U.S. patent application number 15/957835 was filed with the patent office on 2018-11-08 for pcsk9 vaccine.
The applicant listed for this patent is Pfizer Vaccines LLC. Invention is credited to Brian Robert Champion, Leonard Gabriel Contillo, Jr., Michael Dale Eisenbraun, James Downey Fraser, Julie Jia Li Hawkins, James Richard Merson, Brian Gregory Pierce, Xiayang Qiu, Jakir Hussain Ullah, David Michael Wyatt.
Application Number | 20180318407 15/957835 |
Document ID | / |
Family ID | 43413463 |
Filed Date | 2018-11-08 |
United States Patent
Application |
20180318407 |
Kind Code |
A1 |
Champion; Brian Robert ; et
al. |
November 8, 2018 |
PCSK9 VACCINE
Abstract
The present invention relates to the provision of novel
immunogens comprising an antigenic PCSK9 peptide linked to an
immunogenic carrier for the prevention, treatment or alleviation of
PCSK9-mediated disorders. The invention further relates to methods
for production of these medicaments, immunogenic compositions and
pharmaceutical compositing thereof and their use in medicine.
Inventors: |
Champion; Brian Robert;
(Abingdon, GB) ; Contillo, Jr.; Leonard Gabriel;
(Groton, CT) ; Eisenbraun; Michael Dale; (San
Diego, CA) ; Fraser; James Downey; (San Diego,
CA) ; Hawkins; Julie Jia Li; (Old Lyme, CT) ;
Merson; James Richard; (Sandwich, GB) ; Pierce; Brian
Gregory; (Marblehead, MA) ; Qiu; Xiayang;
(Mystic, CT) ; Ullah; Jakir Hussain; (Sandwich,
GB) ; Wyatt; David Michael; (Sandwich, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Vaccines LLC |
New York |
NY |
US |
|
|
Family ID: |
43413463 |
Appl. No.: |
15/957835 |
Filed: |
April 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15266812 |
Sep 15, 2016 |
9987341 |
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15957835 |
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14508977 |
Oct 7, 2014 |
9481875 |
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15266812 |
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12872645 |
Aug 31, 2010 |
8889144 |
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14508977 |
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61239541 |
Sep 3, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/14 20180101; A61K
39/0005 20130101; A61P 9/10 20180101; A61P 9/00 20180101; C12N
9/6424 20130101; A61P 37/04 20180101; C12Y 304/21061 20130101; A61P
3/00 20180101; A61P 3/06 20180101; A61P 25/28 20180101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 9/64 20060101 C12N009/64 |
Claims
1-28. (canceled)
29. A method for alleviating a PCSK9-related disorder in an
subject, comprising administering to the subject a therapeutically
effective amount of an immunogen comprising at least one antigenic
PCSK9 peptide linked to an immunogenic carrier, wherein the
antigenic PCSK9 peptide consists of an amino acid sequence selected
from the group consisting of SEQ ID NOs: 62-73, and wherein the
PCSK9-related disorder is selected from the group consisting of an
ateriosclerotic condition, a coronary artery disease, and a
cardiovascular disease.
30. The method according to claim 29, wherein the immunogenic
carrier is Diphtheria Toxoid, CRM197, or a VLP.
31. The method according to claim 30, wherein the antigenic PCSK9
peptide is selected from the group consisting of SEQ ID Nos. 67,
68, 69, 70, 71, 72, and 73.
32. The method according to claim 29, wherein the immunogenic
carrier is a VLP selected from HBcAg, HBsAg, Qbeta, PP7, PPV or
Norwalk Virus VLP.
33. The method according to claim 31, wherein the immunogen further
comprises, between the antigenic peptide and carrier, either: at
the C-terminus of the PCSK9 peptide a linker having the formula
(G).sub.nC, (G).sub.nSC or (G).sub.nK wherein n is 0, 1, 2, or 3;
or at the N-terminus of the PCSK9 peptide a linker having the
formula C(G).sub.n, CS(G).sub.n or K(G).sub.n wherein n is 0, 1, 2,
or 3.
34. The method according to claim 31, wherein the immunogen further
comprises a cysteine between the C-terminus of the PCSK9 peptide
and N-terminus of the carrier.
35. The method according to claim 31, wherein the immunogen further
comprises a CGG between the N-terminus of the PCSK9 peptide and
C-terminus of the carrier.
36. The method according to claim 31, wherein the immunogen further
comprises a GGC between the N-terminus of the PCSK9 peptide and
C-terminus of the carrier.
37. The method according to claim 31, wherein the immunogen is
cyclized and comprises a cysteine, a (G).sub.nC, or a C(G).sub.n
fragment between the antigenic PCSK9 peptide and immunogenic
carrier, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
38. The immunogen according to claim 29, wherein the antigenic
PCSK9 peptide is conformationally constrained.
39. A method for alleviating a PCSK9-related disorder in an
subject, comprising administering to the subject a therapeutically
effective amount of an immunogen comprising at least one antigenic
PCSK9 peptide linked to an immunogenic carrier, wherein the
antigenic PCSK9 peptide is selected from the group consisting of
SEQ ID Nos. 314, 318, and 319, and wherein the PCSK9-related
disorder is selected from the group consisting of an
ateriosclerotic condition, a coronary artery disease, and a
cardiovascular disease.
40. A method for alleviating a PCSK9-related disorder in an
subject, comprising administering to the subject a therapeutically
effective amount of a composition comprising at least two
immunogens, wherein one of the two immunogens comprises at least
one antigenic PCSK9 peptide linked to an immunogenic carrier,
wherein the antigenic PCSK9 peptide consists of an amino acid
sequence selected from the group consisting of SEQ ID NOs: 62-73,
and wherein the PCSK9-related disorder is selected from the group
consisting of an ateriosclerotic condition, a coronary artery
disease, and a cardiovascular disease.
41. The method according to claim 29 further comprises
administering to the subject a therapeutically effective amount of
at least one adjuvant.
42. The method according to claim 41, wherein the adjuvant is
selected from alum, CpG ODN, and QS21.
43. A method of preventing, alleviating, or treating a
PCSK9-related disorder in an subject, comprising administering a
therapeutically effective amount of a pharmaceutical composition
comprising (i) the immunogen and (ii) a pharmaceutically acceptable
excipient, wherein the immunogen comprises at least one antigenic
PCSK9-peptide linked to an immunogenic carrier, wherein the
antigenic PCSK9 peptide consists of an amino acid sequence selected
form the group consisting of SEQ ID NOs: 62-73, and wherein the
PCSK9-related disorder is selected from the group consisting of an
ateriosclerotic condition, a coronary artery disease, and a
cardiovascular disease.
44. The method according to claim 30, wherein the antigenic PCSK9
peptide consists of the amino acid sequence of SEQ ID NO: 72.
45. The method according to claim 43, wherein the antigenic PCSK9
peptide consists of the amino acid sequence of SEQ ID NO: 72
46. A method for reducing plasma cholesterol levels in an subject,
comprising administering to the subject a therapeutically effective
amount of an immunogen, wherein the immunogen comprises an
antigenic PCSK9 peptide linked to an immunogenic carrier, and
wherein the antigenic PCSK9 peptide consists of an amino acid
sequence selected from the group consisting of SEQ ID NOs:
67-73.
47. The method according to claim 46, wherein the immunogenic
carrier is selected form the group consisting of Diphtheria Toxoid,
CRM197, and a VLP.
48. The method according to claim 46, wherein the immunogenic
carrier is a VLP selected from HBcAg, HBsAg, Qbeta, PP7, PPV or
Norwalk Virus VLP.
49. The method according to claim 46, wherein the immunogen further
comprises, between the antigenic peptide and carrier, either: at
the C-terminus of the PCSK9 peptide a linker having the formula
(G).sub.nC, (G).sub.nSC or (G).sub.nK wherein n is 0, 1, 2, or 3;
or at the N-terminus of the PCSK9 peptide a linker having the
formula C(G).sub.n, CS(G).sub.n or K(G).sub.n wherein n is 0, 1, 2,
or 3.
50. The method according to claim 46, wherein the immunogen further
comprises a cysteine between the C-terminus of the PCSK9 peptide
and N-terminus of the carrier.
51. The method according to claim 46, wherein the immunogen further
comprises a CGG between the N-terminus of the PCSK9 peptide and
C-terminus of the carrier.
52. The method according to claim 46, wherein the immunogen further
comprises a GGC between the N-terminus of the PCSK9 peptide and
C-terminus of the carrier.
53. The method according to claim 46, wherein the immunogen is
cyclized and comprises a cysteine, a (G).sub.nC, or a C(G).sub.n
fragment between the antigenic PCSK9 peptide and immunogenic
carrier, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
54. The method according to claim 46, wherein the antigenic PCSK9
peptide is conformationally constrained.
55. The method according to claim 46, wherein the antigenic PCSK9
peptide consists of the amino acid sequence of SEQ ID NO: 72.
56. A method for reducing plasma cholesterol levels in an subject,
comprising administering to the subject a therapeutically effective
amount of an antigenic PCSK9 peptide consisting of an amino add
sequence selected from the group consisting of SEQ ID NOs: 314,
318, 319.
57. The method according to claim 56, wherein the antigenic PCSK9
peptide is linked to an immunogenic carrier selected from the group
consisting of Diphtheria Toxoid, a VLP and CRM197.
58. The method according to claim 56, wherein the immunogenic
carrier is a VLP selected from HBcAg, HBsAg, Qbeta, PP7, PPV or
Norwalk Virus VLP.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 61/239,541 filed on Sep. 3, 2009, which is
incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence entitled
"PC33896A_SequenceListing.txt" created on Aug. 23, 2010 and having
a size of 108 KB. The sequence listing contained in this .txt file
is part of the specticiation and is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the provision of novel
immunogens comprising an antigenic PCSK9 peptide preferably linked
to an immunogenic carrier for the prevention, treatment or
alleviation of PCSK9-related disorders. The invention further
relates to methods for production of these medicaments, immunogenic
compositions and pharmaceutical composition thereof and their use
in medicine.
BACKGROUND
[0004] Proprotein convertase subtilisin-kexin type 9 (hereinafter
called "PCSK9"), also known as neural apoptosis-regulated
convertase 1 ("NARC-I"), is a proteinase K-like subtilase
identified as the 9th member of the mammalian PCSK family; see
Seidah et al, 2003 PNAS 100:928-933. The gene for PCSK9 localizes
to human chromosome 1p33-p34.3. PCSK9 is expressed in cells capable
of proliferation and differentiation including, for example,
hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon
epithelia as well as embryonic brain telencephalon neurons.
[0005] Original synthesis of PCSK9 is in the form of an inactive
enzyme precursor, or zymogen, of .about.72-kDa which undergoes
autocatalytic, intramolecular processing in the endoplasmic
reticulum ("ER") to activate its functionality. The gene sequence
for human PCSK9, which is .about.22-kb long with 12 exons encoding
a 692 amino acid protein, can be found, for example, at Deposit No.
NP_777596.2. Human, mouse and rat PCSK9 nucleic acid sequences have
been deposited; see, e.g., GenBank Accession Nos.: AX127530 (also
AX207686), AX207688, and AX207690, respectively.
[0006] Human PCSK9 is a secreted protein expressed primarily in the
kidneys, liver and intestines. It has three domains: an inhibitory
pro-domain (amino acids 1-152; including a signal sequence at amino
acids 1-30), a catalytic domain (amino acids 153-448), and a
C-terminal domain 210 residues in length (amino acids 449-692),
which is rich in cysteine residues. PCSK9 is synthesized as a
zymogen that undergoes autocatalytic cleavage between the
pro-domain and catalytic domain in the endoplasmic reticulum. The
pro-domain remains bound to the mature protein after cleavage, and
the complex is secreted. The cysteine-rich domain may play a role
analogous to the P-(processing) domains of other
Furin/Kexin/Subtilisin-like serine proteases, which appear to be
essential for folding and regulation of the activated protease.
Mutations in PCSK9 are associated with abnormal levels of low
density lipoprotein cholesterol (LDL-c) in the blood plasma (Horton
et al., 2006 Trends. Biochem. Sci. 32(2):71-77).
[0007] PCSK9 has been ascribed a role in the differentiation of
hepatic and neuronal cells (Seidah et al, supra), is highly
expressed in embryonic liver, and has been strongly implicated in
cholesterol homeostasis.
[0008] The identification of compounds and/or agents effective in
the treatment of cardiovascular affliction is highly desirable.
Reductions in LDL cholesterol levels have already demonstrated in
clinical trials to be directly related to the rate of coronary
events; Law et al, 2003 BMJ 326: 1423-1427. More, recently moderate
lifelong reduction in plasma LDL cholesterol levels has been shown
to be substantially correlated with a substantial reduction in the
incidence of coronary events; Cohen et al, supra. This was found to
be the case even in populations with a high prevalence of
non-lipid-related cardiovascular risk factors; supra.
[0009] Accordingly, it is of great importance to indentify
therapeutic agent permiting the control of LDL cholesterol
levels.
[0010] Accordingly, it would be of great importance to produce a
medicament that inhibits or antagonizes the activity of PCSK9 and
the corresponding role PCSK9 plays in various therapeutic
conditions.
[0011] Expression or upregulation of PCSK9 is associated with
increased plasma levels of LDL cholesterol, and inhibition or the
lack of expression of PCSK9 is associated with low LDL cholesterol
plasma levels. Significantly, lower levels of LDL cholesterol
associated with sequence variations in PCSK9 have conferred
protection against coronary heart disease; Cohen, 2006 N. Engl. J.
Med. 354: 1264-1272.
SUMMARY OF THE INVENTION
[0012] The present invention relates to an immunogen comprising an
antigenic PCSK9 peptide and optionally an immunogenic carrier.
[0013] The invention also relates to methods for producing such
antigenic PCSK9 peptide optionally linked to an immunogenic
carrier.
[0014] The invention also relates to immunogenic compositions
comprising such antigenic PCSK9 peptide optionally linked to an
immunogenic carrier, optionally comprising one or several
adjuvants, preferably one or two adjuvants.
[0015] Another aspect of the invention relates to pharmaceutical
compositions comprising an antigenic PCSK9 peptide according to the
invention, or an immunogenic composition thereof, as well as to
medical uses of such compositions.
[0016] In particular, the invention relates to an antigenic PCSK9
peptide of the invention, or an immunogenic or pharmaceutical
composition thereof, for use as a medicament, preferably in
treatment, alleviation or prophylaxis of PCSK9-related
disorders.
[0017] In particular, the invention relates to an antigenic PCSK9
peptide of the invention, or an immunogenic or pharmaceutical
composition thereof, for use as a medicament preferably in
treatment, alleviation or prophylaxis of diseases associated with
an elevated level of cholesterol.
[0018] The antigenic PCSK9 peptides of the invention are
particularly suitable for treating human patients having, or at
risk for, elevated LDL-cholesterol or a condition associated with
elevated LDL-cholesterol, e.g., a lipid disorder (e.g.,
hyperlipidemia, type I, type II, type III, type IV, or type V
hyperlipidemia, secondary hypertriglyceridemia,
hypercholesterolemia, familial hypercholesterolemia, xanthomatosis,
cholesterol acetyltransferase deficiency). Antigenic PCSK9 peptide
of the invention are also suitable for treating human patients
having arteriosclerotic conditions (e.g., atherosclerosis),
coronary artery disease, cardiovascular disease, and patients at
risk for these disorders, e.g., due to the presence of one or more
risk factors (e.g., hypertension, cigarette smoking, diabetes,
obesity, or hyperhomocysteinemia).
[0019] In yet another aspect, the present invention provides the
use of an antigenic PCSK9 peptide of the invention or of an
immunogenic composition or a pharmaceutical composition thereof, in
the manufacture of a medicament for the treatment of Alzheimer's
disease.
[0020] In one embodiment, the antigenic PCSK9 peptide or an
immunogenic composition or a pharmaceutical composition thereof is
administered together with another agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1: The PDB structure of human PCSK9 bound to the EGF-A
domain of the LDL-R (3BPS) showing 5 peptide sequences in PCSK9
(peptide 1-5) chosen as being involved in the interaction between
these two proteins.
[0022] FIG. 2: Mice were immunized with peptides VR_9.1 to VR_9.9
conjugated to VLPs, using Alum plus CpG as adjuvant and antibody
responses to full-length recombinant human PCSK9 were measured by
titrating sera in an ELISA assay. Results are shown as reciprocal
titres for each of 6 mice per group, with the reciprocal titre
measured as the dilution of serum giving an optical density reading
of 0.5.
[0023] FIG. 3: Antibody responses to full-length recombinant mouse
PCSK9 protein as described in FIG. 2.
[0024] FIG. 4: Plasma cholesterol levels measured in the sera of
vaccinated mice (same samples as used for antibody assays in FIGS.
2 and 3)
[0025] FIG. 5: Plasma samples used in FIGS. 2 to 4 were tested at
different dilutions for their ability to inhibit the interaction
between recombinant PCSK9 and the extracellular domain of the LDL
receptor as measured by FRET assay.
[0026] FIG. 6: Dilutions of plasma samples from peptides VR_9.5 and
VR_9.6 vaccinations in FRET assay, showing dose-responsive
inhibition of interaction between PCSK9 and the LDL receptor.
[0027] FIG. 7: Complex of PCSK9 (ribbons) and EGF-A (space fill)
from PDB:3BPS. Potential regions of PCSK9 that may interact with
domains of LDLR other than EGF-A are indicated by the elipse.
[0028] FIG. 8: Complex of PCSK9 (ribbons) and EGF-A (space fill),
with the amino acids corresponding to peptides VR_13/14 (A) and
VR_15/16 (B) and VR_9.5 (C) is displayed.
[0029] FIGS. 9 and 10: Plasma antibody responses of mice vaccinated
with peptides VR_9.5 and VR_9.10 to VR_9.16. Antibodies to mouse
PCSK9 were measured by ELISA assay of serial plasma dilutions using
full-length mouse PCSK9 protein. Individual titration curves are
shown for 8 mice per group, with ELISA responses of plasma from
mice immunized with unconjugated VLP shown as a control.
[0030] FIG. 11: Serum antibody responses to full-length human PCSK9
protein induced in BALB/c and C57BL/6 mice vaccinated with either
peptide VR_9.5 or VR_9.10 conjugated to VLPs (using Alum+/-CpG as
adjuvant) or CRM197 (using TiterMax as adjuvant). Antibodies to
human PCSK9 were measured by titrating sera in an ELISA assay.
Results are shown as log reciprocal titres determined at an optical
density of 1.0 for each of 8 mice per group.
[0031] FIG. 12: Serum antibody responses to full-length mouse PCSK9
protein induced in BALB/c and C57BL/6 mice vaccinated as described
for FIG. 11. Results are shown as log reciprocal titres determined
at an optical density of 0.5 for each of 8 mice per group.
[0032] FIG. 13: Total cholesterol levels measured in the serum
samples from BALB/c vaccinated mice (same samples used for antibody
assays in FIGS. 11 and 12).
[0033] FIG. 14: Total cholesterol levels measured in the serum
samples from C57BL/6 vaccinated mice (same samples used for
antibody assays in FIGS. 11 and 12).
[0034] FIG. 15: Antibody responses to full-length human PCSK9
induced in BALB/c mice immunized with peptides VR_9.5 or VR_9.17 to
VR_9.35 conjugated to VLPs using Alum plus CpG as adjuvant.
Antibodies to human PCSK9 were measured by titrating sera in an
ELISA assay. Results are shown as log reciprocal titres determined
at an optical density of 1.0 for each of 8 mice per group.
[0035] FIG. 16: Antibody responses to full-length mouse PCSK9
induced in BALB/c mice immunized as described for FIG. 15.
Antibodies to mouse PCSK9 were measured by titrating sera in an
ELISA assay. Results are shown as log reciprocal titres determined
at an optical density of 0.5 for each of 8 mice per group.
[0036] FIG. 17: Total cholesterol levels measured in the serum
samples from BALB/c vaccinated mice (same samples used for antibody
assays in FIGS. 15 and 16).
[0037] FIG. 18: Complex of PCSK9 (ribbons) and EGF-A (space fill)
with regions of PCSK9 containing amino acids sequences linked to
gain- or loss-of-function mutations and/or protein surface exposed
epitopes indicated by elipses.
DETAILED DESCRIPTION OF THE INVENTION
Antigenic PCSK9 Peptide of the Invention
[0038] The present invention relates to an immunogen comprising an
antigenic PCSK9 peptide optionally linked to an immunogenic
carrier.
[0039] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 comprising between 4 to 20 amino acids and, when
administered to a subject, is able to lower the LDL-cholesterol
level in blood of said subject. Preferably, said subject is a
mammal, preferably a human. Preferably, said antigenic PCSK9
peptide is able to lower the LDL-cholesterol level by at least 2%,
5%, 10%, 20%, 30% or 50%.
[0040] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 which participates in the interaction of PCSK9 with the
LDL receptor.
[0041] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 which participates in the interaction of PCSK9 with the
LDL receptor, comprising between 4 and 20 amino acids and, when
administered to a subject is able to lower the LDL-cholesterol
level in blood of said subject. Preferably, said subject is a
mammal, preferably a human. Preferably, said antigenic PCSK9
peptide is able to lower the LDL-cholesterol level by at least 2%,
5%, 10%, 20%, 30% or 50%.
[0042] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 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, 152, 153, 154, 155, 156, 157, 158, 159, 160,
161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,
174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,
239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,
252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,
265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,
278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,
291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,
304, 305, 306, 307, 308, 309, 310, 311, 312, 330, 331, 332, 333,
334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,
347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,
360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398,
420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432,
433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,
446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,
459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,
472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,
485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,
498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,
511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523,
524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536,
537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,
550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562,
563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575,
576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587 and
588.
[0043] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 1 to 312, 330 to 398, 421,
423, 424, 426 and 428 to 588.
[0044] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 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, 152, 153, 154, 155, 156, 157, 158, 159, 160,
161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,
174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,
226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,
239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,
252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,
265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,
278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,
291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,
304, 305, 306, 307, 308, 309, 310, 311, 312, 330, 331, 332, 333,
334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,
347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,
360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,
373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,
386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397 and
398.
[0045] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 which may participate in the interaction with the domain
EGF-A of the LDL receptor. Examples of such portions are
represented on FIG. 1.
[0046] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 which may participate in the interaction with the domain
EGF-A of the LDL receptor, comprising between 4 and 20 amino acids
and, when administered to a subject, is able to lower the
LDL-cholesterol level in blood of said subject. Preferably, said
subject is a mammal, preferably a human. Preferably, said antigenic
PCSK9 peptide is able to lower the LDL-cholesterol level by at
least 2%, 5%, 10%, 20%, 30% or 50%.
[0047] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 1.
[0048] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44 and 45.
[0049] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 15, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 46.
[0050] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 and
101.
[0051] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 14, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 102.
[0052] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 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 and
146.
[0053] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 147.
[0054] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,
165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,
178, 179, 180 and 181.
[0055] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 182.
[0056] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 182, 183, 184, 185, 186,
187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,
213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225 and
226.
[0057] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 330.
[0058] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 330, 331, 332, 333, 334,
335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,
348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358 and 359.
[0059] In a preferred embodiment, the antigenic PCSK9 peptide is
selected from the group consisting of SEQ ID Nos 19, 56, 63, 109,
153, 165, 184, 186, 187, 188, 332 and 424.
[0060] In a preferred embodiment, the antigenic PCSK9 peptide is
selected from the group consisting of SEQ ID Nos 19, 56, 63, 109,
153 and 184.
[0061] In a preferred embodiment, the antigenic PCSK9 peptide is
selected from the group consisting of SEQ ID Nos 56, 184, 186, 187,
188 and 332.
[0062] In a most preferred embodiment, the antigenic PCSK9 peptide
is a peptide of sequence SEQ ID No 56.
[0063] In a more prefered embodiment, the antigenic PCSK9 peptide
is a peptide of sequence SEQ ID No 184 or 187.
[0064] In a most preferred embodiment, the antigenic PCSK9 peptide
is a peptide of sequence SEQ ID No 184.
[0065] In a most preferred embodiment, the antigenic PCSK9 peptide
is a peptide of sequence SEQ ID No 332.
[0066] In one embodiment, the antigenic PCSK9 peptide is selected
in a region of PCSK9 which may participate in the interaction with
a region of the LDL receptor other than the EGF-A domain. Examples
of such portions are represented on FIGS. 7 and 8.
[0067] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 which may participate in the interaction with a region of
the LDL receptor other than the EGF-A domain, comprising between 4
and 20 amino acids and, when administered to a subject, is able to
lower the LDL-cholesterol level in blood of said subject.
Preferably, said subject is a mammal, preferably a human.
Preferably, said antigenic PCSK9 peptide is able to lower the
LDL-cholesterol level by at least 2%, 5%, 10%, 20%, 30% or 50%.
[0068] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 12, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 227.
[0069] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 227, 228, 229, 230, 231,
232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,
245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,
258, 259, 260, 261 and 262.
[0070] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 263.
[0071] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 263, 264, 265, 266, 267,
268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,
281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,
294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 and
307.
[0072] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 360.
[0073] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 360, 361, 362, 363, 364,
365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377,
378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390,
391, 392, 393, 394, 395, 396, 397 and 398.
[0074] In an embodiment, the antigenic PCSK9 peptide is selected in
a region of PCSK9 pro-domain (SEQ ID No 329).
[0075] In one embodiment, the antigenic PCSK9 peptide is a portion
of PCSK9 pro-domain, comprising between 4 and 20 amino acids and,
when administered to a subject, is able to lower the
LDL-cholesterol level in blood of said subject. Preferably, said
subject is a mammal, preferably a human. Preferably, said antigenic
PCSK9 peptide is able to lower the LDL-cholesterol level by at
least 2%, 5%, 10%, 20%, 30% or 50%.
[0076] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 308, 309, 310, 311 and
312.
[0077] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 12, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 309.
[0078] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 309, 429, 430, 431, 432,
433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,
446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,
459, 460, 461, 462 and 463.
[0079] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 12, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 508.
[0080] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 508, 509, 510, 511, 512,
513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525,
526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,
539, 540, 541, 542 and 543.
[0081] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 310.
[0082] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 310, 464, 465, 466, 467,
468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,
481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,
494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506 and
507.
[0083] In one embodiment, the antigenic PCSK9 peptide is a peptide
comprising 5 to 13, preferably 6 to 8, consecutive amino acids of
the PCSK9 fragment of SEQ ID No 544.
[0084] In one embodiment, the antigenic PCSK9 peptide is selected
from the group consisting of SEQ ID Nos 544, 545, 546, 547, 548,
549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561,
562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574,
575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587,
and 588.
[0085] In a preferred embodiment, the antigenic PCSK9 peptide is
selected from the group consisting of SEQ ID Nos 312, 421, 422,
423, 426, 427, 428, 445, 482, 525, and 563.
[0086] In a more preferred embodiment, the antigenic PCSK9 peptide
is selected from the group consisting of SEQ ID Nos 445, 482, 525,
and 563.
[0087] In a most preferred embodiment, the antigenic PCSK9 peptide
is a peptide of sequence SEQ ID No 445.
[0088] Such antigenic PCSK9 peptides may be used alone or in
combination, preferably when conjugated to an immunogenic carrier,
to induce auto anti-PCSK9 antibodies in a subject in order to
treat, prevent or ameliorate PCSK9-related disorders.
[0089] It will be apparent to the man skilled in the art which
techniques may be used to confirm whether a specific construct
falls within the scope of the present invention. Such techniques
include, but are not restricted to, the techniques described in the
Example section of the present application, and also to the
following.
[0090] The ability of the antigenic PCSK9 peptide of the invention
to induce auto anti-PCSK9 antibodies may be measured in mice, using
the test disclosed in example 3 of the present application. The
ability of auto-antibodies induced by the antigenic PCSK9 peptide
of the invention to decrease the level of circulating plasma
cholesterol may be measured in mice, using the test disclosed in
example 3. The ability of auto-antibodies induced by the antigenic
PCSK9 peptide of the invention to inhibit the interaction between
PCSK9 and LDL receptors may be measured directly using the test
disclosed in example 3 (FRET assay) or indirectly by measuring the
upregulation of cell surface LDL receptors which is a consequence
of blocking PCSK9-mediated down-regulation (as well described in
the relevant literature, either using cell lines in vitro or by
measuring LDL receptor levels in liver biopsies of antibody
expressing animals (e.g. by Western blotting)).
[0091] The term "antigenic PCSK9 peptide biological activity", when
used herein, refers to the ability of the antigenic PCSK9 peptides
of the invention to induce auto anti-PCSK9 antibodies in a
patient.
[0092] Preferably said antigenic PCSK9 peptide, when administered
to a subject, is able to lower the LDL-cholesterol level in blood
of said subject. Preferably, said subject is a mammal, preferably a
human. Preferably, said antigenic PCSK9 peptide is able to lower
the LDL-cholesterol level by at least 2%, 5%, 10%, 20%, 30% or
50%.
[0093] In an embodiment the antigenic PCSK9 peptides of the present
invention are of a size such that they mimic a region selected from
the whole PCSK9 domain in which the native epitope is found. In a
particular embodiment, the antigenic PCSK9 peptides of the
invention are less than 100 amino acids in length, preferably
shorter than 75 amino acids, more preferably less than 50 amino
acids, even more preferably less than 40 amino acids. The antigenic
PCSK9 peptides of the invention are typically 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29 or 30 amino acids in length, preferably from 4 to 20 amino
acids, for example 6 to 12, 6 to 8 or 9 to 12 amino acids.
[0094] Specific examples of antigenic PCSK9 peptides of the
invention are provided in the sequence listing and include peptides
ranging from 5 to 17 amino acids in length.
[0095] The antigenic peptides of the invention include an amino
acid sequence derived from a portion of a mammalian PCSK9,
preferably a human PCSK9 (SEQ ID No 399) or mouse PCSK9 (SEQ ID Nos
400), more preferably human PCSK9, such derived portion of PCSK9
either corresponding to the amino acid sequence of naturally
occurring PCSK9 or corresponding to variant PCSK9, i.e. the amino
acid sequence of naturally occurring PCSK9 in which a small number
of amino acids have been substituted, added or deleted but which
retains essentially the same immunological properties. In addition,
such derived PCSK9 portion can be further modified by amino acids,
especially at the N- and C-terminal ends to allow the antigenic
PCSK9 peptide to be conformationally constrained and/or to allow
coupling of the antigenic PCSK9 peptide to an immunogenic carrier
after appropriate chemistry has been carried out.
[0096] The antigenic PCSK9 peptides disclosed herein encompass
functionally active variant peptides derived from the amino acid
sequence of PCSK9 in which amino acids have been deleted, inserted
or substituted without essentially detracting from the
immunological properties thereof, i.e. such functionally active
variant peptides retain a substantial antigenic PCSK9 peptide
biological activity. Typically, such functionally variant peptides
have an amino acid sequence homologous, preferably highly
homologous, to an amino acid sequence selected from the group
consisting of SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588.
[0097] In one embodiment, such functionally active variant peptides
exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity
to an amino acid sequence selected from the group consisting of SEQ
ID Nos: 1 to 312, 330 to 398 and 420 to 588.
[0098] Sequence similarity for polypeptides, which is also referred
to as sequence identity, is typically measured using sequence
analysis software. Protein analysis software matches similar
sequences using measures of similarity assigned to various
substitutions, deletions and other modifications, including
conservative amino acid substitutions. For instance, GCG contains
programs such as "Gap" and "Bestfit" which can be used with default
parameters to determine sequence homology or sequence identity
between closely related polypeptides, such as homologous
polypeptides from different species of organisms or between a wild
type protein and a mutein thereof. See, e.g., GCG Version 6.1.
Polypeptide sequences also can be compared using FASTA using
default or recommended parameters, a program in GCG Version 6.1.
FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent
sequence identity of the regions of the best overlap between the
query and search sequences (Pearson, Methods Enzymol. 183:63-98
(1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). An
alternative algorithm when comparing a sequence of the invention to
a database containing a large number of sequences from different
organisms is the computer program BLAST, especially blastp or
tblastn, using default parameters. See, e.g., Altschul et al., J.
Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.
25:3389-402 (1997).
[0099] Functionally active variants comprise naturally occurring
functionally active variants such as allelic variants and species
variants and non-naturally occurring functionally active variants
that can be produced by, for example, mutagenesis techniques or by
direct synthesis.
[0100] A functionally active variant differs by about, for example,
1, 2, 3, 4 or 5 amino acid residues from any of the peptide shown
at SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588, and yet retain
an antigenic PCSK9 biological activity. Where this comparison
requires alignment the sequences are aligned for maximum homology.
The site of variation can occur anywhere in the peptide, as long as
the biological activity is substantially similar to a peptide shown
in SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588.
[0101] Guidance concerning how to make phenotypically silent amino
acid substitutions is provided in Bowie et al., Science, 247:
1306-1310 (1990), which teaches that there are two main strategies
for studying the tolerance of an amino acid sequence to change.
[0102] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, the amino
acid positions which have been conserved between species can be
identified. These conserved amino acids are likely important for
protein function. In contrast, the amino acid positions in which
substitutions have been tolerated by natural selection indicate
positions which are not critical for protein function. Thus,
positions tolerating amino acid substitution can be modified while
still maintaining specific immunogenic activity of the modified
peptide.
[0103] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example,
site-directed mutagenesis or alanine-scanning mutagenesis can be
used (Cunningham et al., Science, 244: 1081-1085 (1989)). The
resulting variant peptides can then be tested for specific
antigenic PCSK9 biological activity.
[0104] According to Bowie et al., these two strategies have
revealed that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, the most buried or interior (within
the tertiary structure of the protein) amino acid residues require
nonpolar side chains, whereas few features of surface or exterior
side chains are generally conserved.
[0105] Methods of introducing a mutation into amino acids of a
protein is well known to those skilled in the art. See, e. g.,
Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley
and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
laboratory, Cold Spring Harbor, N.Y. (1989)).
[0106] Mutations can also be introduced using commercially
available kits such as "QuikChange.TM. Site-Directed Mutagenesis
Kit" (Stratagene) or directly by peptide synthesis. The generation
of a functionally active variant to an antigenic PCSK9 peptide by
replacing an amino acid which does not significantly influence the
function of said antigenic PCSK9 peptide can be accomplished by one
skilled in the art.
[0107] A type of amino acid substitution that may be made in one of
the peptides according to the invention is a conservative amino
acid substitution. A "conservative amino acid substitution" is one
in which an amino acid residue is substituted by another amino acid
residue having a side chain R group) with similar chemical
properties (e.g., charge or hydrophobicity). In general, a
conservative amino acid substitution will not substantially change
the functional properties of a protein. In cases where two or more
amino acid sequences differ from each other by conservative
substitutions, the percent sequence identity or degree of
similarity may be adjusted upwards to correct for the conservative
nature of the substitution. Means for making this adjustment are
well-known to those of skill in the art. See e.g. Pearson, Methods
Mol. Biol. 243:307-31 (1994).
[0108] Examples of groups of amino acids that have side chains with
similar chemical properties include 1) aliphatic side chains:
glycine, alanine, valine, leucine, and isoleucine; 2)
aliphatic-hydroxyl side chains: serine and threonine; 3)
amide-containing side chains: asparagine and glutamine; 4) aromatic
side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
chains: lysine, arginine, and histidine; 6) acidic side chains:
aspartic acid and glutamic acid; and 7) sulfur-containing side
chains: cysteine and methionine. Preferred conservative amino acids
substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
glutamate-aspartate, and asparagine-glutamine.
[0109] Alternatively, a conservative replacement is any change
having a positive value in the PAM250 log-likelihood matrix
disclosed in Gonnet et al., Science 256:1443-45 (1992). A
"moderately conservative" replacement is any change having a
nonnegative value in the PAM250 log-likelihood matrix.
[0110] A functionally active variant peptide can also be isolated
using a hybridization technique. Briefly, DNA having a high
homology to the whole or part of a nucleic acid sequence encoding
the peptide of interest, e.g. SEQ ID Nos: 1 to 312, 330 to 398 and
420 to 588, is used to prepare a functionally active peptide.
Therefore, an antigenic PCSK9 peptide of the invention also
includes peptides which are functionally equivalent to one or more
of the peptide of SEQ ID Nos: 1 to 312, 330 to 398, and 420 to 588
and which are encoded by a nucleic acid molecule which hybridizes
with a nucleic acid encoding any one of SEQ ID Nos: 1 to 312, 330
to 398 and 420 to 588, or a complement thereof. One of skill in the
art can easily determine nucleic acid sequences that encode
peptides of the invention using readily available codon tables. As
such, these nucleic acid sequences are not presented herein.
[0111] The stringency of hybridization for a nucleic acid encoding
a peptide, polypeptide or protein that is a functionally active
variant is, for example, 10% formamide, 5.times.SSPE, 1.times.
Denhart's solution, and 1.times. salmon sperm DNA (low stringency
conditions). More preferable conditions are, 25% formamide,
5.times.SSPE, 1.times. Denhart's solution, and 1.times. salmon
sperm DNA (moderate stringency conditions), and even more
preferable conditions are, 50% formamide, 5.times.SSPE, 1.times.
Denhart's solution, and 1.times. salmon sperm DNA (high stringency
conditions). However, several factors influence the stringency of
hybridization other than the above-described formamide
concentration, and one skilled in the art can suitably select these
factors to accomplish a similar stringency.
[0112] Nucleic acid molecules encoding a functionally active
variant can also be isolated by a gene amplification method such as
PCR using a portion of a nucleic acid molecule DNA encoding a
peptide, polypeptide or protein of interest, e.g. any one of the
peptides shown SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588, as
the probe.
[0113] In one embodiment of the invention, a peptide of the
invention is derived from a natural source and isolated from a
mammal, such as a human, a primate, a cat, a dog, a horse, a mouse,
or a rat, preferably from a human source. A peptide of the
invention can thus be isolated from cells or tissue sources using
standard protein purification techniques.
[0114] Alternatively, peptides of the invention can be synthesized
chemically or produced using recombinant DNA techniques.
[0115] For example, a peptide of the invention can be synthesized
by solid phase procedures well known in the art. Suitable syntheses
may be performed by utilising "T-boc" or "F-moc" procedures. Cyclic
peptides can be synthesised by the solid phase procedure employing
the well-known "F-moc"procedure and polyamide resin in the fully
automated apparatus. Alternatively, those skilled in the art will
know the necessary laboratory procedures to perform the process
manually. Techniques and procedures for solid phase synthesis are
described in `Solid Phase Peptide Synthesis: A Practical Approach`
by E. Atherton and R. C. Sheppard, published by IRL at Oxford
University Press (1989) and `Methods in Molecular Biology, Vol. 35:
Peptide Synthesis Protocols (ed. M. W. Pennington and B. M. Dunn),
chapter 7, pp 91-171 by D. Andreau et al.
[0116] Alternatively, a polynucleotide encoding a peptide of the
invention can be introduced into an expression vector that can be
expressed in a suitable expression system using techniques well
known in the art, followed by isolation or purification of the
expressed peptide, polypeptide, or protein of interest. A variety
of bacterial, yeast, plant, mammalian, and insect expression
systems are available in the art and any such expression system can
be used. Optionally, a polynucleotide encoding a peptide of the
invention can be translated in a cell-free translation system.
[0117] Antigenic PCSK9 peptides of the invention can also comprise
those that arise as a result of the existence of multiple genes,
alternative transcription events, alternative RNA splicing events,
and alternative translational and postranslational events. A
peptide can be expressed in systems, e.g. cultured cells, which
result in substantially the same postranslational modifications
present as when the peptide is expressed in a native cell, or in
systems that result in the alteration or omission of
postranslational modifications, e.g. glycosylation or cleavage,
present when expressed in a native cell.
[0118] An antigenic PCSK9 peptide of the invention can be produced
as a fusion protein that contains other non-PCSK9 or
non-PCSK9-derived amino acid sequences, such as amino acid linkers
or signal sequences or immunogenic carriers as defined herein, as
well as ligands useful in protein purification, such as
glutathione-S-transferase, histidine tag, and staphylococcal
protein A. More than one antigenic PCSK9 peptide of the invention
can be present in a fusion protein. The heterologous polypeptide
can be fused, for example, to the N-terminus or C-terminus of the
peptide of the invention. A peptide of the invention can also be
produced as fusion proteins comprising homologous amino acid
sequences, i.e., other PCSK9 or PCSK9-derived sequences.
[0119] The antigenic PCSK9 peptides of the invention might be
linear or conformationally constrained. As used herein in reference
to a peptide, the term "conformationally constrained" means a
peptide, in which the three-dimensional structure is maintained
substantially in one spatial arrangement over time.
Conformationally constrained molecules can have improved properties
such as increased affinity, metabolic stability, membrane
permeability or solubility.
[0120] In addition, such conformationally constrained peptides are
expected to present the antigenic PCSK9 epitope in a conformation
similar to their native loop conformation, thereby inducing
anti-PCSK9 antibodies more susceptible to recognize intact, native
self PCSK9 molecules or with an increased affinity to recognize
self PCSK9 molecules. Methods of conformational constraint are well
known in the art and include, without limitation, bridging and
cyclization.
[0121] There are several approaches known in the prior art to
introduce conformational constraints into a linear peptide. For
example, bridging between two neighbouring amino acids in a peptide
leads to a local conformational modification, the flexibility of
which is limited in comparison with that of regular peptides. Some
possibilities for forming such bridges include incorporation of
lactams and piperazinones (for review see Giannis and. Kolter,
Angew. Chem. Int. Ed., 1993, 32: 1244).
[0122] As used herein in reference to a peptide, the term "cyclic"
refers to a structure including an intramolecular bond between two
non-adjacent amino acids or amino acid analogs. The cyclization can
be effected through a covalent or non-covalent bond. Intramolecular
bonds include, but are not limited to, backbone to backbone,
side-chain to backbone, side-chain to side-chain, side chain to
end-group, end-to-end bonds. Methods of cyclization include,
without limitation, formation of an amide bond between the N-term
residue and the C-term residue of a peptide, formation of a
disulfide bond between the side-chains of non-adjacent amino acids
or amino acid analogs; formation of an amide bond between the
side-chains of Lys and Asp/Glu residues; formation of an ester bond
between serine residues and Asp/Glu residues; formation of a lactam
bond, for example, between a side-chain group of one amino acid or
analog thereof to the N-terminal amine of the amino-terminal
residue; and formation of lysinonorleucine and dityrosine bonds.
Carbon versions of a disulfide linkage, for example an ethenyl or
ethyl linkage, could also be used (J. Peptide Sc., 2008, 14,
898-902) as well as alkylation reactions with an appropriately
polysubstituted electrophilic reagent such as a di-, tri- or
tetrahaloalkane (PNAS, 2008, 105(40), 15293-15298; ChemBioChem,
2005, 6, 821-824). Various modified proline analogs can also be
used to incorporate conformational constraints into peptides (Zhang
et al., J. Med Chem., 1996, 39: 2738-2744; Pfeifer and Robinson,
Chem. Comm., 1998, 1977-1978). Chemistries that may be used to
cyclise peptides of the invention result in peptides cyclised with
a bond including, but not limiting to the following: lactam,
hydrazone, oxime, thiazolidine, thioether or sulfonium bonds.
[0123] Yet another approach in the design of conformationally
constrained peptides, which is described in U.S. Ser. No.
10/114,918, is to attach a short amino acid sequence of interest to
a template, to generate a cyclic constrained peptide. Such cyclic
peptides are not only structurally stabilized by their templates,
and thereby offer three-dimensional conformations that may imitate
conformational epitopes on native proteins such as on viruses and
parasites or on self proteins (autologous mammalian proteins such
as PCSK9), but they are also more resistant than linear peptides to
proteolytic degradation in serum. U.S. Ser. No. 10/114,918 further
discloses the synthesis of conformationally constrained
cross-linked peptides by preparation of synthetic amino acids for
backbone coupling to appropriately positioned amino acids in order
to stabilize the supersecondary structure of peptides.
Cross-linking can be achieved by amide coupling of the primary
amino group of an orthogonally protected (2S,3R)-3-aminoproline
residue to a suitably positioned side chain carboxyl group of
glutamate. This approach has been followed in the preparation of
conformationally constrained tetrapeptide repeats of the CS protein
wherein at least one proline has been replaced by
2S,3R)-3-aminoproline and, in order to introduce a side chain
carboxyl group, glutamate has been incorporated as a replacement
for alanine.
[0124] Cross-linking strategies also include the application of the
Grubbs ring-closing metathesis reaction to form `stapled` peptides
designed to mimic alpha-helical conformations (Angew. Int. Ed.
Engl., 1998, 37, 3281; JACS, 2000, 122, 5891); use of
poly-functionalised saccharides; use of a tryptathionine linkage
(Chemistry Eu. J., 2008, 24, 3404-3409); use of `click` reaction of
azides and alkynes which could be incorporated as either a side
chain amino acid residues or located within the backbone of the
peptide sequence (Drug Disc. Today, 2003, 8(24), 1128-1137). It is
also known in the literature that metal ions can stabilise
constrained conformations of linear peptides through sequestering
specific residues e.g. histidine, which co-ordinate to metal
cations (Angew. Int. Ed. Engl., 2003, 42, 421). Similarly,
functionalising a linear peptide sequence with non-natural acid and
amine functionality, or polyamine and polyacid functionality can be
used to allow access to cyclised structures following activation
and amide bond formation.
[0125] According to one embodiment, the antigenic PCSK9 peptide is
conformationally constrained by intramolecular covalent bonding of
two non-adjacent amino acids of the antigenic PCSK9 peptide to each
other, e.g. the N- and C-terminal amino acids. According to another
embodiment, the antigenic PCSK9 peptide of the invention is
conformationally constrained by covalent binding to a scaffold
molecule. According to a further embodiment, the antigenic PCSK9
peptide is simply constrained, i.e. coupled either at one end, (C
or N terminus) or through another amino acid not located at either
end, to the scaffold molecule. According to another embodiment, the
antigenic PCSK9 peptide is doubly constrained, i.e. coupled at both
C and N termini to the scaffold molecule. According to another
embodiment, the antigenic peptide is constrained by cyclising via
the templating effect of a heterochiral Diproline unit
(D-Pro-L-Pro) (Spath et al, 1998, Helvetica Chimica Acta 81, p
1726-1738).
[0126] The scaffold (also called `platform`) can be any molecule
which is capable of reducing, through covalent bonding, the number
of conformations which the antigenic PCSK9 peptide can assume.
Examples of conformation-constraining scaffolds include proteins
and peptides, for example lipocalin-related molecules such as
beta-barrel containing thioredoxin and thioredoxin-like proteins,
nucleases (e.g. RNaseA), proteases (e.g. trypsin), protease
inhibitors (e.g. eglin C), antibodies or structurally-rigid
fragments thereof, fluorescent proteins such as GFP or YFP,
conotoxins, loop regions of fibronectin type III domain, CTL-A4,
and virus-like particles (VLPs).
[0127] Other suitable platform molecules include carbohydrates such
as sepharose. The platform may be a linear or circular molecule,
for example, closed to form a loop. The platform is generally
heterologous with respect to the antigenic PCSK9 peptide. Such
conformationally constrained peptides linked to a platform are
thought to be more resistant to proteolytic degradation than linear
peptide.
[0128] According to a preferred embodiment, the scaffold is an
immunogenic carrier as defined in the present application. In a
further embodiment, the antigenic PCSK9 peptide is simply
constrained onto the immunogenic carrier. In another further
embodiment, the antigenic PCSK9 peptide is doubly constrained onto
the immunogenic carrier. In this manner, the antigenic PCSK9
peptide forms a conformationally constrained loop structure which
has proven to be a particularly suitable structure as an
intracellular recognition molecule.
[0129] The antigenic PCSK9 peptides of the invention may be
modified for the ease of conjugation to a platform, for example by
the addition of a terminal cysteine at one or both ends and/or by
the addition of a linker sequence, such a double glycine head or
tail plus a terminal cysteine, a linker terminating with a lysine
residue or any other linker known to those skilled in the art to
perform such function. Details of such linkers are disclosed
hereafter. Bioorthogonal chemistry (such as the click reaction
described above) to couple the full peptide sequence to the
carrier, thus avoiding any regiochemical and chemoselectivity
issues, might also be used. Rigidified linkers such as the one
described in Jones et al. Angew. Chem. Int. Ed. 2002, 41:4241-4244
are known to elicit an improved immunological response and might
also be used.
[0130] In a further embodiment, the antigenic PCSK9 peptide is
attached to a multivalent template, which itself is coupled to the
carrier, thus increasing the density of the antigen (see below).
The multivalent template could be an appropriately functionalised
polymer or oligomer such as (but not limited to) oligoglutamate or
oligochitosan (see FIG. 19).
Immunogenic Carrier of the Invention
[0131] In an embodiment of the present invention, the antigenic
PCSK9 peptide of the invention is linked to an immunogenic carrier
molecule to form immunogens for vaccination protocols, preferably
wherein the carrier molecule is not related to the native PCSK9
molecule.
[0132] The term "immunogenic carrier" herein includes those
materials which have the property of independently eliciting an
immunogenic response in a host animal and which can be covalently
coupled to a peptide, polypeptide or protein either directly via
formation of peptide or ester bonds between free carboxyl, amino or
hydroxyl groups in the peptide, polypeptide or protein and
corresponding groups on the immunogenic carrier material, or
alternatively by bonding through a conventional bifunctional
linking group, or as a fusion protein.
[0133] The types of carriers used in the immunogens of the present
invention will be readily known to the person skilled in the art.
Examples of such immunogenic carriers are: serum albumins such as
bovine serum albumin (BSA); globulins; thyroglobulins; hemoglobins;
hemocyanins (particularly Keyhole Limpet Hemocyanin [KLH]);
polylysin; polyglutamic acid; lysine-glutamic acid copolymers;
copolymers containing lysine or ornithine; liposome carriers; the
purified protein derivative of tuberculin (PPD); inactivated
bacterial toxins or toxoids such as tetanus or diptheria toxins (TT
and DT) or fragment C of TT, CRM197 (a nontoxic but antigenically
identical variant of diphtheria toxin) other DT point mutants, such
as CRM176, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218;
3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and
other mutations described by Nicholls and Youle in Genetically
Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion
or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and
other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat.
No. 4,950,740; mutation of at least one or more residues Lys 516,
Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in
U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragment
disclosed in U.S. Pat. No. 5,843,711, pneumococcal pneumolysin (Kuo
et al (1995) Infect Immun 63; 2706-13) including ply detoxified in
some fashion for example dPLY-GMBS (WO 04081515, PCT/EP2005/010258)
or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequences
of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00/37105 or WO
00/39299) and fusions of Pht proteins for example PhtDE fusions,
PhtBE fusions, Pht A-E (WO 01/98334, WO 03/54007, WO2009/000826),
OMPC (meningococcal outer membrane protein--usually extracted from
N. meningitidis serogroup B--EP0372501), PorB (from N.
meningitidis), PD (Haemophilus influenzae protein D--see, e.g., EP
0 594 610 B), or immunologically functional equivalents thereof,
synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO
93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471
177), cytokines, lymphokines, growth factors or hormones (WO
91/01146), artificial proteins comprising multiple human CD4+ T
cell epitopes from various pathogen derived antigens (Falugi et al
(2001) Eur J Immunol 31; 3816-3824) such as N19 protein (Baraldoi
et al (2004) Infect Immun 72; 4884-7) pneumococcal surface protein
PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or
B of C. difficile (WO 00/61761).
[0134] In a preferred embodiment, the immunogenic carrier of the
invention is CRM197.
[0135] In another embodiment, the immunogenic carrier is a
virus-like particle (VLPs), preferably a recombinant virus-like
particle.
[0136] As used herein, the term "virus-like particle" refers to a
structure resembling a virus particle but which has been
demonstrated to be non pathogenic. In general, virus-like particles
lack at least part of the viral genome. Also, virus-like particles
can often be produced in large quantities by heterologous
expression and can be easily purified. A virus-like particle in
accordance with the invention may contain nucleic acid distinct
from their genome. A typical and preferred embodiment of a
virus-like particle in accordance with the present invention is a
viral capsid such as the viral capsid of the corresponding virus,
bacteriophage, or RNA-phage.
[0137] As used herein, the term "virus-like particle of a
bacteriophage" refers to a virus-like particle resembling the
structure of a bacteriophage, being non replicative and
noninfectious, and lacking at least the gene or genes encoding for
the replication machinery of the bacteriophage, and typically also
lacking the gene or genes encoding the protein or proteins
responsible for viral attachment to or entry into the host. This
definition should, however, also encompass virus-like particles of
bacteriophages, in which the aforementioned gene or genes are still
present but inactive, and, therefore, also leading to
non-replicative and noninfectious virus-like particles of a
bacteriophage.
[0138] The capsid structure formed from the self-assembly of 180
subunits of RNA phage coat protein and optionally containing host
RNA is herein referred to as a "VLP of RNA phage coat protein".
Specific examples are the VLP of Qbeta, MS2, PP7 or AP205 coat
proteins. In the specific case of Qbeta coat protein, for example,
the VLP may either be assembled exclusively from Qbeta CP subunits
(generated by expression of a Qbeta CP gene containing, for
example, a TAA stop codon precluding any expression of the longer
A1 protein through suppression, see Kozlovska, T. M., et al.,
Intervirology 39: 9-15 (1996)), or additionally contain A1 protein
subunits in the capsid assembly. Generally, the percentage of Qbeta
A1 protein relative to Qbeta CP in the capsid assembly will be
limited, in order to ensure capsid formation.
[0139] Examples of VLPs suitable as immunogenic carriers in the
context of the present invention include, but are not limited to,
VLPs of Qbeta, MS2, PP7, AP205 and other bacteriophage coat
proteins, the capsid and core proteins of Hepatitis B virus
(Ulrich, et al., Virus Res. 50: 141-182 (1998)), measles virus
(Warnes, et al., Gene 160: 173-178 (1995)), Sindbis virus,
rotavirus (U.S. Pat. Nos. 5,071,651 and 5,374,426),
foot-and-mouth-disease virus (Twomey, et al., Vaccine 13:
1603-1610, (1995)), Norwalk virus (Jiang, X., et al., Science 250:
1580-1583 (1990); Matsui, S. M., et al., J Clin. Invest. 87:
1456-1461 (1991)), the retroviral GAG protein (PCT Patent Appl. No.
WO 96/30523), the retrotransposon Ty protein pI, the surface
protein of Hepatitis B virus (WO 92/11291), human papilloma virus
(WO 98/15631), human polyoma virus (Sasnauskas K., et al., Biol.
Chem. 380 (3): 381-386 (1999); Sasnauskas K., et al., Generation of
recombinant virus-like particles of different polyomaviruses in
yeast. 3rd Interational Workshop "Virus-like particles as
vaccines." Berlin, Sep. 26-29 (2001)), RNA phages, Ty, frphage,
GA-phage, AP 205-phage and, in particular, Qbeta-phage, Cowpea
chlorotic mottle virus, cowpea mosaic virus, human papilloma
viruses (HPV), bovine papilloma viruses, porcine parvovirus,
parvoviruses such as B19, porcine (PPV) and canine (CPV)
parvovirues, caliciviruses (e.g. Norwalk virus,rabbit hemorrhagic
disease virus [RHDV]), animal hepadnavirus core Antigen VLPs,
filamentous/rod-shaped plant viruses, including but not limited to
Tobacco Mosaic Virus (TMV), Potato Virus X (PVX), Papaya Mosaic
Virus (PapMV), Alfalfa Mosaic Virus (AIMV), and Johnson Grass
Mosaic Virus (JGMV), insect viruses such as flock house virus (FHV)
and tetraviruses, polyomaviruses such as Murine Polyomavirus
(MPyV), Murine Pneumotropic Virus (MPtV), BK virus (BKV), and JC
virus (JCV).
[0140] As will be readily apparent to those skilled in the art, the
VLP to be used as an immunogenic carrier of the invention is not
limited to any specific form. The particle can be synthesized
chemically or through a biological process, which can be natural or
nonnatural. By way of example, this type of embodiment includes a
virus-like particle or a recombinant form thereof. In a more
specific embodiment, the VLP can comprise, or alternatively consist
of, recombinant polypeptides of any of the virus known to form a
VLP. The virus-like particle can further comprise, or alternatively
consist of, one or more fragments of such polypeptides, as well as
variants of such polypeptides. Variants of polypeptides can share,
for example, at least 80%, 85%, 90%, 95%, 97%, or 99% identity at
the amino acid level with their wild-type counterparts. Variant
VLPs suitable for use in the present invention can be derived from
any organism so long as they are able to form a "virus-like
particle" and can be used as an "immunogenic carrier" as defined
herein.
[0141] Preferred VLPs according to the invention include the capsid
protein or surface antigen of HBV (HBcAg and HBsAg respectively) or
recombinant proteins or fragments thereof, and the coat proteins of
RNA-phages or recombinant proteins or fragments thereof, more
preferably the coat protein of Qbeta or recombinant proteins or
fragments thereof.
[0142] In one embodiment, the immunogic carrier used in combination
with an antigenic PCSK9 peptide of the invention is an HBcAg
protein. Examples of HBcAg proteins that can be used in the context
of the present invention can be readily determined by one skilled
in the art. Examples include, but are limited to, HBV core proteins
described in Yuan et al., (J. Virol. 73: 10122-10128 (1999)), and
in WO00/198333, WO 00/177158, WO 00/214478, WO WO00/32227,
WO01/85208, WO02/056905, WO03/024480, and WO03/024481. HBcAgs
suitable for use in the present invention can be derived from any
organism so long as they are able to form a "virus-like particle"
and can be used as an "immunogenic carrier" as defined herein.
[0143] HBcAg variants of particular interest that could be used in
the context of the present invention are those variants in which
one or more naturally resident cysteine residues have been either
deleted or substituted. It is well known in the art that free
cysteine residues can be involved in a number of chemical side
reactions including disulfide exchanges, reaction with chemical
substances or metabolites that are, for example, injected or formed
in a combination therapy with other substances, or direct oxidation
and reaction with nucleotides upon exposure to UV light. Toxic
adducts could thus be generated, especially considering the fact
that HBcAgs have a strong tendency to bind nucleic acids. The toxic
adducts would thus be distributed between a multiplicity of
species, which individually may each be present at low
concentration, but reach toxic levels when together. In view of the
above, one advantage to the use of HBcAgs in vaccine compositions
which have been modified to remove naturally resident cysteine
residues is that sites to which toxic species can bind when
antigens or antigenic determinants are attached would be reduced in
number or eliminated altogether.
[0144] In addition, the processed form of HBcAg lacking the
N-terminal leader sequence of the Hepatitis B core antigen
precursor protein can also be used in the context of the invention,
especially when HBcAg is produced under conditions where processing
will not occur (e.g. expression in bacterial systems).
[0145] Other HBcAg variants according to the invention include i)
polypeptide sequence having at least 80%, 85%, 90%, 95%, 97% or 99%
identical to one of the wild-type HBcAg amino acid sequences, or a
subportion thereof, using conventionally using known computer
programs, ii) C-terminal truncation mutants including mutants where
1, 5, 10, 15, 20, 25, 30, 34 or 35, amino acids have been removed
from the C-terminus, ii) N-terminal truncation mutants including
mutants where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have
been removed from the N-terminus, iii) mutants truncated in both
N-terminal and C-terminal include HBcAgs where 1, 2, 5, 7, 9, 10,
12, 14, 15 or 17 amino acids have been removed from the N-terminus
and 1, 5, 10, 15, 20, 25, 30, 34 or 35 amino acids have been
removed from the C-terminus.
[0146] Still other HBcAg variant proteins within the scope of the
invention are those variants modified in order to enhance
immunogenic presentation of a foreign epitope wherein one or more
of the four arginine repeats has been deleted, but in which the
C-terminal cysteine is retained (see e.g. WO01/98333), and chimeric
C-terminally truncated HBcAg such as those described in WO02/14478,
WO03/102165 and WO04/053091.
[0147] In another embodiment, the immunogic carrier used in
combination with an antigenic PCSK9 peptide of the invention is an
HBsAg protein. HBsAg proteins that could be used in the context of
the present invention can be readily determined by one skilled in
the art. Examples include, but are limited to, HBV surface proteins
described in U.S. Pat. No. 5,792,463, WO02/10416, and WO08/020331.
HBsAgs suitable for use in the present invention can be derived
from any organism so long as they are able to form a "virus-like
particle" and can be used as an "immunogenic carrier" as defined
herein.
[0148] In still another embodiment, the immunogic carrier used in
combination with an antigenic PCSK9 peptide or polypeptide of the
invention is a Qbeta coat protein.
[0149] Qbeta coat protein was found to self-assemble into capsids
when expressed in E. coli (Kozlovska T M. et al., GENE 137: 133-137
(1993)). The obtained capsids or virus-like particles showed an
icosahedral phage-like capsid structure with a diameter of 25 nm
and T=3 quasi symmetry. Further, the crystal structure of phage Qss
has been solved. The capsid contains 180 copies of the coat
protein, which are linked in covalent pentamers and hexamers by
disulfide bridges (Golmohammadi, R. et al., Structure 4: 5435554
(1996)) leading to a remarkable stability of the capsid of Qbeta
coat protein. Qbeta capsid protein also shows unusual resistance to
organic solvents and denaturing agents. The high stability of the
capsid of Qbeta coat protein is an advantageous feature, in
particular, for its use in immunization and vaccination of mammals
and humans in accordance of the present invention.
[0150] Examples of Qbeta coat proteins that can be used in the
context of the present invention can be readily determined by one
skilled in the art. Examples have been extensively described in
WO02/056905, WO03/024480, WO03/024481 (incorporated by reference in
their entirety) and include, but are not limited to, amino acid
sequences disclosed in PIR database, accession No. VCBPQbeta
referring to Qbeta CP; Accession No. AAA16663 referring to Qbeta AI
protein; and variants thereof including variants proteins in which
the N-terminal methionine is cleaved; C-terminal truncated forms of
Qbeta A1 missing as much as 100, 150 or 180 amino acids; variant
proteins which have been modified by the removal of a lysine
residue by deletion or substitution or by the addition of a lysine
residue by substitution or insertion (see for example Qbeta-240,
Qbeta-243, Qbeta-250, Qbeta-251 and Qbeta-259 disclosed in
WO03/024481, incorporated by reference in its entirety), and
variants exhibiting at least 80%, 85%, 90%, 95%, 97%, or 99%
identity to any of the Qbeta core proteins described above. Variant
Qbeta coat proteins suitable for use in the present invention can
be derived from any organism so long as they are able to form a
"virus-like particle" and can be used as "immunogenic carriers" as
defined herein.
[0151] The antigenic PCSK9 peptides of the invention may be coupled
to immunogenic carriers via chemical conjugation or by expression
of genetically engineered fusion partners. The coupling does not
necessarily need to be direct, but can occur through linker
sequences. More generally, in the case that antigenic peptides
either fused, conjugated or otherwise attached to an immunogenic
carrier, spacer or linker sequences are typically added at one or
both ends of the antigenic peptides. Such linker sequences
generally comprise sequences recognized by the proteasome,
proteases of the endosomes or other vesicular compartment of the
cell.
[0152] In one embodiment, the peptides of the present invention are
expressed as fusion proteins with the immunogenic carrier. Fusion
of the peptide can be effected by insertion into the immunogenic
carrier primary sequence, or by fusion to either the N- or
C-terminus of the immunogenic carrier. Hereinafter, when referring
to fusion proteins of a peptide to an immunogenic carrier, the
fusion to either ends of the subunit sequence or internal insertion
of the peptide within the carrier sequence are encompassed. Fusion,
as referred to hereinafter, may be effected by insertion of the
antigenic peptide into the sequence of carrier, by substitution of
part of the sequence of the carrier with the antigenic peptide, or
by a combination of deletion, substitution or insertions.
[0153] When the immunogenic carrier is a VLP, the chimeric
antigenic peptide-VLP subunit will be in general capable of
self-assembly into a VLP. VLP displaying epitopes fused to their
subunits are also herein referred to as chimeric VLPs. For example,
EP 0 421 635 B describes the use of chimaeric hepadnavirus core
antigen particles to present foreign peptide sequences in a
virus-like particle.
[0154] Flanking amino acid residues may be added to either end of
the sequence of the antigenic peptide to be fused to either end of
the sequence of the subunit of a VLP, or for internal insertion of
such peptidic sequence into the sequence of the subunit of a VLP.
Glycine and serine residues are particularly favored amino acids to
be used in the flanking sequences added to the peptide to be fused.
Glycine residues confer additional flexibility, which may diminish
the potentially destabilizing effect of fusing a foreign sequence
into the sequence of a VLP subunit.
[0155] In a specific embodiment of the invention, the immunogenic
carrier is a HBcAg VLP. Fusion proteins of the antigenic peptide to
either the N-terminus of a HBcAg (Neyrinck, S. et al., Nature Med.
5: 11571163 (1999)) or insertions in the so called major
immunodominant region (MIR) have been described (Pumpens, P. and
Grens, E., Intervirology 44: 98114 (2001)), WO 01/98333), and are
specific embodiments of the invention. Naturally occurring variants
of HBcAg with deletions in the MIR have also been described
(Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), and
fusions to the N- or C-terminus, as well as insertions at the
position of the MIR corresponding to the site of deletion as
compared to a wt HBcAg are further embodiments of the invention.
Fusions to the C-terminus have also been described (Pumpens, P. and
Grens, E., Intervirology 44: 98-114 (2001)). One skilled in the art
will easily find guidance on how to construct fusion proteins using
classical molecular biology techniques. Vectors and plasmids
encoding HBcAg and HBcAg fusion proteins and useful for the
expression of a HBcAg and HBcAg fusion proteins have been described
(Pumpens, P. and #38; Grens, E. Intervirology 44: 98-114 (2001),
Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)) and can be
used in the practice of the invention. An important factor for the
optimization of the efficiency of self-assembly and of the display
of the epitope to be inserted in the MIR of HBcAg is the choice of
the insertion site, as well as the number of amino acids to be
deleted from the HBcAg sequence within the MIR (Pumpens, P. and
Grens, E., Intervirology 44: 98-114 (2001); EP 0 421 635; U.S. Pat.
No. 6,231,864) upon insertion, or in other words, which amino acids
form HBcAg are to be substituted with the new epitope. For example,
substitution of HBcAg amino acids 76-80, 79-81, 79-80, 75-85 or
80-81 with foreign epitopes has been described (Pumpens, P. and
Grens, E., Intervirology 44: 98-114 (2001); EP0421635; U.S. Pat.
No. 6,231,864, WO00/26385). HBcAg contains a long arginine tail
(Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)) which
is dispensable for capsid assembly and capable of binding nucleic
acids (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)).
HBcAg either comprising or lacking this arginine tail are both
embodiments of the invention.
[0156] In another specific embodiment of the invention, the
immunogenic carrier is a VLP of a RNA phage, preferably Qbeta. The
major coat proteins of RNA phages spontaneously assemble into VLPs
upon expression in bacteria, and in particular in E. coli. Fusion
protein constructs wherein antigenic peptides have been fused to
the C-terminus of a truncated form of the A1 protein of Qbeta, or
inserted within the A1 protein have been described (Kozlovska, T.
M., et al., Intervirology, 39: 9-15 (1996)). The A1 protein is
generated by suppression at the UGA stop codon and has a length of
329 aa, or 328 aa, if the cleavage of the N-terminal methionine is
taken into account. Cleavage of the N-terminal methionine before an
alanine (the second amino acid encoded by the Qbeta CP gene)
usually takes place in E. coli, and such is the case for N-termini
of the Qbeta coat proteins. The part of the A1 gene, 3' of the UGA
amber codon encodes the CP extension, which has a length of 195
amino acids. Insertion of the antigenic peptide between position 72
and 73 of the CP extension leads to further embodiments of the
invention (Kozlovska, T. M., et al., Intervirology 39: 9-15
(1996)). Fusion of an antigenic peptide at the C-terminus of a
C-terminally truncated Qbeta A1 protein leads to further preferred
embodiments of the invention. For example, Kozlovska et al.,
(Intervirology, 39: 9-15 (1996)) describe Qbeta A1 protein fusions
where the epitope is fused at the C-terminus of the Qbeta CP
extension truncated at position 19.
[0157] As described by Kozlovska et al. (Intervirology, 39: 9-15
(1996)), assembly of the particles displaying the fused epitopes
typically requires the presence of both the A1 protein-antigen
fusion and the wt CP to form a mosaic particle. However,
embodiments comprising virus-like particles, and hereby in
particular the VLPs of the RNA phage Qbeta coat protein, which are
exclusively composed of VLP subunits having an antigenic peptide
fused thereto, are also within the scope of the present
invention.
[0158] The production of mosaic particles may be effected in a
number of ways. Kozlovska et al., Intervirology, 39: 9-15 (1996),
describe three methods, which all can be used in the practice of
the invention. In the first approach, efficient display of the
fused epitope on the VLPs is mediated by the expression of the
plasmid encoding the Qbeta A1I protein fusion having a UGA stop
codon between CP and CP extension in a E. coli strain harboring a
plasmid encoding a cloned UGA suppressor tRNA which leads to
translation of the UGA codon into Trp (pISM3001 plasmid (Smiley B.
K., et al., Gene 134: 33-40 (1993))). In another approach, the CP
gene stop codon is modified into UAA, and a second plasmid
expressing the A1 protein-antigen fusion is cotransformed. The
second plasmid encodes a different antibiotic resistance and the
origin of replication is compatible with the first plasmid. In a
third approach, CP and the A1 protein-antigen fusion are encoded in
a bicistronic manner, operatively linked to a promoter such as the
Trp promoter, as described in FIG. 1 of Kozlovska et al.,
Intervirology, 39: 9-15 (1996). Further VLPs suitable for fusion of
antigens or antigenic determinants are described in WO03/024481 and
include bacteriophage fr, RNA phase MS-2, capsid proteine of
papillomavirus, retrotransposon Ty, yeast and also
Retrovirus-like-particles, HIV2 Gag, Cowpea Mosaic Virus,
parvovirus VP2 VLP, HBsAg (U.S. Pat. No. 4,722,840, EP0020416B1).
Examples of chimeric VLPs suitable for the practice of the
invention are also those described in Intervirology 39: 1 (1996).
Further examples of VLPs contemplated for use in the invention are:
HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV,
HIV GAG, Tobacco Mosaic Virus. Virus-like particles of SV-40,
Polyomavirus, Adenovirus, Herpes Simplex Virus, Rotavirus and
Norwalk virus.
[0159] For any recombinantly expressed antigenic PCSK9 peptide
according to the invention coupled or not to an immunogenic
carrier, the nucleic acid which encodes said peptide or protein
also forms an aspect of the present invention, as does an
expression vector comprising the nucleic acid, and a host cell
containing the expression vector (autonomously or chromosomally
inserted). A method of recombinantly producing the peptide or
protein by expressing it in the above host cell and isolating the
immunogen therefrom is a further aspect of the invention. The
full-length native PCSK9 molecule or the full-length native DNA
sequence encoding it are not covered by the present invention.
[0160] In another embodiment, the peptide of the invention is
chemically coupled to an immunogenic carrier, using techniques well
known in the art. Conjugation can occur to allow free movement of
peptides via single point conjugation (e.g. either N-terminal or
C-terminal point) or as locked down structure where both ends of
peptides are conjugated to either a immunogenic carrier protein or
to a scaffold structure such as a VLP. Conjugation occurs via
conjugation chemistry known to those skilled in the art such as via
cysteine residues, lysine residues or other carboxy moiety's
commonly known as conjugation points such as glutamic acid or
aspartic acid. Thus, for example, for direct covalent coupling it
is possible to utilise a carbodiimide, glutaraldehyde or
(N-[y-malcimidobutyryloxy] succinimide ester, utilising common
commercially available heterobifunctional linkers such as CDAP and
SPDP (using manufacturers instructions). Examples of conjugation of
peptides, particularly cyclised peptides, to a protein carrier via
acylhydrazine peptide derivatives are described in WO03/092714.
After the coupling reaction, the immunogen can easily be isolated
and purified by means of a dialysis method, a gel filtration
method, a fractionation method etc. Peptides terminating with a
cysteine residue (preferably with a linker outside the cyclised
region) may be conveniently conjugated to a carrier protein via
maleimide chemistry.
[0161] When the immunogenic carrier is a VLP, several antigenic
peptide, either having an identical amino acid sequence or a
different amino acid sequence, may be coupled to a single VLP
molecule, leading preferably to a repetitive and ordered structure
presenting several antigenic determinants in an oriented manner as
described in WO00/32227, WO03/024481, WO02/056905 and
WO04/007538.
[0162] In a preferred embodiment, the antigenic PCSK9 peptide is
bound to the VLP by way of chemical cross-linking, typically and
preferably by using a heterobifunctional cross-linker. Several
hetero-bifunctional cross-linkers are known to the art. In some
embodiments, the hetero-bifunctional crosslinker contains a
functional group which can react with first attachment sites, i. e.
with the side-chain amino group of lysine residues of the VLP or
VLP subunit, and a further functional group which can react with a
preferred second attachment site, i. e. a cysteine residue fused to
the antigenic peptide and optionally also made available for
reaction by reduction. The first step of the procedure, typically
called the derivatization, is the reaction of the VLP with the
cross-linker. The product of this reaction is an activated VLP,
also called activated carrier. In the second step, unreacted
cross-linker is removed using usual methods such as gel filtration
or dialysis. In the third step, the antigenic peptide is reacted
with the activated VLP, and this step is typically called the
coupling step. Unreacted antigenic peptide may be optionally
removed in a fourth step, for example by dialysis. Several
hetero-bifunctional crosslinkers are known to the art. These
include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS,
Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB,
SIA and other cross-linkers available for example from the Pierce
Chemical Company (Rockford, Ill., USA), and having one functional
group reactive towards amino groups and one functional group
reactive towards cysteine residues. The above mentioned
cross-linkers all lead to formation of a thioether linkage.
[0163] Another class of cross-linkers suitable in the practice of
the invention is characterized by the introduction of a disulfide
linkage between the antigenic peptide and the VLP upon coupling.
Preferred cross-linkers belonging to this class include for example
SPDP and Sulfo-LC-SPDP (Pierce). The extent of derivatization of
the VLP with cross-linker can be influenced by varying experimental
conditions such as the concentration of each of the reaction
partners, the excess of one reagent over the other, the pH, the
temperature and the ionic strength. The degree of coupling, i.e.
the amount of antigenic peptide per subunits of the VLP can be
adjusted by varying the experimental conditions described above to
match the requirements of the vaccine.
[0164] Another method of binding of antigenic peptides to the VLP,
is the linking of a lysine residue on the surface of the VLP with a
cysteine residue on the antigenic peptide. In some embodiments,
fusion of an amino acid linker containing a cysteine residue, as a
second attachment site or as a part thereof, to the antigenic
peptide for coupling to the VLP may be required. In general,
flexible amino acid linkers are favored. Examples of the amino acid
linker are selected from the group consisting of: (a) CGG; (b)
N-terminal gamma 1-linker; (c) N-terminal gamma 3-linker; (d) Ig
hinge regions; (e) N-terminal glycine linkers; (f) (G) kC (G) n
with n=0-12 and k=0-5; (g) N-terminal glycine-serine linkers; (h)
(G) kC (G) m (S) i (GGGGS) n with n=0-3, k=0-5, m=0-10, i=0-2; (i)
GGC; (k) GGC-NH2; (1) C-terminal gamma 1-linker; (m) C-terminal
gamma 3-linker; (n) C-terminal glycine linkers; (o) (G) nC (G) k
with n=0-12 and k=0-5; (p) C-terminal glycine-serine linkers; (q)
(G) m (S) t (GGGGS) n (G) oC (G) k with n=0-3, k=0-5, m=0-10,
1=0-2, and o=0-8. Further examples of amino acid linkers are the
hinge region of immunoglobulins, glycine serine linkers (GGGGS) n,
and glycine linkers (G) n all further containing a cysteine residue
as second attachment site and optionally further glycine residues.
Typically preferred examples of said amino acid linkers are
N-terminal gamma 1: CGDKTHTSPP; C-terminal gamma 1: DKTHTSPPCG;
N-terminal gamma 3: CGGPKPSTPPGSSGGAP; C-terminal gamma 3:
PKPSTPPGSSGGAPGGCG; N-terminal glycine linker: GCGGGG and
C-terminal glycine linker: GGGGCG.
[0165] Other amino acid linkers particularly suitable in the
practice of the invention, when a hydrophobic antigenic peptide is
bound to a VLP, are CGKKGG, or CGDEGG for N-terminal linkers, or
GGKKGC and GGEDGC, for the C-terminal linkers. For the C-terminal
linkers, the terminal cysteine is optionally C-terminally
amidated.
[0166] In some embodiments of the present invention, GGCG, GGC or
GGC-NH2 ("NH2" stands for amidation) linkers at the C-terminus of
the peptide or CGG at its N-terminus are preferred as amino acid
linkers. In general, glycine residues will be inserted between
bulky amino acids and the cysteine to be used as second attachment
site, to avoid potential steric hindrance of the bulkier amino acid
in the coupling reaction. In a further embodiment of the invention,
the amino acid linker GGC-NH2 is fused to the C-terminus of the
antigenic peptide.
[0167] The cysteine residue present on the antigenic peptide has to
be in its reduced state to react with the hetero-bifunctional
cross-linker on the activated VLP, that is a free cysteine or a
cysteine residue with a free sulfhydryl group has to be available.
In the instance where the cysteine residue to function as binding
site is in an oxidized form, for example if it is forming a
disulfide bridge, reduction of this disulfide bridge with e.g. DTT,
TCEP or p-mercaptoethanol is required. Low concentrations of
reducing agent are compatible with coupling as described in WO
02/05690, higher concentrations inhibit the coupling reaction, as a
skilled artisan would know, in which case the reductand has to be
removed or its concentration decreased prior to coupling, e.g. by
dialysis, gel filtration or reverse phase HPLC.
[0168] Binding of the antigenic peptide to the VLP by using a
hetero-bifunctional cross-linker according to the methods described
above, allows coupling of the antigenic peptide to the VLP in an
oriented fashion. Other methods of binding the antigenic peptide to
the VLP include methods wherein the antigenic peptide is
cross-linked to the VLP using the carbodiimide EDC, and NHS.
[0169] In other methods, the antigenic peptide is attached to the
VLP using a homo-bifunctional cross-linker such as glutaraldehyde,
DSGBM [PEO] 4, BS3, (Pierce Chemical Company, Rockford, Ill., USA)
or other known homo-bifunctional cross-linkers with functional
groups reactive towards amine groups or carboxyl groups of the
VLP.
[0170] Other methods of binding the VLP to an antigenic peptide
include methods where the VLP is biotinylated, and the antigenic
peptide expressed as a streptavidin-fusion protein, or methods
wherein both the antigenic peptide and the VLP are biotinylated,
for example as described in WO 00/23955. In this case, the
antigenic peptide may be first bound to streptavidin or avidin by
adjusting the ratio of antigenic peptide to streptavidin such that
free binding sites are still available for binding of the VLP,
which is added in the next step. Alternatively, all components may
be mixed in a "one pot" reaction. Other ligand-receptor pairs,
where a soluble form of the receptor and of the ligand is
available, and are capable of being cross-linked to the VLP or the
antigenic peptide, may be used as binding agents for binding
antigenic peptide to the VLP. Alternatively, either the ligand or
the receptor may be fused to the antigenic peptide, and so mediate
binding to the VLP chemically bound or fused either to the
receptor, or the ligand respectively. Fusion may also be effected
by insertion or substitution.
[0171] One or several antigen molecules can be attached to one
subunit of the capsid or VLP of RNA phages coat proteins,
preferably through the exposed lysine residues of the VLP of RNA
phages, if sterically allowable. A specific feature of the VLP of
the coat protein of RNA phages and in particular of the QP coat
protein VLP is thus the possibility to couple several antigens per
subunit. This allows for the generation of a dense antigen array.
VLPs or capsids of Q coat protein display a defined number of
lysine residues on their surface, with a defined topology with
three lysine residues pointing towards the interior of the capsid
and interacting with the RNA, and four other lysine residues
exposed to the exterior of the capsid. These defined properties
favor the attachment of antigens to the exterior of the particle,
rather than to the interior of the particle where the lysine
residues interact with RNA. VLPs of other RNA phage coat proteins
also have a defined number of lysine residues on their surface and
a defined topology of these lysine residues.
[0172] In a further embodiment of the present invention, the first
attachment site is a lysine residue and/or the second attachment
comprises sulfhydryl group or a cysteine residue. In an even
further embodiment of the present invention, the first attachment
site is a lysine residue and the second attachment is a cysteine
residue. In further embodiments of the invention, the antigen or
antigenic determinant is bound via a cysteine residue, to lysine
residues of the VLP of RNA phage coat protein, and in particular to
the VLP of Qbeta coat protein.
[0173] Another advantage of the VLPs derived from RNA phages is
their high expression yield in bacteria that allows production of
large quantities of material at affordable cost. Moreover, the use
of the VLPs as carriers allow the formation of robust antigen
arrays and conjugates, respectively, with variable antigen density.
In particular, the use of VLPs of RNA phages, and hereby in
particular the use of the VLP of RNA phage Qbeta coat protein
allows a very high epitope density to be achieved.
[0174] According to an embodiment of the present invention the
antigenic PCSK9 peptide disclosed herein are linked, preferably
chemically cross linked, to CRM197, either directly or via one of
the peptide linker disclosed herein, to generate an immunogen. In
an embodiment, the antigenic PCSK9 peptide disclosed herein is
linked to CRM197, by way of chemical cross-linking as described
herein and preferably by using a heterobifunctional cross-linker,
as disclosed above.
[0175] Preferred heterobifunctional cross-linkers for use with
CRM197 are BAANS (bromoacetic acid N-hydroxysuccinimide ester),
SMPH (Succinimidyl-6-[ -maleimidopropionamido]hexanoate),
Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB,
Sulfo-SMCC, SVSB, SIA and other cross-linkers available for example
from the Pierce Chemical Company (Rockford, Ill., USA). In a
preferred embodiment of the present invention, the
hetero-bifunctional crosslinker is BAANS or SMPH.
[0176] Alternatively, cross-linkers suitable allowing the
introduction of a disulfide linkage between the antigenic peptide
and CRM197 could also be used in the context of the invention.
Preferred cross-linkers belonging to this class include for example
SPDP and Sulfo-LC-SPDP (Pierce).
[0177] In a particular embodiment, when the sequence of the
antigenic PCSK9 peptide disclosed herein comprises a cysteine, said
antigenic PCSK9 peptide may be covalently linked to CRM197 directly
via said cysteine.
[0178] In some embodiments of the invention, immunogenic
compositions of the invention may comprise mixtures of immunogenic
conjugates, i.e. immunogenic carriers coupled to one or several
antigenic PCSK9 peptides of the invention. Thus, these immunogenic
compositions may be composed of immunogenic carriers which differ
in amino acid sequence. For example, vaccine compositions could be
prepared comprising a "wild-type" VLP and a modified VLP protein in
which one or more amino acid residues have been altered (e. g.,
deleted, inserted or substituted). Alternatively, the same
immunogenic carrier might be used but coupled to antigenic PCSK9
peptides of different amino acid sequences.
[0179] The invention therefore also relates to method for producing
an immunogen according to the invention comprising i) providing an
antigenic PCSK9 peptide according to the invention, ii) providing
an immunogenic carrier according to the invention, preferably a
VLP, and iii) combining said antigenic PCSK9 peptide and said
immunogenic carrier. In one embodiment, said combining step occurs
through chemical cross-linking, preferably through an
heterobifunctional cross-linker.
[0180] In an embodiment of the present invention, the antigenic
PCSK9 peptide disclosed herein is linked to an immunogenic carrier
molecule. In an embodiment said immunogenic carrier is selected
from the group consisting of any of the immunogenic carrier
described herein. In another embodiment said immunogenic carrier is
selected from the group consisting of: serum albumins such as
bovine serum albumin (BSA); globulins; thyroglobulins; hemoglobins;
hemocyanins (particularly Keyhole Limpet Hemocyanin [KLH]) and
virus-like particle (VLPs). In a preferred embodiment said
immunogenic carrier is Diphtheria Toxoid, CRM197 mutant of
diphtheria toxin, Tetanus Toxoid, Keyhole Limpet Hemocyanin or
virus-like particle (VLPs). In an even preferred embodiment, said
immunogenic carrier is DT, CRM197 or a VLP selected from the group
consisting of HBcAg VLP, HBsAg VLP, Qbeta VLP, PP7 VLP, PPV VLP,
Norwalk Virus VLP or any variant disclosed herein. In an even
preferred embodiment, said immunogenic carrier is a bacteriophage
VLP such as Qbeta VLP selected from the group consisting of Qbeta
CP; Qbeta Al, Qbeta-240, Qbeta-243, Qbeta-250, Qbeta-251 and
Qbeta-259 (disclosed in WO03/024481) or PP7.
[0181] In another preferred embodiment, said immunogenic carrier is
CRM197.
[0182] In an embodiment, said immunogenic carrier is covalently
linked to the antigenic PCSK9 peptide disclosed herein either
directly or via a linker. In an embodiment, said immunogenic
carrier is linked to the antigenic PCSK9 peptide disclosed herein
by expression of a fusion protein as described herein. In another
embodiment, the antigenic PCSK9 peptide disclosed herein is linked
to the immunogenic carrier, preferably a VLP, by way of chemical
cross-linking as described herein and preferably by using a
heterobifunctional cross-linker. Several hetero-bifunctional
cross-linkers are known to the art. In some embodiments, the
hetero-bifunctional crosslinker contains a functional group which
can react with first attachment sites, i.e. with the side-chain
amino group of lysine residues of the VLP or VLP subunit, and a
further functional group which can react with a preferred second
attachment site, i.e. a cysteine residue fused to the antigenic
peptide made available for reaction by reduction.
Antigenic PCSK9 Peptide of the Invention Comprising a Linker
[0183] In an embodiment of the present invention the antigenic
PCSK9 peptide disclosed herein further comprise either at its
N-terminus, or at its C-terminus or at both the N-terminus and
C-terminus a linker which is able to react with an attachment site
of the immunogenic carrier in a chemical cross-linking reaction. In
an embodiment, the antigenic PCSK9 peptide disclosed herein further
comprise at its C-terminus a linker having the formula (G).sub.nC,
(G).sub.nSC or (G).sub.nK, preferably (.sub.G).sub.nC wherein n is
an integer chosen in the group consisting of 0, 1, 2, 3, 4, 5, 6,
7, 8, 9 and 10, preferably in the group consisting of 0, 1, 2, 3, 4
and 5, more preferably in the groups consisting of 0, 1, 2 and 3,
most preferably n is 0 or 1 (where n is equal to 0 said formula
represents a cysteine). Preferably the antigenic PCSK9 peptide
disclosed herein further comprise at its C-terminus a linker having
the formula GGGC, GGC, GC or C.
[0184] In another embodiment of the present invention the antigenic
PCSK9 peptide disclosed herein further comprise at its N-terminus a
linker having the formula C(G).sub.n, CS(G).sub.n or K(G).sub.n,
preferably C(G).sub.n wherein n is an integer chosen in the group
consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably in
the group consisting of 0, 1, 2, 3, 4 and 5, more preferably in the
groups consisting of 0, 1, 2 and 3, most preferably n is 0 or 1
(where n is equal to 0, the formula represents a cysteine).
Preferably the antigenic PCSK9 peptide disclosed herein further
comprise at its N-terminus a linker having the formula CGGG, CGG,
CG or C.
[0185] In another embodiment the antigenic PCSK9 peptide disclosed
herein further comprise at its C-terminus a linker having the
formula (G).sub.nC, (G).sub.nSC or (G).sub.nK, preferably
(G).sub.nC wherein n is an integer chosen in the group consisting
of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably in the group
consisting of 0, 1, 2, 3, 4 and 5, more preferably in the groups
consisting of 0, 1, 2 and 3, most preferably n 0 or 1 (where n is
equal to 0 said formula represents a cysteine) and at its
N-terminus a linker having the formula C(G).sub.n, CS(G).sub.n or
K(G).sub.n, preferably C(G).sub.n wherein n is an integer chosen in
the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,
preferably in the group consisting of 0, 1, 2, 3, 4 and 5, more
preferably in the groups consisting of 0, 1, 2 and 3, most
preferably n is 0 or 1 (where n is equal to 0, the formula
represents a cysteine). Preferably the antigenic PCSK9 peptide
disclosed herein further comprise at its N-terminus a linker having
the formula CGGG, CGG, CG or C and at its C-terminus a linker
having the formula GGGC, GGC, GC or C. Preferably, the antigenic
PCSK9 peptide disclosed herein further comprises at its N-terminus
a cysteine and at its C-terminus a cysteine.
[0186] Representative of said antigenic PCSK9 peptides further
comprising such a linker are disclosed at SEQ ID NO 313, 314, 315,
316, 317, 322, 323, 324, 325, 326, 327, 328, 401, 402, 403, 404,
405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,
418 and 419.
[0187] Representative of said antigenic PCSK9 peptides further
comprising such a linker are disclosed at SEQ ID NO 313, 314, 315,
316, 317, 322, 323, 324, 325, 326, 327 and 328.
[0188] Preferred antigenic PCSK9 peptides comprising a linker are
disclosed at SEQ ID Nos 317, 322, 323, 324, 401, 402, 403, 413,
414, 415 and 416.
[0189] Preferred antigenic PCSK9 peptides comprising a linker are
disclosed at SEQ ID Nos 317, 322, 323 and 324.
[0190] Most preferred antigenic PCSK9 peptides comprising a linker
are disclosed at SEQ ID Nos 317, 322, 402 and 413.
[0191] In one embodiment, the antigenic PCSK9 peptide is cyclised.
In one embodiment, the cyclised antigenic PCSK9 peptide is attached
to an immunogenic carrier. In one embodiment, said cyclised
antigenic PCSK9 peptide is attached to an immunogenic carrier by
covalent binding. In one embodiment, said cyclised antigenic PCSK9
peptide is attached to an immunogenic carrier by covalent binding
of one of the side chain of its amino acids to the carrier. In one
embodiment, a cysteine, a GC or a CC fragment comprising a variable
number of glycine residues and one cysteine residue is added to the
cyclised PCSK9 peptides to enable the covalent binding to the
immunogenic carrier through the added cysteine.
[0192] In one embodiment, the antigenic PCSK9 peptide is cyclised
and comprises a a cysteine, a (G).sub.nC or a C(G).sub.n fragment
wherein n is an integer chosen in the group consisting of 0, 1, 2,
3, 4, 5, 6, 7, 8, 9 and 10, preferably in the group consisting of
0, 1, 2, 3, 4 and 5, more preferably in the groups consisting of 0,
1, 2 and 3, most preferably n is 0 or 1 (where n is equal to 0, the
formula represents a cysteine).
[0193] Non limitative examples of such conformationally constrained
antigenic PCSK9 peptide are the peptides of SEQ ID Nos 318, 319,
320 and 321. A preferred cyclised peptide is the peptide of SEQ ID
Nos 318.
[0194] Examples of conjugations of antigenic PCSK9 peptides with
carrier or scaffolds described above, all within the scope of the
present invention and constituting various embodiments, using
various linkers are provided below:
[0195] Peptide-GGGGGC-scaffold, peptide-GGGGC-scaffold,
peptide-GGGC-scaffold, peptide-GGC-scaffold, peptide-GC-scaffold,
peptide-C-scaffold, peptide-GGGGGK-scaffold,
peptide-GGGGK-scaffold, Peptide-GGGK-scaffold,
Peptide-GGK-scaffold, Peptide-GK-scaffold, Peptide-K-scaffold,
Peptide-GGGGSC-scaffold, Peptide-GGGSC-scaffold,
Peptide-GGSC-scaffold, Peptide-GSC-scaffold-Peptide-SC-scaffold,
Scaffold-CSGGGG-Peptide, Scaffold-CSGGG-Peptide,
Scaffold-CSGG-Peptide, Scaffold-CSG-Peptide, Scaffold-CS-Peptide,
Scaffold-KGGGG-Peptide, Scaffold-KGGG-Peptide,
Scaffold-KGG-Peptide, Scaffold-KG0-Peptide, Scaffold-K-Peptide.
[0196] In an embodiment, the peptide consists of any of the
antigenic PCSK9 peptide disclosed herein and the scaffold consists
of any of the immunogenic carrier disclosed herein, preferably a
VLP.
[0197] Exemplary combinations of conjugations using various linkers
and doubly constrained peptides are provided below, where the
carrier can be the identical monomer of a carrier or a differential
monomer of a carrier. (In the example below, the GC linker can be
substituted by any of the GK linker or GSC linker exemplified above
or any other known to those skilled in the art):
[0198] Carrier-CGGGGG-Peptide-GGGGGC-carrier,
Carrier-CGGGG-Peptide-GGGGC-carrier,
Carrier-CGGGG-Peptide-GGGGC-carrier,
Carrier-CGGG-Peptide-GGGC-carrier, Carrier-CG-Peptide-GC-carrier,
Carrier-CG-Peptide-C-carrier, Carrier-C-Peptide-C-carrier.
[0199] In an embodiment, the peptide consists of any of the
antigenic PCSK9 peptide disclosed herein and the carrier consists
of any of the immunogenic carrier disclosed herein, preferably a
VLP.
[0200] In an embodiment, the invention relates to an immunogen
comprising an antigenic PCSK9 peptide consisting of, or consisting
essentially of, an amino acid sequence selected from the group
consisting of SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588,
wherein said antigenic antigenic PCSK9 peptide further comprises at
its C-terminus or at its N-terminus a cysteine which is chemically
cross linked to an immunogenic carrier via a thioether linkage. In
a preferred embodiment, said immunogenic carrier is selected from
the group consisting of DT (Diphtheria toxin), TT (tetanus toxid)
or fragment C of TT, PD (Haemophilus influenzae protein D), CRM197,
other DT point mutants, such as CRM176, CRM228, CRM 45, CRM 9,
CRM102, CRM 103 and CRM107. Preferably said immunogenic carrier is
CRM197.
[0201] In an embodiment, the invention relates to an immunogen
comprising an antigenic PCSK9 peptide consisting of, or consisting
essentially of, an amino acid sequence selected from the group
consisting of 1 to 312, 330 to 398 and 420 to 588, wherein said
antigenic antigenic PCSK9 peptide further comprises at its
C-terminus or at its N-terminus a cysteine which is chemically
cross linked to an immunogenic carrier via a thioether linkage
using SMPH (Succinimidyl-6-[ -maleimidopropionamido]hexanoate) or
BAANS (bromoacetic acid N-hydroxysuccinimide ester) as cross
linker. In a preferred embodiment, said immunogenic carrier is
selected from the group consisting of DT (Diphtheria toxin), TT
(tetanus toxid) or fragment C of TT, PD (Haemophilus influenzae
protein D, CRM197, other DT point mutants, such as CRM176, CRM228,
CRM 45, CRM 9, CRM102, CRM 103 and CRM107. Preferably said
immunogenic carrier is CRM197.
[0202] In an embodiment, the invention relates to an immunogen
comprising an antigenic PCSK9 peptide consisting of, or consisting
essentially of, an amino acid sequence selected from the group
consisting of SEQ ID Nos: 1 to 312, 330 to 398 and 420 to 588,
wherein said antigenic antigenic PCSK9 peptide further comprises at
its C-terminus a cysteine which is chemically cross linked to an
immunogenic carrier via a thioether linkage using SMPH
(Succinimidyl-6-[ -maleimidopropionamido]hexanoate) or BAANS
(bromoacetic acid N-hydroxysuccinimide ester) as cross linker, said
linkage being between a lysine residue of CRM197 and the cysteine
residue of said antigenic peptide.
Compositions Comprising an Antigenic PCSK9 Peptide of the
Invention
[0203] The present invention further relates to compositions,
particularly immunogenic compositions also referred to as "subject
immunogenic compositions", comprising an antigenic PCSK9 peptide of
the invention, preferably linked to an immunogenic carrier, and
optionally at least one adjuvant. Such immunogenic compositions,
particularly when formulated as pharmaceutical compositions, are
deemed useful to prevent, treat or alleviate PCSK9-related
disorders.
[0204] In some embodiments, a subject immunogenic composition
according to the invention comprises an antigenic PCSK9 peptide,
optionally comprising a linker, comprising an amino acid sequence
selected from SEQ ID Nos 1 to 328, 330 to 398, and 401 to 588 and
functionally active variants thereof. In some embodiment, said
antigenic PCSK9 peptide is linked to an immunogenic carrier,
preferably a DT, CRM197 or a VLP, more preferably to a HBcAg,
HBsAg, Qbeta, PP7, PPV or Norwalk Virus VLP.
[0205] In a preferred embodiment, a subject immunogenic composition
according to the invention comprises an antigenic PCSK9 peptide,
optionally comprising a linker, comprising an amino acid sequence
selected from SEQ ID Nos 1 to 328, 330 to 398 and 401 to 588, and
functionally active variants thereof linked to a VLP, preferably a
Qbeta VLP.
[0206] In a preferred embodiment, a subject immunogenic composition
according to the invention comprises an antigenic PCSK9 peptide
optionally comprising a linker, comprising an amino acid sequence
selected from SEQ ID Nos 1 to 328, 330 to 398, and 401 to 588 and
functionally active variants thereof linked to CRM197.
[0207] A subject immunogenic composition comprising an antigenic
PCSK9 peptide according to the invention can be formulated in a
number of ways, as described in more detail below.
[0208] In some embodiments, a subject immunogenic composition
comprises single species of antigenic PCSK9 peptide, e.g., the
immunogenic composition comprises a population of antigenic PCSK9
peptides, substantially all of which have the same amino acid
sequence. In other embodiments, a subject immunogenic composition
comprises two or more different antigenic PCSK9 peptides, e.g., the
immunogenic composition comprises a population of antigenic PCSK9
peptides, the members of which population can differ in amino acid
sequence. A subject immunogenic composition can comprise from two
to about 20 different antigenic PCSK9 peptides, e.g., a subject
immunogenic composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10,
11-15, or 15-20 different antigenic PCSK9 peptides, each having an
amino acid sequence that differs from the amino acid sequences of
the other antigenic PCSK9 peptides.
[0209] In other embodiments, a subject immunogenic composition
comprises a multimerized antigenic PCSK9 polypeptide, as described
above. As used herein, the terms "immunogenic composition
comprising an antigenic PCSK9 peptide" or "immunogenic composition
of the invention" or "subject immunogenic composition" refers to an
immunogenic composition comprising either single species
(multimerized or not) or multiple species of antigenic PCSK9
peptide(s) coupled or not to an immunogenic carrier. Where two or
more peptides are used coupled to a carrier, the peptide may be
coupled to the same carrier molecule or individually coupled to
carrier molecules and then combined to produce an immunogenic
composition.
[0210] Another aspect of the invention relates to methods for
producing an immunogen according to the invention, said method
comprising coupling an antigenic PCSK9 peptide to an immunogenic
carrier. In one embodiment, said coupling is chemical.
Adjuvants
[0211] In some embodiments, a subject immunogenic composition
comprises at least one adjuvant. Suitable adjuvants include those
suitable for use in mammals, preferably in humans. Examples of
known suitable adjuvants that can be used in humans include, but
are not necessarily limited to, alum, aluminum phosphate, aluminum
hydroxide, MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween
80), 0.5% w/v sorbitan trioleate (Span 85)), CpG-containing nucleic
acid (where the cytosine is unmethylated), QS21 (saponin adjuvant),
MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylated MPL), extracts
from Aquilla, ISCOMS (see, e.g., Sjoander et al. (1998) J.
Leukocyte Biol. 64:713; WO90/03184, WO96/11711, WO 00/48630,
WO98/36772, WO00/41720, WO06/134423 and WO07/026190), LT/CT
mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil
A, TiterMax classic, TiterMax Gold, interleukins, and the like. For
veterinary applications including but not limited to animal
experimentation, one can use Freund's adjuvant,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred
to as nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred
to as MTP-PE), and RIBI, which contains three components extracted
from bacteria, monophosphoryl lipid A, trehalose dimycolate and
cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80
emulsion.
[0212] Further exemplary adjuvants to enhance effectiveness of the
composition include, but are not limited to: (1) oil-in-water
emulsion formulations (with or without other specific
immunostimulating agents such as muramyl peptides (see below) or
bacterial cell wall components), such as for example (a) MF59.TM.
(WO90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant
approach, eds. Powell & Newman, Plenum Press 1995), containing
5% Squalene, 0.5% Tween 80 (polyoxyethylene sorbitan mono-oleate),
and 0.5% Span 85 (sorbitan trioleate) (optionally containing
muramyl tri-peptide covalently linked to dipalmitoyl
phosphatidylethanolamine (MTP-PE)) formulated into submicron
particles using a microfluidizer, (b) SAF, containing 10% Squalane,
0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either
microfluidized into a submicron emulsion or vortexed to generate a
larger particle size emulsion, and (c) RIBI.TM. adjuvant system
(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,
0.2% Tween 80, and one or more bacterial cell wall components such
as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (DETOX.TM.); (2) saponin
adjuvants, such as QS21, STIMULON.TM. (Cambridge Bioscience,
Worcester, Mass.), Abisco.RTM. (Isconova, Sweden), or
Iscomatrix.RTM. (Commonwealth Serum Laboratories, Australia), may
be used or particles generated therefrom such as ISCOMs
(immunostimulating complexes), which ISCOMS may be devoid of
additional detergent e.g. WO00/07621; (3) Complete Freund's
Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4)
cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma
interferon), macrophage colony stimulating factor (M-CSF), tumor
necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or
3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454,
optionally in the substantial absence of alum when used with
pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL
with, for example, QS21 and/or oil-in-water emulsions e.g.
EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides
comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr
opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3,
849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et
al, J. Immunol, 1998, 160, 870-876; Chu et al., J. Exp. Med, 1997,
186, 1623-1631; Lipford et al, Ear. J. Immunol., 1997, 27,
2340-2344; Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg
et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996,
93, 2879-2883; Ballas et al, J. Immunol, 1996, 157, 1840-1845;
Cowdery et al, J. Immunol, 1996, 156, 4570-4575; Halpern et al,
Cell Immunol, 1996, 167, 72-78; Yamamoto et al, Jpn. J. Cancer
Res., 1988, 79, 866-873; Stacey et al, J. Immunol., 1996,
157,2116-2122; Messina et al, J. Immunol, 1991, 147, 1759-1764; Yi
et al, J. Immunol, 1996, 157, 4918-4925; Yi et al, J. Immunol,
1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160, 4755-4761;
and Yi et al, J. Immunol, 1998, 160, 5898-5906; International
patent applications WO96/02555, WO98/16247, WO98/18810, WO98/40100,
WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one
CG dinucleotide, where the cytosine is unmethylated; (8) a
polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549;
(9) a polyoxyethylene sorbitan ester surfactant in combination with
an octoxynol (WO01/21207) or a polyoxyethylene alkyl ether or ester
surfactant in combination with at least one additional non-ionic
surfactant such as an octoxynol (WO01/21152); (10) a saponin and an
immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)
(WO00/62800); (11) an immunostimulant and a particle of metal salt
e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g.
WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+a
sterol) e.g. WO98/57659; (14) other substances that act as
immunostimulating agents to enhance the efficacy of the
composition, such as Muramyl peptides include
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25
acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl--
sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), (15) ligands
for toll-like receptors (TLR), natural or synthesized (e.g. as
described in Kanzler et al 2007, Nature Medicine 13, p 1552-9),
including TLR3 ligands such as polyl:C and similar compounds such
as Hiltonol and Ampligen.
[0213] In a particular embodiment, said adjuvant is an
immunostimulatory oligonucleotide and more preferably a CpG
oligonucleotide. A CpG oligonucleotide as used herein refers to an
immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and
accordingly these terms are used interchangeably unless otherwise
indicated. Immunostimulatory CpG oligodeoxynucleotides contain one
or more immunostimulatory CpG motifs that are unmethylated
cytosine-guanine dinucleotides, optionally within certain preferred
base contexts. The methylation status of the CpG immunostimulatory
motif generally refers to the cytosine residue in the dinucleotide.
An immunostimulatory oligonucleotide containing at least one
unmethylated CpG dinucleotide is an oligonucleotide which contains
a 5' unmethylated cytosine linked by a phosphate bond to a 3'
guanine, and which activates the immune system through binding to
Toll-like receptor 9 (TLR-9). In another embodiment the
immunostimulatory oligonucleotide may contain one or more
methylated CpG dinucleotides, which will activate the immune system
through TLR9 but not as strongly as if the CpG motif(s) was/were
unmethylated. CpG immunostimulatory oligonucleotides may comprise
one or more palindromes that in turn may encompass the CpG
dinucleotide. CpG oligonucleotides have been described in a number
of issued patents, published patent applications, and other
publications, including U.S. Pat. Nos. 6,194,388; 6,207,646;
6,214,806; 6,218,371; 6,239,116; and 6,339,068.
[0214] Different classes of CpG immunostimulatory oligonucleotides
have been identified. These are referred to as A, B, C and P class,
and are described in greater detail below. Methods of the invention
embrace the use of these different classes of CpG immunostimulatory
oligonucleotides.
[0215] Any of the classes may be subjugated to an E modification
which enhances its potency. An E modification may be a halogen
substitution for the 5' terminal nucleotide; examples of such
substitutions include but are not limited to bromo-uridine or
iodo-uridine substitutions. An E modification can also include an
ethyl-uridine substituation for the 5' terminal nucleotide.
[0216] The "A class" CpG immunostimulatory oligonucleotides are
characterized functionally by the ability to induce high levels of
interferon-alpha (IFN-.alpha.) from plasmacytoid dendritic cells
(pDC) and inducing NK cell activation while having minimal effects
on B cell activation. Structurally, this class typically has
stabilized poly-G sequences at 5' and 3' ends. It also has a
palindromic phosphodiester CpG dinucleotide-containing sequence of
at least 6 nucleotides, for example but not necessarily, it
contains one of the following hexamer palindromes: GACGTC, AGCGCT,
or AACGTT described by Yamamoto and colleagues. Yamamoto S et al.
J. Immunol 148:4072-6 (1992). A class CpG immunostimulatory
oligonucleotides and exemplary sequences of this class have been
described in U.S. Non-Provisional patent application Ser. No.
09/672,126 and published PCT application PCT/USOO/26527 (WO
01/22990), both filed on Sep. 27, 2000.
[0217] In an embodiment, the "A class" CpG oligonucleotide of the
invention has the following nucleic acid sequence: 5'
GGGGACGACGTCGTGGGGGGG 3'
[0218] Some non-limiting examples of A-Class oligonucleotides
include:
[0219] 5'G*G*G_G_A_C_G_A_C_G_T_C_G_T_G_G*G*G*G*G*G3'; wherein *
refers to a phosphorothioate bond and .sub.-- refers to a
phosphodiester bond.
[0220] The B class CpG oligonucleotide sequences of the invention
are those broadly described above as well as disclosed in published
PCT Patent Applications PCT/US95/01570 and PCT/US97/19791, and in
U.S. Pat. Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371,
6,239,116 and 6,339,068. Exemplary sequences include but are not
limited to those disclosed in these latter applications and
patents.
[0221] In an embodiment, the "B class" CpG oligonucleotide of the
invention has the following nucleic acid sequence:
TABLE-US-00001 (SEQ ID No 589) 5' TCGTCGTTTTTCGGTGCTTTT 3', or (SEQ
ID No 590) 5' TCGTCGTTTTTCGGTCGTTTT 3' or (SEQ ID No 591) 5'
TCGTCGTTTTGTCGTTTTGTCGTT 3' or (SEQ ID No 592) 5'
TCGTCGTTTCGTCGTTTTGTCGTT 3', or (SEQ ID No 593) 5'
TCGTCGTTTTGTCGTTTTTTTCGA 3'.
[0222] In any of these sequences, all of the linkages may be all
phosphorothioate bonds. In another embodiment, in any of these
sequences, one or more of the linkages may be phosphodiester,
preferably between the "C" and the "G" of the CpG motif making a
semi-soft CpG oligonucleotide. In any of these sequences, an
ethyl-uridine or a halogen may substitute for the 5'T; examples of
halogen substitutions include but are not limited to bromo-uridine
or iodo-uridine substitutions.
[0223] Some non-limiting examples of B-Class oligonucleotides
include:
TABLE-US-00002 5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*G*C*T*T*T*T 3', or
5' T*C*G*T*C*G*T*T*T*T*T*C*G*G*T*C*G*T*T*T*T 3' or 5'
T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T* T 3', or 5'
T*C*G*T*C*G*T*T*T*C*G*T*C*G*T*T*T*T*G*T*C*G*T* T 3', or 5'
T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*C*G* A 3'.
wherein * refers to a phosphorothioate bond.
[0224] The "C class" of CpG immunostimulatory oligonucleotides is
characterized functionally by the ability to activate B cells and
NK cells and induce IFN-.alpha.. Structurally, this class typically
includes a region with one or more B class-type immunostimulatory
CpG motifs, and a GC-rich palindrome or near-palindrome region that
allows the molecules to form secondary (e.g., stem-loop) or
tertiary (e.g., dimer) type structures. Some of these
oligonucleotides have both a traditional "stimulatory" CpG sequence
and a "GC-rich" or "B-cell neutralizing" motif. These combination
motif oligonucleotides have immune stimulating effects that fall
somewhere between the effects associated with traditional B class
CpG oligonucleotides (i.e., strong induction of B cell activation
and dendritic cell (DC) activation), and the effects associated
with A class CpG ODN (i.e., strong induction of IFN-.alpha. and NK
cell activation but relatively poor induction of B cell and DC
activation). Krieg A M et al. (1995) Nature 374:546-9; Ballas Z K
et al. (1996) J Immunol 157:1840-5; Yamamoto S et al. (1992) J
Immunol 148:4072-6.
[0225] The C class of combination motif immune stimulatory
oligonucleotides may have either completely stabilized, (e.g., all
phosphorothioate), chimeric (phosphodiester central region), or
semi-soft (e.g., phosphodiester within CpG motif) backbones. This
class has been described in U.S. patent application Ser. No.
10/224,523 filed on Aug. 19, 2002.
[0226] One stimulatory domain or motif of the C class CpG
oligonucleotide is defined by the formula: 5'X.sub.1DCGHX.sub.23'.
D is a nucleotide other than C. C is cytosine. G is guanine. H is a
nucleotide other than G. X.sub.1 and X.sub.2 are any nucleic acid
sequence 0 to 10 nucleotides long. X.sub.1 may include a CG, in
which case there is preferably a T immediately preceding this CG.
In some embodiments, DCG is TCG. X.sub.1 is preferably from 0 to 6
nucleotides in length. In some embodiments, X.sub.2 does not
contain any poly G or poly A motifs. In other embodiments, the
immunostimulatory oligonucleotide has a poly-T sequence at the 5'
end or at the 3' end. As used herein, "poly-A" or "poly-T" shall
refer to a stretch of four or more consecutive A's or T's
respectively, e.g., 5' AAAA 3' or 5' TTTT 3'. As used herein,
"poly-G end" shall refer to a stretch of four or more consecutive
G's, e.g., 5' GGGG 3', occurring at the 5' end or the 3' end of a
nucleic acid. As used herein, "poly-G oligonucleotide" shall refer
to an oligonucleotide having the formula
5'X.sub.1X.sub.2GGGX.sub.3X.sub.4 3' wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides and preferably at least one of
X.sub.3 and X.sub.4 is a G. Some preferred designs for the B cell
stimulatory domain under this formula comprise TTTTTCG, TCG, TTCG,
TTTCG, TTTTCG, TCGT, TTCGT, TTTCGT, TCGTCGT.
[0227] The second motif of the C class CpG oligonucleotide is
referred to as either P or N and is positioned immediately 5' to
X.sub.1 or immediately 3' to X.sub.2.
[0228] N is a B cell neutralizing sequence that begins with a CGG
trinucleotide and is at least 10 nucleotides long. A B cell
neutralizing motif includes at least one CpG sequence in which the
CG is preceded by a C or followed by a G (Krieg A M et al. (1998)
Proc Natl Acad Sd USA 95:12631-12636) or is a CG containing DNA
sequence in which the C of the CG is methylated. Neutralizing
motifs or sequences have some degree of immunostimulatory
capability when present in an otherwise non-stimulatory motif, but
when present in the context of other immunostimulatory motifs serve
to reduce the immunostimulatory potential of the other motifs.
[0229] P is a GC-rich palindrome containing sequence at least 10
nucleotides long.
[0230] As used herein, "palindrome" and equivalently "palindromic
sequence" shall refer to an inverted repeat, i.e., a sequence such
as ABCDEE'D'C'B'A' in which A and A', B and B', etc., are bases
capable of forming the usual Watson-Crick base pairs.
[0231] As used herein, "GC-rich palindrome" shall refer to a
palindrome having a base composition of at least two-thirds G's and
Cs. In some embodiments the GC-rich domain is preferably 3' to the
"B cell stimulatory domain". In the case of a 10-base long GC-rich
palindrome, the palindrome thus contains at least 8 G's and Cs. In
the case of a 12-base long GC-rich palindrome, the palindrome also
contains at least 8 G's and Cs. In the case of a 14-mer GC-rich
palindrome, at least ten bases of the palindrome are G's and Cs. In
some embodiments the GC-rich palindrome is made up exclusively of
G's and Cs.
[0232] In some embodiments the GC-rich palindrome has a base
composition of at least 81% G's and Cs. In the case of such a
10-base long GC-rich palindrome, the palindrome thus is made
exclusively of G's and Cs. In the case of such a 12-base long
GC-rich palindrome, it is preferred that at least ten bases (83%)
of the palindrome are G's and Cs. In some preferred embodiments, a
12-base long GC-rich palindrome is made exclusively of G's and Cs.
In the case of a 14-mer GC-rich palindrome, at least twelve bases
(86%) of the palindrome are G's and Cs. In some preferred
embodiments, a 14-base long GC-rich palindrome is made exclusively
of G's and Cs. The Cs of a GC-rich palindrome can be unmethylated
or they can be methylated.
[0233] In general this domain has at least 3 Cs and Gs, more
preferably 4 of each, and most preferably 5 or more of each. The
number of Cs and Gs in this domain need not be identical. It is
preferred that the Cs and Gs are arranged so that they are able to
form a self-complementary duplex, or palindrome, such as CCGCGCGG.
This may be interrupted by As or Ts, but it is preferred that the
self-complementarity is at least partially preserved as for example
in the motifs CGACGTTCGTCG or CGGCGCCGTGCCG. When complementarity
is not preserved, it is preferred that the non-complementary base
pairs be TG. In a preferred embodiment there are no more than 3
consecutive bases that are not part of the palindrome, preferably
no more than 2, and most preferably only 1. In some embodiments,
the GC-rich palindrome includes at least one CGG trimer, at least
one CCG trimer, or at least one CGCG tetramer. In other
embodiments, the GC-rich palindrome is not CCCCCCGGGGGG or
GGGGGGCCCCCC, CCCCCGGGGG or GGGGGCCCCC.
[0234] At least one of the G's of the GC rich region may be
substituted with an inosine (I). In some embodiments, P includes
more than one I.
[0235] In certain embodiments, the immunostimulatory
oligonucleotide has one of the following formulas 5'
NX.sub.1DCGHX.sub.2 3', 5' X.sub.1DCGHX.sub.2N 3', 5'
PX.sub.1DCGHX.sub.2 3', 5' X.sub.1DCGHX.sub.2P 3', 5'
X.sub.1DCGHX.sub.2PX.sub.3 3', 5' X.sub.1DCGHPX.sub.3 3', 5'
DCGHX.sub.2PX.sub.3 3', 5' TCGHX.sub.2PX.sub.3 3', 5' DCGHPX.sub.3
3' or 5'DCGHP 3'.
[0236] The invention provides other immune stimulatory
oligonucleotides defined by a formula 5' N.sub.1PyGN.sub.2P 3'.
N.sub.1 is any sequence 1 to 6 nucleotides long. Py is a
pyrimidine. G is guanine. N.sub.2 is any sequence 0 to 30
nucleotides long. P is a GC-rich palindrome containing a sequence
at least 10 nucleotides long.
[0237] N.sub.1 and N.sub.2 may contain more than 50% pyrimidines,
and more preferably more than 50% T. N.sub.1 may include a CG, in
which case there is preferably a T immediately preceding this CG.
hi some embodiments, N1PyG is TCG, and most preferably a
TCGN.sub.2, where N.sub.2 is not G.
[0238] N.sub.1PyGN.sub.2P may include one or more inosine (I)
nucleotides. Either the C or the G in N.sub.1 may be replaced by
inosine, but the CpI is preferred to the IpG. For inosine
substitutions such as IpG, the optimal activity may be achieved
with the use of a "semi-soft" or chimeric backbone, where the
linkage between the IG or the CI is phosphodiester. N1 may include
at least one CI, TCI, IG or TIG motif.
[0239] In certain embodiments N.sub.1PyGN.sub.2 is a sequence
selected from the group consisting of TTTTTCG, TCG, TTCG, TTTCG,
TTTTCG, TCGT, TTCGT, TTTCGT, and TCGTCGT.
[0240] In an embodiment, the "C class" CpG oligonucleotide of the
invention has the following nucleic acid sequence:
TABLE-US-00003 (SEQ ID No 594) 5' TCGCGTCGTTCGGCGCGCGCCG 3', or
(SEQ ID No 595) 5' TCGTCGACGTTCGGCGCGCGCCG 3', or (SEQ ID No 596)
5' TCGGACGTTCGGCGCGCGCCG 3', or (SEQ ID No 597) 5'
TCGGACGTTCGGCGCGCCG 3', or (SEQ ID No 598) 5' TCGCGTCGTTCGGCGCGCCG
3', or (SEQ ID No 599) 5' TCGACGTTCGGCGCGCGCCG 3', or (SEQ ID No
600) 5' TCGACGTTCGGCGCGCCG 3', or (SEQ ID No 601) 5'
TCGCGTCGTTCGGCGCCG 3', or (SEQ ID No 602) 5' TCGCGACGTTCGGCGCGCGCCG
3', or (SEQ ID No 603) 5' TCGTCGTTTTCGGCGCGCGCCG 3', or (SEQ ID No
604) 5' TCGTCGTTTTCGGCGGCCGCCG 3', or (SEQ ID No 605) 5'
TCGTCGTTTTACGGCGCCGTGCCG 3', or (SEQ ID No 606) 5'
TCGTCGTTTTCGGCGCGCGCCGT 3'.
[0241] In any of these sequences, all of the linkages may be all
phosphorothioate bonds. In another embodiment, in any of these
sequences, one or more of the linkages may be phosphodiester,
preferably between the "C" and the "G" of the CpG motif making a
semi-soft CpG oligonucleotide.
[0242] Some non-limiting examples of C-Class oligonucleotides
include:
TABLE-US-00004 5' T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3',
or 5' T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or 5'
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or 5'
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3', or 5'
T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3', or 5'
T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or 5'
T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3', or 5'
T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 3', or 5'
T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3', or 5'
T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G 3', or 5'
T*C*G*T*C*G*T*T*T*T*C*G*G*C*G*G*C*C*G*C*C*G 3', or 5'
T*C*G*T*C_G*T*T*T*T*A*C_G*G*C*G*C*C_G*T*G*C*C* G 3', or 5'
T*C_G*T*C*G*T*T*T*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3'
wherein * refers to a phosphorothioate bond and .sub.-- refers to a
phosphodiester bond.
[0243] In any of these sequences, an ethyl-uridine or a halogen may
substitute for the 5' T; examples of halogen substitutions include
but are not limited to bromo-uridine or iodo-uridine
substitutions.
[0244] The "P class" CpG immunostimulatory oligonucleotides have
been described in WO2007/095316 and are characterized by the fact
that they contain duplex forming regions such as, for example,
perfect or imperfect palindromes at or near both the 5' and 3'
ends, giving them the potential to form higher ordered structures
such as concatamers. These oligonucleotides referred to as P-Class
oligonucleotides have the ability in some instances to induce much
high levels of IFN-a secretion than the C-Class. The P-Class
oligonucleotides have the ability to spontaneously self-assemble
into concatamers either in vitro and/or in vivo. Without being
bound by any particular theory for the method of action of these
molecules, one potential hypothesis is that this property endows
the P-Class oligonucleotides with the ability to more highly
crosslink TLR9 inside certain immune cells, inducing a distinct
pattern of immune activation compared to the previously described
classes of CpG oligonucleotides.
[0245] In an embodiment, the CpG oligonucleotide for use in the
present invention is a P class CpG oligonucleotide containing a 5'
TLR activation domain and at least two palindromic regions, one
palindromic region being a 5' palindromic region of at least 6
nucleotides in length and connected to a 3' palindromic region of
at least 8 nucleotides in length either directly or through a
spacer, wherein the oligonucleotide includes at least one YpR
dinucleotide. In an embodiment, said oligoonucleotide is not
T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G. In one embodiment
the P class CpG oligonucleotide includes at least one unmethylated
CpG dinucleotide. In another embodiment the TLR activation domain
is TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or
TTTT. In yet another embodiment the TLR activation domain is within
the 5' palindromic region. In another embodiment the TLR activation
domain is immediately 5' to the 5' palindromic region. In still
another embodiment the 5' palindromic region is at least 8
nucleotides in length. In another embodiment the 3' palindromic
region is at least 10 nucleotides in length. In another embodiment
the 5' palindromic region is at least 10 nucleotides in length. In
yet another embodiment the 3' palindromic region includes an
unmethylated CpG dinucleotide. In another embodiment the 3'
palindromic region includes two unmethylated CpG dinucleotides. In
another embodiment the 5' palindromic region includes an
unmethylated CpG dinucleotide. In yet another embodiment the 5'
palindromic region includes two unmethylated CpG dinucleotides. In
another embodiment the 5' and 3' palindromic regions have a duplex
stability value of at least 25. In another embodiment the 5' and 3'
palindromic regions have a duplex stability value of at least 30.
In another embodiment the 5' and 3' palindromic regions have a
duplex stability value of at least 35. In another embodiment the 5'
and 3' palindromic regions have a duplex stability value of at
least 40. In another embodiment the 5' and 3' palindromic regions
have a duplex stability value of at least 45. In another embodiment
the 5' and 3' palindromic regions have a duplex stability value of
at least 50. In another embodiment the 5' and 3' palindromic
regions have a duplex stability value of at least 55. In another
embodiment the 5' and 3' palindromic regions have a duplex
stability value of at least 60. In another embodiment the 5' and 3'
palindromic regions have a duplex stability value of at least
65.
[0246] In one embodiment the two palindromic regions are connected
directly. In another embodiment the two palindromic regions are
connected via a 3'-3' linkage. In another embodiment the two
palindromic regions overlap by one nucleotide. In yet another
embodiment the two palindromic regions overlap by two nucleotides.
In another embodiment the two palindromic regions do not overlap.
In another embodiment the two palindromic regions are connected by
a spacer. In one embodiment the spacer is a nucleic acid having a
length of 1-50 nucleotides. In another embodiment the spacer is a
nucleic acid having a length of 1 nucleotide. In another embodiment
the spacer is a non-nucleotide spacer. In one embodiment the
non-nucleotide spacer is a D-spacer. In another embodiment the
non-nucleotide spacer is a linker. In one embodiment the
oligonucleotide has the formula 5' XP.sub.1SP.sub.2T 3', wherein X
is the TLR activation domain, P.sub.1 is a palindrome, S is a
spacer, P.sub.2 is a palindrome, and T is a 3' tail of 0-100
nucleotides in length. In one embodiment X is TCG, TTCG, or TTTCG.
In another embodiment T is 5-50 nucleotides in length. In yet
another embodiment T is 5-10 nucleotides in length. In one
embodiment S is a nucleic acid having a length of 1-50 nucleotides.
In another embodiment S is a nucleic acid having a length of 1
nucleotide. In another embodiment S is a non-nucleotide spacer. In
one embodiment the non-nucleotide spacer is a D-spacer. In another
embodiment the non-nucleotide spacer is a linker. In another
embodiment the oligonucleotide is not an antisense oligonucleotide
or a ribozyme. In one embodiment P.sub.1 is A and T rich. In
another embodiment P.sub.1 includes at least 4 Ts. In another
embodiment P.sub.2 is a perfect palindrome. In another embodiment
P2 is G-C rich. In still another embodiment P.sub.2 is
CGGCGCX.sub.1GCGCCG, where X.sub.1 is T or nothing.
[0247] In one embodiment the oligonucleotide includes at least one
phosphorothioate linkage.
[0248] In another embodiment all internucleotide linkages of the
oligonucleotide are phosphorothioate linkages. In another
embodiment the oligonucleotide includes at least one
phosphodiester-like linkage. In another embodiment the
phosphodiester-like linkage is a phosphodiester linkage. In another
embodiment a lipophilic group is conjugated to the oligonucleotide.
In one embodiment the lipophilic group is cholesterol.
[0249] In an embodiment, the TLR-9 agonist for use in the present
invention is a P class CpG oligonucleotide with a 5' TLR activation
domain and at least two complementarity-containing regions, a 5'
and a 3' complementarity-containing region, each
complementarity-containing region being at least 8 nucleotides in
length and connected to one another either directly or through a
spacer, wherein the oligonucleotide includes at least one
pyrimidine-purine (YpR) dinucleotide, and wherein at least one of
the complementarity-containing regions is not a perfect palindrome.
In one embodiment the oligonucleotide includes at least one
unmethylated CpG dinucleotide. In another embodiment the TLR
activation domain is TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG,
UUCG, UUUCG, TTT, or TTTT. In another embodiment the TLR activation
domain is within the 5' complementarity-containing region. In
another embodiment the TLR activation domain is immediately 5' to
the 5' complementarity-containing region. In another embodiment the
3' complementarity-containing region is at least 10 nucleotides in
length. In yet another embodiment the 5' complementarity-containing
region is at least 10 nucleotides in length. In one embodiment the
3' complementarity-containing region includes an unmethylated CpG
dinucleotide. In another embodiment the 3'
complementarity-containing region includes two unmethylated CpG
dinucleotides. In yet another embodiment the 5'
complementarity-containing region includes an unmethylated CpG
dinucleotide. In another embodiment the 5'
complementarity-containing region includes two unmethylated CpG
dinucleotides. In another embodiment the complementarity-containing
regions include at least one nucleotide analog. In another
embodiment the complementarity-containing regions form an
intramolecular duplex. In one embodiment the intramolecular duplex
includes at least one non-Watson Crick base pair. In another
embodiment the non-Watson Crick base pair is G-T, G-A, G-G, or C-A.
In one embodiment the complementarity-containing regions form
intermolecular duplexes. In another embodiment at least one of the
intermolecular duplexes includes at least one non-Watson Crick base
pair. In another embodiment the non-Watson Crick base pair is G-T,
G-A, G-G, or C-A. In yet another embodiment the
complementarity-containing regions contain a mismatch. In still
another embodiment the complementarity-containing regions contain
two mismatches. In another embodiment the
complementarity-containing regions contain an intervening
nucleotide. In another embodiment the complementarity-containing
regions contain two intervening nucleotides.
[0250] In one embodiment the 5' and 3' complementarity-containing
regions have a duplex stability value of at least 25. In another
embodiment the 5' and 3' complementarity-containing regions have a
duplex stability value of at least 30. In another embodiment the 5'
and 3' complementarity-containing regions have a duplex stability
value of at least 35. In another embodiment the
complementarity-containing regions have a duplex stability value of
at least 40. In another embodiment the complementarity-containing
regions have a duplex stability value of at least 45. In another
embodiment the complementarity-containing regions have a duplex
stability value of at least 50. In another embodiment the
complementarity-containing regions have a duplex stability value of
at least 55. In another embodiment the complementarity-containing
regions have a duplex stability value of at least 60. In another
embodiment the complementarity-containing regions have a duplex
stability value of at least 65.
[0251] In another embodiment the two complementarity-containing
regions are connected directly. In another embodiment the two
palindromic regions are connected via a 3'-3' linkage. In yet
another embodiment the two complementarity-containing regions
overlap by one nucleotide. In another embodiment the two
complementarity-containing regions overlap by two nucleotides. In
another embodiment the two complementarity-containing regions do
not overlap. In another embodiment the two
complementarity-containing regions are connected by a spacer. In
another embodiment the spacer is a nucleic acid having a length of
1 -50 nucleotides. In another embodiment the spacer is a nucleic
acid having a length of 1 nucleotide. In one embodiment the spacer
is a non-nucleotide spacer. In another embodiment the
non-nucleotide spacer is a D-spacer. In yet another embodiment the
non-nucleotide spacer is a linker.
[0252] In one embodiment the P-class oligonucleotide has the
formula 5' XNSPT 3', wherein X is the TLR activation domain, N is a
non-perfect palindrome, P is a palindrome, S is a spacer, and T is
a 3' tail of 0-100 nucleotides in length. In another embodiment X
is TCG, TTCG, or TTTCG. In another embodiment T is 5-50 nucleotides
in length. In another embodiment T is 5-10 nucleotides in length.
In another embodiment S is a nucleic acid having a length of 1-50
nucleotides. In another embodiment S is a nucleic acid having a
length of 1 nucleotide. In another embodiment S is a non-nucleotide
spacer. In another embodiment the non-nucleotide spacer is a
D-spacer. In another embodiment the non-nucleotide spacer is a
linker. In another embodiment the oligonucleotide is not an
antisense oligonucleotide or a ribozyme. In another embodiment N is
A and T rich. In another embodiment N is includes at least 4 Ts. In
another embodiment P is a perfect palindrome. In another embodiment
P is G-C rich. In another embodiment P is CGGCGCX.sub.1GCGCCG,
wherein X.sub.1 is T or nothing. In another embodiment the
oligonucleotide includes at least one phosphorothioate linkage. In
another embodiment all interaucleotide linkages of the
oligonucleotide are phosphorothioate linkages. In another
embodiment the oligonucleotide includes at least one
phosphodiester-like linkage. In another embodiment the
phosphodiester-like linkage is a phosphodiester linkage. In another
embodiment a lipophilic group is conjugated to the oligonucleotide.
In one embodiment the lipophilic group is cholesterol.
[0253] In an embodiment, the "P class" CpG oligonucleotides of the
invention has the following nucleic acid sequence: 5'
TCGTCGACGATCGGCGCGCGCCG 3' (SEQ ID No 607).
[0254] In said sequences, all of the linkages may be all
phosphorothioate bonds. In another embodiment, one or more of the
linkages may be phosphodiester, preferably between the "C" and the
"G" of the CpG motif making a semi-soft CpG oligonucleotide. In any
of these sequences, an ethyl-uridine or a halogen may substitute
for the 5' T; examples of halogen substitutions include but are not
limited to bromo-uridine or iodo-uridine substitutions.
[0255] A non-limiting example of P-Class oligonucleotides
include:
TABLE-US-00005 5' T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G
3'
wherein * refers to a phosphorothioate bond and _ refers to a
phosphodiester bond.
[0256] In an embodiment, all the internucleotide linkage of the CpG
oligonucleotides disclosed herein are phosphodiester bonds ("soft"
oligonucleotides, as described in the PCT application
WO2007/026190). In another embodiment, CpG oligonucleotides of the
invention are rendered resistant to degradation (e.g., are
stabilized). A "stabilized oligonucleotide" refers to an
oligonucleotide that is relatively resistant to in vivo degradation
(e.g. via an exo- or endo-nuclease). Nucleic acid stabilization can
be accomplished via backbone modifications. Oligonucleotides having
phosphorothioate linkages provide maximal activity and protect the
oligonucleotide from degradation by intracellular exo- and
endo-nucleases.
[0257] The immunostimulatory oligonucleotides may have a chimeric
backbone, which have combinations of phosphodiester and
phosphorothioate linkages. For purposes of the instant invention, a
chimeric backbone refers to a partially stabilized backbone,
wherein at least one internucleotide linkage is phosphodiester or
phosphodiester-like, and wherein at least one other internucleotide
linkage is a stabilized internucleotide linkage, wherein the at
least one phosphodiester or phosphodiester-like linkage and the at
least one stabilized linkage are different. When the phosphodiester
linkage is preferentially located within the CpG motif such
molecules are called "semi-soft" as described in the PCT
application WO2007/026190.
[0258] Other modified oligonucleotides include combinations of
phosphodiester, phosphorothioate, methylphosphonate,
methylphosphorothioate, phosphorodithioate, and/or p-ethoxy
linkages.
[0259] Since boranophosphonate linkages have been reported to be
stabilized relative to phosphodiester linkages, for purposes of the
chimeric nature of the backbone, boranophosphonate linkages can be
classified either as phosphodiester-like or as stabilized,
depending on the context. For example, a chimeric backbone
according to the instant invention could, in some embodiments,
includes at least one phosphodiester (phosphodiester or
phosphodiester-like) linkage and at least one boranophosphonate
(stabilized) linkage. In other embodiments, a chimeric backbone
according to the instant invention could include boranophosphonate
(phosphodiester or phosphodiester-like) and phosphorothioate
(stabilized) linkages. A "stabilized internucleotide linkage" shall
mean an internucleotide linkage that is relatively resistant to in
vivo degradation (e.g., via an exo- or endo-nuclease), compared to
a phosphodiester internucleotide linkage. Preferred stabilized
internucleotide linkages include, without limitation,
phosphorothioate, phosphorodithioate, methylphosphonate, and
methylphosphorothioate. Other stabilized internucleotide linkages
include, without limitation, peptide, alkyl, dephospho, and others
as described above.
[0260] Modified backbones such as phosphorothioates may be
synthesized using automated techniques employing either
phosphoramidate or H-phosphonate chemistries. Aryl- and
alkyl-phosphonates can be made, e.g., as described in U.S. Pat. No.
4,469,863; and alkylphosphotriesters (in which the charged oxygen
moiety is alkylated as described in U.S. Pat. No. 5,023,243 and
European Patent No. 092,574) can be prepared by automated solid
phase synthesis using commercially available reagents. Methods for
making other DNA backbone modifications and substitutions have been
described. Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J
(1990) Bioconjugate Chem 1:165. Methods for preparing chimeric
oligonucleotides are also known. For instance patents issued to
Uhlmann et al have described such techniques.
[0261] Mixed backbone modified ODN may be synthesized as described
in the PCT application WO2007/026190.
[0262] The oligonucleotides of the invention can also include other
modifications. These include nonionic DNA analogs, such as alkyl-
and aryl-phosphates (in which the charged phosphonate oxygen is
replaced by an alkyl or aryl group), phosphodiester and
alkylphosphotriesters, in which the charged oxygen moiety is
alkylated. Nucleic acids which contain diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
[0263] The size of the CpG oligonucleotide (i.e., the number of
nucleotide residues along the length of the oligonucleotide) also
may contribute to the stimulatory activity of the oligonucleotide.
For facilitating uptake into cells, CpG oligonucleotide of the
invention preferably have a minimum length of 6 nucleotide
residues. Oligonucleotides of any size greater than 6 nucleotides
(even many kb long) are capable of inducing an immune response if
sufficient immunostimulatory motifs are present, because larger
oligonucleotides are degraded inside cells. In certain embodiments,
the CpG oligonucleotides are 6 to 100 nucleotides long,
preferentially 8 to 30 nucleotides long. In important embodiments,
nucleic acids and oligonucleotides of the invention are not
plasmids or expression vectors.
[0264] In an embodiment, the CpG oligonucleotide disclosed herein
comprise substitutions or modifications, such as in the bases
and/or sugars as described at paragraph 134 to 147 of
WO2007/026190.
[0265] In an embodiment, the CpG oligonucleotide of the present
invention is chemically modified. Examples of chemical
modifications are known to the skilled person and are described,
for example in Uhlmann E. et al. (1990), Chem. Rev. 90:543, S.
Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke, S. T. et al.
(1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; and Hunziker J.
et al., (1995), Mod. Synth. Methods 7:331-417. An oligonucleotide
according to the invention may have one or more modifications,
wherein each modification is located at a particular phosphodiester
internucleoside bridge and/or at a particular .beta.-D-ribose unit
and/or at a particular natural nucleoside base position in
comparison to an oligonucleotide of the same sequence which is
composed of natural DNA or RNA.
[0266] In some embodiments of the invention, CpG-containing nucleic
acids might be simply mixed with immunogenic carriers according to
methods known to those skilled in the art (see, e.g.
WO03/024480).
[0267] In a particular embodiment of the present invention, any of
the vaccine disclosed herein comprises from 20 .mu.g to 20 .mu.g of
CpG oligonucleotide, preferably from 0.1 mg to 10 mg CpG
oligonucleotide, preferably from 0.2 mg to 5 mg CpG
oligonucleotide, preferably from 0.3 mg to 3 mg CpG
oligonucleotide, even preferably from 0.4 to 2 mg CpG
oligonucleotide, even preferably from 0.5 to 1.5 mg CpG
oligonucleotide. In a preferred embodiement, any of the vaccine
disclosed herein comprises approximately 0.5 to 1 mg CpG
oligonucleotide.
[0268] Preferred adjuvants for use in the present invention are
alum, QS21, CpG ODN, alum in combination with CpG ODN, Iscomatrix
and Iscomatrix in combination with CpG ODN.
Pharmaceutical Compositions of the Invention
[0269] The invention also provides pharmaceutical compositions
comprising an antigenic PCSK9 peptide of the invention or an
immunogenic composition thereof, in a formulation in association
with one or more pharmaceutically acceptable excipient(s) and
optionally combined with one or more adjuvants (as adjuvant
described above). The term `excipient` is used herein to describe
any ingredient other than the active ingredient, i.e. the antigenic
PCSK9 peptide of the invention eventually coupled to an immunogenic
carrier and optionally combined with one or more adjuvants. The
choice of excipient(s) will to a large extent depend on factors
such as the particular mode of administration, the effect of the
excipient on solubility and stability, and the nature of the dosage
form. As used herein, "pharmaceutically acceptable excipient"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Some examples of pharmaceutically acceptable excipients are water,
saline, phosphate buffered saline, dextrose, glycerol, ethanol and
the like, as well as combinations thereof. In many cases, it will
be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Additional examples of pharmaceutically acceptable
substances are wetting agents or minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
active ingredient.
[0270] Pharmaceutical compositions of the present invention and
methods for their preparation will be readily apparent to those
skilled in the art. Such compositions and methods for their
preparation may be found, for example, in Remington's
Pharmaceutical Sciences, 19th Edition (Mack Publishing Company,
1995). Pharmaceutical compositions are preferably manufactured
under GMP conditions.
[0271] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0272] Any method for administering peptides, or proteins accepted
in the art may suitably be employed for the peptides or proteins of
the invention.
[0273] The pharmaceutical compositions of the invention are
typically suitable for parenteral administration. As used herein,
"parenteral administration" of a pharmaceutical composition
includes any route of administration characterized by physical
breaching of a tissue of a subject and administration of the
pharmaceutical composition through the breach in the tissue, thus
generally resulting in the direct administration into the blood
stream, into muscle, or into an internal organ. Parenteral
administration thus includes, but is not limited to, administration
of a pharmaceutical composition by injection of the composition, by
application of the composition through a surgical incision, by
application of the composition through a tissue-penetrating
non-surgical wound, and the like. In particular, parenteral
administration is contemplated to include, but is not limited to,
subcutaneous, intraperitoneal, intramuscular, intrasternal,
intravenous, intraarterial, intrathecal, intraventricular,
intraurethral, intracranial, intrasynovial injection or infusions;
and kidney dialytic infusion techniques. Preferred embodiments
include the intravenous, subcutaneous, intradermal and
intramuscular routes, even more preferred embodiments are the
intramuscular or the subcutaneous routes.
[0274] Formulations of a pharmaceutical composition suitable for
parenteral administration typically generally comprise the active
ingredient combined with a pharmaceutically acceptable carrier,
such as sterile water or sterile isotonic saline. Such formulations
may be prepared, packaged, or sold in a form suitable for bolus
administration or for continuous administration. Injectable
formulations may be prepared, packaged, or sold in unit dosage
form, such as in ampoules or in multi-dose containers containing a
preservative. Formulations for parenteral administration include,
but are not limited to, suspensions, solutions, emulsions in oily
or aqueous vehicles, pastes, and the like. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, suspending, stabilizing, or dispersing agents. In
one embodiment of a formulation for parenteral administration, the
active ingredient is provided in dry (i.e. powder or granular) form
for reconstitution with a suitable vehicle (e.g. sterile
pyrogen-free water) prior to parenteral administration of the
reconstituted composition. Parenteral formulations also include
aqueous solutions which may contain excipients such as salts,
carbohydrates and buffering agents (preferably to a pH of from 3 to
9), but, for some applications, they may be more suitably
formulated as a sterile non-aqueous solution or as a dried form to
be used in conjunction with a suitable vehicle such as sterile,
pyrogen-free water. Exemplary parenteral administration forms
include solutions or suspensions in sterile aqueous solutions, for
example, aqueous propylene glycol or dextrose solutions. Such
dosage forms can be suitably buffered, if desired. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, microparticles, or in a liposomal preparation. Formulations
for parenteral administration may be formulated to be immediate
and/or modified release. Modified release formulations include
delayed-, sustained-, pulsed-, controlled-, targeted and programmed
release.
[0275] For example, in one aspect, sterile injectable solutions can
be prepared by incorporating the anti-PCSK9 peptide, preferably
coupled to an immunogenic carrier, optionally in combination with
one or more adjuvants, in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying that yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0276] An exemplary, non-limiting pharmaceutical composition of the
invention is a formulation as a sterile aqueous solution having a
pH that ranges from about 5.0 to about 6.5 and comprising from
about 0.1 mg/mL to about 20 mg/mL of a peptide of the invention,
from about 1 millimolar to about 100 millimolar of histidine
buffer, from about 0.01 mg/mL to about 10 mg/mL of polysorbate 80,
from about 100 millimolar to about 400 millimolar of trehalose, and
from about 0.01 millimolar to about 1.0 millimolar of disodium EDTA
dihydrate.
[0277] The antigenic PCSK9 peptides of the invention can also be
administered intranasally or by inhalation, typically in the form
of a dry powder (either alone, as a mixture, or as a mixed
component particle, for example, mixed with a suitable
pharmaceutically acceptable excipient) from a dry powder inhaler,
as an aerosol spray from a pressurised container, pump, spray,
atomiser (preferably an atomiser using electrohydrodynamics to
produce a fine mist), or nebuliser, with or without the use of a
suitable propellant, or as nasal drops.
[0278] The pressurised container, pump, spray, atomizer, or
nebuliser generally contains a solution or suspension of an
antibody of the invention comprising, for example, a suitable agent
for dispersing, solubilising, or extending release of the active, a
propellant(s) as solvent.
[0279] Prior to use in a dry powder or suspension formulation, the
drug product is generally micronised to a size suitable for
delivery by inhalation (typically less than 5 microns). This may be
achieved by any appropriate comminuting method, such as spiral jet
milling, fluid bed jet milling, supercritical fluid processing to
form nanoparticles, high pressure homogenisation, or spray
drying.
[0280] Capsules, blisters and cartridges for use in an inhaler or
insufflator may be formulated to contain a powder mix of the
compound of the invention, a suitable powder base and a performance
modifier.
[0281] A suitable solution formulation for use in an atomiser using
electrohydrodynamics to produce a fine mist may contain a suitable
dose of the antigenic PCSK9 peptide of the invention per actuation
and the actuation volume may for example vary from 1 .mu.L to 100
.mu.L.
[0282] Suitable flavours, such as menthol and levomenthol, or
sweeteners, such as saccharin or saccharin sodium, may be added to
those formulations of the invention intended for inhaled/intranasal
administration.
[0283] Formulations for inhaled/intranasal administration may be
formulated to be immediate and/or modified release. Modified
release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and programmed release.
[0284] In the case of dry powder inhalers and aerosols, the dosage
unit is determined by means of a valve which delivers a metered
amount. Units in accordance with the invention are typically
arranged to administer a metered dose or "puff" of an antibody of
the invention. The overall daily dose will typically be
administered in a single dose or, more usually, as divided doses
throughout the day.
[0285] A pharmaceutical composition comprising an antigenic PCSK9
peptide may also be formulated for an oral route administration.
Oral administration may involve swallowing, so that the compound
enters the gastrointestinal tract, and/or buccal, lingual, or
sublingual administration by which the compound enters the blood
stream directly from the mouth.
[0286] Formulations suitable for oral administration include solid,
semi-solid and liquid systems such as tablets; soft or hard
capsules containing multi- or nano-particulates, liquids, or
powders; lozenges (including liquid-filled); chews; gels; fast
dispersing dosage forms; films; ovules; sprays; and
buccal/mucoadhesive patches.
[0287] Liquid formulations include suspensions, solutions, syrups
and elixirs. Such formulations may be employed as fillers in soft
or hard capsules (made, for example, from gelatin or
hydroxypropylmethylcellulose) and typically comprise a carrier, for
example, water, ethanol, polyethylene glycol, propylene glycol,
methylcellulose, or a suitable oil, and one or more emulsifying
agents and/or suspending agents. Liquid formulations may also be
prepared by the reconstitution of a solid, for example, from a
sachet.
[0288] The compositions of the invention can be used to treat,
alleviate or prevent PCSK9-mediated disorders or symptoms in a
subject at risk or suffering from such disorder or symptom by
stimulating an immune response in said subject by immunotherapy.
Immunotherapy can comprise an initial immunization followed by
additional, e.g. one, two, three, or more boosters.
[0289] An "immunologically effective amount" of an antigenic PCSK9
peptide of the invention, or composition thereof, is an amount that
is delivered to a mammalian subject, either in a single dose or as
part of a series, which is effective for inducing an immune
response against PCSK9 in said subject. This amount varies
depending upon the health and physical condition of the individual
to be treated, the taxonomic group of individual to be treated, the
capacity of the individual's immune system to synthesize
antibodies, the formulation of the vaccine, and other relevant
factors. It is expected that the amount will fall in a relatively
broad range that can be determined through routine trials.
[0290] A "pharmaceutically effective dose" or "therapeutically
effective dose" is that dose required to treat or prevent, or
alleviate one or more PCSK9-related disorder or symptom in a
subject. The pharmaceutically effective dose depends on inter alia
the specific compound to administer, the severity of the symptoms,
the susceptibility of the subject to side effects, the type of
disease, the composition used, the route of administration, the
type of mammal being treated, the physical characteristics of the
specific mammal under consideration such as health and physical
condition, concurrent medication, the capacity of the individual's
immune system to synthesize antibodies, the degree of protection
desired, and other factors that those skilled in the medical arts
will recognize. For prophylaxis purposes, the amount of peptide in
each dose is selected as an amount which induces an
immunoprotective response without significant adverse side effects
in typical vaccinees. Following an initial vaccination, subjects
may receive one or several booster immunisations adequately
spaced.
[0291] It is understood that the specific dose level for any
particular patient depends upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0292] For example, antigenic PCSK9 peptides or pharmaceutical
composition of the invention can be administered to a subject at a
dose of about 0.1 .mu.g to about 5 mg, e.g., from about 0.1 .mu.g
to about 5 .mu.g, from about 5 .mu.g to about 10 .mu.g, from about
10 .mu.g to about 25 .mu.g, from about 25 .mu.g to about 50 .mu.g,
from about 50 .mu.g to about 100 .mu.g, from about 100 .mu.g to
about 500 .mu.g, from about 500 .mu.g to about 1 mg, from about 1
mg to about 2 mg, with optional boosters given at, for example, 1
week, 2 weeks, 3 weeks, 4 weeks, two months, three months, 6 months
and/or a year later.
[0293] In some embodiments, a single dose of an antigenic PCSK9
peptide or pharmaceutical composition according to the invention is
administered. In other embodiments, multiple doses of an antigenic
PCSK9 peptide or pharmaceutical composition according to the
invention are administered. The frequency of administration can
vary depending on any of a variety of factors, e.g., severity of
the symptoms, degree of immunoprotection desired, whether the
composition is used for prophylactic or curative purposes, etc. For
example, in some embodiments, an antigenic PCSK9 peptide or
pharmaceutical composition according to the invention is
administered once per month, twice per month, three times per
month, every other week (qow), once per week (qw), twice per week
(biw), three times per week (tiw), four times per week, five times
per week, six times per week, every other day (qod), daily (qd),
twice a day (qid), or three times a day (tid). When the composition
of the invention is used for prophylaxis purposes, they will be
generally administered for both priming and boosting doses. It is
expected that the boosting doses will be adequately spaced, or
preferably given yearly or at such times where the levels of
circulating antibody fall below a desired level. Boosting doses may
consist of the antigenic PCSK9 peptide in the absence of the
original immunogenic carrier molecule. Such booster constructs may
comprise an alternative immunogenic carrier or may be in the
absence of any carrier. Such booster compositions may be formulated
either with or without adjuvant.
[0294] The duration of administration of an antigenic PCSK9 peptide
according to the invention, e.g., the period of time over which an
antigenic PCSK9 peptide is administered, can vary, depending on any
of a variety of factors, e.g., patient response, etc. For example,
an antigenic PCSK9 peptide can be administered over a period of
time ranging from about one day to about one week, from about two
weeks to about four weeks, from about one month to about two
months, from about two months to about four months, from about four
months to about six months, from about six months to about eight
months, from about eight months to about 1 year, from about 1 year
to about 2 years, or from about 2 years to about 4 years, or
more.
[0295] A variety of treatment methods are also contemplated by the
present disclosure, which methods comprise administering an
antigenic PCSK9 peptide according to the invention. Subject
treatment methods include methods of inducing an immune response in
an individual to self-PCSK9, and methods of preventing, alleviating
or treating a PCSK9-related disorder or symptom in an
individual.
[0296] In one aspect, the present invention provides a method for
treating, preventing or alleviating a PCSK9-related disorder or
symptom in a subject, comprising administering a therapeutically
effective amount of an antigenic PCSK9 peptide of the invention, or
immunogenic or pharmaceutical composition thereof, to said
subject.
[0297] In another aspect, the present invention provides a method
for inducing an immune response against self-PCSK9 in a subject,
comprising administering a therapeutically or immunogenically
effective amount of an antigenic PCSK9 peptide of the invention, or
immunogenic or pharmaceutical composition thereof, to said
subject.
[0298] A PCSK9 related disease or a PCSK9 mediated disease is, for
example, a disease where the inhibition of PCSK9 activity or the
inhibition of the interaction of PCSK9 with the LDL receptor could
be beneficial.
[0299] "Treat", "treating" and "treatment" refer to a method of
alleviating or abrogating a biological disorder and/or at least one
of its attendant symptoms. As used herein, to "alleviate" a
disease, disorder or condition means reducing the severity and/or
occurrence frequency of the symptoms of the disease, disorder, or
condition. Further, references herein to "treatment" include
references to curative, palliative and prophylactic treatment. Said
subject is preferably human, and may be either male or female, of
any age.
[0300] Other aspects of the invention relate to an antigenic PCSK9
peptide according to the invention or of an immunogenic composition
or a pharmaceutical composition thereof, for use as a medicament,
preferably in treatment, alleviation or prophylaxis of
PCSK9-related disorders.
[0301] In yet another aspect, the present invention provides the
use of an antigenic PCSK9 peptide of the invention or of an
immunogenic composition or a pharmaceutical composition thereof, in
the manufacture of a medicament, preferably for treating a
PCSK9-related disorder.
[0302] In particular, the invention relates to an antigenic PCSK9
peptide of the invention, or an immunogenic or pharmaceutical
composition thereof, for use as a medicament preferably in
treatment, alleviation or prophylaxis of diseases associated with
an elevated level of cholesterol.
[0303] In yet another aspect, the present invention provides the
use of an antigenic PCSK9 peptide of the invention or of an
immunogenic composition or a pharmaceutical composition thereof, in
the manufacture of a medicament, preferably for lowering the
LDL-cholesterol level in blood in a subject in need thereof.
[0304] In some aspects of the uses or methods of the invention,
said PCSK9-related disorder is selected from the group consisting
of elevated cholesterol, a condition associated with elevated
LDL-cholesterol, e.g., a lipid disorder (e.g., hyperlipidemia, type
I, type II, type III, type IV, or type V hyperlipidemia, secondary
hypertriglyceridemia, hypercholesterolemia, familial
hypercholesterolemia, xanthomatosis, cholesterol acetyltransferase
deficiency), arteriosclerotic conditions (e.g., atherosclerosis),
coronary artery disease, and cardiovascular disease.
[0305] In yet another aspect, the present invention provides the
use of an antigenic PCSK9 peptide of the invention or of an
immunogenic composition or a pharmaceutical composition thereof, in
the manufacture of a medicament for treating or alleviating
diseases where an up-regulation of the LDL receptor or an
inhibition of the interaction between PCSK9 and the LDL receptor is
beneficial.
[0306] In yet another aspect, the present invention provides the
use of an antigenic PCSK9 peptide of the invention or of an
immunogenic composition or a pharmaceutical composition thereof, in
the manufacture of a medicament for the treatment of Alzheimer's
disease.
[0307] In other aspects of the uses or methods of the invention,
said subject is a mammal, preferably a human subject.
[0308] In still other aspects of the uses or methods of the
invention, said subject suffers from said PSCK9-related disorder.
Alternatively, said subject is at risk of suffering from said
PCSK9-related disorder, e.g., due to the presence of one or more
risk factors (e.g., hypertension, cigarette smoking, diabetes,
obesity, or hyperhomocysteinemia).
[0309] The antigenic PCSK9 peptide of the invention or an
immunogenic composition or a pharmaceutical composition thereof are
useful for subjects who are intolerant to therapy with another
cholesterol-reducing agent, or for whom therapy with another
cholesterol-reducing agent has produced inadequate results (e.g.,
subjects who experience insufficient LDL-c reduction on statin
therapy). The antigenic PCSK9 peptide of the invention described
herein can be administered to a subject with elevated
LDL-cholesterol.
[0310] Preferably a subject with elevated cholesterol is a human
subject with total plasma cholesterol levels of 200 mg/dl or
greater. Preferably a subject with elevated cholesterol is a human
subject with LDL-cholesterol levels of 160 mg/dl or greater.
[0311] Total plasma cholesterol levels and LDL-cholesterol levels
are measured using standard methods on blood samples obtained after
an appropriate fast. Protocols to measure total plasma cholesterol
levels and LDL-cholesterol levels are well-known to the man skilled
in the art.
[0312] In one embodiment the antigenic PCSK9 peptide or an
immunogenic composition or a pharmaceutical composition thereof is
administered together with another agent, the two can be
administered sequentially in either order or simultaneously. In
some embodiments, an antigenic PCSK9 peptide or an immunogenic
composition or a pharmaceutical composition thereof is administered
to a subject who is also receiving therapy with a second agent
(e.g., a second cholesterol-reducing agent). Cholesterol reducing
agents include statins, bile acid sequestrants, niacin, fibric acid
derivatives, and long chain alpha, omego-dicarboxylic acids.
Statins inhibit cholesterol synthesis by blocking HMGCoA, a key
enzyme in cholesterol biosynthesis. Examples of statins are
lovastatin, pravastatin, atorvastatin, cerivastatin, fluvastatin,
and simvastatin. Bile acid sequestrants interrupt the recycling of
bile acids from the intestine to the liver. Examples of these
agents are cholestyramine and colestipol hydrochloride. Examples of
fibric acid derivatives are clofibrate and gemfibrozil. Long chain
alpha, omego-dicarboxylic acids are described, e.g., by Bisgaier et
al., 1998, J. Lipid Res. 39:17-30; WO 98/30530; U.S. Pat. No.
4,689,344; WO 99/001 16; U.S. Pat. No. 5,756,344; U.S. Pat. No.
3,773,946; U.S. Pat. No. 4,689,344; U.S. Pat. No. 4,689,344; U.S.
Pat. No. 4,689,344; and U.S. Pat. No. 3,930,024); ethers (see,
e.g., U.S. Pat. No. 4,711,896; U.S. Pat. No. 5,756,544; U.S. Pat.
No. 6,506,799). Phosphates of dolichol (U.S. Pat. No. 4,613,593),
and azolidinedione derivatives (U.S. Pat. No. 4,287,200) can also
be used to reduce cholesterol levels. A combination therapy regimen
may be additive, or it may produce synergistic results (e.g.,
reductions in cholesterol greater than expected for the combined
use of the two agents). In some embodiments, combination therapy
with an antigenic PCSK9 peptide or an immunogenic composition or a
pharmaceutical composition thereof and a statin produces
synergistic results (e.g., synergistic reductions in cholesterol).
In some subjects, this can allow reduction in statin dosage to
achieve the desired cholesterol levels.
EXAMPLES
[0313] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1
Selection of Antigenic PCSK9 Peptides at the PCSK9-EGF-A Domain of
the LDL Receptor Interface
[0314] The structure of human PCSK9 binding to the EGF-A domain of
the LDL receptor has been solved and published (Kwon et al, PNAS
105, 1820-1825, 2008). This structural information (PDB: 3BPS) was
used together with information from the structure of free PCSK9,
PDB: 2P4E (Cunningham et al, Nature Structural & Molecular
Biology, 14, 413-419, 2007) to design the following peptides which
would correspond to areas of importance for the PCSK9-LDL receptor
interaction (see FIG. 1).
TABLE-US-00006 Peptide 1. (SEQ ID No: 19) ASSDCSTCFV Peptide 2.
(SEQ ID No: 63) GTRFHRQASK Peptide 3. (SEQ ID No: 109) IQSDHREIEGRV
Peptide 4. (SEQ ID No: 153) SGRDAGVAKGA Peptide 5. (SEQ ID No: 184)
SIPWNLERITP
[0315] Since peptides 1-4 represent loops in the PCSK9 structure,
the respective sequences (SEQ ID Nos 19, 63, 109 and 153) were made
with added Cys, Cys-Gly or Lys linkers to allow coupling via both
ends to the VLP carrier to provide a conformational mimetic of the
natural loop structure (VR_9.1 to VR_9.4 in Table 1). In addition,
cyclised versions of peptides 2-4 were also made (VR_9.6 to VR_9.9
in Table 1) which provided a Cys residue for coupling to VLPs.
Peptide 1 was made with a Lys-Gly-Gly N-terminal linker for
coupling purposes so that the two Cys residues were free to
disulphide bond as they do in the native PCSK9 structure. Peptide 5
represents the N-terminal of the mature processed form of human
PCSK9 and was coupled via a C-terminally added cysteine residue to
allow the N-terminus to be free for antibody recognition (VR_9.5 in
Table 1). The following table (Table 1) describes 9 peptides
generated for evaluation as vaccine candidates.
TABLE-US-00007 TABLE 1 Peptide Sequences SEQ ID No VR_9.1
KGGASSDCSTCFV 313 VR_9.2 CGGTRFHRQASKC 314 VR_9.3 CGIQSDHREIEGRVC
315 VR_9.4 CSGRDAGVAKGAC 316 VR_9.5 SIPWNLERITPC 317 VR_9.6
ASK-Cys(H)-GDGTRFHRQ 318 VR_9.7 AG-Cys-(H)-GTRFHRQ 319 VR_9.8
GRV-Cys(H)-IQSDHREIE 320 VR_9.9 AGVAKGAG-Cys(H)-SGRD 321 Underscore
indicates cysteine residues assed for conjugation purposes and
double underscore indicates a GC or KGG linker.
Example 2
Preparation of Peptide-VLP Conjugates for Evaluation as Vaccine
Candidates
[0316] The peptides were synthesised using a standard Fmoc protocol
on CLEAR amide resin. The amino acid coupling reactions were
carried out using 5 fold excess of Fmoc-protected amino acid
activated with 1 eq of HBTU
(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate) in the presence of HOBt (hydroxybenzotriazole)
and NMM (N-methylmorpholine). The deprotection of Fmoc group was
achieved with 20% piperidine/DMF. Resin-bound peptide was then
cleaved and side chain protecting groups removed simultaneously
with Reagent D (TFA/H2O/DODT: 89/3/8). The peptide was made with a
free N-terminus and amidated C-terminus. The crude peptide was
purified to homogeneity by HPLC using a BEH 130 C18 column and a
water/acetonitrile gradient in the presence of 0.1% TFA. The
purified peptide was vacuum-dried using a lyophilizer. The peptide
was analyzed using mass-spectrometry (LC-MS) and gave satisfactory
data.
[0317] The Q.beta. VLP used in this study was produced by bacterial
E. Coli fermentation in a BL21 (DE3) strain incorporating a pET28
plasmid encoding the 14 kD monomer protein:
MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSR
NRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAAL
LASPLLIDAIDQLNPAY (Genbank ID: M99039). The fermentation is induced
at an OD600 of 0.8 with IPTG and allowed to proceed overnight in
terrific broth (TB) with kanamycin. The VLP, which self-assembles
in the host cell, was then purified from the fermentation cell
pellet using the method described in the patent application
EP1736538 with the following differences: after cell disruption,
the clarified homogenate was treated with ammonium sulphate at 50%
saturation and the cell pellet recovered by centrifugation. Then,
the pellet was redissolved in HEPES buffer and dialysed against
HEPES buffer before proceeding to the first column step in the
published method. After the ion-exchange column and hydroxylapatite
column steps, the material was purified using a further
anion-exchange column step and sterile filtered to make the final
VLP bulk material, which was analysed by size-exclusion
chromatography, SDS-PAGE and electron microscopy with acceptable
results.
Conjugation of Peptides Through Cysteine Residues:
[0318] The Q.beta. VLP was activated using either
N-gamma-maleimido-butyryloxy-succinimide ester (GMBS) or the longer
Succinimidyl-6-[ -maleimidopropionamido]hexanoate linking reagent.
The procedure for the usage of both these reagents was similar:
Solid reagent was dissolved in dimethyl sulphoxide (DMSO) and added
to the VLP solution at .gtoreq.10-fold molar excess. The activation
reaction was allowed to proceed for .gtoreq.90 minutes and the
solution was then desalted using a NAP-25 desalting column into
Dulbeccos Phosphate Buffered Saline (DPBS) with 5 mM EDTA or
Dulbeccos Phosphate Buffered Saline (DPBS) that had been modified
by the addition of solid NaCl (14.6 g/L). If necessary, the protein
solution was concentrated slightly using 10 kD spin
microconcentrators prior to the next conjugation reaction.
[0319] Prior to the conjugation reaction, the peptides were
dissolved in an aliquot of pH 7.4 DPBS, with 5 mM EDTA as an
additive. The concentration of the peptide in solution was 10
mg/ml. The solubilised peptide was added to an aliquot of TCEP
immobilised reducing agent (Pierce Chemical) which had been washed
in DPBS containing 5 mM EDTA. The aliquot of peptides was incubated
with mixing in the presence of the TCEP gel for approximately 1
hour, after which time the aliquot was spun down in a microfuge and
the solid pellet discarded. The reduced peptide-containing
supernatant was added directly to the activated VLP which had been
prepared earlier. One alternative to this procedure however is the
addition of solid peptide directly to the sample of activated
Q.beta. VLP. Both methods work equally well for the generation of
peptide-VLP conjugates.
[0320] The reaction between the VLPs and the reduced peptides was
allowed to proceed for at least thirty minutes with very gentle
mixing. At the end of the reaction time each sample was desalted
into Dulbeccos PBS (DPBS) using NAP-10 or NAP-25 desalting columns
(GE Healthcare). The desalted conjugated peptides were analysed for
protein content using the Bradford (Coomassie Brilliant Blue,
Pierce Chemical Co) assay or BCA protein assay (bicinchoninic acid)
(Pierce Chemical Co) as well as by SDS-PAGE and size-exclusion
chromatography. The conjugate products were sterile filtered using
a 0.22 .mu.m filter and stored at 2-8.degree. C. until use. Careful
attention was paid to these samples during storage to prevent
freezing or exposure to extremes in temperature.
[0321] The conjugation of the PCSK9 peptide to CRM197 was performed
by using BAANS (Bromoacetic acid N-hydroxysuccinimide ester, Sigma
B8271). CRM197 was first activated by reacting with BAANS in 0.1 M
sodium carbonate pH 8.3 with a molar ratio of 100 in a cold room
for 90 minutes. The reaction mixture was passed through a Zeba
desalting column, and the flow-through was collected. The PCSK9
peptide at 10 mg/ml was incubated with an equal volume of
immobilized TCEP reducing gel (Thermo Scientific) at room
temperature for 1 hour, and collected after centrifugation through
a 0.2 .mu.m filter. The activated CRM197 was then mixed with the
treated peptide in the cold room overnight, followed by an
extensive dialysis in PBS buffer. The conjugate was recovered,
concentrated, and sterilized through a 0.22 .mu.m filter. The
protein concentration was determined using Coomassie blue assay
(Thermo Scientific).
Conjugation of Peptides via Amines
[0322] For the conjugation of peptides to Q.beta. via amine
residues, specifically peptide VR_9.1, the following procedure was
followed. Q.beta. was initially derivatised by the addition of
solid succinic anhydride at a .gtoreq.10-fold molar excess relative
to the VLP monomer. The succinic anhydride was allowed to dissolve
and the derivatisation reaction was allowed proceed for at least 30
mins. After this time, the sample was then desalted using a NAP-25
desalting column into Dulbeccos Phosphate Buffered Saline (DPBS)
with 5 mM EDTA. Then, the following reagents were added in the
order listed at a .gtoreq.10-fold molar excess relative to the VLP
monomer: Solid peptide, N-hydroxysulfosuccinimide and finally
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide. Following the
addition of reagents in the order listed above, the sample was
incubated at room temperature and the reaction was allowed proceed
for at least 30 mins, after which time the VLP-peptide conjugate
was desalted using NAP-25 desalting columns into Dulbeccos
Phosphate Buffered Saline (DPBS).
[0323] The extent of the conjugation for the VLP-peptide samples
was measured using SDS-PAGE, and a molecular weight increase was
observed for all samples which is consistent with the addition of
the peptide to the VLP protein monomer. In addition, samples were
tested in the HPLC size-exclusion chromatography assay (using a
Tosoh PWXL5000 HPLC column) and found to contain assembled VLP when
compared to unconjugated samples of VLP. VLP-peptide conjugation
features are summarized in Table 2.
TABLE-US-00008 TABLE 2 VLP-Peptide Conjugates Approximate
Percentage substitution Amount yield (ug peptide Activated of of
per mg VLP peptide input of VLP Peptide added added VLP monomer)
VR_9.1 4 mg 5 mg 40-60% 25 ug VR_9.2 3 mg 5 mg 40-60% 125 ug VR_9.3
3 mg 5 mg 40-60% 125 ug VR_9.4 3 mg 5 mg 40-60% 125 ug VR_9.5 3 mg
5 mg 40-60% 50 ug VR_9.6 3 mg 5 mg 40-60% 125 ug VR_9.7 3 mg 5 mg
40-60% 125 ug VR_9.8 3 mg 5 mg 40-60% 125 ug VR_9.9 3 mg 5 mg
40-60% 125 ug VR_9.10 3 mg 5 mg 40-60% 125 ug VR_9.11 3 mg 2 mg
40-60% 125 ug VR_9.12 3 mg 2 mg 40-60% 125 ug VR_9.13 3 mg 2 mg
40-60% 125 ug VR_9.14 3 mg 2 mg 40-60% 125 ug VR_9.15 3 mg 2 mg
40-60% 125 ug VR_9.16 3 mg 2 mg 40-60% 125 ug VR_9.17 10 mg 9.5 mg
95% 82 ug VR_9.18 10 mg 8.7 mg 90% 74 ug VR_9.19 10 mg 7.6 mg 80%
58 ug VR_9.20 10 mg 8.7 mg 95% 86 ug VR_9.21 10 mg 8.8 mg 95% 92 ug
VR_9.22 10 mg 6.6 mg 85% 62 ug VR_9.23 10 mg 10.0 mg 90% 85 ug
VR_9.24 10 mg 10.5 mg 75% 64 ug VR_9.25 10 mg 5.1 mg 40% 1 ug
VR_9.26 10 mg 10.0 mg 60% 123 ug VR_9.27 10 mg 9.6 mg 60% 136 ug
VR_9.28 10 mg 9.4 mg 65% 153 ug VR_9.29 10 mg 4.2 mg 75% 19 ug
VR_9.30 10 mg 4.4 mg 63% 15 ug VR_9.31 10 mg 4.4 mg 70% 13 ug
VR_9.32 10 mg 4.4 mg 63% 15 ug VR_9.33 10 mg 7.4 mg 40% 18 ug
VR_9.34 10 mg 7.3 mg 50% 23 ug VR_9.35 7.5 mg 4.3 mg 61% 16 ug *As
determined by SDS-PAGE and densitometry calculations
Example 3
PCSK9 Peptide Immunogenicity
[0324] This study aimed to evaluate how efficacious peptides
conjugated to a Qbeta VLP (as detailed in Example 2 above) were in
inducing an antibody response that can bind to human and mouse
PCSK9. Female Balb/c (6-8 weeks) were injected by the intramuscular
route (50 microliter volume injected into each Tibialis anterior
muscle) on days 0, 21 and 42 with VLP-peptide conjugates formulated
in Alum with CpG of formula 5' TCGTCGTTTTTCGGTGCTTTT 3'. One group
of control mice was immunized with VLP coupled to a control
(non-PCSK9) peptide following the same protocol and a second
control group was left unimmunised. Necropsy took place on day 49.
At necropsy 400-600 microliter blood was sampled from euthanised
mice by cardiac puncture using an anti-coagulant. Blood was
centrifuged to separate the plasma, which was stored frozen until
testing.
[0325] IgG antibody responses to full length human recombinant
PCSK9 protein were measured using a colorimetric ELISA method.
Serial dilutions were prepared from sera samples and tested in the
assay.
[0326] Human PCSK9 ELISA method 1: 384-well high bind assay plates
(Corning International Cat #3700) were coated with 25 .mu.L/well of
human PCSK9 protein stock diluted to 1 .mu.g/mL with 0.01M PBS pH
7.4 and incubated on a shaker at RT for 3 hours. After washing
.times.2 with PBS pH 7.4, plates were blocked using 80 .mu.L/well
of 0.01M PBS/1% BSA, incubated at RT for 1 hour before a final wash
.times.3 with 0.01M PBS pH 7.4/0.05% Tween 20. The following day,
an 8 point 1/2 log serial dilution of each sample was prepared
starting at 1:25 dilution (PBS/1% BSA diluent), 25 .mu.L/well of
the serial dilution transferred in duplicate into the human PCSK9
coated plate then incubated shaking at RT for 1.5 hours. After
washing .times.3 with 0.01M PBS pH 7.4/0.05% Tween 20, 25
.mu.L/well of Total IgG detection antibody (Rabbit anti-mu IgG-Fc,
Cat #A90-130A Bethyl Laboratories) at a 1:6000 dilution with 0.01M
PBS pH 7.4/1% BSA was added, then incubated shaking at RT for 1
hour. After washing .times.5 with 0.01M PBS pH 7.4/0.05% Tween 20,
added 25 .mu.L/well Bio-Rad kit goat anti-rabbit horseradish
peroxidase conjugate (Bio-Rad Cat #172-1019) 1:3000 with 0.01M PBS
pH 7.4/0.05% Tween 20 pH 7.4, then incubated shaking at RT for 1
hour. After washing .times.4 with 0.01M PBS pH 7.4/0.05% Tween 20
and then .times.1 with 0.01M PBS pH 7.4 only, 25 .mu.L/well Mouse
Typer HRP Substrate (Bio-Rad Cat #172-1064) were added and the
plates were incubated at RT for 30 mins. 25 .mu.L/well 2% oxalic
acid were added and the absorbance then read at Abs 405 nm.
[0327] Mouse PCSK9 ELISA method 1: Thermo Immunolon 4HBX 96-well
ELISA plates were coated with 100 .mu.l of 1 .mu.g/ml recombinant
mouse PCSK9 in PBS overnight at 4.degree. C. After washing, plates
were blocked with 300 ml PBS/0.5% BSA (Sigma A7030) for 1 hr,
washed 4.times. with 3000 PBS/0.01% Tween-20 and 100 .mu.l of
serial dilutions of plasma samples (in PBS/0.5% BSA) added. After
incubation with gentle shaking for 1 hour at room temperature,
plates were washed 4.times. with 3000 PBS/0.01% Tween-20 and 100
.mu.l of a 1:5000 dilution of goat anti-mouse IgG-HRP (horse radish
peroxidase; Pierce 31430) added to each well. The plates were
incubated at room temperature with gentle shaking for 45 minutes
and then washed .times.7 with 300 .mu.l PBS/0.01% Tween-20. 100
.mu.l TMB substrate (Sigma T-4444) was then added, the colorimetric
reaction stopped after 4 minutes by addition of 2N sulphuric acid
and the absorbance read at 450 nm.
[0328] Data analysis: Titration curves were plotted for each test
sample (sample dilution vs absorbance). The sample titer
(subsequently transformed into reciprocal titer) was then taken as
the serum dilution achieving a cut-off optical density (0.D.)
values of 1.0 or 0.5.
Measurement of Plasma/Serum Cholesterol Level
[0329] Cholesterol levels in plasma and serum samples were measured
using a WAKO Cholesterol E Assay kit (Cat #439-17501) following the
manufacturers' instructions. Dilutions of cholesterol standard or
test plasma/serum samples (4 .mu.l volume) were added to wells of a
96-well plate and 196 .mu.l of prepared cholesterol reagent added.
The plate was incubated for 5 minutes at 37.degree. C. and the
absorbance of the developed colour read at 600 nm within 30
minutes.
Measurement of Interaction Between PCSK9 and the Extracellular
Domain of the LDL Receptor
[0330] Interruption of LDLR and PCSK9 binding by mouse plasma was
determined with TR-FRET assay (time-resolved fluorescence resonance
energy transfer assay) using fluorophor-labeled LDLR extracellular
domain (LDLR-ECD) and full length wild type PCSK9 protein. LDLR-ECD
(R&D system, cat #2148-LD/CF)) was labeled with europium (GE
healthcare, Cat #PA99148) according to manufacturer's instruction
(at a Eu:LDLR molar ratio 6:1). PCSK9 was biotinylated with
Biotin-XX-SSE (Pierce, cat #21237) at a Biotin:PCSK9 molar ratio of
8. The TR-FRET assay was conducted with 5 nM LDLR-Eu+3, 30 nM
PCSK9-biotin and 50 nM Alexa Fluor 647 conjugated streptavidin
(SA647, Invitrogen, cat #S21374) in 20 .mu.l of assay buffer (20 mM
Hepes, pH 7.0, 150 mM NaCl, 0.1 mM CaCl2 and 0.05%(w/v) BSA) in
384-well black plates (Corning, Cat #3676). Serial dilution of
mouse plasma were pre-incubated with PCSK9-biotin at RT for 30
minutes in humidified chamber, followed by mixing with LDLR and
streptavidin-SA647. After an additional 60 minute incubation at RT
in a humidified atmosphere in dark, the plates were read on a
Perkin Elmer Victor2 plate reader using a 50 .mu.s delay time and a
400 .mu.s window. Data are reported as the ratio of the signals at
(665 nm/615 nM).times.1000.
[0331] Results: as shown in FIG. 2, all peptides used as immunogens
were able to induce antibody responses to the intact full-length
human PCSK9 protein, some inducing higher responses than others. In
all cases these responses cross-reacted with mouse PCSK9 as shown
in FIG. 3. FIG. 4 shows that peptide VR_9.5 immunization also led
to a decrease in plasma cholesterol levels and FIGS. 5 and 6 show
that VR_9.5 and VR_9.6 induced serum antibody responses that could
inhibit the interaction between PCSK9 and LDL receptors using a
fluorescence resonance energy transfer (FRET) assay.
Example 4
Design of Peptides Corresponding to Regions Distinct from Receptor
EGF-A Domain Present in PDB: 3BPS
[0332] The LDL receptor is a multidomain protein the extracellular
domains of which consist of an N-terminal ligand binding domain,
three EGF like repeats (of which EGF-A is one) a .beta.-propeller
domain and an "O linked sugar domain" (Kwon et al, PNAS 105,
1820-1825, 2008). The PDB file 3GCX details the structure of PCSK9
in complex with a soluble form of only the EGF-A domain of the LDL
receptor, therefore we postulated that further non-obvious
interactions may exist between PCSK9 and the LDL receptor that
cannot be deduced from direct analysis of the molecules represented
in PDB: 3GCX. Examples of these regions are detailed in FIG. 7 and
specifically two sequences in PCSK9 were identified that could act
as additional putative receptor interfaces (NAQDQPVTLGTL and
INEAWFPEDQRVL--see FIG. 8).
TABLE-US-00009 (mouse) (SEQ ID No 332) SIPWNLERIIP (SEQ ID No: 227)
NAQDQPVTLGTL (SEQ ID No: 263) INEAWFPEDQRVL (mouse) (SEQ ID No 360)
INMAWFPEDQQVL
[0333] By using murine PCSK9 sequences as found in public databases
the murine homologues were also identified (as displayed in table
3). We also postulated that part of the amino acid sequence
contained within peptide VR_9.5 (described in Example 1) may also
interact with parts of the LDL receptor not visible in the PDB:
3BPS (FIG. 8). This concept was probed by refining the sequence
VR_9.5 by altering the amino acid linker and the orientation of
conjugation. We also identified a peptide corresponding to the
mouse homologue of VR_9.5 (peptide VR_9.10). The resultant series
of peptide sequences from the approach described above were
modified by the addition of amino acids to permit chemical
conjugation and were evaluated as vaccine candidates (peptides
detailed in table 3).
TABLE-US-00010 TABLE 3 Peptide sequences Peptide Sequence Species
SEQ ID No. VR_9.10 SIPWNLERIIPC Mouse 322 VR_9.11 CGGSIPWNLERIIP
Mouse 323 VR_9.12 SIPWNLERIIPGGC Mouse 324 VR_9.13 CGGNAQDQPVTLGTL
Mouse & 325 Human VR_9.14 NAQDQPVTLGTLGGC Mouse & 326 Human
VR_9.15 CGGINMAWFPEDQQVL Mouse 327 VR_9.16 INMAWFPEDQQVLGGC Mouse
328 Residues underlined indicate amino acids added for conjugation
purposes.
Example 5
[0334] Peptides VR_9.10 to VR_9.16 (as well as VR_9.5 for
comparison) were conjugated to Q.beta. VLP as described in example
2 and used to immunize mice and assess antibody responses to PCSK9
as described in example 3, with an unconjugated VLP used as a
control immunogen. As shown in FIGS. 9 and 10, all peptides induced
antibodies that could recognize intact full-length mouse PCSK9 in
an ELISA assay.
Example 6
[0335] On the basis of our observation of cholesterol lowering
being induced by immunization with peptide VR_9.5, representing the
N-terminus of the mature processed form of human PCSK9 (SEQ ID No:
184), we hypothesize that, since the cleaved prodomain of immature
PCSK9 is known to remain associated with the mature PCSK9 protein,
regions of this prodomain (SEQ ID No. 329) are also candidate
antibody targets for lowing cholesterol levels.
TABLE-US-00011 (SEQ ID No: 329)
MGTVSSRRSINWPLPLLLLLLLLLGPAGARAQEDEDGDYEELVLALRSEE
DGLAEAPEHGTTATFHRCAKDPWRLPGTYVVVLKEETHLSQSERTARRLQ
AQAARRGYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHVDYIEEDSSV FAQ
[0336] Specific, non-restrictive, examples of peptides of interest
are found within the C-terminal sequence of the prodomain region
and surface exposed sequences and loops, including regions
containing known loss-of-function or gain-of-function genetic
mutations in humans:
TABLE-US-00012 (SEQ ID No: 308) VDYIEEDSSVFAQ (SEQ ID No: 309)
RCAKDPWRLPGT (SEQ ID No: 310) AQAARRGYLTKIL (SEQ ID No: 311)
GDYEELVLALRSEEDG (SEQ ID No: 312) FLVKMSGDLLELALKLP
[0337] The above peptides and truncations thereof are synthesized
with N- or C-terminally added linkers (e.g. CGG or GGC), or as
cyclised or otherwise conformationally constrained molecules and
coupled to Q.beta. VLPs as described in example 2 and used to
immunize mice and assess antibody responses to PCSK9 as described
in example 3.
Example 7
[0338] Peptides VR_9.5 and 9.10 were conjugated to Q.beta. VLP or
to CRM197 as described in example 2 and used to immunize mice
(BALB/c or C57BL/6) using TiterMax Gold, alum or alum-CpG as
adjuvant. Hepatitis B virus derived peptide (amino acids 28-39
(IPQSLDSWWTSL)) was conjugated to Q.beta. VLP or to CRM197 and used
as a control immunogen.
[0339] The ELISA method used was slightly different from the one
disclosed in example 3: the ELISA method was performed as follows
for Human and Mouse PCSK9 ELISA (Method 2): 384-well high bind
assay plates (Greiner bio-one 781061) were coated with 25
.mu.L/well of human or mouse PCSK9 protein stock diluted to 1
.mu.g/mL with 1.times. PBS pH 7.4 and incubated at 4.degree. C.
over night. The following day, plates were blocked using 25
.mu.L/well of 1.times. PBS/0.05% Tween-20/1% BSA, incubated on a
shaker at RT for 1 hour. A 10 point 1/2 log serial dilution of each
sample was prepared starting at 1:50 or 1:500 dilution (1.times.
PBS/0.05% Tween-20 diluent), 25 .mu.L/well of the serial dilution
transferred in duplicate into the human or mouse PCSK9 coated plate
then incubated shaking at RT for 1 hour. After washing .times.3
with 1.times. PBS pH 7.4/0.05% Tween-20, 25 .mu.L/well of Total IgG
detection antibody (Goat Anti-Mouse IgG (.gamma.), HRP, Invitrogen
M30107) at a 1:3000 dilution with 1.times. PBS pH 7.4/0.05%
Tween-20 was added then incubated shaking at RT for 1 hour. After
washing .times.5 with 1.times. PBS pH 7.4/0.05% Tween 20, 25
.mu.L/well Bio-Rad TMB Peroxidase EIA Substrate Kit (Bio-Rad Cat
#172-1067) were added and the plates incubated for 15 minutes. 12.5
.mu.L/well of 1N sulfuric acid were added and the absorbance then
read at Abs 450 nm.
[0340] Results: Peptides VR_9.5 and 9.10 conjugated to Q.beta. VLP
or CRM197 were able to induce antibody responses to the intact
full-length human and mouse PCSK9 protein (see FIGS. 11 and 12).
FIGS. 13 and 14 and Table 4 also show that peptide VR_9.5 and 9.10
conjugated to different carriers and in the presence of different
adjuvants led to a decrease in serum cholesterol levels.
TABLE-US-00013 TABLE 4 Total Cholesterol Levels in Immunized Mmice
Mouse Cholesterol (mg/dL) Strain Immunogen Carrier Adjuvant(s) N
Mean SEM Stats.sup.(a) Stats.sup.(b) BALB/c Untreated -- -- 8 106.5
3.0 -- -- Control peptide VLP Alum + CpG 8 99.0 2.8 P > 0.05 --
VR_9.5 VLP Alum + CpG 8 77.5 3.6 P < 0.001 P < 0.001 VR_9.5
VLP Alum 8 65.9 2.3 P < 0.001 P < 0.001 VR_9.10 VLP Alum +
CpG 8 85.3 2.4 P < 0.001 P < 0.05 Control peptide CRM197
TiterMax 8 96.6 2.3 P > 0.05 -- VR_9.5 CRM197 TiterMax 8 75.0
4.1 P < 0.001 P < 0.001 VR_9.10 CRM197 TiterMax 7 70.9 2.8 P
< 0.001 P < 0.001 C57BL/6 Untreated -- -- 8 83.4 5.8 -- --
Control peptide VLP Alum + CpG 8 81.0 3.7 P > 0.05 -- VR_9.5 VLP
Alum + CpG 8 59.7 2.8 P < 0.001 P < 0.01 VR_9.5 VLP Alum 8
59.5 1.5 P < 0.001 P < 0.01 VR_9.10 VLP Alum + CpG 8 64.5 2.7
P < 0.01 P < 0.05 Control peptide CRM197 TiterMax 8 71.5 1.7
P > 0.05 -- VR_9.5 CRM197 TiterMax 8 58.7 4.1 P < 0.001 P
> 0.05 VR_9.10 CRM197 TiterMax 8 63.8 3.4 P < 0.01 P >
0.05 Stats.sup.(a): 1-way ANOVA statistical comparison with Tukey
post-hoc test displaying p values of test groups vs. untreated.
Stats.sup.(b): 1-way ANOVA statistical comparison with Tukey
post-hoc test displaying p values of test groups vs. matched
control peptide-carrier.
Example 8
[0341] Additional peptides (SEQ ID No 312, 420, 421, 422, 423, 425,
426, 427, 428, 445, 482, 525 and 563) were chosen from both the
prodomain and the C-terminal region of the catalytic domain of
PCSK9 for surface exposure and association with gain of function or
loss of function mutations identified in humans. The resultant
series of peptide sequences from the approach described above were
modified by the addition of amino acids to permit chemical
conjugation and were evaluated as vaccine candidates (Table 5
peptides 9.23-9.35).
TABLE-US-00014 TABLE 5 Peptide sequences Peptide Sequence Species
SEQ ID No VR_9.5 SIPWNLERITPC Human 317 VR_9.10 SIPWNLERIIPC Mouse
322 VR_9.17 SIPWNLERIGGC Human & 401 Mouse VR_9.18 SIPWNLERGGC
Human & 402 Mouse VR_9.19 SIPWNLEGGC Human & 403 Mouse
VR_9.20 CGGSGRDAGVAKGA Human 404 VR_9.21 CGGSGRDAGVAKGT Mouse 405
VR_9.22 RDAGVAKGGC Human 406 VR_9.23 CSRHLAQASQELQ Human 407
VR_9.24 CRSRPSAKASWVQ Mouse 408 VR_9.25 CGGDYEELVLALR Human 409
VR_9.26 CGGDYEELMLALP Mouse 410 VR_9.27 LVLALRSEEDGGC Human 411
VR_9.28 LMLALPSQEDGGC Mouse 412 VR_9.29 AKDPWRLPGGC Human 413
VR_9.30 SKEAWRLPGGC Mouse 414 VR_9.31 CGGAARRGYLTK Human 415
VR_9.32 CGGAARRGYVIK Mouse 416 VR_9.33 FLVKMSGDLLELALKLPGGC Human
417 VR_9.34 FLVKMSSDLLGLALKLPGGC Mouse 418 VR_9.35 CGGEEDSSVFAQ
Human 419 Residues underlined indicate amino acids added for
conjugation purposes.
Example 9
[0342] Peptides VR_9.17 to VR_9.35 (as well as VR_9.5) were
conjugated to Q.beta. VLP as described in example 2 and used to
immunize mice to assess antibody responses to PCSK9 as described in
example 3. Hepatitis B virus derived peptide (amino acids 28-39
(IPQSLDSWWTSL)) was conjugated to Q.beta. VLP and used as a control
immunogen. Method No 2 was used for the ELISA.
[0343] Result: As shown in FIGS. 15 and 16, most of the the
peptides conjugated to Qb VLP were able to induce antibody
responses to intact full length PCSK9. No antibody response was
detected for peptides 9.31 and 9.32. In certain cases (peptides
9.23, 9.24, 9.27, 9.28, 9.29, 9.30, 9.33, and 9.34) the antibody
responses were species specific. Immunization with peptides 9.5,
9.18, and 9.29 also resulted in decreased serum cholesterol, while
immunization with peptides 9.17, 9.19, 9.30, 9.31, and 9.32
resulted in a trend to reduced cholesterol (Table 6, FIG. 17).
TABLE-US-00015 TABLE 6 Total Cholesterol Levels in Immunized Mice
Mouse Cholesterol (mg/dL) Strain Immunogen Carrier Adjuvants N Mean
SEM Stats.sup.(a) Stats.sup.(b) BALB/c Untreated -- -- 8 77.7 5.5
-- -- Control peptide VLP Alum + CpG 4 78.1 1.2 P > 0.05 --
VR_9.5 VLP Alum + CpG 8 65.8 1.1 P < 0.05 P > 0.05 VR_9.17
VLP Alum + CpG 8 70.4 2.1 P > 0.05 P > 0.05 VR_9.18 VLP Alum
+ CpG 8 65.9 1.6 P < 0.05 P > 0.05 VR_9.19 VLP Alum + CpG 8
71.7 1.7 P > 0.05 P > 0.05 VR_9.20 VLP Alum + CpG 7 85.2 4.0
P > 0.05 P > 0.05 VR_9.21 VLP Alum + CpG 8 77.1 2.0 P >
0.05 P > 0.05 VR_9.22 VLP Alum + CpG 8 84.5 2.6 P > 0.05 P
> 0.05 VR_9.23 VLP Alum + CpG 8 79.0 1.8 P > 0.05 P > 0.05
VR_9.24 VLP Alum + CpG 8 76.9 2.1 P > 0.05 P > 0.05 VR_9.25
VLP Alum + CpG 8 77.3 2.5 P > 0.05 P > 0.05 VR_9.26 VLP Alum
+ CpG 8 79.2 2.2 P > 0.05 P > 0.05 VR_9.27 VLP Alum + CpG 8
71.8 3.4 P > 0.05 P > 0.05 VR_9.28 VLP Alum + CpG 8 76.6 2.2
P > 0.05 P > 0.05 VR_9.29 VLP Alum + CpG 8 67.0 1.6 P <
0.05 P > 0.05 VR_9.30 VLP Alum + CpG 8 70.4 2.3 P > 0.05 P
> 0.05 VR_9.31 VLP Alum + CpG 8 71.0 1.7 P > 0.05 P > 0.05
VR_9.32 VLP Alum + CpG 8 72.8 1.0 P > 0.05 P > 0.05 VR_9.33
VLP Alum + CpG 8 75.5 2.2 P > 0.05 P > 0.05 VR_9.34 VLP Alum
+ CpG 8 76.0 2.3 P > 0.05 P > 0.05 VR_9.35 VLP Alum + CpG 8
74.5 2.2 P > 0.05 P > 0.05 Stats.sup.(a): 1-way ANOVA
statistical comparison with Bonferroni post-hoc test displaying p
values of test groups vs. untreated group. Stats.sup.(b): 1-way
ANOVA statistical comparison with Bonferroni post-hoc test
displaying p values of test groups vs. control peptide group.
TABLE-US-00016 SEQ ID No 1 IGASSDCSTCFVS SEQ ID No 2 IGASSDCSTCFV
SEQ ID No 3 IGASSDCSTCF SEQ ID No 4 IGASSDCSTC SEQ ID No 5
IGASSDCST SEQ ID No 6 IGASSDCS SEQ ID No 7 IGASSDC SEQ ID No 8
IGASSD SEQ ID No 9 IGASS SEQ ID No 10 GASSDCSTCFVS SEQ ID No 11
GASSDCSTCFV SEQ ID No 12 GASSDCSTCF SEQ ID No 13 GASSDCSTC SEQ ID
No 14 GASSDCST SEQ ID No 15 GASSDCS SEQ ID No 16 GASSDC SEQ ID No
17 GASSD SEQ ID No 18 ASSDCSTCFVS SEQ ID No 19 ASSDCSTCFV SEQ ID No
20 ASSDCSTCF SEQ ID No 21 ASSDCSTC SEQ ID No 22 ASSDCST SEQ ID No
23 ASSDCS SEQ ID No 24 ASSDC SEQ ID No 25 SSDCSTCFVS SEQ ID No 26
SSDCSTCFV SEQ ID No 27 SSDCSTCF SEQ ID No 28 SSDCSTC SEQ ID No 29
SSDCST SEQ ID No 30 SSDCS SEQ ID No 31 SDCSTCFVS SEQ ID No 32
SDCSTCFV SEQ ID No 33 SDCSTCF SEQ ID No 34 SDCSTC SEQ ID No 35
SDCST SEQ ID No 36 DCSTCFVS SEQ ID No 37 DCSTCFV SEQ ID No 38
DCSTCF SEQ ID No 39 DCSTC SEQ ID No 40 CSTCFVS SEQ ID No 41 CSTCFV
SEQ ID No 42 CSTCF SEQ ID No 43 STCFVS SEQ ID No 44 STCFV SEQ ID No
45 TCFVS SEQ ID No 46 EDGTRFHRQASKCDS SEQ ID No 47 EDGTRFHRQASKCD
SEQ ID No 48 EDGTRFHRQASKC SEQ ID No 49 EDGTRFHRQASK SEQ ID No 50
EDGTRFHRQAS SEQ ID No 51 EDGTRFHRQA SEQ ID No 52 EDGTRFHRQ SEQ ID
No 53 DGTRFHRQASKCDS SEQ ID No 54 DGTRFHRQASKCD SEQ ID No 55
DGTRFHRQASKC SEQ ID No 56 DGTRFHRQASK SEQ ID No 57 DGTRFHRQAS SEQ
ID No 58 DGTRFHRQA SEQ ID No 59 DGTRFHRQ SEQ ID No 60 GTRFHRQASKCDS
SEQ ID No 61 GTRFHRQASKCD SEQ ID No 62 GTRFHRQASKC SEQ ID No 63
GTRFHRQASK SEQ ID No 64 GTRFHRQAS SEQ ID No 65 GTRFHRQA SEQ ID No
66 GTRFHRQ SEQ ID No 67 TRFHRQASKCDS SEQ ID No 68 TRFHRQASKCD SEQ
ID No 69 TRFHRQASKC SEQ ID No 70 TRFHRQASK SEQ ID No 71 TRFHRQAS
SEQ ID No 72 TRFHRQA SEQ ID No 73 TRFHRQ SEQ ID No 74 RFHRQASKCDS
SEQ ID No 75 RFHRQASKCD SEQ ID No 76 RFHRQASKC SEQ ID No 77
RFHRQASK SEQ ID No 78 RFHRQAS SEQ ID No 79 RFHRQA SEQ ID No 80
RFHRQ SEQ ID No 81 FHRQASKCDS SEQ ID No 82 FHRQASKCD SEQ ID No 83
FHRQASKC SEQ ID No 84 FHRQASK SEQ ID No 85 FHRQAS SEQ ID No 86
FHRQA SEQ ID No 87 HRQASKCDS SEQ ID No 88 HRQASKCD SEQ ID No 89
HRQASKC SEQ ID No 90 HRQASK SEQ ID No 91 HRQAS SEQ ID No 92
RQASKCDS SEQ ID No 93 RQASKCD SEQ ID No 94 RQASKC SEQ ID No 95
RQASK SEQ ID No 96 QASKCDS SEQ ID No 97 QASKCD SEQ ID No 98 QASKC
SEQ ID No 99 ASKCDS SEQ ID No 100 ASKCD SEQ ID No 101 SKCDS SEQ ID
No 102 SIQSDHREIEGRVM SEQ ID No 103 SIQSDHREIEGRV SEQ ID No 104
SIQSDHREIEGR SEQ ID No 105 SIQSDHREIEG SEQ ID No 106 SIQSDHREIE SEQ
ID No 107 SIQSDHREI SEQ ID No 108 IQSDHREIEGRVM SEQ ID No 109
IQSDHREIEGRV SEQ ID No 110 IQSDHREIEGR SEQ ID No 111 IQSDHREIEG SEQ
ID No 112 IQSDHREIE SEQ ID No 113 IQSDHREI SEQ ID No 114
QSDHREIEGRVM SEQ ID No 115 QSDHREIEGRV SEQ ID No 116 QSDHREIEGR SEQ
ID No 117 QSDHREIEG SEQ ID No 118 QSDHREIE SEQ ID No 119 QSDHREI
SEQ ID No 120 SDHREIEGRVM SEQ ID No 121 SDHREIEGRV SEQ ID No 122
SDHREIEGR SEQ ID No 123 SDHREIEG SEQ ID No 124 SDHREIE SEQ ID No
125 SDHREI
SEQ ID No 126 DHREIEGRVM SEQ ID No 127 DHREIEGRV SEQ ID No 128
DHREIEGR SEQ ID No 129 DHREIEG SEQ ID No 130 DHREIE SEQ ID No 131
DHREI SEQ ID No 132 HREIEGRVM SEQ ID No 133 HREIEGRV SEQ ID No 134
HREIEGR SEQ ID No 135 HREIEG SEQ ID No 136 HREIE SEQ ID No 137
REIEGRVM SEQ ID No 138 REIEGRV SEQ ID No 139 REIEGR SEQ ID No 140
REIEG SEQ ID No 141 EIEGRVM SEQ ID No 142 EIEGRV SEQ ID No 143
EIEGR SEQ ID No 144 IEGRVM SEQ ID No 145 IEGRV SEQ ID No 146 EGRVM
SEQ ID No 147 VSGRDAGVAKGAS SEQ ID No 148 VSGRDAGVAKGA SEQ ID No
149 VSGRDAGVAKG SEQ ID No 150 VSGRDAGVAK SEQ ID No 151 VSGRDAGVA
SEQ ID No 152 SGRDAGVAKGAS SEQ ID No 153 SGRDAGVAKGA SEQ ID No 154
SGRDAGVAKG SEQ ID No 155 SGRDAGVAK SEQ ID No 156 SGRDAGVA SEQ ID No
157 GRDAGVAKGAS SEQ ID No 158 GRDAGVAKGA SEQ ID No 159 GRDAGVAKG
SEQ ID No 160 GRDAGVAK SEQ ID No 161 GRDAGVA SEQ ID No 162
RDAGVAKGAS SEQ ID No 163 RDAGVAKGA SEQ ID No 164 RDAGVAKG SEQ ID No
165 RDAGVAK SEQ ID No 166 RDAGVA SEQ ID No 167 DAGVAKGAS SEQ ID No
168 DAGVAKGA SEQ ID No 169 DAGVAKG SEQ ID No 170 DAGVAK SEQ ID No
171 DAGVA SEQ ID No 172 AGVAKGAS SEQ ID No 173 AGVAKGA SEQ ID No
174 AGVAKG SEQ ID No 175 AGVAK SEQ ID No 176 GVAKGAS SEQ ID No 177
GVAKGA SEQ ID No 178 GVAKG SEQ ID No 179 VAKGAS SEQ ID No 180 VAKGA
SEQ ID No 181 AKGAS SEQ ID No 182 SIPWNLERITPPR SEQ ID No 183
SIPWNLERITPP SEQ ID No 184 SIPWNLERITP SEQ ID No 185 SIPWNLERIT SEQ
ID No 186 SIPWNLERI SEQ ID No 187 SIPWNLER SEQ ID No 188 SIPWNLE
SEQ ID No 189 SIPWNL SEQ ID No 190 SIPWN SEQ ID No 191 IPWNLERITPPR
SEQ ID No 192 IPWNLERITPP SEQ ID No 193 IPWNLERITP SEQ ID No 194
IPWNLERIT SEQ ID No 195 IPWNLERI SEQ ID No 196 IPWNLER SEQ ID No
197 IPWNLE SEQ ID No 198 IPWNL SEQ ID No 199 PWNLERITPPR SEQ ID No
200 PWNLERITPP SEQ ID No 201 PWNLERITP SEQ ID No 202 PWNLERIT SEQ
ID No 203 PWNLERI SEQ ID No 204 PWNLER SEQ ID No 205 PWNLE SEQ ID
No 206 WNLERITPPR SEQ ID No 207 WNLERITPP SEQ ID No 208 WNLERITP
SEQ ID No 209 WNLERIT SEQ ID No 210 WNLERI SEQ ID No 211 WNLER SEQ
ID No 212 NLERITPPR SEQ ID No 213 NLERITPP SEQ ID No 214 NLERITP
SEQ ID No 215 NLERIT SEQ ID No 216 NLERI SEQ ID No 217 LERITPPR SEQ
ID No 218 LERITPP SEQ ID No 219 LERITP SEQ ID No 220 LERIT SEQ ID
No 221 ERITPPR SEQ ID No 222 ERITPP SEQ ID No 223 ERITP SEQ ID No
224 RITPPR SEQ ID No 225 RITPP SEQ ID No 226 ITPPR SEQ ID No 227
NAQDQPVTLGTL SEQ ID No 228 NAQDQPVTLGT SEQ ID No 229 NAQDQPVTLG SEQ
ID No 230 NAQDQPVTL SEQ ID No 231 NAQDQPVT SEQ ID No 232 NAQDQPV
SEQ ID No 233 NAQDQP SEQ ID No 234 NAQDQ SEQ ID No 235 AQDQPVTLGTL
SEQ ID No 236 AQDQPVTLGT SEQ ID No 237 AQDQPVTLG SEQ ID No 238
AQDQPVTL SEQ ID No 239 AQDQPVT SEQ ID No 240 AQDQPV SEQ ID No 241
AQDQP SEQ ID No 242 QDQPVTLGTL SEQ ID No 243 QDQPVTLGT SEQ ID No
244 QDQPVTLG SEQ ID No 245 QDQPVTL SEQ ID No 246 QDQPVT SEQ ID No
247 QDQPV SEQ ID No 248 DQPVTLGTL SEQ ID No 249 DQPVTLGT SEQ ID No
250 DQPVTLG
SEQ ID No 251 DQPVTL SEQ ID No 252 DQPVT SEQ ID No 253 QPVTLGTL SEQ
ID No 254 QPVTLGT SEQ ID No 255 QPVTLG SEQ ID No 256 QPVTL SEQ ID
No 257 PVTLGTL SEQ ID No 258 PVTLGT SEQ ID No 259 PVTLG SEQ ID No
260 VTLGTL SEQ ID No 261 VTLGT SEQ ID No 262 TLGTL SEQ ID No 263
INEAWFPEDQRVL SEQ ID No 264 INEAWFPEDQRV SEQ ID No 265 INEAWFPEDQR
SEQ ID No 266 INEAWFPEDQ SEQ ID No 267 INEAWFPED SEQ ID No 268
INEAWFPE SEQ ID No 269 INEAWFP SEQ ID No 270 INEAWF SEQ ID No 271
INEAW SEQ ID No 272 NEAWFPEDQRVL SEQ ID No 273 NEAWFPEDQRV SEQ ID
No 274 NEAWFPEDQR SEQ ID No 275 NEAWFPEDQ SEQ ID No 276 NEAWFPED
SEQ ID No 277 NEAWFPE SEQ ID No 278 NEAWFP SEQ ID No 279 NEAWF SEQ
ID No 280 EAWFPEDQRVL SEQ ID No 281 EAWFPEDQRV SEQ ID No 282
EAWFPEDQR SEQ ID No 283 EAWFPEDQ SEQ ID No 284 EAWFPED SEQ ID No
285 EAWFPE SEQ ID No 286 EAWFP SEQ ID No 287 AWFPEDQRVL SEQ ID No
288 AWFPEDQRV SEQ ID No 289 AWFPEDQR SEQ ID No 290 AWFPEDQ SEQ ID
No 291 AWFPED SEQ ID No 292 AWFPE SEQ ID No 293 WFPEDQRVL SEQ ID No
294 WFPEDQRV SEQ ID No 295 WFPEDQR SEQ ID No 296 WFPEDQ SEQ ID No
297 WFPED SEQ ID No 298 FPEDQRVL SEQ ID No 299 FPEDQRV SEQ ID No
300 FPEDQR SEQ ID No 301 FPEDQ SEQ ID No 302 PEDQRVL SEQ ID No 303
PEDQRV SEQ ID No 304 PEDQR SEQ ID No 305 EDQRVL SEQ ID No 306 EDQRV
SEQ ID No 307 DQRVL SEQ ID No 308 VDYIEEDSSVFAQ SEQ ID No 309
RCAKDPWRLPGT SEQ ID No 310 AQAARRGYLTKIL SEQ ID No 311
GDYEELVLALRSEEDG SEQ ID No 312 FLVKMSGDLLELALKLP SEQ ID No 313
KGGASSDCSTCFV SEQ ID No 314 CGGTRFHRQASKC SEQ ID No 315
CGIQSDHREIEGRVC SEQ ID No 316 CSGRDAGVAKGAC SEQ ID No 317
SIPWNLERITPC SEQ ID No 318 ASKCGDGTRFHRQ SEQ ID No 319 AGCGTRFHRQ
SEQ ID No 320 GRVCIQSDHREIE SEQ ID No 321 AGVAKGAGCSGRD SEQ ID No
322 SIPWNLERIIPC SEQ ID No 323 CGGSIPWNLERIIP SEQ ID No 324
SIPWNLERIIPGGC SEQ ID No 325 CGGNAQDQPVTLGTL SEQ ID No 326
NAQDQPVTLGTLGGC SEQ ID No 327 CGGINMAWFPEDQQVL SEQ ID No 328
NMAWFPEDQQVLGGC SEQ ID No 329 MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDED
GDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKD
PWRLPGTYVVVLKEETHLSQSERTARRLQAQAARR
GYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHV DYIEEDSSVFAQ SEQ ID No 330
SIPWNLERIIPAW SEQ ID No 331 SIPWNLERIIPA SEQ ID No 332 SIPWNLERIIP
SEQ ID No 333 SIPWNLERII SEQ ID No 334 IPWNLERIIPAW SEQ ID No 335
IPWNLERIIPAW SEQ ID No 336 IPWNLERIIP SEQ ID No 337 IPWNLERII SEQ
ID No 338 PWNLERIIPAW SEQ ID No 339 PWNLERIIPA SEQ ID No 340
PWNLERIIP SEQ ID No 341 PWNLERII SEQ ID No 342 WNLERIIPAW SEQ ID No
343 WNLERIIPA SEQ ID No 344 WNLERIIP SEQ ID No 345 WNLERII SEQ ID
No 346 NLERIIPAW SEQ ID No 347 NLERIIPA SEQ ID No 348 NLERIIP SEQ
ID No 349 NLERII SEQ ID No 350 LERIIPAW SEQ ID No 351 LERIIPA SEQ
ID No 352 LERIIP SEQ ID No 353 LERII SEQ ID No 354 ERIIPAW SEQ ID
No 355 ERIIPA SEQ ID No 356 ERIIP SEQ ID No 357 RIIPAW SEQ ID No
358 RIIPA SEQ ID No 359 IIPAW SEQ ID No 360 INMAWFPEDQQVL SEQ ID No
361 INMAWFPEDQQV SEQ ID No 362 INMAWFPEDQQ SEQ ID No 363 INMAWFPEDQ
SEQ ID No 364 INMAWFPED SEQ ID No 365 INMAWFPE SEQ ID No 366
INMAWFP SEQ ID No 367 INMAWF SEQ ID No 368 INMAW SEQ ID No 369
NMAWFPEDQQVL SEQ ID No 370 NMAWFPEDQQV SEQ ID No 371 NMAWFPEDQQ SEQ
ID No 372 NMAWFPEDQ SEQ ID No 373 NMAWFPED SEQ ID No 374
NMAWFPE
SEQ ID No 375 NMAWFP SEQ ID No 376 NMAWF SEQ ID No 377 MAWFPEDQQVL
SEQ ID No 378 MAWFPEDQQV SEQ ID No 379 MAWFPEDQQ SEQ ID No 380
MAWFPEDQ SEQ ID No 381 MAWFPED SEQ ID No 382 MAWFPE SEQ ID No 383
MAWFP SEQ ID No 384 AWFPEDQQVL SEQ ID No 385 AWFPEDQQV SEQ ID No
386 AWFPEDQQ SEQ ID No 387 WFPEDQQVL SEQ ID No 388 WFPEDQQV SEQ ID
No 389 WFPEDQQ SEQ ID No 390 FPEDQQVL SEQ ID No 391 FPEDQQV SEQ ID
No 392 FPEDQQ SEQ ID No 393 PEDQQVL SEQ ID No 394 PEDQQV SEQ ID No
395 PEDQQ SEQ ID No 396 EDQQVL SEQ ID No 397 EDQQV SEQ ID No 398
DQQVL SEQ ID No 399 MGTVSSRRSWWPLPLLLLLLLLLGPAGARAQEDED
GDYEELVLALRSEEDGLAEAPEHGTTATFHRCAKD
PWRLPGTYVVVLKEETHLSQSERTARRLQAQAARR
GYLTKILHVFHGLLPGFLVKMSGDLLELALKLPHV
DYIEEDSSVFAQSIPWNLERITPPRYRADEYQPPD
GGSLVEVYLLDTSIQSDHREIEGRVMVTDFENVPE
EDGTRFHRQASKCDSHGTHLAGVVSGRDAGVAKGA
SMRSLRVLNCQGKGTVSGTLIGLEFIRKSQLVQPV
GPLVVLLPLAGGYSRVLNAACQRLARAGVVLVTAA
GNFRDDACLYSPASAPEVITVGATNAQDQPVTLGT
LGTNFGRCVDLFAPGEDIIGASSDCSTCFVSQSGT
SQAAAHVAGIAAMMLSAEPELTLAELRQRLIHFSA
KDVINEAWFPEDQRVLTPNLVAALPPSTHGAGWQL
FCRTVWSAHSGPTRMATAVARCAPDEELLSCSSFS
RSGKRRGERMEAQGGKLVCRAHNAFGGEGVYAIAR
CCLLPQANCSVHTAPPAEASMGTRVHCHQQGHVLT
GCSSHWEVEDLGTHKPPVLRPRGQPNQCVGHREAS
IHASCCHAPGLECKVKEHGIPAPQEQVTVACEEGW
TLTGCSALPGTSHVLGAYAVDNTCVVRSRDVSTTG STSEGAVTAVAICCRSRHLAQASQELQ SEQ
ID No 400 MGTHCSAWLRWPLLPLLPPLLLLLLLLCPTGAGAQ
DEDGDYEELMLALPSQEDGLADEAAHVATATFRRC
SKEAWRLPGTYIVVLMEETQRLQIEQTAHRLQTRA
ARRGYVIKVLHIFYDLFPGFLVKMSSDLLGLALKL
PHVEYIEEDSFVFAQSIPWNLERIIPAWHQTEEDR
SPDGSSQVEVYLLDTSIQGAHREIEGRVTITDFNS
VPEEDGTRFHRQASKCDSHGTHLAGVVSGRDAGVA
KGTSLHSLRVLNCQGKGTVSGTLIGLEFIRKSQLI
QPSGPLVVLLPLAGGYSRILNAACRHLARTGVVLV
AAAGNFRDDACLYSPASAPEVITVGATNAQDQPVT
LGTLGTNFGRCVDLFAPGKDIIGASSDCSTCFMSQ
SGTSQAAAHVAGIVARMLSREPTLTLAELRQRLIH
FSTKDVINMAWFPEDQQVLTPNLVATLPPSTHETG
GQLLCRTVWSAHSGPTRTATATARCAPEEELLSCS
SFSRSGRRRGDWIEAIGGQQVCKALNAFGGEGVYA
VARCCLVPRANCSIHNTPAARAGLETHVHCHQKDH
VLTGCSFHWEVEDLSVRRQPALRSRRQPGQCVGHQ
AASVYASCCHAPGLECKIKEHGISGPSEQVTVACE
AGWTLTGCNVLPGASLTLGAYSVDNLCVARVHDTA RADRTSGEATVAAAICCRSRPSAKASWVQ
SEQ ID No 401 SIPWNLERIGGC SEQ ID No 402 SIPWNLERGGC SEQ ID No 403
SIPWNLEGGC SEQ ID No 404 CGGSGRDAGVAKGA SEQ ID No 405
CGGSGRDAGVAKGT SEQ ID No 406 RDAGVAKGGC SEQ ID No 407 CSRHLAQASQELQ
SEQ ID No 408 CRSRPSAKASWVQ SEQ ID No 409 CGGDYEELVLALR SEQ ID No
410 CGGDYEELMLALP SEQ ID No 411 LVLALRSEEDGGC SEQ ID No 412
LMLALPSQEDGGC SEQ ID No 413 AKDPWRLPGGC SEQ ID No 414 SKEAWRLPGGC
SEQ ID No 415 CGGAARRGYLTK SEQ ID No 416 CGGAARRGYVIK SEQ ID No 417
FLVKMSGDLLELALKLPGGC SEQ ID No 418 FLVKMSSDLLGLALKLPGGC SEQ ID No
419 CGGEEDSSVFAQ SEQ ID No 420 SRHLAQASQELQ SEQ ID No 421
DYEELVLALR SEQ ID No 422 LVLALRSEEDG SEQ ID No 423 EEDSSVFAQ SEQ ID
No 424 SGRDAGVAKGT SEQ ID No 425 RSRPSAKASWVQ SEQ ID No 426
GDYEELMLALP SEQ ID No 427 LMLALPSQED SEQ ID No 428
FLVKMSSDLLGLALKLP SEQ ID No 429 RCAKDPWRLPG SEQ ID No 430
RCAKDPWRLP SEQ ID No 431 RCAKDPWRL SEQ ID No 432 RCAKDPWR SEQ ID No
433 RCAKDPW SEQ ID No 434 RCAKDP SEQ ID No 435 RCAKD SEQ ID No 436
CAKDPWRLPGT SEQ ID No 437 CAKDPWRLPG SEQ ID No 438 CAKDPWRLP SEQ ID
No 439 CAKDPWRL SEQ ID No 440 CAKDPWR SEQ ID No 441 CAKDPW SEQ ID
No 442 CAKDP SEQ ID No 443 AKDPWRLPGT SEQ ID No 444 AKDPWRLPG SEQ
ID No 445 AKDPWRLP SEQ ID No 446 AKDPWRL SEQ ID No 447 AKDPWR SEQ
ID No 448 AKDPW SEQ ID No 449 KDPWRLPGT SEQ ID No 450 KDPWRLPG SEQ
ID No 451 KDPWRLP SEQ ID No 452 KDPWRL SEQ ID No 453 KDPWR SEQ ID
No 454 DPWRLPGT SEQ ID No 455 DPWRLPG SEQ ID No 456 DPWRLP SEQ ID
No 457 DPWRL SEQ ID No 458 PWRLPGT SEQ ID No 459 PWRLPG SEQ ID No
460 PWRLP SEQ ID No 461 WRLPGT SEQ ID No 462 WRLPG SEQ ID No 464
AQAARRGYLTKI SEQ ID No 465 AQAARRGYLTK SEQ ID No 466 AQAARRGYLT SEQ
ID No 467 AQAARRGYL SEQ ID No 468 AQAARRGY SEQ ID No 469 AQAARRG
SEQ ID No 470 AQAARR SEQ ID No 471 AQAAR SEQ ID No 472 QAARRGYLTKIL
SEQ ID No 473 QAARRGYLTKI SEQ ID No 474 QAARRGYLTK SEQ ID No 475
QAARRGYLT SEQ ID No 476 QAARRGYL SEQ ID No 477 QAARRGY SEQ ID No
478 QAARRG SEQ ID No 479 QAARR SEQ ID No 480 AARRGYLTKIL SEQ ID No
481 AARRGYLTKI
SEQ ID No 482 AARRGYLTK SEQ ID No 483 AARRGYLT SEQ ID No 484
AARRGYL SEQ ID No 485 AARRGY SEQ ID No 486 AARRG SEQ ID No 487
ARRGYLTKIL SEQ ID No 488 ARRGYLTKI SEQ ID No 489 ARRGYLTK SEQ ID No
490 ARRGYLT SEQ ID No 491 ARRGYL SEQ ID No 492 ARRGY SEQ ID No 493
RRGYLTKIL SEQ ID No 494 RRGYLTKI SEQ ID No 495 RRGYLTK SEQ ID No
496 RRGYLT SEQ ID No 497 RRGYL SEQ ID No 498 RGYLTKIL SEQ ID No 499
RGYLTKI SEQ ID No 500 RGYLTK SEQ ID No 501 RGYLT SEQ ID No 502
GYLTKIL SEQ ID No 503 GYLTKI SEQ ID No 504 GYLTK SEQ ID No 505
YLTKIL SEQ ID No 506 YLTKI SEQ ID No 507 LTKIL SEQ ID No 508
RCSKEAWRLPGT SEQ ID No 509 RCSKEAWRLPG SEQ ID No 510 RCSKEAWRLP SEQ
ID No 511 RCSKEAWRL SEQ ID No 512 RCSKEAWR SEQ ID No 513 RCSKEAW
SEQ ID No 514 RCSKEA SEQ ID No 515 RCSKE SEQ ID No 516 CSKEAWRLPGT
SEQ ID No 517 CSKEAWRLPG SEQ ID No 518 CSKEAWRLP SEQ ID No 519
CSKEAWRL SEQ ID No 520 CSKEAWR SEQ ID No 521 CSKEAW SEQ ID No 522
CSKEA SEQ ID No 523 SKEAWRLPGT SEQ ID No 524 SKEAWRLPG SEQ ID No
525 SKEAWRLP SEQ ID No 526 SKEAWRL SEQ ID No 527 SKEAWR SEQ ID No
528 SKEAW SEQ ID No 529 KEAWRLPGT SEQ ID No 530 KEAWRLPG SEQ ID No
531 KEAWRLP SEQ ID No 532 KEAWRL SEQ ID No 533 KEAWR SEQ ID No 534
EAWRLPGT SEQ ID No 535 EAWRLPG SEQ ID No 536 EAWRLP SEQ ID No 537
EAWRL SEQ ID No 538 AWRLPGT SEQ ID No 539 AWRLPG SEQ ID No 540
AWRLP SEQ ID No 541 WRLPGT SEQ ID No 542 WRLPG SEQ ID No 543 RLPGT
SEQ ID No 544 TRAARRGYVIKVL SEQ ID No 545 TRAARRGYVIKV SEQ ID No
546 TRAARRGYVIK SEQ ID No 547 TRAARRGYVI SEQ ID No 548 TRAARRGYV
SEQ ID No 549 TRAARRGY SEQ ID No 550 TRAARRG SEQ ID No 551 TRAARR
SEQ ID No 552 TRAAR SEQ ID No 553 RAARRGYVIKVL SEQ ID No 554
RAARRGYVIKV SEQ ID No 555 RAARRGYVIK SEQ ID No 556 RAARRGYVI SEQ ID
No 557 RAARRGYV SEQ ID No 558 RAARRGY SEQ ID No 559 RAARRG SEQ ID
No 560 RAARR SEQ ID No 561 AARRGYVIKVL SEQ ID No 562 AARRGYVIKV SEQ
ID No 563 AARRGYVIK SEQ ID No 564 AARRGYVI SEQ ID No 565 AARRGYV
SEQ ID No 566 AARRGY SEQ ID No 567 AARRG SEQ ID No 568 ARRGYVIKVL
SEQ ID No 569 ARRGYVIKV SEQ ID No 570 ARRGYVIK SEQ ID No 571
ARRGYVI SEQ ID No 572 ARRGYV SEQ ID No 573 ARRGY SEQ ID No 574
RRGYVIKVL SEQ ID No 575 RRGYVIKV SEQ ID No 576 RRGYVIK SEQ ID No
577 RRGYVI SEQ ID No 578 RRGYV SEQ ID No 579 RGYVIKVL SEQ ID No 580
RGYVIKV SEQ ID No 581 RGYVIK SEQ ID No 582 RGYVI SEQ ID No 583
GYVIKVL SEQ ID No 584 GYVIKV SEQ ID No 585 GYVIK SEQ ID No 586
YVIKVL SEQ ID No 587 YVIKV SEQ ID No 588 VIKVL SEQ ID No 589
TCGTCGTTTTTCGGTGCTTTT SEQ ID No 590 TCGTCGTTTTTCGGTCGTTTT SEQ ID No
591 TCGTCGTTTTGTCGTTTTGTCGTT SEQ ID No 592 TCGTCGTTTCGTCGTTTTGTCGTT
SEQ ID No 593 TCGTCGTTTTGTCGTTTTTTTCGA SEQ ID No 594
TCGCGTCGTTCGGCGCGCGCCG SEQ ID No 595 TCGTCGACGTTCGGCGCGCGCCG SEQ ID
No 596 TCGGACGTTCGGCGCGCGCCG SEQ ID No 597 TCGGACGTTCGGCGCGCCG SEQ
ID No 598 TCGCGTCGTTCGGCGCGCCG SEQ ID No 599 TCGACGTTCGGCGCGCGCCG
SEQ ID No 600 TCGACGTTCGGCGCGCCG SEQ ID No 601 TCGCGTCGTTCGGCGCCG
SEQ ID No 602 TCGCGACGTTCGGCGCGCGCCG SEQ ID No 603
TCGTCGTTTTCGGCGCGCGCCG SEQ ID No 604 TCGTCGTTTTCGGCGGCCGCCG SEQ ID
No 605 TCGTCGTTTTACGGCGCCGTGCCG SEQ ID No 606
TCGTCGTTTTCGGCGCGCGCCGT SEQ ID No 607 TCGTCGACGATCGGCGCGCGCCG
Sequence CWU 1
1
607113PRTArtificialsynthetic 1Ile Gly Ala Ser Ser Asp Cys Ser Thr
Cys Phe Val Ser 1 5 10 212PRTArtificialsynthetic 2Ile Gly Ala Ser
Ser Asp Cys Ser Thr Cys Phe Val 1 5 10 311PRTArtificialsynthetic
3Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe 1 5 10
410PRTArtificialsynthetic 4Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys
1 5 10 59PRTArtificialsynthetic 5Ile Gly Ala Ser Ser Asp Cys Ser
Thr 1 5 68PRTArtificialsynthetic 6Ile Gly Ala Ser Ser Asp Cys Ser 1
5 77PRTArtificialsynthetic 7Ile Gly Ala Ser Ser Asp Cys 1 5
86PRTArtificialsynthetic 8Ile Gly Ala Ser Ser Asp 1 5
95PRTArtificialsynthetic 9Ile Gly Ala Ser Ser 1 5
1012PRTArtificialsynthetic 10Gly Ala Ser Ser Asp Cys Ser Thr Cys
Phe Val Ser 1 5 10 1111PRTArtificialsynthetic 11Gly Ala Ser Ser Asp
Cys Ser Thr Cys Phe Val 1 5 10 1210PRTArtificialsynthetic 12Gly Ala
Ser Ser Asp Cys Ser Thr Cys Phe 1 5 10 139PRTArtificialsynthetic
13Gly Ala Ser Ser Asp Cys Ser Thr Cys 1 5 148PRTArtificialsynthetic
14Gly Ala Ser Ser Asp Cys Ser Thr 1 5 157PRTArtificialsynthetic
15Gly Ala Ser Ser Asp Cys Ser 1 5 166PRTArtificialsynthetic 16Gly
Ala Ser Ser Asp Cys 1 5 175PRTArtificialsynthetic 17Gly Ala Ser Ser
Asp 1 5 1811PRTArtificialsynthetic 18Ala Ser Ser Asp Cys Ser Thr
Cys Phe Val Ser 1 5 10 1910PRTArtificialsynthetic 19Ala Ser Ser Asp
Cys Ser Thr Cys Phe Val 1 5 10 209PRTArtificialsynthetic 20Ala Ser
Ser Asp Cys Ser Thr Cys Phe 1 5 218PRTArtificialsynthetic 21Ala Ser
Ser Asp Cys Ser Thr Cys 1 5 227PRTArtificialsynthetic 22Ala Ser Ser
Asp Cys Ser Thr 1 5 236PRTArtificialsynthetic 23Ala Ser Ser Asp Cys
Ser 1 5 245PRTArtificialsynthetic 24Ala Ser Ser Asp Cys 1 5
2510PRTArtificialsynthetic 25Ser Ser Asp Cys Ser Thr Cys Phe Val
Ser 1 5 10 269PRTArtificialsynthetic 26Ser Ser Asp Cys Ser Thr Cys
Phe Val 1 5 278PRTArtificialsynthetic 27Ser Ser Asp Cys Ser Thr Cys
Phe 1 5 287PRTArtificialsynthetic 28Ser Ser Asp Cys Ser Thr Cys 1 5
296PRTArtificialsynthetic 29Ser Ser Asp Cys Ser Thr 1 5
305PRTArtificialsynthetic 30Ser Ser Asp Cys Ser 1 5
319PRTArtificialsynthetic 31Ser Asp Cys Ser Thr Cys Phe Val Ser 1 5
328PRTArtificialsynthetic 32Ser Asp Cys Ser Thr Cys Phe Val 1 5
337PRTArtificialsynthetic 33Ser Asp Cys Ser Thr Cys Phe 1 5
346PRTArtificialsynthetic 34Ser Asp Cys Ser Thr Cys 1 5
355PRTArtificialsynthetic 35Ser Asp Cys Ser Thr 1 5
368PRTArtificialsynthetic 36Asp Cys Ser Thr Cys Phe Val Ser 1 5
377PRTArtificialsynthetic 37Asp Cys Ser Thr Cys Phe Val 1 5
386PRTArtificialsynthetic 38Asp Cys Ser Thr Cys Phe 1 5
395PRTArtificialsynthetic 39Asp Cys Ser Thr Cys 1 5
407PRTArtificialsynthetic 40Cys Ser Thr Cys Phe Val Ser 1 5
416PRTArtificialsynthetic 41Cys Ser Thr Cys Phe Val 1 5
425PRTArtificialsynthetic 42Cys Ser Thr Cys Phe 1 5
436PRTArtificialsynthetic 43Ser Thr Cys Phe Val Ser 1 5
445PRTArtificialsynthetic 44Ser Thr Cys Phe Val 1 5
455PRTArtificialsynthetic 45Thr Cys Phe Val Ser 1 5
4615PRTArtificialsynthetic 46Glu Asp Gly Thr Arg Phe His Arg Gln
Ala Ser Lys Cys Asp Ser 1 5 10 15 4714PRTArtificialsynthetic 47Glu
Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp 1 5 10
4813PRTArtificialsynthetic 48Glu Asp Gly Thr Arg Phe His Arg Gln
Ala Ser Lys Cys 1 5 10 4912PRTArtificialsynthetic 49Glu Asp Gly Thr
Arg Phe His Arg Gln Ala Ser Lys 1 5 10 5011PRTArtificialsynthetic
50Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser 1 5 10
5110PRTArtificialsynthetic 51Glu Asp Gly Thr Arg Phe His Arg Gln
Ala 1 5 10 529PRTArtificialsynthetic 52Glu Asp Gly Thr Arg Phe His
Arg Gln 1 5 5314PRTArtificialsynthetic 53Asp Gly Thr Arg Phe His
Arg Gln Ala Ser Lys Cys Asp Ser 1 5 10 5413PRTArtificialsynthetic
54Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp 1 5 10
5512PRTArtificialsynthetic 55Asp Gly Thr Arg Phe His Arg Gln Ala
Ser Lys Cys 1 5 10 5611PRTArtificialsynthetic 56Asp Gly Thr Arg Phe
His Arg Gln Ala Ser Lys 1 5 10 5710PRTArtificialsynthetic 57Asp Gly
Thr Arg Phe His Arg Gln Ala Ser 1 5 10 589PRTArtificialsynthetic
58Asp Gly Thr Arg Phe His Arg Gln Ala 1 5 598PRTArtificialsynthetic
59Asp Gly Thr Arg Phe His Arg Gln 1 5 6013PRTArtificialsynthetic
60Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp Ser 1 5 10
6112PRTArtificialsynthetic 61Gly Thr Arg Phe His Arg Gln Ala Ser
Lys Cys Asp 1 5 10 6211PRTArtificialsynthetic 62Gly Thr Arg Phe His
Arg Gln Ala Ser Lys Cys 1 5 10 6310PRTArtificialsynthetic 63Gly Thr
Arg Phe His Arg Gln Ala Ser Lys 1 5 10 649PRTArtificialsynthetic
64Gly Thr Arg Phe His Arg Gln Ala Ser 1 5 658PRTArtificialsynthetic
65Gly Thr Arg Phe His Arg Gln Ala 1 5 667PRTArtificialsynthetic
66Gly Thr Arg Phe His Arg Gln 1 5 6712PRTArtificialsynthetic 67Thr
Arg Phe His Arg Gln Ala Ser Lys Cys Asp Ser 1 5 10
6811PRTArtificialsynthetic 68Thr Arg Phe His Arg Gln Ala Ser Lys
Cys Asp 1 5 10 6910PRTArtificialsynthetic 69Thr Arg Phe His Arg Gln
Ala Ser Lys Cys 1 5 10 709PRTArtificialsynthetic 70Thr Arg Phe His
Arg Gln Ala Ser Lys 1 5 718PRTArtificialsynthetic 71Thr Arg Phe His
Arg Gln Ala Ser 1 5 727PRTArtificialsynthetic 72Thr Arg Phe His Arg
Gln Ala 1 5 736PRTArtificialsynthetic 73Thr Arg Phe His Arg Gln 1 5
7411PRTArtificialsynthetic 74Arg Phe His Arg Gln Ala Ser Lys Cys
Asp Ser 1 5 10 7510PRTArtificialsynthetic 75Arg Phe His Arg Gln Ala
Ser Lys Cys Asp 1 5 10 769PRTArtificialsynthetic 76Arg Phe His Arg
Gln Ala Ser Lys Cys 1 5 778PRTArtificialsynthetic 77Arg Phe His Arg
Gln Ala Ser Lys 1 5 787PRTArtificialsynthetic 78Arg Phe His Arg Gln
Ala Ser 1 5 796PRTArtificialsynthetic 79Arg Phe His Arg Gln Ala 1 5
805PRTArtificialsynthetic 80Arg Phe His Arg Gln 1 5
8110PRTArtificialsynthetic 81Phe His Arg Gln Ala Ser Lys Cys Asp
Ser 1 5 10 829PRTArtificialsynthetic 82Phe His Arg Gln Ala Ser Lys
Cys Asp 1 5 838PRTArtificialsynthetic 83Phe His Arg Gln Ala Ser Lys
Cys 1 5 847PRTArtificialsynthetic 84Phe His Arg Gln Ala Ser Lys 1 5
856PRTArtificialsynthetic 85Phe His Arg Gln Ala Ser 1 5
865PRTArtificialsynthetic 86Phe His Arg Gln Ala 1 5
879PRTArtificialsynthetic 87His Arg Gln Ala Ser Lys Cys Asp Ser 1 5
888PRTArtificialsynthetic 88His Arg Gln Ala Ser Lys Cys Asp 1 5
897PRTArtificialsynthetic 89His Arg Gln Ala Ser Lys Cys 1 5
906PRTArtificialsynthetic 90His Arg Gln Ala Ser Lys 1 5
915PRTArtificialsynthetic 91His Arg Gln Ala Ser 1 5
928PRTArtificialsynthetic 92Arg Gln Ala Ser Lys Cys Asp Ser 1 5
937PRTArtificialsynthetic 93Arg Gln Ala Ser Lys Cys Asp 1 5
946PRTArtificialsynthetic 94Arg Gln Ala Ser Lys Cys 1 5
955PRTArtificialsynthetic 95Arg Gln Ala Ser Lys 1 5
967PRTArtificialsynthetic 96Gln Ala Ser Lys Cys Asp Ser 1 5
976PRTArtificialsynthetic 97Gln Ala Ser Lys Cys Asp 1 5
985PRTArtificialsynthetic 98Gln Ala Ser Lys Cys 1 5
996PRTArtificialsynthetic 99Ala Ser Lys Cys Asp Ser 1 5
1005PRTArtificialsynthetic 100Ala Ser Lys Cys Asp 1 5
1015PRTArtificialsynthetic 101Ser Lys Cys Asp Ser 1 5
10214PRTArtificialsynthetic 102Ser Ile Gln Ser Asp His Arg Glu Ile
Glu Gly Arg Val Met 1 5 10 10313PRTArtificialsynthetic 103Ser Ile
Gln Ser Asp His Arg Glu Ile Glu Gly Arg Val 1 5 10
10412PRTArtificialsynthetic 104Ser Ile Gln Ser Asp His Arg Glu Ile
Glu Gly Arg 1 5 10 10511PRTArtificialsynthetic 105Ser Ile Gln Ser
Asp His Arg Glu Ile Glu Gly 1 5 10 10610PRTArtificialsynthetic
106Ser Ile Gln Ser Asp His Arg Glu Ile Glu 1 5 10
1079PRTArtificialsynthetic 107Ser Ile Gln Ser Asp His Arg Glu Ile 1
5 10813PRTArtificialsynthetic 108Ile Gln Ser Asp His Arg Glu Ile
Glu Gly Arg Val Met 1 5 10 10912PRTArtificialsynthetic 109Ile Gln
Ser Asp His Arg Glu Ile Glu Gly Arg Val 1 5 10
11011PRTArtificialsynthetic 110Ile Gln Ser Asp His Arg Glu Ile Glu
Gly Arg 1 5 10 11110PRTArtificialsynthetic 111Ile Gln Ser Asp His
Arg Glu Ile Glu Gly 1 5 10 1129PRTArtificialsynthetic 112Ile Gln
Ser Asp His Arg Glu Ile Glu 1 5 1138PRTArtificialsynthetic 113Ile
Gln Ser Asp His Arg Glu Ile 1 5 11412PRTArtificialsynthetic 114Gln
Ser Asp His Arg Glu Ile Glu Gly Arg Val Met 1 5 10
11511PRTArtificialsynthetic 115Gln Ser Asp His Arg Glu Ile Glu Gly
Arg Val 1 5 10 11610PRTArtificialsynthetic 116Gln Ser Asp His Arg
Glu Ile Glu Gly Arg 1 5 10 1179PRTArtificialsynthetic 117Gln Ser
Asp His Arg Glu Ile Glu Gly 1 5 1188PRTArtificialsynthetic 118Gln
Ser Asp His Arg Glu Ile Glu 1 5 1197PRTArtificialsynthetic 119Gln
Ser Asp His Arg Glu Ile 1 5 12011PRTArtificialsynthetic 120Ser Asp
His Arg Glu Ile Glu Gly Arg Val Met 1 5 10
12110PRTArtificialsynthetic 121Ser Asp His Arg Glu Ile Glu Gly Arg
Val 1 5 10 1229PRTArtificialsynthetic 122Ser Asp His Arg Glu Ile
Glu Gly Arg 1 5 1238PRTArtificialsynthetic 123Ser Asp His Arg Glu
Ile Glu Gly 1 5 1247PRTArtificialsynthetic 124Ser Asp His Arg Glu
Ile Glu 1 5 1256PRTArtificialsynthetic 125Ser Asp His Arg Glu Ile 1
5 12610PRTArtificialsynthetic 126Asp His Arg Glu Ile Glu Gly Arg
Val Met 1 5 10 1279PRTArtificialsynthetic 127Asp His Arg Glu Ile
Glu Gly Arg Val 1 5 1288PRTArtificialsynthetic 128Asp His Arg Glu
Ile Glu Gly Arg 1 5 1297PRTArtificialsynthetic 129Asp His Arg Glu
Ile Glu Gly 1 5 1306PRTArtificialsynthetic 130Asp His Arg Glu Ile
Glu 1 5 1315PRTArtificialsynthetic 131Asp His Arg Glu Ile 1 5
1329PRTArtificialsynthetic 132His Arg Glu Ile Glu Gly Arg Val Met 1
5 1338PRTArtificialsynthetic 133His Arg Glu Ile Glu Gly Arg Val 1 5
1347PRTArtificialsynthetic 134His Arg Glu Ile Glu Gly Arg 1 5
1356PRTArtificialsynthetic 135His Arg Glu Ile Glu Gly 1 5
1365PRTArtificialsynthetic 136His Arg Glu Ile Glu 1 5
1378PRTArtificialsynthetic 137Arg Glu Ile Glu Gly Arg Val Met 1 5
1387PRTArtificialsynthetic 138Arg Glu Ile Glu Gly Arg Val 1 5
1396PRTArtificialsynthetic 139Arg Glu Ile Glu Gly Arg 1 5
1405PRTArtificialsynthetic 140Arg Glu Ile Glu Gly 1 5
1417PRTArtificialsynthetic 141Glu Ile Glu Gly Arg Val Met 1 5
1426PRTArtificialsynthetic 142Glu Ile Glu Gly Arg Val 1 5
1435PRTArtificialsynthetic 143Glu Ile Glu Gly Arg 1 5
1446PRTArtificialsynthetic 144Ile Glu Gly Arg Val Met 1 5
1455PRTArtificialsynthetic 145Ile Glu Gly Arg Val 1 5
1465PRTArtificialsynthetic 146Glu Gly Arg Val Met 1 5
14713PRTArtificialsynthetic 147Val Ser Gly Arg Asp Ala Gly Val Ala
Lys Gly Ala Ser 1 5 10 14812PRTArtificialsynthetic 148Val Ser Gly
Arg Asp Ala Gly Val Ala Lys Gly Ala 1 5 10
14911PRTArtificialsynthetic 149Val Ser Gly Arg Asp Ala Gly Val Ala
Lys Gly 1 5 10 15010PRTArtificialsynthetic 150Val Ser Gly Arg Asp
Ala Gly Val Ala Lys 1 5 10 1519PRTArtificialsynthetic 151Val Ser
Gly Arg Asp Ala Gly Val Ala 1 5 15212PRTArtificialsynthetic 152Ser
Gly Arg Asp Ala Gly Val Ala Lys Gly Ala Ser 1 5 10
15311PRTArtificialsynthetic 153Ser Gly Arg Asp Ala Gly Val Ala Lys
Gly Ala 1 5 10 15410PRTArtificialsynthetic 154Ser Gly Arg Asp Ala
Gly Val Ala Lys Gly 1 5 10 1559PRTArtificialsynthetic 155Ser Gly
Arg Asp Ala Gly Val Ala Lys 1 5 1568PRTArtificialsynthetic 156Ser
Gly Arg Asp Ala Gly Val Ala 1 5 15711PRTArtificialsynthetic 157Gly
Arg Asp Ala Gly Val Ala Lys Gly Ala Ser 1 5 10
15810PRTArtificialsynthetic 158Gly Arg Asp Ala Gly Val Ala Lys Gly
Ala 1 5 10 1599PRTArtificialsynthetic 159Gly Arg Asp Ala Gly Val
Ala Lys Gly 1 5 1608PRTArtificialsynthetic 160Gly Arg Asp Ala Gly
Val Ala Lys 1 5 1617PRTArtificialsynthetic 161Gly Arg Asp Ala Gly
Val Ala 1 5 16210PRTArtificialsynthetic 162Arg Asp Ala Gly Val Ala
Lys Gly Ala Ser 1 5 10 1639PRTArtificialsynthetic 163Arg Asp Ala
Gly Val Ala Lys Gly Ala 1 5 1648PRTArtificialsynthetic 164Arg Asp
Ala Gly Val Ala Lys Gly 1 5 1657PRTArtificialsynthetic 165Arg Asp
Ala Gly Val Ala Lys 1 5 1666PRTArtificialsynthetic 166Arg Asp Ala
Gly Val Ala 1 5 1679PRTArtificialsynthetic 167Asp Ala Gly Val Ala
Lys Gly Ala Ser 1 5 1688PRTArtificialsynthetic 168Asp Ala Gly Val
Ala Lys Gly Ala 1 5 1697PRTArtificialsynthetic 169Asp Ala Gly Val
Ala Lys Gly 1 5 1706PRTArtificialsynthetic 170Asp Ala Gly Val Ala
Lys 1 5 1715PRTArtificialsynthetic 171Asp Ala Gly Val Ala 1 5
1728PRTArtificialsynthetic 172Ala Gly Val Ala Lys Gly Ala Ser 1 5
1737PRTArtificialsynthetic 173Ala Gly Val Ala Lys Gly Ala 1 5
1746PRTArtificialsynthetic 174Ala Gly Val Ala Lys Gly 1 5
1755PRTArtificialsynthetic 175Ala Gly Val Ala Lys 1 5
1767PRTArtificialsynthetic 176Gly Val Ala Lys Gly Ala Ser 1 5
1776PRTArtificialsynthetic 177Gly Val Ala Lys Gly Ala 1 5
1785PRTArtificialsynthetic 178Gly Val Ala Lys Gly 1 5
1796PRTArtificialsynthetic 179Val Ala Lys Gly Ala Ser 1 5
1805PRTArtificialsynthetic 180Val Ala Lys Gly Ala 1 5
1815PRTArtificialsynthetic 181Ala Lys Gly Ala Ser 1 5
18213PRTArtificialsynthetic 182Ser Ile Pro Trp Asn Leu Glu Arg Ile
Thr Pro Pro Arg 1 5 10 18312PRTArtificialsynthetic 183Ser Ile Pro
Trp Asn Leu Glu Arg Ile Thr
Pro Pro 1 5 10 18411PRTArtificialsynthetic 184Ser Ile Pro Trp Asn
Leu Glu Arg Ile Thr Pro 1 5 10 18510PRTArtificialsynthetic 185Ser
Ile Pro Trp Asn Leu Glu Arg Ile Thr 1 5 10
1869PRTArtificialsynthetic 186Ser Ile Pro Trp Asn Leu Glu Arg Ile 1
5 1878PRTArtificialsynthetic 187Ser Ile Pro Trp Asn Leu Glu Arg 1 5
1887PRTArtificialsynthetic 188Ser Ile Pro Trp Asn Leu Glu 1 5
1896PRTArtificialsynthetic 189Ser Ile Pro Trp Asn Leu 1 5
1905PRTArtificialsynthetic 190Ser Ile Pro Trp Asn 1 5
19112PRTArtificialsynthetic 191Ile Pro Trp Asn Leu Glu Arg Ile Thr
Pro Pro Arg 1 5 10 19211PRTArtificialsynthetic 192Ile Pro Trp Asn
Leu Glu Arg Ile Thr Pro Pro 1 5 10 19310PRTArtificialsynthetic
193Ile Pro Trp Asn Leu Glu Arg Ile Thr Pro 1 5 10
1949PRTArtificialsynthetic 194Ile Pro Trp Asn Leu Glu Arg Ile Thr 1
5 1958PRTArtificialsynthetic 195Ile Pro Trp Asn Leu Glu Arg Ile 1 5
1967PRTArtificialsynthetic 196Ile Pro Trp Asn Leu Glu Arg 1 5
1976PRTArtificialsynthetic 197Ile Pro Trp Asn Leu Glu 1 5
1985PRTArtificialsynthetic 198Ile Pro Trp Asn Leu 1 5
19911PRTArtificialsynthetic 199Pro Trp Asn Leu Glu Arg Ile Thr Pro
Pro Arg 1 5 10 20010PRTArtificialsynthetic 200Pro Trp Asn Leu Glu
Arg Ile Thr Pro Pro 1 5 10 2019PRTArtificialsynthetic 201Pro Trp
Asn Leu Glu Arg Ile Thr Pro 1 5 2028PRTArtificialsynthetic 202Pro
Trp Asn Leu Glu Arg Ile Thr 1 5 2037PRTArtificialsynthetic 203Pro
Trp Asn Leu Glu Arg Ile 1 5 2046PRTArtificialsynthetic 204Pro Trp
Asn Leu Glu Arg 1 5 2055PRTArtificialsynthetic 205Pro Trp Asn Leu
Glu 1 5 20610PRTArtificialsynthetic 206Trp Asn Leu Glu Arg Ile Thr
Pro Pro Arg 1 5 10 2079PRTArtificialsynthetic 207Trp Asn Leu Glu
Arg Ile Thr Pro Pro 1 5 2088PRTArtificialsynthetic 208Trp Asn Leu
Glu Arg Ile Thr Pro 1 5 2097PRTArtificialsynthetic 209Trp Asn Leu
Glu Arg Ile Thr 1 5 2106PRTArtificialsynthetic 210Trp Asn Leu Glu
Arg Ile 1 5 2115PRTArtificialsynthetic 211Trp Asn Leu Glu Arg 1 5
2129PRTArtificialsynthetic 212Asn Leu Glu Arg Ile Thr Pro Pro Arg 1
5 2138PRTArtificialsynthetic 213Asn Leu Glu Arg Ile Thr Pro Pro 1 5
2147PRTArtificialsynthetic 214Asn Leu Glu Arg Ile Thr Pro 1 5
2156PRTArtificialsynthetic 215Asn Leu Glu Arg Ile Thr 1 5
2165PRTArtificialsynthetic 216Asn Leu Glu Arg Ile 1 5
2178PRTArtificialsynthetic 217Leu Glu Arg Ile Thr Pro Pro Arg 1 5
2187PRTArtificialsynthetic 218Leu Glu Arg Ile Thr Pro Pro 1 5
2196PRTArtificialsynthetic 219Leu Glu Arg Ile Thr Pro 1 5
2205PRTArtificialsynthetic 220Leu Glu Arg Ile Thr 1 5
2217PRTArtificialsynthetic 221Glu Arg Ile Thr Pro Pro Arg 1 5
2226PRTArtificialsynthetic 222Glu Arg Ile Thr Pro Pro 1 5
2235PRTArtificialsynthetic 223Glu Arg Ile Thr Pro 1 5
2246PRTArtificialsynthetic 224Arg Ile Thr Pro Pro Arg 1 5
2255PRTArtificialsynthetic 225Arg Ile Thr Pro Pro 1 5
2265PRTArtificialsynthetic 226Ile Thr Pro Pro Arg 1 5
22712PRTArtificialsynthetic 227Asn Ala Gln Asp Gln Pro Val Thr Leu
Gly Thr Leu 1 5 10 22811PRTArtificialsynthetic 228Asn Ala Gln Asp
Gln Pro Val Thr Leu Gly Thr 1 5 10 22910PRTArtificialsynthetic
229Asn Ala Gln Asp Gln Pro Val Thr Leu Gly 1 5 10
2309PRTArtificialsynthetic 230Asn Ala Gln Asp Gln Pro Val Thr Leu 1
5 2318PRTArtificialsynthetic 231Asn Ala Gln Asp Gln Pro Val Thr 1 5
2327PRTArtificialsynthetic 232Asn Ala Gln Asp Gln Pro Val 1 5
2336PRTArtificialsynthetic 233Asn Ala Gln Asp Gln Pro 1 5
2345PRTArtificialsynthetic 234Asn Ala Gln Asp Gln 1 5
23511PRTArtificialsynthetic 235Ala Gln Asp Gln Pro Val Thr Leu Gly
Thr Leu 1 5 10 23610PRTArtificialsynthetic 236Ala Gln Asp Gln Pro
Val Thr Leu Gly Thr 1 5 10 2379PRTArtificialsynthetic 237Ala Gln
Asp Gln Pro Val Thr Leu Gly 1 5 2388PRTArtificialsynthetic 238Ala
Gln Asp Gln Pro Val Thr Leu 1 5 2397PRTArtificialsynthetic 239Ala
Gln Asp Gln Pro Val Thr 1 5 2406PRTArtificialsynthetic 240Ala Gln
Asp Gln Pro Val 1 5 2415PRTArtificialsynthetic 241Ala Gln Asp Gln
Pro 1 5 24210PRTArtificialsynthetic 242Gln Asp Gln Pro Val Thr Leu
Gly Thr Leu 1 5 10 2439PRTArtificialsynthetic 243Gln Asp Gln Pro
Val Thr Leu Gly Thr 1 5 2448PRTArtificialsynthetic 244Gln Asp Gln
Pro Val Thr Leu Gly 1 5 2457PRTArtificialsynthetic 245Gln Asp Gln
Pro Val Thr Leu 1 5 2466PRTArtificialsynthetic 246Gln Asp Gln Pro
Val Thr 1 5 2475PRTArtificialsynthetic 247Gln Asp Gln Pro Val 1 5
2489PRTArtificialsynthetic 248Asp Gln Pro Val Thr Leu Gly Thr Leu 1
5 2498PRTArtificialsynthetic 249Asp Gln Pro Val Thr Leu Gly Thr 1 5
2507PRTArtificialsynthetic 250Asp Gln Pro Val Thr Leu Gly 1 5
2516PRTArtificialsynthetic 251Asp Gln Pro Val Thr Leu 1 5
2525PRTArtificialsynthetic 252Asp Gln Pro Val Thr 1 5
2538PRTArtificialsynthetic 253Gln Pro Val Thr Leu Gly Thr Leu 1 5
2547PRTArtificialsynthetic 254Gln Pro Val Thr Leu Gly Thr 1 5
2556PRTArtificialsynthetic 255Gln Pro Val Thr Leu Gly 1 5
2565PRTArtificialsynthetic 256Gln Pro Val Thr Leu 1 5
2577PRTArtificialsynthetic 257Pro Val Thr Leu Gly Thr Leu 1 5
2586PRTArtificialsynthetic 258Pro Val Thr Leu Gly Thr 1 5
2595PRTArtificialsynthetic 259Pro Val Thr Leu Gly 1 5
2606PRTArtificialsynthetic 260Val Thr Leu Gly Thr Leu 1 5
2615PRTArtificialsynthetic 261Val Thr Leu Gly Thr 1 5
2625PRTArtificialsynthetic 262Thr Leu Gly Thr Leu 1 5
26313PRTArtificialsynthetic 263Ile Asn Glu Ala Trp Phe Pro Glu Asp
Gln Arg Val Leu 1 5 10 26412PRTArtificialsynthetic 264Ile Asn Glu
Ala Trp Phe Pro Glu Asp Gln Arg Val 1 5 10
26511PRTArtificialsynthetic 265Ile Asn Glu Ala Trp Phe Pro Glu Asp
Gln Arg 1 5 10 26610PRTArtificialsynthetic 266Ile Asn Glu Ala Trp
Phe Pro Glu Asp Gln 1 5 10 2679PRTArtificialsynthetic 267Ile Asn
Glu Ala Trp Phe Pro Glu Asp 1 5 2688PRTArtificialsynthetic 268Ile
Asn Glu Ala Trp Phe Pro Glu 1 5 2697PRTArtificialsynthetic 269Ile
Asn Glu Ala Trp Phe Pro 1 5 2706PRTArtificialsynthetic 270Ile Asn
Glu Ala Trp Phe 1 5 2715PRTArtificialsynthetic 271Ile Asn Glu Ala
Trp 1 5 27212PRTArtificialsynthetic 272Asn Glu Ala Trp Phe Pro Glu
Asp Gln Arg Val Leu 1 5 10 27311PRTArtificialsynthetic 273Asn Glu
Ala Trp Phe Pro Glu Asp Gln Arg Val 1 5 10
27410PRTArtificialsynthetic 274Asn Glu Ala Trp Phe Pro Glu Asp Gln
Arg 1 5 10 2759PRTArtificialsynthetic 275Asn Glu Ala Trp Phe Pro
Glu Asp Gln 1 5 2768PRTArtificialsynthetic 276Asn Glu Ala Trp Phe
Pro Glu Asp 1 5 2777PRTArtificialsynthetic 277Asn Glu Ala Trp Phe
Pro Glu 1 5 2786PRTArtificialsynthetic 278Asn Glu Ala Trp Phe Pro 1
5 2795PRTArtificialsynthetic 279Asn Glu Ala Trp Phe 1 5
28011PRTArtificialsynthetic 280Glu Ala Trp Phe Pro Glu Asp Gln Arg
Val Leu 1 5 10 28110PRTArtificialsynthetic 281Glu Ala Trp Phe Pro
Glu Asp Gln Arg Val 1 5 10 2829PRTArtificialsynthetic 282Glu Ala
Trp Phe Pro Glu Asp Gln Arg 1 5 2838PRTArtificialsynthetic 283Glu
Ala Trp Phe Pro Glu Asp Gln 1 5 2847PRTArtificialsynthetic 284Glu
Ala Trp Phe Pro Glu Asp 1 5 2856PRTArtificialsynthetic 285Glu Ala
Trp Phe Pro Glu 1 5 2865PRTArtificialsynthetic 286Glu Ala Trp Phe
Pro 1 5 28710PRTArtificialsynthetic 287Ala Trp Phe Pro Glu Asp Gln
Arg Val Leu 1 5 10 2889PRTArtificialsynthetic 288Ala Trp Phe Pro
Glu Asp Gln Arg Val 1 5 2898PRTArtificialsynthetic 289Ala Trp Phe
Pro Glu Asp Gln Arg 1 5 2907PRTArtificialsynthetic 290Ala Trp Phe
Pro Glu Asp Gln 1 5 2916PRTArtificialsynthetic 291Ala Trp Phe Pro
Glu Asp 1 5 2925PRTArtificialsynthetic 292Ala Trp Phe Pro Glu 1 5
2939PRTArtificialsynthetic 293Trp Phe Pro Glu Asp Gln Arg Val Leu 1
5 2948PRTArtificialsynthetic 294Trp Phe Pro Glu Asp Gln Arg Val 1 5
2957PRTArtificialsynthetic 295Trp Phe Pro Glu Asp Gln Arg 1 5
2966PRTArtificialsynthetic 296Trp Phe Pro Glu Asp Gln 1 5
2975PRTArtificialsynthetic 297Trp Phe Pro Glu Asp 1 5
2988PRTArtificialsynthetic 298Phe Pro Glu Asp Gln Arg Val Leu 1 5
2997PRTArtificialsynthetic 299Phe Pro Glu Asp Gln Arg Val 1 5
3006PRTArtificialsynthetic 300Phe Pro Glu Asp Gln Arg 1 5
3015PRTArtificialsynthetic 301Phe Pro Glu Asp Gln 1 5
3027PRTArtificialsynthetic 302Pro Glu Asp Gln Arg Val Leu 1 5
3036PRTArtificialsynthetic 303Pro Glu Asp Gln Arg Val 1 5
3045PRTArtificialsynthetic 304Pro Glu Asp Gln Arg 1 5
3056PRTArtificialsynthetic 305Glu Asp Gln Arg Val Leu 1 5
3065PRTArtificialsynthetic 306Glu Asp Gln Arg Val 1 5
3075PRTArtificialsynthetic 307Asp Gln Arg Val Leu 1 5
30813PRTArtificialsynthetic 308Val Asp Tyr Ile Glu Glu Asp Ser Ser
Val Phe Ala Gln 1 5 10 30912PRTArtificialsynthetic 309Arg Cys Ala
Lys Asp Pro Trp Arg Leu Pro Gly Thr 1 5 10
31013PRTArtificialsynthetic 310Ala Gln Ala Ala Arg Arg Gly Tyr Leu
Thr Lys Ile Leu 1 5 10 31116PRTArtificialsynthetic 311Gly Asp Tyr
Glu Glu Leu Val Leu Ala Leu Arg Ser Glu Glu Asp Gly 1 5 10 15
31217PRTArtificialsynthetic 312Phe Leu Val Lys Met Ser Gly Asp Leu
Leu Glu Leu Ala Leu Lys Leu 1 5 10 15 Pro
31313PRTArtificialsynthetic 313Lys Gly Gly Ala Ser Ser Asp Cys Ser
Thr Cys Phe Val 1 5 10 31413PRTArtificialsynthetic 314Cys Gly Gly
Thr Arg Phe His Arg Gln Ala Ser Lys Cys 1 5 10
31515PRTArtificialsynthetic 315Cys Gly Ile Gln Ser Asp His Arg Glu
Ile Glu Gly Arg Val Cys 1 5 10 15 31613PRTArtificialsynthetic
316Cys Ser Gly Arg Asp Ala Gly Val Ala Lys Gly Ala Cys 1 5 10
31712PRTArtificialsynthetic 317Ser Ile Pro Trp Asn Leu Glu Arg Ile
Thr Pro Cys 1 5 10 31813PRTArtificialsynthetic 318Ala Ser Lys Cys
Gly Asp Gly Thr Arg Phe His Arg Gln 1 5 10
31910PRTArtificialsynthetic 319Ala Gly Cys Gly Thr Arg Phe His Arg
Gln 1 5 10 32013PRTArtificialsynthetic 320Gly Arg Val Cys Ile Gln
Ser Asp His Arg Glu Ile Glu 1 5 10 32113PRTArtificialsynthetic
321Ala Gly Val Ala Lys Gly Ala Gly Cys Ser Gly Arg Asp 1 5 10
32212PRTArtificialsynthetic 322Ser Ile Pro Trp Asn Leu Glu Arg Ile
Ile Pro Cys 1 5 10 32314PRTArtificialsynthetic 323Cys Gly Gly Ser
Ile Pro Trp Asn Leu Glu Arg Ile Ile Pro 1 5 10
32414PRTArtificialsynthetic 324Ser Ile Pro Trp Asn Leu Glu Arg Ile
Ile Pro Gly Gly Cys 1 5 10 32515PRTArtificialsynthetic 325Cys Gly
Gly Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu 1 5 10 15
32615PRTArtificialsynthetic 326Asn Ala Gln Asp Gln Pro Val Thr Leu
Gly Thr Leu Gly Gly Cys 1 5 10 15 32716PRTArtificialsynthetic
327Cys Gly Gly Ile Asn Met Ala Trp Phe Pro Glu Asp Gln Gln Val Leu
1 5 10 15 32815PRTArtificialsynthetic 328Asn Met Ala Trp Phe Pro
Glu Asp Gln Gln Val Leu Gly Gly Cys 1 5 10 15
329152PRTArtificialsynthetic 329Met Gly Thr Val Ser Ser Arg Arg Ser
Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu
Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25 30 Asp Glu Asp Gly Asp
Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu 35 40 45 Glu Asp Gly
Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 50 55 60 His
Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val 65 70
75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala
Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr
Lys Ile Leu 100 105 110 His Val Phe His Gly Leu Leu Pro Gly Phe Leu
Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu Leu Ala Leu Lys Leu
Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp Ser Ser Val Phe Ala
Gln 145 150 33013PRTArtificialsynthetic 330Ser Ile Pro Trp Asn Leu
Glu Arg Ile Ile Pro Ala Trp 1 5 10 33112PRTArtificialsynthetic
331Ser Ile Pro Trp Asn Leu Glu Arg Ile Ile Pro Ala 1 5 10
33211PRTArtificialsynthetic 332Ser Ile Pro Trp Asn Leu Glu Arg Ile
Ile Pro 1 5 10 33310PRTArtificialsynthetic 333Ser Ile Pro Trp Asn
Leu Glu Arg Ile Ile 1 5 10 33412PRTArtificialsynthetic 334Ile Pro
Trp Asn Leu Glu Arg Ile Ile Pro Ala Trp 1 5 10
33512PRTArtificialsynthetic 335Ile Pro Trp Asn Leu Glu Arg Ile Ile
Pro Ala Trp 1 5 10 33610PRTArtificialsynthetic 336Ile Pro Trp Asn
Leu Glu Arg Ile Ile Pro 1 5 10 3379PRTArtificialsynthetic 337Ile
Pro Trp Asn Leu Glu Arg Ile Ile 1 5 33811PRTArtificialsynthetic
338Pro Trp Asn Leu Glu Arg Ile Ile Pro Ala Trp 1 5 10
33910PRTArtificialsynthetic 339Pro Trp Asn Leu Glu Arg Ile Ile Pro
Ala 1 5 10 3409PRTArtificialsynthetic 340Pro Trp Asn Leu Glu Arg
Ile Ile Pro 1 5 3418PRTArtificialsynthetic 341Pro Trp Asn Leu Glu
Arg Ile Ile 1 5 34210PRTArtificialsynthetic 342Trp Asn Leu Glu Arg
Ile Ile Pro Ala Trp 1 5 10 3439PRTArtificialsynthetic 343Trp Asn
Leu Glu Arg Ile Ile Pro Ala 1 5 3448PRTArtificialsynthetic 344Trp
Asn Leu Glu Arg Ile Ile Pro 1 5 3457PRTArtificialsynthetic 345Trp
Asn Leu Glu Arg Ile Ile 1 5 3469PRTArtificialsynthetic 346Asn Leu
Glu Arg Ile Ile Pro Ala Trp 1 5 3478PRTArtificialsynthetic 347Asn
Leu Glu Arg Ile Ile Pro Ala 1 5 3487PRTArtificialsynthetic 348Asn
Leu Glu Arg Ile Ile Pro 1 5 3496PRTArtificialsynthetic 349Asn Leu
Glu Arg Ile Ile 1 5 3508PRTArtificialsynthetic 350Leu Glu Arg Ile
Ile Pro Ala
Trp 1 5 3517PRTArtificialsynthetic 351Leu Glu Arg Ile Ile Pro Ala 1
5 3526PRTArtificialsynthetic 352Leu Glu Arg Ile Ile Pro 1 5
3535PRTArtificialsynthetic 353Leu Glu Arg Ile Ile 1 5
3547PRTArtificialsynthetic 354Glu Arg Ile Ile Pro Ala Trp 1 5
3556PRTArtificialsynthetic 355Glu Arg Ile Ile Pro Ala 1 5
3565PRTArtificialsynthetic 356Glu Arg Ile Ile Pro 1 5
3576PRTArtificialsynthetic 357Arg Ile Ile Pro Ala Trp 1 5
3585PRTArtificialsynthetic 358Arg Ile Ile Pro Ala 1 5
3595PRTArtificialsynthetic 359Ile Ile Pro Ala Trp 1 5
36013PRTArtificialsynthetic 360Ile Asn Met Ala Trp Phe Pro Glu Asp
Gln Gln Val Leu 1 5 10 36112PRTArtificialsynthetic 361Ile Asn Met
Ala Trp Phe Pro Glu Asp Gln Gln Val 1 5 10
36211PRTArtificialsynthetic 362Ile Asn Met Ala Trp Phe Pro Glu Asp
Gln Gln 1 5 10 36310PRTArtificialsynthetic 363Ile Asn Met Ala Trp
Phe Pro Glu Asp Gln 1 5 10 3649PRTArtificialsynthetic 364Ile Asn
Met Ala Trp Phe Pro Glu Asp 1 5 3658PRTArtificialsynthetic 365Ile
Asn Met Ala Trp Phe Pro Glu 1 5 3667PRTArtificialsynthetic 366Ile
Asn Met Ala Trp Phe Pro 1 5 3676PRTArtificialsynthetic 367Ile Asn
Met Ala Trp Phe 1 5 3685PRTArtificialsynthetic 368Ile Asn Met Ala
Trp 1 5 36912PRTArtificialsynthetic 369Asn Met Ala Trp Phe Pro Glu
Asp Gln Gln Val Leu 1 5 10 37011PRTArtificialsynthetic 370Asn Met
Ala Trp Phe Pro Glu Asp Gln Gln Val 1 5 10
37110PRTArtificialsynthetic 371Asn Met Ala Trp Phe Pro Glu Asp Gln
Gln 1 5 10 3729PRTArtificialsynthetic 372Asn Met Ala Trp Phe Pro
Glu Asp Gln 1 5 3738PRTArtificialsynthetic 373Asn Met Ala Trp Phe
Pro Glu Asp 1 5 3747PRTArtificialsynthetic 374Asn Met Ala Trp Phe
Pro Glu 1 5 3756PRTArtificialsynthetic 375Asn Met Ala Trp Phe Pro 1
5 3765PRTArtificialsynthetic 376Asn Met Ala Trp Phe 1 5
37711PRTArtificialsynthetic 377Met Ala Trp Phe Pro Glu Asp Gln Gln
Val Leu 1 5 10 37810PRTArtificialsynthetic 378Met Ala Trp Phe Pro
Glu Asp Gln Gln Val 1 5 10 3799PRTArtificialsynthetic 379Met Ala
Trp Phe Pro Glu Asp Gln Gln 1 5 3808PRTArtificialsynthetic 380Met
Ala Trp Phe Pro Glu Asp Gln 1 5 3817PRTArtificialsynthetic 381Met
Ala Trp Phe Pro Glu Asp 1 5 3826PRTArtificialsynthetic 382Met Ala
Trp Phe Pro Glu 1 5 3835PRTArtificialsynthetic 383Met Ala Trp Phe
Pro 1 5 38410PRTArtificialsynthetic 384Ala Trp Phe Pro Glu Asp Gln
Gln Val Leu 1 5 10 3859PRTArtificialsynthetic 385Ala Trp Phe Pro
Glu Asp Gln Gln Val 1 5 3868PRTArtificialsynthetic 386Ala Trp Phe
Pro Glu Asp Gln Gln 1 5 3879PRTArtificialsynthetic 387Trp Phe Pro
Glu Asp Gln Gln Val Leu 1 5 3888PRTArtificialsynthetic 388Trp Phe
Pro Glu Asp Gln Gln Val 1 5 3897PRTArtificialsynthetic 389Trp Phe
Pro Glu Asp Gln Gln 1 5 3908PRTArtificialsynthetic 390Phe Pro Glu
Asp Gln Gln Val Leu 1 5 3917PRTArtificialsynthetic 391Phe Pro Glu
Asp Gln Gln Val 1 5 3926PRTArtificialsynthetic 392Phe Pro Glu Asp
Gln Gln 1 5 3937PRTArtificialsynthetic 393Pro Glu Asp Gln Gln Val
Leu 1 5 3946PRTArtificialsynthetic 394Pro Glu Asp Gln Gln Val 1 5
3955PRTArtificialsynthetic 395Pro Glu Asp Gln Gln 1 5
3966PRTArtificialsynthetic 396Glu Asp Gln Gln Val Leu 1 5
3975PRTArtificialsynthetic 397Glu Asp Gln Gln Val 1 5
3985PRTArtificialsynthetic 398Asp Gln Gln Val Leu 1 5
399692PRTArtificialsynthetic 399Met Gly Thr Val Ser Ser Arg Arg Ser
Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu
Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25 30 Asp Glu Asp Gly Asp
Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu 35 40 45 Glu Asp Gly
Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 50 55 60 His
Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val 65 70
75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala
Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr
Lys Ile Leu 100 105 110 His Val Phe His Gly Leu Leu Pro Gly Phe Leu
Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu Leu Ala Leu Lys Leu
Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp Ser Ser Val Phe Ala
Gln Ser Ile Pro Trp Asn Leu Glu Arg 145 150 155 160 Ile Thr Pro Pro
Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly 165 170 175 Gly Ser
Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp 180 185 190
His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val 195
200 205 Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys
Asp 210 215 220 Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg
Asp Ala Gly 225 230 235 240 Val Ala Lys Gly Ala Ser Met Arg Ser Leu
Arg Val Leu Asn Cys Gln 245 250 255 Gly Lys Gly Thr Val Ser Gly Thr
Leu Ile Gly Leu Glu Phe Ile Arg 260 265 270 Lys Ser Gln Leu Val Gln
Pro Val Gly Pro Leu Val Val Leu Leu Pro 275 280 285 Leu Ala Gly Gly
Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu 290 295 300 Ala Arg
Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp 305 310 315
320 Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val
325 330 335 Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr
Leu Gly 340 345 350 Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro
Gly Glu Asp Ile 355 360 365 Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys
Phe Val Ser Gln Ser Gly 370 375 380 Thr Ser Gln Ala Ala Ala His Val
Ala Gly Ile Ala Ala Met Met Leu 385 390 395 400 Ser Ala Glu Pro Glu
Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 405 410 415 His Phe Ser
Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp 420 425 430 Gln
Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr 435 440
445 His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His
450 455 460 Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala
Pro Asp 465 470 475 480 Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg
Ser Gly Lys Arg Arg 485 490 495 Gly Glu Arg Met Glu Ala Gln Gly Gly
Lys Leu Val Cys Arg Ala His 500 505 510 Asn Ala Phe Gly Gly Glu Gly
Val Tyr Ala Ile Ala Arg Cys Cys Leu 515 520 525 Leu Pro Gln Ala Asn
Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 530 535 540 Ser Met Gly
Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr 545 550 555 560
Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro 565
570 575 Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His
Arg 580 585 590 Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly
Leu Glu Cys 595 600 605 Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln
Glu Gln Val Thr Val 610 615 620 Ala Cys Glu Glu Gly Trp Thr Leu Thr
Gly Cys Ser Ala Leu Pro Gly 625 630 635 640 Thr Ser His Val Leu Gly
Ala Tyr Ala Val Asp Asn Thr Cys Val Val 645 650 655 Arg Ser Arg Asp
Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val 660 665 670 Thr Ala
Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser 675 680 685
Gln Glu Leu Gln 690 400694PRTArtificialsynthetic 400Met Gly Thr His
Cys Ser Ala Trp Leu Arg Trp Pro Leu Leu Pro Leu 1 5 10 15 Leu Pro
Pro Leu Leu Leu Leu Leu Leu Leu Leu Cys Pro Thr Gly Ala 20 25 30
Gly Ala Gln Asp Glu Asp Gly Asp Tyr Glu Glu Leu Met Leu Ala Leu 35
40 45 Pro Ser Gln Glu Asp Gly Leu Ala Asp Glu Ala Ala His Val Ala
Thr 50 55 60 Ala Thr Phe Arg Arg Cys Ser Lys Glu Ala Trp Arg Leu
Pro Gly Thr 65 70 75 80 Tyr Ile Val Val Leu Met Glu Glu Thr Gln Arg
Leu Gln Ile Glu Gln 85 90 95 Thr Ala His Arg Leu Gln Thr Arg Ala
Ala Arg Arg Gly Tyr Val Ile 100 105 110 Lys Val Leu His Ile Phe Tyr
Asp Leu Phe Pro Gly Phe Leu Val Lys 115 120 125 Met Ser Ser Asp Leu
Leu Gly Leu Ala Leu Lys Leu Pro His Val Glu 130 135 140 Tyr Ile Glu
Glu Asp Ser Phe Val Phe Ala Gln Ser Ile Pro Trp Asn 145 150 155 160
Leu Glu Arg Ile Ile Pro Ala Trp His Gln Thr Glu Glu Asp Arg Ser 165
170 175 Pro Asp Gly Ser Ser Gln Val Glu Val Tyr Leu Leu Asp Thr Ser
Ile 180 185 190 Gln Gly Ala His Arg Glu Ile Glu Gly Arg Val Thr Ile
Thr Asp Phe 195 200 205 Asn Ser Val Pro Glu Glu Asp Gly Thr Arg Phe
His Arg Gln Ala Ser 210 215 220 Lys Cys Asp Ser His Gly Thr His Leu
Ala Gly Val Val Ser Gly Arg 225 230 235 240 Asp Ala Gly Val Ala Lys
Gly Thr Ser Leu His Ser Leu Arg Val Leu 245 250 255 Asn Cys Gln Gly
Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu 260 265 270 Phe Ile
Arg Lys Ser Gln Leu Ile Gln Pro Ser Gly Pro Leu Val Val 275 280 285
Leu Leu Pro Leu Ala Gly Gly Tyr Ser Arg Ile Leu Asn Ala Ala Cys 290
295 300 Arg His Leu Ala Arg Thr Gly Val Val Leu Val Ala Ala Ala Gly
Asn 305 310 315 320 Phe Arg Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser
Ala Pro Glu Val 325 330 335 Ile Thr Val Gly Ala Thr Asn Ala Gln Asp
Gln Pro Val Thr Leu Gly 340 345 350 Thr Leu Gly Thr Asn Phe Gly Arg
Cys Val Asp Leu Phe Ala Pro Gly 355 360 365 Lys Asp Ile Ile Gly Ala
Ser Ser Asp Cys Ser Thr Cys Phe Met Ser 370 375 380 Gln Ser Gly Thr
Ser Gln Ala Ala Ala His Val Ala Gly Ile Val Ala 385 390 395 400 Arg
Met Leu Ser Arg Glu Pro Thr Leu Thr Leu Ala Glu Leu Arg Gln 405 410
415 Arg Leu Ile His Phe Ser Thr Lys Asp Val Ile Asn Met Ala Trp Phe
420 425 430 Pro Glu Asp Gln Gln Val Leu Thr Pro Asn Leu Val Ala Thr
Leu Pro 435 440 445 Pro Ser Thr His Glu Thr Gly Gly Gln Leu Leu Cys
Arg Thr Val Trp 450 455 460 Ser Ala His Ser Gly Pro Thr Arg Thr Ala
Thr Ala Thr Ala Arg Cys 465 470 475 480 Ala Pro Glu Glu Glu Leu Leu
Ser Cys Ser Ser Phe Ser Arg Ser Gly 485 490 495 Arg Arg Arg Gly Asp
Trp Ile Glu Ala Ile Gly Gly Gln Gln Val Cys 500 505 510 Lys Ala Leu
Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Val Ala Arg 515 520 525 Cys
Cys Leu Val Pro Arg Ala Asn Cys Ser Ile His Asn Thr Pro Ala 530 535
540 Ala Arg Ala Gly Leu Glu Thr His Val His Cys His Gln Lys Asp His
545 550 555 560 Val Leu Thr Gly Cys Ser Phe His Trp Glu Val Glu Asp
Leu Ser Val 565 570 575 Arg Arg Gln Pro Ala Leu Arg Ser Arg Arg Gln
Pro Gly Gln Cys Val 580 585 590 Gly His Gln Ala Ala Ser Val Tyr Ala
Ser Cys Cys His Ala Pro Gly 595 600 605 Leu Glu Cys Lys Ile Lys Glu
His Gly Ile Ser Gly Pro Ser Glu Gln 610 615 620 Val Thr Val Ala Cys
Glu Ala Gly Trp Thr Leu Thr Gly Cys Asn Val 625 630 635 640 Leu Pro
Gly Ala Ser Leu Thr Leu Gly Ala Tyr Ser Val Asp Asn Leu 645 650 655
Cys Val Ala Arg Val His Asp Thr Ala Arg Ala Asp Arg Thr Ser Gly 660
665 670 Glu Ala Thr Val Ala Ala Ala Ile Cys Cys Arg Ser Arg Pro Ser
Ala 675 680 685 Lys Ala Ser Trp Val Gln 690
40112PRTArtificialsynthetic 401Ser Ile Pro Trp Asn Leu Glu Arg Ile
Gly Gly Cys 1 5 10 40211PRTArtificialsynthetic 402Ser Ile Pro Trp
Asn Leu Glu Arg Gly Gly Cys 1 5 10 40310PRTArtificialsynthetic
403Ser Ile Pro Trp Asn Leu Glu Gly Gly Cys 1 5 10
40414PRTArtificialsynthetic 404Cys Gly Gly Ser Gly Arg Asp Ala Gly
Val Ala Lys Gly Ala 1 5 10 40514PRTArtificialsynthetic 405Cys Gly
Gly Ser Gly Arg Asp Ala Gly Val Ala Lys Gly Thr 1 5 10
40610PRTArtificialsynthetic 406Arg Asp Ala Gly Val Ala Lys Gly Gly
Cys 1 5 10 40713PRTArtificialsynthetic 407Cys Ser Arg His Leu Ala
Gln Ala Ser Gln Glu Leu Gln 1 5 10 40813PRTArtificialsynthetic
408Cys Arg Ser Arg Pro Ser Ala Lys Ala Ser Trp Val Gln 1 5 10
40913PRTArtificialsynthetic 409Cys Gly Gly Asp Tyr Glu Glu Leu Val
Leu Ala Leu Arg 1 5 10 41013PRTArtificialsynthetic 410Cys Gly Gly
Asp Tyr Glu Glu Leu Met Leu Ala Leu Pro 1 5 10
41113PRTArtificialsynthetic 411Leu Val Leu Ala Leu Arg Ser Glu Glu
Asp Gly Gly Cys 1 5 10 41213PRTArtificialsynthetic 412Leu Met Leu
Ala Leu Pro Ser Gln Glu Asp Gly Gly Cys 1 5 10
41311PRTArtificialsynthetic 413Ala Lys Asp Pro Trp Arg Leu Pro Gly
Gly Cys 1 5 10 41411PRTArtificialsynthetic 414Ser Lys Glu Ala Trp
Arg Leu Pro Gly Gly Cys 1 5 10 41512PRTArtificialsynthetic 415Cys
Gly Gly Ala Ala Arg Arg Gly Tyr Leu Thr Lys 1 5 10
41612PRTArtificialsynthetic 416Cys Gly Gly Ala Ala Arg Arg Gly Tyr
Val Ile Lys 1 5 10 41720PRTArtificialsynthetic 417Phe Leu Val Lys
Met Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu 1 5 10 15 Pro Gly
Gly Cys 20 41820PRTArtificialsynthetic 418Phe Leu Val Lys Met Ser
Ser Asp Leu Leu Gly Leu Ala Leu Lys Leu 1 5 10 15 Pro Gly Gly
Cys
20 41912PRTArtificialsynthetic 419Cys Gly Gly Glu Glu Asp Ser Ser
Val Phe Ala Gln 1 5 10 42012PRTArtificialsynthetic 420Ser Arg His
Leu Ala Gln Ala Ser Gln Glu Leu Gln 1 5 10
42110PRTArtificialsynthetic 421Asp Tyr Glu Glu Leu Val Leu Ala Leu
Arg 1 5 10 42211PRTArtificialsynthetic 422Leu Val Leu Ala Leu Arg
Ser Glu Glu Asp Gly 1 5 10 4239PRTArtificialsynthetic 423Glu Glu
Asp Ser Ser Val Phe Ala Gln 1 5 42411PRTArtificialsynthetic 424Ser
Gly Arg Asp Ala Gly Val Ala Lys Gly Thr 1 5 10
42512PRTArtificialsynthetic 425Arg Ser Arg Pro Ser Ala Lys Ala Ser
Trp Val Gln 1 5 10 42611PRTArtificialsynthetic 426Gly Asp Tyr Glu
Glu Leu Met Leu Ala Leu Pro 1 5 10 42710PRTArtificialsynthetic
427Leu Met Leu Ala Leu Pro Ser Gln Glu Asp 1 5 10
42817PRTArtificialsynthetic 428Phe Leu Val Lys Met Ser Ser Asp Leu
Leu Gly Leu Ala Leu Lys Leu 1 5 10 15 Pro
42911PRTArtificialsynthetic 429Arg Cys Ala Lys Asp Pro Trp Arg Leu
Pro Gly 1 5 10 43010PRTArtificialsynthetic 430Arg Cys Ala Lys Asp
Pro Trp Arg Leu Pro 1 5 10 4319PRTArtificialsynthetic 431Arg Cys
Ala Lys Asp Pro Trp Arg Leu 1 5 4328PRTArtificialsynthetic 432Arg
Cys Ala Lys Asp Pro Trp Arg 1 5 4337PRTArtificialsynthetic 433Arg
Cys Ala Lys Asp Pro Trp 1 5 4346PRTArtificialsynthetic 434Arg Cys
Ala Lys Asp Pro 1 5 4355PRTArtificialsynthetic 435Arg Cys Ala Lys
Asp 1 5 43611PRTArtificialsynthetic 436Cys Ala Lys Asp Pro Trp Arg
Leu Pro Gly Thr 1 5 10 43710PRTArtificialsynthetic 437Cys Ala Lys
Asp Pro Trp Arg Leu Pro Gly 1 5 10 4389PRTArtificialsynthetic
438Cys Ala Lys Asp Pro Trp Arg Leu Pro 1 5
4398PRTArtificialsynthetic 439Cys Ala Lys Asp Pro Trp Arg Leu 1 5
4407PRTArtificialsynthetic 440Cys Ala Lys Asp Pro Trp Arg 1 5
4416PRTArtificialsynthetic 441Cys Ala Lys Asp Pro Trp 1 5
4425PRTArtificialsynthetic 442Cys Ala Lys Asp Pro 1 5
44310PRTArtificialsynthetic 443Ala Lys Asp Pro Trp Arg Leu Pro Gly
Thr 1 5 10 4449PRTArtificialsynthetic 444Ala Lys Asp Pro Trp Arg
Leu Pro Gly 1 5 4458PRTArtificialsynthetic 445Ala Lys Asp Pro Trp
Arg Leu Pro 1 5 4467PRTArtificialsynthetic 446Ala Lys Asp Pro Trp
Arg Leu 1 5 4476PRTArtificialsynthetic 447Ala Lys Asp Pro Trp Arg 1
5 4485PRTArtificialsynthetic 448Ala Lys Asp Pro Trp 1 5
4499PRTArtificialsynthetic 449Lys Asp Pro Trp Arg Leu Pro Gly Thr 1
5 4508PRTArtificialsynthetic 450Lys Asp Pro Trp Arg Leu Pro Gly 1 5
4517PRTArtificialsynthetic 451Lys Asp Pro Trp Arg Leu Pro 1 5
4526PRTArtificialsynthetic 452Lys Asp Pro Trp Arg Leu 1 5
4535PRTArtificialsynthetic 453Lys Asp Pro Trp Arg 1 5
4548PRTArtificialsynthetic 454Asp Pro Trp Arg Leu Pro Gly Thr 1 5
4557PRTArtificialsynthetic 455Asp Pro Trp Arg Leu Pro Gly 1 5
4566PRTArtificialsynthetic 456Asp Pro Trp Arg Leu Pro 1 5
4575PRTArtificialsynthetic 457Asp Pro Trp Arg Leu 1 5
4587PRTArtificialsynthetic 458Pro Trp Arg Leu Pro Gly Thr 1 5
4596PRTArtificialsynthetic 459Pro Trp Arg Leu Pro Gly 1 5
4605PRTArtificialsynthetic 460Pro Trp Arg Leu Pro 1 5
4616PRTArtificialsynthetic 461Trp Arg Leu Pro Gly Thr 1 5
4625PRTArtificialsynthetic 462Trp Arg Leu Pro Gly 1 5
4635PRTArtificialsynthetic 463Arg Leu Pro Gly Thr 1 5
46412PRTArtificialsynthetic 464Ala Gln Ala Ala Arg Arg Gly Tyr Leu
Thr Lys Ile 1 5 10 46511PRTArtificialsynthetic 465Ala Gln Ala Ala
Arg Arg Gly Tyr Leu Thr Lys 1 5 10 46610PRTArtificialsynthetic
466Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr 1 5 10
4679PRTArtificialsynthetic 467Ala Gln Ala Ala Arg Arg Gly Tyr Leu 1
5 4688PRTArtificialsynthetic 468Ala Gln Ala Ala Arg Arg Gly Tyr 1 5
4697PRTArtificialsynthetic 469Ala Gln Ala Ala Arg Arg Gly 1 5
4706PRTArtificialsynthetic 470Ala Gln Ala Ala Arg Arg 1 5
4715PRTArtificialsynthetic 471Ala Gln Ala Ala Arg 1 5
47212PRTArtificialsynthetic 472Gln Ala Ala Arg Arg Gly Tyr Leu Thr
Lys Ile Leu 1 5 10 47311PRTArtificialsynthetic 473Gln Ala Ala Arg
Arg Gly Tyr Leu Thr Lys Ile 1 5 10 47410PRTArtificialsynthetic
474Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys 1 5 10
4759PRTArtificialsynthetic 475Gln Ala Ala Arg Arg Gly Tyr Leu Thr 1
5 4768PRTArtificialsynthetic 476Gln Ala Ala Arg Arg Gly Tyr Leu 1 5
4777PRTArtificialsynthetic 477Gln Ala Ala Arg Arg Gly Tyr 1 5
4786PRTArtificialsynthetic 478Gln Ala Ala Arg Arg Gly 1 5
4795PRTArtificialsynthetic 479Gln Ala Ala Arg Arg 1 5
48011PRTArtificialsynthetic 480Ala Ala Arg Arg Gly Tyr Leu Thr Lys
Ile Leu 1 5 10 48110PRTArtificialsynthetic 481Ala Ala Arg Arg Gly
Tyr Leu Thr Lys Ile 1 5 10 4829PRTArtificialsynthetic 482Ala Ala
Arg Arg Gly Tyr Leu Thr Lys 1 5 4838PRTArtificialsynthetic 483Ala
Ala Arg Arg Gly Tyr Leu Thr 1 5 4847PRTArtificialsynthetic 484Ala
Ala Arg Arg Gly Tyr Leu 1 5 4856PRTArtificialsynthetic 485Ala Ala
Arg Arg Gly Tyr 1 5 4865PRTArtificialsynthetic 486Ala Ala Arg Arg
Gly 1 5 48710PRTArtificialsynthetic 487Ala Arg Arg Gly Tyr Leu Thr
Lys Ile Leu 1 5 10 4889PRTArtificialsynthetic 488Ala Arg Arg Gly
Tyr Leu Thr Lys Ile 1 5 4898PRTArtificialsynthetic 489Ala Arg Arg
Gly Tyr Leu Thr Lys 1 5 4907PRTArtificialsynthetic 490Ala Arg Arg
Gly Tyr Leu Thr 1 5 4916PRTArtificialsynthetic 491Ala Arg Arg Gly
Tyr Leu 1 5 4925PRTArtificialsynthetic 492Ala Arg Arg Gly Tyr 1 5
4939PRTArtificialsynthetic 493Arg Arg Gly Tyr Leu Thr Lys Ile Leu 1
5 4948PRTArtificialsynthetic 494Arg Arg Gly Tyr Leu Thr Lys Ile 1 5
4957PRTArtificialsynthetic 495Arg Arg Gly Tyr Leu Thr Lys 1 5
4966PRTArtificialsynthetic 496Arg Arg Gly Tyr Leu Thr 1 5
4975PRTArtificialsynthetic 497Arg Arg Gly Tyr Leu 1 5
4988PRTArtificialsynthetic 498Arg Gly Tyr Leu Thr Lys Ile Leu 1 5
4997PRTArtificialsynthetic 499Arg Gly Tyr Leu Thr Lys Ile 1 5
5006PRTArtificialsynthetic 500Arg Gly Tyr Leu Thr Lys 1 5
5015PRTArtificialsynthetic 501Arg Gly Tyr Leu Thr 1 5
5027PRTArtificialsynthetic 502Gly Tyr Leu Thr Lys Ile Leu 1 5
5036PRTArtificialsynthetic 503Gly Tyr Leu Thr Lys Ile 1 5
5045PRTArtificialsynthetic 504Gly Tyr Leu Thr Lys 1 5
5056PRTArtificialsynthetic 505Tyr Leu Thr Lys Ile Leu 1 5
5065PRTArtificialsynthetic 506Tyr Leu Thr Lys Ile 1 5
5075PRTArtificialsynthetic 507Leu Thr Lys Ile Leu 1 5
50812PRTArtificialsynthetic 508Arg Cys Ser Lys Glu Ala Trp Arg Leu
Pro Gly Thr 1 5 10 50911PRTArtificialsynthetic 509Arg Cys Ser Lys
Glu Ala Trp Arg Leu Pro Gly 1 5 10 51010PRTArtificialsynthetic
510Arg Cys Ser Lys Glu Ala Trp Arg Leu Pro 1 5 10
5119PRTArtificialsynthetic 511Arg Cys Ser Lys Glu Ala Trp Arg Leu 1
5 5128PRTArtificialsynthetic 512Arg Cys Ser Lys Glu Ala Trp Arg 1 5
5137PRTArtificialsynthetic 513Arg Cys Ser Lys Glu Ala Trp 1 5
5146PRTArtificialsynthetic 514Arg Cys Ser Lys Glu Ala 1 5
5155PRTArtificialsynthetic 515Arg Cys Ser Lys Glu 1 5
51611PRTArtificialsynthetic 516Cys Ser Lys Glu Ala Trp Arg Leu Pro
Gly Thr 1 5 10 51710PRTArtificialsynthetic 517Cys Ser Lys Glu Ala
Trp Arg Leu Pro Gly 1 5 10 5189PRTArtificialsynthetic 518Cys Ser
Lys Glu Ala Trp Arg Leu Pro 1 5 5198PRTArtificialsynthetic 519Cys
Ser Lys Glu Ala Trp Arg Leu 1 5 5207PRTArtificialsynthetic 520Cys
Ser Lys Glu Ala Trp Arg 1 5 5216PRTArtificialsynthetic 521Cys Ser
Lys Glu Ala Trp 1 5 5225PRTArtificialsynthetic 522Cys Ser Lys Glu
Ala 1 5 52310PRTArtificialsynthetic 523Ser Lys Glu Ala Trp Arg Leu
Pro Gly Thr 1 5 10 5249PRTArtificialsynthetic 524Ser Lys Glu Ala
Trp Arg Leu Pro Gly 1 5 5258PRTArtificialsynthetic 525Ser Lys Glu
Ala Trp Arg Leu Pro 1 5 5267PRTArtificialsynthetic 526Ser Lys Glu
Ala Trp Arg Leu 1 5 5276PRTArtificialsynthetic 527Ser Lys Glu Ala
Trp Arg 1 5 5285PRTArtificialsynthetic 528Ser Lys Glu Ala Trp 1 5
5299PRTArtificialsynthetic 529Lys Glu Ala Trp Arg Leu Pro Gly Thr 1
5 5308PRTArtificialsynthetic 530Lys Glu Ala Trp Arg Leu Pro Gly 1 5
5317PRTArtificialsynthetic 531Lys Glu Ala Trp Arg Leu Pro 1 5
5326PRTArtificialsynthetic 532Lys Glu Ala Trp Arg Leu 1 5
5335PRTArtificialsynthetic 533Lys Glu Ala Trp Arg 1 5
5348PRTArtificialsynthetic 534Glu Ala Trp Arg Leu Pro Gly Thr 1 5
5357PRTArtificialsynthetic 535Glu Ala Trp Arg Leu Pro Gly 1 5
5366PRTArtificialsynthetic 536Glu Ala Trp Arg Leu Pro 1 5
5375PRTArtificialsynthetic 537Glu Ala Trp Arg Leu 1 5
5387PRTArtificialsynthetic 538Ala Trp Arg Leu Pro Gly Thr 1 5
5396PRTArtificialsynthetic 539Ala Trp Arg Leu Pro Gly 1 5
5405PRTArtificialsynthetic 540Ala Trp Arg Leu Pro 1 5
5416PRTArtificialsynthetic 541Trp Arg Leu Pro Gly Thr 1 5
5425PRTArtificialsynthetic 542Trp Arg Leu Pro Gly 1 5
5435PRTArtificialsynthetic 543Arg Leu Pro Gly Thr 1 5
54413PRTArtificialsynthetic 544Thr Arg Ala Ala Arg Arg Gly Tyr Val
Ile Lys Val Leu 1 5 10 54512PRTArtificialsynthetic 545Thr Arg Ala
Ala Arg Arg Gly Tyr Val Ile Lys Val 1 5 10
54611PRTArtificialsynthetic 546Thr Arg Ala Ala Arg Arg Gly Tyr Val
Ile Lys 1 5 10 54710PRTArtificialsynthetic 547Thr Arg Ala Ala Arg
Arg Gly Tyr Val Ile 1 5 10 5489PRTArtificialsynthetic 548Thr Arg
Ala Ala Arg Arg Gly Tyr Val 1 5 5498PRTArtificialsynthetic 549Thr
Arg Ala Ala Arg Arg Gly Tyr 1 5 5507PRTArtificialsynthetic 550Thr
Arg Ala Ala Arg Arg Gly 1 5 5516PRTArtificialsynthetic 551Thr Arg
Ala Ala Arg Arg 1 5 5525PRTArtificialsynthetic 552Thr Arg Ala Ala
Arg 1 5 55312PRTArtificialsynthetic 553Arg Ala Ala Arg Arg Gly Tyr
Val Ile Lys Val Leu 1 5 10 55411PRTArtificialsynthetic 554Arg Ala
Ala Arg Arg Gly Tyr Val Ile Lys Val 1 5 10
55510PRTArtificialsynthetic 555Arg Ala Ala Arg Arg Gly Tyr Val Ile
Lys 1 5 10 5569PRTArtificialsynthetic 556Arg Ala Ala Arg Arg Gly
Tyr Val Ile 1 5 5578PRTArtificialsynthetic 557Arg Ala Ala Arg Arg
Gly Tyr Val 1 5 5587PRTArtificialsynthetic 558Arg Ala Ala Arg Arg
Gly Tyr 1 5 5596PRTArtificialsynthetic 559Arg Ala Ala Arg Arg Gly 1
5 5605PRTArtificialsynthetic 560Arg Ala Ala Arg Arg 1 5
56111PRTArtificialsynthetic 561Ala Ala Arg Arg Gly Tyr Val Ile Lys
Val Leu 1 5 10 56210PRTArtificialsynthetic 562Ala Ala Arg Arg Gly
Tyr Val Ile Lys Val 1 5 10 5639PRTArtificialsynthetic 563Ala Ala
Arg Arg Gly Tyr Val Ile Lys 1 5 5648PRTArtificialsynthetic 564Ala
Ala Arg Arg Gly Tyr Val Ile 1 5 5657PRTArtificialsynthetic 565Ala
Ala Arg Arg Gly Tyr Val 1 5 5666PRTArtificialsynthetic 566Ala Ala
Arg Arg Gly Tyr 1 5 5675PRTArtificialsynthetic 567Ala Ala Arg Arg
Gly 1 5 56810PRTArtificialsynthetic 568Ala Arg Arg Gly Tyr Val Ile
Lys Val Leu 1 5 10 5699PRTArtificialsynthetic 569Ala Arg Arg Gly
Tyr Val Ile Lys Val 1 5 5708PRTArtificialsynthetic 570Ala Arg Arg
Gly Tyr Val Ile Lys 1 5 5717PRTArtificialsynthetic 571Ala Arg Arg
Gly Tyr Val Ile 1 5 5726PRTArtificialsynthetic 572Ala Arg Arg Gly
Tyr Val 1 5 5735PRTArtificialsynthetic 573Ala Arg Arg Gly Tyr 1 5
5749PRTArtificialsynthetic 574Arg Arg Gly Tyr Val Ile Lys Val Leu 1
5 5758PRTArtificialsynthetic 575Arg Arg Gly Tyr Val Ile Lys Val 1 5
5767PRTArtificialsynthetic 576Arg Arg Gly Tyr Val Ile Lys 1 5
5776PRTArtificialsynthetic 577Arg Arg Gly Tyr Val Ile 1 5
5785PRTArtificialsynthetic 578Arg Arg Gly Tyr Val 1 5
5798PRTArtificialsynthetic 579Arg Gly Tyr Val Ile Lys Val Leu 1 5
5807PRTArtificialsynthetic 580Arg Gly Tyr Val Ile Lys Val 1 5
5816PRTArtificialsynthetic 581Arg Gly Tyr Val Ile Lys 1 5
5825PRTArtificialsynthetic 582Arg Gly Tyr Val Ile 1 5
5837PRTArtificialsynthetic 583Gly Tyr Val Ile Lys Val Leu 1 5
5846PRTArtificialsynthetic 584Gly Tyr Val Ile Lys Val 1 5
5855PRTArtificialsynthetic 585Gly Tyr Val Ile Lys 1 5
5866PRTArtificialsynthetic 586Tyr Val Ile Lys Val Leu 1 5
5875PRTArtificialsynthetic 587Tyr Val Ile Lys Val 1 5
5885PRTArtificialsynthetic 588Val Ile Lys Val Leu 1 5
58921DNAArtificialsynthetic 589tcgtcgtttt tcggtgcttt t
2159021DNAArtificialsynthetic 590tcgtcgtttt tcggtcgttt t
2159124DNAArtificialsynthetic 591tcgtcgtttt gtcgttttgt cgtt
2459224DNAArtificialsynthetic 592tcgtcgtttc gtcgttttgt cgtt
2459324DNAArtificialsynthetic 593tcgtcgtttt gtcgtttttt tcga
2459422DNAArtificialsynthetic 594tcgcgtcgtt cggcgcgcgc cg
2259523DNAArtificialsynthetic 595tcgtcgacgt tcggcgcgcg ccg
2359621DNAArtificialsynthetic 596tcggacgttc ggcgcgcgcc g
2159719DNAArtificialsynthetic 597tcggacgttc ggcgcgccg
1959820DNAArtificialsynthetic 598tcgcgtcgtt cggcgcgccg
2059920DNAArtificialsynthetic 599tcgacgttcg gcgcgcgccg
2060018DNAArtificialsynthetic 600tcgacgttcg gcgcgccg
1860118DNAArtificialsynthetic 601tcgcgtcgtt cggcgccg
1860222DNAArtificialsynthetic 602tcgcgacgtt cggcgcgcgc cg
2260322DNAArtificialsynthetic 603tcgtcgtttt cggcgcgcgc cg
2260422DNAArtificialsynthetic 604tcgtcgtttt cggcggccgc cg
2260524DNAArtificialsynthetic 605tcgtcgtttt acggcgccgt gccg
2460623DNAArtificialsynthetic 606tcgtcgtttt cggcgcgcgc cgt
2360723DNAArtificialsynthetic 607tcgtcgacga tcggcgcgcg ccg 23
* * * * *