1.
7. The monoclonal antibody of claim 2, wherein said monoclonal
antibody is an IgG.
8. The monoclonal antibody of claim 2, wherein said light chain
variable region is connected to a human kappa amino acid
sequence.
9. The monoclonal antibody of claim 2, wherein the monoclonal
antibody comprises an intact immunoglobulin.
10. The monoclonal antibody of claim 2, wherein the monoclonal
antibody comprises a fragment of an intact immunoglobulin.
11. The monoclonal antibody of claim 2, wherein the monoclonal
antibody comprises the amino acid sequence of SEQ ID NO: 157.
12. A monoclonal antibody that comprises a heavy chain variable
region and a light chain variable region and binds to PCSK9,
wherein the antibody comprises a paratope, wherein the paratope
comprises: at least one amino acid in a specified position of a
light chain variable region selected from the group consisting of:
G30, G31, Y32, N33, S34, E52, Y93, T94, S95, T96, and S97.
13. The monoclonal antibody of claim 12, wherein the light chain
variable region comprises at least two amino acids in the specified
position selected from the group consisting of: G30, G31, Y32, N33,
S34, E52, Y93, T94, S95, T96, and S97.
14. The monoclonal antibody of claim 12, wherein said monoclonal
antibody is a human or a humanized monoclonal antibody.
15. The monoclonal antibody of claim 12, wherein said monoclonal
antibody binds to a D374Y variant of a PCSK9 protein comprising SEQ
ID NO: 1.
16. The monoclonal antibody of claim 12, wherein said monoclonal
antibody is an IgG.
17. The monoclonal antibody of claim 12, wherein said light chain
variable region is connected to a human kappa amino acid
sequence.
18. The monoclonal antibody of claim 12, wherein the monoclonal
antibody comprises an intact immunoglobulin.
19. The monoclonal antibody of claim 12, wherein the monoclonal
antibody comprises a fragment of an intact immunoglobulin.
20. The monoclonal antibody of claim 12, wherein the monoclonal
antibody comprises the amino acid sequence of SEQ ID NO: 157.
21. A monoclonal antibody that comprises a heavy chain variable
region and a light chain variable region and binds to PCSK9,
wherein the antibody comprises a paratope, wherein the paratope
comprises: at least one amino acid in a specified position of a
light chain variable region selected from the group consisting of:
G30, G31, Y32, N33, S34, E52, Y93, T94, S95, T96, and S97; and at
least one amino acid in a specified position of a heavy chain
variable region selected from the group consisting of T30, S31,
Y32, G33, W50, S52, F53, Y54, N55, N57, N59, R98, G99, Y100, and
G101.
22. The monoclonal antibody of claim 21, wherein the light chain
variable region comprises at least two amino acids in the specified
position selected from the group consisting of: G30, G31, Y32, N33,
S34, E52, Y93, T94, S95, T96, and S97.
23. The monoclonal antibody of claim 21, wherein the heavy chain
variable region comprises at least two amino acids in the specified
position selected from the group consisting of T30, S31, Y32, G33,
W50, S52, F53, Y54, N55, N57, N59, R98, G99, Y100, and G101.
24. The monoclonal antibody of claim 23, wherein the light chain
variable region comprises at least two amino acids in the specified
position selected from the group consisting of: G30, G31, Y32, N33,
S34, E52, Y93, T94, S95, T96, and S97.
25. A monoclonal antibody that comprises a heavy chain variable
region and a light chain variable region and binds to PCSK9,
wherein the antibody comprises an antigen binding region, wherein
the antigen binding region comprises: at least one amino acid in a
specified position of a light chain variable region selected from
the group consisting of: G30, G31, Y32, N33, S34, E52, Y93, T94,
S95, T96, and S97; and at least one amino acid in a specified
position of a heavy chain variable region selected from the group
consisting of T30, S31, Y32, G33, W50, S52, F53, Y54, N55, N57,
N59, R98, G99, Y100, and G101.
26. The monoclonal antibody of claim 25, wherein said monoclonal
antibody is a human or a humanized monoclonal antibody.
27. The monoclonal antibody of claim 25, wherein the monoclonal
antibody blocks binding of human PCSK9 to LDLR by at least 20%.
28. The monoclonal antibody of claim 25, wherein the monoclonal
antibody blocks binding of human PCSK9 to LDLR by at least 80%.
29. The monoclonal antibody of claim 25, wherein the monoclonal
antibody competes for binding to human PCSK9 with an antibody
comprising: a heavy chain variable region of SEQ ID NO: 49; and a
light chain variable region of SEQ ID NO: 23.
30. The monoclonal antibody of claim 25, wherein the monoclonal
antibody comprises the amino acid sequence of SEQ ID NO: 157.
31. The monoclonal antibody of claim 25, wherein the monoclonal
antibody inhibits binding of PCSK9 to LDLR.
Description
RELATED APPLICATIONS
[0001] This application is a divisional and claims priority to U.S.
Non-Provisional application Ser. No. 12/197,093, filed Aug. 22,
2008, and U.S. Provisional Application Ser. No. 61/086,133, filed
Aug. 4, 2008, Ser. No. 60/957,668, filed Aug. 23, 2007, Ser No.
61/008,965, filed Dec. 21, 2007, and Ser. No. 61/010,630, filed
Jan. 9, 2008, hereby incorporated by reference in their
entireties.
SEQUENCE LISTING AND TABLES IN ELECTRONIC FORMAT
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled Sequence_Listing_APMOL-003D2.txt, created and last
saved Sep. 30, 2011 which is 296,708 bytes in size. The information
in the electronic format of the Sequence Listing is incorporated
herein by reference in its entirety. The present application is
being filed along with a collection of Tables in electronic format.
The collection of Tables is provided as a file entitled
Table.sub.--35-1-4_APMOL-003D2.txt, created and last saved on Sep.
30, 2011, which is 2,024,359 bytes in size. The information in the
electronic format of the collection of Tables is incorporated
herein by reference in its entirety.
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140357854A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
FIELD OF THE INVENTION
[0003] The present invention relates to antigen binding proteins
that bind to proprotein convertase subtilisin kexin type 9 (PCSK9)
and methods of using and making the antigen binding proteins.
BACKGROUND OF VARIOUS EMBODIMENTS
[0004] Proprotein convertase subtilisin kexin type 9 (PCSK9) is a
serine protease involved in regulating the levels of the low
density lipoprotein receptor (LDLR) protein (Horton et al., 2007;
Seidah and Prat, 2007). In vitro experiments have shown that adding
PCSK9 to HepG2 cells lowers the levels of cell surface LDLR
(Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005;
Park et al., 2004). Experiments with mice have shown that
increasing PCSK9 protein levels decreases levels of LDLR protein in
the liver (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et
al., 2005; Park et al., 2004), while PCSK9 knockout mice have
increased levels of LDLR in the liver (Rashid et al., 2005).
Additionally, various human PCSK9 mutations that result in either
increased or decreased levels of plasma LDL have been identified
(Kotowski et al., 2006; Zhao et al, 2006). PCSK9 has been shown to
directly interact with the LDLR protein, be endocytosed along with
the LDLR, and co-immunofluoresce with the LDLR throughout the
endosomal pathway (Lagace et al., 2006). Degradation of the LDLR by
PCSK9 has not been observed and the mechanism through which it
lowers extracellular LDLR protein levels is uncertain.
[0005] PCSK9 is a prohormone-proprotein convertase in the
subtilisin (S8) family of serine proteases (Seidah et al., 2003).
Humans have nine prohormone-proprotein convertases that can be
divided between the S8A and S8B subfamilies (Rawlings et al.,
2006). Furin, PC1/PC3, PC2, PACE4, PC4, PC5/PC6 and
PC7/PC8/LPC/SPC7 are classified in subfamily S8B. Crystal and NMR
structures of different domains from mouse furin and PC1 reveal
subtilisin-like pro- and catalytic domains, and a P domain directly
C-terminal to the catalytic domain (Henrich et al., 2003; Tangrea
et al., 2002). Based on the amino acid sequence similarity within
this subfamily, all seven members are predicted to have similar
structures (Henrich et al., 2005). SKI-1/S1P and PCSK9 are
classified in subfamily S8A Sequence comparisons with these
proteins also suggest the presence of subtilisin-like pro- and
catalytic domains (Sakai et al., 1998; Seidah et al., 2003; Seidah
et al., 1999). In these proteins the amino acid sequence C-terminal
to the catalytic domain is more variable and does not suggest the
presence of a P domain.
[0006] Prohormone-proprotein convertases are expressed as zymogens
and they mature through a multi step process. The function of the
pro-domain in this process is two-fold. The pro-domain first acts
as a chaperone and is required for proper folding of the catalytic
domain (Ikemura et al., 1987). Once the catalytic domain is folded,
autocatalysis occurs between the pro-domain and catalytic domain.
Following this initial cleavage reaction, the pro-domain remains
bound to the catalytic domain where it then acts as an inhibitor of
catalytic activity (Fu et al., 2000). When conditions are correct,
maturation proceeds with a second autocatalytic event at a site
within the pro-domain (Anderson et al., 1997). After this second
cleavage event occurs the pro-domain and catalytic domain
dissociate, giving rise to an active protease.
[0007] Autocatalysis of the PCSK9 zymogen occurs between Gln152 and
Ser153 (VFAQ|SIP) (Naureckiene et al., 2003), and has been shown to
be required for its secretion from cells (Seidah et al., 2003). A
second autocatalytic event at a site within PCSK9's pro-domain has
not been observed. Purified PCSK9 is made up of two species that
can be separated by non-reducing SDS-PAGE; the pro-domain at 17 Kd,
and the catalytic plus C-terminal domains at 65 Kd. PCSK9 has not
been isolated without its inhibitory pro-domain, and measurements
of PCSK9's catalytic activity have been variable (Naureckiene et
al., 2003; Seidah et al., 2003).
SUMMARY OF VARIOUS EMBODIMENTS
[0008] In some embodiments, the invention comprises an antigen
binding protein to PCSK9.
[0009] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9 comprising: A) one or more heavy
chain complementary determining regions (CDRHs) selected from the
group consisting of: (i) a CDRH1 from a CDRH1 in a sequence
selected from the group consisting of SEQ ID NO: 74, 85, 71, 72,
67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,
65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii) a CDRH2 from a
CDRH2 in a sequence selected from the group consisting of SEQ ID
NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57,
50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60;
(iii) a CDRH3 from a CDRH3 in a sequence selected from the group
consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,
48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78,
83, 69, 81, and 60; and (iv) a CDRH of (i), (ii), and (iii) that
contains one or more amino acid substitutions, deletions or
insertions of no more than 4 amino acids; B) one or more light
chain complementary determining regions (CDRLs) selected from the
group consisting of: (i) a CDRL1 from a CDRL1 in a sequence
selected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12,
13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,
35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 from a CDRL2
in a sequence selected from the group consisting of SEQ ID NO: 5,
7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,
30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) a
CDRL3 from a CDRL3 in a sequence selected from the group consisting
of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and
46; and (iv) a CDRL of (i), (ii) and (iii) that contains one or
more amino acid substitutions, deletions or insertions of no more
than 4 amino acids; or C) one or more heavy chain CDRHs of A) and
one or more light chain CDRLs of B). In some embodiments, the
isolated antigen binding protein comprises at least one CDRH of A)
and at least one CDRL of B). In some embodiments, the isolated
antigen binding protein comprises at least two CDRH of A) and at
least two CDRL of B). In some embodiments, the isolated antigen
binding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2
and CDRL3. In some embodiments, the CDRH of A) is selected from at
least one of the group consisting of: (i) a CDRH1 amino acid
sequence selected from the CDRH1 in a sequence selected from the
group consisting of SEQ ID NO: 67, 79, 89, and 49; (ii) a CDRH2
amino acid sequence selected from the CDRH2 in a sequence selected
from the group consisting of SEQ ID NO: 67, 79, 89, and 49; (iii) a
CDRH3 amino acid sequence selected from the CDRH3 in a sequence
selected from the group consisting of SEQ ID NO: 67, 79, 89, and
49; and (iv) a CDRH of (i), (ii) and (iii) that contains one or
more amino acid substitutions, deletions or insertions of no more
than 2 amino acids. In addition, the CDRL of B) is selected from at
least one of the group consisting of: (i) a CDRL1 amino acid
sequence selected from the CDRL1 in a sequence selected from the
group consisting of SEQ ID NO: 12, 35, 32, and 23; (ii) a CDRL2
amino acid sequence selected from the CDRL2 in a sequence selected
from the group consisting of SEQ ID NO: 12, 35, 32, and 23; (iii) a
CDRL3 amino acid sequence selected from the CDRL3 in a sequence
selected from the group consisting of SEQ ID NO: 12, 35, 32, and
23; and (iv) a CDRL of (i), (ii) and (iii) that contains one or
more amino acid substitutions, deletions or insertions of no more
than 2 amino acids; or C) one or more heavy chain CDRHs of A) and
one or more light chain CDRLs of B. In some embodiments, the CDRHs
of A) is selected from at least one of the group consisting of: (i)
a CDRH1 amino acid sequence of the CDRH1 amino acid sequence in SEQ
ID NO: 67; (ii) a CDRH2 amino acid sequence of the CDRH2 amino acid
sequence in SEQ ID NO: 67; (iii) a CDRH3 amino acid sequence of the
CDRH3 amino acid sequence in SEQ ID NO: 67; and (iv) a CDRH of (i),
(ii) and (iii) that contains one or more amino acid substitutions,
deletions or insertions of no more than 2 amino acids; said CDRL of
B) is selected from at least one of the group consisting of: (i) a
CDRL1 amino acid sequence of the CDRL1 amino acid sequence in SEQ
ID NO: 12; (ii) a CDRL2 amino acid sequence of the CDRL2 amino acid
sequence in SEQ ID NO: 12; (iii) a CDRL3 amino acid sequence of the
CDRL3 amino acid sequence in SEQ ID NO: 12; and (iv) a CDRL of (i),
(ii) and (iii) that contains one or more amino acid substitutions,
deletions or insertions of no more than 2 amino acids; or C) one or
more heavy chain CDRHs of A) and one or more light chain CDRLs of
B). In some embodiments, the antigen binding protein comprises A) a
CDRH1 of the CDRH1 sequence in SEQ ID NO: 67, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 67, and a CDRH3 of the CDRH3 sequence in SEQ
ID NO: 67, and B) a CDRL1 of the CDRL1 sequence in SEQ ID NO: 12, a
CDRL2 of the CDRL2 sequence in SEQ ID NO: 12, and a CDRL3 of the
CDRL3 sequence in SEQ ID NO: 12. In some embodiments, the antigen
binding protein comprises a heavy chain variable region (VH) having
at least 80% sequence identity with an amino acid sequence selected
from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58,
52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80,
76, 77, 78, 83, 69, 81, and 60, and/or a light chain variable
region (VL) having at least 80% sequence identity with an amino
acid sequence selected from the group consisting of SEQ ID NO: 5,
7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,
30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some
embodiments, the VH has at least 90% sequence identity with an
amino acid sequence selected from the group consisting of SEQ ID
NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57,
50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60,
and/or the VL has at least 90% sequence identity with an amino acid
sequence selected from the group consisting of SEQ ID NO: 5, 7, 9,
10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31,
32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some
embodiments, the VH is selected from the group consisting of SEQ ID
NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57,
50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60,
and/or the VL is selected from the group consisting of SEQ ID NO:
5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26,
28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.
[0010] In some aspects, the invention comprises an isolated antigen
binding protein that specifically binds to an epitope that is bound
by any of the ABPs disclosed herein.
[0011] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, wherein the antigen binding
protein comprises: A) one or more heavy chain CDRs (CDRHs) selected
from at least one of the group consisting of: (i) a CDRH1 with at
least 80% sequence identity to a CDRH1 in one of the sequences
selected from the group consisting of SEQ ID NO: 74, 85, 71, 72,
67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,
65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii) a CDRH2 with at
least 80% sequence identity to a CDRH2 in one of the sequences
selected from the group consisting of SEQ ID NO: 74, 85, 71, 72,
67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,
65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; and (iii) a CDRH3 with
at least 80% sequence identity to a CDRH3 in one of the sequences
selected from the group consisting of SEQ ID NO: 74, 85, 71, 72,
67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,
65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; B) one or more light
chain CDRs (CDRLs) selected from at least one of the group
consisting of (i) a CDRL1 with at least 80% sequence identity to a
CDRL1 in one of the sequences selected from the group consisting of
SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46;
(ii) a CDRL2 with at least 80% sequence identity to a CDRL2 in one
of the sequences selected from the group consisting of SEQ ID NO:
5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26,
28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and
(iii) a CDRL3 with at least 80% sequence identity to a CDRL3 in one
of the sequences selected from the group consisting of SEQ ID NO:
5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26,
28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; or C)
one or more heavy chain CDRHs of A) and one or more light chain
CDRLs of B). In some embodiments, the antigen binding protein
comprises: A) one or more CDRHs selected from at least one of the
group consisting of: (i) a CDRH1 with at least 90% sequence
identity to a CDRH1 in one of the sequences selected from the group
consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,
48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78,
83, 69, 81, and 60; (ii) a CDRH2 with at least 90% sequence
identity to a CDRH2 in one of the sequences selected from the group
consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,
48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78,
83, 69, 81, and 60; and (iii) a CDRH3 with at least 90% sequence
identity to a CDRH3 in one of the sequences selected from the group
consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,
48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78,
83, 69, 81, and 60; B) one or more CDRLs selected from at least one
of the group consisting of: (i) a CDRL1 with at least 90% sequence
identity to a CDRL1 in one of the sequences selected from the group
consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40,
42, 44, and 46; (ii) a CDRL2 with at least 90% sequence identity to
a CDRL2 in one of the sequences selected from the group consisting
of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and
46; and (iii) a CDRL3 with at least 90% sequence identity to a
CDRL3 in one of the sequences selected from the group consisting of
SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46;
or C) one or more heavy chain CDRHs of A) and one or more light
chain CDRLs of B).
[0012] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprises: A) a heavy chain complementary determining region (CDRH)
selected from at least one of the group consisting of: (i) a CDRH3
selected from the CDRH3 within the sequences selected from the
group consisting of SEQ ID NOs: 67, 79, and 49, (ii) a CDRH3 that
differs in amino acid sequence from the CDRH3 of (i) by an amino
acid addition, deletion or substitution of not more than two amino
acids; and (iii)
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14 (SEQ ID NO: 404), wherein X.sub.1 is
selected from the group consisting of D, A, R, and not amino acid,
X.sub.2 is selected from the group consisting of Y, I, G, and no
amino acid, X.sub.3 is selected from the group consisting of D, A,
G, and no amino acid, X.sub.4 is selected from the group consisting
of F, A, L, and no amino acid, X.sub.5 is selected from the group
consisting of W, L, A, and no amino acid, X.sub.6 is selected from
the group consisting of S, Y, A, and no amino acid, X.sub.7 is
selected from the group consisting of A, Y, R, and no amino acid,
X.sub.8 is selected from the group consisting of Y, P, and no amino
acid, X.sub.9 is selected from the group consisting of Y, G, and no
amino acid, X.sub.10 is selected from the group consisting of D, G,
and no amino acid, X.sub.11 is selected from the group consisting
of A, M, and no amino acid, X.sub.12 is selected from the group
consisting of F, D, and no amino acid, X.sub.13 is selected from
the group consisting of D, V, and no amino acid, X.sub.14 is
selected from the group consisting of V and no amino acid; B) a
light chain complementary determining region (CDRL) selected from
at least one of the group consisting of: (i) a CDRL3 selected from
the CDRL3 within the sequences selected from the group consisting
of SEQ ID NOs: 12, 35, and 23, (ii) a CDRL3 that differs in amino
acid sequence from the CDRL3 of (i) by an amino acid addition,
deletion or substitution of not more than two amino acids; and
(iii) a CDRL3 amino acid sequence selected from the group
consisting of:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11 (SEQ ID NO: 405), wherein X.sub.1 is selected from the group
consisting of Q and G, X.sub.2 is selected from the group
consisting of S, T, A, and no amino acid, X.sub.3 is selected from
the group consisting of Y, no amino acid, and W, X.sub.4 is
selected from the group consisting of D and no amino acid, X.sub.5
is selected from the group consisting of S and no amino acid,
X.sub.6 is selected from the group consisting of S and no amino
acid, X.sub.7 is selected from the group consisting of L, T, and no
amino acid, X.sub.8 is selected from the group consisting of no
amino acid, A, and S, X.sub.9 is selected from the group consisting
of no amino acid, G, A, and V, X.sub.10 is selected from the group
consisting of no amino acid, S, Y, and V, X.sub.11 is selected from
the group consisting of no amino acid and V.
[0013] In some aspects, the invention comprises an isolated antigen
binding protein comprising a light chain having the amino acid
sequence selected from the group consisting of: 5, 7, 9, 10, 12,
13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,
35, 36, 37, 38, 39, 40, 42, 44, 46, and some combination
thereof.
[0014] In some embodiments, the antigen binding protein
specifically binds to an epitope that is bound by at least one of
the antigen binding proteins disclosed herein. In some embodiments,
the isolated antigen binding protein further comprises a heavy
chain having the amino acid sequence selected from the group
consisting of: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55,
56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81,
60, and some combination thereof. In some embodiments, the amino
acid sequence of the ABP is selected from the group consisting of
SEQ ID NO: 12, 35, 23, and some combination thereof. In some
embodiments, the heavy chain of the ABP comprises a CDRH3 of SEQ ID
NO: 67, a CDRH2 of SEQ ID NO: 67, and a CDRH1 of SEQ ID NO:67, and
said light chain comprises a CDRL3 of SEQ ID NO: 12, a CDRL2 of SEQ
ID NO: 12, and a CDRL1 of SEQ ID NO: 12. In some embodiments, the
isolated antigen binding protein is a monoclonal antibody, a
polyclonal antibody, a recombinant antibody, a human antibody, a
humanized antibody, a chimeric antibody, a multispecific antibody,
or an antibody fragment thereof. In some embodiments, the isolated
antigen binding protein is a Fab fragment, a Fab' fragment, a
F(ab').sub.2 fragment, a Fv fragment, a diabody, or a single chain
antibody molecule. In some embodiments, the isolated antigen
binding protein is a human antibody. In some embodiments, the
isolated antigen binding protein is a monoclonal antibody. In some
embodiments, the isolated antigen binding protein is of the IgG1-,
IgG2- IgG3- or IgG4-type. In some embodiments, the isolated antigen
binding protein is of the IgG4- or IgG2-type. In some embodiments,
the isolated antigen binding protein is coupled to a labeling
group. In some embodiments, the isolated antigen binding protein
competes for binding to PCSK9 with an antigen binding protein
described herein. In some embodiments, the isolated antigen binding
protein is a monoclonal antibody, a polyclonal antibody, a
recombinant antibody, a human antibody, a humanized antibody, a
chimeric antibody, a multispecific antibody, or an antibody
fragment thereof. In some embodiments, the isolated antigen binding
protein is a Fab fragment, a Fab' fragment, a F(ab').sub.2
fragment, a Fv fragment, a diabody, or a single chain antibody
molecule. In some embodiments, the isolated antigen binding protein
is coupled to a labeling group. In some embodiments, the isolated
antigen binding protein reduces binding of PCSK9 to LDLR. In some
embodiments, the isolated antigen binding protein the antigen
binding protein decreases an amount of LDL present in a subject
when administered to the subject. In some embodiments, the isolated
antigen binding protein decreases an amount of serum cholesterol
present in a subject when administered to the subject. In some
embodiments, the isolated antigen binding protein increases an
amount of LDLR present in a subject when administered to the
subject.
[0015] In some aspects, the invention comprises a vector comprising
a nucleic acid molecule as described herein. In some embodiments,
the invention comprises a host cell comprising a nucleic acid
molecule as described herein.
[0016] In some aspects, the invention comprises an isolated antigen
binding protein that competes for binding to PCSK9 with an antigen
binding protein disclosed herein.
[0017] In some aspects, the invention comprises a nucleic acid
molecule encoding the antigen binding protein according disclosed
herein.
[0018] In some aspects, the invention comprises a pharmaceutical
composition comprising at least one antigen binding protein
described herein.
[0019] In some aspects, the invention comprises a method for
treating or preventing a condition associated with elevated serum
cholesterol levels in a patient, comprising administering to a
patient in need thereof an effective amount of at least one
isolated antigen binding protein disclosed herein.
[0020] In some aspects, the invention comprises a method of
inhibiting binding of PCSK9 to LDLR in a subject comprising
administering an effective amount of at least one antigen binding
protein disclosed herein.
[0021] In some aspects, the invention comprises an antigen binding
protein that selectively binds to PCSK9, wherein the antigen
binding protein binds to PCSK9 with a K.sub.d that is smaller than
100 pM.
[0022] In some aspects, the invention comprises a method for
treating or preventing a condition associated with elevated serum
cholesterol levels in a subject, the method comprising
administering to a subject in need thereof an effective amount of
at least one isolated antigen binding protein disclosed herein
simultaneously or sequentially with an agent that elevates the
availability of LDLR protein.
[0023] In some aspects, the invention comprises a method of
lowering serum cholesterol level in a subject, the method
comprising administering to a subject an effective amount of at
least one isolated antigen binding protein as disclosed herein.
[0024] In some aspects, the invention comprises a method of
lowering serum cholesterol level in a subject, the method
comprising administering to a subject an effective amount of at
least one isolated antigen binding protein as disclosed herein,
simultaneously or sequentially with an agent that elevates the
availability of LDLR protein.
[0025] In some aspects, the invention comprises a method of
increasing LDLR protein level in a subject, the method comprising
administering to a subject an effective amount of at least one
isolated antigen binding protein as disclosed herein.
[0026] In some aspects, the invention comprises a method of
increasing LDLR protein levels in a subject, the method comprising
administering to a subject an effective amount of at least one
isolated antigen binding protein as disclosed herein simultaneously
or sequentially with an agent that elevates the availability of
LDLR protein.
[0027] In some aspects, the invention comprises a pharmaceutical
composition comprising an ABP as disclosed herein and an agent that
elevates the availability of LDLR protein levels. In some
embodiments, the agent that elevates the availability of LDLR
protein comprises a statin. In some embodiments, the statin is
selected from the group consisting of atorvastatin, cerivastatin,
fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin,
rosuvastatin, simvastatin, and some combination thereof.
[0028] In some aspect, the invention comprises a method of making
the antigen binding protein as described herein, comprising the
step of preparing said antigen binding protein from a host cell
that secretes said antigen binding protein.
[0029] In some aspect, the invention comprises a pharmaceutical
composition comprising at least one antigen binding protein as
described herein and a pharmaceutically acceptable excipient. In
some embodiments, the pharmaceutical composition further comprises
an additional active agent. In some embodiments, said additional
active agent is selected from the group consisting of a
radioisotope, radionuclide, a toxin, or a therapeutic and a
chemotherapeutic group.
[0030] In some aspects, the invention comprises a method for
treating or preventing a condition associated with an elevated
serum cholesterol level in a patient. The method comprises
administering to a patient in need thereof an effective amount of
at least one isolated antigen binding protein as disclosed herein.
In some embodiments, the condition is hypercholesterolemia.
[0031] In some aspects, the invention comprises a method of
inhibiting binding of PCSK9 to LDLR in a patient comprising
administering an effective amount of at least one antigen binding
protein according as described herein.
[0032] In some aspect, the invention comprises an antigen binding
protein that binds to PCSK9 with a K.sub.d that is smaller than 100
pM. In some embodiments, the antigen binding protein binds with a
K.sub.d that is smaller than 10 pM. In some embodiments, the
antigen binding protein binds with a K.sub.d that is less than 5
pM.
[0033] In some aspects, the invention comprises a method for
treating or preventing a condition associated with elevated serum
cholesterol levels in a subject, said method comprising
administering to a subject in need thereof an effective amount of
at least one isolated antigen binding protein described herein
simultaneously or sequentially with an agent that elevates the
availability of LDLR protein. In some embodiments, the agent that
elevates the availability of LDLR protein comprises a statin. In
some embodiments, the statin is selected from the group consisting
of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,
pitavastatin, pravastatin, rosuvastatin, simvastatin, and some
combination thereof.
[0034] In some aspects, the invention comprises a method of
lowering the serum cholesterol level in a subject. The method
comprises administering to a subject an effective amount of at
least one isolated antigen binding protein as described herein.
[0035] In some aspects, the invention comprises a method of
lowering serum cholesterol levels in a subject comprising
administering to a subject an effective amount of at least one
isolated antigen binding protein, as described herein,
simultaneously or sequentially with an agent that elevates the
availability of LDLR protein. In some embodiments, the agent that
elevates the availability of LDLR protein comprises a statin. In
some embodiments, the statin is selected from the group consisting
of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,
pitavastatin, pravastatin, rosuvastatin, simvastatin, and some
combination thereof.
[0036] In some aspects, the invention comprises a method of
increasing LDLR protein levels in a subject by administering to a
subject an effective amount of at least one isolated antigen
binding protein as provided herein.
[0037] In some aspects, the invention comprises a method of
increasing LDLR protein levels in a subject by administering to a
subject an effective amount of at least one isolated antigen
binding protein, as described herein, simultaneously or
sequentially with an agent that elevates the availability of LDLR
protein. In some embodiments, the agent that elevates the
availability of LDLR protein levels comprises a statin. In some
embodiments, the statin is selected from the group consisting of
atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,
pitavastatin, pravastatin, rosuvastatin, simvastatin, and some
combination thereof.
[0038] In some aspects, the invention comprises a neutralizing
antibody that binds to PCSK9 and reduces a low density lipoprotein
receptor (LDLR) lowering effect of PCSK9 on LDLR. In some
embodiments, the antibody specifically binds to PCSK9. In some
embodiments, the antibody binds to the catalytic domain of PCSK9.
In some embodiments, the antibody binds to an epitope within
residues 31-447 of SEQ ID NO: 3. In some embodiments, the antibody
binds to PCSK9 having an amino acid sequence that is at least 90%
identical to SEQ ID NO: 3.
[0039] In some aspects, the invention comprises a neutralizing
antigen binding protein that binds to PCSK9, wherein the antigen
binding protein binds to PCSK9 at a location within residues 31-447
of SEQ ID NO: 3. In some embodiments, when the antigen binding
protein is bound to PCSK9, the antibody is positioned 8 angstroms
or less from at least one of the following residues of PCSK9: S153,
I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378,
F379, V380, S381, W156, N157, L158, E159, H193, E195, H229, R237,
G240, K243, D367, I368, G370, A371, S373, S376, Q382, W72, F150,
A151, Q152, T214, R215, F216, H217, A220, S221, K222, S225, H226,
C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350, L351,
E366, D367, D374, V380, S381, Q382, S383, G384, K69, D70, P71,
S148, V149, D186, T187, E211, D212, G213, R218, Q219, C223, D224,
G227, H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325,
V346, G352, T353, G365, I368, I369, S372, S373, C378, F379, T385,
S386, Q387, S153, S188, I189, Q190, S191, D192, R194, E197, G198,
R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379,
I154, T187, H193, E195, I196, M201, V202, C223, T228, S235, G236,
A239, G244, M247, I369, S372, C375, or C378. In some embodiments,
the antibody is positioned 8 angstroms or less from at least one of
the following residues of PCSK9: S153, I154, P155, R194, D238,
A239, I369, S372, D374, C375, T377, C378, F379, V380, S381, W156,
N157, L158, E159, H193, E195, H229, R237, G240, K243, D367, I368,
G370, A371, S373, S376, or Q382. In some embodiments, the antibody
is positioned 5 angstroms or less from at least one of the
following residues of PCSK9: S153, I154, P155, R194, D238, A239,
I369, S372, D374, C375, T377, C378, F379, V380, or S381. In some
embodiments, the antibody is positioned 5 angstroms or less from at
least two of the following residues of PCSK9: S153, I154, P155,
R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380,
or S381. In some embodiments, the antibody is 5 angstroms or less
from at least four of the following residues of PCSK9: S153, I154,
P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379,
V380, or S381. In some embodiments, the antibody is positioned 8
angstroms or less from at least one of the following residues of
PCSK9: W72, F150, A151, Q152, T214, R215, F216, H217, A220, S221,
K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348,
G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383, G384,
K69, D70, P71, S148, V149, D186, T187, E211, D212, G213, R218,
Q219, C223, D224, G227, H229, L253, N254, G259, P288, A290, G291,
G316, R319, Y325, V346, G352, T353, G365, I368, I369, S372, S373,
C378, F379, T385, S386, or Q387. In some embodiments, the antibody
is positioned 5 angstroms or less from at least one of the
following residues of PCSK9: W72, F150, A151, Q152, T214, R215,
F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258,
N317, F318, T347, L348, G349, T350, L351, E366, D367, D374, V380,
S381, Q382, S383, or G384. In some embodiments, the antibody is
positioned 5 angstroms or less from at least two of the following
residues of PCSK9: W72, F150, A151, Q152, T214, R215, F216, H217,
A220, S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318,
T347, L348, G349, T350, L351, E366, D367, D374, V380, S381, Q382,
S383, or G384. In some embodiments, the antibody is positioned 5
angstroms or less from at least four of the following residues of
PCSK9: W72, F150, A151, Q152, T214, R215, F216, H217, A220, S221,
K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348,
G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383, or
G384. In some embodiments, the antibody is positioned 8 angstroms
or less from at least one of the following residues of PCSK9: S153,
S188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224,
R237, D238, K243, S373, D374, S376, T377, F379, I154, T187, H193,
E195, I196, M201, V202, C223, T228, S235, G236, A239, G244, M247,
I369, 5372, C375, or C378. In some embodiments, the antibody is
positioned 5 angstroms or less from at least one of the following
residues of PCSK9: S153, S188, I189, Q190, S191, D192, R194, E197,
G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377,
or F379. In some embodiments, the antibody is positioned 5
angstroms or less from at least two of the following residues of
PCSK9: S153, S188, I189, Q190, S191, D192, R194, E197, G198, R199,
V200, D224, R237, D238, K243, S373, D374, S376, T377, or F379. In
some embodiments, the antibody is positioned 5 angstroms or less
from at least four of the following residues of PCSK9: S153, S188,
I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237,
D238, K243, S373, D374, S376, T377, or F379.
[0040] In some aspects, the invention comprises a neutralizing
antibody that binds to PCSK9, wherein the antibody binds to PCSK9
and reduces the likelihood that PCSK9 binds to LDLR.
[0041] In some embodiments, an antibody or antigen binding molecule
that binds to PCSK9 is contemplated. The antibody binds to PCSK9 at
a location within residues 31-447 of SEQ ID NO: 3. In some
embodiments, the antibody or antigen binding molecule, when bound
to PCSK9, is positioned 8 angstroms or less from at least one of
the following residues of PCSK9: S153, I154, P155, R194, D238,
A239, I369, S372, D374, C375, T377, C378, F379, V380, S381, W156,
N157, L158, E159, H193, E195, H229, R237, G240, K243, D367, I368,
G370, A371, S373, S376, Q382, W72, F150, A151, Q152, T214, R215,
F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258,
N317, F318, T347, L348, G349, T350, L351, E366, D367, D374, V380,
S381, Q382, S383, G384, K69, D70, P71, S148, V149, D186, T187,
E211, D212, G213, R218, Q219, C223, D224, G227, H229, L253, N254,
G259, P288, A290, G291, G316, R319, Y325, V346, G352, T353, G365,
I368, I369, S372, S373, C378, F379, T385, S386, Q387, S153, S188,
I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237,
D238, K243, S373, D374, S376, T377, F379, I154, T187, H193, E195,
I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369,
S372, C375, or C378.
[0042] In some embodiments, an isolated antibody or antigen binding
molecule that blocks an antibody to PCSK9 from binding within 8
angstroms of a residue of PCSK9 is provided. In some embodiments
the residue of PCSK9 is selected from at least one of the following
PCSK9 residues: S153, I154, P155, R194, D238, A239, I369, S372,
D374, C375, T377, C378, F379, V380, S381, W156, N157, L158, E159,
H193, E195, H229, R237, G240, K243, D367, I368, G370, A371, S373,
S376, Q382, W72, F150, A151, Q152, T214, R215, F216, H217, A220,
S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347,
L348, G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383,
G384, K69, D70, P71, S148, V149, D186, T187, E211, D212, G213,
R218, Q219, C223, D224, G227, H229, L253, N254, G259, P288, A290,
G291, G316, R319, Y325, V346, G352, T353, G365, I368, I369, S372,
S373, C378, F379, T385, S386, Q387, S153, S188, I189, Q190, S191,
D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373,
D374, S376, T377, F379, I154, T187, H193, E195, I196, M201, V202,
C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, or
C378.
[0043] In some embodiments, an isolated antibody or antigen binding
molecule that binds to PCSK9 at a location that overlaps with a
location that LDLR binds to PCSK9 is provided. In some embodiments,
the location that LDLR binds to PCSK9 includes at least one amino
acid residue selected from the group consisting of: S153, I154,
P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379,
V380, and S381.
[0044] In some embodiments, an isolated antibody or antigen binding
molecule that binds to PCSK9 is provided. In some embodiments, the
antibody or antigen binding molecule reduces the likelihood that
EGFa will bind to PCSK9 within 8 angstroms of at least one of the
following residues on PCSK9: S153, I154, P155, R194, D238, A239,
I369, S372, D374, C375, T377, C378, F379, V380, S381, W156, N157,
L158, E159, H193, E195, H229, R237, G240, K243, D367, I368, G370,
A371, S373, S376, or Q382.
[0045] In some embodiments, an antibody, antigen binding protein,
or antigen binding molecule that binds to a surface of PCSK9 that
overlaps with a surface that EGFa binds, Ab 21B12 binds, and/or
31H4 binds is provided. In some embodiments, an antibody, antigen
binding protein, or antigen binding molecule that binds to PCSK9 in
a manner that is similar to that depicted in the figures is
provided.
[0046] In some embodiments, the above embodiments are neutralizing
antibodies or antigen binding proteins. In some embodiments, the
antigen binding protein is not LDLR or a fragment thereof (such as
EGFa).
[0047] In some aspects, the invention comprises an isolated
neutralizing antibody, wherein when the antibody is bound to PCSK9,
the antibody is positioned 8 angstroms or less from at least one of
the following residues of PCSK9: T468, R469, M470, A471, T472,
R496, R499, E501, A502, Q503, R510, H512, F515, P540, P541, A542,
E543, H565, W566, E567, V568, E569, R592, E593, S465, G466, P467,
A473, I474, R476, G497, E498, M500, G504, K506, L507, V508, A511,
N513, A514, G516, V536, T538, A539, A544, T548, D570, L571, H591,
A594, S595, and H597 of SEQ ID NO: 3. In some embodiments, the
antibody is positioned 5 angstroms or less from at least one of the
following residues of PCSK9: T468, R469, M470, A471, T472, R496,
R499, E501, A502, Q503, R510, H512, F515, P540, P541, A542, E543,
H565, W566, E567, V568, E569, R592, and E593 of SEQ ID NO: 3.
[0048] In some aspects, the invention comprises an isolated antigen
binding protein. The antigen binding protein comprises: A) a CDRH1
of the CDRH1 sequence in SEQ ID NO: 89, a CDRH2 of the CDRH2
sequence in SEQ ID NO: 89, and a CDRH3 of the CDRH3 sequence in SEQ
ID NO: 89, and B) a CDRL1 of the CDRL1 sequence in SEQ ID NO:32, a
CDRL2 of the CDRL2 sequence in SEQ ID NO:32, and a CDRL3 of the
CDRL3 sequence in SEQ ID NO:32.
[0049] In some aspects, the invention comprises an isolated antigen
binding protein that binds to a PCSK9 protein of SEQ ID NO: 1 where
the binding between said isolated antigen binding protein and a
variant PCSK9 protein is less than 50% of the binding between the
isolated antigen binding protein and the PCSK9 protein of SEQ ID
NO: 1 and/or SEQ ID NO: 303. In some embodiments, the variant PCSK9
protein comprises at least one mutation of a residue at a position
selected from the group consisting or comprising 207, 208, 185,
181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167,
123, 129, 311, 313, 337, 519, 521, and 554, as shown in SEQ ID NO:
1. In some embodiments, the at least one mutation selected from the
group comprising or consisting of R207E, D208R, E181R, R185E,
R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R, and E582R.
In some embodiments, the at least one mutation is selected from the
group consisting of D162R, R164E, E167R, S123R, E129R, A311R,
D313R, D337R, R519E, H521R, and Q554R.
[0050] In some aspects, the invention comprises an antigen binding
protein that binds to a PCSK-9 protein of SEQ ID NO: 303 in a first
manner and binds to a variant of PCSK9 in a second manner. The
PCSK9 variant has at least one point mutation at a position
selected from the group comprising or consisting of 207, 208, 185,
181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167,
123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 303 and/or
SEQ ID NO: 1. In some embodiments, the first Manner comprises a
first EC50, a first Bmax, or a first EC50 and a first Bmax. In some
embodiments, the second manner comprises a second EC50, a second
Bmax, or a second EC50 and a second Bmax. The value for the first
manner is different from the value for the second manner. In some
embodiments, the first manner comprises a first EC50, wherein the
second manner involves a second EC50, and wherein the point
mutation is selected from the group consisting or comprising:
R207E, D208R, E181R, R185E, R439E, E513R, V538R, E539R, T132R,
S351R, A390R, A413R, and E582R. In some embodiments, the first EC50
is at least 20% different from the second EC50. In some
embodiments, the first EC50 is at least 50% different from the
second EC50. In some embodiments, the second EC50 is a larger
numerical value than the first EC50. In some embodiments, the first
EC50 is determined by a multiplex bead binding assay. In some
embodiments, the second EC50 is greater than 1 um. In some
embodiments, the antigen binding protein is a neutralizing antigen
binding protein. In some embodiments, the neutralizing antigen
binding protein is a competitive neutralizing antigen binding
protein. In some embodiments, the neutralizing antigen binding
protein is a non-competitive neutralizing antigen binding protein.
In some embodiments, the first manner comprises a first Bmax and
the second manner comprises a second Bmax that is different from
the first Bmax. The PCSK9 variant has at least one point mutation
selected from the group consisting or comprising: D162R, R164E,
E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R.
In some embodiments, the second Bmax is about 10% of the first
Bmax. In some embodiments, the first Bmax is at least 20% different
from the second Bmax. In some embodiments, the first Bmax is at
least 50% different from the second Bmax.
[0051] In some aspects, the invention comprises an isolated antigen
binding protein that binds to a PCSK9 protein of SEQ ID NO: 3,
wherein the epitope of the antigen binding protein includes at
least one of the following amino acids of SEQ ID NO: 1: 207, 208,
181, 185, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164,
167, 123, 129, 311, 313, 337, 519, 521, and 554.
[0052] In some aspects, the invention comprises an isolated
neutralizing antigen binding protein that binds to a PCSK9 protein
comprising the amino acid sequence of SEQ ID NO: 1, wherein the
neutralizing antigen binding protein decreases the LDLR lowering
effect of PCSK9 on LDLR. In some embodiments, the antigen binding
protein is a LDLR non-competitive neutralizing antigen binding
protein. In some embodiments, the antigen binding protein is a LDLR
competitive neutralizing antigen binding protein.
[0053] In some aspects, the invention comprises an isolated antigen
binding protein, wherein said antigen binding protein comprises: A)
a CDRH1 of the CDRH1 sequence in SEQ ID NO: 49, a CDRH2 of the
CDRH2 sequence in SEQ ID NO: 49, and a CDRH3 of the CDRH3 sequence
in SEQ ID NO: 49, and B) a CDRL1 of the CDRL1 sequence in SEQ ID
NO:23, a CDRL2 of the CDRL2 sequence in SEQ ID NO:23, and a CDRL3
of the CDRL3 sequence in SEQ ID NO:23.
[0054] In some aspects, the invention comprises a composition
comprising a crystallized PCSK9 protein and an antigen binding
protein that binds to PCSK9. The composition comprises the
crystallized PCSK9 protein is such that the three dimensional
structure of the PCSK9 protein can be determined to a resolution of
about 2.2 angstroms or better. In some embodiments, the antigen
binding protein is an antibody or a fragment thereof.
[0055] In some aspects, the invention comprises a crystallized
PCSK9 protein and at least an EGFa section of a LDLR protein,
wherein the EGFa section of the LDLR protein is bound by a PCSK9
protein, wherein said crystallized PCSK9 protein is such that the
three dimensional structure of the PCSK9 protein can be determined
to a resolution of about 2.2 angstroms or better. In some
embodiments, the molecular model is on a computer readable
medium.
[0056] In some aspects, the invention comprises the use of an
antigen binding protein as described herein, in the preparation of
a medicament for the lowering of serum cholesterol.
[0057] In some aspects, the invention comprises the use of an
antigen binding protein as described herein, in the preparation of
a medicament for treating or preventing a condition associated with
elevated serum cholesterol levels in a subject.
[0058] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprising: A) a heavy chain complementary determining region
(CDRH) selected from at least one of the group consisting of: (i) a
CDRH1 selected from the CDRH1 within the sequences selected from
the group consisting of SEQ ID NOs: 67, 79, 89, and 49, (ii) a
CDRH1 that differs in amino acid sequence from the CDRH1 of (i) by
an amino acid addition, deletion or substitution of not more than
two amino acids; and (iii) a CDRH1 amino acid sequence selected
from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10
(SEQ ID NO: 406), wherein X.sub.1 is selected from the group
consisting of G, X.sub.2 is selected from the group consisting of
Y, F, and G, X.sub.3 is selected from the group consisting of T and
S, X.sub.4 is selected from the group consisting of L and F,
X.sub.5 is selected from the group consisting of T, S, and N,
X.sub.6 is selected from the group consisting of S and A, X.sub.7
is selected from the group consisting of Y and F, X.sub.8 is
selected from the group consisting of G, S, and Y, X.sub.9 is
selected from the group consisting of I, M, and W, X.sub.10 is
selected from the group consisting of S, N and H, B) a light chain
complementary determining region (CDRL) selected from at least one
of the group consisting of: (i) a CDRL1 selected from the CDRL1
within the sequences selected from the group consisting of SEQ ID
NOs: 12, 32, 35, and 23, (ii) a CDRL1 that differs in amino acid
sequence from the CDRL3 of (i) by an amino acid addition, deletion
or substitution of not more than two amino acids; and (iii) a CDRL1
amino acid sequence selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14 (SEQ ID NO: 407), wherein X.sub.1 is
selected from the group consisting of T and no amino acid, X.sub.2
is selected from the group consisting of G and S, X.sub.3 is
selected from the group consisting of S, T, and G, X.sub.4 is
selected from the group consisting of S, X.sub.5 is selected from
the group consisting of S, X.sub.6 is selected from the group
consisting of N, D, and S, X.sub.7 is selected from the group
consisting of I, V, and N, X.sub.8 is selected from the group
consisting of G and I, X.sub.9 is selected from the group
consisting of A and G, X.sub.10 is selected from the group
consisting of G, Y, S, and N, X.sub.11 is selected from the group
consisting of Y and N, X.sub.12 is selected from the group
consisting of D, S, T, and F, X.sub.13 is selected from the group
consisting of V, X.sub.14 is selected from the group consisting of
S, N, and H. One of skill in the art will appreciate that a single
ABP or antibody can meet one or more of the above options and still
fall within the described invention for this embodiment.
[0059] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprising: A) a heavy chain complementary determining region
(CDRH) selected from at least one of the group consisting of the
following: (i) a CDRH2 selected from the CDRH2 within the sequences
selected from the group consisting of SEQ ID NOs: 67, 79, 89, and
49, (ii) a CDRH2 that differs in amino acid sequence from the CDRH2
of (i) by an amino acid addition, deletion or substitution of not
more than two amino acids; and (iii) a CDRH2 amino acid sequence
selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17 (SEQ ID NO:
408), wherein X.sub.1 is selected from the group consisting of W,
S, L and no amino acid, X.sub.2 is selected from the group
consisting of V, I, and E, X.sub.3 is selected from the group
consisting of S, W, and I, X.sub.4 is selected from the group
consisting of F, S, and N, X.sub.5 is selected from the group
consisting of Y, S, D, and H, X.sub.6 is selected from the group
consisting of N, S, and G, X.sub.7 is selected from the group
consisting of S and G, X.sub.8 is selected from the group
consisting of N, Y, D, and R, X.sub.9 is selected from the group
consisting of T, I, and E, X.sub.10 is selected from the group
consisting of N, S, Y, and D, X.sub.11 is selected from the group
consisting of Y, X.sub.12 is selected from the group consisting of
A and N, X.sub.13 is selected from the group consisting of Q, D,
and P, X.sub.14 is selected from the group consisting of K and S,
X.sub.15 is selected from the group consisting of L, and V,
X.sub.16 is selected from the group consisting of Q and K, X.sub.17
is selected from the group consisting of G and S, B) a light chain
complementary determining region (CDRL) selected from at least one
of the group consisting of the following: (i) a CDRL2 selected from
the CDRL3 within the sequences selected from the group consisting
of SEQ ID NOs: 12, 32, 35, and 23, (ii) a CDRL2 that differs in
amino acid sequence from the CDRL3 of (i) by an amino acid
addition, deletion or substitution of not more than two amino
acids; and (iii) a CDRL2 amino acid sequence selected from the
group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7 (SEQ ID NO: 409),
wherein X.sub.1 is selected from the group consisting of G, E, S,
and D, X.sub.2 is selected from the group consisting of N, V, and
Y, X.sub.3 is selected from the group consisting of S and N,
X.sub.4 is selected from the group consisting of N, Q, and K,
X.sub.5 is selected from the group consisting of R, X.sub.6 is
selected from the group consisting of P, X.sub.7 is selected from
the group consisting of S.
[0060] In some aspects, the invention comprises An isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprising: A) a heavy chain complementary determining region
(CDRH) selected from at least one of the group consisting of the
following: (i) a CDRH3 selected from the CDRH3 within the sequences
selected from the group consisting of SEQ ID NOs: 67, 79, 89, and
49, (ii) a CDRH3 that differs in amino acid sequence from the CDRH3
of (i) by an amino acid addition, deletion or substitution of not
more than two amino acids; and (iii) a CDRH3 amino acid sequence
selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14 (SEQ ID NO: 410), wherein X.sub.1 is
selected from the group consisting of D, and no amino acid, X.sub.2
is selected from the group consisting of Y, A, and no amino acid,
X.sub.3 is selected from the group consisting of D, I, and no amino
acid, X.sub.4 is selected from the group consisting of F, A, and no
amino acid, X.sub.5 is selected from the group consisting of W, A,
and no amino acid, X.sub.6 is selected from the group consisting of
S, L, and no amino acid, X.sub.7 is selected from the group
consisting of A, Y, G, and no amino acid, X.sub.8 is selected from
the group consisting of Y, Q, and no amino acid, X.sub.9 is
selected from the group consisting of G, Y, and L, X.sub.10 is
selected from the group consisting of Y, D, and V, X.sub.11 is
selected from the group consisting of G, A, and P, X.sub.12 is
selected from the group consisting of M and F, X.sub.13 is selected
from the group consisting of D, X.sub.14 is selected from the group
consisting of V and Y, and B) a light chain complementary
determining region (CDRL) selected from at least one of the group
consisting of the following: (i) a CDRL3 selected from the CDRL3
within the sequences selected from the group consisting of SEQ ID
NOs: 12, 32, 35, and 23, (ii) a CDRL3 that differs in amino acid
sequence from the CDRL3 of (i) by an amino acid addition, deletion
or substitution of not more than two amino acids; and (iii) a CDRL3
amino acid sequence selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11 (SEQ ID NO: 411), wherein X.sub.1 is selected from the group
consisting of Q, A, G, and no amino acid, X.sub.2 is selected from
the group consisting of S, V, T, and no amino acid, X.sub.3 is
selected from the group consisting of Y, N, and W, X.sub.4 is
selected from the group consisting of S and D, X.sub.5 is selected
from the group consisting of S, Y, and D, X.sub.6 is selected from
the group consisting of S and T, X.sub.7 is selected from the group
consisting of L and S, X.sub.8 is selected from the group
consisting of S, T, and N, X.sub.9 is selected from the group
consisting of G, S, and A, X.sub.10 is selected from the group
consisting of S, M, W, and Y, and X.sub.11 is selected from the
group consisting of V. In some embodiments, any of the above amino
acids can be replaced by a conservative amino acid
substitution.
[0061] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprises A) a heavy chain complementary determining region (CDRH)
selected from at least one of the group consisting of (i) a CDRH1
selected from the CDRH1 within the sequences selected from the
group consisting of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, and 58, (ii) a CDRH1 that differs in amino acid sequence
from the CDRH1 of (i) by an amino acid addition, deletion or
substitution of not more than two amino acids; and (iii) a CDRH1
amino acid sequence selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10
(SEQ ID NO: 412), wherein X.sub.1 is selected from the group
consisting of G, P, and A, X.sub.2 is selected from the group
consisting of Y, W, F, T, and S, X.sub.3 is selected from the group
consisting of T, P, S and A, C, V, L, and I, X.sub.4 is selected
from the group consisting of L, F, I, V, M, A, and Y, X.sub.5 is
selected from the group consisting of T, P, S, and A, X.sub.6 is
selected from the group consisting of S, T, A, and C, X.sub.7 is
selected from the group consisting of Y, W, F, T, and S, X.sub.8 is
selected from the group consisting of G, P, and A, X.sub.9 is
selected from the group consisting of I, L, V, M, A, and F,
X.sub.10 is selected from the group consisting of S, T, A, and C,
B) a light chain complementary determining region (CDRL) selected
from at least one of the group consisting of: (i) a CDRL1 selected
from the CDRL1 within the sequences selected from the group
consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
and 24, (ii) a CDRL1 that differs in amino acid sequence from the
CDRL3 of (i) by an amino acid addition, deletion or substitution of
not more than two amino acids; and (iii) a CDRL1 amino acid
sequence selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14 (SEQ ID NO: 413), wherein, X.sub.1
is selected from the group consisting of T and S, X.sub.2 is
selected from the group consisting of G, P, and A, X.sub.3 is
selected from the group consisting of T, and S, X.sub.4 is selected
from the group consisting of S N, T, A, C, and Q, X.sub.5 is
selected from the group consisting of S, T, A, and C, X.sub.6 is
selected from the group consisting of D, and E, X.sub.7 is selected
from the group consisting of V, I, M, L, F, and A, X.sub.8 is
selected from the group consisting of G, P, and A, X.sub.9 is
selected from the group consisting of G, A, R, P, V, L, I, K, Q,
and N, X.sub.10 is selected from the group consisting of Y, W, F,
T, and S, X.sub.11 is selected from the group consisting of N, and
Q, X.sub.12 is selected from the group consisting of Y, S, W, F, T,
A, and C, X.sub.13 is selected from the group consisting of V, I,
M, L, F, and A, X.sub.14 is selected from the group consisting of
S, T, A, and C.
[0062] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprising: A) a heavy chain complementary determining region
(CDRH) selected from at least one of the group consisting of: (i) a
CDRH2 selected from the CDRH2 within the sequences selected from
the group consisting of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, and 58, (ii) a CDRH2 that differs in amino acid
sequence from the CDRH2 of (i) by an amino acid addition, deletion
or substitution of not more than two amino acids; and (iii) a CDRH2
amino acid sequence selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17, (SEQ ID NO:
414), wherein X.sub.1 is selected from the group consisting of W,
Y, and F, X.sub.2 is selected from the group consisting of V, I, M,
L, F, and A, X.sub.3 is selected from the group consisting of S, T,
A, and C, X.sub.4 is selected from the group consisting of A, F, V,
L, I, Y, and M, X.sub.5 is selected from the group consisting of Y,
W, F, T, and S, X.sub.6 is selected from the group consisting of N
and Q, X.sub.7 is selected from the group consisting of G, P, and
A, X.sub.8 is selected from the group consisting of N, and Q,
X.sub.9 is selected from the group consisting of T, and S, X.sub.10
is selected from the group consisting of N, and Q, X.sub.11 is
selected from the group consisting of Y, W, F, T, and S, X.sub.12
is selected from the group consisting of A, V, L, and I, X.sub.13
is selected from the group consisting of Q, E, N, and D, X.sub.14
is selected from the group consisting of K, R, Q, and N, X.sub.15
is selected from the group consisting of L, F, V, I, M, A, and Y,
X.sub.16 is selected from the group consisting of Q, and N,
X.sub.17 is selected from the group consisting of G, P, and A, B) a
light chain complementary determining region (CDRL) selected from
at least one of the group consisting of: (i) a CDRL2 selected from
the CDRL3 within the sequences selected from the group consisting
of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, (ii)
a CDRL2 that differs in amino acid sequence from the CDRL3 of (i)
by an amino acid addition, deletion or substitution of not more
than two amino acids; and (iii) a CDRL2 amino acid sequence
selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7 (SEQ ID NO: 415),
wherein X.sub.1 is selected from the group consisting of E, and D,
X.sub.2 is selected from the group consisting of V, I, M, L, F, and
A, X.sub.3 is selected from the group consisting of S, T, A, and C,
X.sub.4 is selected from the group consisting of N, and Q, X.sub.5
is selected from the group consisting of R, K, Q, and N, X.sub.6 is
selected from the group consisting of P, and A, X.sub.7 is selected
from the group consisting of S, T, A, and C.
[0063] In some aspects, the invention comprises an isolated antigen
binding protein that binds PCSK9, the antigen binding protein
comprising: A) a heavy chain complementary determining region
(CDRH) selected from at least one of the group consisting of (i) a
CDRH3 selected from the CDRH3 within the sequences selected from
the group consisting of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, and 58, (ii) a CDRH3 that differs in amino acid
sequence from the CDRH3 of (i) by an amino acid addition, deletion
or substitution of not more than two amino acids; and (iii) a CDRH3
amino acid sequence selected from the group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6 (SEQ ID NO: 416),
wherein X.sub.1 is selected from the group consisting of G, P, A
and no amino acid, X.sub.2 is selected from the group consisting of
Y, W, F, T, and S, X.sub.3 is selected from the group consisting of
G, V, P, A, I, M, L, and F, X.sub.4 is selected from the group
consisting of M, L, F, and I, X.sub.5 is selected from the group
consisting of D, and E, X.sub.6 is selected from the group
consisting of V, I, M, L, F, and A, B) a light chain complementary
determining region (CDRL) selected from at least one of the group
consisting of (i) a CDRL3 selected from the CDRL3 within the
sequences selected from the group consisting of SEQ ID NOs: 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, and 24, (ii) a CDRL3 that differs
in amino acid sequence from the CDRL3 of (i) by an amino acid
addition, deletion or substitution of not more than two amino
acids; and (iii) a CDRL3 amino acid sequence selected from the
group consisting of
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9
(SEQ ID NO: 417), wherein X.sub.1 is selected from the group
consisting of S, N, T, A, C, and Q, X.sub.2 is selected from the
group consisting of S, T, A, and C, X.sub.3 is selected from the
group consisting of Y, W, F, T, and S, X.sub.4 is selected from the
group consisting of T, and S, X.sub.5 is selected from the group
consisting of S, T, A, and C, X.sub.6 is selected from the group
consisting of S, T, A, and C, X.sub.7 is selected from the group
consisting of N, S, Q, T, A, and C, X.sub.8 is selected from the
group consisting of M, V, L, F, I, and A, X.sub.9 is selected from
the group consisting of V, I, M, L, F, and A.
BRIEF DESCRIPTION OF THE FIGURES
[0064] FIG. 1A depicts an amino acid sequence of the mature form of
the PCSK9 with the pro-domain underlined.
[0065] FIGS. 1B.sub.1-1B.sub.4 depict amino acid and nucleic acid
sequences of PCSK9 with the pro-domain underlined and the signal
sequence in bold.
[0066] FIGS. 2A-2D are sequence comparison tables of various light
chains of various antigen binding proteins. FIG. 2C continues the
sequence started in FIG. 2A. FIG. 2D continues the sequence started
on FIG. 2B.
[0067] FIGS. 3A-3D are sequence comparison tables of various heavy
chains of various antigen binding proteins. FIG. 3C continues the
sequence started in FIG. 3A. FIG. 3D continues the sequence started
on FIG. 3B.
[0068] FIGS. 3E-3JJ depict the amino acid and nucleic acid
sequences for the variable domains of some embodiments of the
antigen binding proteins.
[0069] FIG. 3KK depicts the amino acid sequences for various
constant domains.
[0070] FIGS. 3LL-3BBB depict the amino acid and nucleic acid
sequences for the variable domains of some embodiments of the
antigen binding proteins.
[0071] FIGS. 3CCC-3JJJ are sequence comparison tables of various
heavy and light chains of some embodiments of the antigen binding
proteins.
[0072] FIG. 4A is a binding curve of an antigen binding protein to
human PCSK9.
[0073] FIG. 4B is a binding curve of an antigen binding protein to
human PCSK9.
[0074] FIG. 4C is a binding curve of an antigen binding protein to
cynomolgus PCSK9.
[0075] FIG. 4D is a binding curve of an antigen binding protein to
cynomolgus PCSK9.
[0076] FIG. 4E is a binding curve of an antigen binding protein to
mouse PCSK9.
[0077] FIG. 4F is a binding curve of an antigen binding protein to
mouse PCSK9.
[0078] FIG. 5A depicts the results of an SDS PAGE experiment
involving PCSK9 and various antigen binding proteins demonstrating
the relative purity and concentration of the proteins.
[0079] FIGS. 5B and 5C depict graphs from biacore solution
equilibrium assays for 21B12.
[0080] FIG. 5D depicts the graph of the kinetics from a biacore
capture assay.
[0081] FIG. 5E depicts a bar graph depicting binding results for
three ABPs.
[0082] FIG. 6A is an inhibition curve of antigen binding protein
31H4 IgG2 to PCSK9 in an in vitro PCSK9:LDLR binding assay.
[0083] FIG. 6B is an inhibition curve of antigen binding protein
31H4 IgG4 to PCSK9 in an in vitro PCSK9:LDLR binding assay.
[0084] FIG. 6C is an inhibition curve of antigen binding protein
21B12 IgG2 to PCSK9 in an in vitro PCSK9:LDLR binding assay.
[0085] FIG. 6D is an inhibition curve of antigen binding protein
21B12 IgG4 to PCSK9 in an in vitro PCSK9:LDLR binding assay.
[0086] FIG. 7A is an inhibition curve of antigen binding protein
31H4 IgG2 in the cell LDL uptake assay showing the effect of the
ABP to reduce the LDL uptake blocking effects of PCSK9
[0087] FIG. 7B is an inhibition curve of antigen binding protein
31H4 IgG4 in the cell LDL uptake assay showing the effect of the
ABP to reduce the LDL uptake blocking effects of PCSK9
[0088] FIG. 7C is an inhibition curve of antigen binding protein
21B12 IgG2 in the cell LDL uptake assay showing the effect of the
ABP to reduce the LDL uptake blocking effects of PCSK9
[0089] FIG. 7D is an inhibition curve of antigen binding protein
21B12 IgG4 in the cell LDL uptake assay showing the effect of the
ABP to reduce the LDL uptake blocking effects of PCSK9
[0090] FIG. 8A is a graph depicting the serum cholesterol lowering
ability in mice of ABP 31H4, changes relative to the IgG control
treated mice (* p<0.01).
[0091] FIG. 8B is a graph depicting the serum cholesterol lowering
ability in mice of ABP 31H4, changes relative to time=zero hours (#
p, 0.05).
[0092] FIG. 8C is a graph depicting the effect of ABP 31H4 on HDL
cholesterol levels in C57Bl/6 mice (* p<0.01).
[0093] FIG. 8D is a graph depicting the effect of ABP 31H4 on HDL
cholesterol levels in C57Bl/6 mice (# p<0.05).
[0094] FIG. 9 depicts a western blot analysis of the ability of ABP
31H4 to enhance the amount of liver LDLR protein present after
various time points.
[0095] FIG. 10A is a graph depicting the ability of an antigen
binding protein 31H4 to lower total serum cholesterol in wild type
mice, relative.
[0096] FIG. 10B is a graph depicting the ability of an antigen
binding protein 31H4 to lower HDL in wild type mice.
[0097] FIG. 10C is a graph depicting the serum cholesterol lowering
ability of various antigen binding proteins 31H4 and 16F12.
[0098] FIG. 11A depicts an injection protocol for testing the
duration and ability of antigen binding proteins to lower serum
cholesterol.
[0099] FIG. 11B is a graph depicting the results of the protocol in
FIG. 11A.
[0100] FIG. 12A depicts LDLR levels in response to the combination
of a statin and ABP 21B12 in HepG2 cells.
[0101] FIG. 12B depicts LDLR levels in response to the combination
of a statin and ABP 31H4 in HepG2 cells.
[0102] FIG. 12C depicts LDLR levels in response to the combination
of a statin and ABP 25A7.1, a nonneutralizing antibody, (in
contrast the "25A7" a neutralizing antibody) in HepG2 cells.
[0103] FIG. 12D depicts LDLR levels in response to the combination
of a statin and ABP 21B12 in HepG2 cells overexpressing PCSK9.
[0104] FIG. 12E depicts LDLR levels in response to the combination
of a statin and ABP 31H4 in HepG2 cells overexpressing PCSK9.
[0105] FIG. 12F depicts LDLR levels in response to the combination
of a statin and ABP 25A7.1, a nonneutralizing antibody, (in
contrast the "25A7" a neutralizing antibody) in HepG2 cells
overexpressing PCSK9.
[0106] FIG. 13A depicts the various light chain amino acid
sequences of various ABPs to PCSK9. The dots (.) indicate no amino
acid.
[0107] FIG. 13B depicts a light chain cladogram for various ABPs to
PCSK9.
[0108] FIG. 13C depicts the various heavy chain amino acid
sequences of various ABPs to PCSK9. The dots (.) indicate no amino
acid.
[0109] FIG. 13D depicts a heavy chain dendrogram for various ABPs
to PCSK9.
[0110] FIG. 13E depicts a comparison of light and heavy CDRs and
designation of groups from which to derive consensus.
[0111] FIG. 13F depicts the consensus sequences for Groups 1 and
2.
[0112] FIG. 13G depicts the consensus sequences for Groups 3 and
4.
[0113] FIG. 13H depicts the consensus sequences for Groups 1 and 2.
The dots (.) indicated identical residues.
[0114] FIG. 13I depicts the consensus sequences for Group 2. The
dots (.) indicated identical residues.
[0115] FIG. 13J depicts the consensus sequences for Groups 3 and 4.
The dots (.) indicated identical residues.
[0116] FIG. 14A is a graph depicting in vivo LDL lowering ability
of various ABPs (at 10 mg/kg).
[0117] FIG. 14B is a graph depicting in vivo LDL lowering ability
of various ABPs (at 30 mg/kg).
[0118] FIG. 15A and FIG. 15B are sequence comparison tables of
various light chains of various embodiments of antigen binding
proteins. FIG. 15B continues the sequence started in FIG. 15A.
[0119] FIG. 15C and FIG. 15D are sequence comparison tables of
various light chains of various embodiments of antigen binding
proteins. FIG. 15D continues the sequence started in FIG. 15C.
[0120] FIG. 16A is a depiction of a gel used to test the ability of
Ab 21B12 to bind to the ProCat or VD sections of PCSK9.
[0121] FIG. 16B is a depiction of a gel used to test the ability of
Ab 31H4 to bind to the ProCat or VD sections of PCSK9.
[0122] FIG. 17 is a depiction of the structure of PCSK9 and the
EGFa section of LDLR.
[0123] FIG. 18A is a depiction of the structure of PCSK9 and the
31H4 Ab.
[0124] FIG. 18B is a depiction of the structure of PCSK9 and the
31H4 Ab.
[0125] FIG. 19A is a depiction of the structure of PCSK9, the 31H4
Ab, and the 21B12 Ab.
[0126] FIG. 19B is a depiction of the structure of PCSK9 and the
21B12 Ab.
[0127] FIG. 20A is a depiction of the structure of PCSK9 and EGFa
from the LDLR superimposed with the structure of antibodies 31H4
and 21B12 bound to PCSK9.
[0128] FIG. 20B is a depiction of the structural model of PCSK9 and
LDLR.
[0129] FIG. 20C is a depiction of the structural model of PCSK9 and
LDLR from an alternative perspective.
[0130] FIG. 20D is a depiction of the structural model of PCSK9 and
LDLR with structural representations of 31H4 and 21B12
included.
[0131] FIG. 20E is a depiction of the structural model in FIG. 20D,
rotated 90 degrees about the noted axis.
[0132] FIG. 20F is a depiction of the structural model in FIG. 20D
rotated 180 degrees about the noted axis.
[0133] FIG. 21A is a depiction of the structure of PCSK9 and
31A4.
[0134] FIG. 21B is a depiction of the structure of PCSK9 and
31A4.
[0135] FIG. 21C is a depiction of the structure of PCSK9 and
31A4.
[0136] FIG. 21D is a depiction of the structural model of full
length PCSK9 and 31A4.
[0137] FIG. 22 is a set of ABP sequences identifying various
differences between the human ABP sequences and the ABP sequences
that were raised in E. coli and used for the crystal
structures.
[0138] FIG. 23 is a table depicting the various binning
results.
[0139] FIG. 23A is a first part of a table depicting the various
binning results.
[0140] FIG. 23B is a second part of a table depicting the various
binning results.
[0141] FIG. 23C is a third part of a table depicting the various
binning results.
[0142] FIG. 23D is a fourth part of a table depicting the various
binning results.
[0143] FIG. 24A is a depiction of a western blot under non-reduced
conditions.
[0144] FIG. 24B is a depiction of a western blot under reduced
conditions.
[0145] FIG. 25A is a depiction of the surface coverage of
PCSK9.
[0146] FIG. 25B is a depiction of the surface coverage of
PCSK9.
[0147] FIG. 25C is a depiction of the surface coverage of
PCSK9.
[0148] FIG. 25D is a depiction of the surface coverage of
PCSK9.
[0149] FIG. 25E is a depiction of the surface coverage of
PCSK9.
[0150] FIG. 25F is a depiction of the surface coverage of
PCSK9.
[0151] FIG. 26 is a sequence comparison of the PCSK9 amino acid
sequence and all of the residues that were mutated in PCSK9
variants to examine the epitopes of the various antibodies.
[0152] FIG. 27A depicts the 21B12 epitope hits, as mapped onto a
crystal structure of PCSK9 with the 21B12.
[0153] FIG. 27B depicts the 31H4 epitope hits, as mapped onto a
crystal structure of PCSK9 with 31H4 and 21B1.
[0154] FIG. 27C depicts the 31A4 epitope hits, as mapped onto a
crystal structure of PCSK9 with 31H4 and 21B12.
[0155] FIG. 27D depicts the 12H11 epitope hits, as mapped onto the
crystal structure of PCSK9 with 31H4 and 21B12.
[0156] FIG. 27E depicts the 3C4 epitope hits, as mapped onto the
crystal structure of PCSK9 with 31H4 and 21B12.
[0157] FIG. 28A is a graph demonstrating the binding ability of the
various ABPs to various parts of PCSK9.
[0158] FIG. 28B is a graph demonstrating the binding ability of the
various ABPs to various parts of PCSK9.
[0159] FIG. 28C is a graph comparing the LDLR binding ability of
two ABPs.
[0160] FIG. 28D is a graph comparing the cell LDL uptake activity
of two ABPs.
DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS
[0161] Antigen binding proteins (such as antibodies and functional
binding fragments thereof) that bind to PCSK9 are disclosed herein.
In some embodiments, the antigen binding proteins bind to PCSK9 and
prevent PCSK9 from functioning in various ways. In some
embodiments, the antigen binding proteins block or reduce the
ability of PCSK9 to interact with other substances. For example, in
some embodiments, the antigen binding protein binds to PCSK9 in a
manner that prevents or reduces the likelihood that PCSK9 will bind
to LDLR. In other embodiments, antigen binding proteins bind to
PCSK9 but do not block PCSK9's ability to interact with LDLR. In
some embodiments, the antigen binding proteins are human monoclonal
antibodies.
[0162] As will be appreciated by one of skill in the art, in light
of the present disclosure, altering the interactions between PCSK9
and LDLR can increase the amount of LDLR available for binding to
LDL, which in turn decreases the amount of serum LDL in a subject,
resulting in a reduction in the subject's serum cholesterol level.
As such, antigen binding proteins to PCSK9 can be used in various
methods and compositions for treating subjects with elevated serum
cholesterol levels, at risk of elevated serum cholesterol levels,
or which could benefit from a reduction in their serum cholesterol
levels. Thus, various methods and techniques for lowering,
maintaining, or preventing an increase in serum cholesterol are
also described herein. In some embodiments, the antigen binding
protein allows for binding between PCSK9 and LDLR, but the antigen
binding protein prevents or reduces the adverse activity of PCSK9
on LDLR. In some embodiments, the antigen binding protein prevents
or reduces the binding of PCSK9 to LDLR.
[0163] For convenience, the following sections generally outline
the various meanings of the terms used herein. Following this
discussion, general aspects regarding antigen binding proteins are
discussed, followed by specific examples demonstrating the
properties of various embodiments of the antigen binding proteins
and how they can be employed.
DEFINITIONS AND EMBODIMENTS
[0164] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements and
components comprising one unit and elements and components that
comprise more than one subunit unless specifically stated
otherwise. Also, the use of the term "portion" can include part of
a moiety or the entire moiety.
[0165] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose. As
utilized in accordance with the present disclosure, the following
terms, unless otherwise indicated, shall be understood to have the
following meanings:
[0166] The term "proprotein convertase subtilisin kexin type 9" or
"PCSK9" refers to a polypeptide as set forth in SEQ ID NO: 1 and/or
3 or fragments thereof, as well as related polypeptides, which
include, but are not limited to, allelic variants, splice variants,
derivative variants, substitution variants, deletion variants,
and/or insertion variants including the addition of an N-terminal
methionine, fusion polypeptides, and interspecies homologs. In
certain embodiments, a PCSK9 polypeptide includes terminal
residues, such as, but not limited to, leader sequence residues,
targeting residues, amino terminal methionine residues, lysine
residues, tag residues and/or fusion protein residues. "PCSK9" has
also been referred to as FH3, NARC1, HCHOLA3, proprotein convertase
subtilisin/kexin type 9, and neural apoptosis regulated convertase
1. The PCSK9 gene encodes a proprotein convertase protein that
belongs to the proteinase K subfamily of the secretory subtilase
family. The term "PCSK9" denotes both the proprotein and the
product generated following autocatalysis of the proprotein. When
only the autocatalyzed product is being referred to (such as for an
antigen binding protein that selectively binds to the cleaved
PCSK9), the protein can be referred to as the "mature," "cleaved",
"processed" or "active" PCSK9. When only the inactive form is being
referred to, the protein can be referred to as the "inactive",
"pro-form", or "unprocessed" form of PCSK9. The term PCSK9 as used
herein also includes naturally occurring alleles, such as the
mutations D374Y, S127R and F216L. The term PCSK9 also encompasses
PCSK9 molecules incorporating post-translational modifications of
the PCSK9 amino acid sequence, such as PCSK9 sequences that have
been glycosylated, PEGylated, PCSK9 sequences from which its signal
sequence has been cleaved, PCSK9 sequence from which its pro domain
has been cleaved from the catalytic domain but not separated from
the catalytic domain (e.g., FIGS. 1A and 1B).
[0167] The term "PCSK9 activity" 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 some
embodiments, PCSK9 activity is represented by the ability of PCSK9
to bind to a LDL receptor (LDLR). In some embodiments, PCSK9 binds
to and catalyzes a reaction involving LDLR. In some embodiments,
PCSK9 activity includes the ability of PCSK9 to alter (e.g.,
reduce) the availability of LDLR. In some embodiments, PCSK9
activity includes the ability of PCSK9 to increase the amount of
LDL in a subject. In some embodiments, PCSK9 activity includes the
ability of PCSK9 to decrease the amount of LDLR that is available
to bind to LDL. In some embodiments, "PCSK9 activity" includes any
biological activity resulting from PCSK9 signaling. Exemplary
activities include, but are not limited to, PCSK9 binding to LDLR,
PCSK9 enzyme activity that cleaves LDLR or other proteins, PCSK9
binding to proteins other than LDLR that facilitate PCSK9 action,
PCSK9 altering APOB secretion (Sun X-M et al, "Evidence for effect
of mutant PCSK9 on apoliprotein B secretion as the cause of
unusually severe dominant hypercholesterolemia, Human Molecular
Genetics 14: 1161-1169, 2005 and Ouguerram K et al, "Apolipoprotein
B100 metabolism in autosomal-dominant hypercholesterolemia related
to mutations in PCSK9, Arterioscler thromb Vasc Biol. 24:
1448-1453, 2004), PCSK9's role in liver regeneration and neuronal
cell differentiation (Seidah N G et al, "The secretory proprotein
convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver
regeneration and neuronal differentiation" PNAS 100: 928-933,
2003), and PCSK9s role in hepatic glucose metabolism (Costet et
al., "Hepatic PCSK9 expression is regulated by nutritional status
via insulin and sterol regulatory element-binding protein 1c" J.
Biol. Chem. 281(10):6211-18, 2006).
[0168] The term "hypercholesterolemia," as used herein, refers to a
condition in which cholesterol levels are elevated above a desired
level. In some embodiments, this denotes that serum cholesterol
levels are elevated. In some embodiments, the desired level takes
into account various "risk factors" that are known to one of skill
in the art (and are described or referenced herein).
[0169] The term "polynucleotide" or "nucleic acid" includes both
single-stranded and double-stranded nucleotide polymers. The
nucleotides comprising the polynucleotide can be ribonucleotides or
deoxyribonucleotides or a modified form of either type of
nucleotide. Said modifications include base modifications such as
bromouridine and inosine derivatives, ribose modifications such as
2',3'-dideoxyribose, and internucleotide linkage modifications such
as phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and
phosphoroamidate.
[0170] The term "oligonucleotide" means a polynucleotide comprising
200 or fewer nucleotides. In some embodiments, oligonucleotides are
10 to 60 bases in length. In other embodiments, oligonucleotides
are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in
length. Oligonucleotides can be single stranded or double stranded,
e.g., for use in the construction of a mutant gene.
Oligonucleotides can be sense or antisense oligonucleotides. An
oligonucleotide can include a label, including a radiolabel, a
fluorescent label, a hapten or an antigenic label, for detection
assays. Oligonucleotides can be used, for example, as PCR primers,
cloning primers or hybridization probes.
[0171] An "isolated nucleic acid molecule" means a DNA or RNA of
genomic, mRNA, cDNA, or synthetic origin or some combination
thereof which is not associated with all or a portion of a
polynucleotide in which the isolated polynucleotide is found in
nature, or is linked to a polynucleotide to which it is not linked
in nature. For purposes of this disclosure, it should be understood
that "a nucleic acid molecule comprising" a particular nucleotide
sequence does not encompass intact chromosomes. Isolated nucleic
acid molecules "comprising" specified nucleic acid sequences can
include, in addition to the specified sequences, coding sequences
for up to ten or even up to twenty other proteins or portions
thereof, or can include operably linked regulatory sequences that
control expression of the coding region of the recited nucleic acid
sequences, and/or can include vector sequences.
[0172] Unless specified otherwise, the left-hand end of any
single-stranded polynucleotide sequence discussed herein is the 5'
end; the left-hand direction of double-stranded polynucleotide
sequences is referred to as the 5' direction. The direction of 5'
to 3' addition of nascent RNA transcripts is referred to as the
transcription direction; sequence regions on the DNA strand having
the same sequence as the RNA transcript that are 5' to the 5' end
of the RNA transcript are referred to as "upstream sequences;"
sequence regions on the DNA strand having the same sequence as the
RNA transcript that are 3' to the 3' end of the RNA transcript are
referred to as "downstream sequences."
[0173] The term "control sequence" refers to a polynucleotide
sequence that can affect the expression and processing of coding
sequences to which it is ligated. The nature of such control
sequences can depend upon the host organism. In particular
embodiments, control sequences for prokaryotes can include a
promoter, a ribosomal binding site, and a transcription termination
sequence. For example, control sequences for eukaryotes can include
promoters comprising one or a plurality of recognition sites for
transcription factors, transcription enhancer sequences, and
transcription termination sequence. "Control sequences" can include
leader sequences and/or fusion partner sequences.
[0174] The term "vector" means any molecule or entity (e.g.,
nucleic acid, plasmid, bacteriophage or virus) used to transfer
protein coding information into a host cell.
[0175] The term "expression vector" or "expression construct"
refers to a vector that is suitable for transformation of a host
cell and contains nucleic acid sequences that direct and/or control
(in conjunction with the host cell) expression of one or more
heterologous coding regions operatively linked thereto. An
expression construct can include, but is not limited to, sequences
that affect or control transcription, translation, and, if introns
are present, affect RNA splicing of a coding region operably linked
thereto.
[0176] As used herein, "operably linked" means that the components
to which the term is applied are in a relationship that allows them
to carry out their inherent functions under suitable conditions.
For example, a control sequence in a vector that is "operably
linked" to a protein coding sequence is ligated thereto so that
expression of the protein coding sequence is achieved under
conditions compatible with the transcriptional activity of the
control sequences.
[0177] The term "host cell" means a cell that has been transformed,
or is capable of being transformed, with a nucleic acid sequence
and thereby expresses a gene of interest. The term includes the
progeny of the parent cell, whether or not the progeny is identical
in morphology or in genetic make-up to the original parent cell, so
long as the gene of interest is present.
[0178] The term "transfection" means the uptake of foreign or
exogenous DNA by a cell, and a cell has been "transfected" when the
exogenous DNA has been introduced inside the cell membrane. A
number of transfection techniques are well known in the art and are
disclosed herein. See, e.g., Graham et al, 1973, Virology 52:456;
Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual,
supra; Davis et al, 1986, Basic Methods in Molecular Biology,
Elsevier; Chu et al, 1981, Gene 13:197. Such techniques can be used
to introduce one or more exogenous DNA moieties into suitable host
cells.
[0179] The term "transformation" refers to a change in a cell's
genetic characteristics, and a cell has been transformed when it
has been modified to contain new DNA or RNA. For example, a cell is
transformed where it is genetically modified from its native state
by introducing new genetic material via transfection, transduction,
or other techniques. Following transfection or transduction, the
transforming DNA can recombine with that of the cell by physically
integrating into a chromosome of the cell, or can be maintained
transiently as an episomal element without being replicated, or can
replicate independently as a plasmid. A cell is considered to have
been "stably transformed" when the transforming DNA is replicated
with the division of the cell.
[0180] The terms "polypeptide" or "protein" means a macromolecule
having the amino acid sequence of a native protein, that is, a
protein produced by a naturally-occurring and non-recombinant cell;
or it is produced by a genetically-engineered or recombinant cell,
and comprise molecules having the amino acid sequence of the native
protein, or molecules having deletions from, additions to, and/or
substitutions of one or more amino acids of the native sequence.
The term also includes amino acid polymers in which one or more
amino acids are chemical analogs of a corresponding
naturally-occurring amino acid and polymers. The terms
"polypeptide" and "protein" specifically encompass PCSK9 antigen
binding proteins, antibodies, or sequences that have deletions
from, additions to, and/or substitutions of one or more amino acid
of antigen-binding protein. The term "polypeptide fragment" refers
to a polypeptide that has an amino-terminal deletion, a
carboxyl-terminal deletion, and/or an internal deletion as compared
with the full-length native protein. Such fragments can also
contain modified amino acids as compared with the native protein.
In certain embodiments, fragments are about five to 500 amino acids
long. For example, fragments can be at least 5, 6, 8, 10, 14, 20,
50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids
long. Useful polypeptide fragments include immunologically
functional fragments of antibodies, including binding domains. In
the case of a PCSK9-binding antibody, useful fragments include but
are not limited to a CDR region, a variable domain of a heavy
and/or light chain, a portion of an antibody chain or just its
variable region including two CDRs, and the like.
[0181] The term "isolated protein" referred means that a subject
protein (1) is free of at least some other proteins with which it
would normally be found, (2) is essentially free of other proteins
from the same source, e.g., from the same species, (3) is expressed
by a cell from a different species, (4) has been separated from at
least about 50 percent of polynucleotides, lipids, carbohydrates,
or other materials with which it is associated in nature, (5) is
operably associated (by covalent or noncovalent interaction) with a
polypeptide with which it is not associated in nature, or (6) does
not occur in nature. Typically, an "isolated protein" constitutes
at least about 5%, at least about 10%, at least about 25%, or at
least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other
RNA, of synthetic origin, or any combination thereof can encode
such an isolated protein. Preferably, the isolated protein is
substantially free from proteins or polypeptides or other
contaminants that are found in its natural environment that would
interfere with its therapeutic, diagnostic, prophylactic, research
or other use.
[0182] The term "amino acid" includes its normal meaning in the
art.
[0183] A "variant" of a polypeptide (e.g., an antigen binding
protein, or an antibody) comprises an amino acid sequence wherein
one or more amino acid residues are inserted into, deleted from
and/or substituted into the amino acid sequence relative to another
polypeptide sequence. Variants include fusion proteins.
[0184] The term "identity" refers to a relationship between the
sequences of two or more polypeptide molecules or two or more
nucleic acid molecules, as determined by aligning and comparing the
sequences. "Percent identity" means the percent of identical
residues between the amino acids or nucleotides in the compared
molecules and is calculated based on the size of the smallest of
the molecules being compared. For these calculations, gaps in
alignments (if any) are preferably addressed by a particular
mathematical model or computer program (i.e., an "algorithm").
Methods that can be used to calculate the identity of the aligned
nucleic acids or polypeptides include those described in
Computational Molecular Biology, (Lesk, A. M., ed.), 1988, New
York: Oxford University Press; Biocomputing Informatics and Genome
Projects, (Smith, D. W., ed.), 1993, New York: Academic Press;
Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and
Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje,
G., 1987, Sequence Analysis in Molecular Biology, New York:
Academic Press; Sequence Analysis Primer, (Gribskov, M. and
Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo
et al., 1988, SIAM J. Applied Math. 48:1073.
[0185] In calculating percent identity, the sequences being
compared are typically aligned in a way that gives the largest
match between the sequences. One example of a computer program that
can be used to determine percent identity is the GCG program
package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.
12:387; Genetics Computer Group, University of Wisconsin, Madison,
Wis.). The computer algorithm GAP is used to align the two
polypeptides or polynucleotides for which the percent sequence
identity is to be determined. The sequences are aligned for optimal
matching of their respective amino acid or nucleotide (the "matched
span", as determined by the algorithm). A gap opening penalty
(which is calculated as 3.times. the average diagonal, wherein the
"average diagonal" is the average of the diagonal of the comparison
matrix being used; the "diagonal" is the score or number assigned
to each perfect amino acid match by the particular comparison
matrix) and a gap extension penalty (which is usually 1/10 times
the gap opening penalty), as well as a comparison matrix such as
PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In
certain embodiments, a standard comparison matrix (see, Dayhoff et
al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for
the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl.
Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison
matrix) is also used by the algorithm.
[0186] Examples of parameters that can be employed in determining
percent identity for polypeptides or nucleotide sequences using the
GAP program are the following:
[0187] Algorithm: Needleman et al., 1970, J. Mol. Biol.
48:443-453
[0188] Comparison matrix: BLOSUM 62 from Henikoff et al., 1992,
supra
[0189] Gap Penalty: 12 (but with no penalty for end gaps)
[0190] Gap Length Penalty: 4
[0191] Threshold of Similarity: 0
[0192] Certain alignment schemes for aligning two amino acid
sequences may result in matching of only a short region of the two
sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full-length sequences. Accordingly, the selected
alignment method (GAP program) can be adjusted if so desired to
result in an alignment that spans at least 50 or other number of
contiguous amino acids of the target polypeptide.
[0193] As used herein, the twenty conventional (e.g., naturally
occurring) amino acids and their abbreviations follow conventional
usage. See Immunology--A Synthesis (2nd Edition, E. S. Golub and D.
R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which
is incorporated herein by reference for any purpose. Stereoisomers
(e.g., D-amino acids) of the twenty conventional amino acids,
unnatural amino acids such as .alpha.-,.alpha.-disubstituted amino
acids, N-alkyl amino acids, lactic acid, and other unconventional
amino acids can also be suitable components for polypeptides of the
present invention. Examples of unconventional amino acids include:
4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetyl serine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxy-terminal direction, in accordance with standard usage and
convention.
[0194] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end; the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction; sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences"; sequence
regions on the DNA strand having the same sequence as the RNA and
which are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences."
[0195] Conservative amino acid substitutions can encompass
non-naturally occurring amino acid residues, which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics and other
reversed or inverted forms of amino acid moieties.
[0196] Naturally occurring residues can be divided into classes
based on common side chain properties:
[0197] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0198] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0199] 3) acidic: Asp, Glu;
[0200] 4) basic: His, Lys, Arg;
[0201] 5) residues that influence chain orientation: Gly, Pro;
and
[0202] 6) aromatic: Trp, Tyr, Phe.
For example, non-conservative substitutions can involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues can be introduced, for
example, into regions of a human antibody that are homologous with
non-human antibodies, or into the non-homologous regions of the
molecule.
[0203] In making changes to the antigen binding protein or the
PCSK9 protein, according to certain embodiments, the hydropathic
index of amino acids can be considered. Each amino acid has been
assigned a hydropathic index on the basis of its hydrophobicity and
charge characteristics. They are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0204] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157:105-131
(1982). It is known that certain amino acids can be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, in certain embodiments, the
substitution of amino acids whose hydropathic indices are within
.+-.2 is included. In certain embodiments, those which are within
.+-.1 are included, and in certain embodiments, those within
.+-.0.5 are included.
[0205] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functional
protein or peptide thereby created is intended for use in
immunological embodiments, as in the present case. In certain
embodiments, the greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0206] The following hydrophilicity values have been assigned to
these amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3);
asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine
(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, in certain embodiments, the substitution of
amino acids whose hydrophilicity values are within .+-.2 is
included, in certain embodiments, those which are within .+-.1 are
included, and in certain embodiments, those within .+-.0.5 are
included. One can also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0207] Exemplary amino acid substitutions are set forth in Table
1.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Residues
Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile
Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser
Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg
Ile Leu, Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile,
Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric Arg Acid, Gln,
Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala
Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,
Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine
[0208] The term "derivative" refers to a molecule that includes a
chemical modification other than an insertion, deletion, or
substitution of amino acids (or nucleic acids). In certain
embodiments, derivatives comprise covalent modifications,
including, but not limited to, chemical bonding with polymers,
lipids, or other organic or inorganic moieties. In certain
embodiments, a chemically modified antigen binding protein can have
a greater circulating half-life than an antigen binding protein
that is not chemically modified. In certain embodiments, a
chemically modified antigen binding protein can have improved
targeting capacity for desired cells, tissues, and/or organs. In
some embodiments, a derivative antigen binding protein is
covalently modified to include one or more water soluble polymer
attachments, including, but not limited to, polyethylene glycol,
polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S.
Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and
4,179,337. In certain embodiments, a derivative antigen binding
protein comprises one or more polymer, including, but not limited
to, monomethoxy-polyethylene glycol, dextran, cellulose, or other
carbohydrate based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of such polymers.
[0209] In certain embodiments, a derivative is covalently modified
with polyethylene glycol (PEG) subunits. In certain embodiments,
one or more water-soluble polymer is bonded at one or more specific
position, for example at the amino terminus, of a derivative. In
certain embodiments, one or more water-soluble polymer is randomly
attached to one or more side chains of a derivative. In certain
embodiments, PEG is used to improve the therapeutic capacity for an
antigen binding protein. In certain embodiments, PEG is used to
improve the therapeutic capacity for a humanized antibody. Certain
such methods are discussed, for example, in U.S. Pat. No.
6,133,426, which is hereby incorporated by reference for any
purpose.
[0210] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics." Fauchere, J., Adv.
Drug Res., 15:29 (1986); Veber & Freidinger, TINS, p. 392
(1985); and Evans et al., J. Med. Chem., 30:1229 (1987), which are
incorporated herein by reference for any purpose. Such compounds
are often developed with the aid of computerized molecular
modeling. Peptide mimetics that are structurally similar to
therapeutically useful peptides can be used to produce a similar
therapeutic or prophylactic effect. Generally, peptidomimetics are
structurally similar to a paradigm polypeptide (i.e., a polypeptide
that has a biochemical property or pharmacological activity), such
as human antibody, but have one or more peptide linkages optionally
replaced by a linkage selected from: --CH.sub.2 NH--, --CH.sub.2
S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-(cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods
well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) can be used in certain
embodiments to generate more stable peptides. In addition,
constrained peptides comprising a consensus sequence or a
substantially identical consensus sequence variation can be
generated by methods known in the art (Rizo and Gierasch, Ann. Rev.
Biochem., 61:387 (1992), incorporated herein by reference for any
purpose); for example, by adding internal cysteine residues capable
of forming intramolecular disulfide bridges which cyclize the
peptide.
[0211] The term "naturally occurring" as used throughout the
specification in connection with biological materials such as
polypeptides, nucleic acids, host cells, and the like, refers to
materials which are found in nature or a form of the materials that
is found in nature.
[0212] An "antigen binding protein" ("ABP") as used herein means
any protein that binds a specified target antigen. In the instant
application, the specified target antigen is the PCSK9 protein or
fragment thereof "Antigen binding protein" includes but is not
limited to antibodies and binding parts thereof, such as
immunologically functional fragments. Peptibodies are another
example of antigen binding proteins. The term "immunologically
functional fragment" (or simply "fragment") of an antibody or
immunoglobulin chain (heavy or light chain) antigen binding
protein, as used herein, is a species of antigen binding protein
comprising a portion (regardless of how that portion is obtained or
synthesized) of an antibody that lacks at least some of the amino
acids present in a full-length chain but which is still capable of
specifically binding to an antigen. Such fragments are biologically
active in that they bind to the target antigen and can compete with
other antigen binding proteins, including intact antibodies, for
binding to a given epitope. In some embodiments, the fragments are
neutralizing fragments. In some embodiments, the fragments can
block or reduce the likelihood of the interaction between LDLR and
PCSK9. In one aspect, such a fragment will retain at least one CDR
present in the full-length light or heavy chain, and in some
embodiments will comprise a single heavy chain and/or light chain
or portion thereof. These biologically active fragments can be
produced by recombinant DNA techniques, or can be produced by
enzymatic or chemical cleavage of antigen binding proteins,
including intact antibodies. Immunologically functional
immunoglobulin fragments include, but are not limited to, Fab, a
diabody (heavy chain variable domain on the same polypeptide as a
light chain variable domain, connected via a short peptide linker
that is too short to permit pairing between the two domains on the
same chain), Fab', F(ab).sub.2, Fv, domain antibodies and
single-chain antibodies, and can be derived from any mammalian
source, including but not limited to human, mouse, rat, camelid or
rabbit. It is further contemplated that a functional portion of the
antigen binding proteins disclosed herein, for example, one or more
CDRs, could be covalently bound to a second protein or to a small
molecule to create a therapeutic agent directed to a particular
target in the body, possessing bifunctional therapeutic properties,
or having a prolonged serum half-life. As will be appreciated by
one of skill in the art, an antigen binding protein can include
nonprotein components. In some sections of the present disclosure,
examples of ABPs are described herein in terms of
"number/letter/number" (e.g., 25A7). In these cases, the exact name
denotes a specific antibody. That is, an ABP named 25A7 is not
necessarily the same as an antibody named 25A7.1, (unless they are
explicitly taught as the same in the specification, e.g., 25A7 and
25A7.3). As will be appreciated by one of skill in the art, in some
embodiments LDLR is not an antigen binding protein. In some
embodiments, binding subsections of LDLR are not antigen binding
proteins, e.g., EGFa. In some embodiments, other molecules through
which PCSK9 signals in vivo are not antigen binding proteins. Such
embodiments will be explicitly identified as such.
[0213] Certain antigen binding proteins described herein are
antibodies or are derived from antibodies. In certain embodiments,
the polypeptide structure of the antigen binding proteins is based
on antibodies, including, but not limited to, monoclonal
antibodies, bispecific antibodies, minibodies, domain antibodies,
synthetic antibodies (sometimes referred to herein as "antibody
mimetics"), chimeric antibodies, humanized antibodies, human
antibodies, antibody fusions (sometimes referred to herein as
"antibody conjugates"), and fragments thereof, respectively. In
some embodiments, the ABP comprises or consists of avimers (tightly
binding peptide). These various antigen binding proteins are
further described herein.
[0214] An "Fc" region comprises two heavy chain fragments
comprising the C.sub.H1 and C.sub.H2 domains of an antibody. The
two heavy chain fragments are held together by two or more
disulfide bonds and by hydrophobic interactions of the C.sub.H3
domains.
[0215] A "Fab fragment" comprises one light chain and the C.sub.H1
and variable regions of one heavy chain. The heavy chain of a Fab
molecule cannot form a disulfide bond with another heavy chain
molecule.
[0216] A "Fab' fragment" comprises one light chain and a portion of
one heavy chain that contains the VH domain and the C.sub.H1 domain
and also the region between the C.sub.H1 and C.sub.H2 domains, such
that an interchain disulfide bond can be formed between the two
heavy chains of two Fab' fragments to form an F(ab').sub.2
molecule.
[0217] A "F(ab').sub.2 fragment" contains two light chains and two
heavy chains containing a portion of the constant region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond is formed between the two heavy chains. A
F(ab').sub.2 fragment thus is composed of two Fab' fragments that
are held together by a disulfide bond between the two heavy
chains.
[0218] The "Fv region" comprises the variable regions from both the
heavy and light chains, but lacks the constant regions.
[0219] "Single-chain antibodies" are Fv molecules in which the
heavy and light chain variable regions have been connected by a
flexible linker to form a single polypeptide chain, which forms an
antigen binding region. Single chain antibodies are discussed in
detail in International Patent Application Publication No. WO
88/01649 and U.S. Pat. No. 4,946,778 and No. 5,260,203, the
disclosures of which are incorporated by reference.
[0220] A "domain antibody" is an immunologically functional
immunoglobulin fragment containing only the variable region of a
heavy chain or the variable region of a light chain. In some
instances, two or more V.sub.H regions are covalently joined with a
peptide linker to create a bivalent domain antibody. The two
V.sub.H regions of a bivalent domain antibody can target the same
or different antigens.
[0221] A "bivalent antigen binding protein" or "bivalent antibody"
comprises two antigen binding sites. In some instances, the two
binding sites have the same antigen specificities. Bivalent antigen
binding proteins and bivalent antibodies can be bispecific, see,
infra. A bivalent antibody other than a "multispecific" or
"multifunctional" antibody, in certain embodiments, typically is
understood to have each of its binding sites identical.
[0222] A "multispecific antigen binding protein" or "multispecific
antibody" is one that targets more than one antigen or epitope.
[0223] A "bispecific," "dual-specific" or "bifunctional" antigen
binding protein or antibody is a hybrid antigen binding protein or
antibody, respectively, having two different antigen binding sites.
Bispecific antigen binding proteins and antibodies are a species of
multispecific antigen binding protein antibody and can be produced
by a variety of methods including, but not limited to, fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and
Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al.,
1992, J. Immunol. 148:1547-1553. The two binding sites of a
bispecific antigen binding protein or antibody will bind to two
different epitopes, which can reside on the same or different
protein targets.
[0224] An antigen binding protein is said to "specifically bind"
its target antigen when the dissociation constant (K.sub.d) is
.ltoreq.10.sup.-7 M. The ABP specifically binds antigen with "high
affinity" when the K.sub.d is .ltoreq.5.times.10.sup.-9 M, and with
"very high affinity" when the K.sub.d is .ltoreq.5.times.10.sup.-10
M. In one embodiment, the ABP has a K.sub.d of .ltoreq.10.sup.-9 M.
In one embodiment, the off-rate is <1.times.10.sup.-5 In other
embodiments, the ABPs will bind to human PCSK9 with a K.sub.d of
between about 10.sup.-9 M and 10.sup.-13 M, and in yet another
embodiment the ABPs will bind with a
K.sub.d.ltoreq.5.times.10.sup.-10. As will be appreciated by one of
skill in the art, in some embodiments, any or all of the antigen
binding fragments can specifically bind to PCSK9.
[0225] An antigen binding protein is "selective" when it binds to
one target more tightly than it binds to a second target.
[0226] "Antigen binding region" means a protein, or a portion of a
protein, that specifically binds a specified antigen (e.g., a
paratope). For example, that portion of an antigen binding protein
that contains the amino acid residues that interact with an antigen
and confer on the antigen binding protein its specificity and
affinity for the antigen is referred to as "antigen binding
region." An antigen binding region typically includes one or more
"complementary binding regions" ("CDRs"). Certain antigen binding
regions also include one or more "framework" regions. A "CDR" is an
amino acid sequence that contributes to antigen binding specificity
and affinity. "Framework" regions can aid in maintaining the proper
conformation of the CDRs to promote binding between the antigen
binding region and an antigen. Structurally, framework regions can
be located in antibodies between CDRs. Examples of framework and
CDR regions are shown in FIGS. 2A-3D, 3CCC-3JJJ, and 15A-15D. In
some embodiments, the sequences for CDRs for the light chain of
antibody 3B6 are as follows: CDR1 TLSSGYSSYEVD (SEQ ID NO: 279);
CDR2 VDTGGIVGSKGE (SEQ ID NO: 280); CDR3 GADHGSGTNFVVV (SEQ ID NO:
281), and the FRs are as follows: FR1 QPVLTQPLFASASLGASVTLTC (SEQ
ID NO: 282); FR2 WYQQRPGKGPRFVMR (SEQ ID NO: 283); FR3
GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC (SEQ ID NO: 284); and FR4
FGGGTKLTVL (SEQ ID NO: 285).
[0227] In certain aspects, recombinant antigen binding proteins
that bind PCSK9, for example human PCSK9, are provided. In this
context, a "recombinant antigen binding protein" is a protein made
using recombinant techniques, i.e., through the expression of a
recombinant nucleic acid as described herein. Methods and
techniques for the production of recombinant proteins are well
known in the art.
[0228] The term "antibody" refers to an intact immunoglobulin of
any isotype, or a fragment thereof that can compete with the intact
antibody for specific binding to the target antigen, and includes,
for instance, chimeric, humanized, fully human, and bispecific
antibodies. An "antibody" is a species of an antigen binding
protein. An intact antibody will generally comprise at least two
full-length heavy chains and two full-length light chains, but in
some instances can include fewer chains such as antibodies
naturally occurring in camelids which can comprise only heavy
chains. Antibodies can be derived solely from a single source, or
can be "chimeric," that is, different portions of the antibody can
be derived from two different antibodies as described further
below. The antigen binding proteins, antibodies, or binding
fragments can be produced in hybridomas, by recombinant DNA
techniques, or by enzymatic or chemical cleavage of intact
antibodies. Unless otherwise indicated, the term "antibody"
includes, in addition to antibodies comprising two full-length
heavy chains and two full-length light chains, derivatives,
variants, fragments, and muteins thereof, examples of which are
described below. Furthermore, unless explicitly excluded,
antibodies include monoclonal antibodies, bispecific antibodies,
minibodies, domain antibodies, synthetic antibodies (sometimes
referred to herein as "antibody mimetics"), chimeric antibodies,
humanized antibodies, human antibodies, antibody fusions (sometimes
referred to herein as "antibody conjugates"), and fragments
thereof, respectively. In some embodiments, the term also
encompasses peptibodies.
[0229] Naturally occurring antibody structural units typically
comprise a tetramer. Each such tetramer typically is composed of
two identical pairs of polypeptide chains, each pair having one
full-length "light" (in certain embodiments, about 25 kDa) and one
full-length "heavy" chain (in certain embodiments, about 50-70
kDa). The amino-terminal portion of each chain typically includes a
variable region of about 100 to 110 or more amino acids that
typically is responsible for antigen recognition. The
carboxy-terminal portion of each chain typically defines a constant
region that can be responsible for effector function. Human light
chains are typically classified as kappa and lambda light chains.
Heavy chains are typically classified as mu, delta, gamma, alpha,
or epsilon, and define the antibody's isotype as IgM, IgD, IgG,
IgA, and IgE, respectively. IgG has several subclasses, including,
but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses
including, but not limited to, IgM1 and IgM2. IgA is similarly
subdivided into subclasses including, but not limited to, IgA1 and
IgA2. Within full-length light and heavy chains, typically, the
variable and constant regions are joined by a "J" region of about
12 or more amino acids, with the heavy chain also including a "D"
region of about 10 more amino acids. See, e.g., Fundamental
Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989))
(incorporated by reference in its entirety for all purposes). The
variable regions of each light/heavy chain pair typically form the
antigen binding site.
[0230] The variable regions typically exhibit the same general
structure of relatively conserved framework regions (FR) joined by
three hyper variable regions, also called complementarity
determining regions or CDRs. The CDRs from the two chains of each
pair typically are aligned by the framework regions, which can
enable binding to a specific epitope. From N-terminal to
C-terminal, both light and heavy chain variable regions typically
comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is typically in accordance
with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol.,
196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).
[0231] In certain embodiments, an antibody heavy chain binds to an
antigen in the absence of an antibody light chain. In certain
embodiments, an antibody light chain binds to an antigen in the
absence of an antibody heavy chain. In certain embodiments, an
antibody binding region binds to an antigen in the absence of an
antibody light chain. In certain embodiments, an antibody binding
region binds to an antigen in the absence of an antibody heavy
chain. In certain embodiments, an individual variable region
specifically binds to an antigen in the absence of other variable
regions.
[0232] In certain embodiments, definitive delineation of a CDR and
identification of residues comprising the binding site of an
antibody is accomplished by solving the structure of the antibody
and/or solving the structure of the antibody-ligand complex. In
certain embodiments, that can be accomplished by any of a variety
of techniques known to those skilled in the art, such as X-ray
crystallography. In certain embodiments, various methods of
analysis can be employed to identify or approximate the CDR
regions. Examples of such methods include, but are not limited to,
the Kabat definition, the Chothia definition, the AbM definition
and the contact definition.
[0233] The Kabat definition is a standard for numbering the
residues in an antibody and is typically used to identify CDR
regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8
(2000). The Chothia definition is similar to the Kabat definition,
but the Chothia definition takes into account positions of certain
structural loop regions. See, e.g., Chothia et al., J. Mol. Biol.,
196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). The
AbM definition uses an integrated suite of computer programs
produced by Oxford Molecular Group that model antibody structure.
See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272
(1989); "AbM.TM., A Computer Program for Modeling Variable Regions
of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM
definition models the tertiary structure of an antibody from
primary sequence using a combination of knowledge databases and ab
initio methods, such as those described by Samudrala et al., "Ab
Initio Protein Structure Prediction Using a Combined Hierarchical
Approach," in PROTEINS, Structure, Function and Genetics Suppl.,
3:194-198 (1999). The contact definition is based on an analysis of
the available complex crystal structures. See, e.g., MacCallum et
al, J. Mol. Biol., 5:732-45 (1996).
[0234] By convention, the CDR regions in the heavy chain are
typically referred to as H1, H2, and H3 and are numbered
sequentially in the direction from the amino terminus to the
carboxy terminus. The CDR regions in the light chain are typically
referred to as L1, L2, and L3 and are numbered sequentially in the
direction from the amino terminus to the carboxy terminus.
[0235] The term "light chain" includes a full-length light chain
and fragments thereof having sufficient variable region sequence to
confer binding specificity. A full-length light chain includes a
variable region domain, V.sub.L, and a constant region domain,
C.sub.L. The variable region domain of the light chain is at the
amino-terminus of the polypeptide. Light chains include kappa
chains and lambda chains.
[0236] The term "heavy chain" includes a full-length heavy chain
and fragments thereof having sufficient variable region sequence to
confer binding specificity. A full-length heavy chain includes a
variable region domain, V.sub.H, and three constant region domains,
C.sub.H1, C.sub.H2, and C.sub.H3. The V.sub.H domain is at the
amino-terminus of the polypeptide, and the C.sub.H domains are at
the carboxyl-terminus, with the C.sub.H3 being closest to the
carboxy-terminus of the polypeptide. Heavy chains can be of any
isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4
subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
[0237] A bispecific or bifunctional antibody typically is an
artificial hybrid antibody having two different heavy/light chain
pairs and two different binding sites. Bispecific antibodies can be
produced by a variety of methods including, but not limited to,
fusion of hybridomas or linking of Fab' fragments. See, e.g.,
Songsivilai et al., Clin. Exp. Immunol., 79: 315-321 (1990);
Kostelny et al., J. Immunol., 148:1547-1553 (1992).
[0238] Some species of mammals also produce antibodies having only
a single heavy chain.
[0239] Each individual immunoglobulin chain is typically composed
of several "immunoglobulin domains," each consisting of roughly 90
to 110 amino acids and having a characteristic folding pattern.
These domains are the basic units of which antibody polypeptides
are composed. In humans, the IgA and IgD isotypes contain four
heavy chains and four light chains; the IgG and IgE isotypes
contain two heavy chains and two light chains; and the IgM isotype
contains five heavy chains and five light chains. The heavy chain C
region typically comprises one or more domains that can be
responsible for effector function. The number of heavy chain
constant region domains will depend on the isotype. IgG heavy
chains, for example, contain three C region domains known as
C.sub.H1, C.sub.H2 and C.sub.H3. The antibodies that are provided
can have any of these isotypes and subtypes. In certain embodiments
of the present invention, an anti-PCSK9 antibody is of the IgG2 or
IgG4 subtype.
[0240] The term "variable region" or "variable domain" refers to a
portion of the light and/or heavy chains of an antibody, typically
including approximately the amino-terminal 120 to 130 amino acids
in the heavy chain and about 100 to 110 amino terminal amino acids
in the light chain. In certain embodiments, variable regions of
different antibodies differ extensively in amino acid sequence even
among antibodies of the same species. The variable region of an
antibody typically determines specificity of a particular antibody
for its target
[0241] The term "neutralizing antigen binding protein" or
"neutralizing antibody" refers to an antigen binding protein or
antibody, respectively, that binds to a ligand and prevents or
reduces the biological effect of that ligand. This can be done, for
example, by directly blocking a binding site on the ligand or by
binding to the ligand and altering the ligand's ability to bind
through indirect means (such as structural or energetic alterations
in the ligand). In some embodiments, the term can also denote an
antigen binding protein that prevents the protein to which it is
bound from performing a biological function. In assessing the
binding and/or specificity of an antigen binding protein, e.g., an
antibody or immunologically functional fragment thereof, an
antibody or fragment can substantially inhibit binding of a ligand
to its binding partner when an excess of antibody reduces the
quantity of binding partner bound to the ligand by at least about
1-20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%,
85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured in an
in vitro competitive binding assay). In some embodiments, in the
case of PCSK9 antigen binding proteins, such a neutralizing
molecule can diminish the ability of PCSK9 to bind the LDLR. In
some embodiments, the neutralizing ability is characterized and/or
described via a competition assay. In some embodiments, the
neutralizing ability is described in terms of an IC.sub.50 or
EC.sub.50 value. In some embodiments, ABPs 27B2, 13H1, 13B5 and 3C4
are non-neutralizing ABPs, 3B6, 9C9 and 31A4 are weak neutralizers,
and the remaining ABPs in Table 2 are strong neutralizers. In some
embodiments, the antibodies or antigen binding proteins neutralize
by binding to PCSK9 and preventing PCSK9 from binding to LDLR (or
reducing the ability of PCSK9 to bind to LDLR). In some
embodiments, the antibodies or ABPs neutralize by binding to PCSK9,
and while still allowing PCSK9 to bind to LDLR, preventing or
reducing the PCSK9 mediated degradation of LDLR. Thus, in some
embodiments, a neutralizing ABP or antibody can still permit
PCSK9/LDLR binding, but will prevent (or reduce) subsequent PCSK9
involved degradation of LDLR.
[0242] The term "target" refers to a molecule or a portion of a
molecule capable of being bound by an antigen binding protein. In
certain embodiments, a target can have one or more epitopes. In
certain embodiments, a target is an antigen. The use of "antigen"
in the phrase "antigen binding protein" simply denotes that the
protein sequence that comprises the antigen can be bound by an
antibody. In this context, it does not require that the protein be
foreign or that it be capable of inducing an immune response.
[0243] The term "compete" when used in the context of antigen
binding proteins (e.g., neutralizing antigen binding proteins or
neutralizing antibodies) that compete for the same epitope means
competition between antigen binding proteins as determined by an
assay in which the antigen binding protein (e.g., antibody or
immunologically functional fragment thereof) being tested prevents
or inhibits (e.g., reduces) specific binding of a reference antigen
binding protein (e.g., a ligand, or a reference antibody) to a
common antigen (e.g., PCSK9 or a fragment thereof). Numerous types
of competitive binding assays can be used to determine if one
antigen binding protein 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 some
instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%,
95-97%, or 97% or more.
[0244] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antigen binding protein (including, e.g., an antibody or
immunological functional fragment thereof). In some embodiments,
the antigen is capable of being used in an animal to produce
antibodies capable of binding to that antigen. An antigen can
possess one or more epitopes that are capable of interacting with
different antigen binding proteins, e.g., antibodies.
[0245] The term "epitope" includes any determinant capable being
bound by an antigen binding protein, such as an antibody or to a
T-cell receptor. An epitope is a region of an antigen that is bound
by an antigen binding protein that targets that antigen, and when
the antigen is a protein, includes specific amino acids that
directly contact the antigen binding protein. Most often, epitopes
reside on proteins, but in some instances can reside on other kinds
of molecules, such as nucleic acids. Epitope determinants can
include chemically active surface groupings of molecules such as
amino acids, sugar side chains, phosphoryl or sulfonyl groups, and
can have specific three dimensional structural characteristics,
and/or specific charge characteristics. Generally, antibodies
specific for a particular target antigen will preferentially
recognize an epitope on the target antigen in a complex mixture of
proteins and/or macromolecules.
[0246] As used herein, "substantially pure" means that the
described species of molecule is the predominant species present,
that is, on a molar basis it is more abundant than any other
individual species in the same mixture. In certain embodiments, a
substantially pure molecule is a composition wherein the object
species comprises at least 50% (on a molar basis) of all
macromolecular species present. In other embodiments, a
substantially pure composition will comprise at least 80%, 85%,
90%, 95%, or 99% of all macromolecular species present in the
composition. In other embodiments, the object species is purified
to essential homogeneity wherein contaminating species cannot be
detected in the composition by conventional detection methods and
thus the composition consists of a single detectable macromolecular
species.
[0247] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0248] As used herein, the terms "label" or "labeled" refers to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotin
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or colorimetric methods). In certain
embodiments, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and can be used. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I) fluorescent
labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic
labels (e.g., horseradish peroxidase, .beta.-galactosidase,
luciferase, alkaline phosphatase), chemiluminescent, biotinyl
groups, predetermined polypeptide epitopes recognized by a
secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, epitope
tags). In certain embodiments, labels are attached by spacer arms
of various lengths to reduce potential steric hindrance.
[0249] The term "biological sample", as used herein, includes, but
is not limited to, any quantity of a substance from a living thing
or formerly living thing. Such living things include, but are not
limited to, humans, mice, monkeys, rats, rabbits, and other
animals. Such substances include, but are not limited to, blood,
serum, urine, cells, organs, tissues, bone, bone marrow, lymph
nodes, and skin.
[0250] The term "pharmaceutical agent composition" (or agent or
drug) as used herein refers to a chemical compound, composition,
agent or drug capable of inducing a desired therapeutic effect when
properly administered to a patient. It does not necessarily require
more than one type of ingredient.
[0251] The term "therapeutically effective amount" refers to the
amount of a PCSK9 antigen binding protein determined to produce a
therapeutic response in a mammal. Such therapeutically effective
amounts are readily ascertained by one of ordinary skill in the
art.
[0252] The term "modulator," as used herein, is a compound that
changes or alters the activity or function of a molecule. For
example, a modulator can cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Certain exemplary activities and
functions of a molecule include, but are not limited to, binding
affinity, enzymatic activity, and signal transduction. Certain
exemplary inhibitors include, but are not limited to, proteins,
peptides, antibodies, peptibodies, carbohydrates or small organic
molecules. Peptibodies are described in, e.g., U.S. Pat. No.
6,660,843 (corresponding to PCT Application No. WO 01/83525).
[0253] The terms "patient" and "subject" are used interchangeably
and include human and non-human animal subjects as well as those
with formally diagnosed disorders, those without formally
recognized disorders, those receiving medical attention, those at
risk of developing the disorders, etc.
[0254] The term "treat" and "treatment" includes therapeutic
treatments, prophylactic treatments, and applications in which one
reduces the risk that a subject will develop a disorder or other
risk factor. Treatment does not require the complete curing of a
disorder and encompasses embodiments in which one reduces symptoms
or underlying risk factors.
[0255] The term "prevent" does not require the 100% elimination of
the possibility of an event. Rather, it denotes that the likelihood
of the occurrence of the event has been reduced in the presence of
the compound or method.
[0256] Standard techniques can be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques can be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
can be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See, e.g., Sambrook et al., Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated
herein by reference for any purpose. Unless specific definitions
are provided, the nomenclatures utilized in connection with, and
the laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques can be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
Antigen Binding Proteins to PCSK9
[0257] Proprotein convertase subtilisin kexin type 9 (PCSK9) is a
serine protease involved in regulating the levels of the low
density lipoprotein receptor (LDLR) protein (Horton et al., 2007;
Seidah and Prat, 2007). PCSK9 is a prohormone-proprotein convertase
in the subtilisin (S8) family of serine proteases (Seidah et al.,
2003). An exemplary human PCSK9 amino acid sequence is presented as
SEQ ID NOs: 1 and 3 in FIG. 1A (depicting the "pro" domain of the
protein as underlined) and FIG. 1B (depicting the signal sequence
in bold and the pro domain underlined). An exemplary human PCSK9
coding sequence is presented as SEQ ID NO: 2 (FIG. 1B). As
described herein, PCSK9 proteins can also include fragments of the
full length PCSK9 protein. The structure of the PCSK9 protein has
recently been solved by two groups (Cunningham et al., Nature
Structural & Molecular Biology, 2007, and Piper et al.,
Structure, 15:1-8, 2007), the entireties of both of which are
herein incorporated by reference. PCSK9 includes a signal sequence,
a N-terminal prodomain, a subtilisin-like catalytic domain and a
C-terminal domain.
[0258] Antigen binding proteins (ABPs) that bind PCSK9, including
human PCSK9, are provided herein. In some embodiments, the antigen
binding proteins provided are polypeptides which comprise one or
more complementary determining regions (CDRs), as described herein.
In some antigen binding proteins, the CDRs are embedded into a
"framework" region, which orients the CDR(s) such that the proper
antigen binding properties of the CDR(s) is achieved. In some
embodiments, antigen binding proteins provided herein can interfere
with, block, reduce or modulate the interaction between PCSK9 and
LDLR. Such antigen binding proteins are denoted as "neutralizing."
In some embodiments, binding between PCSK9 and LDLR can still
occur, even though the antigen binding protein is neutralizing and
bound to PCSK9. For example, in some embodiments, the ABP prevents
or reduces the adverse influence of PCSK9 on LDLR without blocking
the LDLR binding site on PCSK9. Thus, in some embodiments, the ABP
modulates or alters PCSK9's ability to result in the degradation of
LDLR, without having to prevent the binding interaction between
PCSK9 and LDLR. Such ABPs can be specifically described as
"non-competitively neutralizing" ABPs. In some embodiments, the
neutralizing ABP binds to PCSK9 in a location and/or manner that
prevents PCSK9 from binding to LDLR. Such ABPs can be specifically
described as "competitively neutralizing" ABPs. Both of the above
neutralizers can result in a greater amount of free LDLR being
present in a subject, which results in more LDLR binding to LDL
(thereby reducing the amount of LDL in the subject). In turn, this
results in a reduction in the amount of serum cholesterol present
in a subject.
[0259] In some embodiments, the antigen binding proteins provided
herein are capable of inhibiting PCSK9-mediated activity (including
binding). In some embodiments, antigen binding proteins binding to
these epitopes inhibit, inter alia, interactions between PCSK9 and
LDLR and other physiological effects mediated by PCSK9. In some
embodiments, the antigen binding proteins are human, such as fully
human antibodies to PCSK9.
[0260] In some embodiments, the ABP binds to the catalytic domain
of PCSK9. In some embodiments, the ABP binds to the mature form of
PCSK9. In some embodiments the ABP binds in the prodomain of PCSK9.
In some embodiments, the ABP selectively binds to the mature form
of PCSK9. In some embodiments, the ABP binds to the catalytic
domain in a manner such that PCSK9 cannot bind or bind as
efficiently to LDLR. In some embodiments, the antigen binding
protein does not bind to the c-terminus of the catalytic domain. In
some embodiments, the antigen binding protein does not bind to the
n-terminus of the catalytic domain. In some embodiments, the ABP
does not bind to the n- or c-terminus of the PCSK9 protein. In some
embodiments, the ABP binds to any one of the epitopes bound by the
antibodies discussed herein. In some embodiments, this can be
determined by competition assays between the antibodies disclosed
herein and other antibodies. In some embodiments, the ABP binds to
an epitope bound by one of the antibodies described in Table 2. In
some embodiments, the antigen binding proteins bind to a specific
conformational state of PCSK9 so as to prevent PCSK9 from
interacting with LDLR. In some embodiments, the ABP binds to the V
domain of PCSK9. In some embodiments, the ABP binds to the V domain
of PCSK9 and prevents (or reduces) PCSK9 from binding to LDLR. In
some embodiments, the ABP binds to the V domain of PCSK9, and while
it does not prevent (or reduce) the binding of PCSK9 to LDLR, the
ABP prevents or reduces the adverse activities mediated through
PCSK9 on LDLR.
[0261] The antigen binding proteins that are disclosed herein have
a variety of utilities. Some of the antigen binding proteins, for
instance, are useful in specific binding assays, affinity
purification of PCSK9, in particular human PCSK9 or its ligands and
in screening assays to identify other antagonists of PCSK9
activity. Some of the antigen binding proteins are useful for
inhibiting binding of PCSK9 to LDLR, or inhibiting PCSK9-mediated
activities.
[0262] The antigen binding proteins can be used in a variety of
therapeutic applications, as explained herein. For example, in some
embodiments the PCSK9 antigen binding proteins are useful for
treating conditions associated with PCSK9, such as cholesterol
related disorders (or "serum cholesterol related disorders") such
as hypercholesterolemia, as further described herein. Other uses
for the antigen binding proteins include, for example, diagnosis of
PCSK9-associated diseases or conditions and screening assays to
determine the presence or absence of PCSK9. Some of the antigen
binding proteins described herein are useful in treating
consequences, symptoms, and/or the pathology associated with PCSK9
activity.
[0263] In some embodiments, the antigen binding proteins that are
provided comprise one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs).
In some embodiments, the antigen binding protein comprises (a) a
polypeptide structure and (b) one or more CDRs that are inserted
into and/or joined to the polypeptide structure. The polypeptide
structure can take a variety of different forms. For example, it
can be, or comprise, the framework of a naturally occurring
antibody, or fragment or variant thereof, or can be completely
synthetic in nature. Examples of various polypeptide structures are
further described below.
[0264] In certain embodiments, the polypeptide structure of the
antigen binding proteins is an antibody or is derived from an
antibody, including, but not limited to, monoclonal antibodies,
bispecific antibodies, minibodies, domain antibodies, synthetic
antibodies (sometimes referred to herein as "antibody mimetics"),
chimeric antibodies, humanized antibodies, antibody fusions
(sometimes referred to as "antibody conjugates"), and portions or
fragments of each, respectively. In some instances, the antigen
binding protein is an immunological fragment of an antibody (e.g.,
a Fab, a Fab', a F(ab').sub.2, or a scFv). The various structures
are further described and defined herein.
[0265] Certain of the antigen binding proteins as provided herein
specifically and/or selectively bind to human PCSK9. In some
embodiments, the antigen binding protein specifically and/or
selectively binds to human PCSK9 protein having and/or consisting
of residues 153-692 of SEQ ID NO: 3. In some embodiments the ABP
specifically and/or selectively binds to human PCSK9 having and/or
consisting of residues 31-152 of SEQ ID NO: 3. In some embodiments,
the ABP selectively binds to a human PCSK9 protein as depicted in
FIG. 1A (SEQ ID NO: 1). In some embodiments, the antigen binding
protein specifically binds to at least a fragment of the PCSK9
protein and/or a full length PCSK9 protein, with or without a
signal sequence.
[0266] In embodiments where the antigen binding protein is used for
therapeutic applications, an antigen binding protein can inhibit,
interfere with or modulate one or more biological activities of
PCSK9. In one embodiment, an antigen binding protein 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). Some of the antigen binding proteins
that are provided herein are antibodies. In some embodiments, the
ABP has a K.sub.d of less (binding more tightly) than 10.sup.-7,
10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12,
10.sup.-13 M. In some embodiments, the ABP has an IC.sub.50 for
blocking the binding of LDLR to PCSK9 (D374Y, high affinity
variant) of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10 nM,
10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200
pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1
pM.
[0267] One example of an IgG2 heavy chain constant domain of an
anti-PCSK9 antibody of the present invention has the amino acid
sequence as shown in SEQ ID NO: 154, FIG. 3KK.
[0268] One example of an IgG4 heavy chain constant domain of an
anti-PCSK9 antibody of the present invention has the amino acid
sequence as shown in SEQ ID NO: 155, FIG. 3KK.
[0269] One example of a kappa light chain constant domain of an
anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID
NO: 157, FIG. 3KK.
[0270] One example of a lambda light chain constant domain of an
anti-PCSK9 antibody has the amino acid sequence as shown in SEQ ID
NO: 156, FIG. 3KK.
[0271] Variable regions of immunoglobulin chains generally exhibit
the same overall structure, comprising relatively conserved
framework regions (FR) joined by three hypervariable regions, more
often called "complementarity determining regions" or CDRs. The
CDRs from the two chains of each heavy chain/light chain pair
mentioned above typically are aligned by the framework regions to
form a structure that binds specifically with a specific epitope on
the target protein (e.g., PCSK9). From N-terminal to C-terminal,
naturally-occurring light and heavy chain variable regions both
typically conform with the following order of these elements: FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been
devised for assigning numbers to amino acids that occupy positions
in each of these domains. This numbering system is defined in Kabat
Sequences of Proteins of Immunological Interest (1987 and 1991,
NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol.
196:901-917; Chothia et al., 1989, Nature 342:878-883.
[0272] Various heavy chain and light chain variable regions are
provided herein and are depicted in FIGS. 2A-3JJ and 3LL-3BBB. In
some embodiments, each of these variable regions can be attached to
the above heavy and light chain constant regions to form a complete
antibody heavy and light chain, respectively. Further, each of the
so generated heavy and light chain sequences can be combined to
form a complete antibody structure.
[0273] Specific examples of some of the variable regions of the
light and heavy chains of the antibodies that are provided and
their corresponding amino acid sequences are summarized in TABLE
2.
TABLE-US-00002 TABLE 2 Exemplary Heavy and Light Chain Variable
Regions Light/Heavy Antibody SEQ ID NO 30A4 5/74 3C4 7/85 23B5 9/71
25G4 10/72 31H4 12/67 27B2 13/87 25A7 15/58 27H5 16/52 26H5 17/51
31D1 18/53 20D10 19/48 27E7 20/54 30B9 21/55 19H9 22/56 26E10 23/49
21B12 23/49 17C2 24/57 23G1 26/50 13H1 28/91 9C9 30/64 9H6 31/62
31A4 32/89 1A12 33/65 16F12 35/79 22E2 36/80 27A6 37/76 28B12 38/77
28D6 39/78 31G11 40/81 13B5 42/69 31B12 44/81 3B6 46/60
[0274] Again, each of the exemplary variable heavy chains listed in
Table 2 can be combined with any of the exemplary variable light
chains shown in Table 2 to form an antibody. Table 2 shows
exemplary light and heavy chain pairings found in several of the
antibodies disclosed herein. In some instances, the antibodies
include at least one variable heavy chain and one variable light
chain from those listed in Table 2. In other instances, the
antibodies contain two identical light chains and two identical
heavy chains. As an example, an antibody or antigen binding protein
can include a heavy chain and a light chain, two heavy chains, or
two light chains. In some embodiments the antigen binding protein
comprises (and/or consists) of 1, 2, and/or 3 heavy and/or light
CDRs from at least one of the sequences listed in Table 2 (CDRs for
the sequences are outlined in FIGS. 2A-3D, and other embodiments in
FIGS. 3CCC-3JJJ and 15A-15D). In some embodiments, all 6 CDRs
(CDR1-3 from the light (CDRL1, CDRL2, CDRL3) and CDR1-3 from the
heavy (CDRH1, CDRH2, and CDRH3)) are part of the ABP. In some
embodiments, 1, 2, 3, 4, 5, or more CDRs are included in the ABP.
In some embodiments, one heavy and one light CDR from the CDRs in
the sequences in Table 2 is included in the ABP (CDRs for the
sequences in table 2 are outlined in FIGS. 2A-3D). In some
embodiments, additional sections (e.g., as depicted in FIG. 2A-2D,
3A-3D, and other embodiments in 3CCC-3JJJ and 15A-15D) are also
included in the ABP. Examples of CDRs and FRs for the heavy and
light chains noted in Table 2 are outlined in FIGS. 2A-3D (and
other embodiments in FIGS. 3CCC-3JJJ and 15A-15D). Optional light
chain variable sequences (including CDR1, CDR2, CDR3, FR1, FR2,
FR3, and FR4) can be selected from the following: 5, 7, 9, 10, 12,
13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,
35, 36, 37, 38, 39, 40, 42, 44, and 46. Optional heavy chain
variable sequences (including CDR1, CDR2, CDR3, FR1, FR2, FR3, and
FR4) can be selected from the following: 74, 85, 71, 72, 67, 87,
58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79,
80, 76, 77, 78, 83, 69, 81, and 60. In some of the entries in FIG.
2A-3D, variations of the sequences or alternative boundaries of the
CDRs and FRs are identified. These alternatives are identified with
a "v1" following the ABP name. As most of these alternatives are
minor in nature, only sections with differences are displayed in
the table. It is understood that the remaining section of the light
or heavy chain is the same as shown for the base ABP in the other
panels. Thus, for example, 19H9v1 in FIG. 2C has the same FR1,
CDR1, and FR2 as 19H9 in FIG. 2A as the only difference is noted in
FIG. 2C. For three of the nucleic acid sequences (ABPs 26E10, 30B9,
and 31B12), additional alternative nucleic acid sequences are
provided in the figures. As will be appreciated by one of skill in
the art, no more than one such sequence need actually be used in
the creation of an antibody or ABP. Indeed, in some embodiments,
only one or neither of the specific heavy or light chain nucleic
acids need be present.
[0275] In some embodiments, the ABP is encoded by a nucleic acid
sequence that can encode any of the protein sequences in Table
2.
[0276] In some embodiments, the ABP binds selectively to the form
of PCSK9 that binds to LDLR (e.g., the autocatalyzed form of the
molecule). In some embodiments, the antigen binding protein does
not bind to the c-terminus of the cataylytic domain (e.g., the 5.
5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most amino acids in the
c-terminus). In some embodiments, the antigen binding protein does
not bind to the n-terminus of the catalytic domain (e.g., the 5.
5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most amino acids in the
n-terminus). In some embodiments, the ABP binds to amino acids
within amino acids 1-100 of the mature form of PCSK9. In some
embodiments, the ABP binds to amino acids within (and/or amino acid
sequences consisting of) amino acids 31-100, 100-200, 31-152,
153-692, 200-300, 300-400, 452-683, 400-500, 500-600, 31-692,
31-449, and/or 600-692. In some embodiments, the ABP binds to the
catalytic domain. In some embodiments, the neutralizing and/or
non-neutralizing ABP binds to the prodomain. In some embodiments,
the ABP binds to both the catalytic and pro domains. In some
embodiments, the ABP binds to the catalytic domain so as to
obstruct an area on the catalytic domain that interacts with the
pro domain. In some embodiments, the ABP binds to the catalytic
domain at a location or surface that the pro-domain interacts with
as outlined in Piper et al. (Structure 15:1-8 (2007), the entirety
of which is hereby incorporated by reference, including the
structural representations therein). In some embodiments, the ABP
binds to the catalytic domain and restricts the mobility of the
prodomain. In some embodiments, the ABP binds to the catalytic
domain without binding to the pro-domain. In some embodiments, the
ABP binds to the catalytic domain, without binding to the
pro-domain, while preventing the pro-domain from reorienting to
allow PCSK9 to bind to LDLR. In some embodiments, the ABP binds in
the same epitope as those surrounding residues 149-152 of the
pro-domain in Piper et al. In some embodiments, the ABPs bind to
the groove (as outlined in Piper et al.) on the V domain. In some
embodiments, the ABPs bind to the histidine-rich patch proximal to
the groove on the V domain. In some embodiments, such antibodies
(that bind to the V domain) are not neutralizing. In some
embodiments, antibodies that bind to the V domain are neutralizing.
In some embodiments, the neutralizing ABPs prevent the binding of
PCSK9 to LDLR. In some embodiments, the neturalizing ABPs, while
preventing the PCSK9 degradation of LDLR, do not prevent the
binding of PCSK9 to LDLR (for example ABP 31A4). In some
embodiments, the ABP binds to or blocks at least one of the
histidines depicted in FIG. 4 of the Piper et al. paper. In some
embodiments, the ABP blocks the catalytic triad in PCSK9.
[0277] In some embodiments, the antibody binds selectively to
variant PCSK9 proteins, e.g., D374Y over wild type PCSK9. In some
embodiments, these antibodies bind to the variant at least twice as
strongly as the wild type, and preferably 2-5, 5-10, 10-100,
100-1000, 1000-10,000 fold or more to the mutant than the wild type
(as measured via a K.sub.a). In some embodiments, the antibody
selectively inhibits variant D374Y PCSK9 from interacting with LDLR
over wild type PCSK9's ability to interact with LDLR. In some
embodiments, these antibodies block the variant's ability to bind
to LDLR more strongly than the wild type's ability, e.g., at least
twice as strongly as the wild type, and preferably 2-5, 5-10,
10-100, 100-1000 fold or more to the mutant than the wild type (as
measured via an IC.sub.50). In some embodiments, the antibody binds
to and neutralizes both wild type PCSK9 and variant forms of PCSK9,
such as D374Y at similar levels. In some embodiments, the antibody
binds to PCSK9 to prevent variants of LDLR from binding to PCSK9.
In some embodiments, the variants of LDLR are at least 50%
identical to human LDLR. It is noted that variants of LDLR are
known to those of skill in the art (e.g., Brown M S et al, "Calcium
cages, acid baths and recycling receptors" Nature 388: 629-630,
1997). In some embodiments, the ABP can raise the level of
effective LDLR in heterozygote familial hypercholesterolemia (where
a loss-of function variant of LDLR is present).
[0278] In some embodiments, the ABP binds to (but does not block)
variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80,
80-90, 90-95, 95-99, or greater percent identity to the form of
PCSK9 depicted in FIG. 1A and/or FIG. 1B. In some embodiments, the
ABP binds to (but does not block) variants of PCSK9 that are at
least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, or greater
percent identity to the mature form of PCSK9 depicted in FIG. 1A
and/or FIG. 1B. In some embodiments, the ABP binds to and prevents
variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80,
80-90, 90-95, 95-99, or greater percent identity to the form of
PCSK9 depicted in FIG. 1A and/or FIG. 1B from interacting with
LDLR. In some embodiments, the ABP binds to and prevents variants
of PCSK9 that are at least 50, 50-60, 60-70, 70-80, 80-90, 90-95,
95-99, or greater percent identity to the mature form of PCSK9
depicted in FIG. 1B from interacting with LDLR. In some
embodiments, the variant of PCSK9 is a human variant, such as
variants at position 474, E620G, and/or E670G. In some embodiments,
the amino acid at position 474 is valine (as in other humans) or
threonine (as in cyno and mouse). Given the cross-reactivity data
presented herein, it is believed that the present antibodies will
readily bind to the above variants.
[0279] In some embodiments, the ABP binds to an epitope bound by
one of the antibodies described in Table 2. In some embodiments,
the antigen binding proteins bind to a specific conformational
state of PCSK9 so as to prevent PCSK9 from interacting with
LDLR.
Humanized Antigen Binding Proteins (e.g., Antibodies)
[0280] As described herein, an antigen binding protein to PCSK9 can
comprise a humanized antibody and/or part thereof. An important
practical application of such a strategy is the "humanization" of
the mouse humoral immune system.
[0281] In certain embodiments, a humanized antibody is
substantially non-immunogenic in humans. In certain embodiments, a
humanized antibody has substantially the same affinity for a target
as an antibody from another species from which the humanized
antibody is derived. See, e.g., U.S. Pat. No. 5,530,101, U.S. Pat.
No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No.
5,585,089.
[0282] In certain embodiments, amino acids of an antibody variable
domain that can be modified without diminishing the native affinity
of the antigen binding domain while reducing its immunogenicity are
identified. See, e.g., U.S. Pat. Nos. 5,766,886 and 5,869,619.
[0283] In certain embodiments, modification of an antibody by
methods known in the art is typically designed to achieve increased
binding affinity for a target and/or to reduce immunogenicity of
the antibody in the recipient. In certain embodiments, humanized
antibodies are modified to eliminate glycosylation sites in order
to increase affinity of the antibody for its cognate antigen. See,
e.g., Co et al., Mol. Immunol., 30:1361-1367 (1993). In certain
embodiments, techniques such as "reshaping," "hyperchimerization,"
or "veneering/resurfacing" are used to produce humanized
antibodies. See, e.g., Vaswami et al., Annals of Allergy, Asthma,
& Immunol. 81:105 (1998); Roguska et al., Prot. Engineer.,
9:895-904 (1996); and U.S. Pat. No. 6,072,035. In certain such
embodiments, such techniques typically reduce antibody
immunogenicity by reducing the number of foreign residues, but do
not prevent anti-idiotypic and anti-allotypic responses following
repeated administration of the antibodies. Certain other methods
for reducing immunogenicity are described, e.g., in Gilliland et
al., J. Immunol., 62(6): 3663-71 (1999).
[0284] In certain instances, humanizing antibodies results in a
loss of antigen binding capacity. In certain embodiments, humanized
antibodies are "back mutated." In certain such embodiments, the
humanized antibody is mutated to include one or more of the amino
acid residues found in the donor antibody. See, e.g., Saldanha et
al., Mol Immunol 36:709-19 (1999).
[0285] In certain embodiments the complementarity determining
regions (CDRs) of the light and heavy chain variable regions of an
antibody to PCSK9 can be grafted to framework regions (FRs) from
the same, or another, species. In certain embodiments, the CDRs of
the light and heavy chain variable regions of an antibody to PCSK9
can be grafted to consensus human FRs. To create consensus human
FRs, in certain embodiments, FRs from several human heavy chain or
light chain amino acid sequences are aligned to identify a
consensus amino acid sequence. In certain embodiments, the FRs of
an antibody to PCSK9 heavy chain or light chain are replaced with
the FRs from a different heavy chain or light chain. In certain
embodiments, rare amino acids in the FRs of the heavy and light
chains of an antibody to PCSK9 are not replaced, while the rest of
the FR amino acids are replaced. Rare amino acids are specific
amino acids that are in positions in which they are not usually
found in FRs. In certain embodiments, the grafted variable regions
from an antibody to PCSK9 can be used with a constant region that
is different from the constant region of an antibody to PCSK9. In
certain embodiments, the grafted variable regions are part of a
single chain Fv antibody. CDR grafting is described, e.g., in U.S.
Pat. Nos. 6,180,370, 6,054,297, 5,693,762, 5,859,205, 5,693,761,
5,565,332, 5,585,089, and 5,530,101, and in Jones et al., Nature,
321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988), Winter, FEBS
Letts., 430:92-94 (1998), which are hereby incorporated by
reference for any purpose.
Human Antigen Binding Proteins (e.g., Antibodies)
[0286] As described herein, an antigen binding protein that binds
to PCSK9 can comprise a human (i.e., fully human) antibody and/or
part thereof. In certain embodiments, nucleotide sequences
encoding, and amino acid sequences comprising, heavy and light
chain immunoglobulin molecules, particularly sequences
corresponding to the variable regions are provided. In certain
embodiments, sequences corresponding to complementarity determining
regions (CDR's), specifically from CDR1 through CDR3, are provided.
According to certain embodiments, a hybridoma cell line expressing
such an immunoglobulin molecule is provided. According to certain
embodiments, a hybridoma cell line expressing such a monoclonal
antibody is provided. In certain embodiments a hybridoma cell line
is selected from at least one of the cell lines described in Table
2, e.g., 21B12, 16F12 and 31H4. In certain embodiments, a purified
human monoclonal antibody to human PCSK9 is provided.
[0287] One can engineer mouse strains deficient in mouse antibody
production with large fragments of the human Ig loci in
anticipation that such mice would produce human antibodies in the
absence of mouse antibodies. Large human Ig fragments can preserve
the large variable gene diversity as well as the proper regulation
of antibody production and expression. By exploiting the mouse
machinery for antibody diversification and selection and the lack
of immunological tolerance to human proteins, the reproduced human
antibody repertoire in these mouse strains can yield high affinity
fully human antibodies against any antigen of interest, including
human antigens. Using the hybridoma technology, antigen-specific
human MAbs with the desired specificity can be produced and
selected. Certain exemplary methods are described in WO 98/24893,
U.S. Pat. No. 5,545,807, EP 546073, and EP 546073.
[0288] In certain embodiments, one can use constant regions from
species other than human along with the human variable
region(s).
[0289] The ability to clone and reconstruct megabase sized human
loci in yeast artificial chromosomes (YACs) and to introduce them
into the mouse germline provides an approach to elucidating the
functional components of very large or crudely mapped loci as well
as generating useful models of human disease. Furthermore, the
utilization of such technology for substitution of mouse loci with
their human equivalents could provide insights into the expression
and regulation of human gene products during development, their
communication with other systems, and their involvement in disease
induction and progression.
[0290] Human antibodies avoid some of the problems associated with
antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a patient.
In order to avoid the utilization of murine or rat derived
antibodies, fully human antibodies can be generated through the
introduction of functional human antibody loci into a rodent, other
mammal or animal so that the rodent, other mammal or animal
produces fully human antibodies.
[0291] Humanized antibodies are those antibodies that, while
initially starting off containing antibody amino acid sequences
that are not human, have had at least some of these nonhuman
antibody amino acid sequences replaced with human antibody
sequences. This is in contrast with human antibodies, in which the
antibody is encoded (or capable of being encoded) by genes
possessed a human.
Antigen Binding Protein Variants
[0292] Other antibodies that are provided are variants of the ABPs
listed above formed by combination or subparts of the variable
heavy and variable light chains shown in Table 2 and comprise
variable light and/or variable heavy chains that each have at least
50%, 50-60, 60-70, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%,
or above 99% identity to the amino acid sequences of the sequences
in Table 2 (either the entire sequence or a subpart of the
sequence, e.g., one or more CDR). In some instances, such
antibodies include at least one heavy chain and one light chain,
whereas in other instances the variant forms contain two identical
light chains and two identical heavy chains (or subparts thereof).
In some embodiments, the sequence comparison in FIG. 2A-3D (and
13A-13J and other embodiments in 15A-15D) can be used in order to
identify sections of the antibodies that can be modified by
observing those variations that impact binding and those variations
that do not appear to impact binding. For example, by comparing
similar sequences, one can identify those sections (e.g.,
particular amino acids) that can be modified and how they can be
modified while still retaining (or improving) the functionality of
the ABP. In some embodiments, variants of ABPs include those
consensus groups and sequences depicted in FIGS. 13A, 13C, 13F,
13G, 13H, 13I and/or 13J and variations are allowed in the
positions identified as variable in the figures. The CDRs shown in
FIGS. 13A, 13C, 13F, and 13G were defined based upon a hybrid
combination of the Chothia method (based on the location of the
structural loop regions, see, e.g., "Standard conformations for the
canonical structures of immunoglobulins," Bissan Al-Lazikani,
Arthur M. Lesk and Cyrus Chothia, Journal of Molecular Biology,
273(4): 927-948, 7 November (1997)) and the Kabat method (based on
sequence variability, see, e.g., Sequences of Proteins of
Immunological Interest, Fifth Edition. NIH Publication No. 91-3242,
Kabat et al., (1991)). Each residue determined by either method,
was included in the final list of CDR residues (and is presented in
FIGS. 13A, 13C, 13F, and 13G). The CDRs in FIGS. 13H, 13I, and 13J
were obtained by the Kabat method alone. Unless specified
otherwise, the defined consensus sequences, CDRs, and FRs in FIGS.
13H-13J will define and control the noted CDRs and FRs for the
referenced ABPs in FIG. 13.
[0293] In certain embodiments, an antigen binding protein comprises
a heavy chain comprising a variable region comprising an amino acid
sequence at least 90% identical to an amino acid sequence selected
from at least one of the sequences of SEQ ID NO: 74, 85, 71, 72,
67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,
65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In certain embodiments,
an antigen binding protein comprises a heavy chain comprising a
variable region comprising an amino acid sequence at least 95%
identical to an amino acid sequence selected from at least one of
the sequences of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,
48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78,
83, 69, 81, and 60. In certain embodiments, an antigen binding
protein comprises a heavy chain comprising a variable region
comprising an amino acid sequence at least 99% identical to an
amino acid sequence selected from at least one of the sequences of
SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56,
49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and
60.
[0294] In some embodiments, the antigen binding protein comprises a
sequence that is at least 90%, 90-95%, and/or 95-99% identical to
one or more CDRs from the CDRs in at least one of sequences of SEQ
ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,
57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60.
In some embodiments, 1, 2, 3, 4, 5, or 6 CDR (each being at least
90%, 90-95%, and/or 95-99% identical to the above sequences) is
present.
[0295] In some embodiments, the antigen binding protein comprises a
sequence that is at least 90%, 90-95%, and/or 95-99% identical to
one or more FRs from the FRs in at least one of sequences of SEQ ID
NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57,
50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In
some embodiments, 1, 2, 3, or 4 FR (each being at least 90%,
90-95%, and/or 95-99% identical to the above sequences) is
present.
[0296] In certain embodiments, an antigen binding protein comprises
a light chain comprising a variable region comprising an amino acid
sequence at least 90% identical to an amino acid sequence selected
from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10, 12,
13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,
35, 36, 37, 38, 39, 40, 42, 44, and 46. In certain embodiments, an
antigen binding protein comprises a light chain comprising a
variable region comprising an amino acid sequence at least 95%
identical to an amino acid sequence selected from at least one of
the sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39,
40, 42, 44, and 46. In certain embodiments, an antigen binding
protein comprises a light chain comprising a variable region
comprising an amino acid sequence at least 99% identical to an
amino acid sequence selected from at least one of the sequences of
SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and
46.
[0297] In some embodiments, the antigen binding protein comprises a
sequence that is at least 90%, 90-95%, and/or 95-99% identical to
one or more CDRs from the CDRs in at least one of sequences of SEQ
ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In
some embodiments, 1, 2, 3, 4, 5, or 6 CDR (each being at least 90%,
90-95%, and/or 95-99% identical to the above sequences) is
present.
[0298] In some embodiments, the antigen binding protein comprises a
sequence that is at least 90%, 90-95%, and/or 95-99% identical to
one or more FRs from the FRs in at least one of sequences of SEQ ID
NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In
some embodiments, 1, 2, 3, or 4 FR (each being at least 90%,
90-95%, and/or 95-99% identical to the above sequences) is
present.
[0299] In light of the present disclosure, a skilled artisan will
be able to determine suitable variants of the ABPs as set forth
herein using well-known techniques. In certain embodiments, one
skilled in the art can identify suitable areas of the molecule that
may be changed without destroying activity by targeting regions not
believed to be important for activity. In certain embodiments, one
can identify residues and portions of the molecules that are
conserved among similar polypeptides. In certain embodiments, even
areas that can be important for biological activity or for
structure can be subject to conservative amino acid substitutions
without destroying the biological activity or without adversely
affecting the polypeptide structure.
[0300] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a protein that correspond to amino acid residues which
are important for activity or structure in similar proteins. One
skilled in the art can opt for chemically similar amino acid
substitutions for such predicted important amino acid residues.
[0301] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar ABPs. In view of such information, one
skilled in the art can predict the alignment of amino acid residues
of an antibody with respect to its three dimensional structure. In
certain embodiments, one skilled in the art can choose not to make
radical changes to amino acid residues predicted to be on the
surface of the protein, since such residues can be involved in
important interactions with other molecules. Moreover, one skilled
in the art can generate test variants containing a single amino
acid substitution at each desired amino acid residue. The variants
can then be screened using activity assays known to those skilled
in the art. Such variants can be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change can be
avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0302] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol.,
47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and
Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer
programs are currently available to assist with predicting
secondary structure. One method of predicting secondary structure
is based upon homology modeling. For example, two polypeptides or
proteins which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural database
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(1):244-247 (1999). It has been suggested (Brenner et al., Curr.
Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will become dramatically more accurate.
[0303] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87
(1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile
analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et
al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci. USA, 84(13):4355-4358 (1987)), and "evolutionary
linkage" (See Holm, supra (1999), and Brenner, supra (1997)).
[0304] In certain embodiments, antigen binding protein variants
include glycosylation variants wherein the number and/or type of
glycosylation site has been altered compared to the amino acid
sequences of a parent polypeptide. In certain embodiments, protein
variants comprise a greater or a lesser number of N-linked
glycosylation sites than the native protein. An N-linked
glycosylation site is characterized by the sequence: Asn-X-Ser or
Asn-X-Thr, wherein the amino acid residue designated as X can be
any amino acid residue except proline. The substitution of amino
acid residues to create this sequence provides a potential new site
for the addition of an N-linked carbohydrate chain. Alternatively,
substitutions which eliminate this sequence will remove an existing
N-linked carbohydrate chain. Also provided is a rearrangement of
N-linked carbohydrate chains wherein one or more N-linked
glycosylation sites (typically those that are naturally occurring)
are eliminated and one or more new N-linked sites are created.
Additional preferred antibody variants include cysteine variants
wherein one or more cysteine residues are deleted from or
substituted for another amino acid (e.g., serine) as compared to
the parent amino acid sequence. Cysteine variants can be useful
when antibodies must be refolded into a biologically active
conformation such as after the isolation of insoluble inclusion
bodies. Cysteine variants generally have fewer cysteine residues
than the native protein, and typically have an even number to
minimize interactions resulting from unpaired cysteines.
[0305] According to certain embodiments, amino acid substitutions
are those which: (1) reduce susceptibility to proteolysis, (2)
reduce susceptibility to oxidation, (3) alter binding affinity for
forming protein complexes, (4) alter binding affinities, and/or (4)
confer or modify other physiocochemical or functional properties on
such polypeptides. According to certain embodiments, single or
multiple amino acid substitutions (in certain embodiments,
conservative amino acid substitutions) can be made in the
naturally-occurring sequence (in certain embodiments, in the
portion of the polypeptide outside the domain(s) forming
intermolecular contacts). In certain embodiments, a conservative
amino acid substitution typically may not substantially change the
structural characteristics of the parent sequence (e.g., a
replacement amino acid should not tend to break a helix that occurs
in the parent sequence, or disrupt other types of secondary
structure that characterizes the parent sequence). Examples of
art-recognized polypeptide secondary and tertiary structures are
described in Proteins, Structures and Molecular Principles
(Creighton, Ed., W. H. Freeman and Company, New York (1984));
Introduction to Protein Structure (C. Branden & J. Tooze, eds.,
Garland Publishing, New York, N.Y. (1991)); and Thornton et al.,
Nature, 354:105 (1991), which are each incorporated herein by
reference.
[0306] In some embodiments, the variants are variants of the
nucleic acid sequences of the ABPs disclosed herein. One of skill
in the art will appreciate that the above discussion can be used
for identifying, evaluating, and/creating ABP protein variants and
also for nucleic acid sequences that can encode for those protein
variants. Thus, nucleic acid sequences encoding for those protein
variants (as well as nucleic acid sequences that encode for the
ABPs in Table 2, but are different from those explicitly disclosed
herein) are contemplated. For example, an ABP variant can have at
least 80, 80-85, 85-90, 90-95, 95-97, 97-99 or greater identity to
at least one nucleic acid sequence described in SEQ ID NOs: 152,
153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,
132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,
145, 146, 147, 148, 149, 150, 151 or at least one to six (and
various combinations thereof) of the CDR(s) encoded by the nucleic
acid sequences in SEQ ID NOs: 152, 153, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
and 151.
[0307] In some embodiments, the antibody (or nucleic acid sequence
encoding it) is a variant if the nucleic acid sequence that encodes
the particular ABP (or the nucleic acid sequence itself) can
selectively hybridize to any of the nucleic acid sequences that
encode the proteins in Table 2 (such as, but not limited to SEQ ID
NO: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, and 151) under stringent
conditions. In one embodiment, suitable moderately stringent
conditions include prewashing in a solution of 5.times.SSC; 0.5%
SDS, 1.0 mM EDTA (pH 8:0); hybridizing at 50.degree. C.,
-65.degree. C., 5.times.SSC, overnight or, in the event of
cross-species homology, at 45.degree. C. with 0.5.times.SSC;
followed by washing twice at 65.degree. C. for 20 minutes with each
of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS. Such
hybridizing DNA sequences are also within the scope of this
invention, as are nucleotide sequences that, due to code
degeneracy, encode an antibody polypeptide that is encoded by a
hybridizing DNA sequence and the amino acid sequences that are
encoded by these nucleic acid sequences. In some embodiments,
variants of CDRs include nucleic acid sequences and the amino acid
sequences encoded by those sequences, that hybridize to one or more
of the CDRs within the sequences noted above (individual CDRs can
readily be determined in light of FIGS. 2A-3D, and other
embodiments in FIGS. 3CCC-3JJJ and 15A-15D). The phrase
"selectively hybridize" referred to in this context means to
detectably and selectively bind. Polynucleotides, oligonucleotides
and fragments thereof in accordance with the invention selectively
hybridize to nucleic acid strands under hybridization and wash
conditions that minimize appreciable amounts of detectable binding
to nonspecific nucleic acids. High stringency conditions can be
used to achieve selective hybridization conditions as known in the
art and discussed herein. Generally, the nucleic acid sequence
homology between the polynucleotides, oligonucleotides, and
fragments of the invention and a nucleic acid sequence of interest
will be at least 80%, and more typically with preferably increasing
homologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid
sequences are homologous if there is a partial or complete identity
between their sequences. For example, 85% homology means that 85%
of the amino acids are identical when the two sequences are aligned
for maximum matching. Gaps (in either of the two sequences being
matched) are allowed in maximizing matching; gap lengths of 5 or
less are preferred with 2 or less being more preferred.
Alternatively and preferably, two protein sequences (or polypeptide
sequences derived from them of at least 30 amino acids in length)
are homologous, as this term is used herein, if they have an
alignment score of at more than 5 (in standard deviation units)
using the program ALIGN with the mutation data matrix and a gap
penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein
Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical
Research Foundation (1972)) and Supplement 2 to this volume, pp.
1-10. The two sequences or parts thereof are more preferably
homologous if their amino acids are greater than or equal to 50%
identical when optimally aligned using the ALIGN program. The term
"corresponds to" is used herein to mean that a polynucleotide
sequence is homologous (i.e., is identical, not strictly
evolutionarily related) to all or a portion of a reference
polynucleotide sequence, or that a polypeptide sequence is
identical to a reference polypeptide sequence. In
contradistinction, the term "complementary to" is used herein to
mean that the complementary sequence is homologous to all or a
portion of a reference polynucleotide sequence. For illustration,
the nucleotide sequence "TATAC" corresponds to a reference sequence
"TATAC" and is complementary to a reference sequence "GTATA".
Preparation of Antigen Binding Proteins (e.g., Antibodies)
[0308] In certain embodiments, antigen binding proteins (such as
antibodies) are produced by immunization with an antigen (e.g.,
PCSK9). In certain embodiments, antibodies can be produced by
immunization with full-length PCSK9, a soluble form of PCSK9, the
catalytic domain alone, the mature form of PCSK9 shown in FIG. 1A,
a splice variant form of PCSK9, or a fragment thereof. In certain
embodiments, the antibodies of the invention can be polyclonal or
monoclonal, and/or can be recombinant antibodies. In certain
embodiments, antibodies of the invention are human antibodies
prepared, for example, by immunization of transgenic animals
capable of producing human antibodies (see, for example, PCT
Published Application No. WO 93/12227).
[0309] In certain embodiments, certain strategies can be employed
to manipulate inherent properties of an antibody, such as the
affinity of an antibody for its target. Such strategies include,
but are not limited to, the use of site-specific or random
mutagenesis of the polynucleotide molecule encoding an antibody to
generate an antibody variant. In certain embodiments, such
generation is followed by screening for antibody variants that
exhibit the desired change, e.g. increased or decreased
affinity.
[0310] In certain embodiments, the amino acid residues targeted in
mutagenic strategies are those in the CDRs. In certain embodiments,
amino acids in the framework regions of the variable domains are
targeted. In certain embodiments, such framework regions have been
shown to contribute to the target binding properties of certain
antibodies. See, e.g., Hudson, Curr. Opin. Biotech., 9:395-402
(1999) and references therein.
[0311] In certain embodiments, smaller and more effectively
screened libraries of antibody variants are produced by restricting
random or site-directed mutagenesis to hyper-mutation sites in the
CDRs, which are sites that correspond to areas prone to mutation
during the somatic affinity maturation process. See, e.g.,
Chowdhury & Pastan, Nature Biotech., 17: 568-572 (1999) and
references therein. In certain embodiments, certain types of DNA
elements can be used to identify hyper-mutation sites including,
but not limited to, certain direct and inverted repeats, certain
consensus sequences, certain secondary structures, and certain
palindromes. For example, such DNA elements that can be used to
identify hyper-mutation sites include, but are not limited to, a
tetrabase sequence comprising a purine (A or G), followed by
guainine (G), followed by a pyrimidine (C or T), followed by either
adenosine or thymidine (A or T) (i.e., A/G-G-C/T-A/T). Another
example of a DNA element that can be used to identify
hyper-mutation sites is the serine codon, A-G-C/T.
Preparation of Fully Human ABPs (e.g., Antibodies)
[0312] In certain embodiments, a phage display technique is used to
generate monoclonal antibodies. In certain embodiments, such
techniques produce fully human monoclonal antibodies. In certain
embodiments, a polynucleotide encoding a single Fab or Fv antibody
fragment is expressed on the surface of a phage particle. See,
e.g., Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et
al., J Mol Biol 222: 581 (1991); U.S. Pat. No. 5,885,793. In
certain embodiments, phage are "screened" to identify those
antibody fragments having affinity for target. Thus, certain such
processes mimic immune selection through the display of antibody
fragment repertoires on the surface of filamentous bacteriophage,
and subsequent selection of phage by their binding to target. In
certain such procedures, high affinity functional neutralizing
antibody fragments are isolated. In certain such embodiments
(discussed in more detail below), a complete repertoire of human
antibody genes is created by cloning naturally rearranged human V
genes from peripheral blood lymphocytes. See, e.g., Mullinax et
al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990).
[0313] According to certain embodiments, antibodies of the
invention are prepared through the utilization of a transgenic
mouse that has a substantial portion of the human antibody
producing genome inserted but that is rendered deficient in the
production of endogenous, murine antibodies. Such mice, then, are
capable of producing human immunoglobulin molecules and antibodies
and are deficient in the production of murine immunoglobulin
molecules and antibodies. Technologies utilized for achieving this
result are disclosed in the patents, applications and references
disclosed in the specification, herein. In certain embodiments, one
can employ methods such as those disclosed in PCT Published
Application No. WO 98/24893 or in Mendez et al., Nature Genetics,
15:146-156 (1997), which are hereby incorporated by reference for
any purpose.
[0314] Generally, fully human monoclonal ABPs (e.g., antibodies)
specific for PCSK9 can be produced as follows. Transgenic mice
containing human immunoglobulin genes are immunized with the
antigen of interest, e.g. PCSK9, lymphatic cells (such as B-cells)
from the mice that express antibodies are obtained. Such recovered
cells are fused with a myeloid-type cell line to prepare immortal
hybridoma cell lines, and such hybridoma cell lines are screened
and selected to identify hybridoma cell lines that produce
antibodies specific to the antigen of interest. In certain
embodiments, the production of a hybridoma cell line that produces
antibodies specific to PCSK9 is provided.
[0315] In certain embodiments, fully human antibodies are produced
by exposing human splenocytes (B or T cells) to an antigen in
vitro, and then reconstituting the exposed cells in an
immunocompromised mouse, e.g. SCID or nod/SCID. See, e.g., Brams et
al., J. Immunol. 160: 2051-2058 (1998); Carballido et al., Nat.
Med., 6: 103-106 (2000). In certain such approaches, engraftment of
human fetal tissue into SCID mice (SCID-hu) results in long-term
hematopoiesis and human T-cell development. See, e.g., McCune et
al., Science, 241:1532-1639 (1988); Ifversen et al., Sem. Immunol.,
8:243-248 (1996). In certain instances, humoral immune response in
such chimeric mice is dependent on co-development of human T-cells
in the animals. See, e.g., Martensson et al., Immunol., 83:1271-179
(1994). In certain approaches, human peripheral blood lymphocytes
are transplanted into SCID mice. See, e.g., Mosier et al., Nature,
335:256-259 (1988). In certain such embodiments, when such
transplanted cells are treated either with a priming agent, such as
Staphylococcal Enterotoxin A (SEA), or with anti-human CD40
monoclonal antibodies, higher levels of B cell production is
detected. See, e.g., Martensson et al., Immunol., 84: 224-230
(1995); Murphy et al., Blood, 86:1946-1953 (1995).
[0316] Thus, in certain embodiments, fully human antibodies can be
produced by the expression of recombinant DNA in host cells or by
expression in hybridoma cells. In other embodiments, antibodies can
be produced using the phage display techniques described
herein.
[0317] The antibodies described herein were prepared through the
utilization of the XenoMouse.RTM. technology, as described herein.
Such mice, then, are capable of producing human immunoglobulin
molecules and antibodies and are deficient in the production of
murine immunoglobulin molecules and antibodies. Technologies
utilized for achieving the same are disclosed in the patents,
applications, and references disclosed in the background section
herein. In particular, however, a preferred embodiment of
transgenic production of mice and antibodies therefrom is disclosed
in U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996
and International Patent Application Nos. WO 98/24893, published
Jun. 11, 1998 and WO 00/76310, published Dec. 21, 2000, the
disclosures of which are hereby incorporated by reference. See also
Mendez et al., Nature Genetics, 15:146-156 (1997), the disclosure
of which is hereby incorporated by reference.
[0318] Through the use of such technology, fully human monoclonal
antibodies to a variety of antigens have been produced.
Essentially, XenoMouse.RTM. lines of mice are immunized with an
antigen of interest (e.g. PCSK9), lymphatic cells (such as B-cells)
are recovered from the hyper-immunized mice, and the recovered
lymphocytes are fused with a myeloid-type cell line to prepare
immortal hybridoma cell lines. These hybridoma cell lines are
screened and selected to identify hybridoma cell lines that
produced antibodies specific to the antigen of interest. Provided
herein are methods for the production of multiple hybridoma cell
lines that produce antibodies specific to PCSK9 Further, provided
herein are characterization of the antibodies produced by such cell
lines, including nucleotide and amino acid sequence analyses of the
heavy and light chains of such antibodies.
[0319] The production of the XenoMouse.RTM. strains of mice is
further discussed and delineated in U.S. patent application Ser.
No. 07/466,008, filed Jan. 12, 1990, Ser. No. 07/610,515, filed
Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24, 1992, Ser. No.
07/922,649, filed Jul. 30, 1992, Ser. No. 08/031,801, filed Mar.
15, 1993, Ser. No. 08/112,848, filed Aug. 27, 1993, Ser. No.
08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279, filed Jan.
20, 1995, Ser. No. 08/430,938, filed Apr. 27, 1995, Ser. No.
08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5,
1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837,
filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser.
No. 08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun.
5, 1995, Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No.
08/724,752, filed Oct. 2, 1996, Ser. No. 08/759,620, filed Dec. 3,
1996, U.S. Publication 2003/0093820, filed Nov. 30, 2001 and U.S.
Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598
and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507
B2. See also European Patent No., EP 0 463 151 B1, grant published
Jun. 12, 1996, International Patent Application No., WO 94/02602,
published Feb. 3, 1994, International Patent Application No., WO
96/34096, published Oct. 31, 1996, WO 98/24893, published Jun. 11,
1998, WO 00/76310, published Dec. 21, 2000. The disclosures of each
of the above-cited patents, applications, and references are hereby
incorporated by reference in their entirety.
[0320] In an alternative approach, others, including GenPharm
International, Inc., have utilized a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more D.sub.H genes, one or more
J.sub.H genes, a mu constant region, and usually a second constant
region (preferably a gamma constant region) are formed into a
construct for insertion into an animal. This approach is described
in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.
5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,
5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to
Lonberg & Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to
Krimpenfort & Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and
5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi
& Dunn, and GenPharm International U.S. patent application Ser.
No. 07/574,748, filed Aug. 29, 1990, Ser. No. 07/575,962, filed
Aug. 31, 1990, Ser. No. 07/810,279, filed Dec. 17, 1991, Ser. No.
07/853,408, filed Mar. 18, 1992, Ser. No. 07/904,068, filed Jun.
23, 1992, Ser. No. 07/990,860, filed Dec. 16, 1992, Ser. No.
08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762, filed Jul.
22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.
08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,
1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of
which are hereby incorporated by reference. See also European
Patent No. 0 546 073 B1, International Patent Application Nos. WO
92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO
94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884
and U.S. Pat. No. 5,981,175, the disclosures of which are hereby
incorporated by reference in their entirety. See further Taylor et
al., 1992, Chen et al, 1993, Tuaillon et al., 1993, Choi et al.,
1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon
et al., (1995), Fishwild et al, (1996), the disclosures of which
are hereby incorporated by reference in their entirety.
[0321] Kirin has also demonstrated the generation of human
antibodies from mice in which, through microcell fusion, large
pieces of chromosomes, or entire chromosomes, have been introduced.
See European Patent Application Nos. 773 288 and 843 961, the
disclosures of which are hereby incorporated by reference.
Additionally, KM.TM. mice, which are the result of cross-breeding
of Kirin's Tc mice with Medarex's minilocus (Humab) mice have been
generated. These mice possess the human IgH transchromosome of the
Kirin mice and the kappa chain transgene of the Genpharm mice
(Ishida et al., Cloning Stem Cells, (2002) 4:91-102).
[0322] Human antibodies can also be derived by in vitro methods.
Suitable examples include but are not limited to phage display
(CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion
(formerly Proliferon), Affimed) ribosome display (CAT), yeast
display, and the like.
[0323] In some embodiments, the antibodies described herein possess
human IgG4 heavy chains as well as IgG2 heavy chains. Antibodies
can also be of other human isotypes, including IgG1. The antibodies
possessed high affinities, typically possessing a Kd of from about
10.sup.-6 through about 10.sup.-13 M or below, when measured by
various techniques.
[0324] As will be appreciated, antibodies can be expressed in cell
lines other than hybridoma cell lines. Sequences encoding
particular antibodies can be used to transform a suitable mammalian
host cell. Transformation can be by any known method for
introducing polynucleotides into a host cell, including, for
example packaging the polynucleotide in a virus (or into a viral
vector) and transducing a host cell with the virus (or vector) or
by transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which
patents are hereby incorporated herein by reference). The
transformation procedure used depends upon the host to be
transformed. Methods for introducing heterologous polynucleotides
into mammalian cells are well known in the art and include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0325] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), human
epithelial kidney 293 cells, and a number of other cell lines. Cell
lines of particular preference are selected through determining
which cell lines have high expression levels and produce antibodies
with constitutive PCSK9 binding properties.
[0326] In certain embodiments, antibodies and/or ABP are produced
by at least one of the following hybridomas: 21B12, 31H4, 16F12,
any the other hybridomas listed in Table 2 or disclosed in the
examples. In certain embodiments, antigen binding proteins bind to
PCSK9 with a dissociation constant (K.sub.D) of less than
approximately 1 nM, e.g., 1000 pM to 100 pM, 100 pM to 10 pM, 10 pM
to 1 pM, and/or 1 pM to 0.1 pM or less.
[0327] In certain embodiments, antigen binding proteins comprise an
immunoglobulin molecule of at least one of the IgG1, IgG2, IgG3,
IgG4, Ig E, IgA, IgD, and IgM isotype. In certain embodiments,
antigen binding proteins comprise a human kappa light chain and/or
a human heavy chain. In certain embodiments, the heavy chain is of
the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgM isotype. In
certain embodiments, antigen binding proteins have been cloned for
expression in mammalian cells. In certain embodiments, antigen
binding proteins comprise a constant region other than any of the
constant regions of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and
IgM isotype.
[0328] In certain embodiments, antigen binding proteins comprise a
human lambda light chain and a human IgG2 heavy chain. In certain
embodiments, antigen binding proteins comprise a human lambda light
chain and a human IgG4 heavy chain. In certain embodiments, antigen
binding proteins comprise a human lambda light chain and a human
IgG1, IgG3, IgE, IgA, IgD or IgM heavy chain. In other embodiments,
antigen binding proteins comprise a human kappa light chain and a
human IgG2 heavy chain. In certain embodiments, antigen binding
proteins comprise a human kappa light chain and a human IgG4 heavy
chain. In certain embodiments, antigen binding proteins comprise a
human kappa light chain and a human IgG1, IgG3, IgE, IgA, IgD or
IgM heavy chain. In certain embodiments, antigen binding proteins
comprise variable regions of antibodies ligated to a constant
region that is neither the constant region for the IgG2 isotype,
nor the constant region for the IgG4 isotype. In certain
embodiments, antigen binding proteins have been cloned for
expression in mammalian cells.
[0329] In certain embodiments, conservative modifications to the
heavy and light chains of antibodies from at least one of the
hybridoma lines: 21B12, 31H4 and 16F12 (and corresponding
modifications to the encoding nucleotides) will produce antibodies
to PCSK9 having functional and chemical characteristics similar to
those of the antibodies from the hybridoma lines: 21B12, 31H4 and
16F12. In contrast, in certain embodiments, substantial
modifications in the functional and/or chemical characteristics of
antibodies to PCSK9 can be accomplished by selecting substitutions
in the amino acid sequence of the heavy and light chains that
differ significantly in their effect on maintaining (a) the
structure of the molecular backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[0330] For example, a "conservative amino acid substitution" can
involve a substitution of a native amino acid residue with a
normative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide can also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis."
[0331] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. In certain embodiments,
amino acid substitutions can be used to identify important residues
of antibodies to PCSK9, or to increase or decrease the affinity of
the antibodies to PCSK9 as described herein.
[0332] In certain embodiments, antibodies of the present invention
can be expressed in cell lines other than hybridoma cell lines. In
certain embodiments, sequences encoding particular antibodies can
be used for transformation of a suitable mammalian host cell.
According to certain embodiments, transformation can be by any
known method for introducing polynucleotides into a host cell,
including, for example packaging the polynucleotide in a virus (or
into a viral vector) and transducing a host cell with the virus (or
vector) or by transfection procedures known in the art, as
exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and
4,959,455 (which patents are hereby incorporated herein by
reference for any purpose). In certain embodiments, the
transformation procedure used can depend upon the host to be
transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and
include, but are not limited to, dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei.
[0333] Mammalian cell lines available as hosts for expression are
well known in the art and include, but are not limited to, many
immortalized cell lines available from the American Type Culture
Collection (ATCC), including but not limited to Chinese hamster
ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,
monkey kidney cells (COS), human hepatocellular carcinoma cells
(e.g., Hep G2), and a number of other cell lines. In certain
embodiments, cell lines can be selected through determining which
cell lines have high expression levels and produce antibodies with
constitutive HGF binding properties. Appropriate expression vectors
for mammalian host cells are well known.
[0334] In certain embodiments, antigen binding proteins comprise
one or more polypeptides. In certain embodiments, any of a variety
of expression vector/host systems can be utilized to express
polynucleotide molecules encoding polypeptides comprising one or
more ABP components or the ABP itself. Such systems include, but
are not limited to, microorganisms, such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transfected with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic
virus, TMV) or transformed with bacterial expression vectors (e.g.,
Ti or pBR322 plasmid); or animal cell systems.
[0335] In certain embodiments, a polypeptide comprising one or more
ABP components or the ABP itself is recombinantly expressed in
yeast. Certain such embodiments use commercially available
expression systems, e.g., the Pichia Expression System (Invitrogen,
San Diego, Calif.), following the manufacturer's instructions. In
certain embodiments, such a system relies on the pre-pro-alpha
sequence to direct secretion. In certain embodiments, transcription
of the insert is driven by the alcohol oxidase (AOX1) promoter upon
induction by methanol.
[0336] In certain embodiments, a secreted polypeptide comprising
one or more ABP components or the ABP itself is purified from yeast
growth medium. In certain embodiments, the methods used to purify a
polypeptide from yeast growth medium is the same as those used to
purify the polypeptide from bacterial and mammalian cell
supernatants.
[0337] In certain embodiments, a nucleic acid encoding a
polypeptide comprising one or more ABP components or the ABP itself
is cloned into a baculovirus expression vector, such as pVL1393
(PharMingen, San Diego, Calif.). In certain embodiments, such a
vector can be used according to the manufacturer's directions
(PharMingen) to infect Spodoptera frugiperda cells in sF9
protein-free media and to produce recombinant polypeptide. In
certain embodiments, a polypeptide is purified and concentrated
from such media using a heparin-Sepharose column (Pharmacia).
[0338] In certain embodiments, a polypeptide comprising one or more
ABP components or the ABP itself is expressed in an insect system.
Certain insect systems for polypeptide expression are well known to
those of skill in the art. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes in Spodoptera frugiperda cells or in
Trichoplusia larvae. In certain embodiments, a nucleic acid
molecule encoding a polypeptide can be inserted into a nonessential
gene of the virus, for example, within the polyhedrin gene, and
placed under control of the promoter for that gene. In certain
embodiments, successful insertion of a nucleic acid molecule will
render the nonessential gene inactive. In certain embodiments, that
inactivation results in a detectable characteristic. For example,
inactivation of the polyhedrin gene results in the production of
virus lacking coat protein.
[0339] In certain embodiments, recombinant viruses can be used to
infect S. frugiperda cells or Trichoplusia larvae. See, e.g., Smith
et al., J. Virol., 46: 584 (1983); Engelhard et al., Proc. Nat.
Acad. Sci. (USA), 91: 3224-7 (1994).
[0340] In certain embodiments, polypeptides comprising one or more
ABP components or the ABP itself made in bacterial cells are
produced as insoluble inclusion bodies in the bacteria. In certain
embodiments, host cells comprising such inclusion bodies are
collected by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH
8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma, St.
Louis, Mo.) for 15 minutes at room temperature. In certain
embodiments, the lysate is cleared by sonication, and cell debris
is pelleted by centrifugation for 10 minutes at 12,000.times.g. In
certain embodiments, the polypeptide-containing pellet is
resuspended in 50 mM Tris, pH 8, and 10 mM EDTA; layered over 50%
glycerol; and centrifuged for 30 minutes at 6000.times.g. In
certain embodiments, that pellet can be resuspended in standard
phosphate buffered saline solution (PBS) free of Mg.sup.++ and
Ca.sup.++. In certain embodiments, the polypeptide is further
purified by fractionating the resuspended pellet in a denaturing
SDS polyacrylamide gel (See, e.g., Sambrook et al., supra). In
certain embodiments, such a gel can be soaked in 0.4 M KCl to
visualize the protein, which can be excised and electroeluted in
gel-running buffer lacking SDS. According to certain embodiments, a
Glutathione-S-Transferase (GST) fusion protein is produced in
bacteria as a soluble protein. In certain embodiments, such GST
fusion protein is purified using a GST Purification Module
(Pharmacia).
[0341] In certain embodiments, it is desirable to "refold" certain
polypeptides, e.g., polypeptides comprising one or more ABP
components or the ABP itself. In certain embodiments, such
polypeptides are produced using certain recombinant systems
discussed herein. In certain embodiments, polypeptides are
"refolded" and/or oxidized to form desired tertiary structure
and/or to generate disulfide linkages. In certain embodiments, such
structure and/or linkages are related to certain biological
activity of a polypeptide. In certain embodiments, refolding is
accomplished using any of a number of procedures known in the art.
Exemplary methods include, but are not limited to, exposing the
solubilized polypeptide agent to a pH typically above 7 in the
presence of a chaotropic agent. An exemplary chaotropic agent is
guanidine. In certain embodiments, the refolding/oxidation solution
also contains a reducing agent and the oxidized form of that
reducing agent. In certain embodiments, the reducing agent and its
oxidized form are present in a ratio that will generate a
particular redox potential that allows disulfide shuffling to
occur. In certain embodiments, such shuffling allows the formation
of cysteine bridges. Exemplary redox couples include, but are not
limited to, cysteine/cystamine, glutathione/dithiobisGSH, cupric
chloride, dithiothreitol DTT/dithiane DTT, and 2-mercaptoethanol
(bME)/dithio-bME. In certain embodiments, a co-solvent is used to
increase the efficiency of refolding. Exemplary cosolvents include,
but are not limited to, glycerol, polyethylene glycol of various
molecular weights, and arginine.
[0342] In certain embodiments, one substantially purifies a
polypeptide comprising one or more ABP components or the ABP
itself. Certain protein purification techniques are known to those
of skill in the art. In certain embodiments, protein purification
involves crude fractionation of polypeptide fractionations from
non-polypeptide fractions. In certain embodiments, polypeptides are
purified using chromatographic and/or electrophoretic techniques.
Exemplary purification methods include, but are not limited to,
precipitation with ammonium sulphate; precipitation with PEG;
immunoprecipitation; heat denaturation followed by centrifugation;
chromatography, including, but not limited to, affinity
chromatography (e.g., Protein-A-Sepharose), ion exchange
chromatography, exclusion chromatography, and reverse phase
chromatography; gel filtration; hydroxyapatite chromatography;
isoelectric focusing; polyacrylamide gel electrophoresis; and
combinations of such and other techniques. In certain embodiments,
a polypeptide is purified by fast protein liquid chromatography or
by high pressure liquid chromotography (HPLC). In certain
embodiments, purification steps can be changed or certain steps can
be omitted, and still result in a suitable method for the
preparation of a substantially purified polypeptide.
[0343] In certain embodiments, one quantitates the degree of
purification of a polypeptide preparation. Certain methods for
quantifying the degree of purification are known to those of skill
in the art. Certain exemplary methods include, but are not limited
to, determining the specific binding activity of the preparation
and assessing the amount of a polypeptide within a preparation by
SDS/PAGE analysis. Certain exemplary methods for assessing the
amount of purification of a polypeptide preparation comprise
calculating the binding activity of a preparation and comparing it
to the binding activity of an initial extract. In certain
embodiments, the results of such a calculation are expressed as
"fold purification." The units used to represent the amount of
binding activity depend upon the particular assay performed.
[0344] In certain embodiments, a polypeptide comprising one or more
ABP components or the ABP itself is partially purified. In certain
embodiments, partial purification can be accomplished by using
fewer purification steps or by utilizing different forms of the
same general purification scheme. For example, in certain
embodiments, cation-exchange column chromatography performed
utilizing an HPLC apparatus will generally result in a greater
"fold purification" than the same technique utilizing a
low-pressure chromatography system. In certain embodiments, methods
resulting in a lower degree of purification can have advantages in
total recovery of polypeptide, or in maintaining binding activity
of a polypeptide.
[0345] In certain instances, the electrophoretic migration of a
polypeptide can vary, sometimes significantly, with different
conditions of SDS/PAGE. See, e.g., Capaldi et al., Biochem.
Biophys. Res. Comm., 76: 425 (1977). It will be appreciated that
under different electrophoresis conditions, the apparent molecular
weights of purified or partially purified polypeptide can be
different.
Exemplary Epitopes
[0346] Epitopes to which anti-PCSK9 antibodies bind are provided.
In some embodiments, epitopes that are bound by the presently
disclosed antibodies are particularly useful. In some embodiments,
antigen binding proteins that bind to any of the epitopes that are
bound by the antibodies described herein are useful. In some
embodiments, the epitopes bound by any of the antibodies listed in
Table 2 and FIGS. 2 and 3 are especially useful. In some
embodiments, the epitope is on the catalytic domain PCSK9.
[0347] In certain embodiments, a PCSK9 epitope can be utilized to
prevent (e.g., reduce) binding of an anti-PCSK9 antibody or antigen
binding protein to PCSK9. In certain embodiments, a PCSK9 epitope
can be utilized to decrease binding of an anti-PCSK9 antibody or
antigen binding protein to PCSK9. In certain embodiments, a PCSK9
epitope can be utilized to substantially inhibit binding of an
anti-PCSK9 antibody or antigen binding protein to PCSK9.
[0348] In certain embodiments, a PCSK9 epitope can be utilized to
isolate antibodies or antigen binding proteins that bind to PCSK9.
In certain embodiments, a PCSK9 epitope can be utilized to generate
antibodies or antigen binding proteins which bind to PCSK9. In
certain embodiments, a PCSK9 epitope or a sequence comprising a
PCSK9 epitope can be utilized as an immunogen to generate
antibodies or antigen binding proteins that bind to PCSK9. In
certain embodiments, a PCSK9 epitope can be administered to an
animal, and antibodies that bind to PCSK9 can subsequently be
obtained from the animal. In certain embodiments, a PCSK9 epitope
or a sequence comprising a PCSK9 epitope can be utilized to
interfere with normal PCSK9-mediated activity, such as association
of PCSK9 with the LDLR.
[0349] In some embodiments, antigen binding proteins disclosed
herein bind specifically to N-terminal prodomain, a subtilisin-like
catalytic domain and/or a C-terminal domain. In some embodiments,
the antigen binding protein binds to the substrate-binding groove
of PCSK-9 (described in Cunningham et al., incorporated herein in
its entirety by reference).
[0350] In some embodiments, the domain(s)/region(s) containing
residues that are in contact with or are buried by an antibody can
be identified by mutating specific residues in PCSK9 (e.g., a
wild-type antigen) and determining whether the antigen binding
protein can bind the mutated or variant PCSK9 protein. By making a
number of individual mutations, residues that play a direct role in
binding or that are in sufficiently close proximity to the antibody
such that a mutation can affect binding between the antigen binding
protein and antigen can be identified. From a knowledge of these
amino acids, the domain(s) or region(s) of the antigen that contain
residues in contact with the antigen binding protein or covered by
the antibody can be elucidated. Such a domain can include the
binding epitope of an antigen binding protein. One specific example
of this general approach utilizes an arginine/glutamic acid
scanning protocol (see, e.g., Nanevicz, T., et al., 1995, J. Biol.
Chem., 270:37, 21619-21625 and Zupnick, A., et al., 2006, J. Biol.
Chem., 281:29, 20464-20473). In general, arginine and glutamic
acids are substituted (typically individually) for an amino acid in
the wild-type polypeptide because these amino acids are charged and
bulky and thus have the potential to disrupt binding between an
antigen binding protein and an antigen in the region of the antigen
where the mutation is introduced. Arginines that exist in the
wild-type antigen are replaced with glutamic acid. A variety of
such individual mutants are obtained and the collected binding
results analyzed to determine what residues affect binding.
[0351] Example 39 describes one such arginine/glutamic acid
scanning of PCSK9 for PCSK9 antigen binding proteins provided
herein. A series of mutant PCSK9 antigens were created, with each
mutant antigen having a single mutation. Binding of each mutant
PCSK9 antigen with various PCSK9 ABPs was measured and compared to
the ability of the selected ABPs to bind wild-type PCSK9 (SEQ ID
NO: 303).
[0352] An alteration (for example a reduction or increase) in
binding between an antigen binding protein and a variant PCSK9 as
used herein means that there is a change in binding affinity (e.g.,
as measured by known methods such as Biacore testing or the bead
based assay described below in the examples), EC.sub.50, and/or a
change (for example a reduction) in the total binding capacity of
the antigen binding protein (for example, as evidenced by a
decrease in Bmax in a plot of antigen binding protein concentration
versus antigen concentration). A significant alteration in binding
indicates that the mutated residue is directly involved in binding
to the antigen binding protein or is in close proximity to the
binding protein when the binding protein is bound to antigen.
[0353] In some embodiments, a significant reduction in binding
means that the binding affinity, EC50, and/or capacity between an
antigen binding protein and a mutant PCSK9 antigen is reduced by
greater than 10%, greater than 20%, greater than 40%, greater than
50%, greater than 55%, greater than 60%, greater than 65%, greater
than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90% or greater than 95% relative to binding between
the antigen binding protein and a wild type PCSK9 (e.g., shown in
SEQ ID NO: 1 and/or SEQ ID NO: (303). In certain embodiments,
binding is reduced below detectable limits. In some embodiments, a
significant reduction in binding is evidenced when binding of an
antigen binding protein to a variant PCSK9 protein is less than 50%
(for example, less than 40%, 35%, 30%, 25%, 20%, 15% or 10%) of the
binding observed between the antigen binding protein and a
wild-type PCSK9 protein (for example, the protein of SEQ ID NO: 1
and/or SEQ ID NO: (303). Such binding measurements can be made
using a variety of binding assays known in the art. A specific
example of one such assay is described in Example 39.
[0354] In some embodiments, antigen binding proteins are provided
that exhibit significantly lower binding for a variant PCSK9
protein in which a residue in a wild-type PCSK9 protein (e.g., SEQ
ID NO: 1 or SEQ ID NO: 303 is substituted with arginine or glutamic
acid. In some embodiments, binding of an antigen binding protein is
significantly reduced or increased for a variant PCSK9 protein
having any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
244) of the following mutations: R207E, D208R, R185E, R439E, E513R,
V538R, E539R, T132R, S351R, A390R, A413R, E582R, D162R, R164E,
E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R
as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ
ID NO: 303. In the shorthand notation used here, the format is:
Wild type residue: Position in polypeptide: Mutant residue, with
the numbering of the residues as indicated in SEQ ID NO: 1 for SEQ
ID NO: 303.
[0355] In some embodiments, binding of an antigen binding protein
is significantly reduced or increased for a mutant PCSK9 protein
having one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at the
following positions: 207, 208, 185, 181, 439, 513, 538, 539, 132,
351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519,
521, and 554, as shown in SEQ ID NO: 1 as compared to a wild-type
PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303. In some
embodiments, binding of an antigen binding protein is reduced or
increased for a mutant PCSK9 protein having one or more (e.g., 1,
2, 3, 4, 5, or more) mutations at the following positions: 207,
208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162,
164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as shown in
SEQ ID NO: 1 as compared to a wild-type PCSK9 protein (e.g., SEQ ID
NO: 1 or SEQ ID NO: 303. In some embodiments, binding of an antigen
binding protein is substantially reduced or increased for a mutant
PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more)
mutations at the following positions: 207, 208, 185, 181, 439, 513,
538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311,
313, 337, 519, 521, and 554, within SEQ ID NO: 1 as compared to a
wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303.
[0356] In some embodiments, binding of an ABP is significantly
reduced or increased for a mutant PCSK9 protein having one or more
(e.g., 1, 2, 3, 4, 5, etc.) of the following mutations: R207E,
D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R,
A413R, E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R,
D337R, R519E, H521R, and Q554R within SEQ ID NO: 1 or SEQ ID NO:
303, as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1
or SEQ ID NO: 303).
[0357] In some embodiments, binding of an ABP is significantly
reduced or increased for a mutant PCSK9 protein having one or more
(e.g., 1, 2, 3, 4, 5, etc.) of the following mutations: R207E,
D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R,
A413R, and E582R within SEQ ID NO: 1 or SEQ ID NO: 303, as compared
to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO:
303). In some embodiments, the binding is reduced. In some
embodiments, the reduction in binding is observed as a change in
EC50. In some embodiments, the change in EC50 is an increase in the
numerical value of the EC50 (and thus is a decrease in
binding).
[0358] In some embodiments, binding of an ABP is significantly
reduced or increased for a mutant PCSK9 protein having one or more
(e.g., 1, 2, 3, 4, 5, etc.) of the following mutations: D162R,
R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and
Q554R within SEQ ID NO: 1, as compared to a wild-type PCSK9 protein
(e.g., SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the
binding is reduced. In some embodiments, the reduction in binding
is observed as a change in Bmax. In some embodiments, the shift in
Bmax is a reduction of the maximum signal generated by the ABP. In
some embodiments, for an amino acid to be part of an epitope, the
Bmax is reduced by at least 10%, for example, reductions of at
least any of the following amounts: 20, 30, 40, 50, 60, 70, 80, 90,
95, 98, 99, or 100 percent can, in some embodiments, indicate that
the residue is part of the epitope.
[0359] Although the variant forms just listed are referenced with
respect to the wild-type sequence shown in SEQ ID NO: 1 or SEQ ID
NO: 303, it will be appreciated that in an allelic variant of PCSK9
the amino acid at the indicated position could differ. Antigen
binding proteins showing significantly lower binding for such
allelic forms of PCSK9 are also contemplated. Accordingly, in some
embodiments, any of the above embodiments can be compared to an
allelic sequence, rather than purely the wild-type sequence shown
in FIG. 1A
[0360] In some embodiments, binding of an antigen binding protein
is significantly reduced for a variant PCSK9 protein in which the
residue at a selected position in the wild-type PCSK9 protein is
mutated to any other residue. In some embodiments, the herein
described arginine/glutamic acid replacements are used for the
identified positions. In some embodiments, alanine is used for the
identified positions.
[0361] As noted above, residues directly involved in binding or
covered by an antigen binding protein can be identified from
scanning results. These residues can thus provide an indication of
the domains or regions of SEQ ID NO: 1 (or SEQ ID NO: 303 or SEQ ID
NO: 3) that contain the binding region(s) to which antigen binding
proteins bind. As can be seen from the results summarized in
Example 39, in some embodiments an antigen binding protein binds to
a domain containing at least one of amino acids: 207, 208, 185,
181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167,
123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 1 or SEQ
ID NO: 303. In some embodiments, the antigen binding protein binds
to a region containing at least one of amino acids 207, 208, 185,
181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167,
123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 1 or SEQ
ID NO: 303.
[0362] In some embodiments, the antigen binding protein binds to a
region containing at least one of amino acids 162, 164, 167, 207
and/or 208 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments,
more than one (e.g., 2, 3, 4, or 5) of the identified residues are
part of the region that is bound by the ABP. In some embodiments,
the ABP competes with ABP 21B12.
[0363] In some embodiments, the antigen binding protein binds to a
region containing at least one of amino acid 185 of SEQ ID NO: 1 or
SEQ ID NO: 303. In some embodiments, the ABP competes with ABP
31H4.
[0364] In some embodiments, the antigen binding protein binds to a
region containing at least one of amino acids 439, 513, 538, and/or
539 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more
than one (e.g., 2, 3, or 4) of the identified residues are part of
the region that is bound by the ABP. In some embodiments, the ABP
competes with ABP 31A4.
[0365] In some embodiments, the antigen binding protein binds to a
region containing at least one of amino acids 123, 129, 311, 313,
337, 132, 351, 390, and/or 413 of SEQ ID NO: 1 or SEQ ID NO: 303.
In some embodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or
9) of the identified residues are part of the region that is bound
by the ABP. In some embodiments, the ABP competes with ABP
12H11.
[0366] In some embodiments, the antigen binding protein binds to a
region containing at least one of amino acid 582, 519, 521, and/or
554 of SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more
than one (e.g., 2, 3, or 4) of the identified residues are part of
the region that is bound by the ABP. In some embodiments, the ABP
competes with ABP 3C4.
[0367] In some embodiments, the antigen binding proteins binds to
the foregoing regions within a fragment or the full length sequence
of SEQ ID NO: 1 or SEQ ID NO: 303. In other embodiments, antigen
binding proteins bind to polypeptides consisting of these regions.
The reference to "SEQ ID NO: 1 or SEQ ID NO: 303" denotes that one
or both of these sequences can be employed or relevant. The phrase
does not denote that only one should be employed.
[0368] As noted above, the above description references specific
amino acid positions with reference to SEQ ID NO: 1. However,
throughout the specification generally, reference is made to a
Pro/Cat domain that commences at position 31, which is provided in
SEQ ID NO: 3. As noted below, SEQ ID NO: 1 and SEQ ID NO: 303 lack
the signal sequence of PCSK9. As such, any comparison between these
various disclosures should take this difference in numbering into
account. In particular, any amino acid position in SEQ ID NO: 1,
will correspond to an amino acid position 30 amino acids further
into the protein in SEQ ID NO: 3. For example, position 207 of SEQ
ID NO: 1, corresponds to position 237 of SEQ ID NO: 3 (the full
length sequence, and the numbering system used in the present
specification generally). Table 39.6 outlines how the above noted
positions, which reference SEQ ID NO: 1 (and/or SEQ ID NO: 303)
correspond to SEQ ID NO: 3 (which includes the signal sequence).
Thus, any of the above noted embodiments that are described in
regard to SEQ ID NO: 1 (and/or SEQ ID NO: 303), are described in
reference to SEQ ID NO: 3, by the noted corresponding
positions.
[0369] In some embodiments, ABP 21B12 binds to an epitope including
residues 162-167 (e.g., residues D162-E167 of SEQ ID NO: 1). In
some embodiments, ABP 12H11 binds to an epitope that includes
residues 123-132 (e.g., S123-T132 of SEQ ID NO: 1). In some
embodiments, ABP 12H11 binds to an epitope that includes residues
311-313 (e.g., A311-D313 of SEQ ID NO: 1). In some embodiments,
ABPs can bind to an epitope that includes any one of these strands
of sequences.
Competing Antigen Binding Proteins
[0370] In another aspect, antigen binding proteins are provided
that compete with one of the exemplified antibodies or functional
fragments binding to the epitope described herein for specific
binding to PCSK9. Such antigen binding proteins can also bind to
the same epitope as one of the herein exemplified antigen binding
proteins, or an overlapping epitope. Antigen binding proteins and
fragments that compete with or bind to the same epitope as the
exemplified antigen binding proteins are expected to show similar
functional properties. The exemplified antigen binding proteins and
fragments include those described above, including those with the
heavy and light chains, variable region domains and CDRs included
in TABLE 2 And/or FIGS. 2-3 and 15. Thus, as a specific example,
the antigen binding proteins that are provided include those that
compete with an antibody or antigen binding protein having:
[0371] (a) all 6 of the CDRs listed for an antibody listed in FIGS.
2-3 and 15;
[0372] (b) a VH and a VL listed for an antibody listed in Table 2;
or
[0373] (c) two light chains and two heavy chains as specified for
an antibody listed in Table 2.
Certain Therapeutic Uses and Pharmaceutical Compositions
[0374] In certain instances, PCSK9 activity correlates with a
number of human disease states. For example, in certain instances,
too much or too little PCSK9 activity correlates with certain
conditions, such as hypercholesterolemia. Therefore, in certain
instances, modulating PCSK9 activity can be therapeutically useful.
In certain embodiments, a neutralizing antigen binding protein to
PCSK9 is used to modulate at least one PCSK9 activity (e.g.,
binding to LDLR). Such methods can treat and/or prevent and/or
reduce the risk of disorders that relate to elevated serum
cholesterol levels or in which elevated cholesterol levels are
relevant.
[0375] As will be appreciated by one of skill in the art, in light
of the present disclosure, disorders that relate to, involve, or
can be influenced by varied cholesterol, LDL, or LDLR levels can be
addressed by various embodiments of the antigen binding proteins.
In some embodiments, a "cholesterol related disorder" (which
includes "serum cholesterol related disorders") 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
ABP, 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. ABP
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 some embodiments, the
ABP is 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 some
embodiments, the ABP and methods can be used to reduce the risk of
recurrent cardiovascular events.
[0376] As will be appreciated by one of skill in the art, diseases
or disorders that are generally addressable (either treatable or
preventable) through the use of statins can also benefit from the
application of the instant antigen binding proteins. In addition,
in some embodiments, disorders or disease that can benefit from the
prevention of cholesterol synthesis or increased LDLR expression
can also be treated by various embodiments of the antigen binding
proteins. In addition, as will be appreciated by one of skill in
the art, the use of the anti-PCSK9 antibodies can be especially
useful in the treatment of Diabetes. Not only is Diabetes a risk
factor for coronary heart disease, but insulin increases the
expression of PCSK9. That is, people with Diabetes have elevated
plasma lipid levels (which can be related to high PCSK9 levels) and
can benefit from lowering those levels. This is generally discussed
in more detail in Costet et al. ("Hepatic PCSK9 Expression is
Regulated by Nutritional Status via Insulin and Sterol Regulatory
Element-binding Protein 1C", J. Biol. Chem., 281: 6211-6218, 2006),
the entirety of which is incorporated herein by reference.
[0377] In some embodiments, the antigen binding protein is
administered to those who have diabetes mellitus, abdominal aortic
aneurysm, atherosclerosis and/or peripheral vascular disease in
order to decrease their serum cholesterol levels to a safer range.
In some embodiments, the antigen binding protein is administered to
patients at risk of developing any of the herein described
disorders. In some embodiments, the ABPs are administered to
subjects that smoke, have hypertension or a familial history of
early heart attacks.
[0378] In some embodiments, a subject is administered an ABP if
they are at a moderate risk or higher on the 2004 NCEP treatment
goals. In some embodiments, the ABP is administered to a subject if
the subject's LDL cholesterol level is greater than 160 mg/dl. In
some embodiments, the ABP is administered if the subjects LDL
cholesterol level is greater than 130 (and they have a moderate or
moderately high risk according to the 2004 NCEP treatment goals).
In some embodiments, the ABP is administered if the subjects LDL
cholesterol level is greater than 100 (and they have a high or very
high risk according to the 2004 NCEP treatment goals).
[0379] A physician will be able to select an appropriate treatment
indications and target lipid levels depending on the individual
profile of a particular patient. One well-accepted standard for
guiding treatment of hyperlipidemia is the Third Report of the
National Cholesterol Education Program (NCEP) Expert Panel on
Detection, Evaluation, and Treatment of the High Blood Cholesterol
in Adults (Adult Treatment Panel III) Final Report, National
Institutes of Health, NIH Publication No. 02-5215 (2002), the
printed publication of which is hereby incorporated by reference in
its entirety.
[0380] In some embodiments, antigen binding proteins to PCSK9 are
used to decrease the amount of PCSK9 activity from an abnormally
high level or even a normal level. In some embodiments, antigen
binding proteins to PCSK9 are used to treat or prevent
hypercholesterolemia and/or in the preparation of medicaments
therefore and/or for other cholesterol related disorders (such as
those noted herein). In certain embodiments, an antigen binding
protein to PCSK9 is used to treat or prevent conditions such as
hypercholesterolemia in which PCSK9 activity is normal. In such
conditions, for example, reduction of PCSK9 activity to below
normal can provide a therapeutic effect.
[0381] In some embodiments, more than one antigen binding protein
to PCSK9 is used to modulate PCSK9 activity.
[0382] In certain embodiments, methods are provided of treating a
cholesterol related disorder, such as hypercholesterolemia
comprising administering a therapeutically effective amount of one
or more antigen binding proteins to PCSK9 and another therapeutic
agent.
[0383] In certain embodiments, an antigen binding protein to PCSK9
is administered alone. In certain embodiments, an antigen binding
protein to PCSK9 is administered prior to the administration of at
least one other therapeutic agent. In certain embodiments, an
antigen binding protein to PCSK9 is administered concurrent with
the administration of at least one other therapeutic agent. In
certain embodiments, an antigen binding protein to PCSK9 is
administered subsequent to the administration of at least one other
therapeutic agent. In other embodiments, an antigen binding protein
to PCSK9 is administered prior to the administration of at least
one other therapeutic agent. Therapeutic agents (apart from the
antigen binding protein), include, but are not limited to, at least
one other cholesterol-lowering (serum and/or total body
cholesterol) agent or an agent. In some embodiments, the agent
increases the expression of LDLR, have been observed to increase
serum HDL levels, lower LDL levels or lower triglyceride levels.
Exemplary agents include, but are not limited to, statins
(atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,
pitavastatin, pravastatin, rosuvastatin, simvastatin), Nicotinic
acid (Niacin) (NIACOR, NIASPAN (slow release niacin), SLO-NIACIN
(slow release niacin)), Fibric acid (LOPID (Gemfibrozil), TRICOR
(fenofibrate), Bile acid sequestrants (QUESTRAN (cholestyramine),
colesevelam (WELCHOL), COLESTID (colestipol)), Cholesterol
absorption inhibitors (ZETIA (ezetimibe)), Combining nicotinic acid
with statin (ADVICOR (LOVASTATIN and NIASPAN), Combining a statin
with an absorption inhibitor (VYTORIN (ZOCOR and ZETIA) and/or
lipid modifying agents. In some embodiments, the ABP is combined
with PPAR gamma agonsits, PPAR alpha/gamma agonists, squalene
synthase inhibitors, CETP inhibitors, anti-hypertensives,
anti-diabetic agents (such as sulphonyl ureas, insulin, GLP-1
analogs, DDPIV inhibitors), ApoB modulators, MTP inhibitoris and/or
arteriosclerosis obliterans treatments. In some embodiments, the
ABP is combined with an agent that increases the level of LDLR
protein in a subject, such as statins, certain cytokines like
oncostatin M, estrogen, and/or certain herbal ingredients such as
berberine. In some embodiments, the ABP is combined with an agent
that increases serum cholesterol levels in a subject (such as
certain anti-psycotic agents, certain HIV protease inhibitors,
dietary factors such as high fructose, sucrose, cholesterol or
certain fatty acids and certain nuclear receptor agonists and
antagonists for RXR, RAR, LXR, FXR). In some embodiments, the ABP
is combined with an agent that increases the level of PCSK9 in a
subject, such as statins and/or insulin. The combination of the two
can allow for the undesirable side-effects of other agents to be
mitigated by the ABP. As will be appreciated by one of skill in the
art, in some embodiments, the ABP is combined with the other
agent/compound. In some embodiments, the ABP and other agent are
administered concurrently. In some embodiments, the ABP and other
agent are not administered simultaneously, with the ABP being
administered before or after the agent is administered. In some
embodiments, the subject receives both the ABP and the other agent
(that increases the level of LDLR) during a same period of
prevention, occurrence of a disorder, and/or period of
treatment.
[0384] Pharmaceutical compositions of the invention can be
administered in combination therapy, i.e., combined with other
agents. In certain embodiments, the combination therapy comprises
an antigen binding protein capable of binding PCSK9, in combination
with at least one anti-cholesterol agent. Agents include, but are
not limited to, in vitro synthetically prepared chemical
compositions, antibodies, antigen binding regions, and combinations
and conjugates thereof. In certain embodiments, an agent can act as
an agonist, antagonist, allosteric modulator, or toxin. In certain
embodiments, an agent can act to inhibit or stimulate its target
(e.g., receptor or enzyme activation or inhibition), and thereby
promote increased expression of LDLR or decrease serum cholesterol
levels.
[0385] In certain embodiments, an antigen binding protein to PCSK9
can be administered prior to, concurrent with, and subsequent to
treatment with a cholesterol-lowering (serum and/or total
cholesterol) agent. In certain embodiments, an antigen binding
protein to PCSK9 can be administered prophylactically to prevent or
mitigate the onset of hypercholesterolemia, heart disease,
diabetes, and/or any of the cholesterol related disorder. In
certain embodiments, an antigen binding protein to PCSK9 can be
administered for the treatment of an existing hypercholesterolemia
condition. In some embodiments, the ABP delays the onset of the
disorder and/or symptoms associated with the disorder. In some
embodiments, the ABP is provided to a subject lacking any symptoms
of any one of the cholesterol related disorders or a subset
thereof.
[0386] In certain embodiments, an antigen binding protein to PCSK9
is used with particular therapeutic agents to treat various
cholesterol related disorders, such as hypercholesterolemia. In
certain embodiments, in view of the condition and the desired level
of treatment, two, three, or more agents can be administered. In
certain embodiments, such agents can be provided together by
inclusion in the same formulation. In certain embodiments, such
agent(s) and an antigen binding protein to PCSK9 can be provided
together by inclusion in the same formulation. In certain
embodiments, such agents can be formulated separately and provided
together by inclusion in a treatment kit. In certain embodiments,
such agents and an antigen binding protein to PCSK9 can be
formulated separately and provided together by inclusion in a
treatment kit. In certain embodiments, such agents can be provided
separately. In certain embodiments, when administered by gene
therapy, the genes encoding protein agents and/or an antigen
binding protein to PCSK9 can be included in the same vector. In
certain embodiments, the genes encoding protein agents and/or an
antigen binding protein to PCSK9 can be under the control of the
same promoter region. In certain embodiments, the genes encoding
protein agents and/or an antigen binding protein to PCSK9 can be in
separate vectors.
[0387] In certain embodiments, the invention provides for
pharmaceutical compositions comprising an antigen binding protein
to PCSK9 together with a pharmaceutically acceptable diluent,
carrier, solubilizer, emulsifier, preservative and/or adjuvant.
[0388] In certain embodiments, the invention provides for
pharmaceutical compositions comprising an antigen binding protein
to PCSK9 and a therapeutically effective amount of at least one
additional therapeutic agent, together with a pharmaceutically
acceptable diluent, carrier, solubilizer, emulsifier, preservative
and/or adjuvant.
[0389] In certain embodiments, an antigen binding protein to PCSK9
can be used with at least one therapeutic agent for inflammation.
In certain embodiments, an antigen binding protein to PCSK9 can be
used with at least one therapeutic agent for an immune disorder.
Exemplary therapeutic agents for inflammation and immune disorders
include, but are not limited to cyclooxygenase type 1 (COX-1) and
cyclooxygenase type 2 (COX-2) inhibitors small molecule modulators
of 38 kDa mitogen-activated protein kinase (p38-MAPK); small
molecule modulators of intracellular molecules involved in
inflammation pathways, wherein such intracellular molecules
include, but are not limited to, jnk, IKK, NF-.kappa.B, ZAP70, and
lck. Certain exemplary therapeutic agents for inflammation are
described, e.g., in C. A. Dinarello & L. L. Moldawer
Proinflammatory and Anti-Inflammatory Cytokines in Rheumatoid
Arthritis: A Primer for Clinicians Third Edition (2001) Amgen Inc.
Thousand Oaks, Calif.
[0390] In certain embodiments, pharmaceutical compositions will
include more than one different antigen binding protein to PCSK9.
In certain embodiments, pharmaceutical compositions will include
more than one antigen binding protein to PCSK9 wherein the antigen
binding proteins to PCSK9 bind more than one epitope. In some
embodiments, the various antigen binding proteins will not compete
with one another for binding to PCSK9. In some embodiments, any of
the antigen binding proteins depicted in Table 2 and FIGS. 2 and/or
3 can be combined together in a pharmaceutical composition.
[0391] In certain embodiments, acceptable formulation materials
preferably are nontoxic to recipients at the dosages and
concentrations employed. In some embodiments, the formulation
material(s) are for s.c. and/or I.V. administration. In certain
embodiments, the pharmaceutical composition can contain formulation
materials for modifying, maintaining or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or
penetration of the composition. In certain embodiments, suitable
formulation materials include, but are not limited to, amino acids
(such as glycine, glutamine, asparagine, arginine or lysine);
antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite
or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate,
Tris-HCl, citrates, phosphates or other organic acids); bulking
agents (such as mannitol or glycine); chelating agents (such as
ethylenediamine tetraacetic acid (EDTA)); complexing agents (such
as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, mannose or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences,
18.sup.th Edition, A. R. Gennaro, ed., Mack Publishing Company
(1995). In some embodiments, the formulation comprises PBS; 20 mM
NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9%
Sucrose.
[0392] In certain embodiments, an antigen binding protein to PCSK9
and/or a therapeutic molecule is linked to a half-life extending
vehicle known in the art. Such vehicles include, but are not
limited to, polyethylene glycol, glycogen (e.g., glycosylation of
the ABP), and dextran. Such vehicles are described, e.g., in U.S.
application Ser. No. 09/428,082, now U.S. Pat. No. 6,660,843 and
published PCT Application No. WO 99/25044, which are hereby
incorporated by reference for any purpose.
[0393] In certain embodiments, the optimal pharmaceutical
composition will be determined by one skilled in the art depending
upon, for example, the intended route of administration, delivery
format and desired dosage. See, for example, Remington's
Pharmaceutical Sciences, supra. In certain embodiments, such
compositions may influence the physical state, stability, rate of
in vivo release and rate of in vivo clearance of the antibodies of
the invention.
[0394] In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition can be either aqueous or non-aqueous in
nature. For example, in certain embodiments, a suitable vehicle or
carrier can be water for injection, physiological saline solution
or artificial cerebrospinal fluid, possibly supplemented with other
materials common in compositions for parenteral administration. In
some embodiments, the saline comprises isotonic phosphate-buffered
saline. In certain embodiments, neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. In certain
embodiments, pharmaceutical compositions comprise Tris buffer of
about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can
further include sorbitol or a suitable substitute therefore. In
certain embodiments, a composition comprising an antigen binding
protein to PCSK9, with or without at least one additional
therapeutic agents, can be prepared for storage by mixing the
selected composition having the desired degree of purity with
optional formulation agents (Remington's Pharmaceutical Sciences,
supra) in the form of a lyophilized cake or an aqueous solution.
Further, in certain embodiments, a composition comprising an
antigen binding protein to PCSK9, with or without at least one
additional therapeutic agents, can be formulated as a lyophilizate
using appropriate excipients such as sucrose.
[0395] In certain embodiments, the pharmaceutical composition can
be selected for parenteral delivery. In certain embodiments, the
compositions can be selected for inhalation or for delivery through
the digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the ability of
one skilled in the art.
[0396] In certain embodiments, the formulation components are
present in concentrations that are acceptable to the site of
administration. In certain embodiments, buffers are used to
maintain the composition at physiological pH or at a slightly lower
pH, typically within a pH range of from about 5 to about 8.
[0397] In certain embodiments, when parenteral administration is
contemplated, a therapeutic composition can be in the form of a
pyrogen-free, parenterally acceptable aqueous solution comprising a
desired antigen binding protein to PCSK9, with or without
additional therapeutic agents, in a pharmaceutically acceptable
vehicle. In certain embodiments, a vehicle for parenteral injection
is sterile distilled water in which an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agent,
is formulated as a sterile, isotonic solution, properly preserved.
In certain embodiments, the preparation can involve the formulation
of the desired molecule with an agent, such as injectable
microspheres, bio-erodible particles, polymeric compounds (such as
polylactic acid or polyglycolic acid), beads or liposomes, that can
provide for the controlled or sustained release of the product
which can then be delivered via a depot injection. In certain
embodiments, hyaluronic acid can also be used, and can have the
effect of promoting sustained duration in the circulation. In
certain embodiments, implantable drug delivery devices can be used
to introduce the desired molecule.
[0398] In certain embodiments, a pharmaceutical composition can be
formulated for inhalation. In certain embodiments, an antigen
binding protein to PCSK9, with or without at least one additional
therapeutic agent, can be formulated as a dry powder for
inhalation. In certain embodiments, an inhalation solution
comprising an antigen binding protein to PCSK9, with or without at
least one additional therapeutic agent, can be formulated with a
propellant for aerosol delivery. In certain embodiments, solutions
can be nebulized. Pulmonary administration is further described in
PCT application no. PCT/US94/001875, which describes pulmonary
delivery of chemically modified proteins.
[0399] In certain embodiments, it is contemplated that formulations
can be administered orally. In certain embodiments, an antigen
binding protein to PCSK9, with or without at least one additional
therapeutic agents, that is administered in this fashion can be
formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. In
certain embodiments, a capsule can be designed to release the
active portion of the formulation at the point in the
gastrointestinal tract when bioavailability is maximized and
pre-systemic degradation is minimized. In certain embodiments, at
least one additional agent can be included to facilitate absorption
of an antigen binding protein to PCSK9 and/or any additional
therapeutic agents. In certain embodiments, diluents, flavorings,
low melting point waxes, vegetable oils, lubricants, suspending
agents, tablet disintegrating agents, and binders can also be
employed.
[0400] In certain embodiments, a pharmaceutical composition can
involve an effective quantity of an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agents,
in a mixture with non-toxic excipients which are suitable for the
manufacture of tablets. In certain embodiments, by dissolving the
tablets in sterile water, or another appropriate vehicle, solutions
can be prepared in unit-dose form. In certain embodiments, suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0401] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving antigen
binding proteins to PCSK9, with or without at least one additional
therapeutic agent(s), in sustained- or controlled-delivery
formulations. In certain embodiments, techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art. See
for example, PCT Application No. PCT/US93/00829 which describes the
controlled release of porous polymeric microparticles for the
delivery of pharmaceutical compositions. In certain embodiments,
sustained-release preparations can include semipermeable polymer
matrices in the form of shaped articles, e.g. films, or
microcapsules. Sustained release matrices can include polyesters,
hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman
et al., Biopolymers, 22:547-556 (1983)), poly
(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)),
ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain
embodiments, sustained release compositions can also include
liposomes, which can be prepared by any of several methods known in
the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA,
82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
[0402] The pharmaceutical composition to be used for in vivo
administration typically is sterile. In certain embodiments, this
can be accomplished by filtration through sterile filtration
membranes. In certain embodiments, where the composition is
lyophilized, sterilization using this method can be conducted
either prior to or following lyophilization and reconstitution. In
certain embodiments, the composition for parenteral administration
can be stored in lyophilized form or in a solution. In certain
embodiments, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0403] In certain embodiments, once the pharmaceutical composition
has been formulated, it can be stored in sterile vials as a
solution, suspension, gel, emulsion, solid, or as a dehydrated or
lyophilized powder. In certain embodiments, such formulations can
be stored either in a ready-to-use form or in a form (e.g.,
lyophilized) that is reconstituted prior to administration.
[0404] In certain embodiments, kits are provided for producing a
single-dose administration unit. In certain embodiments, the kit
can contain both a first container having a dried protein and a
second container having an aqueous formulation. In certain
embodiments, kits containing single and multi-chambered pre-filled
syringes (e.g., liquid syringes and lyosyringes) are included.
[0405] In certain embodiments, the effective amount of a
pharmaceutical composition comprising an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agent,
to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment,
according to certain embodiments, will thus vary depending, in
part, upon the molecule delivered, the indication for which an
antigen binding protein to PCSK9, with or without at least one
additional therapeutic agent, is being used, the route of
administration, and the size (body weight, body surface or organ
size) and/or condition (the age and general health) of the patient.
In certain embodiments, the clinician can titer the dosage and
modify the route of administration to obtain the optimal
therapeutic effect. In certain embodiments, a typical dosage can
range from about 0.1 .mu.g/kg to up to about 100 mg/kg or more,
depending on the factors mentioned above. In certain embodiments,
the dosage can range from 0.1 .mu.g/kg up to about 100 mg/kg; or 1
.mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg up to about 100
mg/kg.
[0406] In certain embodiments, the frequency of dosing will take
into account the pharmacokinetic parameters of an antigen binding
protein to PCSK9 and/or any additional therapeutic agents in the
formulation used. In certain embodiments, a clinician will
administer the composition until a dosage is reached that achieves
the desired effect. In certain embodiments, the composition can
therefore be administered as a single dose, or as two or more doses
(which may or may not contain the same amount of the desired
molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. In certain embodiments, appropriate dosages can be
ascertained through use of appropriate dose-response data. In some
embodiments, the amount and frequency of administration can take
into account the desired cholesterol level (serum and/or total) to
be obtained and the subject's present cholesterol level, LDL level,
and/or LDLR levels, all of which can be obtained by methods that
are well known to those of skill in the art.
[0407] In certain embodiments, the route of administration of the
pharmaceutical composition is in accord with known methods, e.g.
orally, through injection by intravenous, intraperitoneal,
intracerebral (intra-parenchymal), intracerebroventricular,
intramuscular, subcutaneously, intra-ocular, intraarterial,
intraportal, or intralesional routes; by sustained release systems
or by implantation devices. In certain embodiments, the
compositions can be administered by bolus injection or continuously
by infusion, or by implantation device.
[0408] In certain embodiments, the composition can be administered
locally via implantation of a membrane, sponge or another
appropriate material onto which the desired molecule has been
absorbed or encapsulated. In certain embodiments, where an
implantation device is used, the device can be implanted into any
suitable tissue or organ, and delivery of the desired molecule can
be via diffusion, timed-release bolus, or continuous
administration.
[0409] In certain embodiments, it can be desirable to use a
pharmaceutical composition comprising an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agent,
in an ex vivo manner. In such instances, cells, tissues and/or
organs that have been removed from the patient are exposed to a
pharmaceutical composition comprising an antigen binding protein to
PCSK9, with or without at least one additional therapeutic agent,
after which the cells, tissues and/or organs are subsequently
implanted back into the patient.
[0410] In certain embodiments, an antigen binding protein to PCSK9
and/or any additional therapeutic agents can be delivered by
implanting certain cells that have been genetically engineered,
using methods such as those described herein, to express and
secrete the polypeptides. In certain embodiments, such cells can be
animal or human cells, and can be autologous, heterologous, or
xenogeneic. In certain embodiments, the cells can be immortalized.
In certain embodiments, in order to decrease the chance of an
immunological response, the cells can be encapsulated to avoid
infiltration of surrounding tissues. In certain embodiments, the
encapsulation materials are typically biocompatible, semi-permeable
polymeric enclosures or membranes that allow the release of the
protein product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
[0411] Based on the ability of ABPs to significantly neutralize
PCSK9 activity (as demonstrated in the Examples below), these ABPs
will have therapeutic effects in treating and preventing symptoms
and conditions resulting from PCSK9-mediated activity, such as
hypercholesterolemia.
Diagnostic Applications
[0412] In some embodiments, the ABP is used as a diagnostic tool.
The ABP can be used to assay the amount of PCSK9 present in a
sample and/or subject. As will be appreciated by one of skill in
the art, such ABPs need not be neutralizing ABPs. In some
embodiments, the diagnostic ABP is not a neutralizing ABP. In some
embodiments, the diagnostic ABP binds to a different epitope than
the neutralizing ABP binds to. In some embodiments, the two ABPs do
not compete with one another.
[0413] In some embodiments, the ABPs disclosed herein are used or
provided in an assay kit and/or method for the detection of PCSK9
in mammalian tissues or cells in order to screen/diagnose for a
disease or disorder associated with changes in levels of PCSK9. The
kit comprises an ABP that binds PCSK9 and means for indicating the
binding of the ABP with PCSK9, if present, and optionally PCSK9
protein levels. Various means for indicating the presence of an ABP
can be used. For example, fluorophores, other molecular probes, or
enzymes can be linked to the ABP and the presence of the ABP can be
observed in a variety of ways. The method for screening for such
disorders can involve the use of the kit, or simply the use of one
of the disclosed ABPs and the determination of whether the ABP
binds to PCSK9 in a sample. As will be appreciated by one of skill
in the art, high or elevated levels of PCSK9 will result in larger
amounts of the ABP binding to PCSK9 in the sample. Thus, degree of
ABP binding can be used to determine how much PCSK9 is in a sample.
Subjects or samples with an amount of PCSK9 that is greater than a
predetermined amount (e.g., an amount or range that a person
without a PCSK9 related disorder would have) can be characterized
as having a PCSK9 mediated disorder. In some embodiments, the ABP
is administered to a subject taking a statin, in order to determine
if the statin has increased the amount of PCSK9 in the subject.
[0414] In some embodiments, the ABP is a non-neutralizing ABP and
is used to determine the amount of PCSK9 in a subject receiving an
ABP and/or statin treatment.
EXAMPLES
[0415] The following examples, including the experiments conducted
and results achieved, are provided for illustrative purposes only
and are not to be construed as limiting the present invention.
Example 1
Immunization and Titering
Generation of Anti-PCSK9 Antibodies and Hybridomas
[0416] Antibodies to the mature form of PCSK9 (depicted as the
sequence in FIG. 1A, with the pro-domain underlined), were raised
in XenoMouse.RTM. mice (Abgenix, Fremont, Calif.), which are mice
containing human immunoglobulin genes. Two groups of XenoMouse.RTM.
mice, group 1 and 2, were used to produce antibodies to PCSK9.
Group 1 included mice of the XenoMouse.RTM. strain XMG2-KL, which
produces fully human IgG2.sub..kappa. and IgG2.lamda. antibodies.
Group 1 mice were immunized with human PCSK9. PCSK9 was prepared
using standard recombinant techniques using the GenBank sequence as
reference (NM.sub.--174936). Group 2 involved mice of the
XenoMouse.RTM. strain XMG4-KL, which produce fully human
IgG4.sub..kappa. and IgG4.lamda. antibodies. Group 2 mice were also
immunized with human PCSK9.
[0417] The mice of both groups were injected with antigen eleven
times, according to the schedule in Table 3. In the initial
immunizations, each mouse was injected with a total of 10 .mu.g of
antigen delivered intraperitoneally into the abdomen. Subsequent
boosts are 5 ug doses and injection method is staggered between
intraperitoneal injections into the abdomen and sub-cutaneous
injections at the base of the tail. For intraperitoneal injections
antigen is prepared as an emulsion with TiterMax.RTM. Gold (Sigma,
Cat # T2684) and for subcutaneous injections antigen is mixed with
Alum (aluminum phosphate) and CpG oligos. In injections 2 through 8
and 10, each mouse was injected with a total of 5 .mu.g of antigen
in the adjuvant alum gel. A final injection of 5 .mu.g of antigen
per mouse is delivered in Phospho buffered saline and delivered
into 2 sites 50% IP into the abdomen and 50% SQ at the base of
tail. The immunization programs are summarized in Table 3, shown
below.
TABLE-US-00003 TABLE 3 mouse strain XMG2/kl XMG4/kl # of animals 10
10 immunogen PCSK9-V5/His PCSK9-V5/His 1st boost IP injection IP
injection 10 ug each 10 ug each Titermax Gold Titermax Gold 2nd
boost tail injection tail injection 5 ug each 5 ug each Alum/CpG
ODN Alum/CpG ODN 3rd boost IP injection IP injection 5 ug each 5 ug
each Titermax Gold Titermax Gold 4th boost tail injection tail
injection 5 ug each 5 ug each Alum/CpG ODN Alum/CpG ODN 5th boost
IP injection IP injection 5 ug each 5 ug each Titermax Gold
Titermax Gold 6th boost tail injection tail injection 5 ug each 5
ug each Alum/CpG ODN Alum/CpG ODN 7th boost IP injection IP
injection 5 ug each 5 ug each Titermax Gold Titermax Gold 8th boost
tail injection tail injection 5 ug each 5 ug each Alum/CpG ODN
Alum/CpG ODN bleed 9th boost IP injection IP injection 5 ug each 5
ug each Titermax Gold Titermax Gold 10th boost tail injection tail
injection 5 ug each 5 ug each Alum/CpG ODN Alum/CpG ODN 11th boost
BIP BIP 5 ug each 5 ug each PBS PBS harvest
[0418] The protocol used to titer the XenoMouse animals was as
follows: Costar 3368 medium binding plates were coated with
neutravadin @ 8 ug/ml (50 ul/well) and incubated at 4.degree. C. in
1.times.PBS/0.05% azide overnight. They were washed using TiterTek
3-cycle wash with RO water. Plates were blocked using 250 ul of
1.times.PBS/1% milk and incubated for at least 30 minutes at RT.
Block was washed off using TiterTek 3-cycle wash with RO water. One
then captured b-human PCSK9 @ 2 ug/ml in 1.times.PBS/1% milk/10 mM
Ca2+ (assay diluent) 50 ul/well and incubated for 1 hr at RT. One
then washed using TiterTek 3-cycle wash with RO water. For the
primary antibody, sera was titrated 1:3 in duplicate from 1:100.
This was done in assay diluent 50 ul/well and incubated for 1 hr at
RT. One then washed using TiterTek 3-cycle wash with RO water. The
secondary antibody was goat anti Human IgG Fc HRP @ 400 ng/ml in
assay diluent at 50 ul/well. This was incubated for 1 hr at RT.
This was then washed using TiterTek 3-cycle wash with RO water and
patted dry on paper towels. For the substrate, one-step TMB
solution (Neogen, Lexington, Ky.) was used (50 ul/well) and it was
allowed to develop for 30 min at RT.
[0419] The protocols followed in the ELISA assays was as follows:
For samples comprising b-PCSK9 with no V5H is tag the following
protocol was employed: Costar 3368 medium binding plates (Corning
Life Sciences) were employed. The plates were coated with
neutravadin at 8 .mu.g/ml in 1.times.PBS/0.05% Azide, (50
.mu.l/well). The plates were incubated at 4.degree. C. overnight.
The plates were then washed using a Titertek M384 plate washer
(Titertek, Huntsville, Ala.). A 3-cycle wash was performed. The
plates were blocked with 250 .mu.l of 1.times.PBS/1% milk and
incubated approximately 30 minutes at room temperature. The plates
were then washed using the M384 plate washer. A 3-cycle wash was
performed. The capture was b-hu PCSK9, without a V5 tag, and was
added at 2 .mu.g/ml in 1.times.PBS/1% milk/10 mM Ca.sup.2+ (40
.mu.l/well). The plates were then incubated for 1 hour at room
temperature. A 3-cycle wash was performed. Sera were titrated 1:3
in duplicate from 1:100, and row H was blank for sera. The
titration was done in assay diluent, at a volume of 50 .mu.l/well.
The plates were incubated for 1 hour at room temperature. Next, a
3-cycle wash was performed. Goat anti Human IgG Fc HRP at 100 ng/ml
(1:4000) in 1.times.PBS/1% milk/10 mM Ca.sup.2+ (50 .mu.l/well) was
added to the plate and was incubated 1 hour at room temperature.
The plates were washed once again, using a 3-cycle wash. The plates
were then patted dry with paper towel. Finally, 1 step TMB (Neogen,
Lexington, Ky.) (50 .mu.l/well) was added to the plate and was
quenched with 1N hydrochloric acid (50 .mu.l/well) after 30 minutes
at room temperature. OD's were read immediately at 450 nm using a
Titertek plate reader.
[0420] Positive controls to detect plate bound PCSK9 were soluble
LDL receptor (R&D Systems, Cat #2148LD/CF) and a polyclonal
rabbit anti-PCSK9 antibody (Caymen Chemical #10007185) titrated 1:3
in duplicate from 3 .mu.g/ml in assay diluent. LDLR was detected
with goat anti LDLR (R&D Systems, Cat #AF2148) and rabbit anti
goat IgGFc HRP at a concentration of 400 ng/ml; the rabbit
polyclonal was detected with goat anti-rabbit IgG Fc at a
concentration of 400 ng/ml in assay diluent. Negative control was
naive XMG2-KL and XMG4-KL sera titrated 1:3 in duplicate from 1:100
in assay diluent.
[0421] For samples comprising b-PCSK9 with a V5H is tag the
following protocol was employed: Costar 3368 medium binding plates
(Corning Life Sciences) were employed. The plates were coated with
neutravadin at 8 .mu.g/ml in 1.times.PBS/0.05% Azide, (50
.mu.l/well). The plates were incubated at 4.degree. C. overnight.
The plates were then washed using a Titertek M384 plate washer
(Titertek, Huntsville, Ala.). A 3-cycle wash was performed. The
plates were blocked with 250 .mu.l of 1.times.PBS/1% milk and
incubated approximately 30 minutes at room temperature. The plates
were then washed using the M384 plate washer. A 3-cycle wash was
performed. The capture was b-hu PCSK9, with a V5 tag, and was added
at 2 .mu.g/ml in 1.times.PBS/1% milk/10 mM Ca.sup.2+ (40
.mu.l/well). The plates were then incubated for 1 hour at room
temperature. A 3-cycle wash was performed. Sera were titrated 1:3
in duplicate from 1:100, and row H was blank for sera. The
titration was done in assay diluent, at a volume of 50 .mu.l/well.
The plates were incubated for 1 hour at room temperature. Next, the
plates were washed using the M384 plate washer operated using a
3-cycle wash. Goat anti Human IgG Fc HRP at 400 ng/ml in
1.times.PBS/1% milk/10 mM Ca.sup.2+ was added at 50 .mu.l/well to
the plate and the plate was incubated 1 hour at room temperature.
The plates were washed once again, using a 3-cycle wash. The plates
were then patted dry with paper towel. Finally, 1 step TMB (Neogen,
Lexington, Ky.) (50 .mu.l/well) was added to the plate and the
plate was quenched with 1N hydrochloric acid (50 .mu.l/well) after
30 minutes at room temperature. OD's were read immediately at 450
nm using a Titertek plate reader.
[0422] Positive control was LDLR, rabbit anti-PCSK9 titrated 1:3 in
duplicate from 3 .mu.g/ml in assay diluent. LDLR detect with goat
anti-LDLR (R&D Systems, Cat #AF2148) and rabbit anti-goat IgG
Fc HRP at a concentration of 400 ng/ml; rabbit poly detected with
goat anti-rabbit IgG Fc at a concentration of 400 ng/ml in assay
diluent. Human anti-His 1.2, 3 and anti-V5 1.7.1 titrated 1:3 in
duplicate from 1 .mu.g/ml in assay diluent; both detected with goat
anti-human IgG Fc HRP at a concentration of 400 ng/ml in assay
diluent. Negative control was naive XMG2-KL and XMG4-KL sera
titrated 1:3 in duplicate from 1:100 in assay diluent.
[0423] Titers of the antibody against human PCSK9 were tested by
ELISA assay for mice immunized with soluble antigen as described.
Table 4 summarizes the ELISA data and indicates that there were
some mice which appeared to be specific for PCSK9. See, e.g., Table
4. Therefore, at the end of the immunization program, 10 mice (in
bold in Table 4) were selected for harvest, and splenocytes and
lymphocytes were isolated from the spleens and lymph nodes
respectively, as described herein.
TABLE-US-00004 TABLE 4 Summary of ELISA Results Titer Titer Animal
b-hu PCSK9 b-hu PCSK9 @ ID (V5His) @ 2 ug/ml 2 ug/ml Group 1 -
P175807 >72900 @ OD 2.2 68359 IgG2k/l P175808 >72900 @ OD 2.3
>72900 @ OD 2.5 P175818 >72900 @ OD 3.2 >72900 @ OD 3.0
P175819 >72900 @ OD 3.4 >72900 @ OD 3.2 P175820 >72900 @
OD 2.4 >72900 @ OD 2.5 P175821 >72900 @ OD 3.4 >72900 @ OD
3.0 P175830 >72900 @ OD 2.6 >72900 @ OD 2.5 P175831 >72900
@ OD 3.1 >72900 @ OD 3.1 P175832 >72900 @ OD 3.8 >72900 @
OD 3.6 P175833 >72900 @ OD 2.6 >72900 @ OD 2.3 Group 2 -
P174501 19369 17109 IgG4k/l P174503 31616 23548 P174508 48472 30996
P174509 23380 21628 P174510 15120 9673 P175773 19407 15973 P175774
54580 44424 P175775 60713 55667 P175776 30871 22899 P175777 16068
12532 Naive <100 @ OD 0.54 <100 @ OD 0.48 G2 Naive <100 @
OD 1.57 <100 @ OD 1.32 G4
Example 2
Recovery of Lymphocytes, B-Cell Isolations, Fusions and Generation
of Hybridomas
[0424] This example outlines how the immune cells were recovered
and the hybridomas were generated. Selected immunized mice were
sacrificed by cervical dislocation and the draining lymph nodes
were harvested and pooled from each cohort. The B cells were
dissociated from lymphoid tissue by grinding in DMEM to release the
cells from the tissues, and the cells were suspended in DMEM. The
cells were counted, and 0.9 ml DMEM per 100 million lymphocytes was
added to the cell pellet to resuspend the cells gently but
completely.
[0425] Lymphocytes were mixed with nonsecretory myeloma
P3X63Ag8.653 cells purchased from ATCC, cat.# CRL 1580 (Kearney et
al., (1979) J. Immunol. 123, 1548-1550) at a ratio of 1:4. The cell
mixture was gently pelleted by centrifugation at 400.times.g 4 min.
After decanting of the supernatant, the cells were gently mixed
using a 1 ml pipette. Preheated PEG/DMSO solution from Sigma (cat#
P7306) (1 ml per million of B-cells) was slowly added with gentle
agitation over 1 min followed by 1 min of mixing. Preheated IDMEM
(2 ml per million of B cells) (DMEM without glutamine, L-glutamine,
pen/strep, MEM non-essential amino acids (all from Invitrogen), was
then added over 2 minutes with gentle agitation. Finally preheated
IDMEM (8 ml per 10.sup.6 B-cells) was added over 3 minutes.
[0426] The fused cells were spun down 400.times.g 6 min and
resuspended in 20 ml selection media (DMEM (Invitrogen), 15 FBS
(Hyclone), supplemented with L-glutamine, pen/strep, MEM
Non-essential amino acids, Sodium Pyruvate, 2-Mercaptoethanol (all
from Invitrogen), HA-Azaserine Hypoxanthine and OPI (oxaloacetate,
pyruvate, bovine insulin) (both from Sigma) and IL-6 (Boehringer
Mannheim)) per million B-cells. Cells were incubated for 20-30 min
at 37 C and then resuspended in 200 ml selection media and cultured
for 3-4 days in T175 flask prior to 96 well plating. Thus,
hybridomas that produced antigen binding proteins to PCSK9 were
produced.
Example 3
Selection of PCSK9 Antibodies
[0427] The present example outlines how the various PCSK9 antigen
binding proteins were characterized and selected. The binding of
secreted antibodies (produced from the hybridomas produced in
Examples 1 and 2) to PCSK9 was assessed. Selection of antibodies
was based on binding data and inhibition of PCSK9 binding to LDLR
and affinity. Binding to soluble PCSK9 was analyzed by ELISA, as
described below. BIAcore.RTM. (surface plasmon resonance) was used
to quantify binding affinity.
Primary Screen
[0428] A primary screen for antibodies which bind to wild-type
PCSK9 was performed. The primary screen was performed on two
harvests. The primary screen comprised an ELISA assay and was
performed using the following protocol:
[0429] Costar 3702 medium binding 384 well plates (Corning Life
Sciences) were employed. The plates were coated with neutravadin at
a concentration of 4 .mu.g/ml in 1.times.PBS/0.05% Azide, at a
volume of 40 .mu.l/well. The plates were incubated at 4.degree. C.
overnight. The plates were then washed using a Titertek plate
washer (Titertek, Huntsville, Ala.). A 3-cycle wash was performed.
The plates were blocked with 90 .mu.l of 1.times.PBS/1% milk and
incubated approximately 30 minutes at room temperature. The plates
were then washed. Again, a 3-cycle wash was performed. The capture
sample was biotinylated-PCSK9, without a V5 tag, and was added at
0.9 .mu.g/ml in 1.times.PBS/1% milk/10 mM Ca.sup.2+ at a volume of
40 .mu.l/well. The plates were then incubated for 1 hour at room
temperature. Next, the plates were washed using the Titertek plate
washer operated using a 3-cycle wash. 10 .mu.l of supernatant was
transferred into 40 .mu.l of 1.times.PBS/1% milk/10 mM Ca.sup.2+
and incubated 1.5 hours at room temperature. Again the plates were
washed using the Titertek plate washer operated using a 3-cycle
wash. 40 .mu.l/well of Goat anti-Human IgG Fc POD at a
concentration of 100 ng/ml (1:4000) in 1.times.PBS/1% milk/10 mM
Ca.sup.2+ was added to the plate and was incubated 1 hour at room
temperature. The plates were washed once again, using a 3-cycle
wash. Finally, 40 .mu.l/well of One-step TMB (Neogen, Lexington,
Ky.) was added to the plate and quenching with 40 .mu.l/well of 1N
hydrochloric acid was performed after 30 minutes at room
temperature. OD's were read immediately at 450 nm using a Titertek
plate reader.
[0430] The primary screen resulted in a total of 3104 antigen
specific hybridomas being identified from the two harvests. Based
on highest ELISA OD, 1500 hybridomas per harvest were advanced for
a total of 3000 positives.
Confirmatory Screen
[0431] The 3000 positives were then rescreened for binding to
wild-type PCSK9 to confirm stable hybridomas were established. The
screen was performed as follows: Costar 3702 medium binding 384
well plates (Corning Life Sciences) were employed. The plates were
coated with neutravadin at 3 .mu.g/ml in 1.times.PBS/0.05% Azide at
a volume of 40 Owen. The plates were incubated at 4.degree. C.
overnight. The plates were then washed using a Titertek plate
washer (Titertek, Huntsville, Ala.). A 3-cycle wash was performed.
The plates were blocked with 90 .mu.l of 1.times.PBS/1% milk and
incubated approximately 30 minutes at room temperature. The plates
were then washed using the M384 plate washer. A 3-cycle wash was
performed. The capture sample was b-PCSK9, without a V5 tag, and
was added at 0.9 .mu.g/ml in 1.times.PBS/1% milk/10 mM Ca.sup.2+ at
a volume of 40 .mu.l/well. The plates were then incubated for 1
hour at room temperature. Next, the plates were washed using a
3-cycle wash. 10 .mu.l of supernatant was transferred into 40 .mu.l
of 1.times.PBS/1% milk/10 mM Ca.sup.2+ and incubated 1.5 hours at
room temperature. Again the plates were washed using the Titertek
plate washer operated using a 3-cycle wash. 40 .mu.l/well of Goat
anti-Human IgG Fc POD at a concentration of 100 ng/ml (1:4000) in
1.times.PBS/1% milk/10 mM Ca.sup.2+ was added to the plate, and the
plate was incubated 1 hour at room temperature. The plates were
washed once again, using the Titertek plate washer operated using a
3-cycle wash. Finally, 40 .mu.l/well of One-step TMB (Neogen,
Lexington, Ky.) was added to the plate and was quenched with 40
.mu.l/well of 1N hydrochloric acid after 30 minutes at room
temperature. OD's were read immediately at 450 nm using a Titertek
plate reader. A total of 2441 positives repeated in the second
screen. These antibodies were then used in the subsequent
screenings.
Mouse Cross-Reactivity Screen
[0432] The panel of hybridomas was then screened for
cross-reactivity to mouse PCSK9 to make certain that the antibodies
could bind to both human and mouse PCSK9. The following protocol
was employed in the cross-reactivity screen: Costar 3702 medium
binding 384 well plates (Corning Life Sciences) were employed. The
plates were coated with neutravadin at 3 .mu.g/ml in
1.times.PBS/0.05% Azide at a volume of 40 .mu.l/well. The plates
were incubated at 4.degree. C. overnight. The plates were then
washed using a Titertek plate washer (Titertek, Huntsville, Ala.).
A 3-cycle wash was performed. The plates were blocked with 90 .mu.l
of 1.times.PBS/1% milk and incubated approximately 30 minutes at
room temperature. The plates were then washed using the Titertek
plate washer. A 3-cycle wash was performed. The capture sample was
biotinylated-mouse PCSK9, and was added at 1 .mu.g/ml in
1.times.PBS/1% milk/10 mM Ca.sup.2+ at a volume of 40 .mu.l/well.
The plates were then incubated for 1 hour at room temperature.
Next, the plates were washed using the Titertek plate washer
operated using a 3-cycle wash. 50 .mu.l of supernatant was
transferred to the plates and incubated 1 hour at room temperature.
Again the plates were washed using a 3-cycle wash. 40 .mu.l/well of
Goat anti-Human IgG Fc POD at a concentration of 100 ng/ml (1:4000)
in 1.times.PBS/1% milk/10 mM Ca.sup.2+ was added to the plate and
the plate was incubated 1 hour at room temperature. The plates were
washed once again, using a 3-cycle wash. Finally, 40 .mu.l/well
One-step TMB (Neogen, Lexington, Ky.) was added to the plate and
was quenched with 40 .mu.l/well of 1N hydrochloric acid after 30
minutes at room temperature. OD's were read immediately at 450 nm
using a Titertek plate reader. 579 antibodies were observed to
cross-react with mouse PCSK9. These antibodies were then used in
the subsequent screenings.
D374Y Mutant Binding Screen
[0433] The D374Y mutation in PCSK9 has been documented in the human
population (e.g., Timms K M et al, "A mutation in PCSK9 causing
autosomal-dominant hypercholesterolemia in a Utah pedigree", Hum.
Genet. 114: 349-353, 2004). In order to determine if the antibodies
were specific for the wild type or also bound to the D374Y form of
PCSK9, the samples were then screened for binding to the mutant
PCSK9 sequence comprising the mutation D374Y. The protocol for the
screen was as follows: Costar 3702 medium binding 384 well plates
(Corning Life Sciences) were employed in the screen. The plates
were coated with neutravadin at 4 .mu.g/ml in 1.times.PBS/0.05%
Azide at a volume of 40 .mu.l/well. The plates were incubated at
4.degree. C. overnight. The plates were then washed using a
Titertek plate washer (Titertek, Huntsville, Ala.). A 3-cycle wash
was performed. The plates were blocked with 90 .mu.l of
1.times.PBS/1% milk and incubated approximately 30 minutes at room
temperature. The plates were then washed using the Titertek plate
washer. A 3-cycle wash was performed. The plates were coated with
biotinylated human PCSK9 D374Y at a concentration of 1 .mu.g/ml in
1.times.PBS/1% milk/10 mM Ca.sup.2+ and incubated for 1 hour at
room temperature. The plates were then washed using a Titertek
plate washer. A 3-cycle wash was performed. Late exhaust hybridoma
culture supernatant was diluted 1:5 in PBS/milk/Ca.sup.2+ (10 ml
plus 40 ml) and incubated for 1 hour at room temperature. Next, 40
.mu.l/well of rabbit anti-human PCSK9 (Cayman Chemical) and human
anti-His 1.2.3 1:2 at 1 ug/ml in 1.times.PBS/1% milk/10 mM
Ca.sup.2+ was titrated onto the plates, which were then incubated
for 1 hour at room temperature. The plates were then washed using a
Titertek plate washer. A 3-cycle wash was performed. 40 .mu.l/well
of Goat anti-Human IgG Fc HRP at a concentration of 100 ng/ml
(1:4000) in 1.times.PBS/1% milk/10 mM Ca.sup.2+ was added to the
plate and the plate was incubated 1 hour at room temperature. 40
.mu.l/well of Goat anti-rabbit IgG Fc HRP at a concentration of 100
ng/ml (1:4000) in 1.times.PBS/1% milk/10 mM Ca.sup.2+ was added to
the plate and the plate was incubated 1 hour at room temperature.
The plates were then washed using a Titertek plate washer. A
3-cycle wash was performed. Finally, 40 .mu.l/well of One-step TMB
(Neogen, Lexington, Ky.) was added to the plate and was quenched
with 40 .mu.l/well of 1N hydrochloric acid after 30 minutes at room
temperature. OD's were read immediately at 450 nm using a Titertek
plate reader. Over 96% of the positive hits on the wild-type PCSK9
also bound mutant PCSK9.
Large Scale Receptor Ligand Blocking Screen
[0434] To screen for the antibodies that block PCSK9 binding to
LDLR an assay was developed using the D374Y PCSK9 mutant. The
mutant was used for this assay because it has a higher binding
affinity to LDLR allowing a more sensitive receptor ligand blocking
assay to be developed. The following protocol was employed in the
receptor ligand blocking screen: Costar 3702 medium binding 384
well plates (Corning Life Sciences) were employed in the screen.
The plates were coated with goat anti-LDLR (R&D Cat #AF2148) at
2 .mu.g/ml in 1.times.PBS/0.05% Azide at a volume of 40 .mu.l/well.
The plates were incubated at 4.degree. C. overnight. The plates
were then washed using a Titertek plate washer (Titertek,
Huntsville, Ala.). A 3-cycle wash was performed. The plates were
blocked with 90 .mu.l of 1.times.PBS/1% milk and incubated
approximately 30 minutes at room temperature. The plates were then
washed using the Titertek plate washer. A 3-cycle wash was
performed. The capture sample was LDLR (R&D, Cat #2148LD/CF),
and was added at 0.4 .mu.g/ml in 1.times.PBS/1% milk/10 mM
Ca.sup.2+ at a volume of 40 .mu.l/well. The plates were then
incubated for 1 hour and 10 minutes at room temperature.
Contemporaneously, 20 ng/ml of biotinylated human D374Y PCSK9 was
incubated with 15 microliters of hybridoma exhaust supernatant in
Nunc polypropylene plates and the exhaust supernatant concentration
was diluted 1:5. The plates were then pre-incubated for about 1
hour and 30 minutes at room temperature. Next, the plates were
washed using the Titertek plate washer operated using a 3-cycle
wash. 50 .mu.l/well of the pre-incubated mixture was transferred
onto the LDLR coated ELISA plates and incubated for 1 hour at room
temperature. To detect LDLR-bound b-PCSK9, 40 .mu.l/well
streptavidin HRP at 500 ng/ml in assay diluent was added to the
plates. The plates were incubated for 1 hour at room temperature.
The plates were again washed using a Titertek plate washer. A
3-cycle wash was performed. Finally, 40 .mu.l/well of One-step TMB
(Neogen, Lexington, Ky.) was added to the plate and was quenched
with 40 .mu.l/well of 1N hydrochloric acid after 30 minutes at room
temperature. OD's were read immediately at 450 nm using a Titertek
plate reader. The screen identified 384 antibodies that blocked the
interaction between PCSK9 and the LDLR well, 100 antibodies blocked
the interaction strongly (OD<0.3). These antibodies inhibited
the binding interaction of PCSK9 and LDLR greater than 90% (greater
than 90% inhibition).
Receptor Ligand Binding Assay on Blocker Subset
[0435] The receptor ligand assay was then repeated using the mutant
enzyme on the 384 member subset of neutralizers identified in the
first large scale receptor ligand inhibition assay. The same
protocol was employed in the screen of the 384 member blocker
subset assay as was done in the large scale receptor ligand
blocking screen. This repeat screen confirmed the initial screening
data.
[0436] This screen of the 384 member subset identified 85
antibodies that blocked interaction between the PCSK9 mutant enzyme
and the LDLR greater than 90%.
Receptor Ligand Binding Assay of Blockers that Bind the Wild Type
PCSK9 but not the D374Y Mutant
[0437] In the initial panel of 3000 sups there were 86 antibodies
shown to specifically bind to the wild-type PCSK9 and not to the
huPCSK9(D374Y) mutant. These 86 sups were tested for the ability to
block wild-type PCSK9 binding to the LDLR receptor. The following
protocol was employed: Costar 3702 medium binding 384 well plates
(Corning Life Sciences) were employed in the screen. The plates
were coated with anti-His 1.2.3 at 10 .mu.g/ml in 1.times.PBS/0.05%
Azide at a volume of 40 .mu.l/well. The plates were incubated at
4.degree. C. overnight. The plates were then washed using a
Titertek plate washer (Titertek, Huntsville, Ala.). A 3-cycle wash
was performed. The plates were blocked with 90 .mu.l of
1.times.PBS/1% milk and incubated approximately 30 minutes at room
temperature. The plates were then washed using the Titertek plate
washer. A 3-cycle wash was performed. LDLR (R&D Systems,
#2148LD/CF or R&D Systems, #2148LD) was added at 5 .mu.g/ml in
1.times.PBS/1% milk/10 mM Ca.sup.2+ at a volume of 40 .mu.l/well.
The plates were then incubated for 1 hour at room temperature.
Next, the plates were washed using the Titertek plate washer
operated using a 3-cycle wash. Contemporaneously, biotinylated
human wild-type PCSK9 was pre-incubated with hybridoma exhaust
supernatant in Nunc polypropylene plates. 22 .mu.l of hybridoma sup
was transferred into 33 .mu.l of b-PCSK9 at a concentration of 583
ng/ml in 1.times.PBS/1% milk/10 mM Ca2+, giving a final b-PCSK9
concentration=350 ng/ml and the exhaust supernatant at a final
dilution of 1:2.5. The plates were pre-incubated for approximately
1 hour and 30 minutes at room temperature. 50 .mu.l/well of the
preincubated mixture was transferred onto LDLR captured ELISA
plates and incubated for 1 hour at room temperature. The plates
were then washed using the Titertek plate washer. A 3-cycle wash
was performed. 40 .mu.l/well streptavidin HRP at 500 ng/ml in assay
diluent was added to the plates. The plates were incubated for 1
hour at room temperature. The plates were then washed using a
Titertek plate washer. A 3-cycle wash was performed. Finally, 40
.mu.l/well of One-step TMB (Neogen, Lexington, Ky.) was added to
the plate and was quenched with 40 .mu.l/well of 1N hydrochloric
acid after 30 minutes at room temperature. OD's were read
immediately at 450 nm using a Titertek plate reader.
Screening Results
[0438] Based on the results of the assays described, several
hybridoma lines were identified as producing antibodies with
desired interactions with PCSK9. Limiting dilution was used to
isolate a manageable number of clones from each line. The clones
were designated by hybridoma line number (e.g. 21B12) and clone
number (e.g. 21B12.1). In general, no difference among the
different clones of a particular line were detected by the
functional assays described herein. In a few cases, clones were
identified from a particular line that behaved differently in the
functional assays, for example, 25A7.1 was found not to block
PCSK9/LDLR but 25A7.3 (referred to herein as 25A7) was
neutralizing. The isolated clones were each expanded in 50-100 ml
of hybridoma media and allowed to grow to exhaustion, (i.e., less
than about 10% cell viability). The concentration and potency of
the antibodies to PCSK9 in the supernatants of those cultures were
determined by ELISA and by in vitro functional testing, as
described herein. As a result of the screening described herein,
the hybridomas with the highest titer of antibodies to PCSK9 were
identified. The selected hybridomas are shown in FIGS. 2A-3D and
Table 2.
Example 4.1
Production of Human 31H4 IgG4 Antibodies from Hybridomas
[0439] This example generally describes how one of the antigen
binding proteins was produced from a hybridoma line. The production
work used 50 ml exhaust supernatant generation followed by protein
A purification. Integra production was for scale up and was
performed later. Hybridoma line 31H4 was grown in T75 flasks in 20
ml of media (Integra Media, Table 5). When the hybridoma was nearly
confluent in the T75 flasks, it was transferred to an Integra flask
(Integra Biosciences, Integra CL1000, cat#90 005).
[0440] The Integra flask is a cell culture flask that is divided by
a membrane into two chambers, a small chamber and a large chamber.
A volume of 20-30 ml hybridoma cells at a minimum cell density of
1.times.10.sup.6 cells per ml from the 31H4 hybridoma line was
placed into the small chamber of an Integra flask in Integra media
(see Table 5 for components of Integra media). Integra media alone
(1 L) was placed in the large chambers of the Integra flasks. The
membrane separating the two chambers is permeable to small
molecular weight nutrients but is impermeable to hybridoma cells
and to antibodies produced by those cells. Thus, the hybridoma
cells and the antibodies produced by those hybridoma cells were
retained in the small chamber.
[0441] After one week, media was removed from both chambers of the
Integra flask and was replaced with fresh Integra media. The
collected media from the small chambers was separately retained.
After a second week of growth, the media from the small chamber was
again collected. The collected media from week 1 from the hybridoma
line was combined with the collected media from week 2 from the
hybridoma line. The resulting collected media sample from the
hybridoma line was spun to remove cells and debris (15 minutes at
3000 rpm) and the resulting supernatant was filtered (0.22 um).
Clarified conditioned media was loaded onto a Protein A-Sepharose
column. Optionally, the media can be first concentrated and then
loaded onto a Protein A Sepharose column. Non-specific bindings
were removed by an extensive PBS wash. Bound antibody proteins on
the Protein A column were recovered by standard acidic antibody
elution from Protein A columns (such as 50 mM Citrate, pH 3.0).
Aggregated antibody proteins in the Protein A Sepharose pool were
removed by size exclusion chromatography or binding ion exchange
chromatography on anion exchanger resin such as Q Sepharose resin.
The specific IEX conditions for the 31H4 proteins are Q-Sepharose
HP at pH 7.8-8.0. Antibody was eluted with a NaCl gradient of 10
mM-500 mM in 25 column volumes.
TABLE-US-00005 TABLE 5 Composition of Media INTEGRA MEDIA HSFM 10%
Ultra Low IgG serum 2 mmol/L L-glutamine 1% NEAA 4 g/L glucose
Example 4.2
Production of Recombinant 31H4 Human IgG2 Antibodies from
Transfected Cells
[0442] The present example outlines how 31H4 IgG2 antibodies were
produced from transfected cells. 293 cells for transient expression
and CHO cells for stable expression were transfected with plasmids
that encode 31H4 heavy and light chains. Conditioned media from
transfected cells was recovered by removing cells and cell debris.
Clarified conditioned media was loaded onto a Protein A-Sepharose
column. Optionally, the media can first be concentrated and then
loaded onto a Protein A Sepharose column. Non-specific bindings
were removed by extensive PBS wash. Bound antibody proteins on the
Protein A column were recovered by standard acidic antibody elution
from Protein A columns (such as 50 mM citrate, pH 3.0). Aggregated
antibody proteins in the Protein A Sepharose pool were removed by
size exclusion chromatography or binding ion exchange
chromatography on anion exchanger resin such as Q Sepharose resin.
The specific IEX conditions for the 31H4 proteins are Q-Sepharose
HP at pH 7.8-8.0. The antibody was eluted with a NaCl gradient of
10 mM-500 mM in 25 column volumes.
Example 5
Production of Human 21B12 IgG4 Antibodies from Hybridomas
[0443] The present example outlines how antibody 21B12 IgG4 was
produced from hybridomas. Hybridoma line 21B12 was grown in T75
flasks in media (Integra Media, Table 5). When the hybridomas were
nearly confluent in the T75 flasks, they were transferred to
Integra flasks (Integra Biosciences, Integra CL1000, cat#90
005).
[0444] The Integra flask is a cell culture flask that is divided by
a membrane into two chambers, a small chamber and a large chamber.
A volume of 20-30 ml hybridoma cells at a minimum cell density of
1.times.10.sup.6 cells per ml from the 31H4 hybridoma line was
placed into the small chamber of an Integra flask in Integra media
(see Table 5 for components of Integra media). Integra media alone
(1 L) was placed in the large chambers of the Integra flasks. The
membrane separating the two chambers is permeable to small
molecular weight nutrients but is impermeable to hybridoma cells
and to antibodies produced by those cells. Thus, the hybridoma
cells and the antibodies produced by those hybridoma cells were
retained in the small chamber. After one week, media was removed
from both chambers of the Integra flask and was replaced with fresh
Integra media. The collected media from the small chambers was
separately retained. After a second week of growth, the media from
the small chamber was again collected. The collected media from
week 1 from the hybridoma line was combined with the collected
media from week 2 from the hybridoma line. The resulting collected
media sample from the hybridoma line was spun to remove cells and
debris (15 minutes at 3000 rpm) and the resulting supernatant was
filtered (0.22 .mu.m). Clarified conditioned media were loaded onto
a Protein A Sepharose column. Optionally, the media are first
concentrated and then loaded onto a Protein A Sepharose column.
Non-specific bindings were removed by an extensive PBS wash. Bound
antibody proteins on the Protein A column were recovered by
standard acidic antibody elution from Protein A columns (such as 50
mM Citrate, pH 3.0). Aggregated antibody proteins in the Protein A
Sepharose pool were removed by size exclusion chromatography or
binding ion exchange chromatography on anion exchanger resin such
as Q Sepharose resin. The specific IEX conditions for the 21B12
proteins are Q-Sepharose HP at pH 7.8-8.0. The antibody was eluted
with a NaCl gradient of 10 mM-500 mM in 25 column volumes.
Example 6
Production of Human 21B12 IgG2 Antibodies from Transfected
Cells
[0445] The present example outlines how 21B12 IgG2 antibodies were
produced from transfected cells. Cells (293 cells for transient
expression and CHO cells for stable expression) were transfected
with plasmids that encode 21B12 heavy and light chains. Conditioned
media from hybridoma cells were recovered by removing cells and
cell debris. Clarified conditioned media were loaded onto a Protein
A-Sepharose column. Optionally, the media can first be concentrated
and then loaded onto a Protein A Sepharose column. Non-specific
bindings were removed by extensive PBS wash. Bound antibody
proteins on the Protein A column were recovered by standard acidic
antibody elution from Protein A columns (50 mM Citrate, pH 3.0).
Aggregated antibody proteins in the Protein A Sepharose pool were
removed by size exclusion chromatography or binding ion exchange
chromatography on cation exchanger resin such as SP-Sepharose
resin. The specific IEX conditions for the 21B12 proteins were
SP-Sepharose HP at pH 5.2. Antibodies were eluted with 25 column
volumes of buffer that contains a NaCl gradient of 10 mM-500 mM in
20 mM sodium acetate buffer.
Example 7
Production of Human 16F12 IgG4 Antibodies from Hybridomas
[0446] The present example outlines how antibody 16F12 IgG4 was
produced from hybridomas. Hybridoma line 16F12 was grown in T75
flasks in media (see Table 5). When the hybridomas were nearly
confluent in the T75 flasks, they were transferred to Integra
flasks (Integra Biosciences, Integra CL1000, cat#90 005).
[0447] The Integra flask is a cell culture flask that is divided by
a membrane into two chambers, a small chamber and a large chamber.
A volume of 20-30 ml Hybridoma cells at a minimum cell density of
1.times.10.sup.6 cells per ml from the 31H4 hybridoma line was
placed into the small chamber of an Integra flask in Integra media
(see Table 5 for components of Integra media). Integra media alone
(1 L) was placed in the large chambers of the Integra flasks. The
membrane separating the two chambers is permeable to small
molecular weight nutrients but is impermeable to hybridoma cells
and to antibodies produced by those cells. Thus, the hybridoma
cells and the antibodies produced by those hybridoma cells were
retained in the small chamber.
[0448] After one week, media was removed from both chambers of the
Integra flask and was replaced with fresh Integra media. The
collected media from the small chambers was separately retained.
After a second week of growth, the media from the small chamber was
again collected. The collected media from week 1 from the hybridoma
line was combined with the collected media from week 2 from the
hybridoma line. The resulting collected media sample from the
hybridoma line were spun to remove cells and debris (15 minutes at
3000 rpm) and the resulting supernatants were filtered (0.22
.mu.m). Clarified conditioned media were loaded onto a Protein A
Sepharose column. Optionally, the media can be first concentrated
and then loaded onto a Protein A Sepharose column. Non-specific
bindings were removed by extensive PBS wash. Bound antibody
proteins on the Protein A column were recovered by standard acidic
antibody elution from Protein A columns (50 mM Citrate, pH 3.0).
Aggregated antibody proteins in the Protein A Sepharose pool were
removed by size exclusion chromatography or binding ion exchange
chromatography on anion exchanger resin such as Q Sepharose resin.
The specific IEX conditions for the 16F12 proteins are Q Sepharose
HP at pH 7.8-8.0. Antibody was eluted with a NaCl gradient of 10
mM-500 mM in 25 column volumes.
Example 8
Production of Human 16F12 IgG2 Antibodies from Transfected
Cells
[0449] The present example outlines how 16F12 IgG2 antibodies were
produced from transfected cells. Cells (293 cells for transient
expression and CHO cells for stable expression) were transfected
with plasmids that encode 16F12 heavy and light chains. Conditioned
media from hybridoma cells were recovered by removing cells and
cell debris. Clarified conditioned media were loaded onto a Protein
A-Sepharose. Optionally, the media can be first concentrated and
then loaded onto a Protein A Sepharose column. Non-specific
bindings were removed by extensive PBS wash. Bound antibody
proteins on the Protein A column were recovered by standard acidic
antibody elution from Protein A columns (50 mM Citrate, pH 3.0).
Aggregated antibody proteins in the Protein A Sepharose pool were
removed by size exclusion chromatography or binding ion exchange
chromatography on cation exchanger resin such as SP Sepharose
resin. The specific IEX conditions for the 16F12 proteins are SP
Sepharose HP at pH 5.2. Antibody is eluted with 25 column volumes
of buffer that contains a NaCl gradient of 10 mM-500 mM in 20 mM
sodium acetate buffer.
Example 9
Sequence Analysis of Antibody Heavy and Light Chains
[0450] The nucleic acid and amino acid sequences for the light and
heavy chains of the above antibodies were then determined by Sanger
(dideoxy) nucleotide sequencing. Amino acid sequences were then
deduced for the nucleic acid sequences. The nucleic acid sequences
for the variable domains are depicted in FIGS. 3E-3JJ.
[0451] The cDNA sequences for the lambda light chain variable
regions of 31H4, 21B12, and 16F12 were determined and are disclosed
as SEQ ID NOs: 153, 95, and 105 respectively.
[0452] The cDNA sequences for the heavy chain variable regions of
31H4, 21B12, and 16F12 were determined and are disclosed as SEQ ID
NOs: 152, 94, and 104 respectively.
[0453] The lambda light chain constant region (SEQ ID NO: 156), and
the IgG2 and IgG4 heavy chain constant regions (SEQ ID NOs: 154 and
155) are shown in FIG. 3KK.
[0454] The polypeptide sequences predicted from each of those cDNA
sequences were determined. The predicted polypeptide sequences for
the lambda light chain variable regions of 31H4, 21B12, and 16F12
were predicted and are disclosed as SEQ ID NOs: 12, 23, and 35
respectively, the lambda light chain constant region (SEQ ID NO:
156), the heavy chain variable regions of 31H4, 21B12, and 16F12
were predicted and are disclosed as (SEQ. ID NOs. 67, 49, and 79
respectively. The IgG2 and IgG4 heavy chain constant regions (SEQ
ID NOs: 154 and 155).
[0455] The FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 divisions are shown
in FIG. 2A-3D.
[0456] Based on the sequence data, the germline genes from which
each heavy chain or light chain variable region was derived was
determined. The identity of the germline genes are indicated next
to the corresponding hybridoma line in FIGS. 2A-3D and each is
represented by a unique SEQ ID NO. FIGS. 2A-3D also depict the
determined amino acid sequences for additional antibodies that were
characterized.
Example 8
Determination of Isoelectric Points of Three Antibodies
[0457] The theoretical pIs of the antibodies based on amino acid
sequence were determined to be 7.36 for 16F12; 8.47 for 21B12; and
6.84 for 31H4.
Example 9
Characterization of Binding of Antibodies to PCSK9
[0458] Having identified a number of antibodies that bind to PCSK9,
several approaches were employed to quantify and further
characterize the nature of the binding. In one aspect of the study,
a Biacore affinity analysis was performed. In another aspect of the
study a KinExA.RTM. affinity analysis was performed. The samples
and buffers employed in these studies are presented in Table 6
below.
TABLE-US-00006 TABLE 6 [sample] [sample] sample mg/ml Buffer uM
hPCSK9 1.26 PBS 16.6 mPCSK9-8xHIS 1.44 PBS 18.9 cPCSK9-V5-6xHIS
0.22 PBS 2.9 16F12, anti-PCSK9 huIgG4 4.6 20 mM NaOAC, 31.9 pH 5.2,
50 mM NaCl 21B12, anti-PCSK9 huIgG4 3.84 10 mM NAOAC, 27.0 pH 5.2,
9% Sucrose 31H4, anti-PCSK9 huIgG4 3.3 10 mM NAOAC, 22.9 pH 5.2, 9%
Sucrose
BIAcore.RTM. Affinity Measurements
[0459] A BIAcore.RTM. (surface plasmon resonance device, Biacore,
Inc., Piscataway, N.J.) affinity analysis of the 21B12 antibodies
to PCSK9 described in this Example was performed according to the
manufacturer's instructions.
[0460] Briefly, the surface plasmon resonance experiments were
performed using Biacore 2000 optical biosensors (Biacore, GE
Healthcare, Piscataway, N.J.). Each individual anti-PCSK9 antibody
was immobilized to a research-grade CM5 biosensor chip by
amine-coupling at levels that gave a maximum analyte binding
response (Rmax) of no more than 200 resonance units (RU). The
concentration of PCSK9 protein was varied at 2 fold intervals (the
analyte) and was injected over the immobilized antibody surface (at
a flow rate of 100 .mu.l/min for 1.5 minutes). Fresh BBS-P buffer
(pH 7.4, 0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant P-20,
Biacore) supplemented with 0.01% BSA was used as binding buffer.
Binding affinities of each anti-PCSK9 antibody were measured in
separate experiments against each of the human, mouse, and
cynomolgus monkey PCSK9 proteins at pH 7.4 (the concentrations used
were 100, 50, 25, 12.5, 6.25, 3.125, and 0 nM).
[0461] In addition, the binding affinities of antibody to human
PCSK9 were also measured at pH 6.0 with the pH 6.0 BBS-P buffer (pH
6.0, 0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant P-20, Biacore)
supplemented with 0.01% BSA. The binding signal obtained was
proportional to the free PCSK9 in solution. The dissociation
equilibrium constant (K.sub.D) was obtained from nonlinear
regression analysis of the competition curves using a dual-curve
one-site homogeneous binding model (KinExA.RTM. software, Sapidyne
Instruments Inc., Boise, Id.) (n=1 for the 6.0 pH runs).
Interestingly, the antibodies appeared to display a tighter binding
affinity at the lower pH (where the Kd was 12.5, 7.3, and 29 pM for
31H4, 21B12, and 16F12 respectively).
[0462] Antibody binding kinetic parameters including k.sub.a
(association rate constant), k.sub.d (dissociation rate constant),
and K.sub.D (dissociation equilibrium constant) were determined
using the BIA evaluation 3.1 computer program (BIAcore, Inc.
Piscataway, N.J.). Lower dissociation equilibrium constants
indicate greater affinity of the antibody for PCSK9. The K.sub.D
values determined by the BIAcore.RTM. affinity analysis are
presented in Table 7.1, shown below.
TABLE-US-00007 TABLE 7.1 Antibody hPCSK9 CynoPCSK9 mPCSK9 31H4 210
pM 190 pM 6 nM 21B12 190 pM 360 pM 460 nM 16F12 470 pM 870 pM 6.4
nM
Table 7.2 depicts the k.sub.on and k.sub.off rates.
TABLE-US-00008 TABLE 7.2 -- K.sub.on (M-1 s-1) K.sub.off (s-1)
K.sub.D 31H4.1, pH 7.4 2.45e+5 5.348e-5 210 pM 31H4.1, pH 6
5.536e+6 6.936e-5 12.5 pM 21B12.1, pH 7.4 3.4918e+4 6.634e-6 190 pM
21B12.1, pH 6 2.291e+6 1.676e-5 7.3 pM 16F12.1, pH 7.4 1.064e+5
4.983e-5 470 pM 16F12.1, pH 6 2.392e+6 7.007e-5 29 pM
KinExA.RTM. Affinity Measurements
[0463] A KinExA.RTM. (Sapidyne Instruments, Inc., Boise, Id.)
affinity analysis of 16F12 and 31H4 was performed according to the
manufacturer's instructions. Briefly, Reacti-Gel.TM. (6.times.)
(Pierce) was pre-coated with one of human, V5-tagged cyno or
His-tagged mouse PCSK9 proteins and blocked with BSA. 10 or 100 pM
of either antibody 16F12 or antibody 31H4 and one of the PCSK9
proteins was then incubated with various concentrations (0.1 pM-25
nM) of PCSK9 proteins at room temperature for 8 hours before being
passed through the PCSK9-coated beads. The amount of the bead-bound
16F12 or 31H4 was quantified by fluorescently (Cy5) labeled goat
anti-human IgG (H+L) antibody (Jackson Immuno Research). The
binding signal is proportional to the concentration of free 16F12
or 31H4 at binding equilibrium. Equilibrium dissociation constant
(K.sub.D) were obtained from nonlinear regression of the two sets
of competition curves using a one-site homogeneous binding model.
The KinExA.RTM. Pro software was employed in the analysis. Binding
curves generated in this analysis are presented as FIGS. 4A-4F.
[0464] Both the 16F12 and 31H4 antibodies showed similar affinity
to human and cyno PCSK9, but approximately 10-250 fold lower
affinity to mouse PCSK9. Of the two antibodies tested using the
KinExA.RTM. system, antibody 31H4 showed higher affinity to both
human and cyno PCSK9 with 3 and 2 pM K.sub.D, respectively. 16F12
showed slightly weaker affinity at 15 pM K.sub.D to human PCSK9 and
16 pM K.sub.D to cyno PCSK9.
[0465] The results of the KinExA.RTM. affinity analysis are
summarized in Table 8.1, shown below.
TABLE-US-00009 TABLE 8.1 hPCSK9 cPCSK mPCSK K.sub.D 95% K.sub.D 95%
K.sub.D 95% Sample (pM) CI (pM) CI (pM) CI 16F12 15 11~22 16 14~19
223 106~410 31H4.1 3 1~5 2 1~3 500 400~620
[0466] In addition, a SDS PAGE was run to check the quality and
quantity of the samples and is shown in FIG. 5A. cPCSK9 showed
around 50% less on the gel and also from the active binding
concentration calculated from KinExA.RTM. assay. Therefore, the
K.sub.D of the mAbs to cPCSK9 was adjusted as 50% of the active
cPCSK9 in the present.
[0467] A BIAcore solution equilibrium binding assay was used to
measure the Kd values for ABP 21B12. 21B12.1 showed little signal
using KinExA assay, therefore, biacore solution equilibrium assay
was applied. Since no significant binding was observed on binding
of antibodies to immobilized PCSK9 surface, 21B12 antibody was
immobilized on the flow cell 4 of a CM5 chip using amine coupling
with density around 7000 RU. Flow cell 3 was used as a background
control. 0.3, 1, and 3 nM of human PCSK9 or cyno PCSK9 were mixed
with a serial dilutions of 21B12.1 antibody samples (ranged from
0.001.about.25 nM) in PBS plus 0.1 mg/ml BSA, 0.005% P20. Binding
of the free PCSK9 in the mixed solutions were measured by injecting
over the 21B12.1 antibody surface. 100% PCSK9 binding signal on
21B12.1 surface was determined in the absence of mAb in the
solution. A decreased PCSK9 binding response with increasing
concentrations of mAb indicated that PCSK9 binding to mAb in
solution, which blocked PCSK9 from binding to the immobilized
peptibody surface. Plotting the PCSK9 binding signal versus mAb
concentrations, K.sub.D was calculated from three sets of curves
(0.3, 1 and 3 nM fixed PCSK9 concentration) using a one-site
homogeneous binding model in KinExA Pro.TM. software. Although
cPCSK9 has lower protein concentration observed from KinExA assay
and SDS-gel, its concentration was not adjusted here since the
concentration of cPCSK9 was not used for calculation of K.sub.D.
The results are displayed in Table 8.2 below and in FIGS. 5B-5D.
FIG. 5B depicts the results from the solution equilibrium assay at
three different hPCSK9 concentrations for hPCSK9. FIG. 5C depicts a
similar set of results for mPCSK9. FIG. 5D depicts the results from
the above biacore capture assay.
TABLE-US-00010 TABLE 8.2 hPCSK9 cPCSK mPCSK K.sub.D 95% K.sub.D 95%
K.sub.D 95% Sample (pM) CI (pM) CI (pM) CI 21B12.1 15 9~23 11 7~16
17000 --
Example 10
Epitope Binning
[0468] Competition ELISA was used for anti-PCSK9 antibody binning.
Briefly, to determine if two antibodies belong to the same epitope
bin, one of the antibodies (mAb1) was first coated onto an ELISA
plate (NUNC) at 2 .mu.g/ml by overnight incubation. The plate was
then washed and blocked with 3% BSA. Meanwhile, 30 ng/ml of
biotinylated hPCSK9 was incubated with the second antibody (mAb2)
for 2 hours at room temperature. The mixture was applied to coated
mAb1 and incubated for 1 hour at room temperature. The ELISA plate
was then washed and incubated with Neutravidin-HRP (Pierce) at
1:5000 dilutions for 1 hour. After another wash, the plate was
incubated with TMB substrate and signal was detected at 650 nm
using a Titertek plate reader. Antibodies with the same binding
profiles were grouped together into the same epitope bin. The
results of the antibody binning studies are presented in Table
8.3.
TABLE-US-00011 TABLE 8.3 Clone Bin 21B12.2 1 31H4 3 20D10 1 25A7.1
2 25A7.3 1 23G1 1 26H5 1 31D1 1 16F12 3 28D6 3 27A6 3 31G11 3 27B2
ND 28B12 3 22E2 3 1A12.2 1 3B6 1 3C4 4 9C9 1 9H6 1 13B5 6 13H1 7
17C2 1 19H9.2 1 23B5 1 25G4 1 26E10 1 27E7 1 27H5 1 30A4 1 30B9 1
31A4 5 31B12 5
[0469] Additional examination of the epitope binning was performed
using BIAcore. Three mAbs, 16F12, 21B12 and 31H4, were immobilized
on flow cells 2, 3 and 4 with density around 8000 RU. 5 nM PCSK9
from human, mouse and cyno were injected over the mAb surfaces to
reach around 100 to 500 RU. 10 nM mAbs were then injected over the
PCSK9 surface. Binding of three mAbs to three different PCSK9
proteins over the three mAbs were then recorded.
[0470] If the two mAbs had a similar epitope on the antigen, mAb 1
will not show the binding to the antigen already bound to the mAb
2. If the two mAbs have the different epitope on the antigen, mAb1
will show the binding to the antigen bound to the mAb2. FIG. 5E
depicts these epitope binning results in graph form for three mAbs
on human PCSk9. A similar pattern was observed for mPCSK9 and
cPCSK9. As shown in the graph, 16F12 and 31H4 appear to share a
similar epitope, while 21B12 appears to have a different
epitope.
Example 11
Efficacy of 31H4 and 21B12 for Blocking D374Y PCSK9/LDLR
Binding
[0471] This example provides the IC50 values for two of the
antibodies in blocking PCSK9 D374Y's ability to bind to LDLR. Clear
384 well plates (Costar) were coated with 2 micrograms/ml of goat
anti-LDL receptor antibody (R&D Systems) diluted in buffer A
(100 mM sodium cacodylate, pH 7.4). Plates were washed thoroughly
with buffer A and then blocked for 2 hours with buffer B (1% milk
in buffer A). After washing, plates were incubated for 1.5 hours
with 0.4 micrograms/ml of LDL receptor (R&D Systems) diluted in
buffer C (buffer B supplemented with 10 mM CaCl2). Concurrent with
this incubation, 20 ng/ml of biotinylated D374Y PCSK9 was incubated
with various concentrations of the 31H4 IgG2, 31H4 IgG4, 21B12 IgG2
or 21B12 IgG4 antibody, which was diluted in buffer A, or buffer A
alone (control). The LDL receptor containing plates were washed and
the biotinylated D374Y PCSK9/antibody mixture was transferred to
them and incubated for 1 hour at room temperature. Binding of the
biotinylated D374Y to the LDL receptor was detected by incubation
with streptavidin-HRP (Biosource) at 500 ng/ml in buffer C followed
by TMB substrate (KPL). The signal was quenched with 1N HCl and the
absorbance read at 450 nm.
[0472] The results of this binding study are shown in FIGS. 6A-6D.
Summarily, IC.sub.50 values were determined for each antibody and
found to be 199 pM for 31H4 IgG2 (FIG. 6A), 156 pM for 31H4 IgG4
(FIG. 6B), 170 pM for 21B12 IgG2 (FIG. 6C), and 169 pM for 21B12
IgG4 (FIG. 6D).
[0473] The antibodies also blocked the binding of wild-type PCSK9
to the LDLR in this assay.
Example 12
Cell LDL Uptake Assay
[0474] This example demonstrates the ability of various antigen
binding proteins to reduce LDL uptake by cells. Human HepG2 cells
were seeded in black, clear bottom 96-well plates (Costar) at a
concentration of 5.times.10.sup.5 cells per well in DMEM medium
(Mediatech, Inc) supplemented with 10% FBS and incubated at
37.degree. C. (5% CO2) overnight. To form the PCSK9 and antibody
complex, 2 .mu.g/ml of D374Y human PCSK9 was incubated with various
concentrations of antibody diluted in uptake buffer (DMEM with 1%
FBS) or uptake buffer alone (control) for 1 hour at room
temperature. After washing the cells with PBS, the D374Y
PCSK9/antibody mixture was transferred to the cells, followed by
LDL-BODIPY (Invitrogen) diluted in uptake buffer at a final
concentration of 6 .mu.g/ml. After incubation for 3 hours at
37.degree. C. (5% CO2), cells were washed thoroughly with PBS and
the cell fluorescence signal was detected by Safire.TM. (TECAN) at
480-520 nm (excitation) and 520-600 nm (emission).
[0475] The results of the cellular uptake assay are shown in FIGS.
7A-7D. Summarily, IC.sub.50 values were determined for each
antibody and found to be 16.7 nM for 31H4 IgG2 (FIG. 7A), 13.3 nM
for 31H4 IgG4 (FIG. 7B), 13.3 nM for 21B12 IgG2 (FIG. 7C), and 18
nM for 21B12 IgG4 (FIG. 7D). These results demonstrate that the
applied antigen binding proteins can reduce the effect of PCSK9
(D374Y) to block LDL updtake by cells The antibodies also blocked
the effect of wild-type PCSK9 in this assay.
Example 13
Serum Cholesterol Lowering Effect of the 31H4 Antibody in 6 Day
Study
[0476] In order to assess total serum cholesterol (TC) lowering in
wild type (WT) mice via antibody therapy against PCSK9 protein, the
following procedure was performed.
[0477] Male WT mice (C57BL/6 strain, aged 9-10 weeks, 17-27 g)
obtained from Jackson Laboratory (Bar Harbor, Me.) were fed a
normal chow (Harland-Teklad, Diet 2918) through out the duration of
the experiment. Mice were administered either anti-PCSK9 antibody
31H4 (2 mg/ml in PBS) or control IgG (2 mg/ml in PBS) at a level of
10 mg/kg through the mouse's tail vein at T=0. Naive mice were also
set aside as a naive control group. Dosing groups and time of
sacrifice are shown in Table 9.
TABLE-US-00012 TABLE 9 Group Treatment Time point after dosing
Number 1 IgG 8 hr 7 2 31H4 8 hr 7 3 IgG 24 hr 7 4 31H4 24 hr 7 5
IgG 72 hr 7 6 31H4 72 hr 7 7 IgG 144 hr 7 8 31H4 144 hr 7 9 Naive
n/a 7
[0478] Mice were sacrificed with CO2 asphyxiation at the
pre-determined time points shown in Table 9. Blood was collected
via vena cava into eppendorf tubes and was allowed to clot at room
temperature for 30 minutes. The samples were then spun down in a
table top centrifuge at 12,000.times.g for 10 minutes to separate
the serum. Serum total cholesterol and HDL-C were measured using
Hitachi 912 clinical analyzer and Roche/Hitachi TC and HDL-C
kits.
[0479] The results of the experiment are shown in FIGS. 8A-8D.
Summarily, mice to which antibody 31H4 was administered showed
decreased serum cholesterol levels over the course of the
experiment (FIG. 8A and FIG. 8B). In addition, it is noted that the
mice also showed decreased HDL levels (FIG. 8C and FIG. 8D). For
FIG. 8A and FIG. 8C, the percentage change is in relation to the
control IgG at the same time point (*P<0.01, #P<0.05). For
FIG. 8B and FIG. 8D, the percentage change is in relation to total
serum cholesterol and HDL levels measured in naive animals at t=0
hrs (*P<0.01, #P<0.05).
[0480] In respect to the lowered HDL levels, it is noted that one
of skill in the art will appreciate that the decrease in HDL in
mice is not indicative that an HDL decrease will occur in humans
and merely further reflects that the serum cholesterol level in the
organism has decreased. It is noted that mice transport the
majority of serum cholesterol in high density lipoprotein (HDL)
particles which is different to humans who carry most serum
cholesterol on LDL particles. In mice the measurement of total
serum cholesterol most closely resembles the level of serum HDL-C.
Mouse HDL contains apolipoprotein E (apoE) which is a ligand for
the LDL receptor (LDLR) and allows it to be cleared by the LDLR.
Thus, examining HDL is an appropriate indicator for the present
example, in mice (with the understanding that a decrease in HDL is
not expected for humans). For example, human HDL, in contrast, does
not contain apoE and is not a ligand for the LDLR. As PCSK9
antibodies increase LDLR expression in mouse, the liver can clear
more HDL and therefore lowers serum HDL-C levels.
Example 14
Effect of Antibody 31H4 on LDLR Levels in a 6 Day Study
[0481] The present example demonstrates that an antigen binding
protein alters the level of LDLR in a subject, as predicted, over
time. A Western blot analysis was performed in order to ascertain
the effect of antibody 31H4 on LDLR levels. 50-100 mg of liver
tissue obtained from the sacrified mice described in Example 13 was
homogenized in 0.3 ml of RIPA buffer (Santa Cruz Biotechnology
Inc.) containing complete protease inhibitor (Roche). The
homogenate was incubated on ice for 30 minutes and centrifuged to
pellet cellular debris. Protein concentration in the supernatant
was measured using BioRad protein assay reagents (BioRad
laboratories). 100 .mu.g of protein was denatured at 70.degree. C.
for 10 minutes and separated on 4-12% Bis-Tris SDS gradient gel
(Invitrogen). Proteins were transferred to a 0.45 .mu.m PVDF
membrane (Invitrogen) and blocked in washing buffer (50 mM Tris
PH7.5, 150 mM NaCL, 2 mM CaCl.sub.2 and 0.05% Tween 20) containing
5% non-fat milk for 1 hour at room temperature. The blot was then
probed with goat anti-mouse LDLR antibody (R&D system) 1:2000
or anti-B actin (sigma) 1:2000 for 1 hour at room temperature. The
blot was washed briefly and incubated with bovine anti-goat IgG-HRP
(Santa Cruz Biotechnology Inc.) 1:2000 or goat anti-mouse IgG-HRP
(Upstate) 1:2000. After a 1 hour incubation at room temperature,
the blot was washed thoroughly and immunoreactive bands were
detected using ECL plus kit (Amersham biosciences). The Western
blot showed an increase in LDLR protein levels in the presence of
antibody 31H4, as depicted in FIG. 9.
Example 15
Serum Cholesterol Lowering Effect of Antibody 31H4 in a 13 Day
Study
[0482] In order to assess total serum cholesterol (TC) lowering in
wild type (WT) mice via antibody therapy against PCSK9 protein in a
13 day study, the following procedure was performed.
[0483] Male WT mice (C57BL/6 strain, aged 9-10 weeks, 17-27 g)
obtained from Jackson Laboratory (Bar Harbor, Me.) were fed a
normal chow (Harland-Teklad, Diet 2918) through out the duration of
the experiment. Mice were administered either anti-PCSK9 antibody
31H4 (2 mg/ml in PBS) or control IgG (2 mg/ml in PBS) at a level of
10 mg/kg through the mouse's tail vein at T=0. Naive mice were also
set aside as naive control group.
[0484] Dosing groups and time of sacrifice are shown in Table 10.
Animals were sacrificed and livers were extracted and prepared as
in Example 13.
TABLE-US-00013 TABLE 10 Group Treatment Time point after dosing
Number Dose 1 IgG 72 hr 6 10 mg/kg 2 31H4 72 hr 6 10 mg/kg 3 31H4
72 hr 6 1 mg/kg 4 IgG 144 hr 6 10 mg/kg 5 31H4 144 hr 6 10 mg/kg 6
31H4 144 hr 6 1 mg/kg 7 IgG 192 hr 6 10 mg/kg 8 31H4 192 hr 6 10
mg/kg 9 31H4 192 hr 6 1 mg/kg 10 IgG 240 hr 6 10 mg/kg 11 31H4 240
hr 6 10 mg/kg 12 31H4 240 hr 6 1 mg/kg 13 IgG 312 hr 6 10 mg/kg 14
31H4 312 hr 6 10 mg/kg 15 31H4 312 hr 6 1 mg/kg 16 Naive n/a 6
n/a
[0485] When the 6 day experiment was extended to a 13 day study,
the same serum cholesterol lowering effect observed in the 6 day
study was also observed in the 13 day study. More specifically,
animals dosed at 10 mg/kg demonstrated a 31% decrease in serum
cholesterol on day 3, which gradually returned to pre-dosing levels
by day 13. FIG. 10A depicts the results of this experiment. FIG.
10C depicts the results of repeating the above procedure with the
10 mg/kg dose of 31H4, and with another antibody, 16F12, also at 10
mg/kg. Dosing groups and time of sacrifice are shown in Table
11.
TABLE-US-00014 TABLE 11 Group Treatment Time point after dosing
Number Dose 1 IgG 24 hr 6 10 mg/kg 2 16F12 24 hr 6 10 mg/kg 3 31H4
24 hr 6 10 mg/kg 4 IgG 72 hr 6 10 mg/kg 5 16F12 72 hr 6 10 mg/kg 6
31H4 72 hr 6 10 mg/kg 7 IgG 144 hr 6 10 mg/kg 8 16F12 144 hr 6 10
mg/kg 9 31H4 144 hr 6 10 mg/kg 10 IgG 192 hr 6 10 mg/kg 11 16F12
192 hr 6 10 mg/kg 12 31H4 192 hr 6 10 mg/kg 13 IgG2 240 hr 6 10
mg/kg 14 16F12 240 hr 6 10 mg/kg 15 31H4 240 hr 6 10 mg/kg 16 IgG2
312 hr 6 10 mg/kg 17 16F12 312 hr 6 10 mg/kg 18 31H4 312 hr 6 10
mg/kg 19 Naive n/a 6 10 mg/kg
[0486] As shown in FIG. 10C both 16F12 and 31H4 resulted in
significant and substantial decreases in total serum cholesterol
after just a single dose and provided benefits for over a week (10
days or more). The results of the repeated 13 day study were
consistent with the results of the first 13 day study, with a
decrease in serum cholesterol levels of 26% on day 3 being
observed. For FIG. 10A and FIG. 10B, the percentage change is in
relation to the control IgG at the same time point (*P<0.01).
For FIG. 10C, the percentage change is in relation to the control
IgG at the same time point (*P<0.05).
Example 16
Effect of Antibody 31H4 on HDL Levels in a 13 Day Study
[0487] The HDL levels for the animals in Example 15 were also
examined. HDL levels decreased in the mice. More specifically,
animals dosed at 10 mg/kg demonstrated a 33% decrease in HDL levels
on day 3, which gradually returned to pre-dosing levels by day 13.
FIG. 10B depicts the results of the experiment. There was a
decrease in HDL levels of 34% on day 3. FIG. 10B depicts the
results of the repeated 13 day experiment.
[0488] As will be appreciated by one of skill in the art, while the
antibodies will lower mouse HDL, this is not expected to occur in
humans because of the differences in HDL in humans and other
organisms (such as mice). Thus, the decrease in mouse HDL is not
indicative of a decrease in human HDL.
Example 17
Repeated Administration of Antibodies Produce Continued Benefits of
Antigen Binding Peptides
[0489] In order to verify that the results obtained in the Examples
above can be prolonged for further benefits with additional doses,
the Experiments in Examples 15 and 16 were repeated with the dosing
schedule depicted in FIG. 11A. The results are displayed in FIG.
11B. As can be seen in the graph in FIG. 11B, while both sets of
mice displayed a significant decrease in total serum cholesterol
because all of the mice received an initial injection of the 31H4
antigen binding protein, the mice that received additional
injections of the 31H4 ABP displayed a continued reduction in total
serum cholesterol, while those mice that only received the control
injection eventually displayed an increase in their total serum
cholesterol. For FIG. 11, the percentage change is in relation to
the naive animals at t=0 hours (*P<0.01, **P<0.001).
[0490] The results from this example demonstrate that, unlike other
cholesterol treatment methods, in which repeated applications lead
to a reduction in efficacy because of biological adjustments in the
subject, the present approach does not seem to suffer from this
issue over the time period examined. Moreover, this suggests that
the return of total serum cholesterol or HDL cholesterol levels to
baseline, observed in the previous examples is not due to some
resistance to the treatment being developed by the subject, but
rather the depletion of the antibody availability in the
subject.
Example 18
Epitope Mapping of Human Anti PCSK9 Antibodies
[0491] This example outlines methods for determining which residues
in PCSK9 are involved in forming or part of the epitope for the
antigen binding proteins disclosed herein to PCSK9.
[0492] In order to determine the epitopes to which certain of the
ABPs of the present invention bind, the epitopes of the ABPs can be
mapped using synthetic peptides derived from the specific PCSK9
peptide sequence.
[0493] A SPOTs peptide array (Sigma Genosys) can be used to study
the molecular interaction of the human anti-PCSK9 antibodies with
their peptide epitope. SPOTs technology is based on the solid-phase
synthesis of peptides in a format suitable for the systematic
analysis of antibody epitopes. Synthesis of custom arrayed
oligopeptides is commerically available from Sigma-Genosys. A
peptide array of overlapping oligopeptides derived from the
amino-acid sequence of the PCSK9 peptide can be obtained. The array
can comprise a series of 12-mer peptides as spots on a
polypropylene membrane sheets. The peptide array can span the
entire length of the PCSK9 mature sequence. Each consecutive
peptide can be offset by 1 residue from the previous one, yielding
a nested, overlapping library of arrayed oligopeptides. The
membrane carrying the peptides can be reacted with different
anti-PCSK9 antibodies (1 micrograms/ml). The binding of the mAbs to
the membrane-bound peptides can be assessed by an enzyme-linked
immunosorbent assay using HRP-conjugated secondary antibody
followed by enhanced chemiluminescence (ECL).
[0494] In addition, 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 ABPs. To accomplish this, a second set of SPOTs arrays
can be used for alanine scanning. A panel of variant peptides with
alanine substitutions in each of the 12 residues can be scanned as
above. This will allow for the epitopes for the ABPs to the human
PCSK9 to be mapped and identified.
[0495] In the alternative, given that it is possible that the
epitope is conformational, a combination of alanine scanning and/or
arginine scanning, antibody FAB/PCSK9 co-crystallization, and
limited proteolysis/LC-MS (liquid chromatography mass spec.) can be
employed to identify the epitopes.
Example 19
Uses of PCSK9 Antibodies for the Treatment of Cholesterol Related
Disorders
[0496] A human patient exhibiting a Cholesterol Related Disorder
(in which a reduction in cholesterol (such as serum cholesterol)
can be beneficial) is administered a therapeutically effective
amount of PCSK9 antibody, 31H4 (or, for example, 21B12 or 16F12).
At periodic times during the treatment, the patient is monitored to
determine whether the symptoms of the disorder has subsided.
Following treatment, it is found that patients undergoing treatment
with the PCSK9 antibody have reduced serum cholesterol levels, in
comparison to patients that are not treated.
Example 20
Uses of PCSK9 Antibodies for the Treatment of
Hypercholesterolemia
[0497] A human patient exhibiting symptoms of hypercholesterolemia
is administered a therapeutically effective amount of PCSK9
antibody, such as 31H4 (or, for example, 21B12 or 16F12). At
periodic times during the treatment, the human patient is monitored
to determine whether the serum cholesterol level has declined.
Following treatment, it is found that the patient receiving the
treatment with the PCSK9 antibodies has reduced serum cholesterol
levels in comparison to arthritis patients not receiving the
treatment.
Example 21
Uses of PCSK9 Antibodies for the Prevention of Coronary Heart
Disease and/or Recurrent Cardiovascular Events
[0498] A human patient at risk of developing coronary heart disease
is identified. The patient is administered a therapeutically
effective amount of PCSK9 antibody, such as 31H4 (or, for example,
21B12 or 16F12), either alone, concurrently or sequentially with a
statin, e.g., simvastatin. At periodic times during the treatment,
the human patient is monitored to determine whether the patient's
total serum cholesterol level changes. Throughout the preventative
treatment, it is found that the patient receiving the treatment
with the PCSK9 antibodies has reduced serum cholesterol thereby
reducing their risk to coronary heart diseases or recurrent
cardiovascular events in comparison to patients not receiving the
treatment.
Example 22
Use of PCSK9 Antibodies as a Diagnostic Agent
[0499] An Enzyme-Linked Immunosorbent Assay (ELISA) for the
detection of PCSK9 antigen in a sample can used to diagnose
patients exhibiting high levels of PCSK9 production. In the assay,
wells of a microtiter plate, such as a 96-well microtiter plate or
a 384-well microtiter plate, are adsorbed for several hours with a
first fully human monoclonal antibody directed against PCSK9. The
immobilized antibody serves as a capture antibody for any of the
PCSK9 that may be present in a test sample. The wells are rinsed
and treated with a blocking agent such as milk protein or albumin
to prevent nonspecific adsorption of the analyte.
[0500] Subsequently the wells are treated with a test sample
suspected of containing the PCSK9, or with a solution containing a
standard amount of the antigen. Such a sample may be, for example,
a serum sample from a subject suspected of having levels of
circulating antigen considered to be diagnostic of a pathology.
[0501] After rinsing away the test sample or standard, the wells
are treated with a second fully human monoclonal PCSK9 antibody
that is labeled by conjugation with biotin. A monoclonal or mouse
or other species origin can also be used. The labeled PCSK9
antibody serves as a detecting antibody. After rinsing away excess
second antibody, the wells are treated with avidin-conjugated
horseradish peroxidase (HRP) and a suitable chromogenic substrate.
The concentration of the antigen in the test samples is determined
by comparison with a standard curve developed from the standard
samples.
[0502] This ELISA assay provides a highly specific and very
sensitive assay for the detection of the PCSK9 antigen in a test
sample.
Determination of PCSK9 Protein Concentration in Subjects
[0503] A sandwich ELISA can quantify PCSK9 levels in human serum.
Two fully human monoclonal PCSK9 antibodies from the sandwich
ELISA, recognize different epitopes on the PCSK9 molecule.
Alternatively, monoclonal antibodies of mouse or other species
origin may be used. The ELISA is performed as follows: 50 .mu.L of
capture PCSK9 antibody in coating buffer (0.1 M NaHCO.sub.3, pH
9.6) at a concentration of 2 .mu.g/mL is coated on ELISA plates
(Fisher). After incubation at 4.degree. C. overnight, the plates
are treated with 200 .mu.L of blocking buffer (0.5% BSA, 0.1% Tween
20, 0.01% Thimerosal in PBS) for 1 hour at 25.degree. C. The plates
are washed (3.times.) using 0.05% Tween 20 in PBS (washing buffer,
WB). Normal or patient sera (Clinomics, Bioreclaimation) are
diluted in blocking buffer containing 50% human serum. The plates
are incubated with serum samples overnight at 4.degree. C., washed
with WB, and then incubated with 100 .mu.L/well of biotinylated
detection PCSK9 antibody for 1 hour at 25.degree. C. After washing,
the plates are incubated with HRP-Streptavidin for 15 minutes,
washed as before, and then treated with 100 .mu.L/well of
o-phenylenediamine in H.sub.2O.sub.2 (Sigma developing solution)
for color generation. The reaction is stopped with 50 .mu.L/well of
H.sub.2SO.sub.4 (2M) and analyzed using an ELISA plate reader at
492 nm. Concentration of PCSK9 antigen in serum samples is
calculated by comparison to dilutions of purified PCSK9 antigen
using a four parameter curve fitting program.
Determination of PCSK9 Variant Protein Concentration in
Subjects
[0504] The steps outlined above can be performed using antibodies
noted herein that bind to both the wild type PCSK9 and the variant
PCSK9 (D374Y). Next, antibodies that bind to the wild type but not
the mutant can be used (again using a similar protocol as outlined
above) to determine if the PCSK9 present in the subject is wild
type or the D374Y variant. As will be appreciated by one of skill
in the art, results that are positive for both rounds will be
wild-type, while those that are positive for the first round, but
not the second round of antibodies, will include the D374Y
mutation. There are high frequency mutations in the population that
are known and the could benefit particularly from an agent such as
the ABPs disclosed herein.
Example 23
Use of PCSK9 Antigen Binding Protein for the Prevention of
Hypercholesterolemia
[0505] A human patient exhibiting a risk of developing
hypercholesterolemia is identified via family history analysis
and/or lifestyle, and/or current cholesterol levels. The subject is
regularly administered (e.g., one time weekly) a therapeutically
effective amount of PCSK9 antibody, 31H4 (or, for example, 21B12 or
16F12). At periodic times during the treatment, the patient is
monitored to determine whether serum cholesterol levels have
decreased. Following treatment, it is found that subjects
undergoing preventative treatment with the PCSK9 antibody have
lowered serum cholesterol levels, in comparison to subjects that
are not treated.
Example 24
PCSK9 ABPs Further Upregulated LDLR in the Presence of Statins
[0506] This example demonstrates that ABPs to PCSK9 produced
further increases in LDLR availability when used in the presence of
statins, demonstrating that further benefits can be achieved by the
combined use of the two.
[0507] HepG2 cells were seeded in DMEM with 10% fetal bovine serum
(FBS) and grown to .about.90% confluence. The cells were treated
with indicated amounts of mevinolin (a statin, Sigma) and PCSK9
ABPs (FIGS. 12A-12C) in DMEM with 3% FBS for 48 hours. Total cell
lysates were prepared. 50 mg of total proteins were separated by
gel electrophoresis and transferred to PVDF membrane. Immunoblots
were performed using rabbit anti-human LDL receptor antibody
(Fitzgerald) or rabbit anti-human b-actin antibody. The enhanced
chemiluminescent results are shown in the top panels of FIGS.
12A-12C. The intensity of the bands were quantified by ImageJ
software and normalized by b-actin. The relative levels of LDLR are
shown in the lower panels of FIGS. 12A-12C. ABPs 21B12 and 31H4 are
PCSK9 neutralizing antibodies, while 25A7.1 is a non-neutralizing
antibody.
[0508] HepG2-PCSK9 cells were also created. These were stable HepG2
cell line transfected with human PCSK9. The cells were seeded in
DMEM with 10% fetal bovine serum (FBS) and grew to .about.90%
confluence. The cells were treated with indicated amounts of
mevinolin (Sigma) and PCSK9 ABPs (FIGS. 12D-12F) in DMEM with 3%
FBS for 48 hours. Total cell lysates were prepared. 50 mg of total
proteins were separated by gel electrophoresis and transferred to
PVDF membrane. Immunoblots were performed using rabbit anti-human
LDL receptor antibody (Fitzgerald) or rabbit anti-human b-actin
antibody. The enhanced chemiluminescent results are shown in the
top panels. The intensity of the bands were quantified by ImageJ
software and normalized by b-actin.
[0509] As can be seen in the results depicted in FIGS. 12A-12F,
increasing amounts of the neutralizing antibody and increasing
amounts of the statin generally resulted in increases in the level
of LDLR. This increase in effectiveness for increasing levels of
the ABP is especially evident in FIGS. 12D-12F, in which the cells
were also transfected with PCSK9, allowing the ABPs to demonstrate
their effectiveness to a greater extent.
[0510] Interestingly, as demonstrated by the results in the
comparison of FIGS. 12D-12F to 12A-12C, the influence of the ABP
concentrations on LDLR levels increased dramatically when PCSK9 was
being produced by the cells. In addition, it is clear that the
neutralizing ABPs (21B12 and 31H4) resulted in a greater increase
in LDLR levels, even in the presence of statins, than the 25A7.1
ABP (a non-neutralizer), demonstrating that additional benefits can
be achieved by the use of both statins and ABPs to PCSK9.
Example 25
Consensus Sequences
[0511] Consensus sequences were determined using standard
phylogenic analyses of the CDRs corresponding to the V.sub.H and
V.sub.L of anti-PCSK9 ABPs. The consensus sequences were determined
by keeping the CDRs contiguous within the same sequence
corresponding to a V.sub.H or V.sub.L. Briefly, amino acid
sequences corresponding to the entire variable domains of either
V.sub.H or V.sub.L were converted to FASTA formatting for ease in
processing comparative alignments and inferring phylogenies. Next,
framework regions of these sequences were replaced with an
artificial linker sequence ("bbbbbbbbbb" placeholders, non-specific
nucleic acid construct) so that examination of the CDRs alone could
be performed without introducing any amino acid position weighting
bias due to coincident events (e.g., such as unrelated antibodies
that serendipitously share a common germline framework heritage)
while still keeping CDRs contiguous within the same sequence
corresponding to a V.sub.H or V.sub.L. V.sub.H or V.sub.L sequences
of this format were then subjected to sequence similarity alignment
interrogation using a program that employs a standard ClutalW-like
algorithm (see, Thompson et al., 1994, Nucleic Acids Res.
22:4673-4680). A gap creation penalty of 8.0 was employed along
with a gap extension penalty of 2.0. This program likewise
generated phylograms (phylogenic tree illustrations) based on
sequence similarity alignments using either UPGMA (unweighted pair
group method using arithmetic averages) or Neighbor-Joining methods
(see, Saitou and Nei, 1987, Molecular Biology and Evolution
4:406-425) to construct and illustrate similarity and distinction
of sequence groups via branch length comparison and grouping. Both
methods produced similar results but UPGMA-derived trees were
ultimately used as the method employs a simpler and more
conservative set of assumptions. UPGMA-derived trees were generated
where similar groups of sequences were defined as having fewer than
15 substitutions per 100 residues (see, legend in tree
illustrations for scale) amongst individual sequences within the
group and were used to define consensus sequence collections. The
results of the comparisons are depicted in FIGS. 13A-13J. In FIG.
13E, the groups were chosen so that sequences in the light chain
that clade are also a clade in the heavy chain and have fewer than
15 substitutions.
[0512] As will be appreciated by one of skill in the art, the
results presented in FIGS. 13A-13J present a large amount of
guidance as to the importance of particular amino acids (for
example, those amino acids that are conserved) and which amino acid
positions can likely be altered (for example, those positions that
have different amino acids for different ABPs).
Example 26
Mouse Model for PCSK9 and ABP Ability to Lower LDL In Vivo
[0513] To generate mice which over-expressed human PCSK9, three
week old WT C57Bl/6 mice were injected via tail vein administration
with various concentrations of adenoassociated virus (AAV),
recombinantly modified to express human PCSK9, to determine the
correct titer which would provide a measurable increase of
LDL-cholesterol in the mice. Using this particular virus that
expressed human PCSK9, it was determined that 4.5.times.10E12 pfu
of virus would result in an LDL-cholesterol level of approximately
40 mg/dL in circulating blood (normal levels of LDL in a WT mice
are approximately 10 mg/dL). The human PCSK9 levels in these
animals was found to be approximately 13 ug/mL. A colony of mice
were generated using this injection criteria.
[0514] One week after injection, mice were assessed for
LDL-cholesterol levels, and randomized into different treatment
groups. Animals were then administered, via tail vein injection, a
single bolus injection of either 10 mg/kg or 30 mg/kg of 16F12,
21B12, or 31H4 antigen binding proteins. IgG2 ABP was administered
in a separate group of animals as a dosing control. Subgroups of
animals (n=6-7) were then euthanized at 24 and 48 hours after ABP
administration. There were no effects on LDL-cholesterol levels
following IgG2 administration at either dose. Both 31H4 and 21B12
demonstrated significant LDL-cholesterol lowering up to and
including 48 hours post-administration, as compared to IgG2 control
(shown in FIGS. 14A and 14B at two different doses). 16F12 shows an
intermediary LDL-cholesterol lowering response, with levels
returning to baseline of approximately 40 mg/dL by the 48 hour time
point. This data is consistent with in vitro binding data (Biacore
and Kinexa), which shows near equivalent binding affinity between
31H4 and 21B12, and a lesser affinity of 16F12 to human PCSK9.
[0515] As can be seen in the results, total cholesterol and
HDL-cholesterol were reduced by the PCSK9 ABPs in the model (both
total and HDL-C are elevated above WT mice due to the
overexpression of PCSK9). While cholesterol lowering in this model
appears to occur over a relatively short period of time, this is
believed to be due to the levels of human PCSK9 that are present,
which are supraphysiologically high in this model. In addition,
given that the expression is governed by AAV, there is no
regulation of PCSK9 expression. In these figures, (*) denotes a
P<0.05, and (**) denotes a P<0.005 as compared to
LDL-cholesterol levels observed in IgG2 control injected animals at
the same time point. The 13 microgram/ml level of serum human PCSK9
in the mice corresponds to an approximately 520-fold increase above
the endogenous mouse PCSK9 levels (.about.25 ng/ml), and an
approximately 75-fold increase above average human serum levels
(.about.175 ng/ml). Thus, the antigen binding proteins should be
even more effective in humans.
[0516] As will be appreciated by one of skill in the art, the above
results demonstrate that appropriateness of the mouse model for
testing the antigen binding protein's ability to alter serum
cholesterol in a subject. One of skill in the art will also
recognize that the use of mouse HDL to monitor serum cholesterol
levels in a mouse, while useful for monitoring mouse serum
cholesterol levels, is not indicative of the ABPs impact on human
HDL in humans. For example, Cohen et al. ("Sequence variations in
PCSK9, low LDL, and protection against coronary heart disease", N
Engl J Med, 354:1264-1272, 2006) demonstrated the lack of any
effect of the PCSK9 loss-of-function mutations on human HDL levels
(the entirety of which is incorporated by reference). Thus, one of
skill in the art will appreciate that the ability of the ABP to
lower mouse HDL (which lack LDL) is not indicative of the ABP's
ability to lower human HDL. Indeed, as shown by Cohen, this is
unlikely to occur for neutralizing antibodies in humans.
Example 27
31H4 and 21B12 Bind to the ProCat Region of PCSK9
[0517] The present example describes one method for determining
where various antibodies bind to PCSK9.
[0518] The ProCat (31-449 of SEQ ID NO: 3) or V domain (450-692 of
SEQ ID NO: 3) of the PCSK9 protein was combined with either
antibody 31H4 or 21B12. The samples were analyzed by Native PAGE
for complex formation. As can be seen in FIG. 16A and FIG. 16B, gel
shifts were present for the ProCat/31H4 and ProCat/21B12 samples,
demonstrating that the antibodies bound to the ProCat domain.
Example 28
The LDLR EGFa Domain Binds to the Catalytic Domain of PCSK9
[0519] The present example presents the solved crystal structure of
PCSK9 ProCat (31-454 of SEQ ID NO: 3) bound to the LDLR EGFa domain
(293-334) at 2.9 .ANG. resolution (the conditions for which are
described in the below Examples).
[0520] A representation of the structure of PCSK9 bound to EGFa is
shown in FIG. 17. The crystal structure (and its depiction in FIG.
17) reveals that the EGFa domain of LDLR binds to the catalytic
domain of PCSK9. In addition, the interaction of PCSK9 and EGFa
appears to occur across a surface of PCSK9 that is between residues
D374 and S153 in the structure depicted in FIG. 17.
[0521] Specific core PCSK9 amino acid residues of the interaction
interface with the LDLR EGFa domain were defined as PCSK9 residues
that are within 5 .ANG. of the EGFa domain. The core residues are
as follows: 5153, 1154, P155, R194, D238, A239, I369, 5372, D374,
C375, T377, C378, F379, V380, and S381.
[0522] Boundary PCSK9 amino acid residues of the interaction
interface with the LDLR EGFa domain were defined as PCSK9 residues
that are 5-8 .ANG. from the EGFa domain. The boundary residues are
as follows: W156, N157, L158, E159, H193, E195, H229, R237, G240,
K243, D367, I368, G370, A371, 5373, 5376, and Q382. Residues that
are underlined are nearly or completely buried within PCSK9.
[0523] As will be appreciated by one of skill in the art, the
results from this example demonstrate where PCSK9 and EGFa
interact. Thus, antibodies that interact with or block any of these
residues can be useful as antibodies that inhibit the interaction
between PCSK9 and the EGFa domain of LDLR (and/or LDLR generally).
In some embodiments, antibodies that, when bound to PCSK9, interact
with or block any of the above residues or are within 15-8,8, 8-5,
or 5 angstroms of the above residues are contemplated to provide
useful inhibition of PCSK9 binding to LDLR.
Example 29
31H4 Interacts with Amino Acid Residues from Both the Pro- and
Catalytic Domains of PCSK9
[0524] The present example presents the crystal structure of full
length PCSK9 (N533A mutant of SEQ ID NO: 3) bound to the Fab
fragment of 31H4, determined to 2.3 .ANG. resolution (the
conditions for which are described in the below Examples). This
structure, depicted in FIGS. 18A and 18B, shows that 31H4 binds to
PCSK9 in the region of the catalytic site and makes contacts with
amino acid residues from both the prodomain and catalytic
domain.
[0525] The depicted structure also allows one to identify specific
core PCSK9 amino acid residues for the interaction interface of
31H4 with PCSK9. This was defined as residues that are within 5
.ANG. of the 31H4 protein. The core residues are as follows: W72,
F150, A151, Q152, T214, R215, F216, H217, A220, S221, K222, S225,
H226, C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350,
L351, E366, D367, D374, V380, S381, Q382, S383, and G384.
[0526] The structures were also used to identify boundary PCSK9
amino acid residues for the interaction interface with 31H4. These
residues were PCSK9 residues that were 5-8 .ANG. from the 31H4
protein. The boundary residues are as follows: K69, D70, P71, S148,
V149, D186, T187, E211, D212, G213, R218, Q219, C223, D224, G227,
H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325, V346,
G352, T353, G365, I368, I369, S372, S373, C378, F379, T385, S386,
and Q387. Amino acid residues completely buried within the PCSK9
protein are underlined.
[0527] As will be appreciated by one of skill in the art, FIG. 18B
depicts the interaction between the CDRs on the antigen binding
protein and PCSK9. As such, the model allows one of skill in the
art to identify the residues and/or CDRs that are especially
important in the paratope, and which residues are less critical to
the paratope. As can be seen in FIG. 18B, the heavy chain CDR1,
CDR2, and CDR3 are most directly involved in the antigen binding
protein's binding to the epitope, with the CDRs from the light
chain being relatively far away from the epitope. As such, it is
probable that larger variations in the light chain CDRs are
possible, without unduly interfering with the binding of the
antigen binding protein to PCSK9. In some embodiments, residues in
the structures that directly interact are conserved (or
alternatively conservatively replaced) while residues that are not
directly interacting with one another can be altered to a greater
extent. As such, one of skill in the art, given the present
teachings, can predict which residues and areas of the antigen
binding proteins can be varied without unduly interfering with the
antigen binding protein's ability to bind to PCSK9. For example,
those residues that are located closest to PCSK9 when the antigen
binding protein is bound to PCSK9 are those that likely play a more
important role in the binding of the antigen binding protein to
PCSK9. As above, these residues can be divided into those that are
within 5 angstroms of PCSK9 and those that are between 5 and 8
angstroms. Specific core 31H4 amino acid residues of the
interaction interface with PCSK9 were defined as 31H4 residues that
are within 5 .ANG. of the PCSK9 protein. For the heavy chain, the
residues that are within 5 angstroms include the following: T28,
S30, S31, Y32, S54, S55, S56, Y57, I58, S59, Y60, N74, A75, R98,
Y100, F102, W103, S104, A105, Y106, Y107, D108, A109, and D111. For
the light chain, those residues that are within 5 angstroms include
the following: L48, S51, Y93, and S98. For the heavy chain, those
residues that are 5-8 .ANG. from the PCSK9 protein include the
following: G26, F27, F29, W47, S50, I51, S52, S53, K65, F68, T69,
I70, S71, R72, D73, K76, N77, D99, D101, F110, and V112. For the
light chain, those residues that are within 5-8 angstroms of PCSK9
include A31, G32, Y33, D34, H36, Y38, I50, G52, N55, R56, P57, S58,
D94, S95, S96, L97, G99, and S100.
[0528] As will be appreciated by one of skill in the art, the
results from Example 29 demonstrate where antibodies to PCSK9 can
interact on PCSK9 and still block PCSK9 from interacting with EGFa
(and thus LDLR). Thus, antigen binding proteins that interact with
any of these PCSK9 residues, or that block any of these residues
(e.g., from other antigen binding proteins that bind to these
residues), can be useful as antibodies that inhibit the interaction
of PCSK9 and EGFa (and LDLR accordingly). Thus, in some
embodiments, antigen binding proteins that interact with any of the
above residues or interact with residues that are within 5 .ANG. of
the above residues are contemplated to provide useful inhibition
PCSK9 binding to LDLR. Similarly, antigen binding proteins that
block any of the above residues (which can be determined, for
example, via a competition assay) can also be useful for inhibition
of the PCSK9/LDLR interaction.
Example 30
21B12 Binds to the Catalytic Domain of PCSK9, has a Distinct
Binding Site from 31H4 and can Bind to PCSK9 Simultaneously with
31H4
[0529] The present example presents the crystal structure of PCSK9
ProCat (31-449 of SEQ ID NO: 3) bound to the Fab fragments of 31H4
and 21B12, determined at 2.8 .ANG. resolution (the conditions for
which are described in the below Examples). This crystal structure,
depicted in FIG. 19A and FIG. 19B, shows that 31H4 and 21B12 have
distinct binding sites on PCSK9 and that both antigen binding
proteins can bind to PCSK9 simultaneously. The structure shows that
21B12 interacts with amino acid residues from PCSK9's catalytic
domain. In this structure, the interaction between PCSK9 and 31H4
is similar to what was observed above.
[0530] Specific core PCSK9 amino acid residues of the interaction
interface with 21B12 were defined as PCSK9 residues that are within
5 .ANG. of the 21B12 protein. The core residues are as follows:
S153, S188, I189, Q190, S191, D192, R194, E197, G198, R199, V200,
D224, R237, D238, K243, S373, D374, S376, T377, and F379.
[0531] Boundary PCSK9 amino acid residues of the interaction
interface with 21B12 were defined as PCSK9 residues that were 5-8
.ANG. from the 21B12 protein. The boundary residues are as follows:
1154, T187, H193, E195, I196, M201, V202, C223, T228, S235, G236,
A239, G244, M247, I369, S372, C375, and C378. Amino acid residues
nearly or completely buried within the PCSK9 protein are
underlined.
[0532] As will be appreciated by one of skill in the art, FIG. 19B
depicts the interaction between the CDRs on the antigen binding
protein and PCSK9. As such, the model allows one of skill in the
art to identify the residues and/or CDRs which are especially
important for the paratope and which residues are less critical to
the paratope. As can be seen in the structure, heavy chain CDR2 and
light chain CDR1 appear to closely interact with the epitope. Next,
heavy chain CDR1, heavy chain CDR3 and light chain CDR3, appear to
be close to the epitope, but not as close as the first set of CDRs.
Finally, light chain CDR2 appears to be some distance from the
epitope. As such, it is probable that larger variations in the more
distant CDRs are possible without unduly interfering with the
binding of the antigen binding protein to PCSK9. In some
embodiments, residues in the structures that directly interact are
conserved (or alternatively conservatively replaced) while residues
that are not directly interacting with one another can be altered
to a greater extent. As such, one of skill in the art, given the
present teachings, can predict which residues and areas of the
antigen binding proteins can be varied without unduly interfering
with the antigen binding protein's ability to bind to PCSK9. For
example, those residues that are located closest to PCSK9 when the
antigen binding protein is bound to PCSK9 are those that likely
play a more important role in the binding of the antigen binding
protein to PCSK9. As above, these residues can be divided into
those that are within 5 angstroms of PCSK9 and those that are
between 5 and 8 angstroms. Specific core 21B12 amino acid residues
of the interaction interface with PCSK9 were defined as 21B12
residues that are within 5 .ANG. of the PCSK9 protein. For the
heavy chain, the residues that are within 5 angstroms include the
following: T30, S31, Y32, G33, W50, S52, F53, Y54, N55, N57, N59,
R98, G99, Y100, and G101. For the light chain, those residues that
are within 5 angstroms include the following: G30, G31, Y32, N33,
S34, E52, Y93, T94, S95, T96, and S97. For the heavy chain, those
residues that are 5-8 .ANG. from the PCSK9 protein include the
following: T28, L29, I34, S35, W47, V51, G56, T58, Y60, T72, M102,
and D103. For the light chain, those residues that are within 5-8
angstroms of PCSK9 include the following: S26, V29, V35, Y51, N55,
S92, M98, and V99.
[0533] As will be appreciated by one of skill in the art, the
results from Example 30 demonstrate where antigen binding proteins
to PCSK9 can interact on PCSK9 and still block PCSK9 from
interacting with EGFa (and thus LDLR). Thus, antigen binding
proteins that interact with any of these PCSK9 residues or that
block any of these residues can be useful as antibodies that
inhibit the interaction of PCSK9 and EGFa (and LDLR accordingly).
Thus, in some embodiments, antibodies that interact with any of the
above residues or interact with residues that are within 5 .ANG. of
the above residues are contemplated to provide useful inhibition
PCSK9 binding to LDLR. Similarly, antigen binding proteins that
block any of the above residues (which can be determined, for
example, via a competition assay) can also be useful for inhibition
of PCSK9/LDLR interaction.
Example 31
Interaction Between EGFa, PCSK9, and the Antibodies
[0534] The structure of the ternary complex (PCSK9/31H4/21B12) from
the above example was overlaid on the PCSK9/EGFa structure
(determined as described in Example 28) and the result of this
combination is depicted in FIG. 20A. This figure demonstrates areas
on PCSK9 which can be usefully targeted to inhibit PCSK9
interaction with EGFa. The figure shows that both 31H4 and 21B12
partially overlap with the position of the EGFa domain of LDLR and
sterically interfere with its binding to PCSK9. In addition, as can
be seen in the structures, 21B12 directly interacts with a subset
of amino acid residues that are specifically involved in binding to
the LDLR EGFa domain.
[0535] As noted above, analysis of the crystal structures
identified specific amino acids involved in the interaction between
PCSK9 and the partner proteins (the core and boundary regions of
the interface on the PCSK9 surface) and the spatial requirements of
these partner proteins to interact with PCSK9. The structures
suggest ways to inhibit the interaction between PCSK9 and the LDLR.
First, as noted above, binding an agent to PCSK9 where it shares
residues in common with the binding site of the EGFa domain of the
LDLR would inhibit the interaction between PCSK9 and the LDLR.
Second, an agent that binds outside of the residues in common can
sterically interfere with the EGFa domain or regions of the LDLR
that are either N- or C-terminal to the EGFa domain to prevent the
interaction between PCSK9 and the LDLR.
[0536] In some embodiments, the residues that are involved in both
EGFa binding and are close to the areas where the above noted
antigen binding proteins bind are especially useful for
manipulating PCSK9 binding to LDLR. For example, amino acid
residues from interfaces in common in both the core region and
boundary region for the different binding partners are listed in
Table 12 below. Amino acid residues completely buried within the
PCSK9 protein are underlined.
TABLE-US-00015 TABLE 12 Parameters Amino acid position(s) 31H4/EGFa
both under 5 .ANG. D374, V380, S381 31H4 under 5 .ANG./EGFa 5-8
.ANG. D367, Q382 31H4 at 5-8 .ANG./EGFa under 5 .ANG. I369, S372,
C378, F379 31H4/EGFa both at 5-8 .ANG. H229, S373 21B12/EGFa both
under 5 .ANG. S153, R194, D238, D374, T377, F379 21B12 under 5
.ANG./EGFa 5-8 .ANG. R237, K243, S373, S376 21B12 at 5-8 .ANG./EGFa
under 5 .ANG. I154, A239, I369, S372, C375, C378 21B12/EGFa both at
5-8 .ANG. H193, E195
[0537] As will be appreciated by one of skill in the art, in some
embodiments, the antigen binding proteins bind to and/or block at
least one of the above noted residues.
Example 32
Structural Interaction of LDLR and PCSK9
[0538] A model of full length PCSK9 bound to a full length
representation of the LDLR was made using the PCSK9 ProCat (31-454
of SEQ ID NO: 3)/EGFa complex structure. The structure of full
length PCSK9.sup.1 (Piper, D. E. et al. The crystal structure of
PCSK9: a regulator of plasma LDL-cholesterol. Structure 15, 545-52
(2007)) was overlaid onto the PCSK9 ProCat 31-454 from the complex
and the structure of the LDLR in its low pH conformation (Rudenko,
G. et al. Structure of the LDL receptor extracellular domain at
endosomal pH. Science 298, 2353-8 (2002)) was overlaid onto the
EGFa domain from the complex. Depictions of the model are shown in
FIGS. 20B and 20C. The EGFa domain is indicated by the box in the
figure. The figures show regions of the LDLR outside of the
immediate EGFa binding domain that lie in close proximity to PCSK9.
FIGS. 20D-20F show the above interaction, along with mesh surface
representations of antibody 31H4 and 21B12 from three different
angles. As is clear from the depictions, not only can the antibody
interact and/or interfere with LDLR's interaction with PCSK9 at the
actual binding site, but other steric interactions appear to occur
as well.
[0539] In light of the above results, it is clear that antigen
binding proteins that bind to PCSK9 can also inhibit the
interaction between PCSK9 and the LDLR by clashing with various
regions of the LDLR (not just the site at which LDLR and PCSK9
interact). For example, it can clash with repeat 7 (R7), the EGFb
domain, and/or the .beta.-propeller domain.
Embodiments of Antigen Binding Molecules that Bind to or Block EGFa
Interaction with PCSK9
[0540] As will be appreciated by one of skill in the art, Examples
28-32, and their accompanying figures, provide a detailed
description of how and where EGFa interacts with PCSK9 and how two
representative neutralizing antigen binding proteins, 21B12 and
31H4 interact with PCSK9 and produce their neutralizing effect. As
such, one of skill in the art will readily be able to identify
antigen binding molecules that can similarly reduce the binding
between EGFa (including LDLR) and PCSK9 by identifying other
antigen binding molecules that bind at or near at least one of the
same locations on PCSK9. While the relevant locations (or epitopes)
on PCSK9 are identified in the figures and the present description,
it can also be advantageous to describe these sites as being within
a set distance from residues that have been identified as close to
the EGFa binding site. In some embodiments, an antigen binding
molecule will bind to or within 30 angstroms of one or more of the
following residues (numbering in reference to SEQ ID NO: 3): S153,
I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378,
F379, V380, S381, W156, N157, L158, E159, H193, E195, H229, R237,
G240, K243, D367, I368, G370, A371, S373, S376, Q382, W72, F150,
A151, Q152, T214, R215, F216, H217, A220, 5221, K222, S225, H226,
C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350, L351,
E366, D367, D374, V380, S381, Q382, S383, G384, K69, D70, P71,
S148, V149, D186, T187, E211, D212, G213, R218, Q219, C223, D224,
G227, H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325,
V346, G352, T353, G365, I368, I369, S372, S373, C378, F379, T385,
S386, Q387, S153, S188, I189, Q190, S191, D192, R194, E197, G198,
R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379,
I154, T187, H193, E195, I196, M201, V202, C223, T228, 5235, G236,
A239, G244, M247, I369, S372, C375, or C378. In some embodiments,
the antigen binding molecule binds within 30 angstroms of one or
more of the following residues (numbering in reference to SEQ ID
NO: 3): S153, I154, P155, R194, D238, A239, I369, S372, D374, C375,
T377, C378, F379, V380, S381, W156, N157, L158, E159, H193, E195,
H229, R237, G240, K243, D367, I368, G370, A371, S373, S376, or
Q382. In some embodiments, the antigen binding molecule binds
within 30 angstroms of one or more of the following residues
(numbering in reference to SEQ ID NO: 3): W72, F150, A151, Q152,
T214, R215, F216, H217, A220, S221, K222, S225, H226, C255, Q256,
G257, K258, N317, F318, T347, L348, G349, T350, L351, E366, D367,
D374, V380, S381, Q382, S383, G384, K69, D70, P71, S148, V149,
D186, T187, E211, D212, G213, R218, Q219, C223, D224, G227, H229,
L253, N254, G259, P288, A290, G291, G316, R319, Y325, V346, G352,
T353, G365, I368, I369, S372, S373, C378, F379, T385, S386, or
Q387. In some embodiments, the antigen binding molecule binds
within 30 angstroms of one or more of the following residues
(numbering in reference to SEQ ID NO: 3): S153, S188, I189, Q190,
S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243,
S373, D374, S376, T377, F379, I154, T187, H193, E195, I196, M201,
V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375,
or C378.
[0541] In some embodiments, the antigen binding molecule binds
within 30, 30-25, 25-20, 20-15, 15-8, 8, 8-5, 5, 5-4, 4 or less
angstroms from one or more of the above residues. In some
embodiments, the antigen binding molecule, when bound to PCSK9, is
within at least one of the above distances, for more than one of
the above noted residues. For example, in some embodiments, the
antigen binding molecule is within one of the recited distances
(e.g., 30, 30-25, 25-20, 20-15, 15-8, 8, 8-5, 5, 5-4, 4 or less)
for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55,
55-60, 60-65, 65-70, 70-75 or more of the above residues. In some
embodiments, the antigen binding molecule is within one of the
recited distances for at least 1-10, 10-20, 20-30, 30-40, 40-50,
50-60, 60-70, 70-80, 80-90, 90-95, 95-99, 99-100% of the residues
identified in each group of subgroup thereof (such as only those
surface residues in the group). Unless specifically stated
otherwise, the distance between the antigen binding molecule and
PCSK9 is the shortest distance between the covalently bonded atom
on PCSK9 and the covalently bonded atom of the antigen binding
molecule that are the closest atoms of PCSK9 and the antigen
binding molecule. Similarly, unless specifically stated otherwise,
the distance between a residue (on the antigen binding molecule or
PCSK9) and another protein (either PCSK9 or the antigen binding
molecule respectively), is the distance from the closest point on
the identified residue to the closest covalently bonded part of the
other protein. In some embodiments, the distance can be measured
from the backbone of the amino acid chains. In some embodiments,
the distance can be measured between an edge of the paratope and an
edge (closest to one another) of the epitope. In some embodiments,
the distance can be measured between the center of the surface of
the paratope and the center of the surface of the epitope. As will
be appreciated by one of skill in the art, the present description
is applicable for each of the individual sets of residues listed
herein. For example, the above ranges are contemplated generally
and specifically for the 8 angstrom residues listed in Examples
28-32 and the 5 angstrom residues listed in Examples 28-32.
[0542] In some embodiments, the antigen binding molecule binds to a
surface on PCSK9 that is bound by at least one of EGFa, 21B12, or
31H4. In some embodiments, the antigen binding molecule binds to
PCSK9 at a location that overlaps with the interaction locations
between PCSK9 and EFGa, Ab 31H4, and/or Ab 21B12 (as described in
the above examples and figures). In some embodiments, the antigen
binding molecule binds to PCSK9 at a position that is further away
from one of the above recited residues. In some embodiments, such
an antigen binding molecule can still be an effective neutralizing
antigen binding molecule.
[0543] In some embodiments, the structure of the catalytic domain
of PCSK9 can be described as generally being triangular (as shown
in FIG. 19A). The first side of the triangle is shown as being
bound by 31H4. The second side of the triangle is shown as being
bound by 21B12, and the third side of the triangle is positioned
toward the bottom of the page, immediately above the "FIG. 19A"
label. In some embodiments, antigen binding molecules that bind to
the first and/or second sides of the catalytic domain of PCSK9 can
be useful as neutralizing antibodies as they can either directly or
sterically interfere with EGFa's binding to PCSK9. As will be
appreciated by one of skill in the art, when the antigen binding
molecules are large enough, such as a full antibody, the antigen
binding molecule need not directly bind to the EGFa binding site in
order to interfere with the binding of EGFa to PCSK9.
[0544] As will be appreciated by one of skill in the art, while the
EGFa domain of the LDLR has been used in many of the examples, the
models and structures are still applicable to how the full length
LDLR protein will interact with PCSK9. Indeed, the additional
structure present on the full length LDLR protein presents
additional protein space that can further be blocked by one of the
antigen binding molecules. As such, if the antigen binding molecule
blocks or inhibits binding of EGFa to PCSK9, it will likely be at
least as, if not more, effective with the full length LDLR protein.
Similarly, antigen binding molecules that are within a set distance
or block various residues that are relevant for inhibiting EGFa
binding, will likely be as effective, if not more effective, for
the full length LDLR.
[0545] As will be appreciated by one of skill in the art, any
molecule that blocks or binds to the above noted PCSK9 residues (or
within the recited distances), or that inhibits one or more of the
interactions noted in the above examples and figures, can be used
to inhibit the interaction of EGFa (or LDLR generally) and PCSK9.
As such, the molecule need not be limited to an antigen binding
"protein," as any antigen binding molecule can also serve the
required purpose. Examples of antigen binding molecules include
aptamers, which can be either oligonucleic acid or peptide
molecules. Other examples of antigen binding molecules include
avimers, peptibodies, small molecules and polymers, and modified
versions of EGFa that can increase its affinity to PCSK9 and/or
half-life, such as mutation of amino acids, glycosylation,
pegylation, Fc fusions, and avimer fusions. As will be appreciated
by one of skill in the art, in some embodiments LDLR is not an
antigen binding molecule. In some embodiments, binding subsections
of LDLR are not antigen binding molecules, e.g., EGFa. In some
embodiments, other molecules through which PCSK9 signals in vivo
are not antigen binding molecules. Such embodiments will be
explicitly identified as such.
Example 33
Expression and Purification of Protein Samples
[0546] The present example describes some embodiments for how the
various embodiments of the PCSK9 proteins/variants were made and
purified (including the LDLR EGFa domain). PCSK9 proteins/variants
(e.g., PSCK9 31-692 N533A, PCSK9 449TEV and PCSK9 ProCat 31-454)
were expressed in baculovirus infected Hi-5 insect cells with an
N-terminal honeybee melittin signal peptide followed by a His.sub.6
tag. The PCSK9 proteins were purified by nickel affinity
chromatography, ion exchange chromatography and size exclusion
chromatography. The melittin-His.sub.6 tag was removed during
purification by cleavage with TEV protease. The construct PCSK9
449TEV was used to generate PCSK9 ProCat (31-449) and V domain
(450-692) samples. This construct had a TEV protease cleavage site
inserted between PCSK9 residues 449 and 450. For the full length
N555A variant for crystallography, the PCSK9 31-454 fragment, and
the PCSK9 449TEV variant for crystalography, the post rTEV protein
product also included an initial GAMG sequence. Thus, post rTEV
cleavage, these proteins were GAMG-PCSK9. Furthermore, the PCSK9
449TEV protein included the sequence "ENLYFQ" (SEQ ID NO: 403)
inserted between positions H449 and G450 of SEQ ID NO: 3. After
cleavage with rTEV, the PCSK9 ProCat protein generated from this
construct was GAMG-PCSK9 (31-449)-ENLYFQ and the V domain generated
from this construct was PCSK9 (450-692) of SEQ ID NO: 3.
[0547] The 21B12 and 31H4Fab fragments were expressed in E. coli.
These proteins were purified by nickel affinity chromatography,
size exclusion chromatography and ion exchange chromatography.
[0548] The LDLR EGFa domain (293-334) was expressed as a GST fusion
protein in E. coli. The EGFa domain was purified by ion exchange
chromatography, glutathione sepharose affinity chromatography and
size exclusion chromatography. The GST protein was removed during
the purification by cleavage with PreScission protease.
Example 34
Complex Formation and Crystallization
[0549] The present example describes how complexes and crystals
used in the above structure examination Examples were made.
[0550] The PCSK9 31-692 N533A/31H4 complex was made by mixing a 1.5
molar excess of the 31H4Fab with PCSK9. The complex was purified by
size exclusion chromatography to remove excess 31H4Fab. The PCSK9
31-692 N533A/31H4 complex crystallizes in 0.1 M Tris pH 8.3, 0.2 M
sodium acetate, 15% PEG 4000, 6% dextran sulfate sodium salt (Mr
5000).
[0551] The PCSK9 ProCat 31-449/31H4/21B12 complex was made by first
mixing a 1.5 molar excess of 31H4Fab with PCSK9 31-449. The complex
was separated from excess 31H4 by purification on a size exclusion
chromatography column. A 1.5 molar excess of 21B12 Fab was then
added to the PCSK9 31-449/31H4 complex. The ternary complex was
separated from excess 21B12 by purification on a size exclusion
chromatography column. The PCSK9 ProCat 31-449/31H4/21B12 complex
crystallizes in 0.1 M Tris pH 8.5, 0.2 M ammonium phosphate
monobasic, 50% MPD.
[0552] The PCSK9 ProCat 31-454/EGFa complex was made by mixing a
1.2 molar excess of EGFa domain with PCSK9 31-454. The PCSK9 ProCat
31-454/EGFa domain complex crystallizes in 0.2 M potassium formate,
20% PEG 3350.
Example 35
Data Collection and Structure Determination
[0553] The present example describes how the datasets were
collected and the structures determined for the above structure
examination Examples.
[0554] Initial datasets for the PCSK9 31-692 N533A/31H4 and PCSK9
ProCat 31-449/31H4/21B12 crystals were collected on a Rigaku FR-E
X-ray source. The PCSK9 ProCat 31-454/EGFa dataset and higher
resolution datasets for the PCSK9 31-692 N533A/31H4 and PCSK9
ProCat 31-449/31H4/21B12 crystals were collected at the Berkeley
Advanced Light Source beamline 5.0.2. All datasets were processed
with denzo/scalepack or HKL2000 (Otwinowski, Z., Borek, D.,
Majewski, W. & Minor, W. Multiparametric scaling of diffraction
intensities. Acta Crystallogr A 59, 228-34 (2003)).
[0555] PCSK9/31H4 crystals grew in the C2 space group with unit
cell dimensions a=264.9, b=137.4, c=69.9 .ANG.,
.beta.=102.8.degree. and diffract to 2.3 .ANG. resolution. The
PCSK9/31H4 structure was solved by molecular replacement with the
program MOLREP (The CCP4 suite: programs for protein
crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-3
(1994) using the PCSK9 structure (Piper, D. E. et al. The crystal
structure of PCSK9: a regulator of plasma LDL-cholesterol.
Structure 15, 545-52 (2007)) as the starting search model. Keeping
the PCSK9 31-692 solution fixed, an antibody variable domain was
used as a search model. Keeping the PCSK9 31-692/antibody variable
domain solution fixed, an antibody constant domain was used as a
search model. The complete structure was improved with multiple
rounds of model building with Quanta and refinement with cnx.
(Brunger, A. T. et al. Crystallography & NMR system: A new
software suite for macromolecular structure determination. Acta
Crystallogr D Biol Crystallogr 54, 905-21 (1998)).
[0556] PCSK9/31H4/21B12 crystals grew in the P2.sub.12.sub.12 space
group with unit cell dimensions a=138.7, b=246.2, c=51.3 .ANG. and
diffract to 2.8 .ANG. resolution. The PCSK9/31H4/21B12 structure
was solved by molecular replacement with the program MOLREP using
the PCSK9 ProCat/31H4 variable domain as the starting search model.
Keeping the PCSK9 ProCat/31H4 variable domain fixed, a search for
antibody constant domain was performed. Keeping the PCSK9
ProCat/31H4/21B12 constant domain fixed, an antibody variable
domain was used as a search model. The complete structure was
improved with multiple rounds of model building with Quanta and
refinement with cnx.
[0557] PCSK9/EGFa domain crystals grew in the space group
P6.sub.522 with unit cell dimensions a=b=70.6, c=321.8 .ANG. and
diffract to 2.9 .ANG. resolution. The PCSK9/EGFa domain structure
was solved by molecular replacement with the program MOLREP using
the PCSK9 ProCat as the starting search model. Analysis of the
electron density maps showed clear electron density for the EGFa
domain. The LDLR EGFa domain was fit by hand and the model was
improved with multiple rounds of model building with Quanta and
refinement with cnx.
[0558] Core interaction interface amino acids were determined as
being all amino acid residues with at least one atom less than or
equal to 5 .ANG. from the PCSK9 partner protein. 5 .ANG. was chosen
as the core region cutoff distance to allow for atoms within a van
der Waals radius plus a possible water-mediated hydrogen bond.
Boundary interaction interface amino acids were determined as all
amino acid residues with at least one atom less than or equal to 8
.ANG. from the PCSK9 partner protein but not included in the core
interaction list. Less than or equal to 8 .ANG. was chosen as the
boundary region cutoff distance to allow for the length of an
extended arginine amino acid. Amino acids that met these distance
criteria were calculated with the program PyMOL. (DeLano, W. L. The
PyMOL Molecular Graphics System. (Palo Alto, 2002)).
Example 36
Crystal Structure of PCSK9 and 31A4
[0559] The crystal structure of the 31A4/PCSK9 complex was
determined.
Expression and Purification of Protein Samples
[0560] PCSK9 449TEV (a PCSK9 construct with a TEV protease cleavage
site inserted between residue 449 and 450, numbering according to
SEQ ID NO: 3) was expressed in baculovirus infected Hi-5 insect
cells with an N-terminal honeybee melittin signal peptide followed
by a His.sub.6 tag. The PCSK9 protein was purified by first by
nickel affinity chromatography. TEV protease was used to remove the
melittin-His.sub.6 tag and cleave the PCSK9 protein between the
catalytic domain and V domain. The V domain was further purified by
ion exchange chromatography and size exclusion chromatography. The
31A4 Fab fragment was expressed in E. coli. This protein was
purified by nickel affinity chromatography, size exclusion
chromatography and ion exchange chromatography.
Complex Formation and Crystallization
[0561] The PCSK9 V domain/31A4 complex was made by mixing a 1.5
molar excess of PCSK9 V domain with 31A4 Fab. The complex was
separated from excess PCSK9 V domain by purification on a size
exclusion chromatography column. The PCSK9 V domain/31A4 complex
crystallized in 1.1 M Succinic acid pH 7, 2% PEG MME 2000.
Data Collection and Structure Determination
[0562] The dataset for the PCSK9 V domain/31A4 crystal was
collected on a Rigaku FR-E x-ray source and processed with
denzo/scalepack (Otwinowski, Z., Borek, D., Majewski, W. &
Minor, W. Multiparametric scaling of diffraction intensities. Acta
Crystallogr A 59, 228-34 (2003)).
[0563] PCSK9 V domain/31A4 crystals grow in the
P2.sub.12.sub.12.sub.1 space group with unit cell dimensions
a=74.6, b=131.1, c=197.9 .ANG. with two complex molecules per
asymmetric unit, and diffract to 2.2 .ANG. resolution. The PCSK9 V
domain/31A4 structure was solved by molecular replacement with the
program MOLREP (CCP4. The CCP4 suite: programs for protein
crystallography. Acta Crystallogr D Biol Crystallogr 50, 760-3
(1994)) using the V domain of the PCSK9 structure (Piper, D. E. et
al. The crystal structure of PCSK9: a regulator of plasma
LDL-cholesterol. Structure 15, 545-52 (2007)) as the starting
search model. Keeping the PCSK9 450-692 solution fixed, an antibody
variable domain was used as a search model. After initial
refinement, the antibody constant domains were fit by hand. The
complete structure was improved with multiple rounds of model
building with Quanta and refinement with cnx (Brunger, A. T. et al.
Crystallography & NMR system: A new software suite for
macromolecular structure determination. Acta Crystallogr D Biol
Crystallogr 54, 905-21 (1998)).
[0564] Core interaction interface amino acids were determined as
being all amino acid residues with at least one atom less than or
equal to 5 .ANG. from the PCSK9 partner protein. 5 .ANG. was chosen
as the core region cutoff distance to allow for atoms within a van
der Waals radius plus a possible water-mediated hydrogen bond.
Boundary interaction interface amino acids were determined as all
amino acid residues with at least one atom less than or equal to 8
.ANG. from the PCSK9 partner protein but not included in the core
interaction list. Less than or equal to 8 .ANG. was chosen as the
boundary region cutoff distance to allow for the length of an
extended arginine amino acid. Amino acids that met these distance
criteria were calculated with the program PyMOL (DeLano, W. L. The
PyMOL Molecular Graphics System. (Palo Alto, 2002)). Distances were
calculated using the V domain "A" and 31A4 "L1,H1" complex.
[0565] The crystal structure of the PCSK9 V domain bound to the Fab
fragment of 31A4 was determined at 2.2 .ANG. resolution. The
depictions of the crystal structure are provided in FIGS. 21A-21D.
FIGS. 21A-21C shows that the 31A4 Fab binds to the PCSK9 V domain
in the region of subdomains 1 and 2.
[0566] A model of full length PCSK9 bound the 31A4 Fab was made.
The structure of full length PCSK9 was overlaid onto the PCSK9 V
domain from the complex. A figure of this model is shown in FIG.
21D. The site of the interaction between the EGFa domain of the
LDLR and PCSK9 is highlighted.
[0567] Analysis of the structure shows where this antibody
interacts with PCSK9 and demonstrated that antibodies that do not
bind to the LDLR binding surface of PCSK9 can still inhibit the
degradation of LDLR that is mediated through PCSK9 (when the
results are viewed in combination with Example 40 and 41 below). In
addition, analysis of the crystal structure allows for
identification of specific amino acids involved in the interaction
between PCSK9 and the 31A4 antibody. Furthermore, the core and
boundary regions of the interface on the PCSK9 surface were also
determined. Specific core PCSK9 amino acid residues of the
interaction interface with 31A4 were defined as PCSK9 residues that
are within 5 .ANG. of the 31A4 protein. The core residues are T468,
R469, M470, A471, T472, R496, R499, E501, A502, Q503, R510, H512,
F515, P540, P541, A542, E543, H565, W566, E567, V568, E569, R592,
and E593. Boundary PCSK9 amino acid residues of the interaction
interface with 31A4 were defined as PCSK9 residues that are 5-8
.ANG. from the 31A4 protein. The boundary residues are as follows:
S465, G466, P467, A473, I474, R476, G497, E498, M500, G504, K506,
L507, V508, A511, N513, A514, G516, V536, T538, A539, A544, T548,
D570, L571, H591, A594, S595, and H597. Amino acid residues nearly
or completely buried within the PCSK9 protein are highlighted by
underline. As noted herein, the numbering references the amino acid
positions of SEQ ID NO: 3 (adjusted as noted herein).
[0568] Specific core 31A4 amino acid residues of the interaction
interface with PCSK9 were defined as 31A4 residues that are within
5 .ANG. of the PCSK9 protein. The core residues for the 31A4
antibody are as follows: Heavy Chain: G27, S28, F29, S30, A31, Y32,
Y33, E50, N52, H53, R56, D58, K76, G98, Q99, L100, and V101; Light
Chain: S31, N32, T33, Y50, S51, N52, N53, Q54, W92, and D94.
Boundary 31A4 amino acid residues of the interaction interface with
PCSK9 were defined as 31A4 residues that are 5-8 .ANG. from the
PCSK9 protein. The boundary residues for 31A4 are as follows: Heavy
Chain: V2, G26, W34, N35, W47, I51, S54, T57, Y59, A96, R97, P102,
F103, and D104; Light Chain: S26, S27, N28, G30, V34, N35, R55,
P56, K67, V91, D93, S95, N97, G98, and W99.
[0569] The crystal structure also displayed the spatial
requirements of this ABP in its interaction with PCSK9. As shown in
this structure, surprisingly, antibodies that bind to PCSK9 without
directly preventing PCSK9's interaction with the LDLR can still
inhibit PCSK9's function.
[0570] In some embodiments, any antigen binding protein that binds
to, covers, or prevents 31A4 from interacting with any of the above
residues can be employed to bind to or neutralize PCSK9. In some
embodiments, the ABP binds to or interacts with at least one of the
following PCSK9 (SEQ ID NO: 3) residues: T468, R469, M470, A471,
T472, R496, R499, E501, A502, Q503, R510, H512, F515, P540, P541,
A542, E543, H565, W566, E567, V568, E569, R592, and E593. In some
embodiments, the ABP is within 5 angstroms of one or more of the
above residues. In some embodiments, the ABP binds to or interacts
with at least one of the following PCSK9 (SEQ ID NO: 3) residues:
5465, G466, P467, A473, I474, R476, G497, E498, M500, G504, K506,
L507, V508, A511, N513, A514, G516, V536, T538, A539, A544, T548,
D570, L571, H591, A594, 5595, and H597. In some embodiments, the
ABP is 5 to 8 angstroms from one or more of the above residues. In
some embodiments, the ABP interacts, blocks, or is within 8
angstroms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,
25, 30, 35, 40, 45, or 50 of the above residues.
[0571] The coordinates for the crystal structures discussed in the
above Examples are are presented in Table 35.1 (full length PCSK9
and 31H4), Table 35.2 (PCSK9 and EGFa), Table 35.3 (PCSK9, 31H4,
and 21B12), and Table 35.4 (PCSK9 and 31A4). Antigen binding
proteins and molecules that interact with the relevant areas or
residues of the structure of PCSK9 (including those areas or
residues within 15, 15-8,8,8-5, 5, or fewer angstroms from where
EGFa, or the antibodies, interact with PCSK9) depicted in the
figures and/or their corresponding positions on the structures from
the coordinates are also contemplated.
[0572] The antibodies that are described in the coordinates were
raised in E. coli and thus possess some minor amino acid
differences from the fully human antibodies. The first residue in
the variable region was a glutamic acid instead of a glutamine for
the heavy and light chains of 21B12 and for the light chain for
31H4. In addition to the differences in the sequence of variable
region, there were also some differences in the constant region of
the antibodies described by the coordinates (again due to the fact
that the antibody was raised in E. coli). FIG. 22 highlights (via
underlining shading, or bold) the differences between the constant
regions of the 21B12, 31H4, and 31A4 Fabs (raised in E. coli) when
compared to SEQ ID NOs: 156, and 155. For 21B12 31H4, and 31A4, the
light chain constant sequence is similar to human lambda (SEQ ID
NO: 156). The underlined glycine residue is an insertion between
where the 21B12 and 31H4 variable sequences stop and the lambda
sequence starts.
[0573] For both 21B12 and 31H4, the heavy chain constant is similar
to human IgG4 (SEQ ID NO: 155). The highlighted differences in FIG.
22 are shown in Table 36.1:
TABLE-US-00016 TABLE 36.1 Crystal SEQ ID NO: 155 S C K R G E G S Q
K I T N D K R P S
[0574] In regard to 31A4, while it also has the same distinctions
noted above, there are three additional differences. As shown in
FIG. 22, there are two additional amino acids at the start, which
comes from incomplete processing of the signal peptide in E. coli
expression. In addition, there is one additional substitution in
the 31A4 heavy chain constant region when compared to SEQ ID NO:
155, which is the adjustment of a L (in SEQ ID NO: 155) to a H.
Finally, 31A4 does have a glutamine as the initial amino acid of
the Fab, rather than the adjustment to glutamic acid noted above
for 21B12 and 31H4.
[0575] For all three antibodies, the end of the heavy chain (boxed
in dark grey) differs as well, but the amino acids are not ordered
in the structure so they do not appear in the coordinates. As will
be appreciated by one of skill in the art, his-tags are not a
required part of the ABP and should not be considered as part of
the ABP's sequence, unless explicitly called out by reference to a
specific SEQ ID NO that includes a histidine tag and a statement
that the ABP sequence "includes the Histidine tag."
Example 37
Epitope Mapping--Binning
[0576] An alternative set of binning experiments was conducted in
addition to the set in Example 10. As in Example 10, ABPs that
compete with each other can be thought of as binding to the same
site on the target and in common parlance are said to "bin"
together.
[0577] A modification of the Multiplexed Binning method described
by Jia, et al (J. Immunological Methods, 288 (2004) 91-98) was
used. Individual bead codes of streptavidin-coated Luminex beads
was incubated in 100 ul 0.5 ug/ml biotinylated monovalent
mouse-anti-human IgG capture antibody (BD Pharmingen, #555785) for
1 hour at room temperature in the dark, then washed 3.times. with
PBSA, phosphate buffered saline (PBS) plus 1% bovine serum albumin
(BSA). Each bead code was separately incubated with 100 ul 2 ug/ml
anti-PCSK9 antibody (Coating Antibody) for 1 hour then washed
3.times. with PBSA. The beads were pooled then dispensed to a
96-well filter plate (Millipore, #MSBVN1250). 100 ul of 2 ug/ml
purified PCSK9 protein was added to half the wells. Buffer was
added to the other half as control. The reaction was incubated for
1 hour then washed. 100 ul of a 2 ug/ml anti-PCSK9 antibody
(Detection Ab) was added to all the wells, incubated for 1 hour
then washed. An irrelevant human-IgG (Jackson, #009-000-003) was
run as another control. 20 ul PE-conjugated monovalent
mouse-anti-human IgG (BD Pharmingen, #555787) was added to each
well and incubated for 1 hour then washed. Beads were resuspended
in 100 ul PBSA and a minimum of 100 events/bead code were collected
on the BioPlex instrument (BioRad).
[0578] Median Fluorescent Intensity (MFI) of the antibody pair
without PCSK9 was subtracted from signal of the corresponding
reaction containing PCSK9. For the antibody pair to be considered
bound simultaneously, and therefore in different bins, the
subtracted signal had to be greater than 3 times the signal of the
antibody competing with itself and the 3 times the signal of the
antibody competing with the irrelevant antibody.
[0579] The data from the above is depicted in FIGS. 23A-23D. The
ABPs fell into five bins. The shaded boxes indicate ABPs that can
bind simultaneously to PCSK9. The nonshaded boxes indicate those
ABPs that compete with each other for binding. A summary of the
results is shown in Table 37.1.
TABLE-US-00017 TABLE 37.1 BIN 1 BIN 2 BIN 3 BIN 4 BIN 5 01A12.2
27B2.1 16F12.1 11G1.5 30A4.1 03B6.1 27B2.5 22E2.1 03C4.1 13B5.1
09C9.1 12H11.1 27A6.1 13H1.1 17C2.1 28B12.1 31A4.1 21B12.2 28D6.1
31B12.1 23G1.1 31G11.1 25G4.1 31H4.1 26E10.1 08A1.2 11H4.1 08A3.1
11H8.1 11F1.1 19H9.2 26H5.1 27E7.1 27H5.1 30B9.1 02B5.1 23B5.1
27B2.6 09H6.1
[0580] Bins 1 (competes with ABP 21B12) and 3 (competes with 31H4)
are exclusive of each other; bin 2 competes with bins 1 and 3; and
Bin 4 does not compete with bins 1 and 3. Bin 5, in this example,
is presented as a "catch all" bin to describe those ABPs that do
not fit into the other bins. Thus, the above identified ABPs in
each of the binds are representative of different types of epitope
locations on PCSK9, some of which overlap with each other.
[0581] As will be appreciated by one of skill in the art, if the
reference ABP prevents the binding of the probe ABP then the
antibodies are said to be in the same bin. The order in which the
ABPs are employed can be important. If ABP A is employed as the
reference ABP and blocks the binding of ABP B the converse is not
always true: ABP B used as the reference ABP will not necessarily
block ABP A. There are a number of factors in play here: the
binding of an ABP can cause conformational changes in the target
which prevent the binding of the second ABP, or epitopes which
overlap but do not completely occlude each other may allow for the
second ABP to still have enough high-affinity interactions with the
target to allow binding. ABPs with a much higher affinity may have
a greater ability to bump a blocking ABP out of the way. In
general, if competition is observed in either order the ABPs are
said to bin together, and if both ABPs can block each other then it
is likely that the epitopes overlap more completely.
Example 38
Epitope Mapping--Western Blot
[0582] The present example demonstrates whether or not the epitopes
for the examined ABPs were linear or conformational. Denaturing
reducing and denaturing non-reducing western blots were run to
determine which antibodies have a conformational epitope.
Antibodies that bind to a denaturing reducing western blot have a
linear epitope and are not conformational. The results are
presented in FIG. 24A and FIG. 24B. For the blot, 0.5 ug/lane of
purified full-length human PCSK9 was run on a 4-12% NuPAGE Bis-Tris
gel and MES SDS Running Buffer. 1 ug/ml anti-PCSK9 antibodies,
except 0.5 ug/ml 31G11, were used to probe the blot. 1:5000
donkey-anti-human-IR700 secondary was used and read on a LiCOR
instrument. Antibody 13H1 bound to a linear epitope on the
pro-domain of PCSK9. All other antibodies displayed results that
were consistent with conformational epitopes. These gels split
apart the pro-domain from the rest of the protein, and the pro
domain ran at about 15 kDa. In addition, 3C4 and 31A4 appeared to
bind to conformational epitopes which were preserved by disulfide
bonds, as these antibodies bound to PCSK-9 under denaturing
conditions where the disulfide bonds had been preserved (left) but
reducing the samples (right) eliminated binding.
Example 39
Epitope Mapping--Arginine/Glutamic Acid Scanning
[0583] Representative ABPs from each bin (from Example 37) were
selected for further epitope analysis. An arginine/glutamic
acid-scanning strategy was performed for mapping ABP binding to
PCSK9. By way of background, this method determines if a residue is
part of the structural epitope, meaning those residues in the
antigen which contact or are buried by the antibody. Arginine and
glutamic acid sidechains are charged and bulky and can disrupt
antibody binding even if the mutated residue is not directly
involved in antibody binding.
Residue Selection
[0584] The crystal structure of PCSK9 was used to select the
residues to be mutated for epitope mapping. The method used to
choose residues to mutate involved both computational mechanisms
and interactive structure analysis. The PCSK9 structure contained
gaps of missing residues and was missing 30 amino acids in the N-
(i.e., the signal sequence) and 10 amino acids in the C-termini.
The internal missing residues were modeled onto the structure, but
the N- and C-terminal missing residues were not. The solvent
exposure ratio for each residue was calculated: the surface area of
each residue in the context of the protein (SA1) was divided by the
surface area of the residue in a trimer with flanking glycines
(SA2) with a conserved backbone structure. Residues with solvent
exposure ratio greater than 10% (R10) were selected as well as the
40 missing terminal residues. From these, prolines and glycines
with positive .PHI. angles were excluded to reduce the possibility
of misfolding. The number of residues to be mutated in the V domain
was reduced by using a solvent exposure ratio of 37% along with
visual inspection of the entire protein to bring the total number
of mutations to 285. Various orientations of the surface of PCSK9
with these various classes identifies are shown in FIG. 25A-25F. In
these figures, lightest gray denotes areas that were not selected
or were deselected darker gray denotes those residues
selected).
Cloning and Expression
[0585] Once the residues to be altered were identified, the various
residues were altered. Human PCSK9 was cloned into the pTT5 vector
with a C-terminal Flag-His tag. Mutants were made from this
original construct by site-directed mutagenesis using a QuikChange
II kit from Stratagene. Sense and anti-sense oligonucleotides used
for mutagenesis were designed using Amgen's MutaGenie software. All
PCSK9 constructs were expressed in transiently-transfected 293-6E
cells in 24-well plates and re-racked into three 96-well plates
with a non-mutated PCSK9 control (wild-type, WT) in each plate.
Expression levels and integrity of the recombinant proteins in
conditioned media were checked by Western blot. Of the 285 mutants
originally selected, 41 failed in cloning or expression. 244
mutants were used for epitope mapping. An alignment of the PCSK9
parent sequence and a representative PCSK9 sequence with the 244
mutated residues is shown in FIG. 26. Separate constructs were made
containing a single mutation. For the purposes of the epitope
sequences and the epitope based inventions involving changes in
binding, the sequences are provided in reference to SEQ ID NO: 1
and/or SEQ ID NO: 303. The sequences in FIG. 26 were the sequences
used for the present binding epitope studies. One of skill in the
art will appreciate that the present results apply to other PCSK9
variants disclosed herein as well (e.g., SEQ ID NO: 1 and 3, as
well as the other allelic variants).
[0586] Five antibodies, a representative of each bin, were chosen
for fine epitope mapping. They were 21B12, 31H4, 12H11, 31A4, 3C4.
All conformational epitope antibodies. Three, 21B12, 31H4, and 31A4
were also crystallized with PCSK9, as described above.
Structural and Functional Epitopes
[0587] Epitopes can be further defined as structural or functional.
Functional epitopes are generally a subset of the structural
epitopes and have those residues that directly contribute to the
affinity of the interaction (e.g. hydrogen bonds, ionic
interactions). Structural epitopes can be thought of as the patch
of the target which is covered by the antibody.
[0588] The scanning mutagenesis employed was an arginine and
glutamic acid scan. These two sidechains were chosen due to their
large steric bulk and their charge, which allows mutations that
occur in the structural epitope to have a greater effect on
antibody binding. Arginine was generally employed except when the
WT reside was arginine, and in these cases the residue was mutated
to glutamic acid to switch the charge.
[0589] For the purpose of epitope mapping, a bead-based multiplexed
assay was used to measure antibody binding to PCSK9 and PCSK9
mutants simultaneously. Antibody binding to mutants was then
compared to its binding to the wild-type in the same well. The
variants were split into three groups: Group 1: 81 variants+2 wt
controls+1 negative control+1 other PCSK9 supernatant; Group 2: 81
variants+2 wt controls+2 negative controls; and Group 3: 82
variants+2 wt control+1 negative control.
[0590] The assay was run as follows: 85 sets of color-coded
strepavidin-coated LumAvidin beads (Luminex) were bound with
biotinylated anti-pentaHis antibody (Qiagen, #1019225) for 1 hour
at room temperature (RT) then washed three times in PBS, 1% BSA,
0.1% Tween 20. Each color-coded bead set was then allowed to bind
to a PCSK9 mutant, wild-type, or negative control in 150 ul
supernatant overnight at 4.degree. C.
[0591] The color-coded bead sets, each associated to a specific
protein, were washed and pooled. At this point, there were 3 pools
of 85 bead sets, one pool for each group of mutants and controls.
The beads from each pool were aliquoted to 24 wells (3 columns) of
a 96-well filter plate (Millipore, #MSBVN1250). 100 ul of
anti-PCSK9 antibodies in 4-fold dilutions were added to nine
columns for triplicate points and incubated for 1 hour at RT and
washed. 100 ul of 1:200 dilution phycoerythrin (PE)-conjugated
anti-human IgG Fc (Jackson Immunoresearch, #109-116-170) was added
to each well and incubated for 1 hour at RT and washed.
[0592] Beads were resuspended in 1% BSA in PBS, shaken for 10 mins
and read on the BioPlex instrument (Bio-Rad). The instrument
identifies each bead by its color-code thereby identifying the
specific protein associated with the color code. At the same time,
it measures the amount of antibody bound to the beads by
fluorescence intensity of the PE dye. Antibody binding to each
mutant can then be compared directly its binding to the wild type
in the same pool. IL-17R chimera E was used as a negative control.
A summary of all of the mutants examined is shown in Table 39.1
(with reference to the sequence numbering used in FIGS. 1A and
26).
TABLE-US-00018 TABLE 39.1 1 2 3 4 5 6 7 8 9 10 11 12 A WT PCSK9 Y8R
E18R P26R A38R T56R A70R H83R E102R L128R D145R B Q1R E9R E19R E27R
K39R H57R Q71R V84R L105R E129R S148R C E2R E10R D20R G29R D40R
L58R A73R H86R K106R R130E pcsk9 supe test D D3R L11R G21R T30R
L44R Q60R R74E K95R H109R T132R IL17R chimera E E E4R V12R L22R
T31R T47R E62R R75E S97R D111R D139R WT PCSK9 F D5R A14R A23R A32R
K53R R63E Y77R G98R A121R E140R G G6R L15R E24R T33R E54R R66E L78R
D99R S123R Y141R H D7R S17R A25R H35R E55R R67E L82R L101R W126R
Q142R A WT PCSK9 M171R E181R Q189R K213R R242E G251R L294R L321R
Q352R E380R B L149R V172R D182R A190R G214R K243R G262R A311R E336R
M368R R384E C S158R T173R G183R S191R S216R S244R R265E Q312R D337R
S371R IL17R chimera E D Q160R D174R T184R K192R R221E Q245R A269R
D313R D344R A372R IL17R chimera E E S161R E176R R185E S195R Q226R
L246R Q272R Q314R T347R E373R WT PCSK9 F D162R N177R F186R H196R
K228R V247R R276E T317R F349R E375R G R164E V178R H187R R207E T230R
Q248R A277R L318R V350R T377R H E167R E180R R188E D208R F240R V250R
R289E T320R S351R L378R A WT PCSK9 N395R V405R W423R R446E E513R
Q525R Q554R Q589R S632R A641R B I386R E396R N409R Q424R D450R A514R
E537R N556R Q591R T633R R650E C H387R A397R A413R A433R A472R S515R
V538R K579R A595R T634R R652E D F388R W398R S417R H434R F485R M516R
E539R V580R E597R G635R IL17R chimera E E A390R E401R T418R T438R
G486R R519E L541R K581R E598R S636R WT PCSK9 F K391R D402R H419R
R439E E488R H521R H544R E582R V620R T637R G D392R Q403R G420R M440R
N503R H523R V548R H583R R629E S638R H V393R R404E A421R T442R T508R
Q524R R552E G584R V631R E639R
Bead Variability Study
[0593] Before running the epitope mapping binding assay, a
validation experiment was conducted to assess the "bead region" to
"bead region" (B-B) variability. In the validation experiment, all
beads were conjugated with the same wild type control protein.
Therefore, the difference between beads regions was due to purely
B-B variance and was not confounded by difference between wild type
and mutant proteins. The titration of antibody was run with twelve
replications in different wells.
[0594] The objective of this statistical analysis was to estimate
the B-B variability of the estimated EC50 of binding curves. The
estimated B-B standard deviation (SD) was then used to build the
EC50 confidence intervals of wild type and mutant proteins during
curve comparison experiments.
[0595] A four-parameter logistic model was fitted to the binding
data for each bead region. The resulting file, containing curve
quality control (QC) results and parameter estimates for top (max),
bottom (min), Hillslope (slope), and natural log of EC50 (xmid) of
the curves, was used as the raw data for the analysis. B-B
variability for each parameter was then estimated by fitting mixed
effect model using SAS PROC MIXED procedure. Only curves with
"good" QC status were included in the analysis. The final mixed
effect model included only residual (i.e. individual bead regions)
as random effect. Least squares means (LS-mean) for each parameter
were estimated by the mixed effect model as well. B-B SD was
calculated by taking square root of B-B variance. Fold change
between LS-mean+2SD and LS-mean-2SD, which represent approximately
upper and lower 97.5 percentile of the population, was also
calculated. The results are displayed in Table 39.2
TABLE-US-00019 TABLE 39.2 Least square mean and bead-to-bead
variance estimations Assay Ls B-B Fold ID parname Mean Variance
-2SD +2SD Change* PCSK9 max 15000 997719 13002.3 16997.7 1.3 PCSK9
min 162.09 1919.66 74.5 249.7 3.4 PCSK9 slope 0.8549 0.000599 0.8
0.9 1.1 PCSK9 xmid 3.1715 0.002098 3.1 3.3 1.2 *xmid is natural log
of the EC50. Fold change for xmid was converted back to original
scale.
Identifying Residues in the Structural Epitope
[0596] A residue was considered part of the structural epitope (a
"hit") when mutating it to arginine or glutamic acid alters
antibody binding. This is seen as a shift in the EC50 or a
reduction of maximum signal compared to antibody binding to wild
type. Statistical analyses of antibody binding curves to wild type
and mutants were used to identify statistically significant EC50
shifts. The analysis takes into consideration variation in the
assay and curve fitting.
[0597] Hit Identification Based on EC50 Comparison
[0598] The EC50 and Bmax values were generated from a Weighted
4-Parameter Logistical model fitted to the binding data using
S-PLUS with VarPower software (Insightful Corporation, Seattle
Wash.). The EC50s of the mutant binding curves and wild type
binding curves were compared. Statistically significant differences
were identified as hits for further consideration. The curves with
"nofit" or "badfit" flags were excluded from the analysis.
[0599] The Variations in EC50 Estimates
[0600] Two sources of variations were considered in the comparison
of EC50 estimates, variation from the curve fit and the bead-bead
variation. Wild types and mutants were linked to different beads,
hence their difference are confounded with the bead-bead difference
(described above). The curve fit variation was estimated by the
standard error of the log EC50 estimates. Bead-bead variation was
experimentally determined using an experiment where wild type
controls were linked to each one of the beads (described above).
The bead variation in EC50 estimates of wild type binding curve
from this experiment was used to estimate the bead-bead variation
in the actual epitope mapping experiment.
[0601] Testing for EC50 Shift Between Mutants and Wild Type
[0602] The comparisons of two EC50s (in log scale) was conducted
using Student's t-test. The t-statistic was calculated as the ratio
between delta (the absolute differences between EC50 estimates) and
the standard deviation of delta. The variance of delta was
estimated by the sum, of the three components, variance estimate of
EC50 for mutant and wild type curves in the nonlinear regression
and two times the bead-bead variance estimated from a separate
experiment. The multiple of two for the bead-bead variance was due
to the assumption that both mutant and wild type beads had the same
variance. The degree of freedom of the standard deviation of delta
was calculated using the Satterthwaite's (1946) approximation.
Individual p-values and confidence intervals (95% and 99%) were
derived based on Student's t distribution for each comparison. In
the case of multiple wild type controls, a conservative approach
was taken by picking the wild type control that was most similar to
the mutant, i.e., picking the ones with the largest p-values.
[0603] Multiplicity adjustments were important to control the false
positive(s) while conducting a large number of tests
simultaneously. Two forms of multiplicity adjustment were
implemented for this analysis: family wise error (FWE) control and
false discovery rate (FDR) control. The FWE approach controls the
probability that one or more hits are not real; FDR approach
controls the expected proportion of false positive among the
selected hits. The former approach is more conservative and less
powerful than the latter one. There are many methods available for
both approaches, for this analysis, the Hochberg's (1988) method
for FWE analysis and Benjamini-Hochberg's (1995) FDR method for FDR
analysis were selected. Adjusted p-values for both approaches were
calculated.
[0604] Results
[0605] EC50 Shift
[0606] Mutations whose EC50 is significantly different from wild
type, e.g., having a False Discovery Rate adjusted p-value for the
whole assay of 0.01 or less, were considered part of the structural
epitope. All the hits also had a Familywise type I error rate
adjusted p-value for each antibody of less than 0.01 except residue
R185E for antibody 31H4 which had an FWE adjusted p-value per
antibody of 0.0109. The residues in the structural epitope of the
various antibodies determined by EC50 shift are shown in Table 39.3
(point mutations are with reference to SEQ ID NO: 1 and 303)
TABLE-US-00020 TABLE 39.3 FDR FWE Adjusted Adjusted Antibody
Mutation Pval By Pval Low99 Low95 FoldChange High95 High99 RawPval
21B12 D208R 0.0000 0.0000 0.3628 0.3844 0.4602 0.5509 0.5837 0.0000
21B12 R207E 0.0000 0.0000 1.7148 1.8488 2.3191 2.9090 3.1364 0.0000
31H4 R185E 0.0024 0.0109 1.2444 1.3525 1.7421 2.2439 2.4388 0.0000
31A4 E513R 0.0001 0.0003 1.4764 1.6219 2.1560 2.8660 3.1485 0.0000
31A4 E539R 0.0000 0.0000 1.6014 1.7461 2.2726 2.9578 3.2252 0.0000
31A4 R439E 0.0000 0.0000 3.1565 3.6501 5.5738 8.5113 9.8420 0.0000
31A4 V538R 0.0004 0.0013 1.4225 1.5700 2.1142 2.8471 3.1423 0.0000
12H11 A390R 0.0000 0.0001 1.4140 1.5286 1.9389 2.4594 2.6588 0.0000
12H11 A413R 0.0009 0.0028 1.2840 1.3891 1.7653 2.2434 2.4269 0.0000
12H11 S351R 0.0009 0.0028 1.2513 1.3444 1.6761 2.0896 2.2452 0.0000
12H11 T132R 0.0000 0.0001 1.3476 1.4392 1.7631 2.1599 2.3068 0.0000
3C4 E582R 0.0016 0.0069 1.3523 1.5025 2.0642 2.8359 3.1509
0.0000
Maximum Signal Reduction
[0607] The percent maximum signal was calculated using the maximum
signal from the curve fitting (BmaxPerWT) and raw data point
(RawMaxPerWT). Mutations that reduced the antibody binding maximum
signal by .gtoreq.70% as compared to to wild type signal or that
reduced the signal of one antibody compared to other antibodies by
>50% when all other antibodies are at least 40% of wild type
were considered hits and part of the epitope. Table 39.4 displays
the residues that are in the structural epitope (italics) as
determined by reduction of maximum signal.
TABLE-US-00021 TABLE 39.4 antibody Mutants BmaxPerWT RawMaxPerWT
21B12 A311R 141.6388 139.7010 31H4 A311R 145.2189 147.8244 31A4
A311R 103.4377 96.2214 12H11 A311R 14.9600 3C4 A311R 129.0460
131.2060 21B12 D162R 7.0520 31H4 D162R 108.8308 112.4904 31A4 D162R
98.8873 95.9268 12H11 D162R 94.6280 97.4928 3C4 D162R 101.4281
100.1586 21B12 D313R 45.8356 45.0011 31H4 D313R 45.6242 44.9706
31A4 D313R 47.9728 44.7741 12H11 D313R 16.1811 18.4262 3C4 D313R
58.5269 57.6032 21B12 D337R 61.9070 62.2852 31H4 D337R 63.1604
64.1029 31A4 D337R 62.9124 59.4852 12H11 D337R 10.8443 3C4 D337R
73.0326 73.9961 21B12 E129R 139.9772 138.9671 31H4 E129R 141.6792
139.1764 31A4 E129R 77.3005 74.8946 12H11 E129R 28.6398 29.3751 3C4
E129R 85.7701 85.7802 21B12 E167R 15.1082 31H4 E167R 127.4479
128.2698 31A4 E167R 115.3403 112.6951 12H11 E167R 111.0979 109.6813
3C4 E167R 109.3223 108.7864 21B12 H521R 133.8480 133.9791 31H4
H521R 130.2068 128.4879 31A4 H521R 124.5091 129.3218 12H11 H521R
130.7979 134.4355 3C4 H521R 22.1077 21B12 Q554R 125.9594 125.2103
31H4 Q554R 122.2045 128.7304 31A4 Q554R 113.6769 121.3369 12H11
Q554R 116.1789 118.4170 3C4 Q554R 31.8416 21B12 R164E 17.3807
19.8505 31H4 R164E 97.8218 99.6673 31A4 R164E 98.2595 96.3352 12H11
R164E 88.0067 89.8807 3C4 R164E 105.0589 105.7286 21B12 R519E
139.4598 141.2949 31H4 R519E 135.5609 140.0000 31A4 R519E 134.2303
137.1110 12H11 R519E 135.4755 137.0824 3C4 R519E 44.0091 21B12
S123R 87.6431 88.1356 31H4 S123R 85.5312 84.7668 31A4 S123R 68.4371
66.6131 12H11 S123R 20.8560 20.6910 3C4 S123R 73.6475 71.5959
(Point mutations are with reference to SEQ ID NO: 1 and FIG.
26).
[0608] Table 39.5 displays a summary of all of the hits for the
various antibodies.
TABLE-US-00022 TABLE 39.5 EC50 shift hits Bmax shift hits 21B12
31H4 31A4 12H11 3C4 21B12 31H4 31A4 12H11 3C4 R207E R185E R439E
T132R E582R D162R S123R R519E D208R* E513R S351R R164E E129R H521R
V538R A390R E167R A311R Q554R E539R A413R D313R D337R *decreases
EC50
[0609] To further examine how these residues form part of or all of
the relevant epitopes, the above noted positions were mapped onto
various crystal structure models, the results are shown in FIG. 27A
through 27E. FIG. 27A depicts the 21B12 epitope hits, as mapped
onto a crystal structure of PCSK9 with the 21B12 antibody. The
structure identifies PCSK9 residues as follows: light gray
indicates those residues that were not mutated (with the exception
of those residues that are explicitly indicated on the structure)
and darker gray indicates those residues mutated (a minority of
which failed to express). Residues that are explicitly indicated
were tested (regardless of the shading indicated on the figure) and
resulted in a significant change in EC50 and/or Bmax The epitope
hits were based on Bmax shift. In this figure, 31H4 is behind 21
B12.
[0610] FIG. 27B depicts the 31H4 epitope hits, as mapped onto a
crystal structure of PCSK9 with 31H4 and 21B12 antibodies. The
structure identifies PCSK9 residues as follows: light gray
indicates those residues that were not mutated (with the exception
of those residues that are explicitly indicated on the structure)
and darker gray indicates those residues mutated (a minority of
which failed to express). Residues that are explicitly indicated
were tested (regardless of the shading indicated on the figure) and
resulted in a significant change in EC50 and/or Bmax. The epitope
hits were based on the EC50 shift.
[0611] FIG. 27C depicts the 31A4 epitope hits, as mapped onto a
crystal structure of PCSK9 with 31H4 and 21B12 antibodies. The
structure identifies PCSK9 residues as follows: light gray
indicates those residues that were not mutated (with the exception
of those residues that are explicitly indicated on the structure)
and darker gray indicates those residues mutated (a minority of
which failed to express). Residues that are explicitly indicated
were tested (regardless of the shading indicated on the figure) and
resulted in a significant change in EC50 and/or Bmax. The epitope
hits were based on the EC50 shift. 31A4 antibody is known to bind
to the V-domain of PCSK9, which appears consistent with the results
presented in FIG. 27C.
[0612] FIG. 27D depicts the 12H11 epitope hits, as mapped onto the
crystal structure of PCSK9 with 31H4 and 21B12 antibodies. The
structure identifies PCSK9 residues as follows: light gray
indicates those residues that were not mutated (with the exception
of those residues that are explicitly indicated on the structure)
and darker gray indicates those residues mutated (a minority of
which failed to express). Residues that are explicitly indicated
were tested (regardless of the shading indicated on the figure) and
resulted in a significant change in EC50 and/or Bmax. 12H11
competes with 21B12 and 31H4 in the binning assay described
above.
[0613] FIG. 27E depicts the 3C4 epitope hits, as mapped onto the
crystal structure of PCSK9 with 31H4 and 21B12 antibodies. The
structure identifies PCSK9 residues as follows: light gray
indicates those residues that were not mutated (with the exception
of those residues that are explicitly indicated on the structure)
and darker gray indicates those residues mutated (a minority of
which failed to express). Residues that are explicitly indicated
were tested (regardless of the shading indicated on the figure) and
resulted in a significant change in EC50 and/or Bmax.
[0614] 3C4 does not compete with 21B12 and 31H4 in the binning
assay. 3C4 binds to the V-domain in the domain binding assay (see
results from Example 40, FIGS. 28A and 28B).
[0615] While there were approximately a dozen mutants that could
have been expected to have an effect on binding (based upon the
crystal structure), the present experiment demonstrated that,
surprisingly, they did not. As will be appreciated by one of skill
in the art, the results presented above are in good agreement with
the crystal structures and PCSK-9's binding of these antibodies.
This demonstrates that the provided structural and corresponding
functional data adequately identifies the key residues and areas of
interaction of the neutralizing ABPs and PCSK9. Thus, variants of
the ABPs that possess the ability to bind to the above noted areas
are adequately provided by the present description.
[0616] As will be appreciated by one of skill in the art, while the
B-max drop and EC50 shift hits can be considered manifestations of
the same phenomenon, strictly speaking, a B-max drop alone does not
reflect a loss of affinity per se but, rather, the destruction of
some percentage of the epitope of an antibody. Although there is no
overlap in the hits determined by B-max and EC50, mutations with a
strong affect on binding may not allow for the generation of a
useful binding curve and hence, no EC50 can be determined for such
variants.
[0617] As will be appreciated by one of skill in the art, ABPs in
the same bin (with the exception of bin 5, which as noted above, is
a general catch all bin) likely bind to overlapping sites on the
target protein. As such, the above epitopes and relevant residues
can generally be extended to all such ABPs in the same bin.
[0618] To further examine the above results in regard to ABP 31H4,
position E181R, which, according to the above crystal structure,
was predicted to interact with R185 to form part of the surface
that interacts with the ABP, was also altered (E181R). The results,
while not statistically significant on their own, were, when
combined with the crystal structure, demonstrative of 31H4
interacting with E181R (data not shown). Thus, position 181 also
appears to form part of the epitope for the 31H4 ABP.
[0619] As noted above, the above binding data and epitope
characterization references a PCSK9 sequence (SEQ ID NO: 1) that
does not include the first 30 amino acids of PCSK9. Thus, the
numbering system of this protein fragment, and the SEQ ID NO:s that
refer to this fragment, are shifted by 30 amino acids compared to
the data and experiments that used a full length PCSK9 numbering
system (such as that used in the crystal study data described
above). Thus, to compare these results, an extra 30 amino acids
should be added to the positions in each of the above epitope
mapping results. For example, position 207 of SEQ ID NO: 1 (or SEQ
ID NO: 303), correlates to position 237 of SEQ ID NO: 3 (the full
length sequence, and the numbering system used throughout the rest
of the specification). Table 39.6 outlines how the above noted
positions, which reference SEQ ID NO: 1 (and/or SEQ ID NO: 303)
correlate with SEQ ID NO: 3 (which includes the signal
sequence).
TABLE-US-00023 TABLE 39.6 AMINO ACID POSITION IN AMINO ACID
POSITION SEQ ID NO: 1 IN SEQ ID NO: 3 (EPITOPE DATA) (EPITOPE DATA)
207 237 208 238 185 215 181 211 439 469 513 543 538 568 539 569 132
162 351 381 390 420 413 443 582 612 162 192 164 194 167 197 123 153
129 159 311 341 313 343 337 367 519 549 521 551 554 584
[0620] Thus, those embodiments described herein with reference to
SEQ ID NO: 1 can also be described, by their above noted
corresponding position with reference to SEQ ID NO: 3.
Example 40
PCSK9 Domain Binding Assay
[0621] The present example examined where on PCSK9 the various ABPs
bound.
[0622] Clear, 96 well maxisorp plates (Nunc) were coated overnight
with 2 ug/ml of various anti-PCSK9 antibodies diluted in PBS.
Plates were washed thoroughly with PBS/0.05% Tween-20 and then
blocked for two hours with 3% BSA/PBS. After washing, plates were
incubated for two hours with either full length PCSK9 (aa 31-692
SEQ ID NO: 3, procat PCSK9 (aa 31-449 SEQ ID NO: 3) or v-domain
PCSK9 (aa 450-692 of SEQ ID NO: 3) diluted in general assay diluent
(Immunochemistry Technologies, LLC). Plates were washed and a
rabbit polyclonal biotinylated anti-PCSK9 antibody (D8774), which
recognizes the procat and v-domain as well as full-length PCSK9,
was added at 1 ug/ml (in 1% BSA/PBS). Bound full-length, procat or
v-domain PCSK9 was detected by incubation with neutravidin-HRP
(Thermo Scientific) at 200 ng/ml (in 1% BSA/PBS) followed by TMB
substrate (KPL) and absorbance measurement at 650 nm. The results,
presented in FIGS. 28A and 28B, demonstrate the ability of the
various ABS to bind to various parts of PCSK9. As shown in FIG.
28B, ABP 31A4 binds to the V domain of PCSK9.
Example 41
Neutralizing, Non-Competitive Antigen Binding Proteins
[0623] The present example demonstrates how to identify and
characterize an antigen binding protein that is non-competitive
with LDLR for binding with PCSK9, but is still neutralizing towards
PCSK9 activity. In other words, such an antigen binding protein
will not block PCSK9 from binding to LDLR, but will prevent or
reduce PCSK9 mediated LDLR degradation.
[0624] Clear, 384 well plates (Costar) were coated with 2 ug/ml of
goat anti-LDL receptor antibody (R&D Systems) diluted in buffer
A (100 mM sodium cacodylate, pH 7.4). Plates were washed thoroughly
with buffer A and then blocked for 2 hours with buffer B (1% milk
in buffer A). After washing, plates were incubated for 1.5 hours
with 0.4 ug/ml of LDL receptor (R&D Systems) diluted in buffer
C (buffer B supplemented with 10 mM CaCl.sub.2). Concurrent with
this incubation, 20 ng/ml of biotinylated D374Y PCSK9 was incubated
with 100 ng/ml of antibody diluted in buffer A or buffer A alone
(control). The LDL receptor containing plates were washed and the
biotinylated D374Y PCSK9/antibody mixture was transferred to them
and incubated for 1 hour at room temperature. Binding of the
biotinylated D374Y to the LDL receptor was detected by incubation
with streptavidin-HRP (Biosource) at 500 ng/ml in buffer C followed
by TMB substrate (KPL). The signal was quenched with 1N HCl and the
absorbance read at 450 nm. The results are presented in FIG. 28C,
which shows that while ABP 31H4 inhibits LDLR binding, ABP 31A4
does not inhibit LDLR binding to PCSK9. In combination with the
results from Example 40 and shown in FIGS. 28A and 28B, it is clear
that 31A4 ABP binds to the V domain of PCSK9 and does not block the
interaction of PCSK9 with LDLR.
[0625] Next, the Ability of ABP 31A4 to serve as a neutralizing ABP
was further confirmed via a cell LDL uptake assay (as described in
the examples above). The results of this LDL uptake assay are
presented in FIG. 28D. As shown in FIG. 28D, ABP 31 A4 displays
significant PCSK9 neutralizing ability. Thus, in light of Example
40 and the present results, it is clear that ABPs can bind to PCSK9
without blocking the PCSK9 and LDLR binding interaction, while
still being useful as neutralizing PCSK9 ABPs.
INCORPORATION BY REFERENCE
[0626] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated herein by reference in their entirety. To the extent
that any of the definitions or terms provided in the references
incorporated by reference differ from the terms and discussion
provided herein, the present terms and definitions control.
EQUIVALENTS
[0627] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The foregoing description and examples detail certain
preferred embodiments of the invention and describe the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
5751662PRTHomo sapiens 1Gln Glu Asp Glu Asp Gly Asp Tyr Glu Glu Leu
Val Leu Ala Leu Arg1 5 10 15 Ser Glu Glu Asp Gly Leu Ala Glu Ala
Pro Glu His Gly Thr Thr Ala 20 25 30 Thr Phe His Arg Cys Ala Lys
Asp Pro Trp Arg Leu Pro Gly Thr Tyr 35 40 45 Val Val Val Leu Lys
Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr 50 55 60 Ala Arg Arg
Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys65 70 75 80 Ile
Leu His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met 85 90
95 Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr
100 105 110 Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp
Asn Leu 115 120 125 Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu
Tyr Gln Pro Pro 130 135 140 Asp Gly Gly Ser Leu Val Glu Val Tyr Leu
Leu Asp Thr Ser Ile Gln145 150 155 160 Ser Asp His Arg Glu Ile Glu
Gly Arg Val Met Val Thr Asp Phe Glu 165 170 175 Asn Val Pro Glu Glu
Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys 180 185 190 Cys Asp Ser
His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp 195 200 205 Ala
Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn 210 215
220 Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu
Phe225 230 235 240 Ile Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro
Leu Val Val Leu 245 250 255 Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val
Leu Asn Ala Ala Cys Gln 260 265 270 Arg Leu Ala Arg Ala Gly Val Val
Leu Val Thr Ala Ala Gly Asn Phe 275 280 285 Arg Asp Asp Ala Cys Leu
Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile 290 295 300 Thr Val Gly Ala
Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr305 310 315 320 Leu
Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu 325 330
335 Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln
340 345 350 Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala
Ala Met 355 360 365 Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu
Leu Arg Gln Arg 370 375 380 Leu Ile His Phe Ser Ala Lys Asp Val Ile
Asn Glu Ala Trp Phe Pro385 390 395 400 Glu Asp Gln Arg Val Leu Thr
Pro Asn Leu Val Ala Ala Leu Pro Pro 405 410 415 Ser Thr His Gly Ala
Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser 420 425 430 Ala His Ser
Gly Pro Thr Arg Met Ala Thr Ala Ile Ala Arg Cys Ala 435 440 445 Pro
Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys 450 455
460 Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys
Arg465 470 475 480 Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala
Ile Ala Arg Cys 485 490 495 Cys Leu Leu Pro Gln Ala Asn Cys Ser Val
His Thr Ala Pro Pro Ala 500 505 510 Glu Ala Ser Met Gly Thr Arg Val
His Cys His Gln Gln Gly His Val 515 520 525 Leu Thr Gly Cys Ser Ser
His Trp Glu Val Glu Asp Leu Gly Thr His 530 535 540 Lys Pro Pro Val
Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly545 550 555 560 His
Arg Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu 565 570
575 Glu Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Gly Gln Val
580 585 590 Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser
Ala Leu 595 600 605 Pro Gly Thr Ser His Val Leu Gly Ala Tyr Ala Val
Asp Asn Thr Cys 610 615 620 Val Val Arg Ser Arg Asp Val Ser Thr Thr
Gly Ser Thr Ser Glu Glu625 630 635 640 Ala Val Thr Ala Val Ala Ile
Cys Cys Arg Ser Arg His Leu Ala Gln 645 650 655 Ala Ser Gln Glu Leu
Gln 660 22076DNAHomo sapiens 2atgggcaccg tcagctccag gcggtcctgg
tggccgctgc cactgctgct gctgctgctg 60ctgctcctgg gtcccgcggg cgcccgtgcg
caggaggacg aggacggcga ctacgaggag 120ctggtgctag ccttgcgctc
cgaggaggac ggcctggccg aagcacccga gcacggaacc 180acagccacct
tccaccgctg cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg
240gtgctgaagg aggagaccca cctctcgcag tcagagcgca ctgcccgccg
cctgcaggcc 300caggctgccc gccggggata cctcaccaag atcctgcatg
tcttccatgg ccttcttcct 360ggcttcctgg tgaagatgag tggcgacctg
ctggagctgg ccttgaagtt gccccatgtc 420gactacatcg aggaggactc
ctctgtcttt gcccagagca tcccgtggaa cctggagcgg 480attacccctc
cgcggtaccg ggcggatgaa taccagcccc ccgacggagg cagcctggtg
540gaggtgtatc tcctagacac cagcatacag agtgaccacc gggaaatcga
gggcagggtc 600atggtcaccg acttcgagaa tgtgcccgag gaggacggga
cccgcttcca cagacaggcc 660agcaagtgtg acagtcatgg cacccacctg
gcaggggtgg tcagcggccg ggatgccggc 720gtggccaagg gtgccagcat
gcgcagcctg cgcgtgctca actgccaagg gaagggcacg 780gttagcggca
ccctcatagg cctggagttt attcggaaaa gccagctggt ccagcctgtg
840gggccactgg tggtgctgct gcccctggcg ggtgggtaca gccgcgtcct
caacgccgcc 900tgccagcgcc tggcgagggc tggggtcgtg ctggtcaccg
ctgccggcaa cttccgggac 960gatgcctgcc tctactcccc agcctcagct
cccgaggtca tcacagttgg ggccaccaat 1020gcccaggacc agccggtgac
cctggggact ttggggacca actttggccg ctgtgtggac 1080ctctttgccc
caggggagga catcattggt gcctccagcg actgcagcac ctgctttgtg
1140tcacagagtg ggacatcaca ggctgctgcc cacgtggctg gcattgcagc
catgatgctg 1200tctgccgagc cggagctcac cctggccgag ttgaggcaga
gactgatcca cttctctgcc 1260aaagatgtca tcaatgaggc ctggttccct
gaggaccagc gggtactgac ccccaacctg 1320gtggccgccc tgccccccag
cacccatggg gcaggttggc agctgttttg caggactgtg 1380tggtcagcac
actcggggcc tacacggatg gccacagcca tcgcccgctg cgccccagat
1440gaggagctgc tgagctgctc cagtttctcc aggagtggga agcggcgggg
cgagcgcatg 1500gaggcccaag ggggcaagct ggtctgccgg gcccacaacg
cttttggggg tgagggtgtc 1560tacgccattg ccaggtgctg cctgctaccc
caggccaact gcagcgtcca cacagctcca 1620ccagctgagg ccagcatggg
gacccgtgtc cactgccacc aacagggcca cgtcctcaca 1680ggctgcagct
cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg
1740ccacgaggtc agcccaacca gtgcgtgggc cacagggagg ccagcatcca
cgcttcctgc 1800tgccatgccc caggtctgga atgcaaagtc aaggagcatg
gaatcccggc ccctcagggg 1860caggtgaccg tggcctgcga ggagggctgg
accctgactg gctgcagcgc cctccctggg 1920acctcccacg tcctgggggc
ctacgccgta gacaacacgt gtgtagtcag gagccgggac 1980gtcagcacta
caggcagcac cagcgaagag gccgtgacag ccgttgccat ctgctgccgg
2040agccggcacc tggcgcaggc ctcccaggag ctccag 20763692PRTHomo sapiens
3Met 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 Ile 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 Gly 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 Glu 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
4112PRTHomo sapiens 4Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90
95 Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105 110 5112PRTHomo sapiens 5Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15 Glu Pro Pro Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn
Phe Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Glu Ile65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
Gln Val 85 90 95 Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys 100 105 110 6107PRTHomo sapiens 6Asp 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 Ser Ile Ser Ser Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln 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 Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 7107PRTHomo sapiens 7Asp 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 Arg Ile Ser Asn Tyr 20 25 30 Leu Ser Trp
Tyr Leu Gln Lys Pro Gly Ile Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln 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
Ser65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
Thr Pro Leu 85 90 95 Ile Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 8107PRTHomo sapiens 8Asp 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 Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Ser Leu Gln 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 Ser Thr Pro Ile
85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
9107PRTHomo sapiens 9Asp Ile Leu 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 Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
Ser Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 10107PRTHomo sapiens 10Asp 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 Ser Ile Ser Ile Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Tyr Leu Leu Ile 35 40 45 Tyr Ala Ala
Ala Ser Leu Gln 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 Ser Ala Pro Ile
85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
11111PRTHomo sapiens 11Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Gly Ala Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser
Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn
Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90
95 Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 110 12111PRTHomo sapiens 12Gln Ser Val Leu Thr Gln Pro Pro Ser
Val Ser Gly Ala Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Thr
Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Ser
Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75
80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser
85 90 95 Leu Ser Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 110 13111PRTHomo sapiens 13Gln Ser Val Leu Thr Gln Pro
Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser
Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala His 20 25 30 Tyr Asp Val
His Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu
Ile Tyr Gly Asn Thr Tyr Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr
Asp Asn Ser 85 90 95 Leu Ser Gly Val Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 110 14108PRTHomo sapiens 14Gln Ser Ala Leu
Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30
Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser
Ser Tyr Thr Ser Ser 85 90 95 Ser Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 15109PRTHomo sapiens 15Gln Ser Ala Leu Thr Gln
Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile
Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Arg Tyr 20 25 30 Asn Ser
Val Ser Trp Tyr Gln His His Pro Gly Lys Ala Pro Lys Val 35 40 45
Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Thr Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
Thr Ser Ser 85 90 95 Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 16109PRTHomo sapiens 16Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Ile Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 17109PRTHomo sapiens 17Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Ile Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 18109PRTHomo sapiens 18Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Ala
Val Leu 100 105 19109PRTHomo sapiens 19Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln Tyr Pro Gly Lys Pro Pro Lys Leu 35 40 45 Lys
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 20109PRTHomo sapiens 20Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 21109PRTHomo sapiens 21Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 22109PRTHomo sapiens 22Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Asn Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Ile Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 23109PRTHomo sapiens 23Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr
Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 24109PRTHomo sapiens 24Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Arg 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Asn Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 25108PRTHomo sapiens 25Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
Thr Ser Ser 85 90 95 Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 26109PRTHomo sapiens 26Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Glu Val Thr Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr
Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 27108PRTHomo sapiens 27Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Leu Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala
Gly Ser 85 90 95 Ser Thr Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 28110PRTHomo sapiens 28Leu Ser Ala Leu Thr Gln Pro Ala Ser
Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys Thr
Gly Thr Ser Ser Asp Val Gly Asn Tyr 20 25 30 Asn Leu Val Ser Trp
Tyr Gln Gln Tyr Ser Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr
Glu Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser
Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75
80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser
85 90 95 Ser Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110 29108PRTHomo sapiens 29Gln Ser Val Leu Thr Gln Pro Pro
Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr
Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65
70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
Ser Leu 85 90 95 Asn Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 30109PRTHomo sapiens 30Gln Ser Val Leu Thr Gln Pro Pro Ser
Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Ser Lys 20 25 30
Thr Val Asn Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Leu Gly Val Pro Asp Arg Phe
Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser
Gly Leu Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala
Trp Asp Asp Ser Leu 85 90 95 Asn Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 31109PRTHomo sapiens 31Gln Ser Val Leu Thr
Gln Pro Pro Ser Ala Ser Gly Pro Pro Gly Gln1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr
Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Ser Asn Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
Asp Asp Ser Leu 85 90 95 Asn Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 32110PRTHomo sapiens 32Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Val Trp Asp
Asp Ser Leu 85 90 95 Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 110 33109PRTHomo sapiens 33Gln Ser Val Leu Thr
Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys 20 25 30 Thr
Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Ser Asn Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
Leu Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
Asp Asp Ser Leu 85 90 95 Asn Trp Val Phe Gly Ala Gly Thr Lys Leu
Thr Val Leu 100 105 34110PRTHomo sapiens 34Gln Ser Val Leu Thr Gln
Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15 Lys Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val
Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu
Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp
Ser Ser Leu 85 90 95 Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu 100 105 110 35110PRTHomo sapiens 35Gln Ser Val Leu Thr
Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15 Lys Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Phe
Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40
45 Ile Tyr Asp Tyr Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser
50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly
Leu Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp
Asp Ser Ser Leu 85 90 95 Ser Ala Tyr Val Phe Gly Thr Gly Thr Arg
Val Thr Val Leu 100 105 110 36110PRTHomo sapiens 36Gln Ser Val Leu
Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15 Lys Val
Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30
Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35
40 45 Ile Tyr Asp Tyr Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe
Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr
Gly Leu Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr
Trp Asp Ser Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Thr Gly Thr
Arg Val Thr Val Leu 100 105 110 37110PRTHomo sapiens 37Gln Ser Val
Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15 Lys
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25
30 Phe Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Asp Tyr Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile
Thr Gly Leu Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly
Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ser Tyr Val Phe Gly Thr Gly
Thr Arg Val Thr Val Leu 100 105 110 38110PRTHomo sapiens 38Gln Ser
Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15
Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20
25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Asp Tyr Asn Lys Arg Pro Ser Gly Ile Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly
Ile Thr Gly Leu Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys
Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Thr
Gly Thr Arg Val Thr Val Leu 100 105 110 39110PRTHomo sapiens 39Gln
Ser Val Leu Thr Gln Pro Pro Thr Val Ser Ala Ala Pro Gly Gln1 5 10
15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys
Leu Leu 35 40 45 Ile Tyr Asp Tyr Asn Lys Arg Pro Ser Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu
Gly Ile Thr Gly Leu Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr
Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly
Thr Gly Thr Arg Val Thr Val Leu 100 105 110 40110PRTHomo sapiens
40Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1
5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn
Asn 20 25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45 Ile Tyr Asp Ser Asn Lys Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr
Leu Asp Ile Thr Gly Leu Gln65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr
Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Tyr Val Phe
Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 41110PRTHomo
sapiens 41Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro
Gly Gln1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro
Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65 70 75 80 Thr Gly Asp Glu
Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala
Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
42110PRTHomo sapiens 42Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser
Asn Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn
Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Asn
Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65 70 75 80 Thr
Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90
95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 43107PRTHomo sapiens 43Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val
Ser Val Ser Pro Gly Gln1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly
Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val 85
90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
44106PRTHomo sapiens 44Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser
Val Ser Pro Gly Gln1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp
Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asn Thr Lys Trp
Pro Leu Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly
Asn Thr Val Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65 70 75 80 Asp
Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Val Val 85 90
95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 45116PRTHomo
sapiens 45Gln Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Ala Ser Leu
Gly Ala1 5 10 15 Ser Val Thr Leu Thr Cys Thr Leu Ser Ser Gly Tyr
Ser Asn Tyr Lys 20 25 30 Val Asp Trp Tyr Gln Gln Arg Pro Gly Lys
Gly Pro Arg Phe Val Met 35 40 45 Arg Val Gly Thr Gly Gly Ile Val
Gly Ser Lys Gly Asp Gly Ile Pro 50 55 60 Asp Arg Phe Ser Val Leu
Gly Ser Gly Leu Asn Arg Tyr Leu Thr Ile65 70 75 80 Lys Asn Ile Gln
Glu Glu Asp Glu Ser Asp Tyr His Cys Gly Ala Asp 85 90 95 His Gly
Ser Gly Ser Asn Phe Val Val Val Phe Gly Gly Gly Thr Lys 100 105 110
Leu Thr Val Leu 115 46116PRTHomo sapiens 46Gln Pro Val Leu Thr Gln
Pro Leu Phe Ala Ser Ala Ser Leu Gly Ala1 5 10 15 Ser Val Thr Leu
Thr Cys Thr Leu Ser Ser Gly Tyr Ser Ser Tyr Glu 20 25 30 Val Asp
Trp Tyr Gln Gln Arg Pro Gly Lys Gly Pro Arg Phe Val Met 35 40 45
Arg Val Asp Thr Gly Gly Ile Val Gly Ser Lys Gly Glu Gly Ile Pro 50
55 60 Asp Arg Phe Ser Val Leu Gly Ser Gly Leu Asn Arg Tyr Leu Thr
Ile65 70 75 80 Lys Asn Ile Gln Glu Glu Asp Glu Ser Asp Tyr His Cys
Gly Ala Asp 85 90 95 His Gly Ser Gly Thr Asn Phe Val Val Val Phe
Gly Gly Gly Thr Lys 100 105 110 Leu Thr Val Leu 115 47114PRTHomo
sapiens 47Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly
Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met
Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Arg
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110
Ser Ser48115PRTHomo sapiens 48Gln Ile Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Pro Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile
Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val 50 55 60 Gln
Gly Ser Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr 100 105 110 Val Ser Ser 115 49115PRTHomo sapiens 49Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20
25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala
Gln Lys Leu 50 55 60 Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser
Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
50115PRTHomo sapiens 50Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Val Ser Phe
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg
Gly Thr Met Thr Thr Asp Pro Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
51115PRTHomo sapiens 51Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Phe
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser 115 52115PRTHomo sapiens 52Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys
Val 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Val Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Ser Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
53115PRTHomo sapiens 53Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Phe
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Gly Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser 115 54115PRTHomo sapiens 54Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Leu
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Leu Thr Ser Tyr 20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys
Val 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Val Tyr65 70 75 80 Met Glu Val Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
55115PRTHomo sapiens 55Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Pro Leu Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser 115 56115PRTHomo sapiens 56Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Leu Thr Ser Tyr 20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys
Val 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Val Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
57115PRTHomo sapiens 57Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Val Ser Ala
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Tyr Val Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser 115 58115PRTHomo sapiens 58Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Tyr 20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Glu Lys
Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Met Glu Val Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Phe Tyr Cys 85 90 95 Ala Arg Gly Tyr Val Met Asp Val Trp
Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
59113PRTHomo sapiens 59Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala
Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110 Ser60115PRTHomo sapiens 60Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Trp Ile Ser Thr Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val 50 55
60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Thr Arg Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 61116PRTHomo
sapiens 61Glu 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 Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser
Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu 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
Asn Trp Gly Ala Phe Asp Val Trp Gly Gln Gly Thr Met Val 100 105 110
Thr Val Ser Ser 115 62119PRTHomo sapiens 62Glu 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 Arg Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Asn Ile Lys His Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
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 Glu Ser Asn Trp Gly Phe Ala Phe Asp
Val Trp Gly His Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115
63116PRTHomo sapiens 63Glu 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 Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 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 Asn Trp Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val
100 105 110 Thr Val Ser Ser 115 64119PRTHomo sapiens 64Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser
Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu 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 Glu Ser Asn Trp Gly Phe
Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser
Ser 115 65119PRTHomo sapiens 65Glu 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 Leu Thr Phe Ser Asn Phe 20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile
Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Ser Cys
85 90 95 Thr Arg Glu Ser Asn Trp Gly Phe Ala Phe Asp Ile Trp Gly
Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 66123PRTHomo
sapiens 66Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser
Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu 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
Asp Tyr Asp Phe Trp Ser Gly Tyr Tyr Thr Ala Phe Asp Val 100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 67123PRTHomo
sapiens 67Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser
Tyr Ile Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg
Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val 100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 68112PRTHomo
sapiens 68Glu Val Gln Leu Leu 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 Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110
69117PRTHomo sapiens 69Glu Val Gln Leu Leu 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 Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly
Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Glu Val Gly Ser Pro Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 70117PRTHomo sapiens 70Glu
Val Gln Leu Leu 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 Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Leu Met Val
Tyr Ala Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser 115 71121PRTHomo sapiens 71Glu Val Gln Leu Leu 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 Tyr 20 25 30 Ala Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile
Ser Gly Ser Gly Asp Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Lys Phe Val Leu Met Val Tyr Ala Met Leu Asp Tyr
Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
72121PRTHomo sapiens 72Glu Val Gln Leu Leu 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 Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly
Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Lys Phe Val Leu Met Val Tyr Ala Met Leu Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
73116PRTHomo sapiens 73Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val
100 105 110 Thr Val Ser Ser 115 74123PRTHomo sapiens 74Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asp Lys Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu 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 Glu Thr Gly Pro Leu Lys
Leu Tyr Tyr Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 75116PRTHomo sapiens 75Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu 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 Ile Ala Ala Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115
76122PRTHomo sapiens 76Gln Val His Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Ser
Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Ala Ile Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
77122PRTHomo sapiens 77Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Asn
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Ala Ile Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly His Gly Thr Thr Val Thr Val Ser Ser 115 120
78122PRTHomo sapiens 78Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Asn
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Ala Ile Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
79122PRTHomo sapiens 79Gln Val His Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Ser
Asp Gly Ser Asp Glu Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Ala Ile Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
80122PRTHomo sapiens 80Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Asn
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Ala Ile Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
81122PRTHomo sapiens 81Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Arg Gly Gly Leu Ala Ala Arg Pro Gly Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
82122PRTHomo sapiens 82Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Gly Ile Ala Val Ala Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
83122PRTHomo sapiens 83Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp His
Asp Gly Ser Asn Thr Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Gly Ile Ala Val Ala Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
84117PRTHomo sapiens 84Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile
Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile
Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65 70 75 80 Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90
95 Cys Ala Arg Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
100 105 110 Val Thr Val Ser Ser 115 85122PRTHomo sapiens 85Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20
25 30 Asp Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr
Asn Pro Ser 50 55 60 Leu Lys Ser Arg Ile Thr Ile Ser Val Asp Thr
Ser Lys Asn Leu Phe65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Gly Gly Val Thr
Thr Tyr Tyr Tyr Ala Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 86120PRTHomo sapiens 86Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30
Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35
40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro
Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Asp Thr Ala Met Val
Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 87121PRTHomo sapiens 87Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp
Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile
Gly Tyr Ile Tyr Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65
70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr 85 90 95 Cys Ala Arg Glu Asp Thr Ala Met Val Pro Tyr Phe
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 88115PRTHomo sapiens 88Gln Val Gln Leu Gln Gln Trp Gly Ala Gly
Leu Leu Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn
His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70
75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95 Arg Gly Gln Leu Val Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr 100 105 110 Val Ser Ser 115 89116PRTHomo sapiens 89Gln
Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10
15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Ala Tyr
20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Arg Thr Asp Tyr Asn
Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Lys Gln Phe Ser Leu65 70 75 80 Lys Leu Asn Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gln Leu Val Pro
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser
115 90115PRTHomo sapiens 90Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp
Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg
Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 91121PRTHomo sapiens 91Gln Val Gln
Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15 Thr
Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25
30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu
35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Lys Asn
Tyr Ser 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp
Thr Ser Lys Asn65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Gly Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Gly Gly Pro
Thr Ala Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 92345DNAHomo sapiens 92cagattcagc tggtgcagtc
tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta
ccccttgacc agctatggta tcagctgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggatgg atcagcgctt acaatggtaa cacaaactat
180gcacagaagg tccagggcag cgtcaccatg accacagaca catccacgag
cacagtctac 240atggagctga ggagcctgag atctgacgac acggccgtgt
attactgtgc gagaggctac 300ggtatggacg tctggggcca agggaccacg
gtcaccgtct cctct 34593327DNAHomo sapiens 93cagtctgccc tgactcagcc
tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag
tgacgttggt ggttataact ctgtctcctg gtaccaacag 120tacccaggca
aaccccccaa actcaagatt tatgaggtca gtaatcggcc ctcaggggtt
180tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat
ctctgggctc 240caggctgagg acgaggctga ttatttctgc agctcatata
caagcaccag catggtcttc 300ggcggaggga ccaagctgac cgtccta
32794345DNAHomo sapiens 94caggttcagc tggtgcagtc tggagctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta caccttaacc
agctatggta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg gtcagttttt ataatggtaa cacaaactat 180gcacagaagc
tccagggcag aggcaccatg accacagacc catccacgag cacagcctac
240atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc
gagaggctac 300ggtatggacg tctggggcca agggaccacg gtcaccgtct cctct
34595327DNAHomo sapiens 95cagtctgccc tgactcagcc tgcctccgtg
tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt
ggttataact ctgtctcctg gtaccaacag 120cacccaggca aagcccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240caggctgagg acgaggctga ttattactgc aattcatata caagcaccag
catggtattc 300ggcggaggga ccaagctgac cgtccta 32796345DNAHomo sapiens
96caggttcagc tggtgcagtc tggagctgaa gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggtta caccttgacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagctttt acaatggtaa
cacaaactat 180gcacagaagg tccagggcag agtcaccatg accacagaca
catccacgag cacagtctac 240atggagctga ggagcctgag atctgacgac
acggccgtgt attactgtgc gagaggctac 300ggtatggacg tctggggcca
agggaccacg gtcaccgtct cctct 34597327DNAHomo sapiens 97cagtctgccc
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg
gaaccagcag tgacgttggt ggttataact ctgtctcctg gtaccaacag
120cacccaggca aaccccccaa actcatgatt tatgaggtca gtaatcggcc
ctcaggggtt 180tctattcgct tctctggctc caagtctggc aacacggcct
ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttatttctgc
agctcatata caagcaccag catggtcttc 300ggcggaggga ccaagctgac cgtccta
32798345DNAHomo sapiens 98cagattcagc tggtgcagtc tggagctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta caccttgacc
agctatggta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atcagctttt acaatggtaa cacaaactat 180gcacagaagg
tccagggcag agtcaccatg accacagaca catccacgag cacagtctac
240atggagctga ggagcctgag atctgacgac acggccgtgt atttctgtgc
gagaggttac 300ggtatggacg tctggggcca agggaccacg gtcaccgtct cctca
34599327DNAHomo sapiens 99cagtctgccc tgactcagcc tgcctccgtg
tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt
ggttataact ctgtctcgtg gtaccaacag 120cacccaggca aaccccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240caggctgagg acgaggctga ttatttctgc agctcatata caagcaccag
catggtcttc 300ggcggaggga ccaagctggc cgtccta 327100345DNAHomo
sapiens 100caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggtta caccttaacc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg gtcagttttt
ataatggtaa cacaaactat 180gcacagaagc tccagggcag aggcaccatg
accacagacc catccacgag cacagcctac 240atggagctga ggagcctgag
atctgacgac acggccgtgt attactgtgc gagaggctac 300ggtatggacg
tctggggcca agggaccacg gtcaccgtct cctca 345101327DNAHomo sapiens
101cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt ggttataact ctgtctcctg
gtaccaacag 120cacccaggca aagcccccaa actcatgatt tatgaggtca
ctaatcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc
aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga
ttattactgc aactcatata caagcaccag catggtgttc 300ggcggaggga
ccaagctgac cgtccta 327102363DNAHomo sapiens 102caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc
120cagcacccag ggaagggcct ggagtggatt gggtacatat ataacagtgg
gagcacctac 180tacaacccgt ccctcaagag tcgagttacc atatcagtag
acacgtctaa gaaccagttc 240tccctgaagc tgagctctgt gactgccgcg
gacacggccg tgtattactg tgcgagagag 300gatacagcta tggttcctta
ctttgactac tggggccagg gaaccctggt caccgtctcc 360tca 363103333DNAHomo
sapiens 103cagtctgtac tgacgcagcc gccctcagtg tctggggccc cagggcagag
ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg gcacattatg atgtgcactg
gtaccagcag 120gttccaggaa cagcccccaa actcctcatc tatggtaaca
cctatcggcc ctcaggggtc 180cctgaccgat tctctggctc caagtctggc
acctcagcct ccctggccat cactgggctc 240caggctgagg atgaggctga
ttattactgc cagtcctatg acaacagcct gagtggtgtg 300gtattcggcg
gagggaccaa gctgaccgtc cta 333104366DNAHomo sapiens 104caggtgcacc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cgtctggatt caccttcaac agctttggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcactt atctggtctg atggaagtga
tgaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagccata 300gcagccctct actactacta
cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366105330DNAHomo sapiens 105cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattttg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gactataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag
tgcttatgtc 300ttcggaactg ggaccagggt caccgtccta 330106366DNAHomo
sapiens 106caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagc agctttggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcactt atatggaatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagccata 300gcagccctct
actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366107330DNAHomo sapiens 107cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattttg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gactataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gcagtctgag
tggttatgtc 300ttcggaactg ggaccagggt caccgtccta 330108366DNAHomo
sapiens 108caggtgcacc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcaac agctttggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcactt atatggtctg
atggaagtga taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagccata 300gcagccctct
actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366109330DNAHomo sapiens 109cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagttc caacattggg
aataattttg tatcctggta ccagcagttc 120ccaggaacag cccccaaact
cctcatttat gactataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag
ttcttatgtc 300ttcggaactg ggaccagggt caccgtccta 330110366DNAHomo
sapiens 110caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagc agctttggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcactt atatggaatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagccata 300gcagccctct
actactacta cggtatggac gtctggggcc acgggaccac ggtcaccgtc 360tcctca
366111330DNAHomo sapiens 111cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattttg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gactataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag
tggttatgtc 300ttcggaactg ggaccagggt caccgtccta 330112366DNAHomo
sapiens 112caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagc agctttggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcactt atatggaatg
atggaagtaa taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagccata 300gcagccctct
actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366113330DNAHomo sapiens 113cagtctgtgt tgacgcagcc gcccacagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattttg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gactataata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag
240actggggacg aggccgatta ctactgcgga acatgggata gcagcctgag
tggttatgtc 300ttcggaactg ggaccagggt caccgtccta 330114366DNAHomo
sapiens 114caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagg agctatggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcactt atatggcatg
atggaagtaa tacatactat 180gtagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagaggtata 300gcagtggctt
actactacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366115330DNAHomo sapiens 115cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa ggtcaccatc 60tcctgctctg gaagcagctc caacattggg
aataattttg tatcctggta ccagcagctc 120ccaggaacag cccccaaact
cctcatttat gacagtaata agcgaccctc agggattcct 180gaccgattct
ctggctccaa gtctggcacg tcagccaccc tggacatcac cggactccag
240actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag
tgcttatgtt 300ttcggaactg ggaccaaggt caccgtccta 330116363DNAHomo
sapiens 116gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagc agctatgcca tgaactgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcaact attagtggta
gtggtgataa cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaaaagttt 300gtactaatgg
tgtatgctat gcttgactac tggggccagg gaaccctggt caccgtctcc 360tca
363117321DNAHomo sapiens 117gacatcctga tgacccagtc tccatcctcc
ctgtctgcat ctgttggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc
agttatttaa attggtatca gcagaaacca 120gggaaagccc ctaaggtcct
gatctatgct gcctccagtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcaacag tctgcaacct
240gaagattttg caacttacta ctgtcaacag agttacagtt cccccatcac
cttcggccaa 300gggacacgac tggagattaa a 321118363DNAHomo sapiens
118gaggtgcagc tgttggagtc tgggggaggc ttggtacagc cgggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagc agctatgcca tgaactgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcaact attagtggta
gtggtggtaa cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaaaagttt 300gtactaatgg
tgtatgctat gcttgactac tggggccagg gaaccctggt caccgtctcc 360tca
363119321DNAHomo sapiens 119gacatccaga tgacccagtc tccatcctcc
ctatctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc
atctatttaa attggtatca gcagaagcca 120gggaaagccc cttacctcct
gatctatgct gcagccagtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg caacttacta ctgtcaacag agttacagtg cccccatcac
cttcggccaa 300gggacacgac tggagattaa a 321120345DNAHomo sapiens
120caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
actgaaggtc 60tcctgcaagg cttctggtta cagtttgacc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcagcgctt
acaatggtaa cacaaactat 180gcacagaagg tccagggcag agtcaccatg
accacagaca catccacgag cacagtctac 240atggaggtga ggagtctgag
atctgacgac acggccgtgt attactgtgc gagaggctac 300ggtatggacg
tctggggcca agggaccacg gtcaccgtct cctca 345121327DNAHomo sapiens
121cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt ggttataact ctgtctcctg
gtaccaacag 120cacccaggca aaccccccaa actcatgatt tatgaggtca
gtaatcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc
aatacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga
ttatttctgc agctcatata caagcaccag catggtcttc 300ggcggaggga
ccaagctgac cgtccta 327122345DNAHomo sapiens 122caggttcagc
tggtgcagtc tggagctgag gtgaagaggc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggtta caccttgacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgttt acaatggtaa
cacaaactat 180gcacagaagg tccagggcag agtcaccatg accacagaca
catccacgag cacagtctac 240atggagctga ggagcctgag ctctgacgac
acggccgtgt attactgtgc gagaggctac 300ggtatggacg tctggggcca
agggaccacg gtcaccgtct cctca 345123327DNAHomo sapiens 123cagtctgccc
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc
60tcctgcactg
gaaccagcag tgacgttggt ggttataact ctgtctcctg gtaccaacag
120cacccaggca aaccccccaa actcatgatt tatgaggtca gtaatcggcc
ctcaggggtt 180tctattcgct tctctggctc caagtctggc aacacggcct
ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttatttctgc
agctcatata caagcaccag catggtcttc 300ggcggaggga ccaagctgac cgtccta
327124345DNAHomo sapiens 124caggttcagc tggtgcagtc tggagctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta ccccttgacc
agctatggta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atcagcgctt acaatggtaa cacaaactat 180gcacagaagg
tccagggcag agtcaccatg accacagaca catccacgag cacagtctac
240atggagttga ggagcctgag atctgacgac acggccgtgt attactgtgc
gagaggctac 300ggtatggacg tctggggcca agggaccacg gtcaccgtct cctca
345125327DNAHomo sapiens 125cagtctgccc tgactcagcc tgcctccgtg
tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt
ggttataact ctgtctcctg gtaccaacag 120cacccaggca aaccccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180tctaatcgct
tctctggctc caagtctggc aatacggcct ccctgaccat ctctgggctc
240caggctgagg acgaggctga ttatttctgc agctcatata caagcaccag
catggtcttc 300ggcggaggga ccaagctgac cgtccta 327126345DNAHomo
sapiens 126caggttcagt tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggtta cgccttgacc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcagcgctt
acaatggtaa cacaaactat 180gcacagaagg tccagggcag agtcaccatg
accacagaca catccacgag cacagtctac 240atggagctga ggagcctgag
atctgacgac acggccgtgt attactgtgc gagaggctac 300ggtatggacg
tctggggcca agggaccacg gtcaccgtct cctca 345127327DNAHomo sapiens
127cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccaacag tgacgttggt ggttataact ctgtctcctg
gtaccaacag 120cacccaggca aaccccccaa actcatgatt tatgaggtca
gtaatcggcc ctcagggatt 180tctaatcgct tctctggctc caagtctggc
aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga
ttatttctgc agctcatata caagcaccag catggtcttc 300ggcggaggga
ccaagctgac cgtccta 327128345DNAHomo sapiens 128caggttcagc
tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttctggtta cagctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg gtcagcgctt acaatggtaa
cacaaactat 180gcacagaagt tccagggcag agtcaccatg accacagaca
catccacgag cacagcctac 240atggaactga ggagcctgag atctgacgac
acggccgtgt attactgtgc gagaggctac 300gttatggacg tctggggcca
agggaccacg gtcaccgtct cctca 345129327DNAHomo sapiens 129cagtctgccc
tgactcagcc tgcctccgtt tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg
gaaccagcag tgacgttggt gcttataact ctgtctcctg gtaccaacag
120cacccaggca aagcccccaa acgcatgatt tatgaggtca gtaatcggcc
ctcaggggtt 180tctaatcgct tctctggctc caagtctggc aacacggcct
ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttattactgc
agctcatata caagcaccaa catggtattc 300ggcggaggga ccaagctgac cgtccta
327130363DNAHomo sapiens 130caggtacagt tgcagcagtc aggtccagga
ctggtgaagc cctcgcagac cctctcactc 60acctgtgcca tctccgggga cagtgtctct
agcaacagtg ctgcttggaa ctggatcagg 120cagtccccat cgagaggcct
tgagtggctg ggaaggacat actacaggtc caagtggtat 180aaaaattatt
cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac
240cagttctctc tgcaactgaa ctctgtgact cccggggaca cggctgtgta
ttactgtgca 300agaggggggc caactgctgc ttttgactac tggggccagg
gaaccctggt caccgtctcc 360tca 363131330DNAHomo sapiens 131ctttctgccc
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg
gaaccagcag tgatgttggg aattataacc ttgtctcctg gtaccaacag
120tattcaggca aagcccccaa actcatgatt tatgaggtca gtaagcggcc
ctcaggggtt 180tctaatcgct tctctggctc caagtctggc aacacggcct
ccctgacaat ctctgggctc 240caggctgagg acgaggctga ttattactgc
tgctcatatg caggtagtag cactttggtt 300ttcggcggag ggaccaagct
gaccgtccta 330132357DNAHomo sapiens 132gaggtgcagt tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgtag tctctggatt
cacctttagt agctattgga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaatactat
180gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa
ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtat
attactgtgc gagagagtca 300aactggggat ttgcttttga tatctggggc
caagggacaa tggtcaccgt ctcttca 357133327DNAHomo sapiens
133cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag
ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga agtaagactg taaactggta
ccaacaggtc 120ccaggaacgg cccccaaact cctcatctat aggaataatc
agcggccctt aggggtccct 180gaccgattct ctggctccaa gtctggcacc
tcagcctccc tggccatcag tgggctccag 240tctgaggatg aggctgatta
ttattgtgca gcatgggatg acagcctgaa ttgggtgttc 300ggcggaggga
ccaagctgac cgtccta 327134357DNAHomo sapiens 134gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacctttagt cgctattgga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtggccaac ataaagcatg atggaagtga
gaaatactat 180gtggactctg tgaagggccg attcaccatt tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagagtca 300aactggggat ttgcttttga
tgtctggggc cacgggacaa tggtcaccgt ctcttca 357135327DNAHomo sapiens
135cagtctgtgc tgactcagcc accctcagcg tctgggcccc ccggacagag
ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga agtaatactg taaactggta
ccagcagctc 120ccaggaacgg cccccaaact cctcatctat agtaataatc
ggcggccctc aggggtccct 180gaccgattct ctggctccaa gtctggcacc
tcagcctccc tggccatcag tgggctccag 240tctgaggatg aggctgatta
ttactgtgca gcatgggatg acagcctgaa ttgggtgttc 300ggcggaggga
ccaagctgac cgtccta 327136351DNAHomo sapiens 136gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcaact attagtggta gtggtggtag
gacatattac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagaagtt 300ggcagtccct ttgactactg
gggccaggga accctggtca ccgtctcctc a 351137330DNAHomo sapiens
137cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa
ggtcaccatc 60tcctgctctg gaagcaactc caacattggg aataattatg tatcctggta
ccagcagctc 120ccaggaacag cccccaaact cctcatttat gacaataata
agcgaccctc agggattcct 180gaccgattct ctggctccaa ctctggcacg
tcagccaccc tgggcatcac cggactccag 240actggggacg aggccgatta
ttactgcgga acatgggata gcagcctgag tgctgtggta 300ttcggcggag
ggaccaagct gaccgtccta 330138366DNAHomo sapiens 138caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcaatt atatggtatg atggaagtaa
taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacactgtat 240cttcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gaggaggggg 300ggtctggcag ctcgtccggg
cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366139318DNAHomo sapiens 139tcctatgagc tgactcagcc accctcagtg
tctgtgtccc caggacagac agccagaatc 60acctgctctg gagataaatt gggggataaa
tatgcttgct ggtatcagca gaaaccaggc 120cagtcccctg tgctggtcat
ctatcaaaat accaagtggc ccttagggat ccctgagcga 180ttctctggct
ccaagtctgg gaacacagtc actctgacca tcagcgggac ccaggctatg
240gatgaggctg actattactg tcaggcgtgg gacagcagca ctgtggtatt
cggcggaggg 300accaagctga ccgtccta 318140366DNAHomo sapiens
140caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac
cctgtccctc 60acctgcactg tctctggtgg ctccatcagc agtagtgatt actactggag
ctggatccgc 120cagcacccag ggaagggcct ggagtggatt gggtacatct
attacagtgg gagcacctac 180tacaacccgt ccctcaagag tcgaattacc
atatcagtag acacgtctaa gaacctgttc 240tccctgaagt tgagctctgt
gactgccgcg gacacggccg tgtattactg tgcgagaggg 300ggggtgacta
cgtactacta cgctatggac gtctggggcc aagggaccac ggtcaccgtc 360tcctca
366141321DNAHomo sapiens 141gacatacaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gcgcattagc
aactatttaa gttggtatct gcagaaacca 120gggattgccc ctaagctcct
gatctatgct gcatccagtt tgcagagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaatct
240gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcat
tttcggcgga 300gggaccaagg tggagatcaa a 321142369DNAHomo sapiens
142caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
atggaagtga taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagagact 300ggtcccttga
aactctacta ctacggtatg gacgtctggg gccaagggac cacggtcacc 360gtctcctca
369143336DNAHomo sapiens 143gatattgtga tgactcagtc tccactctcc
ctgtccgtca cccctggaga gccgccctcc 60atctcctgca ggtctagtca gagcctcctg
catagtaatg gatacaactt tttgaattgg 120tacctgcaga agccagggca
gtctccacaa ctcctgatct atttgggttc tcatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac actggaaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagttct
acaaactcca 300ttcactttcg gccctgggac caaagtggat atcaaa
336144357DNAHomo sapiens 144gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggact cacctttagt
aacttttgga tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtggccaac ataaagcaag atggaagtga gaaatactat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ttcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attcctgtac
gagagagtca 300aactggggat ttgcttttga tatctggggc caagggacaa
tggtcaccgt ctcttca 357145327DNAHomo sapiens 145cagtctgtgc
tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg
gaagcagctc caacatcgga agtaaaactg taaactggta ccagcagttc
120ccaggaacgg cccccaaact cctcatctat agtaataatc ggcggccctc
aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcag tgggctccag 240tctgaggatg aggctgatta ttactgtgca
gcatgggatg acagcctgaa ttgggtgttc 300ggcgcaggga ccaagctgac cgtccta
327146345DNAHomo sapiens 146caggttcagc tggtgcagtc tggagctgag
gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta cacctttacc
agctatggta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggatgg atcagcactt acaatggtaa cacaaactat 180gcacagaagg
tccagggcag agtcaccatg accacagaca catccacgag cacagcctac
240atggagctga ggagcctgag atctgacgac acggccgttt attactgtgc
gagagggtat 300actcgggact actggggcca gggaaccctg gtcaccgtct cctca
345147348DNAHomo sapiens 147cagcctgtgc tgactcagcc actttttgca
tcagcctccc tgggagcctc ggtcacactc 60acctgcaccc tgagcagcgg ctacagtagt
tatgaagtgg actggtatca gcagagacca 120gggaagggcc cccggtttgt
catgcgagtg gacactggtg ggattgtggg atccaagggg 180gaaggcatcc
ctgatcgctt ctcagttttg ggctcaggcc tgaatcggta tctgaccatc
240aagaacatcc aggaagagga tgagagtgac taccactgtg gggcagacca
tggcagtggg 300accaacttcg tggtggtatt cggcggaggg accaagctga ccgtccta
348148348DNAHomo sapiens 148caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt
gcgtactact ggaactggat ccgccagccc 120ccagggaagg ggctggagtg
gattggggaa atcaatcata gtggaagaac cgactacaac 180ccgtccctca
agagtcgagt caccatatca gtagacacgt ccaagaagca gttctccctg
240aagctgaact ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag
agggcagctc 300gtcccctttg actactgggg ccagggaacc ctggtcaccg tctcttca
348149330DNAHomo sapiens 149cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga
agtaatactg taaattggta tcagcaactc 120ccaggaacgg cccccaaact
cctcatctat agtaataatc agcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag
240tctgaggatg aggctgatta ttactgtgca gtatgggatg acagcctgaa
tggttgggtg 300ttcggcggag ggaccaagct gaccgtccta 330150345DNAHomo
sapiens 150caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggtta cacctttccc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcagcgctt
acaatggtaa cacaaactat 180gcagagaagc tccagggcag agtcaccatg
accacagaca catccacgag cacagcctac 240atggaggtga ggagcctgag
atctgacgac acggccgtgt tttactgtgc gagaggctac 300gttatggacg
tctggggcca agggaccacg gtcaccgtct cctct 345151327DNAHomo sapiens
151cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt cgttataatt ctgtctcctg
gtaccaacac 120cacccaggca aagcccccaa agtcatgatt tatgaggtca
gtaatcggcc ctcaggggtt 180tctactcgct tctctggctc caagtctggc
aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga
ttattactgc agctcatata caagcagcag cgttgtattc 300ggcggaggga
ccaaactgac cgtccta 327152369DNAHomo sapiens 152gaggtgcagc
tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta
catttcctac 180gcagactcag tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt atttctgtgc gagagattac 300gatttttgga gtgcttacta
tgatgctttt gatgtctggg gccaagggac aatggtcacc 360gtctcttca
369153333DNAHomo sapiens 153cagtctgtgc tgacgcagcc gccctcagtg
tctggggccc cagggcagag ggtcaccatc 60tcctgcactg ggagcagctc caacatcggg
gcaggttatg atgtacactg gtaccagcag 120cttccaggaa cagcccccaa
actcctcatc tctggtaaca gcaatcggcc ctcaggggtc 180cctgaccgat
tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc
240caggctgagg atgaggctga ttattactgc cagtcctatg acagcagcct
gagtggttcg 300gtattcggcg gagggaccaa gctgaccgtc cta 333154326PRTHomo
sapiens 154Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80 Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115
120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235
240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320 Ser Leu Ser Pro Gly
Lys 325 155327PRTHomo sapiens 155Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65
70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val
Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser
Cys Pro Ala Pro 100 105 110 Glu Phe Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135
140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240 Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250
255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser305 310 315 320 Leu Ser Leu Ser Leu Gly Lys
325 156105PRTHomo sapiens 156Gln Pro Lys Ala Ala Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu1 5 10 15 Glu Leu Gln Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75
80 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 157106PRTHomo
sapiens 157Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80 His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 105 15814PRTHomo sapiens 158Thr
Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser1 5 10
15914PRTHomo sapiens 159Thr Gly Thr Asn Ser Asp Val Gly Gly Tyr Asn
Ser Val Ser1 5 10 16014PRTHomo sapiens 160Thr Gly Thr Ser Ser Asp
Val Gly Ala Tyr Asn Ser Val Ser1 5 10 16114PRTHomo sapiens 161Thr
Gly Thr Ser Ser Asp Val Gly Arg Tyr Asn Ser Val Ser1 5 10
1627PRTHomo sapiens 162Glu Val Ser Asn Arg Pro Ser1 5 1637PRTHomo
sapiens 163Glu Val Thr Asn Arg Pro Ser1 5 1649PRTHomo sapiens
164Ser Ser Tyr Thr Ser Thr Ser Met Val1 5 1659PRTHomo sapiens
165Asn Ser Tyr Thr Ser Thr Ser Met Val1 5 1669PRTHomo sapiens
166Ser Ser Tyr Thr Ser Thr Asn Met Val1 5 1679PRTHomo sapiens
167Ser Ser Tyr Thr Ser Ser Ser Val Val1 5 16810PRTHomo sapiens
168Gly Tyr Pro Leu Thr Ser Tyr Gly Ile Ser1 5 10 16910PRTHomo
sapiens 169Gly Tyr Ser Leu Thr Ser Tyr Gly Ile Ser1 5 10
17010PRTHomo sapiens 170Gly Tyr Ala Leu Thr Ser Tyr Gly Ile Ser1 5
10 17110PRTHomo sapiens 171Gly Tyr Thr Leu Thr Ser Tyr Gly Ile Ser1
5 10 17210PRTHomo sapiens 172Gly Tyr Ser Phe Thr Ser Tyr Gly Ile
Ser1 5 10 17310PRTHomo sapiens 173Gly Tyr Thr Phe Pro Ser Tyr Gly
Ile Ser1 5 10 17417PRTHomo sapiens 174Trp Ile Ser Ala Tyr Asn Gly
Asn Thr Asn Tyr Ala Gln Lys Val Gln1 5 10 15 Gly17517PRTHomo
sapiens 175Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys
Leu Gln1 5 10 15 Gly17617PRTHomo sapiens 176Trp Ile Ser Phe Tyr Asn
Gly Asn Thr Asn Tyr Ala Gln Lys Val Gln1 5 10 15 Gly17717PRTHomo
sapiens 177Trp Ile Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys
Val Gln1 5 10 15 Gly17817PRTHomo sapiens 178Trp Val Ser Ala Tyr Asn
Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15 Gly17917PRTHomo
sapiens 179Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Glu Lys
Leu Gln1 5 10 15 Gly1806PRTHomo sapiens 180Gly Tyr Gly Met Asp Val1
5 1816PRTHomo sapiens 181Gly Tyr Val Met Asp Val1 5 18213PRTHomo
sapiens 182Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Phe Val Ser1 5
10 1837PRTHomo sapiens 183Asp Tyr Asn Lys Arg Pro Ser1 5
1847PRTHomo sapiens 184Asp Ser Asn Lys Arg Pro Ser1 5 18511PRTHomo
sapiens 185Gly Thr Trp Asp Ser Ser Leu Ser Gly Tyr Val1 5 10
18611PRTHomo sapiens 186Gly Thr Trp Asp Ser Ser Leu Ser Ala Tyr
Val1 5 10 18711PRTHomo sapiens 187Gly Thr Trp Asp Ser Ser Leu Ser
Ser Tyr Val1 5 10 18810PRTHomo sapiens 188Gly Phe Thr Phe Ser Ser
Phe Gly Met His1 5 10 18910PRTHomo sapiens 189Gly Phe Thr Phe Asn
Ser Phe Gly Met His1 5 10 19010PRTHomo sapiens 190Gly Phe Thr Phe
Arg Ser Tyr Gly Met His1 5 10 19117PRTHomo sapiens 191Leu Ile Trp
Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15
Gly19217PRTHomo sapiens 192Leu Ile Trp Ser Asp Gly Ser Asp Glu Tyr
Tyr Ala Asp Ser Val Lys1 5 10 15 Gly19317PRTHomo sapiens 193Leu Ile
Trp Ser Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15
Gly19417PRTHomo sapiens 194Leu Ile Trp His Asp Gly Ser Asn Thr Tyr
Tyr Val Asp Ser Val Lys1 5 10 15 Gly19513PRTHomo sapiens 195Ala Ile
Ala Ala Leu Tyr Tyr Tyr Tyr Gly Met Asp Val1 5 10 19613PRTHomo
sapiens 196Gly Ile Ala Val Ala Tyr Tyr Tyr Tyr Gly Met Asp Val1 5
10 19713PRTHomo sapiens 197Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
Thr Val Asn1 5 10 19813PRTHomo sapiens 198Ser Gly Ser Ser Ser Asn
Ile Gly Ser Lys Thr Val Asn1 5 10 1997PRTHomo sapiens 199Ser Asn
Asn Arg Arg Pro Ser1 5 2007PRTHomo sapiens 200Arg Asn Asn Gln Arg
Pro Leu1 5 20110PRTHomo sapiens 201Ala Ala Trp Asp Asp Ser Leu Asn
Trp Val1 5 10 20210PRTHomo sapiens 202Gly Phe Thr Phe Ser Arg Tyr
Trp Met Ser1 5 10 20310PRTHomo sapiens 203Gly Leu Thr Phe Ser Asn
Phe Trp Met Ser1 5 10 20410PRTHomo sapiens 204Gly Phe Thr Phe Ser
Ser Tyr Trp Met Ser1 5 10 20517PRTHomo sapiens 205Asn Ile Lys His
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10 15
Gly20617PRTHomo sapiens 206Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val Lys1 5 10 15 Gly20710PRTHomo sapiens 207Glu Ser
Asn Trp Gly Phe Ala Phe Asp Val1 5 10 20810PRTHomo sapiens 208Glu
Ser Asn Trp Gly Phe Ala Phe Asp Ile1 5 10 20911PRTHomo sapiens
209Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn1 5 10 21011PRTHomo
sapiens 210Arg Ala Ser Gln Ser Ile Ser Ile Tyr Leu Asn1 5 10
2117PRTHomo sapiens 211Ala Ala Ser Ser Leu Gln Ser1 5 2127PRTHomo
sapiens 212Ala Ala Ala Ser Leu Gln Ser1 5 2139PRTHomo sapiens
213Gln Gln Ser Tyr Ser Ser Pro Ile Thr1 5 2149PRTHomo sapiens
214Gln Gln Ser Tyr Ser Ala Pro Ile Thr1 5 21510PRTHomo sapiens
215Gly Phe Thr Phe Ser Ser Tyr Ala Met Asn1 5 10 21617PRTHomo
sapiens 216Thr Ile Ser Gly Ser Gly Asp Asn Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15 Gly21717PRTHomo sapiens 217Thr Ile Ser Gly Ser Gly
Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly21812PRTHomo
sapiens 218Lys Phe Val Leu Met Val Tyr Ala Met Leu Asp Tyr1 5 10
21911PRTHomo sapiens 219Arg Ala Ser Gln Arg Ile Ser Asn Tyr Leu
Ser1 5 10 22016PRTHomo sapiens 220Arg Ser Ser Gln Ser Leu Leu His
Ser Asn Gly Tyr Asn Phe Leu Asn1 5 10 15 22114PRTHomo sapiens
221Thr Gly Thr Ser Ser Asp Val Gly Asn Tyr Asn Leu Val Ser1 5 10
22214PRTHomo sapiens 222Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr
Asp Val His1 5 10 22314PRTHomo sapiens 223Thr Gly Ser Ser Ser Asn
Ile Gly Ala His Tyr Asp Val His1 5 10 22413PRTHomo sapiens 224Ser
Gly Ser Asn Ser Asn Ile Gly Asn Asn Tyr Val Ser1 5 10 22511PRTHomo
sapiens 225Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Cys1 5 10
22612PRTHomo sapiens 226Thr Leu Ser Ser Gly Tyr Ser Ser Tyr Glu Val
Asp1 5 10 2277PRTHomo sapiens 227Leu Gly Ser His Arg Ala Ser1 5
2287PRTHomo sapiens 228Glu Val Ser Lys Arg Pro Ser1 5 2297PRTHomo
sapiens 229Gly Asn Ser Asn Arg Pro Ser1 5 2307PRTHomo sapiens
230Gly Asn Thr Tyr Arg Pro Ser1 5 2317PRTHomo sapiens 231Ser Asn
Asn Gln Arg Pro Ser1 5 2327PRTHomo sapiens 232Asp Asn Asn Lys Arg
Pro Ser1 5 2337PRTHomo sapiens 233Gln Asn Thr Lys Trp Pro Leu1 5
2347PRTHomo sapiens 234Val Asp Thr Gly Gly Ile Val1 5 2359PRTHomo
sapiens 235Gln Gln Ser Tyr Ser Thr Pro Leu Ile1 5 2369PRTHomo
sapiens 236Met Gln Val Leu Gln Thr Pro Phe Thr1 5 23710PRTHomo
sapiens 237Cys Ser Tyr Ala Gly Ser Ser Thr Leu Val1 5 10
23811PRTHomo sapiens 238Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser
Val1 5 10 23911PRTHomo sapiens 239Gln Ser Tyr Asp Asn Ser Leu Ser
Gly Val Val1 5 10 24011PRTHomo sapiens 240Ala Val Trp Asp Asp Ser
Leu Asn Gly Trp Val1 5 10 24111PRTHomo sapiens 241Gly Thr Trp Asp
Ser Ser Leu Ser Ala Val Val1 5 10 2429PRTHomo sapiens 242Gln Ala
Trp Asp Ser Ser Thr Val Val1 5 24318PRTHomo sapiens 243Ser Asp Tyr
His Cys Gly Ala Asp His Gly Ser Gly Thr Asn Phe Val1 5 10 15 Val
Val24410PRTHomo sapiens 244Gly Tyr Thr Phe Thr Ser Tyr Gly Ile Ser1
5 10 24510PRTHomo sapiens 245Gly Phe Thr Phe Ser Ser Tyr Ala Met
Ser1 5 10 24610PRTHomo sapiens 246Gly Phe Thr Phe Ser Ser Tyr Gly
Met His1 5 10 24710PRTHomo sapiens 247Gly Phe Thr Phe Ser Ser Tyr
Ser Met Asn1 5 10 24812PRTHomo sapiens 248Gly Gly Ser Ile Ser Ser
Gly Gly Tyr Tyr Trp Ser1 5 10 24912PRTHomo sapiens 249Gly Gly Ser
Ile Ser Ser Ser Asp Tyr Tyr Trp Ser1 5 10 25010PRTHomo sapiens
250Gly Gly Ser Phe Ser Ala Tyr Tyr Trp Asn1 5 10 25112PRTHomo
sapiens 251Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn1 5 10
25217PRTHomo sapiens 252Trp Ile Ser Thr Tyr Asn Gly Asn Thr Asn Tyr
Ala Gln Lys Val Gln1 5 10 15 Gly25317PRTHomo sapiens 253Thr Ile Ser
Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15
Gly25417PRTHomo sapiens 254Val Ile Trp Tyr Asp Gly Ser Asp Lys Tyr
Tyr Ala Asp Ser Val Lys1 5 10 15 Gly25517PRTHomo sapiens 255Ile Ile
Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15
Gly25617PRTHomo sapiens 256Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ser
Tyr Ala Asp Ser Val Lys1 5 10 15 Gly25716PRTHomo sapiens 257Tyr Ile
Tyr Asn Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser1 5 10 15
25816PRTHomo sapiens 258Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn
Pro Ser Leu Lys Ser1 5 10 15 25916PRTHomo sapiens 259Glu Ile Asn
His Ser Gly Arg Thr Asp Tyr Asn Pro Ser Leu Lys Ser1 5 10 15
26018PRTHomo sapiens 260Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Lys Asn
Tyr Ser Val Ser Val1 5 10 15 Lys Ser2616PRTHomo sapiens 261Gly Tyr
Thr Arg Asp Tyr1 5 2628PRTHomo sapiens 262Glu Val Gly Ser Pro Phe
Asp Tyr1 5 26314PRTHomo sapiens 263Glu Thr Gly Pro Leu Lys Leu Tyr
Tyr Tyr Gly Met Asp Val1 5 10 26413PRTHomo sapiens 264Arg Gly Gly
Leu Ala Ala Arg Pro Gly Gly Met Asp Val1 5 10 26514PRTHomo sapiens
265Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val1 5 10
26611PRTHomo sapiens 266Glu Asp Thr Ala Met Val Pro Tyr Phe Asp
Tyr1 5 10 26712PRTHomo sapiens 267Gly Gly Val Thr Thr Tyr Tyr Tyr
Ala Met Asp Val1 5 10 2688PRTHomo sapiens 268Gly Gln Leu Val Pro
Phe Asp Tyr1 5 2699PRTHomo sapiens 269Gly Gly Pro Thr Ala Ala Phe
Asp Tyr1 5 270109PRTHomo sapiens 270Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Phe Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr
Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 271109PRTHomo sapiens 271Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Phe Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ala Tyr 20 25 30 Asn Ser Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Arg 35 40 45 Met
Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly
Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
Thr Ser Thr 85 90 95 Asn Met Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 272109PRTHomo sapiens 272Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Pro Pro Gly Gln1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Ser Asn Asn Arg Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 273109PRTHomo sapiens 273Gln Ser
Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Pro Pro Gly Gln1 5 10 15
Arg Val Thr Ile Phe Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20
25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Ser Asn Asn Arg Arg Pro Ser Gly Val Pro Asp
Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala
Ile Ser Gly Leu Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu 100 105 274106PRTHomo sapiens 274Ser Tyr
Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5 10 15
Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20
25 30 Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile
Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
Gly Thr Gln Ala Met65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala
Trp Asp Ser Ser Thr Val Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 105 275106PRTHomo sapiens 275Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5 10 15 Thr Ala Arg
Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40
45 Gln Asn Thr Lys Trp Pro Leu Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60 Lys Ser Gly Asn Thr Val Thr Leu Thr Ile Ser Gly Thr Gln
Ala Met65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser
Ser Thr Val Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 276107PRTHomo sapiens 276Ser Tyr Glu Leu Thr Gln Pro Pro
Ser Val Ser Val Ser Pro Gly Gln1 5 10 15 Thr Ala Ser Ile Thr Cys
Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp
Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65
70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr
Ala Val 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 277107PRTHomo sapiens 277Ser Tyr Glu Leu Ile Gln Pro Pro Ser
Val Ser Val Ser Pro Gly Gln1 5 10 15 Thr Ala Ser Ile Thr Cys Ser
Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys Trp Tyr Gln Arg
Lys Pro Gly Gln Ser Pro Ile Leu Val Ile Tyr 35 40 45 Gln Asp Thr
Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn
Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65 70 75
80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala Val
85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
278120PRTHomo sapiens 278Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Thr Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85
90 95 Arg Gly Ser Tyr Ser Ser Gly Trp Phe Glu Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
27912PRTHomo sapiens 279Thr Leu Ser Ser Gly Tyr Ser Ser Tyr Glu Val
Asp1 5 10 28012PRTHomo sapiens 280Val Asp Thr Gly Gly Ile Val Gly
Ser Lys Gly Glu1 5 10 28113PRTHomo sapiens 281Gly Ala Asp His Gly
Ser Gly Thr Asn Phe Val Val Val1 5 10 28222PRTHomo sapiens 282Gln
Pro Val Leu Thr Gln Pro Leu Phe Ala Ser Ala Ser Leu Gly Ala1 5 10
15 Ser Val Thr Leu Thr Cys 20 28315PRTHomo sapiens 283Trp Tyr Gln
Gln Arg Pro Gly Lys Gly Pro Arg Phe Val Met Arg1 5 10 15
28432PRTHomo sapiens 284Gly Ile Pro Asp Arg Phe Ser Val Leu Gly Ser
Gly Leu Asn Arg Tyr1 5 10 15 Leu Thr Ile Lys Asn Ile Gln Glu Glu
Asp Glu Ser Asp Tyr His Cys 20 25 30 28510PRTHomo sapiens 285Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu1 5 10 286108PRTHomo sapiens
286Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Arg
Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln His His Pro Gly Lys Ala
Pro Lys Val 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly
Val Ser Thr Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Val Val Phe Gly
Gly Gly Thr Lys Leu Thr Val 100 105 287108PRTHomo sapiens 287Gln
Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30 Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro
Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val
Ser Ile Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Phe Cys Ser Ser Tyr Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly
Gly Thr Lys Leu Thr Val 100 105 288108PRTHomo sapiens 288Gln Ser
Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5 10 15
Ser Ile Thr Ile Ser Cys Thr Gly Thr Asn Ser Asp Val Gly Gly Tyr 20
25 30 Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Pro Pro Lys
Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Ile Ser
Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu
Thr Ile Ser Gly Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Phe
Cys Ser Ser Tyr Thr Ser Thr 85 90 95 Ser Met Val Phe Gly Gly Gly
Thr Lys Leu Thr Val 100 105 289122PRTHomo sapiens 289Gln Val His
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25
30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Leu Ile Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu 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 Ala Ile Ala Ala Leu Tyr
Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly His Gly Thr Thr Val
Thr Val Ser Ser 115 120 290122PRTHomo sapiens 290Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Cys Val 35
40 45 Ala Ile Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu 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 Arg Gly Gly Leu Ala Ala Arg
Pro Gly Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120 291121PRTHomo sapiens 291Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Leu Ile Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu 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 Ala Ile Ala Ala Leu Tyr Tyr Tyr
Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val
Ser 115 120 292119PRTHomo sapiens 292Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Ile Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
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 Arg Gly Gly Leu Pro Gly Gly Met Asp
Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
293327DNAHomo sapiens 293cagtctgccc tgactcagcc tgcctccgtg
tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt
ggttataact ctgtctcctg gtaccaacag 120cacccaggca aagcccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240caggctgagg acgaggctga ttattactgc aactcatata caagcaccag
catggtattc 300ggcggaggga ccaagctgac cgtccta 327294327DNAHomo
sapiens 294cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc
gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt ggttataact ctgtctcctg
gtaccaacag 120cacccaggca aaccccccaa actcatgatt tatgaggtca
gtaatcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc
aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga
ttatttctgc agctcatata caagcaccag catggtcttc 300ggcggaggga
ccaagctgac cgtccta 327295318DNAHomo sapiens 295tcctatgagc
tgactcagcc accctcagtg tccgtgtccc caggacagac agccagaatc 60acctgctctg
gagataaatt gggggataaa tatgcttgct ggtatcagca gaagccaggc
120cagtcccctg tgctggtcat ctatcaaaat accaagtggc ccttagggat
ccctgagcga 180ttctctggct ccaagtctgg gaacacagtc actctgacca
tcagcgggac ccaggctatg 240gatgaggctg actattactg tcaggcgtgg
gacagcagca ctgtggtatt cggcggaggg 300accaagctga ccgtccta
318296327DNAHomo sapiens 296cagtctgccc tgactcagcc tgcctccgtg
tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt
ggttataact ctgtctcctg gtaccaacag 120cacccaggca aagcccccaa
actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180tctaatcgct
tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc
240caggctgagg acgaggctga ttattactgc aattcatata caagcaccag
catggtattc 300ggcggaggga ccaagctgac cgtccta 327297215PRTHomo
sapiens 297Glu Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg
Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr 85 90 95 Ser Met
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115
120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr
Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
Val Lys Ala145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr
Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val
Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr
Glu Cys Ser 210 215 298230PRTHomo sapiens 298Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20 25 30 Gly
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu
50 55 60 Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser Thr Ser Thr
Ala Tyr65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser
Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala145 150 155 160 Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195
200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Ala Ala Asp Glu Val
Asp 210 215 220 His His His His His His225 230 299217PRTHomo
sapiens 299Glu Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro
Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro
Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Ser Gly Asn Ser Asn Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser
Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115
120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser
Ser Pro Val145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser
Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys
Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala
Pro Thr Glu Cys Ser 210 215 300238PRTHomo sapiens 300Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ser Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Tyr Asp Phe Trp Ser
Ala Tyr Tyr Asp Ala Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Met
Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Ala Ala Asp Glu Val Asp
His His His His His His225 230 235 301218PRTHomo sapiens 301Ala Leu
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro1 5 10 15
Gly Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly 20
25 30 Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys 35 40 45 Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val
Pro Asp Arg 50 55 60 Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Ser Gly65 70 75 80 Leu Gln Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Val Trp Asp Asp 85 90 95 Ser Leu Asn Gly Trp Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 Gly Gln Pro Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 115 120 125 Glu Glu Leu
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 130 135 140 Phe
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro145 150
155 160 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn
Asn 165 170 175 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu
Gln Trp Lys 180 185 190 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His
Glu Gly Ser Thr Val 195 200 205 Glu Lys Thr Val Ala Pro Thr Glu Cys
Ser 210 215 302231PRTHomo sapiens 302Gln Val Gln Leu Gln Gln Trp
Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr
Cys Ala Val Tyr Gly Gly Ser Phe Ser Ala Tyr 20 25 30 Tyr Trp Asn
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly
Glu Ile Asn His Ser Gly Arg Thr Asp Tyr Asn Pro Ser Leu Lys 50 55
60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Lys Gln Phe Ser
Leu65 70 75 80 Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Gly Gln Leu Val Pro Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160 Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175
Gly Leu Tyr Ser His Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180
185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Ala Ala
Asp Glu Val 210 215 220 Asp His His His His His His225 230
303680PRTHomo sapiens 303Gln Glu Asp Glu Asp Gly Asp Tyr Glu Glu
Leu Val Leu Ala Leu Arg1 5 10 15 Ser Glu Glu Asp Gly Leu Ala Glu
Ala Pro Glu His Gly Thr Thr Ala 20 25 30 Thr Phe His Arg Cys Ala
Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr 35 40 45 Val Val Val Leu
Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr 50 55 60 Ala Arg
Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys65 70 75 80
Ile Leu His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met 85
90 95 Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp
Tyr 100 105 110 Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro
Trp Asn Leu 115 120 125 Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp
Glu Tyr Gln Pro Pro 130 135 140 Asp Gly Gly Ser Leu Val Glu Val Tyr
Leu Leu Asp Thr Ser Ile Gln145 150 155 160 Ser Asp His Arg Glu Ile
Glu Gly Arg Val Met Val Thr Asp Phe Glu 165 170 175 Asn Val Pro Glu
Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys 180 185 190 Cys Asp
Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp 195 200 205
Ala Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn 210
215 220 Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu
Phe225 230 235 240 Ile Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro
Leu Val Val Leu 245 250 255 Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val
Leu Asn Ala Ala Cys Gln 260 265 270 Arg Leu Ala Arg Ala Gly Val Val
Leu Val Thr Ala Ala Gly Asn Phe 275 280 285 Arg Asp Asp Ala Cys Leu
Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile 290 295 300 Thr Val Gly Ala
Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr305 310 315 320 Leu
Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu 325 330
335 Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln
340 345 350 Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala
Ala Met 355 360 365 Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu
Leu Arg Gln Arg 370 375 380 Leu Ile His Phe Ser Ala Lys Asp Val Ile
Asn Glu Ala Trp Phe Pro385 390 395 400 Glu Asp Gln Arg Val Leu Thr
Pro Asn Leu Val Ala Ala Leu Pro Pro 405 410 415 Ser Thr His Gly Ala
Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser 420 425 430 Ala His Ser
Gly Pro Thr Arg Met Ala Thr Ala Ile Ala Arg Cys Ala 435 440 445 Pro
Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys 450 455
460 Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys
Arg465 470 475 480 Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala
Ile Ala Arg Cys 485 490 495 Cys Leu Leu Pro Gln Ala Asn Cys Ser Val
His Thr Ala Pro Pro Ala 500 505 510 Glu Ala Ser Met Gly Thr Arg Val
His Cys His Gln Gln Gly His Val 515 520 525 Leu Thr Gly Cys Ser Ser
His Trp Glu Val Glu Asp Leu Gly Thr His 530 535 540 Lys Pro Pro Val
Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly545 550 555 560 His
Arg Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu 565 570
575 Glu Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val
580 585 590 Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser
Ala Leu 595 600 605 Pro Gly Thr Ser His Val Leu Gly Ala Tyr Ala Val
Asp Asn Thr Cys 610 615 620 Val Val Arg Ser Arg Asp Val Ser Thr Thr
Gly Ser Thr Ser Glu Glu625 630 635 640 Ala Val Thr Ala Val Ala Ile
Cys Cys Arg Ser Arg His Leu Ala Gln 645 650 655 Ala Ser Gln Glu Leu
Gln Gly Ser Ser Asp Tyr Lys Asp Asp Asp Lys 660 665 670 His His His
His His His His His 675 680 304680PRTHomo sapiens 304Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Arg Arg Arg1 5 10 15 Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg His Arg Arg Arg Arg 20 25
30 Arg Phe Arg Arg Cys Arg Arg Arg Pro Trp Arg Arg Pro Gly Arg Tyr
35 40 45 Val Val Val Leu Arg Arg Arg Arg Arg Arg Ser Arg Ser Arg
Glu Thr 50 55 60 Ala Glu Glu Leu Gln Arg Arg Ala Arg Glu Glu Gly
Arg Arg Thr Lys65 70 75 80 Ile Arg Arg Arg Phe Arg Gly Leu Leu Pro
Gly Phe Leu Val Arg Met 85 90 95 Arg Arg Arg Leu Arg Arg Leu Ala
Arg Arg Leu Pro Arg Val Arg Tyr 100 105 110 Ile Glu Glu Asp Ser Ser
Val Phe Arg Gln Arg Ile Pro Arg Asn Arg 115 120 125 Arg Glu Ile Arg
Pro Pro Arg Tyr Arg Ala Arg Arg Arg Arg Pro Pro 130 135 140 Arg Gly
Gly Arg Arg Val Glu Val Tyr Leu Leu Asp Thr Arg Ile Arg145 150 155
160 Arg Arg His Glu Glu Ile Arg Gly Arg Val Arg Arg Arg Arg Phe Arg
165 170 175 Arg Arg Pro Arg Arg Arg Arg Arg Glu Arg Glu Glu Arg Arg
Arg Arg 180 185 190 Cys Asp Arg Arg Gly Thr His Leu Ala Gly Val Val
Ser Gly Glu Arg 195 200 205 Ala Gly Val Ala Arg Arg Ala Arg Met Arg
Ser Leu Glu Val Leu Asn 210 215 220 Cys Arg Gly Arg Gly Arg Val Ser
Gly Thr Leu Ile Gly Leu Glu Arg225 230 235 240 Ile Glu Arg Arg Arg
Arg Arg Arg Pro Arg Arg Pro Leu Val Val Leu 245 250 255 Leu Pro Leu
Ala Gly Arg Tyr Ser Glu Val Leu Asn Arg Ala Cys Arg 260 265 270 Arg
Leu Ala Glu Arg Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe 275 280
285 Glu Asp Asp Ala Cys Arg Tyr Ser Pro Ala Arg Ala Pro Glu Val Ile
290 295 300 Thr Val Gly Ala Thr Asn Arg Arg Arg Arg Pro Val Arg Arg
Gly Arg305 310 315 320 Arg Gly Thr Asn Phe Gly Arg Cys Val Asp Leu
Phe Ala Pro Gly Arg 325 330 335 Arg Ile Ile Gly Ala Ser Ser Arg Cys
Ser Arg Cys Arg Arg Arg Arg 340 345 350 Ser Gly Thr Ser Gln Ala Ala
Ala His Val Ala Gly Ile Ala Ala Arg 355 360 365 Met Leu Arg Arg Arg
Pro Arg Leu Arg Arg Ala Arg Leu Arg Gln Glu 370 375 380 Leu Arg Arg
Arg Ser Arg Arg Arg Arg Ile Arg Arg Arg Arg Phe Pro385 390 395 400
Arg Arg Arg Glu Arg Leu Thr Pro Arg Leu Val Ala Arg Leu Pro Pro 405
410 415 Arg Arg Arg Arg Arg Gly Arg Arg Leu Phe Cys Arg Thr Val Trp
Ser 420 425 430 Arg Arg Ser Gly Pro Arg Glu Arg Ala Arg Ala Ile Ala
Glu Cys Ala 435 440 445 Pro Arg Glu Glu Leu Leu Ser Cys Ser Ser Phe
Ser Arg Ser Gly Lys 450 455 460 Arg Arg Gly Glu Arg Met Glu Arg Gln
Gly Gly Lys Leu Val Cys Arg465 470 475 480 Ala His Asn Ala Arg Arg
Gly Arg Gly Val Tyr Ala Ile Ala Arg Cys 485 490 495 Cys Leu Leu Pro
Gln Ala Arg Cys Ser Val His Arg Ala Pro Pro Ala 500 505 510 Arg Arg
Arg Arg Gly Thr Glu Val Arg Cys Arg Arg Arg Gly His Val 515 520 525
Leu Thr Gly Cys Ser Ser His Trp Arg Arg Arg Asp Arg Gly Thr Arg 530
535 540 Lys Pro Pro Arg Leu Arg Pro Glu Gly Arg Pro Arg Gln Cys Val
Gly545 550 555 560 His Arg Glu Ala Ser Ile His Ala Ser Cys Cys His
Ala Pro Gly Leu 565 570 575 Glu Cys Arg Arg Arg Arg Arg Arg Ile Pro
Ala Pro Arg Glu Arg Val 580 585 590 Thr Val Arg Cys Arg Arg Gly Trp
Thr Leu Thr Gly Cys Ser Ala Leu 595 600 605 Pro Gly Thr Ser His Val
Leu Gly
Ala Tyr Ala Arg Asp Asn Thr Cys 610 615 620 Val Val Arg Ser Glu Asp
Arg Arg Arg Arg Arg Arg Arg Arg Arg Glu625 630 635 640 Arg Val Thr
Ala Val Ala Ile Cys Cys Glu Ser Glu His Leu Ala Gln 645 650 655 Ala
Ser Gln Glu Leu Gln Gly Ser Ser Asp Tyr Lys Asp Asp Asp Lys 660 665
670 His His His His His His His His 675 680 30514PRTHomo sapiens
305Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Ser Val Ser1 5 10
3067PRTHomo sapiens 306Glu Val Ser Asn Arg Pro Ser1 5 3079PRTHomo
sapiens 307Ser Ser Tyr Thr Ser Thr Ser Met Val1 5 3085PRTHomo
sapiens 308Ser Tyr Gly Ile Ser1 5 30917PRTHomo sapiens 309Trp Ile
Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val Gln1 5 10 15
Gly3106PRTHomo sapiens 310Gly Tyr Gly Met Asp Val1 5 31114PRTHomo
sapiens 311Thr Gly Thr Ser Ser Asp Val Gly Arg Tyr Asn Ser Val Ser1
5 10 3127PRTHomo sapiens 312Glu Val Ser Asn Arg Pro Ser1 5
3139PRTHomo sapiens 313Ser Ser Tyr Thr Ser Ser Ser Val Val1 5
31417PRTHomo sapiens 314Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr
Ala Glu Lys Leu Gln1 5 10 15 Gly3156PRTHomo sapiens 315Gly Tyr Val
Met Asp Val1 5 31614PRTHomo sapiens 316Thr Gly Thr Ser Ser Asp Val
Gly Ala Tyr Asn Ser Val Ser1 5 10 3179PRTHomo sapiens 317Ser Ser
Tyr Thr Ser Thr Asn Met Val1 5 31817PRTHomo sapiens 318Trp Val Ser
Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln1 5 10 15
Gly3199PRTHomo sapiens 319Asn Ser Tyr Thr Ser Thr Ser Met Val1 5
32017PRTHomo sapiens 320Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr
Ala Gln Lys Leu Gln1 5 10 15 Gly3217PRTHomo sapiens 321Glu Val Thr
Asn Arg Pro Ser1 5 32214PRTHomo sapiens 322Thr Gly Thr Asn Ser Asp
Val Gly Gly Tyr Asn Ser Val Ser1 5 10 32317PRTHomo sapiens 323Trp
Ile Ser Val Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Val Gln1 5 10
15 Gly32417PRTHomo sapiens 324Trp Ile Ser Phe Tyr Asn Gly Asn Thr
Asn Tyr Ala Gln Lys Val Gln1 5 10 15 Gly32513PRTHomo sapiens 325Ser
Gly Ser Ser Ser Asn Ile Gly Asn Asn Phe Val Ser1 5 10 3267PRTHomo
sapiens 326Asp Tyr Asn Lys Arg Pro Ser1 5 32711PRTHomo sapiens
327Gly Thr Trp Asp Ser Ser Leu Ser Gly Tyr Val1 5 10 3285PRTHomo
sapiens 328Ser Phe Gly Met His1 5 32917PRTHomo sapiens 329Leu Ile
Trp Asn Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15
Gly33013PRTHomo sapiens 330Ala Ile Ala Ala Leu Tyr Tyr Tyr Tyr Gly
Met Asp Val1 5 10 3317PRTHomo sapiens 331Asp Ser Asn Lys Arg Pro
Ser1 5 33211PRTHomo sapiens 332Gly Thr Trp Asp Ser Ser Leu Ser Ala
Tyr Val1 5 10 3335PRTHomo sapiens 333Ser Tyr Gly Met His1 5
33417PRTHomo sapiens 334Leu Ile Trp His Asp Gly Ser Asn Thr Tyr Tyr
Val Asp Ser Val Lys1 5 10 15 Gly33513PRTHomo sapiens 335Gly Ile Ala
Val Ala Tyr Tyr Tyr Tyr Gly Met Asp Val1 5 10 33617PRTHomo sapiens
336Leu Ile Trp Ser Asp Gly Ser Asp Glu Tyr Tyr Ala Asp Ser Val Lys1
5 10 15 Gly33711PRTHomo sapiens 337Gly Thr Trp Asp Ser Ser Leu Ser
Ser Tyr Val1 5 10 33817PRTHomo sapiens 338Leu Ile Trp Ser Asp Gly
Ser Asp Lys Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly33913PRTHomo
sapiens 339Ser Gly Ser Ser Ser Asn Ile Gly Ser Lys Thr Val Asn1 5
10 3407PRTHomo sapiens 340Ser Asn Asn Arg Arg Pro Ser1 5
34110PRTHomo sapiens 341Ala Ala Trp Asp Asp Ser Leu Asn Trp Val1 5
10 3424PRTHomo sapiens 342Tyr Trp Met Ser1 34317PRTHomo sapiens
343Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1
5 10 15 Gly34410PRTHomo sapiens 344Glu Ser Asn Trp Gly Phe Ala Phe
Asp Ile1 5 10 34513PRTHomo sapiens 345Ser Gly Ser Ser Ser Asn Ile
Gly Ser Asn Thr Val Asn1 5 10 3465PRTHomo sapiens 346Arg Tyr Trp
Met Ser1 5 34717PRTHomo sapiens 347Asn Ile Lys His Asp Gly Ser Glu
Lys Tyr Tyr Val Asp Ser Val Lys1 5 10 15 Gly34810PRTHomo sapiens
348Glu Ser Asn Trp Gly Phe Ala Phe Asp Val1 5 10 3497PRTHomo
sapiens 349Arg Asn Asn Gln Arg Pro Leu1 5 3505PRTHomo sapiens
350Ser Tyr Trp Met Ser1 5 3515PRTHomo sapiens 351Asn Phe Trp Met
Ser1 5 35210PRTHomo sapiens 352Arg Ala Ser Gln Ser Ile Ser Tyr Leu
Asn1 5 10 3536PRTHomo sapiens 353Ala Ala Ser Leu Gln Ser1 5
3548PRTHomo sapiens 354Gln Gln Ser Tyr Ser Pro Ile Thr1 5
35511PRTHomo sapiens 355Arg Ala Ser Gln Ser Ile Ser Ile Tyr Leu
Asn1 5 10 3567PRTHomo sapiens 356Ala Ala Ala Ser Leu Gln Ser1 5
3579PRTHomo sapiens 357Gln Gln Ser Tyr Ser Ala Pro Ile Thr1 5
35811PRTHomo sapiens 358Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu
Asn1 5 10 3597PRTHomo sapiens 359Ala Ala Ser Ser Leu Gln Ser1 5
3609PRTHomo sapiens 360Gln Gln Ser Tyr Ser Ser Pro Ile Thr1 5
3615PRTHomo sapiens 361Ser Tyr Ala Met Asn1 5 36216PRTHomo sapiens
362Thr Ile Ser Gly Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly1
5 10 15 36312PRTHomo sapiens 363Lys Phe Val Leu Met Val Tyr Ala Met
Leu Asp Tyr1 5 10 36417PRTHomo sapiens 364Thr Ile Ser Gly Ser Gly
Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15 Gly36517PRTHomo
sapiens 365Thr Ile Ser Gly Ser Gly Asp Asn Thr Tyr Tyr Ala Asp Ser
Val Lys1 5 10 15 Gly36610PRTHomo sapiens 366Gly Tyr Ser Leu Thr Ser
Tyr Gly Ile Ser1 5 10 36710PRTHomo sapiens 367Gly Tyr Ala Leu Thr
Ser Tyr Gly Ile Ser1 5 10 36810PRTHomo sapiens 368Gly Tyr Thr Leu
Thr Ser Tyr Gly Ile Ser1 5 10 36910PRTHomo sapiens 369Gly Tyr Ser
Phe Thr Ser Tyr Gly Ile Ser1 5 10 37010PRTHomo sapiens 370Gly Tyr
Thr Phe Pro Ser Tyr Gly Ile Ser1 5 10 37110PRTHomo sapiens 371Gly
Phe Thr Phe Ser Ser Tyr Trp Met Ser1 5 10 37210PRTHomo sapiens
372Gly Phe Thr Phe Ser Arg Tyr Trp Met Ser1 5 10 37310PRTHomo
sapiens 373Gly Leu Thr Phe Ser Asn Phe Trp Met Ser1 5 10
37410PRTHomo sapiens 374Gly Phe Thr Phe Ser Ser Tyr Ala Met Asn1 5
10 37510PRTHomo sapiens 375Gly Phe Thr Phe Asn Ser Phe Gly Met His1
5 10 37610PRTHomo sapiens 376Gly Phe Thr Phe Arg Ser Tyr Gly Met
His1 5 10 37716PRTHomo sapiens 377Asn Ile Lys Gln Asp Gly Ser Glu
Lys Tyr Val Asp Ser Val Lys Gly1 5 10 15 37816PRTHomo sapiens
378Asn Ile Lys His Asp Gly Ser Glu Lys Tyr Val Asp Ser Val Lys Gly1
5 10 15 37916PRTHomo sapiens 379Thr Ile Ser Gly Ser Gly Asp Asn Thr
Tyr Ala Asp Ser Val Lys Gly1 5 10 15 38016PRTHomo sapiens 380Thr
Ile Ser Gly Ser Gly Gly Asn Thr Tyr Ala Asp Ser Val Lys Gly1 5 10
15 38116PRTHomo sapiens 381Leu Ile Trp Asn Asp Gly Ser Asn Lys Tyr
Ala Asp Ser Val Lys Gly1 5 10 15 38216PRTHomo sapiens 382Leu Ile
Trp Ser Asp Gly Ser Asp Glu Tyr Ala Asp Ser Val Lys Gly1 5 10 15
38316PRTHomo sapiens 383Leu Ile Trp Ser Asp Gly Ser Asp Lys Tyr Ala
Asp Ser Val Lys Gly1 5 10 15 38416PRTHomo sapiens 384Leu Ile Trp
His Asp Gly Ser Asn Thr Tyr Val Asp Ser Val Lys Gly1 5 10 15
38510PRTHomo sapiens 385Glu Ser Asn Trp Gly Phe Ala Phe Asp Ile1 5
10 38610PRTHomo sapiens 386Glu Ser Asn Trp Gly Phe Ala Phe Asp Val1
5 10 3876PRTHomo sapiens 387Gly Tyr Val Met Asp Val1 5 38811PRTHomo
sapiens 388Arg Ala Ser Gln Ser Ile Ser Ile Tyr Leu Asn1 5 10
38914PRTHomo sapiens 389Thr Gly Thr Asn Ser Asp Val Gly Gly Tyr Asn
Ser Val Ser1 5 10 39014PRTHomo sapiens 390Thr Gly Thr Ser Ser Asp
Val Gly Ala Tyr Asn Ser Val Ser1 5 10 39114PRTHomo sapiens 391Thr
Gly Thr Ser Ser Asp Val Gly Arg Tyr Asn Ser Val Ser1 5 10
3927PRTHomo sapiens 392Arg Asn Asn Gln Arg Pro Leu1 5 3937PRTHomo
sapiens 393Ala Ala Ala Ser Leu Gln Ser1 5 3949PRTHomo sapiens
394Gln Gln Ser Tyr Ser Ala Pro Ile Thr1 5 3959PRTHomo sapiens
395Asn Ser Tyr Thr Ser Thr Ser Met Val1 5 3969PRTHomo sapiens
396Ser Ser Tyr Thr Ser Ser Ser Val Val1 5 39710PRTHomo sapiens
397Ala Ala Trp Asp Asp Ser Leu Asn Trp Val1 5 10 39811PRTHomo
sapiens 398Gly Thr Trp Asp Ser Ser Leu Ser Ser Tyr Val1 5 10
39911PRTHomo sapiens 399Gly Thr Trp Asp Ser Ser Leu Ser Ala Tyr
Val1 5 10 400116PRTHomo sapiens 400Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr
Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65
70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Tyr Ser Ser Gly Trp Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 401118PRTHomo
sapiens 401Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser
Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu 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
Glu Ser Asn Trp Gly Phe Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110
Thr Met Val Thr Val Ser 115 402115PRTHomo sapiens 402Glu 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 Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu 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 Asn Trp Gly Ala Phe Asp
Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser 115
4036PRTHomo sapiens 403Glu Asn Leu Tyr Phe Gln1 5 40414PRTHomo
sapiensVARIANT1Xaa= D, A, R or no amino acid 404Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 40511PRTHomo
sapiensVARIANT1Xaa=Q or G 405Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10 40610PRTHomo sapiensVARIANT1Xaa=G 406Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 40714PRTHomo sapiensVARIANT1Xaa=T or
no amino acid 407Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa1 5 10 40817PRTHomo sapiensVARIANT1Xaa=W, S, L or no amino
acid 408Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 15 Xaa4097PRTHomo sapiensVARIANT1Xaa=G, E, S or D 409Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 41014PRTHomo sapiensVARIANT1Xaa=D or no
amino acid 410Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 41111PRTHomo sapiensVARIANT1Xaa=Q, A, G or no amino acid
411Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 41210PRTHomo
sapiensVARIANT1Xaa=G, P or A 412Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa1 5 10 41314PRTHomo sapiensVARIANT1Xaa=T or S 413Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 41417PRTHomo
sapiensVARIANT1Xaa=W, Y or F 414Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15 Xaa4157PRTHomo
sapiensVARIANT1Xaa=E or D 415Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
4166PRTHomo sapiensVARIANT1Xaa=G, P, A or no amino acid 416Xaa Xaa
Xaa Xaa Xaa Xaa1 5 4179PRTHomo sapiensVARIANT1Xaa=S, N, T, A, C or
Q 417Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 418363DNAHomo sapiens
418caggtgcagg tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc ggctactata tacactgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaaccctc
acagtggtgg cgcaaactat 180gcacagaagt ttcagggcag ggtcaccatg
accagggaca cgtccatcag cacagcctac 240atggagctga gcaggctgag
atctgacgac acggccgtgt attactgtgc gagaggcaac 300tggaactacg
actactacgg tatggacgtc tggggccaag ggaccacggt caccgtctcc 360tca
363419121PRTHomo sapiens 419Gln Val Gln Val Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn
Pro His Ser Gly Gly Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Asn Trp Asn Tyr Asp Tyr Tyr Gly Met Asp Val Trp
Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
420321DNAHomo sapiens 420gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca ggacattagc
aattatttag cctggtatca gcagaaacca 120gggaaagttc ctaagctcct
gatctatgct gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag cctacagcct
240gaagatgttg caacttattt ctgtcaaagg tatcagattg ccccattcac
tttcggccct 300gggaccaagg tggatatcaa a 321421107PRTHomo sapiens
421Asp 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 Ile Ser Asn
Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Thr Leu Gln 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 Val Ala Thr Tyr Phe Cys Gln Arg Tyr
Gln Ile Ala Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys 100 105 422366DNAHomo sapiens 422caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atctggtatg atggaagtac taaatactat
180gcagactccg tgaagggccg atccaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gaggtcagtg 300gctggttacc actactacta cggtatggac
gtctggggcc aagggaccac ggtcaccgtc 360tcctca 366423122PRTHomo sapiens
423Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Thr Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu 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 Ser Val Ala
Gly Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 424324DNAHomo sapiens
424tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccaag gagacagcct cagaggctat tatgcaacct ggtaccagca
gaagccaaga 120caggcccctg tacttgtcat ctatggtaaa aactaccggc
cctcagggat cccagaccga 180ttctctggct ccacctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taactcccgg gacagcattg gtaaccatct ggtgttcggc 300ggagggacca
agctgaccgt ccta 324425108PRTHomo sapiens 425Ser Ser Glu Leu Thr Gln
Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Gly Tyr Tyr Ala 20 25 30 Thr Trp
Tyr Gln Gln Lys Pro Arg Gln Ala Pro Val Leu Val Ile Tyr 35 40 45
Gly Lys Asn Tyr Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50
55 60 Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ile
Gly Asn His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 426366DNAHomo sapiens 426caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggct tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggttag atggaagtaa taaatactat
180gcagactccg tgaagggccg atccaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gaggtcagtg 300gctggttacc actactacta cggtatggac
gtctggggcc aagggaccac ggtcaccgtc 360tcctca 366427122PRTHomo sapiens
427Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Leu Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu 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 Ser Val Ala
Gly Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 428324DNAHomo sapiens
428tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccaag gagacagcct cagaagttat tatggaagct ggtaccagca
gaagccaaga 120caggcccctg tacttgtcat ctttggtaaa aacaaccggc
cctcagggat cccagaccga 180ttctctggct ccacctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taactcacgg gacatcattg gtgaccatct gctgttcggc 300ggagggacca
agctgaccgt ccta 324429108PRTHomo sapiens 429Ser Ser Glu Leu Thr Gln
Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Gly 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Arg Gln Ala Pro Val Leu Val Ile Phe 35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50
55 60 Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Ile
Gly Asp His 85 90 95 Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 430366DNAHomo sapiens 430caggtgcagc tggtggagtc
tgggggaggc gtggtccagt ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagg aactatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtttg atggaagtaa taaatactat
180gcagactccg tgaagggccg atccaccatc tccagagaca attccaagaa
cacgctgtat 240ctgctaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gaggtcagtg 300gctggttacc actactacta cggtatggac
gtctggggcc aagggaccac ggtcaccgtc 360tcctca 366431122PRTHomo sapiens
431Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Ser Gly Arg1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Phe Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Leu Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Ala
Gly Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 432324DNAHomo sapiens
432tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccagg gagacagcct cagaagctat tatgcaagct ggtaccagca
gaagccaaga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc
cctcagggat cccagaccga 180atctctggct ccacctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taaatcccgg gacatcattg gtgaccatct ggtgttcggc 300ggagggacca
aactgaccgt ccta 324433108PRTHomo sapiens 433Ser Ser Glu Leu Thr Gln
Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Arg Gln Ala Pro Val Leu Val Ile Tyr 35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Ile Ser Gly Ser 50
55 60 Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Ile Ile
Gly Asp His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 434342DNAHomo sapiens 434caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgt gagagatcgg 300ggactggact ggggccaggg aaccctggtc
accgtctcct ca 342435114PRTHomo sapiens 435Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Val Arg Asp Arg Gly Leu Asp Trp Gly Gln Gly
Thr Leu Val Thr Val 100 105 110 Ser Ser436324DNAHomo sapiens
436tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccaag gagacagcct cagaggctat tatgcaagct ggtaccagca
gaagccaaga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc
cctcagggat cccagaccga 180ttctctggct ccacctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taagtcccgg gacagcagtg gtgaccatct ggtgttcggc 300ggagggacca
agctgaccgt ccta 324437108PRTHomo sapiens 437Ser Ser Glu Leu Thr Gln
Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Gly Tyr Tyr Ala 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Arg Gln Ala Pro Val Leu Val Ile Tyr 35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50
55 60 Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Ser Ser
Gly Asp His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 438366DNAHomo sapiens 438caggtgcagg tggtggagtc
tgggggaggc gtggtccagc ctggggggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt aactatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atttggtatg atggaagtag taaatactat
180gcagactccg tgaagggccg atccaccatc tccagagaca attccaagaa
cacggtgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gaggtcagtg 300gctggttacc actactacta cggtatggac
gtctggggcc aagggaccac ggtcaccgtc 360tcctca 366439122PRTHomo sapiens
439Gln Val Gln Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Ser Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Val 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 Ser Val Ala
Gly Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 440324DNAHomo sapiens
440tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccaag gagacagcct cagaggctat tatgcaagct ggtaccagca
gaagccaaga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc
cctcagggat cccagaccga 180ttctctggct ccacctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taagtcccgg gacagcagtg gtgaccatct ggtgttcggc 300ggagggacca
agctgaccgt ccta 324441108PRTHomo sapiens 441Ser Ser Glu Leu Thr Gln
Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Gly Tyr Tyr Ala 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Arg Gln Ala Pro Val Leu Val Ile Tyr 35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50
55 60 Thr Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Ser Ser
Gly Asp His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 442366DNAHomo sapiens 442caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagtctc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaagtta taaagactat
180gcagactccg tgaagggccg atccaccatc tccagagaca actccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attattgtgc gaggtcagtg 300gctggttacc actactacta cggtatggac
gtctggggcc aagggaccac ggtcaccgtc 360tcctca 366443122PRTHomo sapiens
443Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Tyr Lys Asp
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Ser Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu 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 Ser Val Ala
Gly Tyr His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 444324DNAHomo sapiens
444tcttctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccaag gagacagcct cagaacctat tatgcaagct ggtaccagca
gaagccaaga 120caggccccta ttcttgtcat ctatggtaaa aacaaccggc
cctcagggat cccagaccga 180ttctctggct ccacctcagg aatcacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taaatcccgg gacatcattg gtaaccatct gctgttcggc 300ggagggacta
agctgaccgt ccta 324445108PRTHomo sapiens 445Ser Ser Glu Leu Thr Gln
Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Thr Tyr Tyr Ala 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Arg Gln Ala Pro Ile Leu Val Ile Tyr 35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50
55 60 Thr Ser Gly Ile Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Ile Ile
Gly Asn His 85 90 95 Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105 446375DNAHomo sapiens 446caggtgcagc tggtggcgtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccctcagt agctatggca tgcactgggt ccgccaggct 120ccaggccagg
ggctggagtg ggtggcagtc atatggtatg atggaagtaa caaatactat
180gcagcctccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagtctgag agccgaggac acggctgtgt
attactgtgc gagagggggt 300ggttcgggga gtcatcgcta ctactactac
ggtatggacg tctggggcca agggaccacg 360gtcaccgtct cctca
375447125PRTHomo sapiens 447Gln Val Gln Leu Val
Ala Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Ala Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu 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 Gly Gly Gly Ser Gly Ser His Arg
Tyr Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 125 448324DNAHomo sapiens 448tcttctgagc
tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag
gagacagcct cagaacctat tatgcaagct ggtaccagca gaagccaaga
120caggccccta ttcttgtcat ctatggtaaa aacaaccggc cctcagggat
cccagaccga 180ttctctggct ccacctcagg aatcacagct tccttgacca
tcactggggc tcaggcggaa 240gatgaggctg actattactg taaatcccgg
gacatcattg gtaaccatct gctgttcggc 300ggagggacta agctgaccgt ccta
324449108PRTHomo sapiens 449Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly
Asp Ser Leu Arg Thr Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys
Pro Arg Gln Ala Pro Ile Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn
Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Thr Ser
Gly Ile Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Ile Ile Gly Asn His 85
90 95 Leu Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
450366DNAHomo sapiens 450caggtgcaag tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt
aactatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaggtaa taaatactat 180gcagactccg
tgaagggccg atccatcatc tccagagaca attccaagag cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgttt attattgtgc
gaggtcagtg 300gctggttacc attattacta cggtatggac gtctggggcc
aagggaccac ggtcaccgtc 360gcctca 366451122PRTHomo sapiens 451Gln Val
Gln Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Gly Asn Lys Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Ser Ile Ile Ser Arg Asp Asn Ser
Lys Ser Thr Leu 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 Ser Val Ala Gly Tyr
His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr
Val Thr Val Ala Ser 115 120 452327DNAHomo sapiens 452cagtctgccc
tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg
gaaccagcag tgacgttggt ggttataact ctgtctcctg gtaccaacag
120cacccaggca aaccccccaa actcatgatt tatgaggtca gtaatcggcc
ctcagggatt 180tctaatcgct tctctggctc caagtctggc aacacggcct
ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttatttctgc
agctcatata caagcaccag catggtcttc 300ggcggaggga ccaagctggc cgtccta
327453109PRTHomo sapiens 453Gln Ser Ala Leu Thr Gln Pro Ala Ser Val
Ser Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly
Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr
Gln Gln His Pro Gly Lys Pro Pro Lys Leu 35 40 45 Met Ile Tyr Glu
Val Ser Asn Arg Pro Ser Gly Ile Ser Asn Arg Phe 50 55 60 Ser Gly
Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu65 70 75 80
Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ser Ser Tyr Thr Ser Thr 85
90 95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu 100 105
454366DNAHomo sapiens 454caggtgcaag tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt
aactatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaggtaa taaatactat 180gcagactccg
tgaagggccg atccatcatc tccagagaca attccaagag cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgttt attattgtgc
gaggtcagtg 300gctggttacc attattacta cggtatggac gtctggggcc
aagggaccac ggtcaccgtc 360gcctca 366455122PRTHomo sapiens 455Gln Val
Gln Val Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20
25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Gly Asn Lys Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Ser Ile Ile Ser Arg Asp Asn Ser
Lys Ser Thr Leu 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 Ser Val Ala Gly Tyr
His Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr
Val Thr Val Ala Ser 115 120 456324DNAHomo sapiens 456tcttctgagc
tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag
gagacagcct cagaggctat tatgcaagct ggtaccagca gaagccaaga
120caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat
cccagaccga 180ttctctggct ccacgtcagg aaacacagct tccttgacca
tcactggggc tcaggcggaa 240gatgaggctg actattactg taactcccgg
gacaacattg gtgaccatct ggtgttcggc 300ggagggacca agctgaccgt ccta
324457108PRTHomo sapiens 457Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly
Asp Ser Leu Arg Gly Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys
Pro Arg Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn
Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Thr Ser
Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Asn Ile Gly Asp His 85
90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
458381DNAHomo sapiens 458gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctccggatt cacctttagt
agctattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtggccagc ataaaacaag atggaagtga gaaatactat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaggaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagagatctt 300gtattaatgg tgtatgatat agactactac tactacggta
tggacgtctg gggccaaggg 360accacggtca ccgtctcctc a 381459127PRTHomo
sapiens 459Glu 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 Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Lys Gln Asp Gly Ser
Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Arg Asn Ser Leu 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
Asp Leu Val Leu Met Val Tyr Asp Ile Asp Tyr Tyr Tyr Tyr 100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
125 460336DNAHomo sapiens 460gatattgtga tgactcagtc tccactctcc
ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg
catagtaatg gatacaacta tttggattgg 120tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct
acaaactccg 300ctcactttcg gcggagggac caaggtagag atcaaa
336461112PRTHomo sapiens 461Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85
90 95 Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 110 462381DNAHomo sapiens 462gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctccggatt
cacctttagt aactattgga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtggccagc ataaaacaag atggaagtga gaaatactat
180gtggactctg tgaagggccg attcgccatc tccagagaca acgccaagaa
ctcactgttt 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagatctt 300gtactaatgg tgtatgatat agactactac
tactacggta tggacgtctg gggccaaggg 360accacggtca ccgtctcctc a
381463127PRTHomo sapiens 463Glu 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 Asn Tyr 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Lys
Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly
Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asp Leu Val Leu Met Val Tyr Asp Ile Asp Tyr Tyr Tyr
Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 125 464336DNAHomo sapiens 464gatattgtga tgactcagtc
tccactctcc ctgcctgtca cccctggaga gccggcctcc 60atctcttgca ggtctagtca
gagcctcctg catagtaatg ggtacaacta tttggattgg 120tacctgcaga
agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc
180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cacatcttac
actgaaaatc 240agcagagtgg aggctgagga tgttggagtt tattactgca
tgcaaactct acaaactccg 300ctcactttcg gcggagggac caaggtggag atcaaa
336465112PRTHomo sapiens 465Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr His Leu Thr Leu Lys Ile65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Thr 85
90 95 Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys 100 105 110 466342DNAHomo sapiens 466caggtgcagc tggtggagtc
tgggggaggc gtggcccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatactatg atggaattaa taaacactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagagatcgg 300ggactggact ggggccaggg aaccctggtc
accgtctcct ca 342467114PRTHomo sapiens 467Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Ala Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Tyr Tyr Asp Gly Ile Asn Lys His Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
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 Asp Arg Gly Leu Asp Trp Gly Gln Gly
Thr Leu Val Thr Val 100 105 110 Ser Ser 468339DNAHomo sapiens
468gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga
gagggccacc 60atcaactgca agtccagcca gagtgtttta tacagctcca acagtaagaa
ctacttagtt 120tggtaccagc agaaaccagg acagcctcct aagctgctca
tttactgggc ctctacccgg 180gaatccgggg tccctgaccg attcagtggc
agcgggtctg ggacagattt cactctcacc 240atcagcagcc tgcaggctga
agatgtggca gtttattact gtcaacaata ttatagtact 300ccgtggacgt
tcggccaagg gaccaaggtg gaaatcaaa 339469113PRTHomo sapiens 469Asp Ile
Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20
25 30 Ser Asn Ser Lys Asn Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly
Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu
Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val
Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys 470357DNAHomo
sapiens 470gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggact cacctttagt aacttttgga tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtggccaac ataaagcaag
atggaaatga taaatactat 180gtggactctg tgaagggccg attcaccatc
tccagagaca acgccaagaa ttcactgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagagtca 300aactggggat
ttgcttttga tatctggggc caagggacaa tggtcaccgt ctcttca
357471119PRTHomo sapiens 471Glu 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 Leu Thr Phe Ser Asn Phe 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys
Gln Asp Gly Asn Asp Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 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 Glu Ser Asn Trp Gly Phe Ala Phe Asp Ile Trp Gly Gln
Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 472327DNAHomo
sapiens 472cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag
ggtcaccatc
60tcttgttctg gaagcagctc caacatcgga agtaaaactg taaactggta ccagcagttc
120ccaggaacgg cccccaaact cctcatctat agtaataatc ggcggccctc
aggggtccct 180gaccgattct ctggctccaa gtctggcacc tcagcctccc
tggccatcag tgggctccag 240tctgaggatg aggctgatta ttactgtgca
gcatgggatg acagcctgaa ttgggtgttc 300ggcgcaggga ccaagctgac cgtccta
327473109PRTHomo sapiens 473Gln Ser Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Lys 20 25 30 Thr Val Asn Trp Tyr Gln
Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn
Asn Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95 Asn Trp Val Phe Gly Ala Gly Thr Lys Leu Thr Val Leu 100 105
474357DNAHomo sapiens 474gaggtgcagc tggtggagtc tgggggaggt
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggact cacctttagt
aacttttgga tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtggccaac ataaagcaag atggaagtga gaaatactat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ttcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagagagtca 300aactggggat ttgcttttga tatctggggc caagggacaa
tggtcaccgt ctcttca 357475119PRTHomo sapiens 475Glu 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 Leu Thr Phe Ser Asn Phe 20 25 30 Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu 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 Glu Ser Asn Trp Gly Phe Ala Phe
Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115
476327DNAHomo sapiens 476cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga
agtaaaactg taaactggta ccagcagttc 120ccaggaacgg cccccaaact
cctcatctat agtaataatc ggcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag
240tctgaggatg aggctgatta ttactgtgca acatgggatg acagactgaa
ttgggtgttc 300ggcgcaggga ccaagctgac cgtccta 327477109PRTHomo
sapiens 477Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro
Gly Gln1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Ser Lys 20 25 30 Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Arg Arg Pro
Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln65 70 75 80 Ser Glu Asp Glu
Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Arg Leu 85 90 95 Asn Trp
Val Phe Gly Ala Gly Thr Lys Leu Thr Val Leu 100 105 478366DNAHomo
sapiens 478caggtcacct tgaaggagtc tggtcctgtg ctggtgaaac ccacagagac
cctcacgctg 60acctgcaccg tctctgggtt ctcactcagc aatgttagaa tgggtgtgag
ctggatccgt 120cagcccccag ggaaggccct ggagtggctt gcacacattt
tttcgaatga cgaaaattcc 180tacagaacat ctctgaagag caggctcacc
atctccaagg acacctccaa aagccaggtg 240gtccttacca tgaccaacat
ggaccctgtg gacacagcca catattactg tgcacggata 300gtgggagcta
caacggatga tgcttttgat atctggggcc aagggacaat ggtcaccgtc 360tcttca
366479122PRTHomo sapiens 479Gln Val Thr Leu Lys Glu Ser Gly Pro Val
Leu Val Lys Pro Thr Glu1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Ser Asn Val 20 25 30 Arg Met Gly Val Ser Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His
Ile Phe Ser Asn Asp Glu Asn Ser Tyr Arg Thr Ser 50 55 60 Leu Lys
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val65 70 75 80
Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85
90 95 Cys Ala Arg Ile Val Gly Ala Thr Thr Asp Asp Ala Phe Asp Ile
Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
480324DNAHomo sapiens 480tcctatgtgc tgactcagcc accctcggtg
tcagtggccc caggacagac ggccaggatt 60acctgtgggg gaaacaacat tggaagtaaa
agtgtgcact ggtaccagca gaagccaggc 120caggcccctg tgctggtcgt
ctatgatgat agcgaccggc cctcagggat ccctgagcga 180ttctctggct
ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg
240gatgaggccg acttttactg tcaggtgtgg gatagtagta gtgatcctgt
ggtattcggc 300ggagggacca agctgaccgt ccta 324481108PRTHomo sapiens
481Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1
5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser
Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu
Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu
Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Arg Val Glu Ala Gly65 70 75 80 Asp Glu Ala Asp Phe Tyr Cys
Gln Val Trp Asp Ser Ser Ser Asp Pro 85 90 95 Val Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 482381DNAHomo sapiens
482gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagt aactattgga tgacctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtggccagc ataaagcaag
atggaagtga gagatactat 180gtggactctg tgaagggccg attcaccatc
tcccgagaca ccgccaagaa ctctctgtat 240ctccaaatga acagcctgcg
agccgaggac acggctgtgt attactgtgc gagacctctt 300gtactaatgg
tgtatgctct acactactac tactacggta tggacgtctg gggccacggg
360accacggtca ccgtctcctc a 381483127PRTHomo sapiens 483Glu 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 Asn Tyr 20 25
30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Ser Ile Lys Gln Asp Gly Ser Glu Arg Tyr Tyr Val Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys
Asn Ser Leu 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 Pro Leu Val Leu Met Val
Tyr Ala Leu His Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly
His Gly Thr Thr Val Thr Val Ser Ser 115 120 125 484336DNAHomo
sapiens 484gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga
gccggcctcc 60atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta
tttggattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct
atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt
ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga
tgttggggtt tattactgca tgcaagctct acaaactccg 300ctcactttcg
gcggagggac caaggtggag atcaaa 336485112PRTHomo sapiens 485Asp Ile
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20
25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 486100PRTHomo
sapiens 486Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro
Thr Glu1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Ser Asn Ala 20 25 30 Arg Met Gly Val Ser Trp Ile Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Phe Ser Asn
Asp Glu Lys Ser Tyr Ser Thr Ser 50 55 60 Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Ser Gln Val65 70 75 80 Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala
Arg Ile 100 48798PRTHomo sapiens 487Glu 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 Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn
Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 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 48898PRTHomo sapiens 488Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu 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 48993PRTHomo sapiens 489Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser
20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys 85 90 49094PRTHomo sapiens 490Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15 Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30
Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35
40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys 85 90 49189PRTHomo sapiens 491Gln Ser Val Leu Thr Gln
Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile
Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val
Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45
Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50
55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu
Gln65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 49287PRTHomo
sapiens 492Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro
Gly Lys1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly
Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asp Ser Asp Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80 Asp Glu Ala Asp
Tyr Tyr Cys 85 49349PRTHomo sapiens 493Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly49498PRTHomo sapiens 494Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg49598PRTHomo sapiens 495Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg49688PRTHomo sapiens 496Asp 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 Gly Ile Ser Asn Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Thr Leu Gln 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 Val Ala Thr Tyr Tyr Cys 85
49790PRTHomo sapiens 497Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 85
90 49887PRTHomo sapiens 498Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly Gln1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly
Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Gly Lys Asn Asn
Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser
Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys 85 49913PRTHomo sapiens 499Ser Gly Ser
Ser Ser Asn Ile Gly Ser Lys Thr Val Asn1 5 10 50010PRTHomo sapiens
500Gly Phe Thr Phe Ser Asn Tyr Trp Met Ser1 5 10 50117PRTHomo
sapiens 501Ser Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser
Val Lys1 5 10 15 Gly50218PRTHomo sapiens 502Asp Leu Val Leu Met Val
Tyr Asp Ile Asp Tyr Tyr Tyr Tyr Gly Met1 5 10 15 Asp
Val50316PRTHomo sapiens 503Arg Ser Ser Gln Ser Leu Leu His Ser Asn
Gly Tyr Asn Tyr Leu Asp1 5 10 15 5047PRTHomo sapiens 504Leu Gly Ser
Asn Arg Ala Ser1 5 5059PRTHomo sapiens 505Met Gln Thr Leu Gln Thr
Pro Leu Thr1 5 50625PRTHomo sapiens 506Gln Val Thr Leu Lys Glu Ser
Gly Pro Val Leu Val Lys Pro Thr Glu1 5 10 15 Thr Leu Thr Leu Thr
Cys Thr Val Ser 20 25 50712PRTHomo sapiens 507Gly Phe Ser Leu Ser
Asn Ala Arg Met Gly Val Ser1 5 10 50812PRTHomo sapiens 508Gly Phe
Ser Leu Ser Asn Val Arg Met Gly Val Ser1 5 10 50914PRTHomo sapiens
509Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala1 5 10
51025PRTHomo sapiens 510Glu 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 20
25 51110PRTHomo sapiens 511Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser1
5 10 51210PRTHomo sapiens 512Gly Leu Thr Phe Ser Asn Phe Trp Met
Ser1 5 10 51310PRTHomo sapiens 513Gly Phe Thr Phe Ser Asn Tyr Trp
Met Thr1 5 10 51414PRTHomo sapiens 514Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ala1 5 10 51525PRTHomo sapiens 515Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 51625PRTHomo sapiens
516Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Ala Gln Pro Gly Arg1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 51710PRTHomo
sapiens 517Gly Phe Thr Phe Ser Ser Tyr Gly Met His1 5 10
51814PRTHomo sapiens 518Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val Ala1 5 10 51916PRTHomo sapiens 519His Ile Phe Ser Asn Asp
Glu Lys Ser Tyr Ser Thr Ser Leu Lys Ser1 5 10 15 52016PRTHomo
sapiens 520His Ile Phe Ser Asn Asp Glu Asn Ser Tyr Arg Thr Ser Leu
Lys Ser1 5 10 15 52133PRTHomo sapiens 521Arg Leu Thr Ile Ser Lys
Asp Thr Ser Lys Ser Gln Val Val Leu Thr1 5 10 15 Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 20 25 30
Ile52211PRTHomo sapiens 522Val Gly Ala Thr Thr Asp Asp Ala Phe Asp
Ile1 5 10 52311PRTHomo sapiens 523Trp Gly Gln Gly Thr Met Val Thr
Val Ser Ser1 5 10 52411PRTHomo sapiens 524Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser1 5 10 52511PRTHomo sapiens 525Trp Gly His Gly
Thr Thr Val Thr Val Ser Ser1 5 10 52617PRTHomo sapiens 526Asn Ile
Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10 15
Gly52717PRTHomo sapiens 527Asn Ile Lys Gln Asp Gly Asn Asp Lys Tyr
Tyr Val Asp Ser Val Lys1 5 10 15 Gly52817PRTHomo sapiens 528Ser Ile
Lys Gln Asp Gly Ser Glu Arg Tyr Tyr Val Asp Ser Val Lys1 5 10 15
Gly52932PRTHomo sapiens 529Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr Leu Gln1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 53032PRTHomo sapiens
530Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ser Leu Tyr Leu Gln1
5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg 20 25 30 53132PRTHomo sapiens 531Arg Phe Ala Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Phe Leu Gln1 5 10 15 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 53232PRTHomo
sapiens 532Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Ser Leu Tyr
Leu Gln1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Arg 20 25 30 53310PRTHomo sapiens 533Glu Ser Asn Trp
Gly Phe Ala Phe Asp Ile1 5 10 53418PRTHomo sapiens 534Pro Leu Val
Leu Met Val Tyr Ala Leu His Tyr Tyr Tyr Tyr Gly Met1 5 10 15 Asp
Val53517PRTHomo sapiens 535Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val Lys1 5 10 15 Gly53617PRTHomo sapiens 536Val Ile
Tyr Tyr Asp Gly Ile Asn Lys His Tyr Ala Asp Ser Val Lys1 5 10 15
Gly53732PRTHomo sapiens 537Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 5385PRTHomo sapiens 538Asp
Arg Gly Leu Asp1 5 53911PRTHomo sapiens 539Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser1 5 10 54023PRTHomo sapiens 540Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys 20 54115PRTHomo sapiens 541Trp Tyr Leu Gln Lys
Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr1 5 10 15 54223PRTHomo
sapiens 542Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15 Glu Arg Ala Thr Ile Asn Cys 20 54317PRTHomo
sapiens 543Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn
Tyr Leu1 5 10 15 Ala54417PRTHomo sapiens 544Lys Ser Ser Gln Ser Val
Leu Tyr Ser Ser Asn Ser Lys Asn Tyr Leu1 5 10 15 Val54515PRTHomo
sapiens 545Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
Tyr1 5 10 15 54622PRTHomo sapiens 546Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly Thr Pro Gly Gln1 5 10 15 Arg Val Thr Ile Ser
Cys 20 54713PRTHomo sapiens 547Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn Thr Val Asn1 5 10 54813PRTHomo sapiens 548Ser Gly Ser Ser Ser
Asn Ile Gly Ser Lys Thr Val Asn1 5 10 54915PRTHomo sapiens 549Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr1 5 10 15
55015PRTHomo sapiens 550Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys
Leu Leu Ile Tyr1 5 10 15 55122PRTHomo sapiens 551Ser Tyr Val Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys1 5 10 15 Thr Ala
Arg Ile Thr Cys 20 55222PRTHomo sapiens 552Ser Tyr Val Leu Thr Gln
Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10 15 Thr Ala Arg Ile
Thr Cys 20 55311PRTHomo sapiens 553Gly Gly Asn Asn Ile Gly Ser Lys
Ser Val His1 5 10 55415PRTHomo sapiens 554Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val Leu Val Ile Tyr1 5 10 15 55515PRTHomo sapiens
555Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr1 5
10 15 55632PRTHomo sapiens 556Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr1 5 10 15 Leu Lys Ile Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 55732PRTHomo sapiens
557Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Leu Thr1
5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys 20 25 30 5589PRTHomo sapiens 558Met Gln Ala Leu Gln Thr Pro Leu
Thr1 5 55910PRTHomo sapiens 559Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys1 5 10 5607PRTHomo sapiens 560Trp Ala Ser Thr Arg Glu Ser1 5
56132PRTHomo sapiens 561Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ala Glu
Asp Val Ala Val Tyr Tyr Cys 20 25 30 5629PRTHomo sapiens 562Gln Gln
Tyr Tyr Ser Thr Pro Trp Thr1 5 56310PRTHomo sapiens 563Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys1 5 10 5647PRTHomo sapiens 564Ser Asn
Asn Gln Arg Pro Ser1 5 5657PRTHomo sapiens 565Ser Asn Asn Arg Arg
Pro Ser1 5 56632PRTHomo sapiens 566Gly Val Pro Asp Arg Phe Ser Gly
Ser Lys Ser Gly Thr Ser Ala Ser1 5 10 15 Leu Ala Ile Ser Gly Leu
Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 30 56710PRTHomo
sapiens 567Ala Ala Trp Asp Asp Ser Leu Asn Trp Val1 5 10
56810PRTHomo sapiens 568Ala Thr Trp Asp Asp Arg Leu Asn Trp Val1 5
10 56910PRTHomo sapiens 569Phe Gly Ala Gly Thr Lys Leu Thr Val Leu1
5 10 5707PRTHomo sapiens 570Tyr Asp Ser Asp Arg Pro Ser1 5
5717PRTHomo sapiens 571Asp Asp Ser Asp Arg Pro Ser1 5 57232PRTHomo
sapiens 572Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr
Ala Thr1 5 10 15 Leu Thr Ile Ser Arg Val Glu Ala Gly Asp Glu Ala
Asp Tyr Tyr Cys 20 25 30 57332PRTHomo sapiens 573Gly Ile Pro Glu
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr1 5 10 15 Leu Thr
Ile Ser Arg Val Glu Ala Gly Asp Glu Ala Asp Phe Tyr Cys 20 25 30
57411PRTHomo sapiens 574Gln Val Trp Asp Ser Ser Ser Asp Pro Val
Val1 5 10 57510PRTHomo sapiens 575Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu1 5 10
* * * * *
References