U.S. patent application number 12/021629 was filed with the patent office on 2008-09-18 for novel peptides that promote lipid efflux.
This patent application is currently assigned to LIPID SCIENCES, INC.. Invention is credited to H. Bryan Brewer.
Application Number | 20080227686 12/021629 |
Document ID | / |
Family ID | 39763317 |
Filed Date | 2008-09-18 |
United States Patent
Application |
20080227686 |
Kind Code |
A1 |
Brewer; H. Bryan |
September 18, 2008 |
Novel Peptides that Promote Lipid Efflux
Abstract
Disclosed herein are peptides with domains that promote lipid
efflux from cells and optionally possess at least one
anti-inflammatory domain or a domain that stimulates LCAT activity.
Provided herein are methods of using the peptides to treat or
inhibit diseases including dyslipidemic disorders, stroke and
myocardial infarction. Also provided are methods of detecting
plaque in vessels using the labeled peptides of the present
invention.
Inventors: |
Brewer; H. Bryan; (Potomac,
MD) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Assignee: |
LIPID SCIENCES, INC.
Pleasanton
CA
|
Family ID: |
39763317 |
Appl. No.: |
12/021629 |
Filed: |
January 29, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11764619 |
Jun 18, 2007 |
|
|
|
12021629 |
|
|
|
|
60814466 |
Jun 16, 2006 |
|
|
|
60847586 |
Sep 26, 2006 |
|
|
|
60858073 |
Nov 10, 2006 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
530/300; 530/324 |
Current CPC
Class: |
A61K 51/08 20130101;
A61P 43/00 20180101; A61P 9/00 20180101; C07K 14/775 20130101 |
Class at
Publication: |
514/2 ; 530/300;
530/324 |
International
Class: |
A61K 38/02 20060101
A61K038/02; C07K 2/00 20060101 C07K002/00; A61P 9/00 20060101
A61P009/00; C07K 14/435 20060101 C07K014/435 |
Claims
1. An isolated peptide comprising Formula I (A-B-C).sub.n, wherein
I A comprises a modified form of Helix 8 of ApoA-I (Helix 8'); C
comprises Helix 8 of ApoA-I; B comprises a linking group between A
and C; and n is an integer from 1 to 10.
2. The isolated peptide of claim 1, wherein: A comprises SEQ ID NO:
2 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys, a reverse orientation thereof (SEQ ID NO: 3 Lys Lys Thr
Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu), or a
substitution of SEQ ID NO: 2 or SEQ ID NO: 3; C comprises SEQ ID
NO: 14 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys, a reverse orientation thereof (SEQ ID NO: 15 Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu),
or a substitution of SEQ ID NO: 14 or SEQ ID NO: 15; B comprises
Pro; SEQ ID NO: 4 Lys Leu Ser Pro Leu; SEQ ID NO: 5 Leu Ser Pro
Leu; SEQ ID NO: 6 Ser Pro Leu; Pro Leu; SEQ ID NO: 7 Lys Leu Ser
Pro; SEQ ID NO: 8 Leu Ser Pro; Ser Pro; SEQ ID NO: 9 Leu Pro Ser
Leu Lys; SEQ ID NO: 10 Leu Pro Ser Leu; SEQ ID NO: 11 Leu Pro Ser;
Leu Pro; SEQ ID NO: 12 Pro Ser Leu Lys; SEQ ID NO: 13 Pro Ser Leu;
or Pro Ser; and n is 1.
3. The isolated peptide of claim 1, further comprising at least one
of G and H to form subgeneric Formula II G-(A-B-C).sub.n--H wherein
II G is absent or present and comrprises SEQ ID NO: 6 Ser Pro Leu;
Pro Leu; Leu; Ser Pro; Ser; Pro; SEQ ID NO: 11 Leu Pro Ser; Pro
Ser; Leu Pro; or a substitution thereof; and H is absent or present
and comprises SEQ ID NO: 16 Leu Asn Thr Gln; SEQ ID NO: 17 Asn Thr
Gln; Thr Gln; Gln; SEQ ID NO: 18 Leu Asn Thr; Leu Asn; Leu; SEQ ID
NO: 19 Gln Thr Asn Leu; SEQ ID NO: 20 Thr Asn Leu; Asn Leu; SEQ ID
NO: 21 Gln Thr Asn; Gln Thr; or a substitution thereof.
4. The isolated peptide of claim 3, wherein: A comprises SEQ ID NO:
2 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys; B comprises SEQ ID NO: 4 Lys Leu Ser Pro Leu; C comprises
SEQ ID NO: 14 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; G comprises SEQ ID NO: 6 Ser Pro Leu; H
comprises SEQ ID NO: 16 Leu Asn Thr Gln; and n is 1.
5. The isolated peptide of claim 3, wherein the peptide comprises
SEQ ID NO: 142 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln or a substitution thereof.
6. The isolated peptide of claim 1, wherein an N-terminal amino
acid is acetylated and/or a C-terminal amino acid is amidated.
7. The isolated peptide of claim 3, wherein an N-terminal amino
acid is acetylated and/or a C-terminal amino acid is amidated.
8. The isolated peptide of claim 7, wherein said isolated peptide
comprises SEQ ID NO: 602 Ac-Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln-NH.sub.2 or a substitution thereof
9. The isolated peptide of claim 1, wherein the isolated peptide
contains at least one D-amino acid.
10. The isolated peptide of claim 3, wherein the isolated peptide
contains at least one D-amino acid.
11. The isolated peptide of claim 1, further comprising a
label.
12. The isolated peptide of claim 3, further comprising a
label.
13. A pharmaceutical composition comprising the isolated peptide of
claim 1 and a pharmaceutically acceptable carrier.
14. A pharmaceutical composition comprising the isolated peptide of
claim 3 and a pharmaceutically acceptable carrier.
15. A method of treating disease in a human or non-human animal,
comprising administering to the human or non-human animal the
pharmaceutical composition of claim 13, thereby treating disease in
the human or non-human animal.
16. A method of treating disease in a human or non-human animal,
comprising administering to the human or non-human animal the
pharmaceutical composition of claim 14, thereby treating disease in
the human or non-human animal.
17. A method of treating or inhibiting a dyslipidemic disorder or a
vascular disorder in a human or non-human animal, comprising
administering to the animal or the human a therapeutically
effective amount of the pharmaceutical composition of claim 13,
thereby treating or inhibiting the dyslipidemic disorder or
vascular disorder in the human or non-human animal.
18. A method of treating or inhibiting a dyslipidemic disorder or a
vascular disorder in a human or non-human animal, comprising
administering to the animal or the human a therapeutically
effective amount of the pharmaceutical composition of claim 14,
thereby treating or inhibiting the dyslipidemic disorder or
vascular disorder in the human or non-human animal.
19. A method of visualizing plaque comprising: a) administering the
labeled peptide of claim 11 in an acceptable carrier to a vascular
system of human or non-human animal; and b) detecting the labeled
peptide bound to the plaque within the vascular system of the
animal or the human.
20. A method of visualizing plaque comprising: a) administering the
labeled peptide of claim 12 in an acceptable carrier to a vascular
system of human or non-human animal; and b) detecting the labeled
peptide bound to the plaque within the vascular system of the
animal or the human.
Description
PRIOR RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
benefit of priority from U.S. Ser. No. 11/764,619 (filed Jun. 18,
2007), which claims benefit of priority from U.S. Provisional
Patent Application Nos. 60/814,466 (filed Jun. 16, 2006),
60/847,586 (filed Sep. 26, 2006), and 60/858,073 (filed Nov. 10,
2006). Each of the preceding applications is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] This present invention relates to peptides or peptide
analogs that contain functional domains and promote lipid efflux.
These peptides or peptide analogs optionally contain one or more
anti-inflammatory domain and one or more domains that affects
lecithin cholesterol acyltransferase (LCAT) activity. The
disclosure further relates to methods for administering these
peptides in the treatment and prevention of dyslipidemic and
vascular disorders. The disclosure further relates to methods for
using these peptides in assays and in methods of imaging sites of
association of these peptides with receptors and with sites of
lipid deposition.
BACKGROUND OF THE INVENTION
[0003] Clearance of excess cholesterol from cells by high density
lipoproteins (HDL) is facilitated by the interaction of HDL
apolipoprotein with cell-surface binding sites or receptors.
Research has demonstrated an inverse correlation between the
occurrence of atherosclerosis events and levels of HDL and its most
abundant protein constituent, apolipoprotein A-I (ApoA-I)
(Panagotopulos et al., J. Biol. Chem. 277:39477-39484, 2002).
ApoA-I has been shown to promote lipid efflux from
ABCA1-transfected cells (Wang et al., J. Biol. Chem.
275:33053-33058, 2000; Hamon et al., Nat. Cell Biol. 2:399-406,
2000; and Remaley et al., Biochem. Biophys. Res. Commun.
280:818-823, 2001). However, the nature of the interaction between
ApoA-I and ABCA1 is not fully understood.
[0004] There exists a need for non-cytotoxic, synthetic peptide
mimetics of apolipoproteins that promote specific lipid efflux from
cells, perhaps by an ABCA1-dependent pathway, for use in the
treatment and prevention of cardiovascular diseases, such as
atherosclerosis.
[0005] Inflammation is believed to contribute to a variety of
disease processes, including vascular disease. Inflammation is
believed to contribute to the process of atherosclerosis, and
physicians often prescribe anti-inflammatory medicine, such as
aspirin, to patients with atherosclerosis, in conjunction with
statins, in an attempt to decrease the ongoing inflammatory process
that contributes to atherosclerosis and vascular disease. What is
needed are compounds that decrease inflammation.
[0006] LCAT is the major enzyme involved in the esterification of
free cholesterol present in circulating plasma lipoproteins, and a
major determinant of plasma HDL concentrations. What is needed are
compounds that increase LCAT activity.
[0007] What is needed are new compositions that promote lipid
efflux. What is also needed are new compositions with functional
domains that promote lipid efflux and have anti-inflammatory
properties and/or activity to modulate LCAT activity, or a
combination of domains that have anti-inflammatory properties and
the activity to modulate LCAT activity.
SUMMARY OF THE INVENTION
[0008] The present invention solves the problems described above by
providing novel peptide compositions with functional domains. In
several embodiments, these novel peptide compositions promote lipid
efflux. In several embodiments, these novel peptide compositions
promote lipid efflux and have anti-inflammatory properties. In
several embodiments, these novel peptide compositions promote lipid
efflux and have one or more anti-inflammatory domains. In several
embodiments, these novel peptide compositions promote lipid efflux
and have one or more domains that affect LCAT activity. In several
embodiments, these novel peptide compositions promote lipid efflux
and have one or more anti-inflammatory domains and one or more
domains that affect LCAT activity.
[0009] These novel peptide compositions may be labeled and used in
a variety of applications including the visualization of plaque in
vessels. These novel peptide compositions also display low
toxicity.
[0010] The peptides of the present invention may be combined with
pharmaceutically acceptable carriers and administered to a human or
an animal as a composition. Administration may be through any means
described herein and includes but is not limited to parenteral and
oral administration and also administration on a coated device such
as a stent or catheter.
[0011] Also described herein is a method of treating dyslipidemic
and vascular disorders in an animal or a human, including
administering to the animal or the human a therapeutically
effective amount of the peptides or peptide analogs thereof
presented herein. Dyslipidemic and vascular disorders amenable to
treatment with the peptides disclosed herein include, but are not
limited to, hyperlipidemia, hyperlipoproteinemia,
hypercholesterolemia, hypertriglyceridemia, HDL deficiency, apoA-I
deficiency, coronary artery disease, atherosclerosis, myocardial
infarction, stroke and inflammation secondary to stroke, ischemia,
ischemic stroke, thrombotic stroke, peripheral vascular disease
including peripheral arterial disease, restenosis, thrombosis,
acute coronary syndrome, and reperfusion myocardial injury.
[0012] The peptides of the present invention may be labeled with
labels known to one of ordinary skill in the art and used for
numerous applications, including but not limited to use in imaging
applications to visualize atherosclerotic plaque. Labels include
but are not limited to colorimetric labels, radiodense labels and
radioisotopic labels. Other uses include but are not limited to use
in assays, such as ELISAs, Western blots, radioimmunoassays and
radioreceptor assays.
[0013] The peptides of the present invention may be used to
generate antisera using techniques known to one of ordinary skill
in the art.
[0014] The amino acid sequences disclosed herein are shown using
standard three letter codes for amino acids, as defined in 37
C.F.R. 1.822 and as commonly known to one of ordinary skill in the
art. When the three letter designation for an amino acid is shown
in three upper case letters, for example SER for serine, the SER is
a D amino acid.
[0015] The generic formulae described herein refer to the helical
region 8 (Helix 8) of ApoA-I and a modified version of Helix 8 that
contains three amino acid substitutions. Helix 8 of ApoA-I contains
eighteen amino acids and consists of amino acid residues 222-239 of
ApoA-I. FIG. 1 shows the numbered amino acid sequence of ApoA-I
(SEQ ID NO: 1).
[0016] In one embodiment, the peptides of the present invention are
described by the following generic formula I:
(A-B-C).sub.n I
[0017] wherein A comprises a modified form of Helix 8 of ApoA-I
(also referred to herein as Helix 8'), C comprises Helix 8 of
ApoA-I, B comprises a linking group between A and C, and n is an
integer from 1 to 10.
[0018] In one embodiment, A (Helix 8') is SEQ ID NO: 2 Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys, or a
substitution thereof. These amino acids may also appear in reverse
orientation such that Lys is at the N-terminus and Leu is at the
C-terminus as in SEQ ID NO: 3 Lys Lys Thr Ala Glu Glu Leu Ala Ser
Leu Ala Ser Val Lys Ala Ser Glu Leu.
[0019] The modification of Helix 8 to yield Helix 8' involves
substitutions at positions 4 (Phe to Ala), 8 (Phe to Ala) and 15
(Tyr to Ala). It is to be understood that the present invention
encompasses other amino acid substitutions at these locations. At
positions 4 and 8, Phe may be substituted with Val, Leu, Gly, Thr,
Ser or gamma aminobutyric acid (GABA: GABA is also designated as
4Abu herein). At position 15, Tyr may be substituted with Val, Leu,
Gly, Thr, Ser or GABA. While not wanting to be bound by the
following statement, it is believed that A, the modified form of
helix 8 of ApoA-I, has a lower lipid affinity than C, the
unmodified form of helix 8 of ApoA-1.
[0020] In one embodiment, B is Pro; SEQ ID NO: 4 Lys Leu Ser Pro
Leu; SEQ ID NO: 5 Leu Ser Pro Leu; SEQ ID NO: 6 Ser Pro Leu; Pro
Leu; SEQ ID NO: 7 Lys Leu Ser Pro; SEQ ID NO: 8 Leu Ser Pro; Ser
Pro; or a substitution thereof. These amino acids may appear in
reverse orientation, for example, as in SEQ ID NO: 9 Leu Pro Ser
Leu Lys; SEQ ID NO: 10 Leu Pro Ser Leu; SEQ ID NO: 11 Leu Pro Ser;
Leu Pro; SEQ ID NO: 12 Pro Ser Leu Lys; SEQ ID NO: 13 Pro Ser Leu;
Pro Ser; or a substitution thereof.
[0021] In one embodiment, C is helix 8 of ApoA-I and is SEQ ID NO:
14 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys, or a substitution thereof. These amino acids may also
appear in reverse orientation such that Lys is at the N-terminus
and Leu is at the C-terminus as SEQ ID NO: 15 Lys Lys Thr Tyr Glu
Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu.
[0022] It is to be understood that A and C may be switched in
location as in C-B-A.
[0023] In a further embodiment, peptides of the present invention
are described by the following subgeneric formula II, in which one
or more additional elements indicated as variables G and H, are
added to formula I to make subgeneric formula II.
G-(A-B-C).sub.n--H II
[0024] (A-B-C), are as described in formula I above,
[0025] G is absent or present and is a peptide as defined in the
present specification. In one embodiment, G is SEQ ID NO: 6 Ser Pro
Leu; Pro Leu; Leu; Ser Pro; Ser; Pro; or a substitution thereof.
These amino acids can also appear in reverse orientation: SEQ ID
NO: 11 Leu Pro Ser; Pro Ser; Leu Pro; or a substitution thereof. It
is to be understood that one or more of the amino acids in the G
peptide may be D amino acids.
[0026] H is absent or present and is a peptide as defined in the
present specification. In one embodiment, H is SEQ ID NO: 16 Leu
Asn Thr Gln; SEQ ID NO: 17 Asn Thr Gln; Thr Gln; Gln; SEQ ID NO: 18
Leu Asn Thr; Leu Asn; Leu; or a substitution thereof. These amino
acids can also appear in reverse orientation: SEQ ID NO: 19 Gln Thr
Asn Leu; SEQ ID NO: 20 Thr Asn Leu; Asn Leu; SEQ ID NO: 21 Gln Thr
Asn; Gln Thr; or a substitution thereof. It is to be understood
that one or more of the amino acids in the H peptide may be D amino
acids.
[0027] The present invention also includes compositions comprising
combinations of individual peptides of the present invention in an
acceptable carrier. It is to be understood that a mixture of
peptides may include different amounts of the individual peptides.
For example, in one embodiment, each peptide component of the
combination may be present in a different relative percentage than
each other peptide component due to differences in relative
efficacy to promote lipid efflux or to provide one or more types of
anti-inflammatory activity.
[0028] It is to be understood that the letters in the generic
formulae I and II or in components thereof are defined by the text
that follows each letter and do not designate an individual amino
acid.
[0029] It is to be understood that in some embodiments, one or more
of the amino acids of the peptides of the present invention are D
amino acids. In one embodiment, the N-terminal amino acid, the
C-terminal amino acid or both are D amino acids. The presence of
these D amino acids can help protect against peptide degradation.
In another embodiment, all the amino acids of the peptides of the
present invention are D amino acids. This embodiment is useful for
protection against degradation following oral administration of a
pharmaceutical composition comprising the peptides of the present
invention.
[0030] The N and/or C-terminal amino acids may also be modified by
amidation, acetylation or other modifications known to one of
ordinary skill in the art. The peptides of the present invention
may optionally be acetylated at the N-terminus or the C-terminus
using techniques known to one of ordinary skill in the art. The
peptides of the present invention may optionally be amidated at the
N-terminus or the C-terminus using techniques known to one of
ordinary skill in the art. In one embodiment, the peptides of the
present invention are acetylated at the N-terminus, amidated at the
C-terminus, or both acetylated at the N-terminus and amidated at
the C-terminus. In some embodiments, the peptides of the present
invention may have both an acetylated N-terminus and a carboxy
terminal amide. In the present application, when a peptide is
acetylated on an N or C terminus, the letters Ac are indicated. In
the present application, when a peptide is amidated on an N or C
terminus, the designation NH.sub.2 is employed.
[0031] The present invention also includes compositions comprising
one or more individual peptides of the present invention in an
acceptable carrier. These peptides are as defined above and may be
labeled or unlabelled. It is to be understood that a mixture of
peptides, may include different amounts of the individual peptides.
For example, in one embodiment, each peptide component of the
combination may be present in a different relative percentage than
each other peptide component due to differences in relative
efficacy to promote lipid efflux or to provide one or more types of
anti-inflammatory activity.
[0032] Accordingly, it is an object of the present invention to
provide novel peptides.
[0033] Accordingly, it is an object of the present invention to
provide novel peptides that facilitate lipid efflux.
[0034] Yet another object of the present invention is to provide
novel peptides that facilitate lipid efflux and possess
anti-inflammatory biological activity.
[0035] Still another object of the present invention is to provide
novel peptides that facilitate lipid efflux and stimulate LCAT
activity.
[0036] Yet another object of the present invention is to provide
novel peptides that facilitate lipid efflux, possess
anti-inflammatory biological activity, and stimulate LCAT
activity.
[0037] It is another object of the present invention to provide new
methods for visualizing plaque using labeled peptides of the
present invention.
[0038] It is yet another object of the present invention to provide
new methods for the treatment of atherosclerosis, cardiovascular
disease and cerebrovascular disease in an animal or a human by
administering pharmaceutical compositions comprising one or more
peptides of the present invention with a pharmaceutically
acceptable carrier, or on a medical device.
[0039] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 shows the amino acid sequence of ApoA-I (SEQ ID NO:
1).
[0041] FIG. 2 is a schematic illustration of the statistically
significant, anti-inflammatory effects of ApoA-I (SEQ ID NO: 1) and
Peptide 1 (SEQ ID NO: 602), (each at 20 ug/ml) to decrease PMA (1
uM) induced expression of CD11b in human monocytes.
[0042] FIG. 3 is a schematic illustration of the dose dependent
stimulation of cholesterol efflux from the cells containing the
ABCA1 pathway (Peptide 1=SEQ ID NO: 602; Peptide 2=SEQ ID NO: 610;
Peptide 3=SEQ ID NO: 610; Peptide 4=SEQ ID NO: 142; and Peptide
5=SEQ ID NO: 602).
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides novel peptides. The present
invention solves the problems described above by providing novel
peptide compositions with functional domains. In some embodiments,
these novel peptide compositions promote lipid efflux. In some
embodiments, these novel peptide compositions promote lipid efflux
and have anti-inflammatory properties. In other embodiments, these
novel peptide compositions promote lipid efflux and have one or
more anti-inflammatory domains. In yet other embodiments, these
novel peptide compositions promote lipid efflux and have one or
more domains that affect activity of lecithin-cholesterol
acetyltransferase (LCAT), an enzyme that converts free cholesterol
to cholesterol ester on high-density lipoprotein (HDL). In several
embodiments, these novel peptide compositions promote lipid efflux
and have one or more anti-inflammatory domains and one or more
domain that affects LCAT activity.
[0044] Any of the peptides of the present invention may optionally
be acetylated at the N-terminus or the C-terminus using techniques
known to one of ordinary skill in the art. The peptides of the
present invention may optionally be amidated at the N-terminus or
the C-terminus using techniques known to one of ordinary skill in
the art. In one embodiment, the peptides of the present invention
are acetylated at the N-terminus, amidated at the C-terminus, or
both acetylated at the N-terminus and amidated at the C-terminus.
In some embodiments, the peptides of the present invention may have
both an acetylated N-terminus and a carboxy terminal amide. In the
present application, when a peptide is acetylated on an N or C
terminus, the letters Ac are indicated. In the present application,
when a peptide is amidated on an N or C terminus, the designation
NH.sub.2 is employed.
[0045] One or more of these peptides may be combined with an
acceptable carrier and administered as compositions to individuals
in order to provide lipid efflux and anti-inflammatory activities.
These compositions may be administered to treat dyslipidemic and
vascular disorders or to delay or prevent the onset or progression
of dyslipidemic and vascular disorders. In one embodiment, these
compositions may be administered to treat atherosclerosis or to
delay or prevent its onset or progression. These novel peptide
compositions may be labeled and used in a variety of applications
including the visualization of plaque in vessels. These novel
peptide compositions also display low toxicity.
I. ABBREVIATIONS
[0046] ABCA1: ATP-binding cassette transporter A1
[0047] apoA-I: apolipoprotein A-I
[0048] DMPC: dimyristoyl phosphatidyl choline
[0049] HDL: high-density lipoprotein
[0050] HPLC: high-pressure liquid chromatography
[0051] LDL: low-density lipoprotein
[0052] RBC: red blood cell
II. TERMS
[0053] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes VII, published by
Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN
0471186341); and other similar references.
[0054] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. Also, as used
herein, the term "comprises" means "includes." Hence "comprising A
or B" means including A, B, or A and B.
[0055] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0056] Analog, derivative or mimetic: An analog is a molecule that
differs in chemical structure from a parent compound, for example a
homolog (differing by an increment in the chemical structure, such
as a difference in the length of an alkyl chain), a molecular
fragment, a structure that differs by one or more functional
groups, a change in ionization. Structural analogs are often found
using quantitative structure activity relationships (QSAR), with
techniques such as those disclosed in Remington (The Science and
Practice of Pharmacology, 19th Edition (1995), chapter 28). A
derivative is a biologically active molecule derived from the base
structure. A mimetic is a molecule that mimics the activity of
another molecule, such as a biologically active molecule.
Biologically active molecules can include chemical structures that
mimic the biological activities of a compound.
[0057] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects, for
example, humans, non-human primates, dogs, cats, horses, and
cows.
[0058] Antibody: A protein (or protein complex) that includes one
or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon, and
mu constant region genes, as well as the myriad immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively.
[0059] The basic immunoglobulin (antibody) structural unit is
generally a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms "variable light chain" (V.sub.L) and "variable heavy chain"
(V.sub.H) refer, respectively, to these light and heavy chains.
[0060] As used herein, the term "antibody" includes intact
immunoglobulins as well as a number of well-characterized
fragments. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs)
that bind to target protein (or epitope within a protein or fusion
protein) would also be specific binding agents for that protein (or
epitope). These antibody fragments are as follows: (1) Fab, the
fragment which contains a monovalent antigen-binding fragment of an
antibody molecule produced by digestion of whole antibody with the
enzyme papain to yield an intact light chain and a portion of one
heavy chain; (2) Fab', the fragment of an antibody molecule
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody molecule;
(3) (Fab').sub.2, the fragment of the antibody obtained by treating
whole antibody with the enzyme pepsin without subsequent reduction;
(4) F(ab').sub.2, a dimer of two Fab' fragments held together by
two disulfide bonds; (5) Fv, a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and (6) single
chain antibody, a genetically engineered molecule containing the
variable region of the light chain, the variable region of the
heavy chain, linked by a suitable polypeptide linker as a
genetically fused single chain molecule. Methods of making these
fragments are routine (see, e.g., Harlow and Lane, Using
Antibodies: A Laboratory Manual, CSHL, New York, 1999).
[0061] Antibodies for use in the methods and compositions of this
disclosure can be monoclonal or polyclonal. Merely by way of
example, monoclonal antibodies can be prepared from murine
hybridomas according to the classical method of Kohler and Milstein
(Nature 256:495-97, 1975) or derivative methods thereof. Detailed
procedures for monoclonal antibody production are described in
Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New
York, 1999.
[0062] Domain: A domain of a protein is a part of a protein that
shares common structural, physiochemical and functional features;
for example hydrophobic, polar, globular, helical domains or
properties, for example a DNA binding domain, an ATP binding
domain, an anti-inflammatory domain, an LCAT activating domain and
the like. Some peptides of the present invention possess a domain
or domains that have more than one functional feature, for example
both lipid efflux activity and anti-inflammatory activity.
[0063] Dyslipidemic disorder: A disorder associated with any
altered amount of any or all of the lipids or lipoproteins in the
blood. Dyslipidemic disorders include, for example, hyperlipidemia,
hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia,
HDL deficiency, apoA-I deficiency, and cardiovascular disease
(e.g., coronary artery disease, atherosclerosis and
restenosis).
[0064] Efflux: The process of flowing out. As applied to the
results described herein, lipid efflux refers to a process whereby
lipid, such as cholesterol and phospholipid, is complexed with an
acceptor, such as an apolipoprotein or apolipoprotein peptide
mimetic, or a peptide of the present invention and removed from
vesicles or cells. "ABCA1-dependent lipid efflux" (or lipid efflux
by an "ABCA1-dependent pathway") refers to a process whereby
apolipoproteins, synthetic peptide mimetics of apolipoproteins, or
a peptide of the present invention, bind to a cell and efflux lipid
from the cell by a process that is facilitated by the ABCA1
transporter.
[0065] Helix: The molecular conformation of a spiral nature,
generated by regularly repeating rotations around the backbone
bonds of a macromolecule. Helix 8 of ApoA-I contains eighteen amino
acids and consists of amino acid residues 222-239 of ApoA-I. FIG. 1
shows the amino acid sequence of ApoA-I (SEQ ID NO: 361).
[0066] Hydrophobic: A hydrophobic (or lipophilic) group is
electrically neutral and nonpolar, and thus prefers other neutral
and nonpolar solvents or molecular environments. Examples of
hydrophobic molecules include alkanes, oils and fats.
[0067] Hydrophilic: A hydrophilic (or lipophobic) group is
electrically polarized and capable of H-bonding, enabling it to
dissolve more readily in water than in oil or other "non-polar"
solvents.
[0068] Inhibiting or treating a disease: Inhibiting the full
development of a disease, disorder or condition, for example, in a
subject who is at risk for a disease such as atherosclerosis and
cardiovascular disease. "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition after it has begun to develop. As used
herein, the term "ameliorating," with reference to a disease,
pathological condition or symptom, refers to any observable
beneficial effect of the treatment. The beneficial effect can be
evidenced, for example, by a delayed onset of clinical symptoms of
the disease in a susceptible subject, a reduction in severity of
some or all clinical symptoms of the disease, a slower progression
of the disease, a reduction in the number of relapses of the
disease, an improvement in the overall health or well-being of the
subject, or by other parameters well known in the art that are
specific to the particular disease.
[0069] Isolated/purified: An "isolated" or "purified" biological
component (such as a nucleic acid, peptide or protein) has been
substantially separated, produced apart from, or purified away from
other biological components in the cell of the organism in which
the component naturally occurs, that is, other chromosomal and
extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides
and proteins that have been "isolated" thus include nucleic acids
and proteins purified by standard purification methods. The term
also embraces nucleic acids, peptides and proteins prepared by
recombinant expression in a host cell as well as chemically
synthesized nucleic acids or proteins. The term "isolated" or
"purified" does not require absolute purity; rather, it is intended
as a relative term. Thus, for example, an isolated biological
component is one in which the biological component is more enriched
than the biological component is in its natural environment within
a cell. Preferably, a preparation is purified such that the
biological component represents at least 50%, such as at least 70%,
at least 90%, at least 95%, or greater of the total biological
component content of the preparation.
[0070] Label: A detectable compound or composition that is
conjugated directly or indirectly to another molecule to facilitate
detection of that molecule. Specific, non-limiting examples of
labels include fluorescent tags, calorimetric labels, dyes, beads,
enzymatic linkages, radiodense materials, and radioactive
isotopes.
[0071] Linker: A molecule that joins two other molecules, either
covalently, or through ionic, van der Waals or hydrogen bonds.
[0072] Lipid: A class of water-insoluble, or partially water
insoluble, oily or greasy organic substances, that are extractable
from cells and tissues by nonpolar solvents, such as chloroform or
ether. Types of lipids include triglycerides (e.g., natural fats
and oils composed of glycerin and fatty acid chains), glycolipids,
phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, and phosphatidylinositol), sphingolipids (e.g.,
sphingomyelin, cerebrosides and gangliosides), and sterols (e.g.,
cholesterol).
[0073] Lipid affinity: A measurement of the relative binding
affinity of an amphipathic .alpha.-helix for lipids. In some
embodiments, the lipid affinity of an amphipathic .alpha.-helix is
determined by one or more functional tests. Specific, non-limiting
examples of functional tests include: retention time on reverse
phase HPLC, surface monolayer exclusion pressure (Palgunachari et
al., Arterioscler. Thromb. Vasc. Biol. 16:328-338, 1996), binding
affinity to phospholipid vesicles (Palgunachari et al.,
Arterioscler. Thromb. Vasc. Biol. 16:328-338, 1996), and DMPC
vesicle solubilization (Remaley et al., J. Lipid Res. 44:828-836,
2003).
[0074] Further non-limiting examples of alternative methods of
calculating the lipid affinity of an amphipathic .alpha.-helix
include: total hydrophobic moment, total peptide hydrophobicity,
total peptide hydrophobicity per residue, hydrophobicity of amino
acids on the hydrophobic face, hydrophobicity per residue of amino
acids on the hydrophobic face, and calculated lipid affinity based
on predicted peptide penetration into phospholipid bilayers
(Palgunachari et al., Arterioscler. Thromb. Vasc. Biol. 16:328-338,
1996).
[0075] Non-cytotoxic: A non-cytotoxic compound is one that does not
substantially affect the viability or growth characteristics of a
cell at a dosage normally used to treat the cell or a subject.
Furthermore, the percentage of cells releasing intracellular
contents, such as LDH or hemoglobin, is low (e.g., about 10% or
less) in cells treated with a non-cytotoxic compound. Lipid efflux
from a cell that occurs by a non-cytotoxic compound results in the
removal of lipid from a cell by a process that maintains the
overall integrity of the cell membrane and does not lead to
significant cell toxicity.
[0076] Non-polar: A non-polar compound is one that does not have
concentrations of positive or negative electric charge. Non-polar
compounds, such as, for example, oil, are not well soluble in
water.
[0077] Peptide: A polymer in which the monomers are amino acid
residues which are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used. The amino acid sequences
disclosed herein are shown using three letter codes for amino
acids, as defined in 37 C.F.R. 1.822 and as commonly known to one
of ordinary skill in the art. When the three letter designation for
an amino acid, for example Ser for serine is shown in upper case,
SER, the serine is a D amino acid. The terms "peptide" or
"polypeptide" as used herein are intended to encompass any amino
acid sequence and include modified sequences such as glycoproteins.
The term "peptide" is specifically intended to cover naturally
occurring peptides, as well as those which are recombinantly or
synthetically produced. The term "residue" or "amino acid residue"
includes reference to an amino acid that is incorporated into a
peptide, polypeptide, or protein. As known to one of skill in the
art, the peptides presented herein are read from the N to the C
terminus i.e., from left to right. Accordingly, the N-terminal
amino acid in Leu Glu Lys is Leu and the C-terminal amino acid is
Lys.
[0078] Substitutions: Peptides of the present invention include
peptides with substitutions for amino acids in the peptide
sequence. Such substitutions may be conservative substitutions,
isosteric substitutions, substitutions between isosteric groups,
and non-conservative substitutions as defined herein.
[0079] Peptides of the present invention include conservatively
substituted peptides, wherein these conservative substitutions
occur at 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of
the amino acid residues. Peptides of the present invention include
peptides that are homologous at 50%, 60%, 70%, 80%, 90%, 95%, 97%,
98%, 99% of the entire sequence of the peptide.
[0080] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic compounds or molecules, such as one or
more peptides or peptide analogs and additional pharmaceutical
agents.
[0081] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0082] Phospholipid: A phospholipid consists of a water-soluble
polar head, linked to two water-insoluble non-polar tails (by a
negatively charged phosphate group). Both tails consist of a fatty
acid, each about 14 to about 24 carbon groups long. When placed in
an aqueous environment, phospholipids form a bilayer or micelle,
where the hydrophobic tails line up against each other. This forms
a membrane with hydrophilic heads on both sides. A phospholipid is
a lipid that is a primary component of animal cell membranes.
[0083] Polar: A polar molecule is one in which the centers of
positive and negative charge distribution do not converge. Polar
molecules are characterized by a dipole moment, which measures
their polarity, and are soluble in other polar compounds and
virtually insoluble in nonpolar compounds.
[0084] Recombinant nucleic acid: A sequence that is not naturally
occurring or has a sequence that is made by an artificial
combination of two otherwise separated segments of sequence. This
artificial combination is often accomplished by chemical synthesis
or, more commonly, by the artificial manipulation of isolated
segments of nucleic acids, for example, by genetic engineering
techniques such as those described in Sambrook et al. (ed.),
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989. The term recombinant includes nucleic acids that have been
altered solely by addition, substitution, or deletion of a portion
of the nucleic acid.
[0085] Therapeutically effective amount: A quantity of a specified
agent sufficient to achieve a desired effect in a subject being
treated with that agent. For example, this can be the amount of a
peptide or peptide analog useful in preventing, ameliorating,
and/or treating a dyslipidemic disorder (e.g., atherosclerosis) in
a subject. Ideally, a therapeutically effective amount of an agent
is an amount sufficient to prevent, ameliorate, and/or treat a
dyslipidemic disorder (e.g., atherosclerosis) in a subject without
causing a substantial cytotoxic effect (e.g., membrane
microsolubilization) in the subject. The effective amount of an
agent useful for preventing, ameliorating, and/or treating a
dyslipidemic disorder (e.g., atherosclerosis) in a subject will be
dependent on the subject being treated, the severity of the
disorder, and the manner of administration of the therapeutic
composition.
[0086] Transformed: A "transformed" cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. The term encompasses all techniques by which a nucleic
acid molecule might be introduced into such a cell, including
transfection with viral vectors, transformation with plasmid
vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration.
III. PEPTIDES OF THE PRESENT INVENTION AND ANALOGS THEREOF
[0087] A. (A-B-C).sub.nPeptides
[0088] In one embodiment, the peptides of the present invention are
described by the following generic formula I:
(A-B-C).sub.n. I
[0089] It is to be understood that A and C can be switched in
location as in C-B-A. One of ordinary skill in the art will
understand that A and/or C can contain one or more substitutions as
described herein.
[0090] One of ordinary skill in the art will also understand that
A, B, and/or C can contain one or more D-amino acids. In one
embodiment, the N-terminal amino acid, the C-terminal amino acid,
or both are D amino acids. The presence of these D amino acids can
help protect against peptide degradation. In another embodiment,
all the amino acids of the peptides of the present invention are D
amino acids. This embodiment is useful for protection against
degradation following oral administration of a pharmaceutical
composition comprising the peptides of the present invention. Any
number of amino acids (e.g., between one and all of the amino
acids) in the peptides of the invention can be D amino acids.
[0091] Peptides of the invention may contain an amino-terminal
acetyl group and/or a carboxy-terminal amide group.
[0092] i. Segment A
[0093] Segment A comprises a modified form of Helix 8 of ApoA-I,
also referred to herein as Helix 8', which is SEQ ID NO: 2 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys, or
a substitution thereof, as described herein. These amino acids may
also appear in reverse orientation such that Lys is at the
N-terminus and Leu is at the C-terminus as in SEQ ID NO: 3 Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu.
The modification of Helix 8 to yield Helix 8' involves
substitutions at positions 4 (Phe to Ala), 8 (Phe to Ala), and 15
(Tyr to Ala). It is to be understood that the present invention
encompasses other amino acid substitutions at these locations. At
positions 4 and 8 of SEQ ID NO: 2, Phe may be substituted with Val,
Leu, Gly, Thr, Ser or gamma-aminobutyric acid (GABA). At position
15 of SEQ ID NO: 2, Tyr may be substituted with Val, Leu, Gly, Thr,
Ser, or GABA. While not wanting to be bound by the following
statement, it is believed that A, the modified form of helix 8 of
ApoA-I, has a lower lipid affinity than segment C, the unmodified
form of helix 8 of ApoA-I. It is to be understood that one or more
of the amino acids in the Segment A peptide may be D amino
acids.
[0094] ii. Segment B
[0095] B comprises a linking peptide that covalently joins A and C
in the peptides of the present invention. In various embodiments
the connecting peptide B, can be: SEQ ID NO: 4 Lys Leu Ser Pro Leu;
SEQ ID NO: 5 Leu Ser Pro Leu; SEQ ID NO: 6 Ser Pro Leu; Pro Leu;
SEQ ID NO: 7 Lys Leu Ser Pro; SEQ ID NO: 8 Leu Ser Pro; Ser Pro; or
a substitution thereof. These amino acids may appear in reverse
orientation, for example, as in SEQ ID NO: 9 Leu Pro Ser Leu Lys;
SEQ ID NO: Leu Pro Ser Leu; SEQ ID NO: 11 Leu Pro Ser; Leu Pro; SEQ
ID NO: 12 Pro Ser Leu Lys; SEQ ID NO: 13 Pro Ser Leu; Pro Ser; or a
substitution thereof. In addition, B can comprise the amino acid
Pro or consist solely of the amino acid Pro. It is to be understood
that one or more of the amino acids in the B peptide may be D amino
acids.
[0096] iii. Segment C
[0097] C comprises Helix 8 of ApoA-I, which is SEQ ID NO: 14 Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys, or a substitution thereof. These amino acids may also appear
in reverse orientation such that Lys is at the N-terminus and Leu
is at the C-terminus as SEQ ID NO: 15 Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu. It is to be understood
that A and C may be switched in location as in C-B-A. It is to be
understood that one or more of the amino acids in the C peptide may
be D amino acids.
[0098] iv. Specific Examples of (A-B-C).sub.n Peptides
[0099] Below is a non-limiting subset of examples of peptides
represented by the basic formula (A-B-C).sub.n, in which A is Helix
8' and C is Helix 8. One of ordinary skill in the art will
understand that the (A-B-C).sub.n peptides of the invention include
any of the B sequences set forth herein. One of ordinary skill in
the art will understand that A and/or C can contain one or more
substitutions, for example but not limited to, isosteric
substitutions, substitutions between isosteric amino acid groups,
conservative substitutions, and nonconservative substitutions, as
described herein. A person of ordinary skill in the art will also
understand that A, B, and/or C can contain one or more D-amino
acids. Peptides of the invention may contain an amino-terminal
acetyl group and/or a carboxy-terminal amide group.
TABLE-US-00001 a. Embodiments Wherein A is Helix 8' and C is Helix
8: Variations in the B Group with A and C Intact as A-B-C SEQ ID
NO: 22 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 23 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro
Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 24 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 25
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; SEQ ID NO: 26 Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys;
SEQ ID NO: 27 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 28 Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys; SEQ ID NO: 29 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
30 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 31 Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 32 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 33 Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser
Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys; SEQ ID NO: 34 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
35 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 36 Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys. b.
Embodiments Wherein A is Helix 8' and C is Helix 8: Variations in
the B Group with A and C Intact but in a Different Orientation as
C-B-A SEQ ID NO: 37 Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 38
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 39 Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Ser Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys; SEQ ID NO: 40 Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 41 Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Lys
Leu Ser Pro Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys; SEQ ID NO: 42 Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Ser Pro Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO:
43 Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Ser Pro Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys; SEQ ID NO: 44 Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Pro Ser Leu
LysLeu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys; SEQ ID NO: 45 Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 46
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Pro Ser Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys; SEQ ID NO: 47 Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Pro Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID
NO: 48 Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Pro Ser Leu Lys Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 49
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Pro Ser Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys; SEQ ID NO: 50 Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Pro Ser Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID
NO: 51 Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Pro Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys. c. Variations in the B Group with A and C
Intact as A-B-C with the Amino Acids in A and C in Reverse
Orientation SEQ ID NO: 52 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu
Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu Lys Lys Thr Tyr
Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO:
53 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser
Glu Leu Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe
Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 54 Lys Lys Thr Ala Glu Glu
Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Ser Pro Leu Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu;
SEQ ID NO: 55 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val
Lys Ala Ser Glu Leu Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 56 Lys Lys Thr Ala Glu
Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro
Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu; SEQ ID NO: 57 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser
Val Lys Ala Ser Glu Leu Leu Ser Pro Lys Lys Thr Tyr Glu Glu Leu Ala
Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 58 Lys Lys Thr
Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Ser Pro
Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu; SEQ ID NO: 59 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser
Val Lys Ala Ser Glu Leu Leu Pro Ser Leu Lys Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 60 Lys
Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu
Leu Pro Ser Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val
Lys Phe Ser Glu Leu; SEQ ID NO: 61 Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Leu Pro Ser Lys Lys Thr Tyr
Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO:
62 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser
Glu Leu Leu Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val
Lys Phe Ser Glu Leu; SEQ ID NO: 63 Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Ser Leu Lys Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID
NO: 64 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala
Ser Glu Leu Pro Ser Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe
Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 65 Lys Lys Thr Ala Glu Glu
Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Ser Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID
NO: 66 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala
Ser Glu Leu Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val
Lys Phe Ser Glu Leu. d. Variations in the B Group with A and C
Intact as C-B-A with the Amino Acids in C and A in Reverse
Orientation SEQ ID NO: 67 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu Lys Leu Ser Pro Leu Lys Lys Thr Ala
Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO:
68 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser
Glu Leu Leu Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala
Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 69 Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Ser Pro Leu Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu;
SEQ ID NO: 70 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val
Lys Phe Ser Glu Leu Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu
Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 71 Lys Lys Thr Tyr Glu
Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Lys Leu Ser Pro
Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu
Leu; SEQ ID NO: 72 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser Glu Leu Leu Ser Pro Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 73 Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Ser Pro
Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu
Leu; SEQ ID NO: 74 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser Glu Leu Leu Pro Ser Leu Lys Lys Lys Thr Ala Glu Glu
Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 75 Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu
Leu Pro Ser Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val
Lys Ala Ser Glu Leu; SEQ ID NO: 76 Lys Lys Thr Tyr Glu Glu Leu Ala
Ser Leu Phe Ser
Val Lys Phe Ser Glu Leu Leu Pro Ser Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 77 Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Pro
Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu
Leu; SEQ ID NO: 78 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser Glu Leu Pro Ser Leu Lys Lys Lys Thr Ala Glu Glu Leu
Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 79 Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Pro
Ser Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala
Ser Glu Leu; SEQ ID NO: 80 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu Pro Ser Lys Lys Thr Ala Glu Glu Leu
Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 81 Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Pro
Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu
Leu. e. Variations in the B Group with A and C Intact as A-B-C with
the Amino Acids in A in Reverse Orientation SEQ ID NO: 82 Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; SEQ ID NO: 83 Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Leu Ser Pro Leu; SEQ ID NO:
84 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser
Glu Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 85 Lys Lys Thr Ala Glu Glu Leu
Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Leu; SEQ ID NO: 86
Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu
Leu Lys Leu Ser Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 87 Lys Lys Thr Ala Glu Glu Leu
Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Leu Ser Pro Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID
NO: 88 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala
Ser Glu Leu Ser Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 89 Lys Lys Thr Ala Glu Glu Leu
Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Leu Pro Ser Leu Lys Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 90 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser
Val Lys Ala Ser Glu Leu Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 91 Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Leu
Pro Ser Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 92 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu
Ala Ser Val Lys Ala Ser Glu Leu Leu Pro Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 93 Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro
Ser Leu Lys Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys; SEQ ID NO: 94 Lys Lys Thr Ala Glu Glu Leu Ala Ser
Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Ser Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 95
Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu
Leu Pro Ser Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys; SEQ ID NO: 96 Lys Lys Thr Ala Glu Glu Leu Ala Ser
Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys. f. Variations in the B
Group with A and C Intact as A-B-C with the Amino Acids in C in
Reverse Orientation SEQ ID NO: 97 Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu;
SEQ ID NO: 98 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala
Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 99 Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro
Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser
Glu Leu; SEQ ID NO: 100 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala
Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 101 Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu
Ser Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe
Ser Glu Leu; SEQ ID NO: 102 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 103 Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Ser Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe
Ser Glu Leu; SEQ ID NO: 104 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu; SEQ ID NO: 105 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 106 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Pro Ser Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe
Ser Glu Leu; SEQ ID NO: 107 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 108 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Ser Leu Lys Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys
Phe Ser Glu Leu; SEQ ID NO: 109 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Lys Thr Tyr Glu
Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 110
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Pro Ser Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys
Phe Ser Glu Leu; SEQ ID NO: 111 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu. g. Variations in the B
Group with A and C Intact as C-B-A with the Amino Acids in A in
Reverse Orientation SEQ ID NO: 112 Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Lys Leu Ser Pro Leu Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu;
SEQ ID NO: 113 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 114 Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Ser Pro
Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser
Glu Leu; SEQ ID NO: 115 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 116 Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Lys Leu
Ser Pro Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala
Ser Glu Leu; SEQ ID NO: 117 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Ser Pro Lys Lys Thr Ala Glu Glu
Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 118 Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Ser Pro Lys Lys Thr Ala Glu Glu Leu Ala Leu Ala Ser Val Lys Ala Ser
Glu Leu; SEQ ID NO: 119 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Pro Ser Leu Lys Lys Lys Thr Ala Glu
Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 120
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Pro Ser Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser
Val Lys Ala Ser Glu Leu; SEQ ID NO: 121 Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Pro Ser Lys Lys Thr
Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID
NO: 122 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Pro Lys Lys Thr Ala Glu Glu Leu Ala Leu Ala Ser Val
Lys Ala Ser Glu Leu; SEQ ID NO: 123 Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Pro Ser Leu Lys Lys Lys Thr
Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID
NO: 124 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Pro Ser Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala
Ser Val Lys Ala Ser Glu Leu; SEQ ID NO: 125 Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Pro Ser Lys Lys Thr
Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu; SEQ ID
NO: 126 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Pro Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val
Lys Ala Ser Glu Leu. h. Variations in the B Group with A and C
Intact as C-B-A with the Amino Acids in C in Reverse Orientation
SEQ ID NO: 127 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val
Lys Phe Ser Glu Leu Lys Leu Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 128 Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu
Ser Pro Leu; SEQ ID NO: 129 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 130 Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys; SEQ ID NO: 131 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu Lys Leu Ser Pro Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO:
132
Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu Leu Ser Pro Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys; SEQ ID NO: 133 Lys Lys Thr Tyr Glu Glu Leu Ala
Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Ser Pro Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 134
Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu Leu Pro Ser Leu Lys Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 135 Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Pro Ser Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys; SEQ ID NO: 136 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser Glu Leu Leu Pro Ser Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 137 Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Pro
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys; SEQ ID NO: 138 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser Glu Leu Pro Ser Leu Lys Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 139 Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Pro
Ser Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys; SEQ ID NO: 140 Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu Pro Ser Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 141 Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Pro
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys.
[0100] B. G-(A-B-C).sub.n--H Peptides
[0101] In a further embodiment, peptides of the present invention
are described by the following subgeneric formula II, in which one
or more additional elements, indicated as variables G and H, are
added to formula I to make subgeneric formula II
G-(A-B-C).sub.n--H. II
[0102] (A-B-C).sub.n are as described in formula I above. G is
absent or present and is a peptide as defined hereinbelow. H is
absent or present and is a peptide as defined hereinbelow. Thus,
within G-(A-B-C).sub.n--H, both G and H may be present or only one
of G and H may be present. The peptide segments may also be
arranged in reverse orientations, such as H-(A-B-C).sub.n-G,
H--(C-B-A).sub.n-G, or G-(C-B-A).sub.n--H).
[0103] One of ordinary skill in the art will understand that A
and/or C can contain one or more substitutions as described herein.
A person of ordinary skill in the art will also understand that A,
B, C, G, and/or H may contain one or more D-amino acids. In some
embodiments, one or more of the amino acids of the peptides of the
present invention are D amino acids. In one embodiment, the
N-terminal amino acid, the C-terminal amino acid, or both are D
amino acids. The presence of these D amino acids can help protect
against peptide degradation. In another embodiment, all the amino
acids of the peptides of the present invention are D amino acids.
This embodiment is useful for protection against degradation
following oral administration of a pharmaceutical composition
comprising the peptides of the present invention. Any number of
amino acids (e.g., between one and all of the amino acids) in the
peptides of the invention can be D amino acids.
[0104] Peptides of the invention may contain an amino-terminal
acetyl group and/or a carboxy-terminal amide group.
[0105] i. Segment G
[0106] G is absent or present and is a peptide as defined in
herein. In one embodiment, G is SEQ ID NO: 6 Ser Pro Leu; Pro Leu;
Leu; Ser Pro; Ser; Pro; or a substitution thereof. These amino
acids can also appear in reverse orientation: SEQ ID NO: 11 Leu Pro
Ser; Pro Ser; Leu Pro; or a conservative substitution thereof. It
is to be understood that one or more of the amino acids in the G
peptide may be D amino acids.
[0107] ii. Segment H
[0108] H is absent or present and is a peptide as defined in the
present specification. In one embodiment, H is SEQ ID NO: 16 Leu
Asn Thr Gln; SEQ ID NO: 17 Asn Thr Gln; Thr Gln; Gln; SEQ ID NO: 18
Leu Asn Thr; Leu Asn; Leu; or a substitution thereof. These amino
acids can also appear in reverse orientation: SEQ ID NO: 19 Gln Thr
Asn Leu; SEQ ID NO: 20 Thr Asn Leu; Asn Leu; SEQ ID NO: 21 Gln Thr
Asn; Gln Thr; or a substitution thereof. It is to be understood
that one or more of the amino acids in the H peptide may be D amino
acids.
[0109] iii. Specific Examples of G-(A-B-C).sub.n--H Peptides
[0110] Below is a non-limiting subset of examples of peptides
represented by the basic formula G-(A-B-C).sub.n--H, in which A is
Helix 8' and C is Helix 8. One of ordinary skill in the art will
understand that the G-(A-B-C).sub.nH peptides of the invention
include any of the B, G, and H sequences set forth herein. One of
ordinary skill in the art will understand that A and/or C can
contain one or more substitutions as described herein. A person of
ordinary skill in the art will also understand that A, B, C, G,
and/or H may contain one or more D-amino acids. Peptides of the
invention may contain an amino-terminal acetyl group and/or a
carboxy-terminal amide group.
TABLE-US-00002 a. G-(A-B-C).sub.n-H with Successive Deletions of G
in Both Orientations from N- and C-Termini (A, B, C, and H are
Intact) SEQ ID NO: 142 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln; SEQ ID NO: 143 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln; SEQ ID NO: 144 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln; SEQ ID NO: 145 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 146 Ser Pro Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 147 Ser Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 148 Pro Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 149 Leu Pro Ser Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln; SEQ ID NO: 150 Pro Ser Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln; SEQ ID NO: 151 Leu Pro Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln. b. G-(A-B-C).sub.n-H with Successive Deletions of H in
Both Orientations from N-and C-Termini (G, A, B, and C are Intact)
SEQ ID NO: 152 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Asn Thr
Gln; SEQ ID NO: 153 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Thr
Gln; SEQ ID NO: 154 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Gln;
SEQ ID NO: 155 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
156 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO:
157 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 158
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 159 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Gln Thr Asn Leu; SEQ ID NO: 160 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Thr Asn Leu; SEQ ID NO: 161 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Asn Leu; SEQ ID NO: 162 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Gln Thr Asn; SEQ ID NO: 163 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Gln Thr. c. G-(A-B-C).sub.n-H with
Successive Deletions of G and H (A, B, and C are Intact) SEQ ID NO:
164 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 165
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 166 Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 167 Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu
Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys; SEQ ID NO: 168 Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr;
SEQ ID NO: 169 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
170 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn SEQ ID NO: 171 Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 172 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys; SEQ ID NO: 173 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr;
SEQ ID NO: 174 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 175
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Lys Leu Ser Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu; d. G-(A-B-C).sub.n-H with Successive
Deletions in the B Group with G, A, C, and H Intact as in G-A-B-C-H
SEQ ID NO: 176 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 177 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 178 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 179
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO:
180 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO:
181 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 182 Ser Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 183 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 184 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 185 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 186 Ser Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser
Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 187 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 188 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 189 Ser Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln. e. G-(A-B-C).sub.n-H with Successive N- and/or
C- Terminal Deletions in the B Group with Successive Deletions of G
SEQ ID NO: 190 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 191 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 192 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln;
SEQ ID NO: 193 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 194 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 195 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln;
SEQ ID NO: 196 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln;
SEQ ID NO: 197 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID
NO: 198 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 199 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 200 Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 201 Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln; SEQ ID NO: 202 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln; SEQ ID NO: 203 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln; SEQ ID NO: 204 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 205 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 206 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln;
SEQ ID NO: 207 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO:
208 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 209 Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 210 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln; SEQ ID NO: 211 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln; SEQ ID NO: 212 Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln; SEQ ID NO: 213 Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln; SEQ ID NO: 214 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln; SEQ ID NO: 215 Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln; SEQ ID NO: 216 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln; SEQ ID NO: 217 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 218 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln;
SEQ ID NO: 219 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO:
220 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 221 Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 222 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln; SEQ ID NO: 223 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln;
SEQ ID NO: 224 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln;
SEQ ID NO: 225 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID
NO: 226 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO:
227 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 228 Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 229 Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 230 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln; SEQ ID NO: 231 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln; SEQ ID NO: 232 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID
NO: 233 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 234 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln. f. G-(A-B-C).sub.n-H with Successive N-
and/or C- Terminal Deletions in the B Group with Successive
Deletions of H SEQ ID NO: 235 Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr; SEQ ID NO: 236 Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn; SEQ ID NO: 237 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 238 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID
NO: 239 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 240 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
241 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 242 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 243 Ser Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 244 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser
Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 245 Ser Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu; SEQ ID NO: 246 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys;
SEQ ID NO: 247 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 248 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 249 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 250 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 251 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr; SEQ ID NO: 252 Ser Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn; SEQ ID NO: 253 Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu; SEQ ID NO: 254 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 255 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr; SEQ ID NO: 256 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn;
SEQ ID NO: 257 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO:
258 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 259 Ser Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 260 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn; SEQ ID NO: 261 Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 262 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 263
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO:
264 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
265 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 266
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 267 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 268 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 269 Ser Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 270 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; SEQ ID NO: 271 Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr; SEQ ID NO: 272 Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn; SEQ ID NO: 273 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu;
SEQ ID NO: 274 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 275
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 276 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 277 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu; SEQ ID NO: 278 Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys;
SEQ ID NO: 279 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr; SEQ ID NO: 280 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 281 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu;
SEQ ID NO: 282 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
283 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 284
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 285 Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 286 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys; SEQ ID NO: 287 Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr; SEQ ID NO: 288 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn;
SEQ ID NO: 289 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 290
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 291 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr; SEQ ID NO: 292 Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 293 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 294
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys. g. G-(A-B-C).sub.n-H with Successive
N- and/or C- Terminal Deletions in the B Group with Successive
Deletions of G and H SEQ ID NO: 295 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr; SEQ ID NO: 296 Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr; SEQ ID NO: 297 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr;
SEQ ID NO: 298 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID
NO: 299 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 300 Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 301 Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu
Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu; SEQ ID NO: 302 Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 303 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 304
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 305 Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu
Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys; SEQ ID NO: 306 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys;
SEQ ID NO: 307 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr;
SEQ ID NO: 308 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 309 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 310 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 311 Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 312 Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn; SEQ ID NO: 313 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 314 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO:
315 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 316 Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; SEQ ID NO: 317 Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 318 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 319 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 320 Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser
Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 321 Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr; SEQ ID NO: 322 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 323 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
324 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 325 Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser
Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu; SEQ ID NO: 326 Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 327 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 328 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 329 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 330 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 331 Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 332 Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr; SEQ ID NO: 333 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr;
SEQ ID NO: 334 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 335 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn; SEQ ID NO: 336 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 337
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 338 Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu; SEQ ID NO: 339 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 340 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 341 Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys;
SEQ ID NO: 342 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Pro Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 343 Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys
Leu Ser ProLeu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 344 Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
ProLeu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr; SEQ ID NO: 345 Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser ProLeu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr; SEQ ID NO: 346 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser ProLeu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 347 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser ProLeu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID
NO: 348 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser ProLeu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 349 Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Lys Leu Ser ProLeu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 350 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
ProLeu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu; SEQ ID NO: 351 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser ProLeu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Tlr Lys Lys Leu;
SEQ ID NO: 352 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser ProLeu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 353
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser ProLeu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 354 Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
ProLeu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 355 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr; SEQ ID NO: 356 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 357 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 358 Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 359 Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn; SEQ ID NO: 360 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn;
SEQ ID NO: 361 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 362
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu
Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 363 Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 364 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 365
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 366 Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 367 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 368 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 369 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr; SEQ ID NO: 370 Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn; SEQ ID NO: 371 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
372 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 373 Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu; SEQ ID NO: 374 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO:
375 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 376 Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 377 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 378 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 379 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr; SEQ ID NO: 380 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr; SEQ ID NO: 381 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr; SEQ ID NO: 382 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 383 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn;
SEQ ID NO: 384 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
385 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 386 Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 387 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser
Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu; SEQ ID NO: 388 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys; SEQ ID NO: 389 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Lys Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID
NO: 390 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Pro Ser Leu Lys Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 391 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr; SEQ ID NO: 392 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr;
SEQ ID NO: 393 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO:
394 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 395 Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 396 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser
Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn; SEQ ID NO: 397 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu; SEQ ID NO: 398 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID
NO: 399 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 400 Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 401 Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 402 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 403 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 404 Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 405 Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr; SEQ ID NO: 406 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 407 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
408 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 409 Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu; SEQ ID NO: 410 Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 411 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 412 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 413 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 414 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 415 Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 416 Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr; SEQ ID NO: 417 Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr;
SEQ ID NO: 418 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn; SEQ ID NO: 419 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO:
420 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 421 Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu; SEQ ID NO: 422 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO:
423 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 424 Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 425 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Leu Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 426 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Leu
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 427 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr; SEQ ID NO: 428 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr; SEQ ID NO: 429 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 430 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 431
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 432 Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser
Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 433 Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu; SEQ ID NO: 434 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu;
SEQ ID NO: 435 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 436 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 437 Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu
Lys Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys; SEQ ID NO: 438 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Lys Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
439 Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 440 Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 441 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr; SEQ ID NO: 442 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn; SEQ ID NO: 443 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID
NO: 444 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 445 Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 446 Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 447
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Pro Ser Ala Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 448 Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 449 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 450 Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Pro Ser Leu Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; SEQ ID NO: 451 Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr; SEQ ID NO: 452 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 453 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lya Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 454 Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 455 Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn; SEQ ID NO: 456 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 457 Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 458 Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu; SEQ ID NO: 459 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 460 Pro Leu
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys
Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys; SEQ ID NO: 461 Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Ser Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID
NO: 462 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 463 Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr; SEQ ID NO: 464 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID
NO: 465 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr; SEQ ID NO: 466 Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn; SEQ ID NO: 467 Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID
NO: 468 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn; SEQ ID NO: 469 Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu; SEQ ID NO: 470 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu; SEQ ID NO: 471 Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu; SEQ ID NO: 472 Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys. SEQ ID NO:
473 Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys. SEQ ID NO: 474 Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys.
[0111] h. Examples of G-(A-B-C)n-H with A and/or C in Reverse
Orientation
TABLE-US-00003 SEQ ID NO: 475 Ser Pro Leu Lys Lys Thr Ala Glu Glu
Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln; SEQ ID NO: 476 Lys Lys Thr Ala Glu Glu Leu Ala
Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln; SEQ ID NO: 477 Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu
Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys; SEQ ID NO: 478 Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser
Leu Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Asn
Thr Gln; SEQ ID NO: 479 Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala
Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu Lys Lys Thr Tyr Glu
Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Asn Thr
Gln; SEQ ID NO: 480 Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser
Leu Ala Ser Val Lys Ala Ser Glu Leu Lys Leu Ser Pro Leu Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID
NO: 481 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Asn Thr Gln;
SEQ ID NO: 482 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu Leu Asn Thr Gln; SEQ ID
NO: 483 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID NO: 484 Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 485 Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln; SEQ ID NO: 486 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 487
Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys
Ala Ser Glu Leu Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 488 Lys Lys Thr
Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln; SEQ ID NO: 489 Ser Pro Leu Lys Lys Thr Ala Glu Glu
Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
490 Ser Pro Leu Lys Lys Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val
Lys Ala Ser Glu Leu Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe
Ser Val Lys Phe Ser Glu Leu Leu Asn Thr Gln; SEQ ID NO: 491 Lys Lys
Thr Ala Glu Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro
Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu Leu Asn Thr Gln; SEQ ID NO: 492 Ser Pro Leu Lys Lys Thr Ala Glu
Glu Leu Ala Ser Leu Ala Ser Val Lys Ala Ser Glu Leu Pro Lys Lys Thr
Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu Leu; SEQ ID
NO: 493 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu
Phe Ser Val Lys Phe Ser Glu Leu Leu Asn Thr Gln; SEQ ID NO: 494 Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser
Glu Leu Leu Asn Thr Gln; and, SEQ ID NO: 495 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Pro
Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser Glu
Leu.
[0112] C. Amino Acid Substitutions within A and/or C of Peptides
(A-B-C).sub.n and G-(A-B-C).sub.n--H
[0113] The present invention includes peptides (specifically,
(A-B-C).sub.n and G-(A-B-C).sub.n--H peptides) containing isosteric
amino acid substitutions at specific amino acid positions within
the peptides. By "isosteric substitution" is meant that an amino
acid at a particular position within a peptide of the invention can
be substituted with another amino acid belonging to the same
isosteric group as described hereinbelow. Amino acids within a
given isosteric group, as set forth hereinbelow, are amino acids
having similar size, shape, polar/nonpolar properties, charge,
and/or steric properties. The invention provides peptides wherein
substitution of an amino acid with an amino acid belonging to the
same isosteric group allows the substituted peptide to retain at
least about 20% of the biological activity of the unsubstituted
peptide, e.g., at least about: 25%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 110%, 125%, 150%, 175%, 200%, 250%, 300%, or more, of
the biological activity of the unsubstituted peptide. By
"biological activity" of the peptide is meant the ability of the
peptide to promote lipid efflux and/or have an anti-inflammatory
effect, as described hereinbelow.
[0114] The invention also provides peptides having an amino acid
substituted with an amino acid belonging to a different isosteric
group from the original amino acid, such that the substitution
allows the peptide to retain at least about 20% of the biological
activity (e.g., lipid efflux and/or anti-inflammatory properties)
of the unsubstituted peptide, e.g., at least about: 25%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 110%, 125%, 150%, 175%, 200%, 250%,
300%, or more, of the biological activity of the unsubstituted
peptide.
[0115] Peptides with an amino acid substitution that retain at
least about 20% or more of the biological activity of an
unsubstituted peptide will be readily recognized by the skilled
artisan using well-known approaches, e.g., the lipid efflux and/or
other biological assays of the invention.
[0116] Isosteric amino acid groupings of the present invention are
as follows:
[0117] a) Isosteric Group 1: Lys, H is, and Arg;
[0118] b) Isosteric Group 2: Asp and Glu;
[0119] c) Isosteric Group 3: Ser, Thr, Leu, Ile, Gly, Ala, Val, and
GABA;
[0120] d) Isosteric Group 4: Phe and Tyr; and
[0121] e) Isosteric Group 5: Pro.
[0122] Tables 1 and 2 below show the numbering that is used herein
to refer to amino acid positions within Helix 8' of A and Helix 8
of C for which isosteric substitutions can be made as described
herein. Both Tables employ the conventional single-letter amino
acid code to refer to the amino acids of each helix. This
single-letter code is well-known in the art (see, e.g., Alberts et
al., Molecular Biology of the Cell, 2.sup.nd Ed., Garland
Publishing, Inc., N.Y., 1989, and similar references). Amino acids
at positions 4, 8, and 15 are in bold and are underlined to
highlight the amino acid sequence differences between Helix 8' and
Helix 8.
TABLE-US-00004 TABLE 1 Numbering of Amino Acid Positions in Helix
8' (Segment A) Amino Acid L E S A K V S A L S A L E E A T K K
Position in Helix 8' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
(Segment A)
TABLE-US-00005 TABLE 2 Numbering of Amino Acid Positions in Helix 8
(Segment C) Amino Acid L E S F K V S F L S A L E E Y T K K Position
in Helix 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 (Segment
C)
[0123] Isosteric substitutions can be made singly or in multiples,
and in any combination. In just one non-limiting example, within
Helix 8' (Segment A) and/or Helix 8 (Segment C) there may be only
one amino acid in Isosteric Group 3 that is substituted.
Alternatively, there may be two, three, four, five, six, etc., up
to substitutions at all amino acid positions assigned to Isosteric
Group 3. One of ordinary skill in the art will understand that the
peptides of the invention can contain one or more isosteric amino
acid substitutions in either of A and C or in both A and C. As
described below, within the peptides of the invention, an amino
acid can be substituted with a different amino acid belonging to
the same isosteric group or with an amino acid belonging to a
different isosteric group.
[0124] For each isosteric grouping described below, any number of
amino acid positions may be substituted, i.e., one position, more
than one position, or all positions in either one or both helices
(i.e., Helix 8' and/or Helix 8) within a given isosteric group can
be substituted. Moreover, when more than one amino acid position
within an isosteric group is substituted, the substitutions need
not be the same within one helix or between the two helices. In one
non-limiting example, in a peptide having three isosteric
substitutions at positions belonging to Isosteric Group 3 (e.g.,
but not limited to, Ser at positions 3 and 7 of Helix 8' (Segment
A) and Leu at position 1 of Helix 8 (Segment C)), one position
could be substituted with a Val, another position could be
substituted with a Thr, and yet another position could be
substituted with a Leu. Moreover, peptides of the invention can
simultaneously contain substitutions at amino acid positions
assigned to different isosteric groups (i.e., within Helix 8',
amino acid positions 1 and 2, which are Leu (Isosteric Group 3) and
Glu (Isosteric Group 2), respectively, can both carry amino acid
substitutions within the same peptide). The substitutions can be
mixed or matched within isosteric groups using the guidance set
forth hereinbelow. The foregoing applies to any and all isosteric
substitutions described herein. The isosteric substitutions
disclosed herein for A and C can be mixed and matched with any
deletion or conservative substitution described herein for B, G,
and/or H).
[0125] i. Substitutions within Helix 8' (Segment A)
[0126] For amino acid positions 1 through 18 in Helix 8' (Segment
A), the following amino acid substitutions can be made.
[0127] Amino acid positions 5, 17, and 18 within Helix 8' (Segment
A) are assigned herein to Isosteric Group 1; thus any one or more
of these positions can be substituted with Lys, H is, or Arg. In
addition, any one or more of these amino acid positions assigned to
Isosteric Group 1 can be substituted with an amino acid from
Isosteric Group 2 (Asp or Glu), Isosteric Group 3 (Ser, Thr, Leu,
Ile, Gly, Ala, Val, or GABA), or Isosteric Group 4 (Phe or
Tyr).
[0128] Amino acid positions 2, 13, and 14 within Helix 8' (Segment
A) are assigned herein to Isosteric Group 2; thus any one or more
of these positions can be substituted with Asp or Glu. In addition,
any one or more of these amino acid positions assigned to Isosteric
Group 2 can also be substituted with amino acids from Isosteric
Group 1 (Lys, H is, or Arg), Isosteric Group 3 (Ser, Thr, Leu, Ile,
Gly, Ala, Val, or GABA), or Isosteric Group 4 (Phe or Tyr).
[0129] Amino acid positions 1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 15,
and 16 within Helix 8' (Segment A) are assigned herein to Isosteric
Group 3; thus any one or more of these positions can be substituted
with Ser, Thr, Leu, Ile, Gly, Ala, Val, or GABA. In addition, any
one or more of these amino acid positions assigned to Isosteric
Group 3 can be substituted with amino acids from Isosteric Group 4
(Phe or Tyr). Moreover, amino acid positions belonging to Isosteric
Group 3 that reside on the hydrophilic surface of Helix 8' (i.e.,
amino acid positions 3, 6, 7, 9, 10, and 16) can be substituted
with amino acids from Isosteric Group 1 (Lys, H is, or Arg) or
Isosteric Group 2 (Asp or Glu).
[0130] The isosteric group for each individual amino acid position
within Helix 8' (Segment A) is provided below.
[0131] Position 1: Leu (Isosteric Group 3) can be substituted with
Ser, Thr, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0132] Position 2: Glu (Isosteric Group 2) can be substituted with
Asp or amino acids from other isosteric groups as provided
above.
[0133] Position 3: Ser (Isosteric Group 3) can be substituted with
Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0134] Position 4: Ala (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0135] Position 5: Lys (Isosteric Group 1) can be substituted with
H is or Arg (Isosteric Group 1).
[0136] Position 6: Val (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Ala, GABA, or amino acids from other
isosteric groups as provided above.
[0137] Position 7: Ser (Isosteric Group 3) can be substituted with
Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0138] Position 8: Ala (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0139] Position 9: Leu (Isosteric Group 3) can be substituted with
Ser, Thr, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0140] Position 10: Ser (Isosteric Group 3) can be substituted with
Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0141] Position 11: Ala (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0142] Position 12: Leu (Isosteric Group 3) can be substituted with
Ser, Thr, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0143] Position 13: Glu (Isosteric Group 1) can be substituted with
Asp or amino acids from other isosteric groups as provided
above.
[0144] Position 14: Glu (Isosteric Group 1) can be substituted with
Asp or amino acids from other isosteric groups as provided
above.
[0145] Position 15: Ala (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0146] Position 16: Thr (Isosteric Group 3) can be substituted with
Ser, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0147] Position 17: Lys (Isosteric Group 1) can be substituted with
H is, Arg, or amino acids from other isosteric groups as provided
above.
[0148] Position 18: Lys (Isosteric Group 1) can be substituted with
H is, Arg, or amino acids from other isosteric groups as provided
above.
[0149] ii. Amino Acid Substitutions Within Segment C (Helix 8)
[0150] For amino acid positions 1 through 18 in Helix 8 of Segment
C, the following amino acid substitutions can be made.
[0151] Amino acid positions 5, 17, and 18 within Helix 8 of Segment
C are assigned herein to Isosteric Group 1; thus any one or more of
these positions can be substituted with Lys, H is, or Arg. In
addition, any one or more of these amino acid positions assigned to
Isosteric Group 1 can be substituted with an amino acid from
Isosteric Group 2 (Asp or Glu), Isosteric Group 3 (Ser, Thr, Leu,
Ile, Gly, Ala, Val, or GABA), or Isosteric Group 4 (Phe or
Tyr).
[0152] Amino acid positions 2, 13, and 14 within Helix 8 Segment C)
are assigned herein to Isosteric Group 2; thus any one or more of
these positions can be substituted with Asp or Glu. In addition,
any one or more of these amino acid positions assigned to Isosteric
Group 2 can also be substituted with amino acids from Isosteric
Group 1 (Lys, H is, or Arg), Isosteric Group 3 (Ser, Thr, Leu, Ile,
Gly, Ala, Val, or GABA), or Isosteric Group 4 (Phe or Tyr).
[0153] Amino acid positions 1, 3, 6, 7, 9, 10, 11, 12, and 16
within Helix 8 (Segment C) are assigned herein to Isosteric Group
3; thus any one or more of these positions can be substituted with
Ser, Thr, Leu, Ile, Gly, Ala, or GABA. In addition, any one or more
of these amino acid positions assigned to Isosteric Group 3 can be
substituted with amino acids from Isosteric Group 4 (Phe or Tyr).
Moreover, amino acid positions belonging to Isosteric Group 3 that
reside on the hydrophilic surface of Helix 8 (i.e., amino acid
positions 3, 6, 7, 9, 10, and 16) can be substituted with amino
acids from Isosteric Group 1 (Lys, H is, or Arg) or Isosteric Group
2 (Asp or Glu).
[0154] Amino acid positions 4, 8, and 15 within Helix 8 (Segment C)
are assigned herein to Isosteric Group 4; thus any one or more of
these positions can be substituted with Phe or Tyr. In addition,
any one or more of these amino acid positions assigned to Isosteric
Group 4 can be substituted with amino acids from Isosteric Group 3
(Ser, Thr, Leu, Ile, Gly, Ala, or GABA).
[0155] The isosteric group for each individual amino acid position
within Helix 8 (Segment C) is provided below.
[0156] Position 1: Leu (Isosteric Group 3) can be substituted with
Ser, Thr, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0157] Position 2: Glu (Isosteric Group 2) can be substituted with
Asp or amino acids from other isosteric groups as provided
above.
[0158] Position 3: Ser (Isosteric Group 3) can be substituted with
Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0159] Position 4: Phe (Isosteric Group 4) can be substituted with
Tyr or amino acids from other isosteric groups as provided
above.
[0160] Position 5: Lys (Isosteric Group 1) can be substituted with
H is or Arg (Isosteric Group 1).
[0161] Position 6: Val (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0162] Position 7: Ser (Isosteric Group 3) can be substituted with
Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0163] Position 8: Phe (Isosteric Group 4) can be substituted with
Tyr or amino acids from other isosteric groups as provided
above.
[0164] Position 9: Leu (Isosteric Group 3) can be substituted with
Ser, Thr, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0165] Position 10: Ser (Isosteric Group 3) can be substituted with
Thr, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0166] Position 11: Ala (Isosteric Group 3) can be substituted with
Ser, Thr, Leu, Ile, Gly, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0167] Position 12: Leu (Isosteric Group 3) can be substituted with
Ser, Thr, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0168] Position 13: Glu (Isosteric Group 1) can be substituted with
Asp or amino acids from other isosteric groups as provided
above.
[0169] Position 14: Glu (Isosteric Group 1) can be substituted with
Asp or amino acids from other isosteric groups as provided
above.
[0170] Position 15: Tyr (Isosteric Group 4) can be substituted with
Phe or amino acids from other isosteric groups as provided
above.
[0171] Position 16: Thr (Isosteric Group 3) can be substituted with
Ser, Leu, Ile, Gly, Ala, Val, GABA, or amino acids from other
isosteric groups as provided above.
[0172] Position 17: Lys (Isosteric Group 1) can be substituted with
H is, Arg, or amino acids from other isosteric groups as provided
above.
[0173] Position 18: Lys (Isosteric Group 1) can be substituted with
H is, Arg, or amino acids from other isosteric groups as provided
above.
[0174] iii. Specific Examples of Isosteric Substitutions in Helices
8' and 8 of A and C
[0175] Below are some examples of isosteric substitutions that can
be made in Helices 8' and 8 in the peptides of the invention.
Isosteric substitutions can be mixed and matched without limit. For
example, Helix 8' and/or 8 in a peptide of the invention can each
contain multiple isosteric substitutions at any position belonging
to any isosteric group.
[0176] a. Examples of Isosteric Group 1 Substitutions in Helix 8'
(SEQ ID NO: 2)
TABLE-US-00006 SEQ ID NO: 2 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 496 Leu Glu Ser Ala His
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 497
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr His
Lys; SEQ ID NO: 498 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys His; SEQ ID NO: 499 Leu Glu Ser Ala His Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr His Lys; SEQ ID NO: 500 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr His His;
SEQ ID NO: 501 Leu Glu Ser Ala His Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys His; SEQ ID NO: 502 Leu Glu Ser Ala His Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr His His; SEQ ID NO: 503 Leu Glu Ser
Ala Arg Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID
NO: 504 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Arg Lys; SEQ ID NO: 505 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Arg; SEQ ID NO: 506 Leu Glu Ser Ala Arg
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Arg Lys; SEQ ID NO: 507
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Arg
Arg; SEQ ID NO: 508 Leu Glu Ser Ala Arg Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Arg; SEQ ID NO: 509 Leu Glu Ser Ala Arg Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr Arg Arg; SEQ ID NO: 510 Leu Glu
Ser Ala Arg Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr His His;
SEQ ID NO: 511 Leu Glu Ser Ala His Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Arg His; SEQ ID NO: 512 Leu Glu Ser Ala His Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr His Arg SEQ ID NO: 513 Leu Glu Ser
Ala Arg Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Arg His; SEQ ID
NO: 514 Leu Glu Ser Ala Arg Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr His Arg; SEQ ID NO: 515 Leu Glu Ser Ala His Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Arg Arg; SEQ ID NO: 516 Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Arg His; SEQ ID NO: 517
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr His
Arg; SEQ ID NO: 518 Leu Glu Ser Ala Arg Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys His; SEQ ID NO: 519 Leu Glu Ser Ala Arg Val Ser
Ala Leu Ser Ala Leu Glu Glu Ala Thr His Lys; SEQ ID NO: 520 Leu Glu
Ser Ala His Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Arg;
SEQ ID NO: 521 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Arg Lys.
[0177] b. Examples of Isosteric Group 1 Substitutions in Helix 8
(SEQ ID NO: 14)
TABLE-US-00007 SEQ ID NO: 14 Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 522 Leu Glu Ser Phe
His Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
523 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
His Lys; SEQ ID NO: 524 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys His; SEQ ID NO: 525 Leu Glu Ser Phe His Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr His Lys; SEQ ID NO: 526 Leu
Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr His
His; SEQ ID NO: 527 Leu Glu Ser Phe His Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys His; SEQ ID NO: 528 Leu Glu Ser Phe His Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr His His; SEQ ID NO: 529 Leu Glu
Ser Phe Arg Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys;
SEQ ID NO: 530 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Arg Lys; SEQ ID NO: 531 Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Arg; SEQ ID NO: 532 Leu Glu Ser
Phe Arg Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Arg Lys; SEQ ID
NO: 533 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Arg Arg; SEQ ID NO: 534 Leu Glu Ser Phe Arg Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Arg; SEQ ID NO: 535 Leu Glu Ser Phe Arg
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Arg Arg; SEQ ID NO: 536
Leu Glu Ser Phe Arg Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr His
His; SEQ ID NO: 537 Leu Glu Ser Phe His Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Arg His; SEQ ID NO: 538 Leu Glu Ser Phe His Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr His Arg; SEQ ID NO: 539 Leu Glu
Ser Phe Arg Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Arg His;
SEQ ID NO: 540 Leu Glu Ser Phe Arg Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr His Arg; SEQ ID NO: 541 Leu Glu Ser Phe His Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Arg Arg; SEQ ID NO: 542 Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Arg His; SEQ ID
NO: 543 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr His Arg; SEQ ID NO: 544 Leu Glu Ser Phe Arg Val Ser PheLeu Ser
Ala Leu Glu Glu Tyr Thr Lys His SEQ ID NO: 545 Leu Glu Ser Phe Arg
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr His Lys; SEQ ID NO: 546
Leu Glu Ser Phe His Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Arg; SEQ ID NO: 547 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Arg Lys.
[0178] c. Examples of Isosteric Group 2 Substitutions in Helix 8'
(SEQ ID NO: 2)
TABLE-US-00008 SEQ ID NO: 2 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 548 Leu Asp Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys; SEQ ID NO: 549
Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Asp Glu Ala Thr Lys
Lys; SEQ ID NO: 550 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Asp Ala Thr Lys Lys; SEQ ID NO: 551 Leu Asp Ser Ala Lys Val Ser
Ala Leu Ser Ala Leu Asp Glu Ala Thr Lys Lys; SEQ ID NO: 552 Leu Asp
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Asp Ala Thr Lys Lys;
SEQ ID NO: 553 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Asp
Asp Ala Thr Lys Lys; SEQ ID NO: 554 Leu Asp Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Asp Asp Ala Thr Lys Lys.
[0179] d. Examples of Isosteric Group 2 Substitutions in Helix 8
(SEQ ID NO: 14)
TABLE-US-00009 SEQ ID NO: 14 Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 555 Leu Asp Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO:
556 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Asp Glu Tyr Thr
Lys Lys; SEQ ID NO: 557 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala
Leu Glu Asp Tyr Thr Lys Lys; SEQ ID NO: 558 Leu Asp Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Asp Glu Tyr Thr Lys Lys; SEQ ID NO: 559 Leu
Asp Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Asp Tyr Thr Lys
Lys; SEQ ID NO: 560 Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Asp Asp Tyr Thr Lys Lys; SEQ ID NO: 561 Leu Asp Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Asp Asp Tyr Thr Lys Lys.
[0180] e. Examples of Isosteric Group 4 Substitutions in Helix 8
(SEQ ID NO: 14)
TABLE-US-00010 SEQ ID NO: 14 Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 562 Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Phe Thr Lys Lys; SEQ ID NO:
563 Leu Glu Ser Phe Lys Val Ser Tyr Leu Ser Ala Leu Glu Glu Phe Thr
Lys Lys; SEQ ID NO: 564 Leu Glu Ser Phe Lys Val Ser Tyr Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 565 Leu Glu Ser Tyr Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 566 Leu
Glu Ser Tyr Lys Val Ser Tyr Leu Ser Ala Leu Glu Glu Phe Thr Lys
Lys; SEQ ID NO: 567 Leu Glu Ser Tyr Lys Val Ser Tyr Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 568 Leu Glu Ser Tyr Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Phe Thr Lys Lys.
[0181] It is to be understood that the letters in the generic
formulae I and II or in components thereof are defined by the text
that follows each letter and do not designate an individual amino
acid.
[0182] It is to be understood that in some embodiments, one or more
of the amino acids of the peptides of the present invention are D
amino acids. In one embodiment, the N-terminal amino acid, the
C-terminal amino acid or both are D amino acids. The presence of
these D amino acids can help protect against peptide degradation.
In another embodiment, all the amino acids of the peptides of the
present invention are D amino acids. This embodiment is useful for
protection against degradation following oral administration of a
pharmaceutical composition comprising the peptides of the present
invention.
[0183] D. Peptides of the Present Invention Containing D Amino
Acids
[0184] One of ordinary skill in the art will understand that any of
the peptides of the present invention may containone or more D
amino acids, for example, a D amino acid at the N-terminus, a D
amino acid at the C-terminus, or a D amino acid at both the N- and
C-termini. The presence of these D amino acids can help protect
against peptide degradation. In some embodiments, all the amino
acids of any peptide of the present invention may be D amino acids.
This embodiment is useful for protection against degradation
following oral administration of a pharmaceutical composition
comprising the peptides of the present invention. Some examples of
peptides of the present invention containing one or more D amino
acids include but are not limited to those shown below. D amino
acids are indicated in all-upper-case letters, e.g., "SER" or
"PRO."
[0185] i. Examples of Peptides with One or More Amino Acids
Replaced by a D Amino Acid
[0186] Below are examples of two peptides (SEQ ID NO: 22 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys and SEQ ID NO: 142 Ser Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln) in which the N-terminal, C-terminal,
N-terminal and C-terminal, or all amino acids are replaced by D
amino acids. One of ordinary skill in the art will understand that
any peptide of the invention can similarly be formulated to contain
one or more D amino acids as exemplified below.
TABLE-US-00011 SEQ ID NO: 569 LEU Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID
NO: 570 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys LYS; SEQ ID NO: 571 LEU Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys LYS; SEQ ID NO: 572 LEU GLU SER ALA LYS VAL SER ALA LEU SER
ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO LEU LEU GLU SER PHE
LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR LYS LYS; SEQ ID NO:
573 SER Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID
NO: 574 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr GLN;
SEQ ID NO: 575 SER Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
GLN; SEQ ID NO: 576 SER PRO LEU LEU GLU SER ALA LYS VAL SER ALA LEU
SER ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO LEU LEU GLU SER
PHE LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR LYS LYS LEU ASN
THR GLN.
[0187] E. N-Terminal Modification and/or C-terminal Modification of
the Peptides of the Present Invention
[0188] The peptides of the present invention may optionally be
acetylated at the amino-terminus. The peptides of the present
invention may optionally have a carboxy-terminal amide. In some
embodiments, the peptides of the present invention may have both an
amino-terminal acetyl group and a carboxy-terminal amide group.
Methods of acetylating the amino-terminus or adding a carboxy
terminal amide are well known to one of ordinary skill in the art.
While it is to be understood that any of the peptides disclosed in
this application may be modified at the N-terminus, at the
C-terminus, or both at the N-terminus and at the C-terminus, the
following sequences are presented as exemplary embodiments.
[0189] Below are examples of two peptides (SEQ ID NO: 22 Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys and SEQ ID NO: 142 Ser Pro Leu Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln) in which the N-terminal is acetylated,
the C-terminal is amidated, or the N-terminal is acetylated and the
C-terminal is amidated. One of ordinary skill in the art will
understand that any peptide of the invention can similarly be
formulated to contain an amino-terminal acetyl group and/or a
carboxy-terminal amide group as exemplified below.
TABLE-US-00012 i. Peptides Containing an Amino-Terminal Acetylation
SEQ ID NO: 577 Ac-Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu
Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 578
Ac-LEU Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys; SEQ ID NO: 579 Ac-Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys LYS; SEQ ID NO: 580 Ac-LEU Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys LYS; SEQ ID
NO: 581 Ac-LEU GLU SER ALA LYS VAL SER ALA LEU SER ALA LEU GLU GLU
ALA THR LYS LYS LYS LEU SER PRO LEU LEU GLU SER PHE LYS VAL SER PHE
LEU SER ALA LEU GLU GLU TYR THR LYS LYS; SEQ ID NO: 582 Ac-Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID NO: 583
Ac-SER Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; SEQ ID
NO: 584 Ac-Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala
Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
GLN; SEQ ID NO: 585 Ac-SER Pro Leu Leu Glu Ser Ala Lys Val Ser Ala
Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr GLN; SEQ ID NO: 586 Ac-SER PRO LEU LEU GLU SER ALA LYS VAL
SER ALA LEU SER ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO LEU
LEU GLU SER PHE LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR LYS
LYS LEU ASN THR GLN. ii. Peptides Containing a Carboxy-Terminal
Amidation SEQ ID NO: 587 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys-NH.sub.2;
SEQ ID NO: 588 LEU Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys-NH.sub.2; SEQ ID NO:
589 Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys LYS-NH.sub.2; SEQ ID NO: 590 LEU Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys LYS-NH.sub.2; SEQ ID NO: 591 LEU GLU SER ALA LYS
VAL SER ALA LEU SER ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO
LEU LEU GLU SER PHE LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR
LYS LYS-NH.sub.2; SEQ ID NO: 592 Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln- NH.sub.2; SEQ ID NO: 593 SER Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln- NH.sub.2; SEQ ID NO: 594
Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr GLN- NH.sub.2;
SEQ ID NO: 595 SER Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
GLN- NH.sub.2; SEQ ID NO: 596 SER PRO LEU LEU GLU SER ALA LYS VAL
SER ALA LEU SER ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO LEU
LEU GLU SER PHE LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR LYS
LYS LEU ASN THR GLN- NH.sub.2. iii. Peptides Containing an
Amino-Terminal Acetylation and a Carboxy-Terminal Amidation SEQ ID
NO: 597 Ac-Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu
Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe
Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys-NH.sub.2; SEQ ID NO: 598
Ac-LEU Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys-NH.sub.2; SEQ ID NO: 599 Ac-Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys LYS-NH.sub.2; SEQ ID NO: 600 Ac-LEU Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys LYS-NH.sub.2; SEQ ID NO: 601 Ac-LEU GLU SER ALA LYS VAL SER ALA
LEU SER ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO LEU LEU GLU
SER PHE LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR LYS
LYS-NH.sub.2;
SEQ ID NO: 602 Ac-Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu
Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln-NH.sub.2; SEQ ID NO: 603 Ac-SER Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln-NH.sub.2; SEQ ID NO: 604 Ac-Ser Pro Leu Leu
Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr GLN-NH.sub.2; SEQ ID NO: 605
Ac-SER Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu
Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
GLN-NH.sub.2; SEQ ID NO: 606 Ac-SER PRO LEU LEU GLU SER ALA LYS VAL
SER ALA LEU SER ALA LEU GLU GLU ALA THR LYS LYS LYS LEU SER PRO LEU
LEU GLU SER PHE LYS VAL SER PHE LEU SER ALA LEU GLU GLU TYR THR LYS
LYS LEU ASN THR GLN-NH.sub.2.
[0190] F. Modified Peptides of the Present Invention
[0191] The present invention may be used for the production of the
peptides or peptide analogs of the present invention. "Proteins",
"peptides," "polypeptides" and "oligopeptides" are chains of amino
acids (typically L-amino acids) whose alpha carbons are linked
through peptide bonds formed by a condensation reaction between the
carboxyl group of the alpha carbon of one amino acid and the amino
group of the alpha carbon of another amino acid. The terminal amino
acid at one end of the chain (i.e., the amino terminal) has a free
amino group, while the terminal amino acid at the other end of the
chain (i.e., the carboxy terminal) has a free carboxyl group. As
such, the term "amino terminus" (abbreviated N-terminus) refers to
the free alpha-amino group on the amino acid at the amino terminal
of the protein, or to the alpha-amino group (imino group when
participating in a peptide bond) of an amino acid at any other
location within the protein. Similarly, the term "carboxy terminus"
(abbreviated C-terminus) refers to the free carboxyl group on the
amino acid at the carboxy terminus of a protein, or to the carboxyl
group of an amino acid at any other location within the
protein.
[0192] Typically, the amino acids making up a protein are numbered
in order, starting at the amino terminal and increasing in the
direction toward the carboxy terminal of the protein. Thus, when
one amino acid is said to "follow" another, that amino acid is
positioned closer to the carboxy terminal of the protein than the
preceding amino acid.
[0193] The term "residue" is used herein to refer to an amino acid
(D or L) or an amino acid mimetic that is incorporated into a
protein by an amide bond. When a D amino acid is present in the
peptides of the present invention, the three letter designation for
the amino acid appears in upper case instead of a capital letter.
For example the amino acid serine, represented as Ser indicates an
L amino acid. The D amino acid form is represented as the upper
case letters SER. This is not to be confused with letters appearing
as subscripts used in generic formula and defined as variables
herein. As such, the amino acid may be a naturally occurring amino
acid or, unless otherwise limited, may encompass known analogs of
natural amino acids that function in a manner similar to the
naturally occurring amino acids (i.e., amino acid mimetics).
Moreover, an amide bond mimetic includes peptide backbone
modifications well known to those skilled in the art.
[0194] Furthermore, one of skill will recognize that, as mentioned
above, individual substitutions, deletions or additions which
alter, add or delete a single amino acid or a small percentage of
amino acids (typically less than about 5%, or typically less than
about 1%) in a sequence are conservatively modified variations
where the alterations result in the substitution of an amino acid
with a chemically similar amino acid. Conservative substitution
tables providing functionally similar amino acids are well known in
the art. The following six groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
[0195] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q), Histidine (H);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0196] A conservative substitution is a substitution in which the
substituting amino acid (naturally occurring or modified) is
structurally related to the amino acid being substituted, i.e., has
about the same size and electronic properties as the amino acid
being substituted. Thus, the substituting amino acid would have the
same or a similar functional group in the side chain as the
original amino acid. A "conservative substitution" also refers to
utilizing a substituting amino acid which is identical to the amino
acid being substituted except that a functional group in the side
chain is protected with a suitable protecting group. Peptides of
the present invention include conservatively substituted peptides,
wherein these conservative substitutions occur at 1%, 3%, 5%, 7%,
10%, 15%, 20%, 25%, 30%, 40%, or 50% of the amino acid residues.
Peptides of the present invention include peptides that are
homologous at 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% of the
entire sequence of the peptide.
[0197] Suitable protecting groups are described in Green and Wuts,
"Protecting Groups in Organic Synthesis", John Wiley and Sons,
Chapters 5 and 7, 1991, the teachings of which are incorporated
herein by reference. Preferred protecting groups are those which
facilitate transport of the peptide through membranes, for example,
by reducing the hydrophilicity and increasing the lipophilicity of
the peptide, and which can be cleaved, either by hydrolysis or
enzymatically (Ditter et al., 1968. J. Pharm. Sci. 57:783; Ditter
et al., 1968. J. Pharm. Sci. 57:828; Ditter et al., 1969. J. Pharm.
Sci. 58:557; King et al., 1987. Biochemistry 26:2294; Lindberg et
al., 1989. Drug Metabolism and Disposition 17:311; Tunek et al.,
1988. Biochem. Pharm. 37:3867; Anderson et al., 1985 Arch. Biochem.
Biophys. 239:538; and Singhal et al., 1987. FASEB J. 1:220).
Suitable hydroxyl protecting groups include ester, carbonate and
carbamate protecting groups. Suitable amine protecting groups
include acyl groups and alkoxy or aryloxy carbonyl groups, as
described above for N-terminal protecting groups. Suitable
carboxylic acid protecting groups include aliphatic, benzyl and
aryl esters, as described below for C-terminal protecting groups.
In one embodiment, the carboxylic acid group in the side chain of
one or more glutamic acid or aspartic acid residues in a peptide of
the present invention is protected, preferably as a methyl, ethyl,
benzyl or substituted benzyl ester, more preferably as a benzyl
ester.
[0198] Provided below are groups of naturally occurring and
modified amino acids in which each amino acid in a group has
similar electronic and steric properties. Thus, a conservative
substitution can be made by substituting an amino acid with another
amino acid from the same group. A "nonconservative substitution"
can be made by substituting an amino acid with another amino acid
from a different group. It is to be understood that these groups
are non-limiting, i.e. that there are additional modified amino
acids which could be included in each group. [0199] Group I
includes leucine, isoleucine, valine, methionine and modified amino
acids having the following side chains: ethyl, n-propyl n-butyl.
Preferably, Group I includes leucine, isoleucine, valine and
methionine. [0200] Group II includes glycine, alanine, valine and a
modified amino acid having an ethyl side chain. Preferably, Group
II includes glycine and alanine. [0201] Group III includes
phenylalanine, phenylglycine, tyrosine, tryptophan,
cyclohexylmethyl glycine, and modified amino residues having
substituted benzyl or phenyl side chains.
[0202] Preferred substituents include one or more of the following:
halogen, methyl, ethyl, nitro, --NH.sub.2, methoxy, ethoxy and
--CN. Preferably, Group III includes phenylalanine, tyrosine and
tryptophan. [0203] Group IV includes glutamic acid, aspartic acid,
a substituted or unsubstituted aliphatic, aromatic or benzylic
ester of glutamic or aspartic acid (e.g., methyl, ethyl, n-propyl
iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine,
asparagine, --CO--NH-- alkylated glutamine or asparagines (e.g.,
methyl, ethyl, n-propyl and iso-propyl) and modified amino acids
having the side chain --(CH.sub.2).sub.3--COOH, an ester thereof
(substituted or unsubstituted aliphatic, aromatic or benzylic
ester), an amide thereof and a substituted or unsubstituted
N-alkylated amide thereof. Preferably, Group IV includes glutamic
acid, aspartic acid, methyl aspartate, ethyl aspartate, benzyl
aspartate and methyl glutamate, ethyl glutamate and benzyl
glutamate, glutamine and asparagine. [0204] Group V includes
histidine, lysine, ornithine, arginine, N-nitroarginine,
.beta.-cycloarginine, .gamma.-hydroxyarginine, N-amidinocitruline
and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs
of arginine and homologs of ornithine. Preferably, Group V includes
histidine, lysine, arginine and ornithine. A homolog of an amino
acid includes from 1 to about 3 additional or subtracted methylene
units in the side chain. [0205] Group VI includes serine,
threonine, and modified amino acids having C.sub.1-C.sub.5 straight
or branched alkyl side chains substituted with --OH or --SH, for
example, --CH.sub.2CH.sub.2OH, --CH.sub.2CH.sub.2CH.sub.2OH or
--CH.sub.2CH.sub.2OHCH.sub.3. Preferably, Group VI includes serine,
or threonine.
[0206] In another aspect, suitable substitutions for amino acid
residues include "severe" substitutions or "nonconservative"
substitutions. These terms are used interchangeably throught the
application. A "severe substitution" or "nonconservative
substitution" is a substitution in which the substituting amino
acid (naturally occurring or modified) has significantly different
size and/or electronic properties compared with the amino acid
being substituted. Thus, the side chain of the substituting amino
acid can be significantly larger (or smaller) than the side chain
of the amino acid being substituted and/or can have functional
groups with significantly different electronic properties than the
amino acid being substituted. Examples of severe substitutions of
this type include the substitution of phenylalanine or
cyclohexylmethyl glycine for alanine, isoleucine for glycine, a D
amino acid for the corresponding L amino acid, or
--NH--CH[(--CH.sub.2).sub.5--COOH]--CO-- for aspartic acid.
Alternatively, a functional group may be added to the side chain,
deleted from the side chain or exchanged with another functional
group. Examples of severe substitutions of this type include adding
of valine, leucine or isoleucine, exchanging the carboxylic acid in
the side chain of aspartic acid or glutamic acid with an amine, or
deleting the amine group in the side chain of lysine or ornithine.
In yet another alternative, the side chain of the substituting
amino acid can have significantly different steric and electronic
properties that the functional group of the amino acid being
substituted. Examples of such modifications include tryptophan for
glycine, lysine for aspartic acid and --(CH.sub.2).sub.4COOH for
the side chain of serine. These examples are not meant to be
limiting.
[0207] In addition to the naturally occurring genetically encoded
amino acids, amino acid residues in the peptides may be substituted
with naturally occurring non-encoded amino acids and synthetic
amino acids. Certain commonly encountered amino acids which provide
useful substitutions include, but are not limited to,
.beta.-alanine and other omega-amino acids, such as
3-aminopropionic acid, 2,3-diaminopropionic acid, 4-aminobutyric
acid and the like; .alpha.-aminoisobutyric acid;
.epsilon.-aminohexanoic acid; .delta.-aminovaleric acid;
N-methylglycine or sarcosine; ornithine; citrulline;
t-butylalanine; t-butylglycine; N-methylisoleucine; phenylglycine;
cyclohexylalanine; norleucine; naphthylalanine;
4-chlorophenylalanine; 2-fluorophenylalanine;
3-fluorophenylalanine; 4-fluorophenylalanine; penicillamine;
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
.beta.2-thienylalanine; methionine sulfoxide; homoarginine;
N-acetyl lysine; 2,4-diaminobutyric acid; 2,3-diaminobutyric acid;
p-aminophenylalanine; N-methyl valine; homocysteine;
homophenylalanine; homoserine; hydroxyproline; homoproline;
N-methylated amino acids; and peptoids (N-substituted
glycines).
[0208] While in certain embodiments, the amino acids of the
peptides will be substituted with L-amino acids, the substitutions
are not limited to L-amino acids. Thus, also encompassed by the
present disclosure are modified forms of the peptides, wherein an
L-amino acid is replaced with an identical D-amino acid (e.g.,
L-Arg.fwdarw.D-Arg) or with a conservatively-substituted D-amino
acid (e.g., LArg.fwdarw.D-Lys), and vice versa.
[0209] Additional aspects of the disclosure include analogs,
variants, derivatives, and mimetics based on the amino acid
sequence of the peptides disclosed herein. Typically, mimetic
compounds are synthetic compounds having a three-dimensional
structure (of at least part of the mimetic compound) that mimics,
for example, the primary, secondary, and/or tertiary structural,
and/or electrochemical characteristics of a selected peptide,
structural domain, active site, or binding region (e.g., a
homotypic or heterotypic binding site, a catalytic active site or
domain, a receptor or ligand binding interface or domain, or a
structural motif) thereof. The mimetic compound will often share a
desired biological activity with a native peptide, as discussed
herein (e.g., the ability to interact with lipids). Typically, at
least one subject biological activity of the mimetic compound is
not substantially reduced in comparison to, and is often the same
as or greater than, the activity of the native peptide on which the
mimetic was modeled.
[0210] A variety of techniques well known to one of skill in the
art are available for constructing synthetic peptide mimetics with
the same, similar, increased, or reduced biological activity as the
corresponding native peptide. Often these analogs, variants,
derivatives and mimetics will exhibit one or more desired
activities that are distinct or improved from the corresponding
native peptide, for example, improved characteristics of
solubility, stability, lipid interaction, and/or susceptibility to
hydrolysis or proteolysis (see, e.g., Morgan and Gainor, Ann. Rep.
Med. Chem. 24:243-252, 1989). In addition, mimetic compounds of the
disclosure can have other desired characteristics that enhance
their therapeutic application, such as increased cell permeability,
greater affinity and/or avidity for a binding partner, and/or
prolonged biological half-life. The mimetic compounds of the
disclosure can have a backbone that is partially or completely
non-peptide, but with side groups identical to the side groups of
the amino acid residues that occur in the peptide on which the
mimetic compound is modeled. Several types of chemical bonds, for
example, ester, thioester, thioamide, retroamide, reduced carbonyl,
dimethylene and ketomethylene bonds, are known in the art to be
generally useful substitutes for peptide bonds in the construction
of protease-resistant mimetic compounds.
[0211] In one embodiment, peptides useful within the disclosure are
modified to produce synthetic peptide mimetics by replacement of
one or more naturally occurring side chains of the genetically
encoded amino acids (or D-amino acids) with other side chains, for
example with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-,
to 7-membered alkyl, amide, amide lower alkyl, amide di(lower
alkyl), lower alkoxy, hydroxy, carboxy and the lower ester
derivatives thereof, and with 4-, 5-, 6-, to 7-membered
heterocyclics. For example, proline analogs can be made in which
the ring size of the proline residue is changed from a 5-membered
ring to a 4-, 6-, or 7-membered ring. Cyclic groups can be
saturated or unsaturated, and if unsaturated, can be aromatic or
non-aromatic. Heterocyclic groups can contain one or more nitrogen,
oxygen, and/or sulphur heteroatoms. Examples of such groups include
furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl,
isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl,
piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl,
piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,
pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g.,
1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,
thienyl, thiomorpholinyl (e.g., thiomorpholino), and thiazolyl
groups. These heterocyclic groups can be substituted or
unsubstituted. Where a group is substituted, the substituent can be
alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted
phenyl. Peptides, as well as peptide analogs and mimetics, can also
be covalently bound to one or more of a variety of nonproteinaceous
polymers, for example, polyethylene glycol, polypropylene glycol,
or polyoxyalkenes, as described in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337.
[0212] Other peptide analogs and mimetics within the scope of the
disclosure include glycosylation variants, and covalent or
aggregate conjugates with other chemical moieties. Covalent
derivatives can be prepared by linkage of functionalities to groups
which are found in amino acid side chains or at the N- or
C-termini, by means which are well known in the art. These
derivatives can include, without limitation, aliphatic esters or
amides of the carboxyl terminus, or of residues containing carboxyl
side chains, O-acyl derivatives of hydroxyl group-containing
residues, and N-acyl derivatives of the amino terminal amino acid
or amino-group containing residues (e.g., lysine or arginine). Acyl
groups are selected from the group of alkyl-moieties including C3
to C18 alkyl, thereby forming alkanoyl aroyl species. Also embraced
are versions of a native primary amino acid sequence which have
other minor modifications, including phosphorylated amino acid
residues, for example, phosphotyrosine, phosphoserine, or
phosphothreonine, or other moieties, including ribosyl groups or
cross-linking reagents.
[0213] In the peptides disclosed herein, the linkage between amino
acid residues can be a peptide bond or amide linkage (e.g.,
--C--C(O)NH--). Alternatively, one or more amide linkages is
optionally replaced with a linkage other than amide, for example, a
substituted amide. Substituted amides generally include, but are
not limited to, groups of the formula --C(O)NR--, where R is
(C.sub.1-C.sub.6) alkyl, substituted (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkenyl, substituted (C.sub.1-C.sub.6) alkenyl,
(C.sub.1-C.sub.6) alkynyl, substituted (C.sub.1-C.sub.6) alkynyl,
(C.sub.5-C.sub.20) aryl, substituted (C.sub.5-C.sub.20) aryl,
(C.sub.6-C.sub.26) alkaryl, substituted (C.sub.6-C.sub.26) alkaryl,
5-20 membered heteroaryl, substituted 5-20 membered heteroaryl,
6-26 membered alkheteroaryl, and substituted 6-26 membered
alkheteroaryl. Additionally, one or more amide linkages can be
replaced with peptidomimetic or amide mimetic moieties which do not
significantly interfere with the structure or activity of the
peptides. Suitable amide mimetic moieties are described, for
example, in Olson et al., J. Med. Chem. 36:3039-3049, 1993.
[0214] The peptides of the present invention may optionally be
acetylated at the N-terminus. The peptides of the present invention
may optionally have a carboxy terminal amide. In some embodiments,
the peptides of the present invention may have both an acetylated
N-terminus and a carboxy terminal amide. Methods of acetylating the
N-terminus or adding a carboxy terminal amide are well known to one
of ordinary skill in the art.
IV. OVERVIEW OF SEVERAL EMBODIMENTS
[0215] Isolated peptides and peptide analogs with domains that
promote lipid efflux from cells are disclosed herein. The isolated
peptides and peptide analogs are believed to stimulate LCAT
activity. In some embodiments, the isolated peptides and peptide
analogs of the present invention contain domains that promote lipid
efflux and also possess anti-inflammatory activity. Domains that
possess lipid efflux and anti-inflammatory activity provide
additional benefit as many vascular conditions are considered by
one of ordinary skill in the art to have inflammation as a
component of the disease etiology.
[0216] For administration to an animal or a human, the peptides and
peptide analogs of the present invention are combined with an
acceptable carrier to form a pharmaceutical composition and are
administered to the animal or the human.
[0217] In another embodiment, a method is provided for treating or
inhibiting dyslipidemic and vascular disorders in an animal or a
human. This method includes administering to the animal or the
human a therapeutically effective amount of a pharmaceutical
composition that includes one or more isolated peptides or peptide
analogs and one or more anti-inflammatory domains. In specific,
non-limiting examples, the dyslipidemic and vascular disorders
include hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia,
hypertriglyceridemia, HDL deficiency, apoA-I deficiency, coronary
artery disease, atherosclerosis, myocardial infarction, stroke,
thrombotic stroke, peripheral vascular disease, restenosis, acute
coronary syndrome, and reperfusion myocardial injury. In yet
another specific example of the provided method, the isolated
peptide includes a domain or domains (A and C) that possess both
anti-inflammatory and lipid efflux activity and has an amino acid
sequence as set forth herein.
[0218] Additionally, in representative peptides disclosed herein,
the amino- and carboxy-terminal ends can be modified by conjugation
with various functional groups. Neutralization of the terminal
charge of synthetic peptide mimetics of apolipoproteins has been
shown to increase their lipid affinity (Yancey et al., Biochem.
34:7955-7965, 1995; Venkatachalapathi et al., Protein: Structure,
Function and Genetics 15:349-359, 1993). For example, acetylation
of the amino terminal end of amphipathic peptides increases the
lipid affinity of the peptide (Mishra et al., J. Biol. Chem.
269:7185-7191, 1994). Other possible end modifications are
described, for example, in Brouillette et al., Biochem. Biophys.
Acta 1256:103-129, 1995: Mishra et al., J. Biol. Chem.
269:7185-7191, 1994; and Mishra et al., J. Biol. Chem.
270:1602-1611, 1995.
[0219] In another embodiment, a detectable moiety can be linked to
any of the peptides disclosed herein, creating a peptide-detectable
moiety conjugate. The peptides or peptide analogs disclosed herein
may be labeled using labels and techniques known to one of ordinary
skill in the art. Some of these labels are described in the
"Handbook of Fluorescent Probes and Research Products", ninth
edition, Richard P. Haugland (ed) Molecular Probes, Inc. Eugene,
Oreg.), which is incorporated herein in its entirety. Detectable
moieties suitable for such use include any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical, magnetic or chemical means. The detectable
moieties contemplated for the present disclosure can include, but
are not limited to, an immunofluorescent moiety (e.g., fluorescein,
rhodamine, Texas red, and the like), a radioactive moiety (e.g.,
.sup.3H, .sup.32P, .sup.125I, .sup.131I, .sup.35S), an enzyme
moiety (e.g., horseradish peroxidase, alkaline phosphatase), a
colorimetric moiety (e.g., colloidal gold, biotin, colored glass or
plastic, and the like). The detectable moiety can be liked to the
peptide or peptide analog at either the N- and/or C-terminus.
Optionally, a linker can be included between the peptide or peptide
analog and the detectable moiety.
[0220] The detectable peptides of the present invention may be
employed in imaging techniques to identify sites of atherosclerotic
plaque and sites of cholesterol efflux. Such imaging techniques may
occur in vivo using IVUS, NMR, CAT, PET or other techniques
commonly known to one of ordinary skill in the art.
[0221] Means of detecting such moieties are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film, gamma counters or scintillation counters.
Fluorescent markers may be detected using a photodetector to detect
emitted illumination. Enzymatic labels are typically detected by
providing the enzyme with a substrate and detecting the reaction
product produced by the action of the enzyme on the substrate, and
calorimetric labels are detected by simply visualizing the colored
label.
[0222] The linkers contemplated by the present disclosure can be
any bifunctional molecule capable of covalently linking two
peptides to one another. Thus, suitable linkers are bifunctional
molecules in which the functional groups are capable of being
covalently attached to the N- and/or C-terminus of a peptide.
Functional groups suitable for attachment to the N- or C-terminus
of peptides are well known in the art, as are suitable chemistries
for effecting such covalent bond formation.
[0223] The linker may be flexible, rigid or semi-rigid. Suitable
linkers include, for example, amino acid residues such as Pro or
Gly or peptide segments containing from about 2 to about 5, 10, 15,
20, or even more amino acids, bifunctional organic compounds such
as H.sub.2N(CH.sub.2).sub.nCOOH where n is an integer from 1 to 12,
and the like. Examples of such linkers, as well as methods of
making such linkers and peptides incorporating such linkers, are
well-known in the art (see, e.g., Hunig et al., Chem. Ber.
100:3039-3044, 1974 and Basak et al, Bioconjug. Chem. 5:301-305,
1994).
[0224] Conjugation methods applicable to the present disclosure
include, by way of non-limiting example, reductive amination, diazo
coupling, thioether bond, disulfide-bond, amidation and
thiocarbamoyl chemistries. In one embodiment, the amphipathic
.alpha.-helical domains are "activated" prior to conjugation.
Activation provides the necessary chemical groups for the
conjugation reaction to occur. In one specific, non-limiting
example, the activation step includes derivatization with adipic
acid dihydrazide. In another specific, non-limiting example, the
activation step includes derivatization with the
N-hydroxysuccinimide ester of 3-(2-pyridyl dithio)-propionic acid.
In yet another specific, non-limiting example, the activation step
includes derivatization with succinimidyl 3-(bromoacetamido)
propionate. Further, non-limiting examples of derivatizing agents
include succinimidylformylbenzoate and succinimidyllevulinate.
V. SYNTHESIS AND PURIFICATION OF THE PEPTIDES
[0225] The peptides or peptide analogs of the disclosure can be
prepared using virtually any technique known to one of ordinary
skill in the art for the preparation of peptides. For example, the
peptides can be prepared using step-wise solution or solid phase
peptide syntheses, or recombinant DNA techniques, or the
equivalents thereof.
[0226] A. Chemical Synthesis
[0227] Peptides of the disclosure containing amino acids having
either the D- or L-configuration can be readily synthesized by
automated solid phase procedures well known in the art. Suitable
syntheses can be performed by utilizing "T-boc" or "F-moc"
procedures. 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, Oxford
University Press, 1989. Alternatively, the peptides may be prepared
by way of segment condensation, as described, for example, in Liu
et al., Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am.
Chem. Soc. 117:1881-1887, 1995; Tam et al., Int. J. Peptide Protein
Res. 45:209-216, 1995; Schnolzer and Kent, Science 256:221-225,
1992; Liu and Tam, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and
Tam, Proc. Natl. Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro
and Li, Int. J. Peptide Protein Res. 31:322-334, 1988). This is
particularly the case with glycine containing peptides. Other
methods useful for synthesizing the peptides of the disclosure are
described in Nakagawa et al., J. Am. Chem. Soc. 107:7087-7092,
1985.
[0228] Additional exemplary techniques known to those of ordinary
skill in the art of peptide and peptide analog synthesis are taught
by Bodanszky, M. and Bodanszky, A., The Practice of Peptide
Synthesis, Springer Verlag, New York, 1994; and by Jones, J., Amino
Acid and Peptide Synthesis, 2nd ed., Oxford University Press, 2002.
The Bodanszky and Jones references detail the parameters and
techniques for activating and coupling amino acids and amino acid
derivatives. Moreover, the references teach how to select, use and
remove various useful functional and protecting groups.
[0229] Peptides of the disclosure having either the D- or
L-configuration can also be readily purchased from commercial
suppliers of synthetic peptides. Such suppliers include, for
example, Advanced ChemTech (Louisville, Ky.), Applied Biosystems
(Foster City, Calif.), Anaspec (San Jose, Calif.), and Cell
Essentials (Boston, Mass.).
[0230] B. Recombinant Synthesis
[0231] If the peptide is composed entirely of gene-encoded amino
acids, or a portion of it is so composed, the peptide or the
relevant portion can also be synthesized using conventional
recombinant genetic engineering techniques. For recombinant
production, a polynucleotide sequence encoding the peptide is
inserted into an appropriate expression vehicle, that is, a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence, or in the case of an
RNA viral vector, the necessary elements for replication and
translation. The expression vehicle is then transfected into a
suitable target cell which will express the peptide. Depending on
the expression system used, the expressed peptide is then isolated
by procedures well-established in the art. Methods for recombinant
protein and peptide production are well known in the art (see,
e.g., Sambrook et al. (ed.), Molecular Cloning: A Laboratory
Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, Ch. 17 and Ausubel et al Short
Protocols in Molecular Biology, 4.sup.th ed., John Wiley &
Sons, Inc., 1999).
[0232] One of ordinary skill in the art will be familiar with the
genetic code by which nucleic acids encode proteins, polypeptides,
and peptides. Each amino acid is encoded by one or more sets of
three nucleotides; each of these sets is called a codon. This
triplet genetic code is degenerate in that all of the amino acids,
except for methionine, are encoded by more than one codon. See,
e.g., Watson et al., Molecular Biology of the Gene, 4.sup.th ed.,
Vol. 1, Benjamin/Cummings Publishing Co., Menlo Park, 1987. For
example, the amino acid serine can be encoded by six different
codons. One of ordinary skill in the art will appreciate that the
particular codons selected to encode a given protein, polypeptide,
or peptide can be based upon the particular type of cell or in
vitro translation system in which the protein, polypeptide, or
peptide is to be produced (i.e., to optimize translation in the
particular translation system selected for production of the
protein, polypeptide, or peptide). Just one example of a DNA
sequence that can encode a peptide of the invention, in this case
SEQ ID NO: 142 Ser Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser
Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln, is (from 5' to 3') SEQ ID NO: 607 tct cct ctt ctt gag tct gct
aag gtt tct gct ctt tct gct ctt gaa gaa gct act aaa aaa aaa ctt tct
cct ctt ctt gaa agt ttt aaa gtt tct ttt ctt tct gct ctt gaa gaa tat
act aaa aaa tta aat act caa. One of ordinary skill will understand
that in an mRNA sequence encoding this peptide, the base thymine
(represented by "t" in the DNA sequence above) would be replaced
with the base uracil ("u").
[0233] It is well-known by one of ordinary skill in the art that
DNA is transcribed into messenger RNA (mRNA), after which the mRNA
is translated into the encoded protein, polypeptide, or peptide.
One of skill in the art will appreciate that, for an mRNA to be
translated into a peptide or polypeptide within a cell or using an
in vitro translation system, there must be a translation start
site, i.e., the codon for the amino acid methionine (aug). Thus,
nucleic acids encoding peptides of the present invention can be
designed to encode fusion proteins with an optional internal
cleavage site, after which the translated protein optionally can be
cleaved using an endopeptidase to release free peptide, as will be
understood by one of ordinary skill in the art. Alternatively,
nucleic acids encoding peptides of the present invention can be
designed to encode an amino-terminal methionine, which optionally
can be cleaved off by an exopeptidase, as will be appreciated by
one of ordinary skill in the art. As one of skill in the art will
appreciate, the nucleic acid encoding the peptide can also contain
a stop codon (e.g., taa, tag, or tga in DNA; uaa, uag, or uga in
RNA) to terminate translation of the peptide.
[0234] To increase efficiency of production, the polynucleotide can
be designed to encode multiple units of the peptide separated by
enzymatic cleavage sites. The resulting polypeptide can be cleaved
(e.g., by treatment with the appropriate enzyme) in order to
recover the peptide units. This can increase the yield of peptides
driven by a single promoter. In one embodiment, a polycistronic
polynucleotide can be designed so that a single mRNA is transcribed
which encodes multiple peptides, each coding region operatively
linked to a cap-independent translation control sequence, for
example, an internal ribosome entry site (IRES). When used in
appropriate viral expression systems, the translation of each
peptide encoded by the mRNA is directed internally in the
transcript, for example, by the IRES. Thus, the polycistronic
construct directs the transcription of a single, large
polycistronic mRNA which, in turn, directs the translation of
multiple, individual peptides. This approach eliminates the
production and enzymatic processing of polyproteins and can
significantly increase yield of peptide driven by a single
promoter.
[0235] A variety of host-expression vector systems may be utilized
to express the peptides described herein. These include, but are
not limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage DNA or plasmid DNA expression vectors
containing an appropriate coding sequence; yeast or filamentous
fungi transformed with recombinant yeast or fungi expression
vectors containing an appropriate coding sequence; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing an appropriate coding sequence; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing an appropriate coding sequence; or animal cell
systems.
[0236] The expression elements of the expression systems vary in
their strength and specificities. Depending on the host/vector
system utilized, any of a number of suitable transcription and
translation elements, including constitutive and inducible
promoters, can be used in the expression vector. For example, when
cloning in bacterial systems, inducible promoters such as pL of
bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter)
and the like can be used. When cloning in insect cell systems,
promoters such as the baculovirus polyhedron promoter can be used.
When cloning in plant cell systems, promoters derived from the
genome of plant cells (e.g., heat shock promoters, the promoter for
the small subunit of RUBISCO, the promoter for the chlorophyll a/b
binding protein) or from plant viruses (e.g., the 35S RNA promoter
of CaMV, the coat protein promoter of TMV) can be used. When
cloning in mammalian cell systems, promoters derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter, the vaccinia
virus 7.5 K promoter) can be used.
[0237] C. Purification
[0238] The peptides or peptide analogs of the disclosure can be
purified by many techniques well known in the art, such as reverse
phase chromatography, high performance liquid chromatography, ion
exchange chromatography, size exclusion chromatography, affinity
chromatography, gel electrophoresis, and the like. The actual
conditions used to purify a particular peptide or peptide analog
will depend, in part, on synthesis strategy and on factors such as
net charge, hydrophobicity, hydrophilicity, and the like, and will
be apparent to those of ordinary skill in the art.
[0239] For affinity chromatography purification, any antibody which
specifically binds the peptide or peptide analog may be used.
[0240] The peptides of the present invention may optionally be
acetylated at the N-terminus. The peptides of the present invention
may optionally have a carboxy terminal amide. In some embodiments,
the peptides of the present invention may have both an acetylated
N-terminus and a carboxy terminal amide. Methods of acetylating the
N-terminus or adding a carboxy terminal amide are well known to one
of ordinary skill in the art.
[0241] D. Antibody Production
[0242] For the production of antibodies, various host animals,
including but not limited to, rabbits, mice, rats, and the like,
may be immunized by injection with a peptide or peptide analog. The
peptide or peptide analog can be attached to a suitable carrier
(e.g., bovine serum albumin (BSA)) by means of a side chain
functional group or linker attached to a side chain functional
group. Various adjuvants may be used to increase the immunological
response, depending on the host species, including but not limited
to, Freund's (complete and incomplete), mineral gels (e.g.,
aluminum hydroxide), surface active substances (e.g., lysolecithin,
pluronic polyols, polyanions, and oil emulsions), keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacilli Calmette-Guerin) and Corynebacterium
parvum.
[0243] Booster injections can be given at regular intervals, and
antiserum harvested when the antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
e.g., Ouchterlony et al., Handbook of Experimental Immunology,
Wier, D. (ed.), Chapter 19, Blackwell, 1973. A plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher (Manual of Clinical Immunology,
Ch. 42, 1980).
[0244] Monoclonal antibodies to a peptide or peptide analog may be
prepared using any technique which provides for the production of
antibody molecules by continuous cell lines in culture, for example
the classic method of Kohler & Milstein (Nature 256:495-97,
1975), or a derivative method thereof. Briefly, a mouse is
repetitively inoculated with a few micrograms of the selected
protein immunogen (e.g., a peptide or peptide analog) over a period
of a few weeks. The mouse is then sacrificed, and the
antibody-producing cells of the spleen isolated. The spleen cells
are fused by means of polyethylene glycol with mouse myeloma cells,
and the excess unfused cells destroyed by growth of the system on
selective media comprising aminopterin (HAT media). The
successfully fused cells are diluted and aliquots of the dilution
placed in wells of a microtiter plate where growth of the culture
is continued. Antibody-producing clones are identified by detection
of antibody in the supernatant fluid of the wells by immunoassay
procedures, such as enzyme-linked immunosorbent assay (ELISA), as
originally described by Engvall (Meth. Enzymol., 70:419-39, 1980),
or a derivative method thereof. Selected positive clones can be
expanded and their monoclonal antibody product harvested for use.
Detailed procedures for monoclonal antibody production are
described in Harlow and Lane, Using Antibodies: A Laboratory
Manual, CSHL, New York, 1999. Polyclonal antiserum containing
antibodies can be prepared by immunizing suitable animals with a
polypeptide comprising at least one peptide or peptide analog,
which can be unmodified or modified, to enhance immunogenicity.
[0245] Antibody fragments may be used in place of whole antibodies
and may be readily expressed in prokaryotic host cells. Methods of
making and using immunologically effective portions of monoclonal
antibodies, also referred to as "antibody fragments," are well
known and include those described in Better & Horowitz, Methods
Enzymol. 178:476-96, 1989; Glockshuber et al., Biochemistry
29:1362-67, 1990; and U.S. Pat. Nos. 5,648,237 (Expression of
Functional Antibody Fragments); 4,946,778 (Single Polypeptide Chain
Binding Molecules); and 5,455,030 (Immunotherapy Using Single Chain
Polypeptide Binding Molecules), and references cited therein.
Conditions whereby a polypeptide/binding agent complex can form, as
well as assays for the detection of the formation of a
polypeptide/binding agent complex and quantitation of binding
affinities of the binding agent and polypeptide, are standard in
the art. Such assays can include, but are not limited to, Western
blotting, immunoprecipitation, immunofluorescence,
immunocytochemistry, immunohistochemistry, fluorescence activated
cell sorting (FACS), fluorescence in situ hybridization (FISH),
immunomagnetic assays, ELISA, ELISPOT (Coligan et al., Current
Protocols in Immunology, Wiley, NY, 1995), agglutination assays,
flocculation assays, cell panning, etc., as are well known to one
of skill in the art.
[0246] E. Peptide Reconstitution
[0247] The peptides of the present invention may be reconstituted
in any pharmaceutically acceptable carrier before use or
administration. In one embodiment, the peptides may be
reconstituted with saline, a lipid or a phospholipid, or a
combination thereof. Some phospholipids that may be employed
include but are not limited to the following:
dipalmitoylphosphatidylcholine (DPPC); dioleoylphosphatidylcholine
(DOPC); 1-palmitoyl-2-oleoylphosphatidylcholine (POPC);
1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC);
1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC);
1-palmitoyl-2-docosahexanoylphosphatidylcholine (PDPC); and
1-palmitoyl-2-myristoylphosphatidylcholine (PMPC). See, e.g., Shah
et al. (Circulation (2001), 103(25):3047-50), in which DPPC was
used to reconstitute peptides. The peptides of the present
invention may be complexed with lipids or phospholipids in weight
ratios ranging from 1:0.5 to 1:10, or 1:1 to 1:5. Any ratio within
these ranges may be employed.
[0248] The phospholipids may also be complexed with other agents,
such as sphingomyelin before complexing with the peptides of the
present invention. Ratios of phospholipids to sphingomyelin include
ratios occurring in the ranges of 1:9 to 9:1, 1:5 to 5:1, 1.2 to
2.1 (all weight %).
[0249] The peptides of the present invention may be complexed with
the combination of phospholipid:sphingomyelin in weight ratios
ranging from 1:0.5 to 1:10, or 1:1 to 1:5. Any ratio within these
ranges may be employed.
VI. PHARMACEUTICAL COMPOSITIONS AND USES THEREOF
[0250] The peptides or peptide analogs of the disclosure can be
used, alone or in combination, together with a pharmaceutically
acceptable carrier, to treat any disorder in animals, especially
mammals (e.g., humans), for which promoting lipid efflux and/or
decreasing inflammation is beneficial. Such conditions include, but
are not limited to, hyperlipidemia (e.g., hypercholesterolemia),
cardiovascular disease (e.g., atherosclerosis), cerebrovascular
disease, restenosis (e.g., atherosclerotic plaques), peripheral
vascular disease, acute coronary syndrome, reperfusion myocardial
injury, and the like. The peptides or peptide analogs of the
disclosure can also be used alone or in combination during the
treatment of thrombotic stroke, infarcts secondary to occlusion of
a vessel and during thrombolytic treatment of occluded coronary
artery disease. The peptides or peptide analogs of the disclosure
can be used to treat tissue following hypoxia, ischemia and
infarction due to impairment of blood supply, and also following
hemorrhage following rupture or trauma of a blood vessel. Such
tissue includes, without limitation, neural tissue in the central
or peripheral nervous system, peripheral vascular tissue, and
cardiac muscle.
[0251] It is to be understood that a mixture of peptides may
include different amounts of the individual peptides. For example,
in one embodiment, each peptide component of the combination may be
present in a different relative percentage than each other peptide
component due to differences in relative efficacy to promote lipid
efflux or to provide one or more types of anti-inflammatory
activity. In one exemplary embodiment, two or more of the peptides
comprising the sequences shown in SEQ ID NO: 22 (Leu Glu Ser Ala
Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys); SEQ ID NO: 142 (Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln); SEQ ID NO: 602 (Ac-Ser Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu
Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln-NH.sub.2); and/or SEQ ID NO: 608
Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala
Phe Pro Asp Trp Ala Lys Ala Ala Tyr Asp Lys Ala Ala Glu Lys Ala Lys
Glu Ala Ala may be combined in a mixture for administration.
[0252] The peptides or peptide analogs can be used alone or in
combination therapy with other lipid lowering compositions or drugs
and/or other anti-inflammatory compositions or drugs used to treat
the foregoing conditions. Such therapies include, but are not
limited to simultaneous or sequential administration of the drugs
involved. For example, in the treatment of hypercholesterolemia or
atherosclerosis, the peptide or peptide analog formulations can be
administered with any one or more of the cholesterol lowering
therapies currently in use, for example, bile-acid resins, niacin,
statins, fat uptake inhibitors, and HDL raising drugs.
[0253] In another embodiment, the peptides or peptide analogs can
be used in conjunction with statins or fibrates to treat
hyperlipidemia, hypercholesterolemia and/or cardiovascular disease,
such as atherosclerosis. In yet another embodiment, the peptides or
peptide analogs of the disclosure can be used in combination with
an anti-microbial agent and/or an anti-inflammatory agent, such as
aspirin. In another embodiment peptides or peptide analogs of the
disclosure can be used in combination with anti-hypertensive
medicines known to one of ordinary skill in the art. It is to be
understood that more than one additional therapy may be combined
with administration of the peptides or peptide analogs of the
disclosure.
[0254] In a further embodiment, the peptides can also be expressed
in vivo, by using any of the available gene therapy approaches.
[0255] In yet another embodiment, the peptides or peptide analogs
can be used in conjunction with medicines used to treat patients
with cerebrovascular and cardiovascular disease resulting in
hypoxia, ischemia and infarction due to impairment of blood supply,
and also following hemorrhage following rupture or trauma of a
blood vessel. Such medicines are commonly known to one of ordinary
skill in the art and include without limitation, modulators of
excitatory amino acids and modulators of platelet aggregation.
[0256] A. Administration of Peptides or Peptide Analogs
[0257] In some embodiments, peptides or peptide analogs can be
isolated from various sources and administered directly to the
animal or human. For example, a peptide or peptide analog can be
expressed in vitro, such as in an E. coli expression system, as is
well known in the art, and isolated in amounts useful for
therapeutic compositions. The peptide or peptide analogs of the
present invention may also be made though peptide synthetic methods
known to one of ordinary skill in the art, such as solid phase
synthesis.
[0258] In exemplary applications, therapeutic compositions
comprising the peptide or peptide analogs in an acceptable carrier
are administered to an animal or a human suffering from a
dyslipidemic or vascular disorder, such as hyperlipidemia,
hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia,
HDL deficiency, apoA-I deficiency, coronary artery disease,
atherosclerosis, stroke, ischemia, infarction, myocardial
infarction, hemorrhage, peripheral vascular disease, restenosis,
acute coronary syndrome, or reperfusion myocardial injury, in an
amount sufficient to inhibit or treat the dyslipidemic or vascular
disorder. Amounts effective for this use will depend upon the
severity of the disorder and the general state of the subject's
health. A therapeutically effective amount of the compound is that
which provides either subjective relief of a symptom(s) or an
objectively identifiable improvement as noted by the clinician or
other qualified observer.
[0259] A peptide or peptide analog can be administered by any means
known to one of skill in the art (see, e.g., Banga, "Parenteral
Controlled Delivery of Therapeutic Peptides and Proteins," in
Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,
Lancaster, Pa., 1995), such as by intramuscular, subcutaneous, or
intravenous injection, but even oral, nasal, or anal administration
is contemplated. In one embodiment, administration is by
subcutaneous or intramuscular injection. To extend the time during
which the peptide or peptide analog is available to inhibit or
treat a dyslipidemic or vascular disorder, the peptide or peptide
analog can be provided as an implant, an oily injection, or as a
particulate system. The particulate system can be a microparticle,
a microcapsule, a microsphere, a nanoparticle, or similar particle
(Banga, "Parenteral Controlled Delivery of Therapeutic Peptides and
Proteins," in Therapeutic Peptides and Proteins, Technomic
Publishing Co., Inc., Lancaster, Pa., 1995). The peptide or peptide
analog may also be applied to a medical device for delivery to a
specific location. For example, a surgical tool, catheter, stent,
balloon, electrode, suture, or an artificial vessel or transplanted
vessel may contain or be coated with the peptide or peptide
analog.
[0260] It is to be understood that in some embodiments, one or more
of the amino acids of the peptides of the present invention are D
amino acids. In one embodiment, the N-terminal amino acid, the
C-terminal amino acid or both are D amino acids. The presence of
these D amino acids can help protect against peptide degradation.
In another embodiment, all the amino acids of the peptides of the
present invention are D amino acids. This embodiment is useful for
protection against degradation following oral administration of a
pharmaceutical composition comprising the peptides of the present
invention.
[0261] In one specific, non-limiting example, a peptide is
administered that includes one or more of the amino acid sequences
disclosed herein.
[0262] B. Representative Methods of Administration Formulations and
Dosage
[0263] The provided peptides or peptide analogs, constructs, or
vectors encoding such peptides, can be combined with a
pharmaceutically acceptable carrier (e.g., a phospholipid or other
type of lipid) or vehicle for administration to human or animal
subjects. As described previously in the application, the peptides
may be reconstituted with acceptable carriers such as saline,
lipid, phospholipid, lipid:sphingomyelin complexes and
phospholipid: sphingomyelin complexes. In some embodiments, more
than one peptide or peptide analog can be combined to form a single
preparation. The peptides or peptide analogs can be conveniently
presented in unit dosage form and prepared using conventional
pharmaceutical techniques. Such techniques include the step of
bringing into association the active ingredient and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers.
Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of a sterile
liquid carrier, for example, water for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
may be prepared from sterile powders, granules and tablets commonly
used by one of ordinary skill in the art.
[0264] In certain embodiments, unit dosage formulations are those
containing a dose or unit, or an appropriate fraction thereof, of
the administered ingredient. It should be understood that in
addition to the ingredients particularly mentioned above,
formulations encompassed herein may include other agents commonly
used by one of ordinary skill in the art.
[0265] The pharmaceutical compositions provided herein, including
those for use in treating dyslipidemic and vascular disorders, may
be administered through different routes, such as oral, including
buccal and sublingual, rectal, parenteral, aerosol, nasal,
intramuscular, intraperitoneal, intravascular, subcutaneous,
intradermal, and topical. They may be administered in different
forms, including but not limited to solutions, emulsions and
suspensions, microspheres, particles, microparticles,
nanoparticles, and liposomes. In one embodiment, peptides or
peptide analogs with suitable features of lipid efflux and low
cytotoxicity can be precomplexed with phospholipids or other lipids
into either discoidal or spherical shape particles prior to
administration to subjects.
[0266] In another embodiment, it may be desirable to administer the
pharmaceutical compositions locally to the area in need of
treatment. This maybe achieved by, for example, and not by way of
limitation, local or regional infusion or perfusion during surgery,
direct perfusion into a vessel, such as an atherosclerotic vessel,
topical application (e.g., wound dressing, peptide coated stent),
injection, catheter, suppository, or implant (e.g., implants formed
from porous, non-porous, or gelatinous materials, including
membranes, such as silastic membranes or fibers), and the like. In
one embodiment, administration can be by direct injection at the
site (or former site) of a tissue that is to be treated, such as
the heart or the peripheral vasculature. In another embodiment, the
pharmaceutical compositions are delivered in a vesicle, in
particular liposomes (see, e.g., Langer, Science 249:1527-1533,
1990; Treat et al., in Liposomes in the Therapy of Infectious
Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y.,
pp. 353-365, 1989). Combinations of administration methods may also
be employed such as a systemic or local infusion of a peptide of
the present invention, before, after or during placement of a stent
coated with a peptide of the present invention.
[0267] In yet another embodiment, the pharmaceutical compositions
can be delivered in a controlled release system. In one embodiment,
a pump can be used (see, e.g., Langer Science 249:1527-1533, 1990;
Sefton Crit. Rev. Biomed. Eng. 14:201-240, 1987; Buchwald et al.,
Surgery 88:507-516, 1980; Saudek et al., N. Engl. J. Med.
321:574-579, 1989). In another embodiment, polymeric materials can
be used (see, e.g., Ranger et al., Macromol. Sci. Rev. Macromol.
Chem. 23:61-64, 1983; Levy et al., Science 228:190-192, 1985;
During et al., Ann. Neurol. 25:351-356, 1989; and Howard et al., J.
Neurosurg. 71:105-112, 1989). Other controlled release systems,
such as those discussed in the review by Langer (Science
249:1527-1533, 1990), can also be used.
[0268] The amount of the pharmaceutical compositions that will be
effective depends on the nature of the disorder or condition to be
treated, as well as the stage of the disorder or condition.
Effective amounts can be determined by standard clinical
techniques. The precise dose to be employed in the formulation will
also depend on the route of administration, and should be decided
according to the judgment of the health care practitioner and each
subject's circumstances. An example of such a dosage range is 0.1
to 200 mg/kg body weight in single or divided doses. Another
example of a dosage range is 1.0 to 100 mg/kg body weight in single
or divided doses.
[0269] The specific dose level and frequency of dosage for any
particular subject may be varied and will depend upon a variety of
factors, including the activity of the specific compound, the
metabolic stability and length of action of that compound, the age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, and severity
of the condition of the subject undergoing therapy.
[0270] The pharmaceutical compositions of the present disclosure
can be administered at about the same dose throughout a treatment
period, in an escalating dose regimen, or in a loading-dose regime
(e.g., in which the loading dose is about two to five times the
maintenance dose). In some embodiments, the dose is varied during
the course of a treatment based on the condition of the subject
being treated, the severity of the disease or condition, the
apparent response to the therapy, and/or other factors as judged by
one of ordinary skill in the art. The volume of administration will
vary depending on the route of administration. By way of example,
intramuscular injections may range from about 0.1 ml to about 1.0
ml. Those of ordinary skill in the art will know appropriate
volumes for different routes of administration.
[0271] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various embodiments,
modifications and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the invention.
[0272] The subject matter of the present disclosure is further
illustrated by the following non-limiting Examples.
EXAMPLES
Example 1
Lipid Efflux From Cells Mediated by Peptides of the Present
Invention
[0273] This example demonstrates a method to test the ability of
peptides of the present invention to efflux lipid from
ABCA1-expressing cells.
[0274] HeLa cells stably transfected with human ABCA1 cDNA (ABCA1
cells) and HeLa cells transfected with only a hygromycin-resistant
control plasmid (control cells) are produced and grown in
.alpha.-modified Eagle's medium (aMEM) plus 10% fetal calf serum,
as described by Remaley et al. (Biochem. Biophys. Res. Commun.
280:818-823, 2001). Cholesterol and phospholipid efflux is
performed for 18 hours on noncholesterol-loaded cells radiolabeled
with either cholesterol or choline (Remaley et al., Arterioscler.
Thromb. Vasc. Biol. 17:1813-1821, 1997). Percentage efflux is
calculated after subtracting the radioactive counts in the blank
media (aMEM plus 1 mg/ml of BSA), and expressed as the percent of
total radioactive counts removed from the cells during the efflux
period.
[0275] Cell fixation is performed by a 10 minute treatment with 3%
paraformaldehyde in phosphate buffered saline (PBS), followed by
three washes with blank media. Lactate dehydrogenase (LDH) release
from cells into the media is measured enzymatically (Roche
Diagnostics, Indianapolis, Ind.) and expressed, after subtraction
of LDH released into blank media, as the percentage of total cell
LDH. Total cell LDH is determined after cell solubilization with 1%
Triton X-100.
[0276] The peptides of the present invention are synthesized by a
solid-phase procedure, using a Fmoc/DIC/HOBt protocol on a
Biosearch 9600 peptide synthesizer (Applied Biosystems, Foster
City, Calif.), or an equivalent instrument. Both L-amino acid and
D-amino acid enantiomers are synthesized. All peptides are purified
to greater than 98% homogeneity by reverse-phase HPLC on an
Aquapore RP-300 column, or similar chromatographic procedure.
[0277] ABCA1 cells are used to assess the ability of apoA-I and
synthetic peptides to efflux lipid from cells. As previously
described (Hamon et al., Nat. Cell Biol. 2:399-406, 2000 and
Remaley et al., Biochem. Biophys. Res. Commun. 280:818-823, 2001),
control cells do not efflux significant amounts of cholesterol and
phospholipid to apoA-I, but do so after transfection with ABCA1.
The peptides of the present invention efflux approximately 2- to
4-fold more cholesterol and phospholipid from ABCA1 cells than from
control cells. Both the peptides of the present invention and
apoA-I began to show saturation for lipid efflux at approximately
the same protein concentration of 10 .mu.g/ml. The peptides of the
present invention remove more cholesterol and phospholipids from
control cells than apoA-I.
Example 2
Lipid Efflux Time Course
[0278] This example demonstrates the cholesterol efflux time course
from ABCA1-expressing cells to apoA-1 and peptides of the present
invention.
[0279] Cholesterol efflux from ABCA1 cells to apoA-1 is first
detectable after 2 hours and increases throughout the 30 hour
efflux period. In contrast, there is no significant increase above
background in cholesterol efflux to apoA-I from control cells.
Overall, the kinetics for cholesterol efflux to peptides of the
present invention from ABCA1 cells is similar to that of apoA-I,
except that cholesterol efflux is first detectable after 30
minutes. The peptides of the present invention, unlike apoA-I, also
promote cholesterol efflux from control cells but at a lower
rate.
Example 3
Identification of Non-Cytotoxic Peptides that Promote
ABCA1-Dependent Lipid Efflux
[0280] This example illustrates a method for identifying
non-cytotoxic peptides that promote ABCA1-dependent lipid efflux
from cells.
[0281] Peptide Design: Based on the principles and procedures
described in the present application, an amino acid sequence can be
designed for a peptide that promotes lipid efflux.
[0282] Peptide production: Peptides to be tested can be produced
synthetically or by recombinant DNA methods, as described in the
present application, and purified by reverse phase HPLC or other
suitable techniques well known to one of skill in the art.
[0283] Peptide Cytotoxicity Testing: Peptides can be tested for
cytotoxicity by any number of methods well known to one of skill in
the art, such as the release of intracellular LDH.
[0284] Peptide ABCA1-specificity for Lipid Efflux: Peptides to be
tested can be added to serum-free cell culture media in the
approximate concentration range of 1-20 micrograms and incubated
with a control cell line that does not express the ABCA1
transporter and the same cell line after transfection with human
cDNA for the ABCA1 transporter, as described herein. Alternatively,
cells, such as macrophages, that either express or do not express
the ABCA1 transporter depending on their cholesterol content and/or
exposure to agents that induce the ABCA1 transporter (e.g., cAMP
and LXR agonists) can also be used. After a suitable period of
approximately 4 to 24 hours, the conditioned media can be removed
from the cells and the amount of cholesterol and or phospholipid
effluxed can be quantified, as described herein. ABCA1-specific
lipid efflux is calculated by subtracting the total lipid efflux of
the cell line that does not express the ABCA1 transporter from the
lipid efflux from the ABCA1 expressing cell line.
Example 4
Peptides of the Present Invention Reduce Atherosclerosis in Animal
Models
[0285] The ability of the peptides of the present invention and
associated fragments are tested in apoe knockout mice on a chow
diet and LDL receptor knockout mice on a western high fat diet to
determine the effect of these peptides to reduce atherosclerosis in
a mouse model system. One or more of the peptides of the present
invention, in a range of concentration of 2 mg/kg to 50 mg/kg, is
injected intravenously (iv) or intraperitoneally (ip) 2 to 3 times
per week over a period of approximately 6 weeks. In one study,
peptides of SEQ ID NO: 142 and/or SEQ ID NO: 602 are tested. Aortic
atherosclerosis is quantitated in the aortic arch before
administration of the peptides and after the 6 week period of
administration. (Wu et al., J. Biol. Chem.; 2004: 279,
22913-22925). The results demonstrate reduced atherosclerosis in
the aortic arch in mice in both treatment groups.
Example 5
Administration of the Peptides of the Present Invention to Treat
Atherosclerosis in Humans
[0286] Individuals with acute coronary syndrome and documented
atherosclerosis have a cardiac catherization with intravascular
ultrasound (IVUS) to document coronary atherosclerosis of 20 to 50%
obstruction in the target artery. Each individual is on stable
hypolipidemic drug therapy and receives an acceptable dose of a
peptide of the present invention and/or an associated fragment iv
weekly for a period of 5 to 8 weeks. In one study, peptides of SEQ
ID NO: 142 and/or SEQ ID NO: 602 are tested. A repeat IVUS
measurement is made at the end of the treatment period to assess
the effect of the peptide infusion on coronary atherosclerosis in
the target vessel. Plaque is reduced in the atherosclerotic
coronary artery following the peptide treatment demonstrating
efficacy of the peptides of the present invention to treat
atherosclerosis.
Example 6
Administration of the Peptides of the Present Invention to Prevent
or Delay the Onset of Atherosclerosis in Humans
[0287] Individuals with documented risk factors for atherosclerosis
and having high plasma cholesterol levels have a ultrasound
analysis of the coronary (IVUS), carotid (IMT) or popliteal
arteries to establish a baseline measurement. A portion of these
individuals are daily administered individual peptides of the
present invention at a dose of 2 mg/kg to 50 mg/kg intravenously
(iv) or intramuscular (im) 1 to 3 times per week over a period of
approximately one to six months. In one study, peptides of SEQ ID
NO: 142 and/or SEQ ID NO: 602 are tested. The other individuals
receive a control peptide. A new ultrasound analysis at the end of
the treatment period indicates higher levels of plaque in the
vessels of individuals receiving the control peptide. This example
indicates that the individual peptides of the present invention are
effective in preventing or reducing atherosclerosis in individuals
at risk for developing atherosclerosis and in reducing plaque
accumulation in coronary, carotid or popliteal arteries.
Example 7
Administration of the Peptides of the Present Invention on Stents
to Reduce Inflammation and Restenosis
[0288] Individuals with acute coronary syndrome and having plaque
in coronary vessels which require a stent to reduce the obstruction
receive an IVUS procedure to document the coronary anatomy. A
representative protocol divides these individuals into three
groups. One group receives a stent coated with a peptide of the
present invention. A second group receives an iv infusion of a
peptide of the present invention at a dose of 2 mg/kg to 50 mg/kg,
1 to 3 times per week over a period of approximately 5 to 10 weeks.
A third group receives a stent coated with a peptide of the present
invention and an iv infusion of a peptide of the present invention
at a dose of 2 mg/kg to 50 mg/kg, 1 to 3 times per week over a
period of approximately 5 to 10 weeks.
[0289] In one study, peptides of SEQ ID NO: 142 and/or SEQ ID NO:
602 are tested.
[0290] All individuals receive a second IVUS procedure at the end
of 5 or 10 weeks. The results demonstrate that individuals
receiving either a peptide coated stent, a peptide coated stent
plus iv peptide infusion, or iv peptide infusion alone, all display
reduced inflammation and restenosis when compared to their
condition at the time of the first IVUS procedure.
Example 8
Blockade of ICAM-1/LFA-1-Mediated T-cell Adhesion to Caco-2 Cell
Monolayers by the Peptides of the Present Invention
[0291] The ability of the peptides of the present invention and
associated fragments are tested to decrease inflammation by their
ability to block the binding of ICAM-1 to LFA-1 using a model cell
adhesion assay of T cells (Mott-3) and Caco-2 cells (Anderson et
al., Bioorganic & Medicinal Chemistry Letters; 2004:14,
1399-1402). Peptide concentrations of from 0 .mu.M to 500 .mu.M are
tested. In one experiment, peptides of SEQ ID NO: 142 and/or SEQ ID
NO: 602 are tested. The results demonstrate dose dependent
inhibition of ICAM-1/LFA-1 mediated T-cell adhesion to Caco-2 cell
monolayers by the peptides of the present invention. While not
wanting to be bound by theory, it is believed that the A and/or C
domains of the peptides of the present invention are involved in
this inhibitory effect.
[0292] These results indicate that the interaction of ICAM-1 and
LFA-1 in the vessel wall can be blocked by the of the peptides of
the present invention, and result in decreased movement of
inflammatory cells, particularly T cells, from the plasma into the
vessel wall. A decrease in the influx of inflammatory cells into
the vessel wall decreases this inflammatory component of the
atherosclerotic process and decreases the frequency of clinical
vascular events (Yusuf-Makagiqansar, Inflammation: 2001;
25,203-213).
Example 9
Blockade of Neutrophils Through Inhibition of the Formyl Peptide
Receptor-like-I (FPRL1) by the Peptides of the Present
Invention
[0293] The anti-inflammatory properties of the peptides of the
present invention and associated fragments are tested by evaluating
the ability of the peptides to block the binding of neutrophils to
the formyl peptide-like 1 receptor using techniques as described by
Bae et al., (Bae et al Journal of Immunology; 2004: 173,607-614;
Bae et al., Journal of Immunology; 2003: 171,6807-6813). The
peptides of the present invention are tested in a range of 1 pM to
10 .mu.M for their ability to inhibit the binding of radiolabelled
SEQ ID NO: 609 Trp Lys Tyr Met Val MET peptide to FPRL1 expressing
RBL-2H3 cells, and for their ability to block SEQ ID NO: 609 Trp
Lys Tyr Met Val MET induced cellular chemotaxis in FPRL 1
expressing RBL-2H3 cells. In one study, peptides of SEQ ID NO: 142
and/or SEQ ID NO: 602 are tested. The peptides of the present
invention are also tested in other assays described in these two
references by Bae et al.
[0294] The results demonstrate that the anti-inflammatory
properties of the peptides of the present invention and associated
fragments to inhibit the binding of radiolabelled SEQ ID NO: 609
Trp Lys Tyr Met Val MET peptide to FPRL1 expressing RBL-2H3 cells,
inhibit SEQ ID NO: 609 Trp Lys Tyr Met Val MET induced cellular
chemotaxis in FPRL1 expressing RBL-2H3 cells, and decrease
superoxide generation.
[0295] While not wanting to be bound by theory, it is believed that
administration of the peptides of the present invention to
individuals decreases the early neutrophil influx into the vessel
wall mediated by the formyl peptide-like 1 receptor in acute
myocardial infarction or acute coronary syndrome resulting in a
decrease in the inflammatory component of atherosclerosis, thereby
reducing subsequent clinical events and post-perfusion injury.
Example 10
Use of Labelled Peptides of the Present Invention to Visualize and
Locate Plaque in Atherosclerotic Vessels
[0296] The peptides of the present invention are complexed with
phospholipids as well as gadolinium or other suitable reagent and
the recombined particle is targeted to cholesterol filled cells
which have increased expression of the ABCA1 transporter in the
vulnerable plaque of the coronary artery. In one experiment,
peptides of SEQ ID NO: 142 and/or SEQ ID NO: 602 are tested. It is
believed that the peptides of the present invention have a high
affinity for the ABCA1 transporter and are anticipated to bind to
only those cells with an increased intracellular level of
cholesterol which induced upregulation of the ABCA1
transporter.
[0297] These studies on the peptides of the present invention and
associated fragments are compared to results from studies employing
ApoA-I protein/phospholipid complex to determine the specificity
and selectivity of the peptides of the present invention versus
ApoA-I in the localization of the label to vulnerable plaque. The
use of the labeled peptides of the present invention to visualize
vulnerable plaque provides a valuable tool for diagnosis and
treatment of patients at risk for developing cardiovascular
disease. (Frias et al., J Am Chem Soc; 2004:126, 16316-7).
Example 11
Synthesis of SEQ ID NO: 602 Ac-Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln-NH.sub.2
[0298] The peptide was synthesized manually on Fmoc-Rink Amide PEG
resin via Fmoc chemistry. Protecting groups used for amino acids
were: t-Butyl group for Ser, Thr, Glu and Tyr, Trt group for Asn
and Gln, Boc group for Lys. Fmoc-protected amino acids were
purchased from EMD Biosciences. Reagents for coupling and cleavage
were purchased from Aldrich. Solvents were purchased from Fisher
Scientific. The peptide chain was assembled on resin by repetitive
removal of the Fmoc protecting group and coupling of protected
amino acid. HBTU and HOBt were used as coupling reagent and NMM was
used as base. 20% piperidine in DMF was used as de-Fmoc-reagent.
After removal of last Fmoc protecting group, resin was treated with
TFA/TIS/H.sub.2O (95:3:2) for cleavage and removal of the side
chain protecting groups.
[0299] Crude peptide was precipitated from cold ether and collected
by filtration. Purification of crude peptide was achieved via
RP-HPLC using 47 mm.times.300 mm column from Waters. Peptide was
purified using TFA Buffer. Pooled fractions were lyophilized. The
peptide has been verified by MS analysis and amino acid analysis.
The peptide purity was determined by analytical HPLC column
(Supelco C18, 4.6.times.250 mm).
Example 12
Analysis of SR-B 1-Mediated Efflux and ABCA 1-Mediated Efflux
[0300] The methods employed in this study have been described in
U.S. Pat. Nos. 7,029,863, 7,060,452, U.S. Patent Application
Publication No. 2005/0191715, and in Moya et al., Arteriosclerosis
& Thrombosis 1994:14:1056-1065 and Liu et al., J. Biol. Chem.,
2003:278(44), 42976-42984. SR-B 1 mediated cholesterol efflux was
examined in FU5AH rat hepatoma cells and ABCA1 mediated cholesterol
efflux was examined in J774 mouse macrophage cells as described in
these references.
TABLE-US-00013 TABLE 3 Efflux Assay with Peptides minus blank SR-BI
ABCA1 Mediated Mediated Efflux Efflux +ABCA1 -ABCA1 Peptides* % Per
4 h % Per 4 h Cells Cells 1 0.15 18.71 20.338 .+-. 0.136 1.624 .+-.
0.126 2 0.16 13.08 14.283 .+-. 0.545 1.205 .+-. 0.211 3 0.16 12.14
13.447 .+-. 0.549 1.306 .+-. 0.261 4 0.10 11.20 12.420 .+-. 1.019
1.224 .+-. 0.121 5 0.25 14.50 15.718 .+-. 0.123 1.215 .+-. 0.288
Legend: 1 = SEQ ID NO: 602, 2 = SEQ ID NO: 610, 3 = SEQ ID NO: 610,
4 = SEQ ID NO: 142, 5 = SEQ ID NO: 602.
TABLE-US-00014 SEQ ID NO: 602 is Ac-Ser Pro Leu Leu Glu Ser Ala Lys
Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln-NH.sub.2; SEQ ID NO: 610 is Ser Pro Leu Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln; and SEQ ID NO: 142 is Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala
Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln.
TABLE-US-00015 TABLE 4 Controls for Efflux Assay with Samples minus
blank SR-BI ABCA1 Mediated Mediated Efflux Efflux +ABCA1 -ABCA1
Controls % Per 4 h % Per 4 h Cells Cells 2% Human 8.47 12.56 24.658
.+-. 0.130 12.100 .+-. 0.485 Serum Pool Apo A-I 0.26 18.97 20.682
.+-. 0.724 1.713 .+-. 0.409 @ 20 .mu.g/ml * Efflux for all peptide
samples was run at 30 .mu.g/ml.
[0301] The results demonstrate that peptides SEQ ID NO: 610, SEQ ID
NO: 142, and the N-terminally acetylated and C-terminally amidated
form of SEQ ID NO: 142, which is SEQ ID NO: 602, each stimulated
efflux of cholesterol from J774 macrophage cells (ABCA1 pathway)
while having negligible or no effect on cholesterol efflux from the
Fu5AH cells (SRB 1 pathway), similar to the effect of Apo AI. These
selective effects of these peptides demonstrates their efficacy to
act as ApoA-I mimetics and selectively efflux cholesterol from
cells.
[0302] These effects were also dose dependent as shown in FIG. 3
with increasing efflux activity demonstrated through the range of 5
ug/ml to 30 ug/ml. Based on these in vitro efflux studies, the
elevation of the Apo AI mimetic peptides of the present invention,
in plasma, is expected to decrease coronary and other forms of
atherosclerosis in high risk patients.
Example 13
Effect of the Peptides of the Present Invention on CD11b Expression
in Monocytes Methods
[0303] Monocyte Isolation Peripheral whole blood (PWB) was drawn
from healthy consenting individuals into syringes containing sodium
citrate (final concentration-19.2 mM). Resting human monocytes were
isolated from PWB by density centrifugation with Lymphoprep (Axis
Shield). Mononuclear cells (MNCs) were collected and monocytes were
further separated to purity using the Dynal negative isolation kit
(Invitrogen). Monocytes were resuspended in phosphate buffered
saline (PBS) and cell number was determined counting cell
suspension on an automated hematology analyzer (Sysmex, KX-21N,
USA).
Purification of HDL and apoA-1 Human plasma apoA-1 was isolated as
previously described and the purity determined using total mass
spectrometry. Flow Cytometry 100 .mu.L of monocytes were stimulated
with either 1 .mu.mol/L phorbol-myristate-acetate (PMA) or 1
.mu.g/ml lipopolysaccharide (LPS) (Sigma, Australia) in the
presence or absence of apoA-1 (20 .mu.g/ml), or 20 .mu.g/ml of the
test peptide SEQ ID NO: 602 (Ac-Ser Pro Leu Leu Glu Ser Ala Lys Val
Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln-NH.sub.2, each tested separately. The cells
were incubated with the FITC conjugated antibody to either the
active epitope of CD11b (eBiosciences, USA, Clone CBRMl/5) or total
CD11b (Serotec, USA, Clone ICRF44) for 15 min at 37.degree. C.
Cells were then fixed with 4% para-formaldehyde. Samples were
controlled for by using the appropriately matched isotype matched
negative control (FITC-anti-mouse IgG) (Serotec, USA, Clone W3/25).
CD11b expression was measured by flow cytometry using FACS Calibur
(Becton Dickinson). Analysis was conducted using the Cell Quest Pro
software. Statistical Analysis Values are presented as the mean
.+-.SD or percentage of control SD. FACS results were analyzed for
statistical significance using one-way ANOVA followed by Bonferroni
post-hoc test. Significance was accepted at P<0.05. Results: As
expected, ApoA1 (SEQ ID NO: 1) significantly reduced PMA-induced
CD11b expression. In addition, SEQ ID NO: 602 significantly reduced
PMA-induced CD11b expression (FIG. 2). This result demonstrates the
anti-inflammatory properties of the peptides of the present
invention. The combined effects of increasing cholesterol efflux
and decreasing inflammation indicate that the peptides of the
present invention effectively mimic the function of Apo AI and will
decrease atherosclerosis.
Example 14
Evaluation of Peptide Utility in ApoE Knockout and LDL Receptor
Knockout Mice
[0304] ApoE knockout and LDL receptor knockout mice, two well
established animal models for the study of atherosclerosis, are
injected with either saline as control or the synthetic peptides of
the present invention to ascertain if these peptides can be used to
increase HDL and decrease atherosclerosis.
[0305] The mice receive 3 injections per week for either 4-6 or
8-10 weeks. After the completion of the injection, the amount of
hardening of the arteries or atherosclerosis is determined in the
control injected animals and peptide injected animals to determine
if the injections of the synthetic peptide decreased development of
atherosclerosis.
[0306] The proposed studies test if intraperitoneal infusions of
the apoA-1 mimetic peptides of the present invention result in
decreased aortic atherosclerosis in apoe and LDL receptor knockout
mice, two well established mouse models of atherosclerosis.
[0307] The mouse is ideal animal species for the proposed study
since well characterized and established mouse models of
atherosclerosis are readily available. In particular, apoe and LDL
receptor knockout mouse models have been universally employed as
animal models for atherosclerosis. Because they are available with
a homogenous genetic background, these knockout mice are ideal
models for analysis of atherosclerotic lesion formation which is
readily impacted by genetic background variability. Additionally,
lesion development in apoe and LDL-receptor knockout mice is
readily modified by changes in plasma lipoproteins, including HDL,
the levels of which are altered by the peptide infusion in this
study.
[0308] The knockout mouse model is a well established and widely
employed animal model for the study of atherosclerosis. Mice are
used because of their homogenous genetic background and are ideal
models for analysis of atherosclerotic lesion formation which is
readily impacted by genetic background variability. Importantly,
lesion development in apoe and LDL-receptor knockout mice is highly
affected by changes in LDL, HDL and other plasma lipoproteins.
[0309] The peptides of the present invention are synthesized
according to standard synthetic techniques using tBOC amino acids.
The peptides are purified for study by high pressure liquid
chromatography. Some peptides are N-acetylated and/or C-terminally
amidated.
Mouse Models of Atherosclerosis
[0310] Four to six week old C57B1/6 mice, apoe knockout (JAX 2052)
and LDL-receptor knockout (JAX'2207) mice, all in the C57B1/6
background, are obtained from Jackson Laboratories. During the
entire study, C57B1/6 and apoe knockout mice are maintained on a
regular chow diet (0.02% cholesterol, 3% fat) and LDL-receptor mice
are maintained on a Western diet (TD88137; Harlan Teklad; Madison,
Wis.- containing 0.20% cholesterol and 21% fat).
Infusion of Synthetic ApoA-I Mimetic Peptides
[0311] Three different infusion studies are conducted.
[0312] Aim A (Infusion Study A) to determine the functional
half-life of the injection of the synthetic peptide on plasma HDL
levels.
[0313] In the first study (Infusion Study A), C57B1/6 mice as well
as apoe knockout and LDL receptor knockout mice are injected by the
intraperitoneal (ip) route or intravenous (iv) route with synthetic
peptides of the present invention mimetic (30 mg/kg) on up to four
different occasions two weeks apart. To evaluate changes in the
plasma lipid and lipoprotein profile associated with injection of
the synthetic peptide, blood for lipid analyses is obtained before
and at 2, 4, 6, 24 and 48 hours after peptide injection. At the end
of the study the animals are sacrificed.
[0314] Aim B (Infusion Study B) To determine whether ip injection
of the synthetic peptide 3.times./wk decreases development of
atherosclerosis when assayed 4-5 weeks after initiation of
treatment.
[0315] Aim C (Infusion Study C) To determine whether ip injection
of the synthetic peptide 3.times./wk decreases development of
atherosclerosis when assayed 8-10 weeks after initiation of
treatment.
[0316] For infusion studies B and C, mice are injected ip with
either placebo or a synthetic peptide of the present invention (30
mg/kg) three times per week for either 4 to 5 weeks (Infusion Study
B) or 8 to 10 weeks (Infusion Study C). Blood for lipid and
lipoprotein analyses is obtained at the beginning of the study (day
0) and every two weeks after placebo/peptide injection and at the
completion of the study. At the completion of the study (4 to 5
weeks for Infusion Study B and 8 to 10 weeks for Infusion Study C),
the animals are sacrificed, organs harvested for analyses of
cholesterol content and for aortic atherosclerosis.
Statistical Methods Used to Analyze Data.
[0317] All statistical analyses are conducted in SAS8.2 (SAS
Institute, NC). After completion of the atherosclerosis study the
mean with standard deviation between the control (C57BI/6) and
treated group (apoE knockout or LDL-receptor knockout) is
calculated. The differences are tested by t test (PROC TTEST) and
p-values less than 0.05 are considered significant. Non-parametric
analysis of aortic atherosclerosis are performed by the
Mann-Whitney test.
[0318] In the first infusion study (Infusion Study A), 5 C57B1/6, 5
apoe knockout and 5 LDL receptor knockout mice are injected (IP)
with a synthetic peptide of the present invention and blood is
obtained for lipid and lipoprotein analyses. A total of 15 mice are
used for Infusion Study A.
[0319] A total of 40 mice (20 control-placebo injected and 20
study-peptide injected mice) are utilized in each of the two other
infusion studies (Infusion Study B-4 to 5 weeks duration as well as
Infusion Study C-8 to 10 weeks duration). Since each infusion study
is conducted in two different mouse lines (i.e.: apoE-KO and LDL
receptor KO), the total number of mice used for both Infusion
Studies B and C is 160.
[0320] Total number of mice used for the entire protocol is 175
(five-C57B1/6, eighty five-apoe KO mice and eighty five-LDL
receptor KO mice) (These animal numbers take into account an
estimated 10% morbidity rate during the course of the study as well
as the number of animals previously required to achieve statistical
significance during analysis of the non-random distribution aortic
lesion pattern that develops in mice).
[0321] For Infusion Study A, 5 four to six week old C57B1/6, apoe
knockout (JAX 2052) and LDL receptor knockout (JAX 2207) control
mice receive ip or iv injections of the synthetic peptide of the
present invention for up to four times two weeks apart. The
sequence of procedures for this study is as follows: [0322] 1) Mice
are first anaesthetized by using 1-3% isoflurane by inhalation
prior to each ip injection to insure appropriate and complete
delivery of placebo/peptide. [0323] 2) Mice are injected with
either placebo (0.2 ml saline) or apoA-I synthetic peptide (30
mg/kg in 0.2 ml saline) via either the intraperitoneal or
intravenous route. [0324] 3) In order to evaluate changes in the
plasma lipids and lipoproteins in the time-frame between
injections, each mouse in this study group is bled from the
retro-orbital sinus following administration of a topical
anesthesia. before and at 2, 4, 6, 24 and 48 hours after the
peptide injection. No more than 300 ul blood is drawn during this
48 hour period. [0325] 4) At the end of infusion study A all mice
are sacrificed by using Avertin (2.5%, 0.011 ml/gm, ip) or ketamine
(80 ug/gm, ip).
[0326] For Infusion Studies B and C, 20 control and 20 study four
to six weeks old apoe knockout (JAX 2052) and LDL-receptor knockout
(JAX 2207) mice receive ip injections of either placebo or the
synthetic peptide of the present invention three times per week for
a total of either 4-5 weeks (Infusion Study B) or 8-10 weeks
(Infusion Study C). [0327] 1) Mice are first anaesthetized by using
1-3% isoflurane by inhalation prior to each IP injection to insure
appropriate and complete delivery of placebo/peptide. [0328] 2)
Mice are injected ip with either placebo (0.2 ml saline) or apoA-I
synthetic peptide (30 mg/kg in 0.2 ml saline) on Monday, Wednesday
and Friday of each study week. [0329] 3) To measure plasma lipids
and lipoproteins, each mouse in the two study groups is fasted for
4 hours in the morning (7AM to 11 AM) and then bled from the
retro-orbital sinus at the start and end of the infusion study as
well as every two weeks after the initial infusion for a total of
either 4 weeks (Infusion Study B) or 8 weeks (Infusion Study C). No
more than 300 ul blood every two weeks is obtained from each mouse.
[0330] 4) At the end of Infusion Study B and C all mice are
sacrificed by cervical dislocation following isoflurane anesthesia
and organs are harvested for analyses of cholesterol as well as
aortic atherosclerosis. Before intraperitoneal injections, brief
inhaled analgesia is obtained by isoflurane utilizing the E-Z
Rodent Anesthesia System in the procedure room. A topical
anesthetic (proparacaine) will be applied prior to obtaining blood
from the retro-orbital sinus.
[0331] The results indicate that the synthetic peptides of the
present invention decrease aortic atherosclerosis compared to
controls. In one test, peptide SEQ ID NO: 142 Ser Pro Leu Leu Glu
Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr Lys Lys Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln or SEQ ID NO: 602 Ac-Ser Pro
Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu Glu Ala Thr
Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln-NH.sub.2 is tested
and is found to decrease aortic atherosclerosis compared to
controls.
Example 15
Evaluation of Peptide Utility in Rabbits
[0332] The isolated peptides of the present invention are examined
for anti-inflammatory activity using an in vivo rabbit model of
acute proinflammatory changes in the carotid artery. This method is
explained in detail by Nicholls et al., (Circulation 2005:111,
1543-1550). Normocholesterolemic rabbits are administered the
isolated peptides of the present invention iv in a dose of from 1
to 50 mg per day for 3 days, optionally contained in unilamellar
vesicles of phosphatidylcholine, with only unilamellar vesicles of
phosphatidylcholine with no peptide, or saline as a control. In one
test, SEQ ID NOs: 142 and 602 are administered. On the second day,
after administration of the peptides, a periarterial collar is
introduced around the carotid artery and filled with saline. Two
days later, the rabbits are humanely sacrificed and the carotid
arteries are processed and analyzed for the presence of reactive
oxygen species, the infiltration of neutrophils, and the expression
of adhesion proteins and chemokines. The administration of the
peptides of the present invention decrease the presence of reactive
oxygen species, the infiltration of neutrophils, and the expression
of adhesion proteins and chemokines compared to controls, thereby
demonstrating anti-inflammatory activity in vivo, which can help
retard the atherogenic process.
Example 16
Evaluation of Peptide Utility to Promote Reverse Cholesterol
Transport In Vivo
[0333] The isolated peptides of the present invention are examined
for the ability to release cholesterol in mice using the method
described by Zhang et al., (Circulation. 2003; 108: 661-663).
Macrophages (J774 cells) are loaded with tritiated cholesterol in
vitro and injected ip into mice. These mice are an administered
isolated peptide of the present invention, iv, at a dose of from 1
ug to 1 mg, or saline as a control. In one test, SEQ ID NO: 142 or
SEQ ID NO: 602 are administered. The peptides are administered
either in saline as a vehicle or in lipid vesicles, such as
vesicles of phosphatidylcholine. The mice receiving the peptides of
the present invention demonstrate increased levels of tritiated
cholesterol in the liver, plasma and feces, than mice receiving
saline. The results demonstrate that the peptides of the present
invention stimulate reverse cholesterol transport from macrophages
to the liver and feces.
Example 17
An Apo A-I Mimetic Peptide (SEQ ID NO: 602) Exhibits ABCA-I
Specific Efflux and Anti-Inflammatory Properties
[0334] Two functions of HDL and apo A-I, efflux of cholesterol via
the reverse cholesterol transport pathway with the ABCA-I
transporter and anti-inflammatory properties, play a pivotal role
in the reduction of atherosclerosis based on studies in mice and
humans. The ability of a synthetic amphipathic peptide, SEQ ID NO:
602, to mimic these two key functions of apoA-I was evaluated.
[0335] J774 macrophages and Fu5AH hematocytes in culture were used
to determine the specificity of SEQ ID NO: 602 to efflux
cholesterol by the ABCA-I or SRB1 pathways using the methods of de
la Llera Moya, M., V., et al. "A cell culture system for screening
human serum for ability to promote cellular cholesterol efflux.
Relations between serum components and efflux, esterification, and
transfer." Arterioscler. Thromb. 14: 1056-1065 (1994).
Anti-inflammatory properties were evaluated by determination of
inhibition of expression of activated CD11B human monocytes and
suppression of cytokine-induced vascular cell adhesion molecule-1,
V-CAM, expression in human coronary artery endothelial cells
(HCAECs). SEQ ID NO: 602 showed significant and reproducible
cholesterol efflux via the ABCA-I pathway (15.34%+2.57) comparable
to apo A-I (15.99%+2.07) with no SRB-1 pathway activity.
Lipid-complexed SEQ ID NO: 602 did not increase cholesterol efflux
by the ABCA-I pathway compared to the uncomplexed peptide, but
activity via the SRB1 pathway did increase. Inhibition of
expression of CD11B on human monocytes by SEQ ID NO: 602 was
statistically significant (p=<0.01) and comparable to apo A-I.
Initial data showed that SEQ ID NO: 602 decreases V-CAM-1 mRNA
levels in HCAECs (p<0.05).
[0336] In conclusion, SEQ ID NO: 602, a proprietary, synthetic,
amphipathic peptide designed to mimic functions of apo A-I,
demonstrated both specific ABCA-I cholesterol efflux and
anti-inflammatory effects comparable to apo A-I. Therapies based
upon an apo A-I mimetic peptide such as SEQ ID NO: 602 can be
administered to individuals in need thereof to decrease
atherosclerotic plaque formation and progression, thereby reducing
morbidity and mortality in high risk patients with cardiovascular
disease.
[0337] All patents, publications and abstracts cited above are
incorporated herein by reference in their entirety. It should be
understood that the foregoing relates only to preferred embodiments
of the present invention and that numerous modifications or
alterations may be made therein without departing from the spirit
and the scope of the present invention as defined in the following
claims. It will be apparent that the precise details of the
constructs, compositions, and methods described herein may be
varied or modified without departing from the spirit of the
described invention. We claim all such modifications and variations
that fall within the scope and spirit of the claims below
Sequence CWU 1
1
169118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn218PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 2Asn Glu Lys Leu Ala Glu
Leu Arg Ala Ala Leu Arg Gln Arg Leu Glu1 5 10 15Asp
Ser35PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Lys Leu Ser Pro Leu1 544PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Leu
Ser Pro Leu153PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Ser Pro Leu164PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Lys
Leu Ser Pro173PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Leu Ser Pro185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Leu
Pro Ser Leu Lys1 594PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 9Leu Pro Ser Leu1104PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Pro
Ser Leu Lys1113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 11Pro Ser Leu1123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Leu
Pro Ser11318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 13Leu Glu Ser Phe Lys Val Ser Phe Leu
Ser Ala Leu Glu Glu Tyr Thr1 5 10 15Lys Lys1418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser1 5 10
15Glu Leu154PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 15Leu Asn Thr Gln1163PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Asn
Thr Gln1173PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Leu Asn Thr1184PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Gln
Thr Asn Leu1193PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 19Gln Thr Asn1203PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Thr
Asn Leu121243PRTHomo sapiens 21Asp Glu Pro Pro Gln Ser Pro Trp Asp
Arg Val Lys Asp Leu Ala Thr1 5 10 15Val Tyr Val Asp Val Leu Lys Asp
Ser Gly Arg Asp Tyr Val Ser Gln20 25 30Phe Glu Gly Ser Ala Leu Gly
Lys Gln Leu Asn Leu Lys Leu Leu Asp35 40 45Asn Trp Asp Ser Val Thr
Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu50 55 60Gly Pro Val Thr Gln
Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu65 70 75 80Gly Leu Arg
Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys85 90 95Val Gln
Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met100 105
110Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln
Glu115 120 125Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu
Ser Pro Leu130 135 140Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His
Val Asp Ala Leu Arg145 150 155 160Thr His Leu Ala Pro Tyr Ser Asp
Glu Leu Arg Gln Arg Leu Ala Ala165 170 175Arg Leu Glu Ala Leu Lys
Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr180 185 190His Ala Lys Ala
Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys195 200 205Pro Ala
Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser210 215
220Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu225 230 235 240Asn Thr Gln2248PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 22Ser Pro Leu Leu Glu Ser
Ala Lys Val Ser Ala Leu Ser Ala Leu Glu1 5 10 15Glu Ala Thr Lys Lys
Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40
452348PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn Thr Gln35 40 452448PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Ser
Pro Leu Leu Glu Ser Ala Lys Val Ser Ala Leu Ser Ala Leu Glu1 5 10
15Glu Ala Thr Lys Lys Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20
25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln35 40 452541PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 25Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr
Lys Lys35 402637PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 26Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Pro Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu20 25 30Glu Tyr Thr Lys
Lys352740PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Leu Ser Pro Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys35
402839PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Ser Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys
Lys352938PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Ser Pro Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu20 25 30Glu Glu Tyr Thr Lys
Lys353038PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Pro Leu Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu20 25 30Glu Glu Tyr Thr Lys
Lys353140PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys35
403239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Leu Ser Pro Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys
Lys353341PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Leu Pro Ser Leu Lys Leu Glu Ser Phe
Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys35
403440PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Leu Pro Ser Leu Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys35
403538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 35Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Pro Ser Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala Leu20 25 30Glu Glu Tyr Thr Lys
Lys353640PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Pro Ser Leu Lys Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys35
403739PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 37Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Pro Ser Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys
Lys353839PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Leu Pro Ser Leu Glu Ser Phe Lys Val
Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys
Lys353941PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Lys Leu Ser Pro Leu Ser Asp Glu Leu
Arg Gln Arg Leu Ala20 25 30Ala Arg Leu Glu Ala Leu Lys Glu Asn35
404040PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Leu Ser Pro Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala20 25 30Arg Leu Glu Ala Leu Lys Glu Asn35
404139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 41Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg20 25 30Leu Glu Ala Leu Lys Glu
Asn354238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Ser Pro Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu20 25 30Glu Ala Leu Lys Glu
Asn354338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Pro Leu Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu20 25 30Glu Ala Leu Lys Glu
Asn354440PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Lys Leu Ser Pro Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala20 25 30Arg Leu Glu Ala Leu Lys Glu Asn35
404539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Leu Ser Pro Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg20 25 30Leu Glu Ala Leu Lys Glu
Asn354641PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Leu Pro Ser Leu Lys Ser Asp Glu Leu
Arg Gln Arg Leu Ala20 25 30Ala Arg Leu Glu Ala Leu Lys Glu Asn35
404740PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Leu Pro Ser Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala20 25 30Arg Leu Glu Ala Leu Lys Glu Asn35
404838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Pro Ser Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu20 25 30Glu Ala Leu Lys Glu
Asn354938PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Leu Pro Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu20 25 30Glu Ala Leu Lys Glu
Asn355040PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Pro Ser Leu Lys Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala20 25 30Arg Leu Glu Ala Leu Lys Glu Asn35
405139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Pro Ser Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg20 25 30Leu Glu Ala Leu Lys Glu
Asn355239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Leu Pro Ser Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg20 25 30Leu Glu Ala Leu Lys Glu
Asn355337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu
Glu Glu Tyr Thr1 5 10 15Lys Lys Pro Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu20 25 30Ala Leu Lys Glu Asn355441PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Asn
Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg Gln Arg Leu Glu1 5 10
15Asp Ser Lys Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser20
25 30Leu Phe Ser Val Lys Phe Ser Glu Leu35 405540PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Asn
Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg Gln Arg Leu Glu1 5 10
15Asp Ser Leu Ser Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu20
25 30Phe Ser Val Lys Phe Ser Glu Leu35 405639PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Asn
Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg Gln Arg Leu Glu1 5 10
15Asp Ser Ser Pro Leu Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe20
25 30Ser Val Lys Phe Ser Glu Leu355738PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Asn
Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg Gln Arg Leu Glu1 5 10
15Asp Ser Ser Pro Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser20
25 30Val Lys Phe Ser Glu Leu355838PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 58Asn Glu Lys Leu Ala Glu
Leu Arg Ala Ala Leu Arg Gln Arg Leu Glu1 5 10 15Asp Ser Pro Leu Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser20 25 30Val Lys Phe Ser
Glu Leu355940PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Asn Glu Lys Leu Ala Glu Leu Arg Ala
Ala Leu Arg Gln Arg Leu Glu1 5 10 15Asp Ser Lys Leu Ser Pro Lys Lys
Thr Tyr Glu Glu Leu Ala Ser Leu20 25 30Phe Ser Val Lys Phe Ser Glu
Leu35 406039PRTArtificial SequenceDescription of Artificial
Sequence
Synthetic peptide 60Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Leu Ser Pro Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe20 25 30Ser Val Lys Phe Ser Glu
Leu356141PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 61Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Leu Pro Ser Leu Lys Lys Lys Thr Tyr
Glu Glu Leu Ala Ser20 25 30Leu Phe Ser Val Lys Phe Ser Glu Leu35
406240PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Leu Pro Ser Leu Lys Lys Thr Tyr Glu
Glu Leu Ala Ser Leu20 25 30Phe Ser Val Lys Phe Ser Glu Leu35
406338PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Pro Ser Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser20 25 30Val Lys Phe Ser Glu
Leu356438PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Leu Pro Lys Lys Thr Tyr Glu Glu Leu
Ala Ser Leu Phe Ser20 25 30Val Lys Phe Ser Glu
Leu356540PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Pro Ser Leu Lys Lys Lys Thr Tyr Glu
Glu Leu Ala Ser Leu20 25 30Phe Ser Val Lys Phe Ser Glu Leu35
406639PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Pro Ser Leu Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe20 25 30Ser Val Lys Phe Ser Glu
Leu356739PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 67Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Leu Pro Ser Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe20 25 30Ser Val Lys Phe Ser Glu
Leu356837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg
Gln Arg Leu Glu1 5 10 15Asp Ser Pro Lys Lys Thr Tyr Glu Glu Leu Ala
Ser Leu Phe Ser Val20 25 30Lys Phe Ser Glu Leu356941PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 69Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser1 5 10
15Glu Leu Lys Leu Ser Pro Leu Asn Glu Lys Leu Ala Glu Leu Arg Ala20
25 30Ala Leu Arg Gln Arg Leu Glu Asp Ser35 407040PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser1 5 10
15Glu Leu Leu Ser Pro Leu Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala20
25 30Leu Arg Gln Arg Leu Glu Asp Ser35 407139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser1 5 10
15Glu Leu Ser Pro Leu Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu20
25 30Arg Gln Arg Leu Glu Asp Ser357238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Lys
Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser Val Lys Phe Ser1 5 10
15Glu Leu Ser Pro Asn Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg20
25 30Gln Arg Leu Glu Asp Ser357338PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 73Lys Lys Thr Tyr Glu Glu
Leu Ala Ser Leu Phe Ser Val Lys Phe Ser1 5 10 15Glu Leu Pro Leu Asn
Glu Lys Leu Ala Glu Leu Arg Ala Ala Leu Arg20 25 30Gln Arg Leu Glu
Asp Ser357440PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 74Lys Lys Thr Tyr Glu Glu Leu Ala Ser
Leu Phe Ser Val Lys Phe Ser1 5 10 15Glu Leu Lys Leu Ser Pro Asn Glu
Lys Leu Ala Glu Leu Arg Ala Ala20 25 30Leu Arg Gln Arg Leu Glu Asp
Ser35 407539PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 75Lys Lys Thr Tyr Glu Glu Leu Ala Ser
Leu Phe Ser Val Lys Phe Ser1 5 10 15Glu Leu Leu Ser Pro Asn Glu Lys
Leu Ala Glu Leu Arg Ala Ala Leu20 25 30Arg Gln Arg Leu Glu Asp
Ser357641PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Leu Pro Ser Leu Lys Asn Glu Lys Leu
Ala Glu Leu Arg Ala20 25 30Ala Leu Arg Gln Arg Leu Glu Asp Ser35
407740PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Leu Pro Ser Leu Asn Glu Lys Leu Ala
Glu Leu Arg Ala Ala20 25 30Leu Arg Gln Arg Leu Glu Asp Ser35
407838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 78Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Pro Ser Asn Glu Lys Leu Ala Glu Leu
Arg Ala Ala Leu Arg20 25 30Gln Arg Leu Glu Asp
Ser357938PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Leu Pro Asn Glu Lys Leu Ala Glu Leu
Arg Ala Ala Leu Arg20 25 30Gln Arg Leu Glu Asp
Ser358040PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Pro Ser Leu Lys Asn Glu Lys Leu Ala
Glu Leu Arg Ala Ala20 25 30Leu Arg Gln Arg Leu Glu Asp Ser35
408139PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Pro Ser Leu Asn Glu Lys Leu Ala Glu
Leu Arg Ala Ala Leu20 25 30Arg Gln Arg Leu Glu Asp
Ser358239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Leu Pro Ser Asn Glu Lys Leu Ala Glu
Leu Arg Ala Ala Leu20 25 30Arg Gln Arg Leu Glu Asp
Ser358337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Lys Lys Thr Tyr Glu Glu Leu Ala Ser Leu Phe Ser
Val Lys Phe Ser1 5 10 15Glu Leu Pro Asn Glu Lys Leu Ala Glu Leu Arg
Ala Ala Leu Arg Gln20 25 30Arg Leu Glu Asp Ser358444PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 84Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20
25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys35
408544PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 85Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln35 408641PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 86Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Pro Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala Leu Glu20 25 30Glu Tyr Thr Lys Lys Leu Asn
Thr Gln35 408740PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 87Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys
Lys35 408847PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 88Pro Leu Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln35 40 458946PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 89Leu
Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10
15Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40
459045PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu
Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe
Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln35 40 459147PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 91Ser Pro Leu Ser Asp Glu Leu
Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr35 40 459246PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20
25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn35 40
459345PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu35 40 459446PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 94Pro Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Lys Leu
Ser Pro Leu Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr35 40 459545PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Pro
Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10
15Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser20
25 30Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn35 40
459644PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu35 409743PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 97Pro Leu Ser Asp Glu Leu Arg Gln Arg
Leu Ala Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Lys Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys35 409845PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr35 40 459944PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 99Leu
Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10
15Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn35
4010043PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 100Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu
Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu35 4010142PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 101Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Lys Leu Ser Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys35 4010244PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 102Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr35 4010343PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10
15Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu20
25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn35
4010442PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 104Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg
Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu35 4010547PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 105Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Leu Ser Pro
Leu Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4510646PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40
4510745PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Ser Pro Leu Glu Ser
Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln35 40 4510845PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 108Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg
Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Leu Leu Glu Ser Phe Lys Val
Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln35 40 4510947PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 109Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser
Pro Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4511046PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 110Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Leu Ser Pro Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40
4511148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Leu Pro Ser Leu Lys
Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln35 40 4511247PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 112Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser20
25 30Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35
40 4511345PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Ser Leu Glu Ser
Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln35 40 4511445PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 114Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Pro Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4511547PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Pro Ser Leu Lys Leu Glu Ser Phe Lys Val Ser20
25 30Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35
40 4511646PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Ser Leu Leu Glu
Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn Thr Gln35 40 4511746PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 117Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Pro Ser Leu Glu Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4511842PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Leu
Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10
15Lys Glu Asn Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu20
25 30Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 4011944PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10
15Glu Asn Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser20
25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35
4012045PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln35 40 4512143PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 121Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Ser Pro
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr
Thr Lys Lys Leu Asn Thr Gln35 4012242PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 122Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10
15Glu Asn Pro Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu20
25 30Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 4012346PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 123Pro
Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10
15Leu Lys Glu Asn Lys Leu Ser Pro Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40
4512444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 124Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Ser Pro Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn Thr Gln35 4012545PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 125Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Leu Pro Ser
Leu Lys Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4512646PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 126Pro
Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10
15Leu Lys Glu Asn Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40
4512743PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 127Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Pro Ser Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln35 4012843PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 128Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Pro Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr Gln35 4012946PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 129Pro Leu Ser Asp Glu Leu
Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Pro
Ser Leu Lys Leu Glu Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4513044PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 130Leu
Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10
15Lys Glu Asn Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser20
25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln35
4013143PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 131Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg
Leu Glu Ala Leu Lys1 5 10 15Glu Asn Leu Pro Ser Leu Glu Ser Phe Lys
Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys Lys Leu Asn
Thr Gln35 4013242PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 132Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn35 4013344PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 133Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala
Leu Glu Glu Tyr Thr Lys Lys Leu35 4013442PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 134Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys35 4013544PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 135Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Ser Pro Leu Glu Ser Phe Lys Val Ser Phe Leu20
25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr35
4013643PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys Lys
Leu Asn35 4013744PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 137Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser
Pro Leu Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu35 4013842PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 138Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Ser Pro Leu Glu Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys35 4013947PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 139Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Pro Ser Leu Lys Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser
Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr35 40
4514045PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Leu Pro Ser Leu Leu
Glu Ser Phe Lys Val Ser20 25 30Phe Leu Ser Ala Leu Glu Glu Tyr Thr
Lys Lys Leu Asn35 40 4514142PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 141Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Pro Ser Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu35 4014244PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 142Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Pro Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr35 4014346PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 143Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Pro Ser Leu Lys Leu Glu Ser Phe Lys Val Ser20
25 30Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr35 40
4514444PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Ser Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Pro Ser Leu Leu Glu
Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys
Lys Leu Asn35 4014543PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 145Ser Pro Leu Ser Asp Glu
Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn
Leu Pro Ser Leu Glu Ser Phe Lys Val Ser Phe20 25 30Leu Ser Ala Leu
Glu Glu Tyr Thr Lys Lys Leu35 4014643PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 146Pro
Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10
15Leu Lys Glu Asn Leu Ser Pro Leu Leu Glu Ser Phe Lys Val Ser Phe20
25 30Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu35
4014741PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu Asn Ser Pro Leu Leu Glu Ser
Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr Lys
Lys35 4014842PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 148Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Ser Pro Leu Glu Ser
Phe Lys Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr Thr Lys Lys
Leu Asn Thr35 4014941PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 149Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Pro Leu
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr
Thr Lys Lys Leu Asn35 4015041PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 150Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Pro
Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala20 25 30Leu Glu Glu Tyr
Thr Lys Lys Leu Asn35 4015142PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 151Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Lys Leu
Ser Pro Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu35 4015240PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 152Leu Ser Asp Glu Leu Arg
Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Ser
Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu
Tyr Thr Lys Lys35 4015344PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 153Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Leu Pro Ser
Leu Lys Leu Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr35 4015442PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 154Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5
10
15Glu Asn Leu Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser20
25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn35 4015539PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 155Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10
15Glu Asn Pro Ser Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu20
25 30Glu Glu Tyr Thr Lys Lys Leu3515643PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 156Leu
Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu1 5 10
15Lys Glu Asn Pro Ser Leu Leu Glu Ser Phe Lys Val Ser Phe Leu Ser20
25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr35
4015742PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 157Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala
Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Pro Ser Leu Glu Ser Phe
Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys Lys Leu
Asn35 4015841PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 158Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Pro Ser Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys
Lys Leu35 4015940PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 159Leu Ser Asp Glu Leu Arg Gln Arg Leu
Ala Ala Arg Leu Glu Ala Leu1 5 10 15Lys Glu Asn Leu Pro Ser Leu Glu
Ser Phe Lys Val Ser Phe Leu Ser20 25 30Ala Leu Glu Glu Tyr Thr Lys
Lys35 4016048PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 160Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4516148PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 161Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20
25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln35 40 4516248PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 162Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4516348PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 163Ser
Pro Leu Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu1 5 10
15Ala Leu Lys Glu Asn Lys Leu Ser Pro Leu Leu Glu Ser Phe Lys Val20
25 30Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln35 40 4516441PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 164Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr
Lys Lys35 4016541PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 165Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr
Lys Lys35 4016641PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 166Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr
Lys Lys35 4016741PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 167Ser Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys1 5 10 15Glu Asn Lys Leu Ser Pro Leu Leu
Glu Ser Phe Lys Val Ser Phe Leu20 25 30Ser Ala Leu Glu Glu Tyr Thr
Lys Lys35 4016848PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 168Ser Pro Leu Ser Asp Glu Leu Arg Gln
Arg Leu Ala Ala Arg Leu Glu1 5 10 15Ala Leu Lys Glu Asn Lys Leu Ser
Pro Leu Leu Glu Ser Phe Lys Val20 25 30Ser Phe Leu Ser Ala Leu Glu
Glu Tyr Thr Lys Lys Leu Asn Thr Gln35 40 4516937PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 169Ser
Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys1 5 10
15Glu Asn Pro Leu Glu Ser Phe Lys Val Ser Phe Leu Ser Ala Leu Glu20
25 30Glu Tyr Thr Lys Lys35
* * * * *